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0004666f-919b-4eaa-ac9b-a02a0e3c589b	# HUMAN CULTURES THROUGH THE SCIENTIFIC LENS Pascal Boyer Human Cultures through the Scientific Lens oBP www.openbookpublishers.com HUMAN CULTURES THROUGH This volume brings together a collection of seven articles previously published by the author, with a new introduction reframing the articles in the context of past and present questions in anthropology, psychology and human evolution. It promotes the perspective of 'integrated' social science, in which social science questions are addressed in a deliberately eclectic manner, combining results and models from evolutionary biology, experimental psychology, economics, anthropology and history. It thus constitutes a welcome contribution to a gradually emerging approach to social science based on E. O. Human Cultures through the Scientific Lens spans a wide range of topics, from an examination of ritual behaviour, integrating neuro-science, ethology and anthropology to explain why humans engage in ritual actions (both cultural and individual), to the motivation of conflicts between groups. As such, the collection gives readers a comprehensive and accessible This volume will be a useful resource for scholars and students in the social sciences (particularly psychology, anthropology, evolutionary biology and the political sciences), as This is the author-approved edition of this Open Access title. As with all Open Book publications, this entire book is available to read for free on the publisher's website. Printed and digital editions, together with supplementary digital material, can also be found at introduction to the applications of an evolutionary paradigm in the social sciences. Photo by Marc-Olivier Jodoin on Unsplash at https://unsplash.com/photos/-TQUERQGUZ8 well as a general readership interested in the social sciences. THE SCIENTIFIC LENS Essays in Evolutionary Cognitive Anthropology Wilson's concept of 'consilience'. Pascal Boyer http://www.openbookpublishers.com Cover Design by Anna Gatti book ebook and OA editions also available e # Essays in Evolutionary Cognitive Anthropology Pascal Boyer # To access digital resources including: blog posts videos online appendices and to purchase copies of this book in: hardback paperback ebook editions Go to: https://www.openbookpublishers.com/product/1424 Open Book Publishers is a non-profit independent initiative. We rely on sales and donations to continue publishing high-quality academic works. # HUMAN CULTURES THROUGH THE SCIENTIFIC LENS # Human Cultures through the Scientific Lens Essays in Evolutionary Cognitive Anthropology Pascal Boyer https://www.openbookpublishers.com © 2021 Pascal Boyer This work is licensed under a Creative Commons Attribution 4.0 International license (CC BY 4.0). This license allows you to share, copy, distribute and transmit the text; to adapt the text and to make commercial use of the text providing attribution is made to the authors (but not in any way that suggests that they endorse you or your use of the work). Attribution should include the following information: Pascal Boyer, Human Cultures through the Scientific Lens: Essays in Evolutionary Cognitive Anthropology. Cambridge, UK: Open Book Publishers, 2021, https://doi.org/10.11647/ OBP.0257 In order to access detailed and updated information on the license, please visit https:// doi.org/10.11647/OBP.0257#copyright. Further details about CC BY licenses are available at http://creativecommons.org/licenses/by/4.0/ All external links were active at the time of publication unless otherwise stated and have been archived via the Internet Archive Wayback Machine at https://archive.org/web Digital material and resources associated with this volume are available at https://doi. org/10.11647/OBP.0257#resources Every effort has been made to identify and contact copyright holders and any omission or error will be corrected if notification is made to the publisher. ISBN Paperback: 9781800642065 ISBN Hardback: 9781800642072 ISBN Digital (PDF): 9781800642089 ISBN Digital ebook (epub): 9781800642096 ISBN Digital ebook (mobi): 9781800642102 ISBN XML: 9781800642119 DOI: 10.11647/OBP.0257 Cover photo: Marc-Olivier Jodoin on Unsplash at https://unsplash.com/photos/-TQU ERQGUZ8 Cover design by Anna Gatti # Contents # 1. Anthropology, Useful and Scientific: # An Introduction The essays gathered in this volume were all intended as contributions to what I would like to call a useful and scientific anthropology, two words that may seem a tad presumptuous and require an explanation. First, the useful part. The essays address specific questions such as the following: These are all questions of some social importance. It is not difficult to see that it would be a Good Thing, so to speak, to make progress in addressing such issues. I do not claim that the essays gathered here are more useful than other attempts in the social sciences, but simply that the main motivation here is indeed to be useful, to provide models and findings that help us move closer to a proper explanation of these phenomena. That is the goal, the ambition, if perhaps not the actuality. What about 'scientific'? In my view, the main way for scholarship to be useful, indeed useable, in these domains, is to proceed in a scientific manner. By using this term, I certainly do not mean to claim or imply that the various statements contained here are true. In fact, making such a claim would be quite the unscientific thing to do. The implication is simpler and more modest, meaning that the models proposed can and should be examined in terms of empirical data, and that they may be found to be false or in serious need of revision on the basis of such data. In all these essays we adopt the perspective of an 'integrated' social science, that addresses questions about cultures and societies in a deliberately eclectic manner, combining results and models from evolutionary biology, experimental psychology, economics, anthropology and history (Morin, 2016; Sperber, 1996; Tooby & Cosmides, 1992). This approach is sometimes derided as 'positivistic' and 'reductionistic', and that is exactly what it is. It is blithely reductionist (explaining what happens at a high level of complexity in terms of the combinations of simpler, lower-level elements) and mostly positivist (if the term simply denotes the scientific aspiration). # Why Science Isn't and Should Not Be True to Life To some people, it may seem that this way of describing and explaining social phenomena robs them of much of their substance. The models may be compelling but they miss out the rich texture and detail of actual social interactions. We talk about rituals in general without considering the particular and highly varied social contexts in which they take place; we examine people's views of economic processes, but we ignore the subtle individual differences in their construction; we consider widespread assumptions about madness, but not how they are modulated in each case… to these objections, the proper reply would be: Yes, YES! We do that, and that is exactly what we should do. Far from being a problem, the exclusion of so much information is precisely the main virtue of this way of proceeding. The point will seem quite obvious to some and strikingly wrongheaded to others. For some people, doing science consists in discovering 'what really happens', beyond error, prejudice and received wisdom. Scientists are seen as people who describe things the way they really are. So it seems that one's theories should always be 'true to life.' That is very misleading. In some sense, of course, scientific theories are 'true to life' because evidence is the only tribunal that judges right and wrong. An embarrassing, unexplained fact carries more weight than a satisfactory, elegant theory, and that is what makes scientific activities so frustrating sometimes. In another sense, scientific theories are not, cannot be, and should not be 'true to life.' Producing a theory does not mean taking into account all possible aspects of the phenomena you describe. On the contrary, it means that you focus on some aspects that can be described in terms of abstract generalizations, assuming, for the sake of simplicity, that all other aspects are 'equal'. The notion of 'all else being equal' seems entirely natural and compelling to some people; and it seems utterly alien to many others. As the Russian writer Alexander Zinoviev put it, the two styles of thinking are diametrically opposed: 'the scientific principle produces abstractions, the anti-scientific principle destroys them on the grounds that such and such has not been considered. The scientific principle establishes strict concepts, the anti-scientific principle makes them ambiguous on the pretext of thus revealing their true variety' (Zinoviev, 1979, p. 209). # Why Social Science Is Impossible (Or Nearly So) Where do we stand, in our understanding of social phenomena? How much do we know? It often seems like we are nowhere near where we should be, given the amount of available information about human cultures and history. Analogies with other sciences are certainly difficult, but it may seem that we are at the same stage as chemistry was, say around the beginning of the nineteenth century. At the time, chemists had at their disposal a vast number of facts about different substances and their interactions, but very little by way of a systematic understanding of these facts. Why would an acid and a base combine to form water and a salt? (For that matter, the distinction between acids and alkali would have been difficult to explain). One obstacle on the way to social science is, as it turns out, human minds themselves. The problem is that, in a sense, we already have all sorts of ideas about societies, what could be called a 'folk-sociology' (Boyer, 2018, pp. 216–237). Folk-sociology consists in a set of partly tacit assumptions, that we all use when trying to describe or explain social facts and processes. For instance, one major feature of our folk-sociology, found in the most diverse societies, is that we spontaneously construe human groups as agents. We talk about villages or social classes or nations as entities that want this, fear that, make decisions, fail to perceive what is happening, reward people or take revenge against them, are hostile towards other groups, and so on. All these terms suggest that, in some implicit way, we consider that what happens in social groups is very much the same as what happens in a human mind. Another assumption of folk-sociology is that power is a kind of substance attached to particular individuals, and its operation is analogous to a physical force. This is manifest in such phrases as 'she has power,' 'she lost power,' 'his power increased,' and so on. This is not just a Western or European way of speaking. Such metaphors are familiar from many tribal societies, chiefdoms, and early states. We say that people 'have' and 'exercise' power. We conceive of someone with power as able to 'push' others toward certain behaviors (as a physical force can move objects), we say that people who did not follow the leader are 'resisting,' that they are not 'swayed', they resent being 'pushed around', etc. These conceptions of social facts and processes are based on loose and misleading conventional metaphors (Lakoff & Johnson, 1980). We vaguely perceive that social groups are not literally agents and that power is not literally a force, but it is very difficult to think outside the metaphors. Try to describe political power without ever using notions like 'pushing' and 'resisting'; or try to describe international relations without ever saying that 'Russia wanted this' and 'England realized that…', and so forth. Indeed, the metaphors are so entrenched that they may seem self-evidently true—which is why some social scientists, in the past, tried to argue that nations really were like agents and political power really was a force. Now folk-sociology is a real hindrance, when you try to think about cultural phenomena in a scientific manner, because it hides the very problems we should try to solve. Seeing the nation or the ethnic group as agents conceals difficult questions, such as: why do people favor their group against others? Why would people behave as loyal members of an ethnic group, rather than defect to another one? In the same way, seeing power as a force makes it impossible to describe the complicated dynamics, whereby the preferences of some people (the leaders) seem to have effects on the behaviors of others (the followers). The notion of power as force indeed makes it impossible to understand how power relations change: why was the East German communist party so powerful in 1988 and so powerless in 1990? Can we really discard folk-sociology? It is difficult for two reasons. The first one is that our social understandings are largely implicit. As the old saying goes, it is difficult to reason people out of something they were not reasoned into. The view that power is a force, for instance, is not usually an explicit, conscious representation of what political power consists of. A second, more familiar reason is that our ideas about society are not just a matter of detached consideration. They guide our own social interaction, and what happens in that interaction does matter to us. While abandoning your folk-theories in the domains of physics or biology does not come at much of a price, giving up on some ill-conceived notion of political power or gender roles may be a more delicate affair. If all this is true, then doing social science in the scientific manner might seem well-nigh impossible. There may be both a natural inclination and some strong incentives not to consider social and cultural processes in scientific terms. On the contrary, there may be powerful reasons to adopt and preserve theories that are not entirely coherent, or do not have much supporting evidence, simply because they fit both our intuitive expectations and our particular projects. That may explain why the results are decidedly mixed, why we are very much in the same position as chemists before Galton. While we can admire the great insights of luminaries like Montesquieu or Ibn Khaldun, the prospect of a cumulative social science seems to recede almost as fast as we proceed. # Why Cultural Stability Is a Mystery A standard answer to many questions in social science, such as those listed at the beginning of this introduction, is that people have particular mental representations, e.g., about what rituals to perform, or what the economy is like, or what is morally repugnant behavior, because those notions 'are in their culture'. So, the fact that you consider, e.g., the economy as a large pie that can be divided in different ways, or a shaman's rituals as required in order to combat witchcraft, these are notions that 'are in the culture' which would explain why people entertain them. That cannot be a very good explanation, because it is not an explanation at all. To adopt a phrase from physicists, it is not even wrong. It makes little sense to say that most Zulu people like spicy foods, or that Mongols consult shamans because those preferences are in their culture—because what we mean when we say that some notion is 'in the culture' is simply that it is common among people in a particular place. So we are in effect saying that many Zulu people like spicy foods because many Zulu people like spicy foods. That is not a good start. The only way that kind of strange statement could make sense would be if we assumed that ideas and values, representations and preferences, are always transmitted identically from generation to generation. That is, we might be implying that Mongols resort to shamanism because previous Mongols did that too. In this sense, 'it is in their culture' would mean 'they adopted whatever their forebears did'. That would be almost reasonable. Of course, it would also be largely false. Cultures change as much as they persist. But at least we are now talking about something that is not entirely tautological, and in fact introduces the most important theme in the study of cultures: What is 'in the culture' depends on what is transmitted from one individual to another. That is of course an old idea, but it is only very recently that social scientists took it seriously enough to build formal models of what is now called 'cultural evolution'. A convenient date of birth for that movement might be the publication of Culture and the Evolutionary Process by Boyd and Richerson (1985). The starting point of the model was that cultural material comes in different packets of information, called memes, transmitted from individual to individual. The notion of memes had originally been proposed by Richard Dawkins (1976), and it then formed the starting point of many attempts to describe cultural material. In this selectionist perspective, trends in cultural evolution, for instance, the persistence of a particular tradition or its downfall, the fact that some ideas can diffuse to large communities or on the contrary remain confined to a few individuals all stems from the relative selective success of different memes. This way of thinking transposed to cultural material the successful models of genetic evolution by random mutation and selective retention. There was a limitation in these selectionist models, however. Memes were construed as abstract realities that replicate by passing from one mind to another, but there was no explanation of how that happened. Or, people assumed that 'imitation' would be the explanation. This was consistent with another one of our folk-sociological assumptions, namely, that human cultures are by default stable. Social scientists for a long time assumed that there was nothing special to explain in the fact that many Venetian and Xhosa customs or ideas were very similar to what the Venetians and the Xhosa of the previous generation had been doing or thinking. In that view, stability is not mysterious, in fact it is invisible! And only change requires a special explanation. But it is stability that is mysterious. The Xhosa views about marriage or agriculture are conveyed through a vast number of communicative interactions between individuals. But human communication is a place of high entropy—it resembles a game of Chinese Whispers more than serial photocopying. What you get at the end is very different from the beginning, not just because of distortion, but mostly because of reconstruction (Morin, 2016). Distortion does happen when you make copies of copies of copies… but in Chinese Whispers, each individual in the chain is trying to construct something that would make sense, given what they heard. Human communication, even about 'cultural' matters like marriage or agriculture, is even more entropic, as people are in many cases not even trying to reproduce what they heard. The 'epidemiology of culture' promoted by Dan Sperber and others (and illustrated in several of the essays in this volume) assumed that these facts about human communication were crucial for understanding the apparent stability of some aspects of human cultures, or the fact that different individuals across space and time seem to have roughly similar mental representations (Sperber, 1985). Human communication has to be reconstructive, because much of what is conveyed is not said and need not be said. That is true of the simplest everyday conversations, as studies in linguistic pragmatics demonstrate (Grice, 1991). Sperber and others argued that this fact was essential to understanding human cultures. What makes them stable or changing is not the 'memes', the explicit statements and gestures, but the way these are completed, in the minds of the receivers, with all sorts of additional content (Sperber, 1985). So, where does stability come from? The main factor here is not imitation or repetition, but similarities in the 'additional content' I just mentioned. That is where the view of communication inherited from pragmatics was combined with a view of the human mind promoted by cognitive psychology (Tooby & Cosmides, 2005). Human mental capacities were no longer described as a unified, multi-purpose computer that would absorb what the environment threw at it, but as a series of learning systems shaped by natural selection, and specialized in handling recurrent challenges of ancestral environments—how to find nutrition and avoid predators, for sure, but how to find the best possible mate, how to recruit social support, how to defend one's group against enemies, and many more, as described by what is now called evolutionary psychology (Buss, 2016). # Why Social Science Is Possible after All: A Field Without a Name I of course assume that, against the odds, we can build scientific accounts and that we are in fact gaining ground in our models of human cultures. In this volume, my co-authors and I consider what could be described as questions of political science (What makes institutions stable, and compelling?), cultural anthropology (Why perform rituals? How do people detect mental illness?), sociology (How does ethnicity impact health?) and economics (Do people's view of the economy match their economic behavior?). The list may seem a tad disparate, but it is not haphazard. These questions all spring from a common way of seeing human cultures, as the product of the interaction of evolved human capacities and preferences with variable environments. We take seriously the fact that natural selection provides not just an explanation for what we know of human nature, but also a source of rich hypotheses for what is still to be discovered. We also take as self-evident that economic models and game theory provide rich models for interactions between agents, that experimental psychology or neuroscience are the best sources for understanding human minds, and that the variation in human norms and concepts provides a wonderful opportunity to describe the envelope of human nature. Is there a discipline that studies all that? Not if the term 'discipline' denotes traditional academic divisions. But those matter less and less to actual scholarly projects. Our field-without-a-name is making great progress, and it will prove both scientific and useful. # References # Introductory Note One of the most enduring and most damaging assumptions in the social sciences is the belief that it makes sense to talk about nature and culture, or to part the 'innate' from the 'acquired' in describing human behavior. Almost as misguided is the recommendation that we should describe behavior as some combination or mixture of these elements—an insipid counsel for moderation that only results in a stubborn incuriosity about what is being 'mixed' and how (Pinker, 2002). Against all this, many biologists, anthropologists and psychologists have, for decades, tried to illustrate how these oppositions dissolve, when we consider human capacities and preferences from an evolutionary standpoint (Ridley, 2003; Tooby & Cosmides, 2010). It is part of mankind's evolved nature that we can acquire from our conspecifics vast amounts of information that constitute our ecological niche (Tooby & DeVore, 1987). This is possible because genetic selection fashioned a whole suite of learning mechanisms that orient the growing mind's attention to specific cues in the environment, and govern that mind's inferences. That is how we can acquire detailed and valuable information about, e.g., the physical relations between solid objects, the invisible beliefs and intentions that explain agents' behaviors, the nature of the social bonds between people around us, the syntax of the local language, the best ways to extract resources from the natural world or to establish cooperation and garner social support. All this requires extensive learning, which requires extensively prepared systems—for a survey, see Boyer (2018, pp. 1–30) and Tooby & Cosmides (1992). How does this relate to the study of institutions? To be more specific, Michael Petersen and I were trying to address the very general question, why do people adopt some institutions as quite 'natural', in the familiar sense, while others are much less compelling? Why is marriage apparently so self-evident, that in most cultures throughout history, no-one needed an explanation for it? Why would the rules of a deliberative democracy be a much more fragile construction? We can describe institutions as the 'rules of the game' in complex social interaction (North, 1990). These rules can be very different, from time to time and place to place. From that diversity, many people would conclude that genetic evolution by natural selection is irrelevant. But historical or cultural differences are, just like commonalities, an outcome of our evolved dispositions (Sperber & Hirschfeld, 2004). That is what Michael Petersen and I tried to illustrate in this article, using the contrasted cases of marriage institutions, criminal justice, and commons management as our examples. These display vast cultural and historical differences, and in fact some institutions are only found in some human societies. But in these different cases and, we would argue, many more besides these, we can see highly intuitive specific expectations at play, which make some parts of the local, historically specific institutional arrangements very easy to acquire, which in turn makes it relatively easy for people to coordinate their behaviors around common rules. The intuitive expectations are shaped by evolved learning systems, and in turn they shape the various institutions. An important consequence of this model is that explanations of institutions are, by necessity, domain-specific. For instance, cultural rules about marriage are strongly constrained by human intuitions about mating, about the ways humans combine sexual access, care for their offspring and economic cooperation. By contrast, judicial rules are influenced by our moral intuitions and expectations concerning cooperation. So, to explain two different domains of institutions, we need to investigate two separate mental systems, each of which has its own domain of application, its computational rules, and its associated emotions. That is why general models or theories of institutions are, in our view, incomplete. True, political scientists and economists have put forward important models of, e.g., the conditions under which there is demand for and supply of institutional rules, especially in complex modern societies—in the article we discuss some of these, especially from the neo-institutional economics literature. But institutions are not just systems of rules, they are also systems of rules mentally represented by individuals—in fact, in many cases they consist in individual mental representations about the mental representations of other individuals (Heintz, 2007). That is why, at some point in our explanations, we must consider the role of evolved domain-specific intuitions, which means that we leave aside a general theory of institutions and produce theories of particular kinds of institutions. # References # The Naturalness of (Many) Social Institutions: Evolved Cognition as their Foundation1 with Michael Bang Petersen Abstract: Most standard social science accounts only offer limited explanations of institutional design, i.e., why institutions have common features observed in many different human groups. Here, we suggest that these features are best explained as the outcome of evolved human cognition, in such domains as mating, moral judgment and social exchange. As empirical illustrations, we show how this evolved psychology makes marriage systems, legal norms and commons management systems intuitively obvious and compelling, thereby ensuring their occurrence and cultural stability. We extend this to propose under what conditions institutions can become 'natural', compelling and legitimate, and outline probable paths for institutional change given human cognitive dispositions. Explaining institutions in terms of these exogenous factors also suggests that a general theory of institutions as such is neither necessary nor in fact possible. What are required are domain-specific accounts of institutional design in different domains of evolved cognition. <sup>1</sup> An earlier version of this chapter was originally published as Boyer, P., & Petersen, MB. (2011). The naturalness of (many) social institutions: Evolutionary and Cognitive Background, Journal of Institutional Economics 8(1): 1–25, https://doi.org/10.1017/ S1744137411000300. Reprinted with permission of Cambridge University Press. # 1. Introduction General accounts of social institutions should provide plausible and testable answers to questions of institutional design, such as, why do social institutions have the specific features that we observe in human societies? Why do we observe common institutional features in otherwise very different cultural environments? Or, why do some institutions seem natural and compelling to participants, while others are considered alien or coercive? Here, we develop the view that present institutional theories do not properly address such design questions, and that this can be remedied only by taking into account what we call the 'naturalness' of institutions, their connection to human expectations and preferences that result from evolution by natural selection. This perspective may help us understand commonalities across cultures, but also why some institutions are more successful and compelling than others and why they change in particular directions. To some extent, this suggestion echoes a defining feature of the neo-institutional approach. From the beginning, neo-institutionalism has been oriented towards developing realistic models of the actors, countering the Homo economicus model inherent in older institutional accounts and emphasizing the cognitive limits of human decision makers (Brousseau & Glachant, 2008). From this perspective, important lines of inquiry have been developed with regards to, first, how institutions carry a range of unintended consequences given the cognitive limits of their designers, and, second, how a function of institutions is to counter such limits (North, 1990). At the same time, however, this perspective of bounded rationality provides only a partial description of human cognition. While one line of research within the cognitive sciences has been preoccupied with the biased and fallible nature of human cognition, a complementary line of research has developed the view that human cognition is in fact 'better than rational' (Cosmides & Tooby, 1994). Evolutionary psychologists have argued that human cognition includes a multitude of domain-specific cognitive programs, each optimally geared (within evolutionary constraints) to solve particular problems in the course of human evolutionary history (Barkow, Cosmides, & Tooby, 1992). The inferential power of these specialized programs comes from their content-rich nature. That is, they are loaded with inbuilt assumptions about their domain. Environments that fit these inbuilt assumptions appear intuitive and readily understandable. Our aim is to outline the argument that institutions are effective not despite human cognition but, in part, because of human cognition. Essentially, we argue that the content-rich nature of evolved intuitions provides a foundation which can be and is often used in the design of many social institutions. Institutions that fit these intuitions, we propose, develop more easily, require less effort to conform to, and are more culturally stable. While evolutionary psychology is increasingly incorporated into social theory (Alford & Hibbing, 2004; Hodgson, 1999; McDermott, 2006; Petersen, 2010), and some economists have been keen to integrate an evolutionary logic into their models (Dopfer, 2005; Enright, 1984), many social scientists may be unfamiliar with the approach. By way of developing our account, we therefore present a series of illustrations of how our knowledge of human evolution and cognition provides the tools for a causal, naturalistic understanding of social institutions such as marriage rules and norms, legal systems and social exchange mechanisms. In each instance, our point of departure will be the existence of specific cross-cultural features in the design of these institutions and how these can be seen as the institutionalization of evolved intuitions. From this, we show how these insights can inform the study of institutions, the naturalness of (many) social institutions and develop a range of novel predictions on how institutions develop and change. # 2. Explaining Common Features We focus here on named social institutions, that is, sets of norms and rules in which all culturally competent members of a group have explicit, accessible mental representations. For instance, football in England, marriage in the USA, potlatch among the Tlingit or meeting for Quakers are social institutions of the kind we consider here. The important point here is that people have some notion that, for example, there is such a thing as potlatch in their social environment and they have some notion of how observed behaviors can be seen as exemplars of these abstract notions, or violations of their rules (Searle, 1995). These named bundles of concepts, norms and behaviors are what we call 'institutions' in the rest of this article (Ostrom, 2005). This is only a subset of 'institutions' in the neo-institutional sense, some of which remain implicit, such as, for example, a sense of fairness or simple habits. Institutional models generally emphasize the contribution of both formal and informal aspects of such institutions, the former including laws, contracts, administrative rules and procedures, while the latter include implicit norms and routines. Here, we want to explore the cognitive processes that underpin both aspects of institutions. An institution such as 'marriage' in the USA combines legal norms and emotional preferences, contracts and moral intuitions; in short, both formal and informal aspects. The question for us is, what makes certain 'packages' of informal and formal norms natural and compelling to participants? In many domains, fairly similar institutions can be found in diverse cultural environments. For instance, despite obvious differences, many human groups know of interaction norms that (seemingly) correspond to what an English speaker would call 'marriage' (we will discuss, presently, whether that similarity is an illusion). An interesting fact is that such diverse institutions share not just very general properties, for example, conditions and limits of sexual relationships and parenting, but also many other features, for example, the association between long-term sexual intimacy and economic solidarity, the fact that the union is in principle exclusive, the fact that its inception requires public ceremonies, etc. These are common features, most of which may not be universal, but all of which are so widespread that this recurrence requires an explanation. In the social sciences, different frameworks suggest very different ways of considering institutions and their common features. For instance, a culturalist account is the default position of much anthropological reflection on institutions. In this view, the latter are the way they are because they are congruent with the particular concepts, values, norms, etc. widespread in a particular place (Gudeman, 1986). A recurrent problem of anthropological culturalism is, of course, the presence of recurrent features of social institutions, which in a purely localist framework would have to constitute massively improbable coincidences. This is true for marriage but also for the emergence of similar patterns in, for example, commons management, sports or political structures. To a large extent, classical functionalist accounts are fraught with similar problems. They require that most institutions emerge as solutions to particular classes of problems or situations, and survive to the extent that they fulfill that role in congruence with other institutions. However, again, this has proved insufficient to account for the recurrence of particular institutions (e.g., raising children in families) compared with other, possibly efficient alternatives (e.g., raising children in kibbutzlike communities) (Merton, 1996). A more promising account of institutions appeared in economics with the development of neo-institutional models. These extended the notion of institution to encompass both formal and informal, tacit or explicit 'rules of the game' that constrain interaction between economic agents (North, 1990). These rules decrease transaction costs and information costs in particular. Within the neo-institutional tradition, there are different accounts of how institutions are created. Since there is not the space to review such models in detail, for such surveys, see Knight and Sened (1995), North (2005) and Brousseau and Glachant (2008). Briefly, conventional accounts assume that institutions emerge out of the recurrent features of repeated economic interactions—they are simply regularities turned into rules (Sugden, 1986). Competitive accounts suggest that institutions develop out of original, small-scale norms by conferring competitive advantages to newcomers who participate in the new arrangement (Greif, 2006). Finally, bargaining models emphasize power asymmetries between parties in the creation or modifications of institutions (Knight, 1995). However, none of these accounts point to easy, natural answers to questions of design. Whether specific institutions are shaped by bargaining or convention is not sufficient to account for highly specific features, such as, for example, the link between intention and responsibility in the law, or the connection between economic sharing and sex in marriage norms. Here, we present a complement to neo-institutional accounts. Institutions comprise rules or norms that most agents obey, expect most others to obey and expect most others to expect them to obey (Bicchieri, 2006). But, for a rule or a norm to become an institution, it must be widely distributed in the minds of the members in a group (Sperber, 1996). In order to explain how institutions are developed and changed, we therefore need to understand how people adopt, modify and transmit rules and norms (Heintz, 2007). Most importantly, we need to understand which types of rules and norms are particularly likely to be transmitted and adopted without much modification, while others require significant effort, skill and special knowledge. This leads to our main contention, that institutions are best understood against the background of a set of human psychological dispositions that influence the effort needed to adopt and accept certain social arrangements. To introduce this cognitive account of institutions, we illustrate how our evolved psychology makes an impact on the developments of common features in three different domains of institutional design. # 3. Illustration (I). Marriage Norms and Mating Strategies #### Institutional Framework In most societies, there is a distinction between occasional or informal sexual encounters and arrangements (which may be approved, tolerated, frowned upon, prohibited, etc.) and more stable and formalized unions. The initiation of the latter kind of union is generally, at least in principle, marked by some public event. There are shared norms about what each party should expect from the other, given such ceremonies, and about how they should behave towards third parties. Finally, sanctions are associated with the violation of these norms. Why is all this so common? A standard, and plausible initial answer would be that such norms reduce uncertainty in social interaction, a general feature of social institutions. Marriage norms and wedding ceremonies certainly have that effect, in several ways. First, marriage between two individuals conveys to third parties that the individuals concerned have rights in each other that (mutatis mutandis the local norms) are not available to other members of the group. There is, for example, a certain amount of resources or help that a husband may expect from a wife or vice versa, or a woman from her in-laws, but not from others. Second, marriage conveys to third parties that the individuals concerned have (again, with local variations) withdrawn from the pool of potential mates. The fact that there is a long-term stable union between the partners modifies third parties' mating strategies and preferences towards either one of the partners. Ceremonies do not just signal this to a large number of people, but also inform them all at the same time in the same way. Third, marriage conveys to each party that the other is (at least overtly) committed to fulfilling their obligations as per the local norms. Regardless of intentions, the public commitment signal creates expectations against which either party can measure behaviors. In other words, the most important effects of weddings seem to consist in signaling. In all human societies, weddings are of interest and great concern to outsiders, which is why for instance the ceremonies are often quite literally very noisy affairs (van Gennep, 1909). Internal signals between the married parties are equally important. The potential benefits of an efficient marriage are in part public goods, and in many cases cannot be achieved without sacrifices, given the spouses' divergent preferences (E. Posner, 2000). So marriage requires honest, hard-tofake signals of commitment. These are provided in many societies by costly conditions for marriage, for example, the obligation for brides to leave their kin groups, for grooms to provide bride wealth, or to show adequate means to support a family, etc. Such conditions serve (in part) as signals, which may explain why, when they are relaxed as happened in many Western societies, they are replaced with informal signals such as occasional gifts (E. Posner, 2000). The occurrence of informal, 'spontaneous' signals varies inversely with the precision of the groupwide representation of marriage roles. All this is fine, but falls short of a satisfactory answer to questions of design. Coordination and uncertainty-reduction effects do not explain why marriage is universally about two parties, so that polygamy is a series of two-party contracts, not a group arrangement; why polygyny is common and polyandry exceedingly rare; why a single institution binds sexual, economic and offspring-related norms in most societies; why divorce is often available but fixed-term marriage is generally not; and other such common features. #### Evolutionary Background The institutions may make more sense in the context of specific preferences and competences that arise from human evolution. Obviously, natural selection results in particular mating preferences and processes in each species, and ours can be no exception (Symons, 1979). It would be surprising if human competences and preferences in this domain had no influence on the design of marriage. Indeed, human reproduction and parenting display expected features given the specific history of our primate lineage (Van Schaik & Van Hooff, 1983). As in most other mammals, there is in humans a large asymmetry of reproductive costs between human males and females. The latter's costs include a long gestation, an even longer nurturing period, with their associated energy and opportunity costs, as compared with the lesser cost incurred by males. This, as in comparable species, means that sexual selection has been important in favoring distinct behaviors and preferences in males and females. Females need to be much choosier than males in mate selection. Also, females should prefer males with demonstrable capacity and willingness to nurture their offspring. Differences between male and female preferences result in an equilibrium that includes relatively long-term paternal investment in children's nurturing, conditional on fathers' certainty that the children are their own biological offspring, as well as a certain but limited amount of philandering and 'mate poaching' in both sexes. These evolutionary factors predict a whole variety of human behaviors which are actually observed in most human societies, such as: the general disposition towards long-term paternal investment; sexual differences in attractiveness criteria (Buss, 1989); differences between criteria for long- and short-term mates (Kaplan & Gangestad, 2005); the ways in which attractiveness is turned off by childhood cohabitation and other cues, leading to incest avoidance (Lieberman, Tooby, & Cosmides, 2003); the specific triggers of and gender differences in sexual and romantic jealousy (Buss, 2000); mechanisms of sexual coercion and attempts by men to control women's sexuality and increase paternal certainty (Wilson & Daly, 1998); the general pattern of serial exclusive monogamy (and polygyny) observed in human societies (Van Schaik & Van Hooff, 1983); the male tendency to reject step-children (Anderson, Kaplan, Lam, & Lancaster, 1999; Anderson, Kaplan, & Lancaster, 1999; Daly & Wilson, 1988); the influence of male presence/absence on young women's choices of reproductive strategies (Ellis, 1993); and many other behaviors. Naturally, most computations required by such behaviors are largely unconscious — only their results are available to conscious inspection. To cite but one example, it seems that women's choice of early sexual activity and early pregnancy are directly affected by paternal presence during a critical period in early childhood (Ellis et al., 2003). This can be explained as learning from the environment which reproductive strategy is most appropriate, given low paternal investment in offspring (Quinlan, 2003). Obviously, young women never represent reproductive choices as a search for optimal fitness. They are responding to such proxies as the attractiveness of particular mates or a desire for children, and other here-and-now preferences for particular kinds of behaviors, all of which are the outcome of non-conscious cognitive processes. #### An Integrated Perspective An important point here is that human preferences and behaviors in the mating domain include the expectation of stable long-term unions between men and women that associate privileged or exclusive sexual access with economic solidarity. Note that this is largely intuitive, that is, most humans hold this expectation without necessarily having the explicit model or principles that would explain it. Also, this expectation is of course more abstract than the norms for such long-term unions in particular societies, which can vary in many respects such as number of people involved (polygyny versus monogamy), exclusiveness (e.g., societies with sanctioned 'visiting' lovers), required paternal investment in offspring (from full responsibility to occasional visits) and, most important, filiation and inheritance rules. All this suggests that human beings are equipped with an evolved, domain-specific learning system that is prepared for and attentive to information about the particular ways in which marriage unions are locally defined and organized. In this perspective, the transmission of culturally specific information about marriage norms 'free-rides' on information supplied by our intuitive expectations. That is, people easily acquire their local marriage norms because the assumptions (e.g., that the union is heterosexual, that it is about long-term mating, that it associates sexual access with resource sharing, etc.) are among the evolved cognitive equipment of the species. This would explain why many aspects of marriage norms are not the object of explicit, deliberate transmission, and seem to 'go without saying' while others are the object of explicit norms. For instance, the assumption that marriage binds a man and a woman is intuitive enough that it is not actually specified in most cultures. By contrast, the permissible number of simultaneous unions, or the precise manner in which they can be broken up, are matters of explicitly transmitted information. The expectations that married people will contribute to each other's welfare, or that an officially declared union must be officially dissolved, do not have to be made explicit. In this perspective, the social institution seems to consist in particular parameter settings of a marriage template that is spontaneously created by normally developing minds. # 4. Illustration (II). Criminal Law and Moral Intuitions #### Institutions The emergence of 'the law' as a separate domain of norms and behaviors, distinct from other social norms, is confined to large polities with literacy (Goody, 1986; Maine, 1963). However, most human groups do have explicit norms for conflict resolution and the punishment of wrongs, even if these are not defined as different from ordinary, non-legal decision making (Hoebel, 1964). From these norms and procedures to the literate, codified legal systems of large states, there is a continuum of social complexification, along which some central aspects of legal norms are preserved. Legal systems all modify personal, faceto-face conflict resolution on the basis of norms that are: (a) explicit; (b) (at least partly) de-contextualized (e.g., construed as the right way to sanction theft, rather than this particular act of theft); (c) (at least partly) impersonal, as they in principle apply to whole classes of agents or even to all possible agents; and (d) therefore more predictable than informal ones. Why these common features? One possible explanation is that legal institutions are just economically efficient sets of conventions. Richard Posner, for instance, considers that standard economic models of utility maximization explain most features of legal systems (R. A. Posner, 1981). Economic rationality would account for differences between the custom-bound legal systems of small-scale traditional societies, and the legal codes of large-scale industrial polities. For instance, the former generally maintain strict (no-fault) liability, so that one (or one's kin group) is responsible for whatever damage one has caused, whether or not one is guilty of a wrong or of negligence. This, as Richard Posner argues, makes sense in economic systems where the cost of information is particularly high, so that long inquiries into circumstances and intentions would be problematic. In the same way, the fact that litigants are generally asked to pay for arbitrage, in other words to hire a judge, makes sense as there is no institution for the public provision of magistrates (R. A. Posner, 1981). Economic efficiency can certainly account for specific differences between the legal norms of various places, but it seems insufficient to explain the common features of these systems and the ways in which people generally find them compelling (Cosmides & Tooby, 2006). This is particularly clear in the domain of criminal justice, where apparently obvious features of the institutions, e.g., tacit assumptions about the relative severity of different crimes, only make sense against the background of cooperation in ancestral conditions. The naturalness of (many) social institutions arrangements are based on complex intuitive assumptions about behavior, intentions and fairness. #### Relevant Cognitive Systems In the last 20 years, convergent findings in developmental psychology, behavioral economics and cross-cultural psychology have suggested that human beings in very different groups evaluate the moral valence of actions on the basis of largely tacit, emotion-laden common intuitions (Haidt, 2007). Intuitive morality is independent from (and only partly affected by) explicit, culturally specific understandings of and teachings about right and wrong (Greene, 2005). Intuitive morality also underpins a sense of fairness that is quite distinct from economic rationality (McCabe & Smith, 2001). Rather than survey these models and findings, we will only mention those points directly relevant to the issue of criminal behavior and appropriate punishment. Human minds in a variety of cultural environments develop the following specific intuitive processes. First, there is a domain of moral principles and norms, distinct from other evaluative dimensions of action. Indeed, even preschool children have definite intuitions about the difference between moral rules and mere social conventions (Elliot Turiel, 1994). Second, the judgment that a behavior is permissible, commendable or wrong occurs as a fast, automatic consequence of representing the specific action and context. These intuitions may then be explicated, nuanced or (more rarely) reversed by explicit reasoning, but the latter is quite literally an afterthought — deliberate, slow and often produced in order to justify a pre-existing intuition. Third, intuitive moral appraisals are generally accompanied by congruent emotions. Emotional appraisal is part of the processes leading to moral evaluation, which is why experimental manipulations of the emotion can trigger significant changes in moral judgment (Haidt, 2001). All this is particularly visible in young children's moral development. In contrast to the classical, Kantian picture of children gradually building moral understandings by acquiring more complex modes of reasoning (Kohlberg, 1981), experimental evidence suggests that moral development consists in the calibration of prior intuitions (E. Turiel, 2002). Experimental evidence also shows that people are intuitively convinced that wrong behaviors vary in seriousness — that much is assumed by young children even for completely novel behaviors (E. Turiel, 2002). Another common intuition is that the punishment should fit the crime, as it were — such that a schedule of graded punishments is required (Nichols & Knobe, 2008). Again, these thoughts are not entertained as the result of deliberate reasoning on moral matters, but as the intuitions that start the process of moral reasoning. Another important aspect of moral intuitions is a motivation to punish norm violators, even in third parties who are not harmed by the transgression. This preference is not based on learning from trial and error, since the potential consequences of either punitive or nonpunitive strategies are manifest only in the long run. Such punitive attitudes are universal in human groups and virtually non-existent in other animals. There are various interpretations for this evolutionary novelty. Punitive sentiments may have helped recruitment to collective action (Yamagishi, 1992). They may also signal cooperative attitudes, as those who punish transgressors are signaling their attachment to local norms and their willingness to incur costs in their defense (Fessler, 2001), which would explain why people tend to be more punitive when observed by others (Robinson, Kurzban, & Jones, 2007). Finally, punitive attitudes may be an attempt to eliminate the fitness advantage enjoyed by free-riders (Price, Cosmides, & Tooby, 2002) or recalibrate their motivations (Petersen, Sell, Tooby, & Cosmides, 2010). What is certain is that the motivation for third-party punishment is general in human groups, and strong enough to override the cost involved. #### An Integrated Perspective In the same way as for marriage, evolved psychological capacities and processes constrain legal norms. They provide a set of understandings that need not be explicitly transmitted as a condition for participation, and therefore make institutions 'learnable' to the extent that they are congruent to intuitive understandings (Cosmides & Tooby, 2006). Legal institutions do not require that one learn concepts of right and wrong, the need for appropriate sanctions, or that one acquire the motivation for third-party punishment. Also, the ways in which legal institutions publicize decision making seems to derive from moral intuitions. As we noted, people have definite intuitions about the role of reputation in cooperation. It may be no surprise that legal institutions turn reasoning and decision making, ordinarily private mental events, into publicly scrutable processes. Courts work in the open, laws are inscribed in stone or in books, and penalties are made visible, for instance, by using stigma as a salient form of punishment (Kurzban & Leary, 2001; E. A. Posner, 2007). All these aspects of the law seem self-evident to most practitioners, as indeed they should be if they are based on common pre-existing intuitions. In return, institutions do modify social interactions in the legal domain. Obviously, the existence of public representations of norms and processes make punishment more predictable and the domain of lawful behavior more easily delineated, which translates as an advantage in transaction costs (R. A. Posner, 1981). But the effects may be even deeper, as most people tend to reify or essentialize the law as independent from actual people's decisions and the workings of their minds. To the extent that the motivations for particular judgments seem both stable and impersonal, they reinforce this tacit form of legal idealism, a notion that laws are not made but discovered, which itself may make them more compelling. # 5. Illustration (III). Commons and Exchange Intuitions The cognitive framework may also make sense of some common features of particular economic institutions. Consider, for instance, Elinor Ostrom's description of the principles that allow efficient management of common-pool resources such as fisheries, water distribution, etc., in which a resource must be pooled and might be depleted by opportunistic unregulated use (Ostrom, 1990). According to Ostrom, the following principles are necessary, though not sufficient, to preserve the semi-formal institutions that manage commons: (1) some rules must clearly define the set of agents authorized to use the commons and the conditions for entry; (2) the rules must be adapted to the specific nature of the resource; (3) the rules must be designed by the users; (4) rule observance must be monitored by the users or agents accountable to the users; and (5) rule violation must be sanctioned by graded punishment (Ostrom, 1990). Why are commons institutions the way they are, and why these recurrent features? An institutional account does not directly address them, as it is focused on different issues, both theoretical (showing how efficient commons-management systems emerge despite collective action problems) and pragmatic (deriving recommendations for efficient commons management). All the rules mentioned above require a complex background of psychological processes and preferences. For one thing, commons management implies definite judgments about distributive justice, about which divisions of resources count as acceptable, given different agents' contributions or needs (Fehr, Schmidt, Kolm, & Ythier, 2006). Psychologists have shown that such judgments are mostly based on early developed intuitions (Enright, 1984). Young children in very diverse cultures use similar principles of distributive justice, combining a principle of equality (equal shares as the best distribution) with context-based intuitions about merit and need (Sigelman & Waitzman, 1991). Obviously, these early judgments are then calibrated during development as a function of local forms of exchange. But the underlying principles subsist. They result in specific fairness intuitions that cannot be explained in terms of standard rational choice models (Fehr et al., 2006). The cognitive mechanisms required for commons management also include the capacity and motivation to identify violators of agreed norms. Experimental findings suggest that people are specifically sensitive to cheating (taking benefits without paying costs in a social contract) and quickly identify which behaviors constitute cheating. The underlying cognitive system is domain specific, in the sense that social contract violations are not processed in the same way as violations of social norms in general, or exceptions to other kinds of rules (Cosmides & Tooby, 2005). As Ostrom and others have demonstrated, efficient use of commons requires a whole lot of specific 'tools' (institutions in the neo-institutional framework) such as rules, norms and models to overcome collective action problems. However, these tools need not be provided by the institutions themselves. To a large extent, norms and rules 'free-ride' on competencies and motivations for fair exchange that are part of our evolved cognitive equipment. # 6. What Are Evolved Domain-Specific Systems? ### Evolved Systems as Specialized Learning and Decision Mechanisms The perspective developed in relation to these three examples highlights how institutional designs are directly facilitated by the structure of human cognition. In this way, they complement the focus of previous accounts of the interplay between cognition and institutions. In the extant literature, the focus has been on the general cognitive limitations of human cognition and how the latter affect the workings of institutions. One strand of argument has been preoccupied with how the fallibility of institutional designs can be traced back to the fallibility of the cognitive capabilities of their designers (Pierson, 2004). Another strand of research has focused on how institutions can buffer the limits of human cognition (Knight & North, 1997). Hence, institutions — refined through trial and error — provide external constraints on behavior which simplify individual choice and guide it toward rational outcomes. A third strand of research has focused less on the limits of fixed cognitive processes but rather argued for the plasticity of cognitive processes and how they are molded by the institutional environment of the individual (Dequech, 2006). While these avenues toward integrating insights on human cognition and institutions are highly important, they are based on an incomplete description of the current state of knowledge in cognitive science. In our view, the content (and not just the limits) of a variety of special and species-typical cognitive systems, as observed by evolutionary anthropologists and psychologists, is relevant to issues of institutional design and maintenance. As we noted above, human beings have an intuitive mating psychology that includes attractiveness judgments, relationship maintenance and reproductive strategies (Buss, 1989; Symons, 1979). They have specialized social exchange mechanisms for cheater- and cooperator-detection (Cosmides & Tooby, 2005) and a highly specific moral psychology (Haidt, 2007). They also have a coalitional psychology which monitors the establishment and maintenance of groups with common interests, vigilance towards defection, rivalry towards other groups, etc. (Kurzban & Neuberg, 2005), as well as systems that monitor ethnic cohesion and attitudes towards others (Schaller, 2006) or gender relations (Sidanius & Veniegas, 2000; Wilson & Daly, 1992). In fact, sketching the range of evolved cognitive mechanisms underlying common human behaviors would be far beyond the scope of this article (for general surveys, see (Buss, 2005); (Dunbar, Barrett, & Lycett, 2005)). Several features of these cognitive systems are of particular relevance here: Cognitive systems are domain specific. Cognitive predispositions are not just general constraints, for example, on the amount of material that can be acquired, on the capacity of attention and memory. Cognitive predispositions also consist in domain-specific expectations about the kinds of objects and agents to be found in the world. Only some items of information trigger operation of a specific system, in much the same way as only molecules of a particular shape and composition trigger the activity of specific enzymes (Barrett, 2005). Many such narrow input–output relationships are species-typical fixations rather than plastic features that can be molded by environmental processes (Tooby & Cosmides, 1992). Each domain-specific system includes its own decision-making procedures. In most standard models of economists and political scientists, one assumes that people's behavior is guided by a domain-general, utilitymaximizing cognitive system. In those cases in which human behavior does not conform to normative models, this is said to result from general limitations of the decision-making system, for instance because of biases (Kahneman, Slovic, & Tversky, 1982) or impulsiveness (Ainslie, 2005; Loewenstein & O'Donoghue, 2005). These putative flaws in decision making are thought to be domain general — they would occur in the same way in, say, keeping friends and keeping lovers, avoiding enemies and avoiding pathogens. However, psychological evidence suggests a different picture, in which each domain-specific system (e.g., concerned with retaining mates, or with recruiting coalitional allies) comes with its own, domain-appropriate decision rules. For instance, if you are dealing with a contractor for house repairs, it may make sense to average the benefits and costs from previous interactions with that specific agent, in order to compute their overall value to you. If dealing with a lover, it would seem intuitively odd to balance cases of infidelity with cases of availability. Even when we use rules of thumb or 'fast and frugal heuristics', these are tailored to the kinds of problems we evolved to encounter (Gigerenzer, 2002; Gigerenzer, Todd, & Group, 1999). Competencies and preferences are integrated. This is a consequence of the previous point. Each domain-specific cognitive system includes its own decision-making procedures which combine specific preferences and specific competencies. There is no reason to consider that preferences are external to decision-making systems. For instance, consider human coalitional psychology, our capacity to form an alliance with genetically unrelated agents, usually against other groups. It includes as part of a single package both a set of preferences (e.g., a strong aversion for other agents' defection, a desire to make the coalition stronger, a willingness to pay a high price of entry, etc.) and a set of competences (e.g., the monitoring skills to detect other agents' commitment, the signaling skills to express one's solidarity, etc.). Cognitive systems are learning mechanisms. Each domain-specific system is specialized in picking up particular kinds of information in the organism's environment. Contrary to widespread assumptions outside evolutionary biology, 'acquired information' and 'genetically specified information' are not a zero-sum system. On the contrary, organisms that can acquire vast amounts of information from their environments (e.g., primates) need vastly more specified initial systems than organisms (e.g., invertebrates) that acquire less. Between species, more learning invariably means more 'instinct', so to speak. Humans have a complex coalitional psychology and a complex mating psychology, which means that in both domains they acquire enormous amounts of information from their social and natural environments precisely because sophisticated learning systems in these domains are specified by their genotypes. #### Implications for Interaction with Environments These features have a number of implications for the operations of human cognition which are highly important to institutional researchers. Here, we focus on two implications. We begin by outlining them in relative broad terms, review some of the evidence for these implications in the cognitive science literature and then specifically apply them to institutional analysis. Cognitive systems operate more reliably in matching environments. Cognitive systems are designed to operate within a specific domain and, therefore, the inbuilt assumptions, categories, competences and learning procedures reflect the evolutionarily recurrent structure of that exact domain. When cognitive systems are applied to problems on the fringes of their proper domain, they will operate less automatically and less reliably. This has been directly shown in cognitive research. For example, we have evolved to hunt prey and protect ourselves against predators and, therefore, most probably have specialized cognitive systems designed to track the movements of animals, their orientation, and their most likely trajectory (Barrett, 2005a). In modern societies, predatory animals constitute less a threat than, for example, cars but this does not mean that we can effortlessly apply the systems designed for tracking the former to the latter. And, in fact, Joshua New et al. showed that subjects are far slower to recognize changes in car orientation than the orientation of animals—even when these animals are visually unfamiliar, unimportant, and barely discernible (New, Cosmides, & Tooby, 2007). Similarly, we detect violations of rules faster and more reliably when detecting violations corresponds to detecting cheaters on social exchanges (Ermer, Cosmides, & Tooby, 2007). As argued above, rule violation in the context of social exchange is the exact domain of a set of highly specialized cognitive systems while, most likely, generic rule violations are not. This insight is also directly applicable to actual design situations. Human–computer interfaces require much less effort on behalf of the user if these interfaces correspond to the structure of cognitive systems designed for handling real-world objects (Nørager, 2009). The structure of cognitive systems creates a baseline motivation to shape environments into a format that matches them. Because environments that fit our cognitive systems can be processed effortlessly (given the possibility for reliably applying evolved categories, competences, etc.), people will find matching environments more 'natural' and their exigencies more compelling. Also, creating and upholding non-matching environments require a level of effort that individuals could be unwilling to pay without special incentives. Again, a number of studies in cognitive science have directly demonstrated this. Most of these studies have focused on an extreme version of the above principle, in the sense that these studies have shown that we simply process and react toward non-matching environments as if they were matching. In the domain of popular culture, one obvious example is pornography that is psychologically represented as if mating opportunities were present, thereby triggering sexual arousal, etc. (Saad & Gill, 2014). Similarly, research shows that people have difficulties in distinguishing between their real friends and people they see on television in the sense that their satisfaction with their friendships is influenced by both (Kanazawa, 2002). These cognitive effects also occur in the direct interaction with others. For example, modern individuals process the anonymous one-shot interactions of mass society (an evolutionary novel phenomenon) as if they were of the iterated kind to which we have most probably adapted (Hagen & Hammerstein, 2006; Price et al., 2002). #### Predictions concerning Institutional Design One may object that this is fine but insufficient. The evolutionary — cognitive model may solve some issues of design, telling us what rules are 'natural', easy to acquire and intuitively compelling for human beings. But, the objection goes, it does not address the major question of institutional development, i.e., why are these cognitive tools and motivations activated in some but not all contexts? Why, as a result, are some commons successful and others less so? Since the cognitive tools are always present, why are they not always used? Institutions are a part of the external environment of individual actors and, hence, are processed with the same cognitive effects as other parts of the environment. The application of the above insights to institutional analysis is, therefore, straightforward. Doing so should enable researchers to build specific predictions about (a) which institutions or aspects of institutions people are more likely to find 'natural', (b) to what extent people can have an intuitive grasp of the actual workings of their institutions, and (c) how institutional participation can recruit motivations that are there anyway, regardless of the institutional environment. In essence, we suggest that the structure of evolved cognitive systems and dispositions create a cognitive 'rubber cage' (Gellner, 1985). That is, human understandings are usually constructed and therefore constrained by the structure of long-evolved cognitive systems, and remain inside the cage, as it were. It is not impossible to think beyond our intuitive assumptions or to build institutions that violate them. However, each such extension requires some effort, and the further one moves away from intuitive expectations, preferences and understandings, the more effort is required (Boyer, 1998; Sperber, 1996). The further away one moves from our evolved understandings, (a) the more effort will be required to get them adopted by large numbers of people, (b) the less people will intuitively grasp how the institution works, and (c) the less motivated they may be to participate. Because divergence from the intuitive set of design features requires effort, such divergence will be less common than convergence (at least, absent other strong environmental pressures for divergence). Of course, this is likely to be a matter of degree. For instance, it is not too difficult for some human groups to extend the scope of marriage-like institutions to, say, encompass homosexual unions. A more radical departure from common intuitions would be to envisage fixed-term marriage contracts or simultaneous polyandric unions. Although such arrangements are not unthinkable or impossible, they are less likely than standard marriagelike systems in human societies, given the intuitive assumptions that normal human minds spontaneously develop about the connections between sex, reproduction and subsistence. Within this approach, it is also possible to specify a number of other specific predictions: People will prefer intuitive to non-intuitive institutions. Intuitive institutions, quite simply, seem more natural and appropriate to people. For people to prefer a non-intuitive solution to a problem that mimics something which our cognitive architecture was designed to solve, effort is required on their behalf and they need good reasons to put in this extra effort. Some research has specifically shown this in the domain of punishment institutions. As argued above, punishment has most probably played a key role for evolution of human social life. Recent studies in neuroscience demonstrate that brain regions related to the production of pleasure are activated when subjects engage in the punishment of free-riders (de Quervain et al., 2004). Also, economic experiments have demonstrated that people prefer to tackle collective action problems in institutional contexts that allow for punishment (Gürerk, Irlenbusch, & Rockenbach, 2006). We can observe such effects outside the laboratory as well. Throughout the twentieth century, criminal justice institutions have shifted from punishing to helping the offender (Garland, 1990). Politically, this shift was legitimized by references to criminologists and other experts' observations that punishment did not work to reduce crime in large societies. In this way, criminal justice institutions were pulled away from their intuitive function — to impose costs on anti-social individuals (Petersen et al., 2010)—and instead designed to simply decrease recidivism in a nonmoralizing manner. Across countries, however, these attempts have now been significantly reversed and an explicit part of this has been public reactions led by, for example, victim movements. When they reverted to more punitive practices, policy makers often made clear that the reversal aimed at placating public sentiment rather than decreasing crime (Balvig, 2005). In our terms, the return to punitive rhetoric and practices made the institution closer to our evolved intuitions. This example also illustrates another point: that intuitiveness is especially important for institutions that are directed toward the public. Worries about rehabilitation-oriented systems did not come from within the penal system itself, whose personnel were quite willing to invest the needed effort to think outside their intuitions. Rather, the pressure came from the general public who have much else on their minds than investing cognitive effort in overwriting their punishment intuitions (Roberts, Stalans, Indermaur, & Hough, 2002). More intuitive institutions are more efficient in influencing behavior. This follows from our argument that intuitive institutions are easier to process. It is important to notice that this is not just because it is easier to learn some kind of institutional rules than others but rather because intuitive institutional rules simply require less learning on behalf of the subjects. For instance, criminal justice practices that rely on evolved concepts of right and wrong influence public behavior, we suggest, not because citizens have uploaded legal knowledge in their minds but precisely because evolved expectations spare them that effort. While laymen, for example, do not know the specific punishments for shoplifting versus grievous assault, their behavior can be guided by an intuitive understanding that the latter is more serious than the first (Robinson et al., 2007). An example from the health sector serves to illustrate this. To avoid poisoning of children, a government-funded health program in the USA encouraged parents to mark poisonous materials with a 'Mr. Yuk' sticker, an emoticon with the facial expression of disgust, to signal that the material should not be ingested. The effectiveness of this program was negligible (Demorest, Posner, Osterhoudt, & Henretig, 2004). One factor is that accidental poisoning does not fall within the evolved domain of disgust but rather within the domain of fear — i.e., hazard management. Adults as well as children react to accidental poisoning with fear rather than disgust (Pooley & Fiddick, 2010). Marking poisonous material with disgust-conveying emoticons does not engage the cognitive machinery for producing the very behavior that the institution aims at activating. Our claim here, it must be stressed, is not that 'natural' institutions are necessarily more efficient in terms of generating optimal outcomes. Efficiency is here strictly understood with reference to their power to influence behavior and not whether the resulting behavior is optimal or rational. In fact, given that our evolved cognitive systems evolved in ancestral environments, institutions that seem 'natural' to the human mind might often be ill-suited to solve the problems of modern-day mass society (Carvalho & Koyama, 2010). More intuitive institutions seem more legitimate. In the domain of social and moral interaction, institutions that promote our welfare in an intuitive way (i.e., by promoting behavior that would have been ancestrally beneficial and sanctioning behavior that would have been ancestrally costly to us) would be, all else being equal, perceived as more legitimate. As classical sociologists have emphasized, legitimacy is at the root of effective governance (Tyler, 2001). If rules are perceived as legitimate, individuals will spontaneously incorporate them into their decisions. Importantly, efficiency in influencing behavior is not necessarily the same as efficiency in solving the problem that the institution is designed for. Our cognitive systems are designed to function within evolutionarily recurrent situations and can be ill suited for solving the problems of large societies. Similarly, there is no guarantee that institutions matching these intuitions are good at solving modern problems. Evidence concerning the management of common-pool resources can be interpreted along these lines. In relation to common-pool resources, the problem is congestion and, hence, people need to be restrained in their use of the resources. Detailed studies have shown that the institutions that facilitate restraint most effectively are institutions that facilitate face-to-face interaction among the participants (Ostrom, 1990). This allows for a social situation that mimics that kind of situation in which we have evolved to deal with such problems of collective actions. In fact, laboratory experiments show that resources are protected by institutions that emerge as a result of between-participant social interaction, better than by externally enforced institutions, even if the latter yield the optimal use of the resource (Cardenas, Stranlund, & Willis, 2002). The reason is that people do not feel intrinsically committed to the optimal-but-enforced institutions and, therefore, cheat on them whenever possible. Clearly, then, the efficiency of institutions in regulating behavior is not a matter of their inherent rationality. Rather, it is the extent to which they allow for appropriate cognitive machinery to become activated. In the case of collective action, a number of studies document that the human mind contains sophisticated machinery for committing ourselves to pro-social decisions but that these are extremely sensitive to the extent to which others are similarly committed (given selection pressures for making cooperation reciprocal) (Frank, 1988). Coordination of commitment is possible when institutions for common-pool resources are endogenously agreed upon rather than exogenously enforced. Another illustration of the importance of 'naturalness' for the regulatory potential of institutions is provided by a series of studies of when people accept specific distributions of costs and benefits (Hibbing & Alford, 2004; Smith, Larimer, Littvay, & Hibbing, 2007). People's reactions are modulated, not just by whether or not they benefit from the allocation, but also by the way the institutions orchestrating the allocation matches evolved moral sentiments. Using the Ultimatum Game, Hibbing and Alford, for example, experimentally varied the institutions governing who would be assigned the role of proposer and, hence, be allowed to divide a pot of money between themselves and the other participant — the receiver — who could accept the division or decline (in which case neither participant received any money). Receivers readily accepted (and felt satisfied with) highly unequal divisions (against their interest), if the institutions governing the allocation of roles focused on merit or chance but not if these institutions focused on preferences, that is, granted a participant the power to propose because he/she wanted this role most (Hibbing & Alford, 2004). We have evolved cognitive devices to resist exploitation (Buss & Duntley, 2008), which is why we spontaneously suspect the motives of eager dictators — and, of particular relevance here, feel more dissatisfied with institutions that allow them to move into power. Non-intuitive institutions will drift towards greater intuitiveness. Nonintuitive institutions require subjects to continuously invest effort to ensure that their rules are correctly recalled. For example, studies in social psychology demonstrate that individuals use effortful cognitive operations to encode and recall expectation-inconsistent information when forming impressions of others (Macrae, Bodenhausen, Milne, & Calvini, 1999). By implication, we expect that popular images and understandings of the rules of non-intuitive institutions will drift towards greater intuitiveness; that is, over time (if countervailing actions are not taken) subjects will be more likely to recall intuition-confirming parts of institutions and mold initially intuition-disconfirming rules into a format that matches intuitions. One example of this comes from religious institutions and, in particular, Max Weber's classical account of the rise of capitalism (Weber, 2002). Before the reformation, Christians could secure salvation by submitting themselves to the authority of the Catholic Church. After the reformation, this possibility was closed and, instead, the dominant theological paradigm described how certain people were predestined to become saved. With predestination, the normal response to a problem as psychologically significant as the prospect of eternal damnation, i.e., action, was effectively removed. As a result, at the popular level, the theological institution of predestination quickly drifted into the more intuitive informal institution of looking for signs for salvation in the form of success in the current life. Through success and, hence, hard work, one could then 'reveal' oneself as chosen for salvation. Divergence between non-intuitive official doctrine and public practice and belief is also widespread in the realm of politics (Kuran, 1995). In the literature on political tolerance, for example, it is often noted that people strongly endorse official doctrines about widespread civil rights for everyone, and at the same time display strong intolerance towards specific groups (McClosky & Brill, 2003). In the domain of criminal justice, there is widespread support for the principle of proportionality, i.e., that punishments should 'fit' the crime (Darley & Pittman, 2003), enshrined in modern criminal justice institutions but at the same time people, when considering specific criminals, allow for a number of exceptions to these principles. While such public beliefs do not necessarily reshape official institutions, they nonetheless influence how the institutions in fact work. As these last examples show, a cognitive science account of institutions does not preclude the possibility that institutions are also shaped by environmental factors that can make them divert from natural focal points. Rather, the point is that such divergence will constantly be put under pressure by processes of institutional drift towards greater fit with our evolved cognitive systems. # 7. Is a General Theory of Institutions Possible (or Desirable)? The framework proposed here implies a substantial departure from common assumptions in theories of institutional design. Neo-institutional models, for instance, describe domain-general processes that should in principle apply in similar ways to marriage, exchange or criminal law, or most other domains of institutional norms (Ostrom, 2005). Also, the aim of such models is to provide general economic or political factors that constrain institutional development. By contrast, we have argued that one should explain institutions in terms of domain-specific psychological systems. Human psychology comes with assumptions, capacities and preferences concerning, for example, reproduction and parenting, distinct and separate from those concerning the punishment of wrongdoing or the establishment of reliable exchange relations. These are exogenous factors in the sense that they stem from the evolved cognitive make-up of the species, independently of social institutions. If this is a valid proposal, then a general theory of institutions as such is not really what social scientists should aim for. That is because a general model, based solely on endogenous factors, should be extremely abstract to be equally applicable to the many disparate domains of institutional development. At such a level of abstraction, the model may not predict or exclude anything in particular, and therefore may not be of great value. An account of institutional design and development is more likely to come from integrated, probably situation-specific, models that bring together economic constraints and human-specific competencies in particular domains of social interaction. # References students. Evolution & Human Behavior, 20, 433–451. https://doi.org/10.1016/ S1090-5138(99)00022-7 ——. (2006). Evolutionary psychology, moral heuristics, and the law. In G. Gigerenzer & C. Engel (Eds.), Heuristics and the law. (pp. 175–205). Cambridge, MA; Berlin: MIT Press; Dahlem University Press. ——. (Eds.). (2005). Neurocognitive Adaptations Designed for Social Exchange. Hoboken, NJ: John Wiley & Sons Inc. Daly, M., & Wilson, M. (1988). Homicide. New York: Aldine. ——. (2007). Social Norms, Nonlegal Sanctions, and the Law. Cheltenham; Northampton, MA: Edward Elgar. Psychology and the Generation of Culture. (pp. 19–136). New York, NY: Oxford University Press. # Introductory Note Why do people perform rituals? Over the world and however far we can go in the past, human groups seem to engage in what we would recognize as, well, 'rituals' of some kind or other, even though we may be very unclear about what that term is supposed to convey. Pierre Liénard nudged me to join forces and re-open that question, which used to be central in classical anthropology, together with the additional query, what is the connection (if any) between the collective ceremonies described by anthropologists or historians, and the compulsive behaviors of obsessive patients? So, why perform rituals? The question of why this (vaguely defined) way of behaving is universal was more often avoided than addressed in anthropology, as quite a few anthropologists have pointed out (Bloch, 1974; Rappaport, 1999). We were told that collective rituals expressed a world-view, or reflected social values or made manifest a social order etc. All such statements raise more questions than they solve. Why would you need a ritual to do any of these things? As for the striking similarities between collective ceremonies and the individual, often pathological rituals observed in obsessive-compulsive disorder (OCD)—the article provides a detailed account of these similarities—the question had been largely abandoned after Sigmund Freud's desultory observation (1948) that religious ritual could be described as collective obsessiveness and obsessiveness as a kind of individual religion… which did not help much. Our work diverged from previous models and theories of rituals in three distinct ways. First, Liénard and I agreed that we should try to describe ritualization rather than rituals. That is not a pedantic distinction. Rituals are the outcome—the collective or private ceremonies found in most human cultures, the routines of individuals with obsessive-compulsive disorder, as well as the repetitive behaviors of many children. Ritualization is the combination of underlying processes that creates all this, making it natural or even compelling individuals to engage in these behaviors. A great deal of confusion in anthropology stems from a focus on the result, on 'rituals', rather than on the processes that cause them. Anthropologists have for instance wasted much time trying to define the term ritual, to demarcate what is and what is not a ritual, and so forth. This is a bit like spending one's time trying to define what counts as a 'fire' and what does not, rather than describing the physics and chemistry of combustion—for a more recent critique of that unfortunate tendency, see Boyer & Liénard (2020). Second, Liénard suggested that we should build our account on the basis of the 'security motivation' model proposed by Szetchman & Woody (2004) to account for the neuro-physiology of obsessivecompulsive disorders. Together with previous evolutionary models by Abed & de Pauw (1998), as well as Fiske & Haslam (1997), this neurophysiological model provided a key to understanding how ordinary actions can become ritualized. Our model is very much a modified version of the Szechtman & Woody account, with a few important twists, as discussed in the paper. (I should point out that the neuro-physiology in the paper is of course partly out of date, although the main points remain valid). Third, Freud was obviously wrong—most anthropologists would agree with that—but it matters to understand exactly why. Consider people performing a collective ritual, e.g., sacrificing a pig to the ancestors as a way to placate them and ward off witches and devils. The reason why people engage in such behaviors is that they receive messages from other people, their elders for instance, that one should engage in this course of action. The content of these messages is the reason why the ritual actions are reiterated. As we describe in the paper, people will follow a ritual recipe if it is (even marginally) more relevant than alternatives available in their social environment. In our account, people receive a description of the cultural ceremonies that is relevant because it activates, however faintly, cognitive systems that evolved to protect us against potential hazards like contagion and predators. That is sufficient. This is a matter of cultural selection and reconstruction—in the same way as people select some stories among the many stories they hear, and store and reconstruct them, whilst abandoning other variants or other stories, a process of cultural 'epidemiology' described in detail by Dan Sperber (1996) and Sperber & Hirschfeld (2004). The re-iteration and performance of such rituals does not, in any way, require that the participants suffered from any special anxieties, that rituals could allay mental states, or any other such functionalist assumptions. Liénard made this even clearer in our subsequent paper, focused on cultural rituals and their dynamics (Liénard & Boyer, 2006). Strikingly, that unfounded assumption (that people participate in collective rituals to assuage their anxieties) is so entrenched, that some of the commentators on the original article took it for granted that we must be defending that explanation, despite our (as we saw it) clear assurances to the contrary. # References Sperber, D. (1996). Explaining Culture: A Naturalistic Approach. Oxford: Blackwell. # Why Ritualized Behavior? Precaution Systems and Action Parsing in Developmental, Pathological and Cultural Rituals1 with Pierre Liénard2 Abstract: Ritualized behavior, intuitively recognizable by its stereotypy, rigidity, repetition, and apparent lack of rational motivation, is found in a variety of life conditions, customs, and everyday practices: in cultural rituals, whether religious or non-religious; in many children's complicated routines; in the pathology of obsessive-compulsive disorders (OCD); in normal adults around certain stages of the life-cycle, birthing in particular. Combining evidence from evolutionary anthropology, neuropsychology and neuroimaging, we propose an explanation of ritualized behavior in terms of an evolved Precaution System geared to the detection of and reaction to inferred threats to fitness. This system, distinct from fear-systems geared to respond to manifest danger, includes a repertoire of clues for potential danger as well as a repertoire of species-typical precautions. In OCD pathology, this system does not supply a negative feedback <sup>1</sup> An earlier version of this chapter was originally published as Boyer, P., & Liénard, P. (2006). Why ritualized behavior? Precaution systems and action parsing in developmental, pathological and cultural rituals. Behavioral and Brain Sciences, 29, 595–613. Republished by permission of Cambridge University Press. <sup>2</sup> We are grateful to Leda Cosmides and John Tooby for initial inspiration, and to Dan Fessler, Thomas Lawson, Robert McCauley, Pascale Michelon, Mayumi Okada, Tom Oltmanns, Ilkka Pyysiäinen, Howard Waldow, Dan Wegner, Harvey Whitehouse, and Jeff Zacks, for detailed comments on a draft version of this article. to the appraisal of potential threats, resulting in doubts about the proper performance of precautions, and repetition of action. Also, anxiety levels focus the attention on low-level gestural units of behavior rather than on the goal-related higher-level units normally used in parsing the action-flow. Normally automatized actions are submitted to cognitive control. This 'swamps' working memory, an effect of which is a temporary relief from intrusions but also their long-term strengthening. Normal activation of this Precaution System explains intrusions and ritual behaviors in normal adults. Gradual calibration of the system occurs through childhood rituals. Cultural mimicry of this system's normal input makes cultural rituals attention-grabbing and compelling. A number of empirical predictions follow from this synthetic model. # 1. Ritualized Behavior In a variety of circumstances, humans produce rituals, intuitively recognizable by their stereotypy, rigidity, repetition, and apparent lack of rational motivation. Behavior of this kind is found in cultural rituals, religious or non-religious; in the complicated routines of many children; in the pathology of obsessive-compulsive disorders; in normal adults around certain stages of the life-cycle, especially during birthing. The common features of these behaviors cry out for explanation. We build on a variety of prior models to describe a core psychological process that we call action ritualization—which is only a part of individual or cultural rituals, but a crucial part. The occurrence of ritualization depends on the conjunction of two specialized cognitive systems. One is a motivational system geared to the detection of and reaction to particular potential threats to fitness. This 'Hazard-Precaution System' includes a repertoire of clues for potential danger as well as a repertoire of species-typical precautions. The other system might be called 'Action Parsing.' It is concerned with the division of the flow of behavior into meaningful units. In some circumstances, specific interaction between these systems creates ritualized actions. The circumstances are different for individual, pathological, and collective rituals, as we will see. But the core ritualization process explains some of their common properties. There is no precise definition of 'ritual' in any of the three fields that deal with its typical manifestations. Cultural anthropologists generally accept a very vague definition of the term as scripted, stereotypic forms of collective action (Gluckman, 1975). Ethologists use criteria such as repetition and stereotypy (Payne, 1998). Clinical psychologists' descriptions of OCD pathology, as in the DSM-IV, mention 'ritualistic behaviors' without more precision (American Psychiatric, 1995). Besides, models of the phenomenon are generally limited to one domain of ritual. There is a large clinical literature about children's OCD but little study of normal childhood ritualization, simply because the latter is not pathological, even though it may be difficult to understand one without the other (Evans, Leckman, Carter, Reznick, et al., 1997). Models of OCD do not usually cover normal episodes of obsessiveness and ritualistic compulsion in the life-cycle although these are probably continuous with the pathology (Mataix-Cols, do Rosario-Campos, & Leckman, 2005). Very few anthropologists have considered the striking similarities between cultural ritualized behavior and individual pathology (Rappaport, 1999). A notable exception is Alan Fiske (Dulaney & Fiske, 1994; Fiske & Haslam, 1997), who re-opened an issue famously framed by Freud a long time ago (Freud, 1928). Following up on Fiske's pioneering work, discussed in Section 8.1 , as well as neuro-physiological (Szechtman & Woody, 2004) and evolutionary (Abed & de Pauw, 1998) models, we aim to provide a model of the different domains of occurrence of ritualized behavior. We certainly do not mean to underestimate the obvious differences, but we do think that the common features of ritualized actions require an explanation. We aim to provide an integrated model that includes not only a cognitive specification of the behavioral patterns and their elicitation conditions, but also the neural correlates of the behaviors and of their pathological distortion, the developmental patterns involved, and the evolutionary background. It might seem imprudent to make any general statements about a disparate set that includes pathological and normal manifestations, and individual as well as collective rituals. Note, however, that our aim here is not to account for all these behaviors. Our aim is to account for the psychological salience of a particular feature they share, namely the performance of what we call here 'Ritualized Behavior,' a precisely defined way of organizing a limited range of actions. In the following sections we outline the diverse domains of ritualized behavior before putting forward an integrated neural-developmental-evolutionary model of ritualization. # 2. Diverse Domains of Ritualization #### 2.1 Obsessive-Compulsive Disorder (OCD) The main features of the pathology of OCD are familiar: intrusive, bothersome thoughts about potential danger, as well as a strong compulsion to engage in stereotyped and repetitive activities with no rational justification. Standard criteria in the DSM-IV include (a) intrusive thoughts that (b) cause distress and (c) are often accompanied by ritualistic behaviors that (d) disturb normal activity and (e) are recognized as irrational by the patient (American Psychiatric, 1995). Typical obsessions include contamination and contagion (i.e., fear of catching other people's germs, of ingesting contaminated substances, of passing on diseases to others), possible harm to others (e.g., handling kitchen utensils and wounding people), as well as social ostracism following shameful or aggressive acts (thoughts about assaulting others, shouting obscenity, exhibitionism, etc.). This is often combined with 'thought-action fusion'—the assumption that having forebodings of possible misfortunes is tantamount to bringing them about—and an exaggerated feeling of responsibility for others (Salkovskis et al., 2000). Obsessions are typically accompanied by rituals. Some patients engage in endlessly repeated sequences of washing hands, cleaning tools or utensils (Hodgson & Rachman, 1972). Others repeatedly verify that they properly locked their door, rolled up the car window, or turned off the gas stove (Hodgson & Rachman, 1977). Still others are engaged in constant counting activities or need to group objects in sets of particular numbers, with specific alignments (Radomsky, Rachman, & Hammond, 2001). Although a categorical division between 'checkers,' 'washers,' and 'hoarders' has become popular in descriptions of OCD and as a descriptive clinical tool, there seems to be a large overlap in these categories (Khanna, Kaliaperumal, & Channabasavanna, 1990). A more accurate description would construe 'contamination,' 'insecurity and doubt,' and 'excessive precautions' as dimensions of the syndrome (Mataix-Cols et al., 2005), with each patient presenting a cluster of symptoms distributed along these dimensions (Calamari et al., 2004). Most patients are aware that their obsessions are unreasonable and their rituals pointless (patients' insight used to be a criterion in the DSM) but they also report that neither is easily controlled (Eisen, Phillips, & Rasmussen, 1999). #### 2.2 Children's Rituals Most young children engage in ritualistic behaviors in a limited range of situations and at a particular stage of development, starting at age 2 and peaking in middle childhood. This developmental phase is characterized by perfectionism, preoccupation with just-right ordering of objects, attachment to a favorite object (imbued with a special value), concerns about dirt and cleanliness, preferred household routines, action repeated over and over or a specific number of times, rituals for eating, awareness of minute details of one's home, hoarding, and bedtime rituals. (Obviously, most children in most situations also create disorder, at least relative to what adults expect; insistence on 'just so' performance is limited to highly specific contexts.) The themes and the age-range are similar among American and other cultural groups (A. H. Zohar & Felz, 2001). In many children, rituals are connected to anxiety states with specific targets. Among them is the fear of strangers, as well as the possibility of inflicting harm to self or others, possible contamination, attack by strangers or animals. The tendency to engage in rituals is correlated with anxiety or fearful traits (A. H. Zohar & Felz, 2001). Both fears and rituals typically evolve with development, from 'just so' insistence to elaborate rituals (Leonard, Goldberger, Rapoport, Cheslow, & Swedo, 1990). Younger children's ritualistic behaviors are related to prepotent fears such as stranger and separation anxieties, whereas the ritualistic behaviors of older ones are related to more specific and contextual fears such as contamination and social hazard (Evans, Gray, & Leckman, 1999). Some children connect their rituals to supposed effects by magical beliefs in ritual efficacy (Evans, Milanak, Medeiros, & Ross, 2002), but this is by no means necessary or even general. Although the facts of childhood ritualization are familiar and impressive, there is no definitive account of the functional basis of such behaviors in young children. This is mostly because OCD pathology is seen as discontinuous with the 'normal' routines of childhood, given both the obvious differences in frequency and emotional intensity and the fact that only very few young ritualists become clinically obsessive (Leonard et al., 1990). However, it seems difficult to understand the pathology in the absence of a proper causal model for this highly recurrent, culturally stable part of the normal developmental process (Evans et al., 1997). #### 2.3 Life-Stage-Relevant Intrusive Thoughts Specific disturbing thoughts occur in many people at particular phases in the lifetime, notably pregnancy, motherhood, and fatherhood. Senseless, intrusive, unacceptable ideas, thoughts, urges, and images about infants are common among healthy parents of newborns, both fathers and mothers (Abramowitz, Schwartz, Moore, & Luenzmann, 2003). The content of intrusions is related to specific stages of the life-cycle. While new fathers and post-partum mothers report fears about harming the infant, pregnant women report heightened fears about contamination (Abramowitz et al., 2003). They also develop rituals of washing and cleaning related to these intrusions. A common underlying theme is uncertainty and doubt concerning possible harm to the infant. Threequarters of the new parents surveyed by Abramowitz et al. reported persistent thoughts about accidents, suffocation, and other possible ways of intentionally harming the infant (Abramowitz et al., 2003). The individuals feel responsible for these intrusive thoughts. Development of specific perinatal anxieties may be part of a 'primary parental preoccupation' complex that includes nesting behaviors, repeated checking, thoughts about the infant's perfection, and fantasies about possible threats to its security (Leckman et al., 2004). Rodent models suggest oxytocin as a major modulator of such maternal behaviors (Leckman et al., 2004). The connection between these non-clinical context-relevant intrusions and OCD is not just a matter of similarity. The onset of OCD in women occurs during pregnancy more than at other life-stages (Maina, Albert, Bogetto, Vaschetto, & Ravizza, 2000; Neziroglu, Anemone, & Yaryura-Tobias, 1992). Note that the development of intrusions and early rituals into OCD is quite distinct from the evolution of post-partum depression (Williams & Koran, 1997). The former triggers very specific, highly consistent obsessive thoughts as opposed to unfocused or frequently shifting depressive ruminations. OCD onset also results in an urge to act (perform specific rituals) very different from the withdrawal from action observed in post-partum depression (Hagen, 2002). Among OCD patients, pregnancy and postpartum result in more severe symptoms (Labad et al., 2005). Activation of the fronto-striatal networks as a result of infant cries is different in new mothers and controls (Lorberbaum et al., 2002), suggesting functional calibration of the circuitry involved in OCD (see Section 3.1 ). #### 2.4 Cultural Rituals A great variety of social occasions are identified as 'rituals' in the anthropological literature. They range from private ceremonies with few participants, or indeed just one person, to large gatherings, and from single acts to long sequences spread over months or years. The general themes range from worship to protection to aggression. The occasions for ritualized behaviors also vary, based either on contingencies such as illness or misfortune, life-stages like birth, initiation, and death, or recurrent occasions such as seasonal changes. Finally, the connections between rituals and religious concepts are crucial in some cases (e.g., ancestor worship, Islamic prayer), or only peripheral (e.g., antiwitchcraft divination), or just absent (as in 'secular' rituals). How do we recognize such actions? As Roy Rappaport argued, it seems that we (anthropologists but also lay folk) use a conjunction of specific criteria that a model of ritual should explain (Rappaport, 1979). Here is a slightly modified list of features he emphasized: 1. First, actions are divorced from their usual goals. In cultural rituals, one typically washes instruments or body parts that are already clean, one enters rooms to exit them straightaway, one talks to interlocutors that are manifestly absent. Also, many rituals include actions for which there could not possibly be any clear empirical goal, such as passing a chicken from hand to hand in a circle, going round a temple seven times, and so forth. 2. Second, cultural rituals are often presented as compulsory, given a particular situation. People are told that a particular ceremony must be performed. More often than not, there is no explanation of why that ritual should be performed given the circumstances. True, a ritual often has a specific overall purpose (e.g., healing a particular person, keeping witches at bay); but the set of sequences that compose the ritual are not connected to this goal in the same way as sub-actions connect to subgoals in ordinary behavior (Boyer, 1994). 3. Third, in many cultural rituals people create an orderly environment that is quite different from the one of everyday interaction. People line up instead of walking, they dance instead of moving, they wear similar clothes or make-up, they build alignments of rocks or logs, they create elaborate color and shape combinations, and so on. Related to this is the recurrent concern with delimiting a particular space (a sacred circle, a taboo territory) often visually distinct from the other, unmarked space. It is important to distinguish 'rituals' from ritualization. There may be lots of different reasons why particular kinds of ceremonies are found in human cultures, why they persist, and why they are relatively stable. We discuss these issues elsewhere (Liénard & Boyer, forthcoming). For instance, one may propose plausible evolutionary scenarios for the existence of birth celebrations and of death rituals in most cultural environments. But these scenarios do not explain why these social occasions all include ritualized behavior in the precise sense intended here. #### 2.5 General Features of Rituals Behavior in these different domains displays obvious similarities: 1. Compulsion. Given certain circumstances, people feel that it would be dangerous or unsafe or improper not to perform ritualized actions. There is an emotional drive to perform the action, often associated with some anxiety at the thought of not performing it (especially in patients and children) and some relief after performance. Naturally, this varies between domains. Anxiety precedes ritual actions or behavior in many personal and pathological rituals but not always in cultural rituals. Common to all domains, though, is the important fact that compulsion does not require any explanation. People feel that they must perform the ritual, otherwise. . . [something might happen], but they require no specific representation of what would happen otherwise. 2. Rigidity, adherence to script. People feel that they should perform a ritual in the precise way it was performed before. They strive to achieve a performance that matches their representation of past performances and attach negative emotion to any deviation from that remembered pattern. This is familiar in childhood rituals and OCD but also in the 'traditionalistic' flavor of most cultural rituals (Bloch, 1974). Deviation from the established pattern is intuitively construed as dangerous, although in most cases the participants have or require no explanation of why that is the case. 3. Goal-demotion. Rituals generally include action-sequences selected from ordinary goal-directed behavior. But the context in which they are performed, or the manner of performance, results in 'goal-demotion,' in performance divorced from observable goals. For instance, people tie shoelaces that were tied already; they touch a specific piece of furniture without trying to move it or use it as support; they wash hands many more times than hygiene would require; and so on. 4. Internal repetition and redundancy. Repeated enactments of the same action or gesture, as well as reiterations of the same utterances, are typical of many rituals. A given sequence is executed three or five or ten times. What matters is the exact number. This makes many ritual sequences clearly distinct from everyday action, in which there is either no repetition of identical sequences (e.g., in assembling a musical instrument, one performs a series of unique actions), or each repeated act has a specific outcome (e.g., in weaving), or repetition is cumulative (the egg-whites rise only after a long period of whipping). 5. A restricted range of themes. Many rituals seem to focus around such themes as: pollution and purification, danger and protection, the possible danger of intrusion from other people, the use of particular colors or specific numbers, the construction of an ordered environment (Dulaney & Fiske, 1994). A ritual space or instruments are described as 'pure' or 'safe' (or, on the contrary, as the locus of concentrated 'pollution') or the point of the ritual is to 'purify' people or objects, to 'cleanse' mind or body, and so on. In collective rituals, this concern with pollution and cleansing is so prevalent that it has been considered a foundation of religious ritual (Douglas, 1982). Is there a common explanation for these different features of ritualized behavior? Here we will start from pathology and summarize what can be safely concluded from the clinical and neuropsychological evidence. This supports a particular model of action ritualization which we will also extend to developmental rituals in children and adults, before proceeding to the distinct case of cultural rituals. # 3. Interpretations of Compulsive Ritualization #### 3.1 Neuropsychological Modeling OCD has been interpreted as a specific dysfunction of the basal ganglia (Rapoport, 1990, 1991). To understand how this would result in the specific symptoms, the impairment should be described in terms of the specific functions of a cortical-striato-pallidal-thalamic circuit (CSPT). This network includes projections from many cortical areas (including medial and orbital frontal cortex) into the striatum (caudate and putamen) and back to the cortex via the substantia nigra and thalamus (Rauch et al., 2001; Saxena, Brody, Schwartz, & Baxter, 1998). This has been confirmed by neuro-imaging studies, as OCD is associated with increased activity of the orbitofrontal cortex (OFC) as well as in the striatum, thalamus, and anterior cingulate cortex (ACC) (Saxena et al., 2004; Saxena et al., 1998). Also, the anatomy of the caudate, putamen, and globus pallidus seems to differ between patients and controls (see, e.g., (Giedd, Rapoport, Garvey, Perlmutter, & Swedo, 2000). One generally distinguishes between a 'direct' and an 'indirect' pathway in the CSPT networks (see Fig. 1 ). The direct pathway links (1) frontal cortices to (2) the striatum, to the globus pallidus (pars interna) and substantia nigra (pars reticulata) to (4) thalamus and (5) cortex. The indirect pathway connects (1) cortex to (2) striatum to (3a) globus pallidus (pars externa) and subthalamic nucleus to (3b) globus pallidus (pars interna) and substantia nigra (pars reticulata) to (4) thalamus to (5) cortex. The basal ganglia are involved in the formation of habits, motor habits in particular (Rauch et al., 1997). The pattern of projections from Fig. 1. A summary of some cortico-striatal pathways relevant to OCD. Continuous line for the 'direct' pathway and dotted line for 'indirect' pathways (both highly simplified). SMA: Supplementary Motor Area, DLPFC: dorso-lateral prefrontal cortex, OFC: orbito-frontal cortex, Caud: caudate nucleus, Put: Putamen, Cing.: Cingulate Cortex, NA: Nucleus Accumbens, GP: globus pallidus (external and internal), SN(pr): substantia nigra pars reticulata, SubTh Nuc: Subthalamic nuclei. (Figure by P Boyer, 2006). the cortex to the striatum suggests that the latter may store summaries or 'chunks' of motor behavior. This is confirmed by involvement of the striatum in the learning and production of habitual responses (Graybiel, 1998). Striatal networks may act as coordinators of cortical input and orchestrators of motor habits. What specific dysfunction would result in OCD symptoms? In animal models, modifying dopamine uptake in the striatum results in stereotypic and repetitive behavior (Canales & Graybiel, 2000; Szechtman, Sulis, & Eilam, 1998). So an imbalance between various parts of the basal ganglia system or a modification in the dynamics of cortico-striatal pathways are probably involved in the condition. Saxena and colleagues identify the 'indirect' pathway as the locus of impairment. In their model, the association of globus pallidus (external) and subthalamic nucleus can be construed as a 'basal ganglia control system' that modulates the projections to the thalamus and cortices (Saxena et al., 1998). The indirect pathway consists of inhibitory (GABAergic) projections from the striatum to the thalamus. To the extent that this pathway becomes less tonic, it would fail to inhibit habitual motor responses and result in unmotivated, stereotypic routines (Saxena et al., 1998). Also important is the regulatory role played by the orbitofrontal cortex (OFC) and the anterior cingulate cortex (ACC). Early neuroimaging studies showed differential activation of these regions in OCD patients in situations of symptom provocation (Adler et al.; Rauch et al., 1994). OFC activation makes sense given its role in the selection, control, and inhibition of behavior as demonstrated both by neuroimaging and by lesions of this area (Happaney, Zelazo, & Stuss, 2004; Ogai, Iyo, Mori, & Takei, 2005; Schnider, Treyer, & Buck, 2005). Anterior cingulate activity is also revealing. Ablation of the area has been used in refractory OCD cases (Kim et al., 2003). ACC hyperactivity is not limited to situations of symptom provocation (Ursu, Stenger, Shear, Jones, & Carter, 2003). In an event-related study of error-processing, Fitzgerald and colleagues found increased ACC activity with error-detection in both patients and controls, with significantly higher increases in patients. The amount of ACC activity also correlated with the severity of the patients' compulsive symptoms (Fitzgerald et al., 2005). The anterior cingulate can be described as an error-detection network that activates top-down responses to situations of conflicting information, for example, between expectation and perception in errors, or between discrepant stimuli (Van Veen & Carter, 2002). All this converges to suggest that OCD may stem from a dysfunction of a neural system involved in the production and inhibition of a particular set of habitual or routinized behaviors. The etiology of the dysfunction includes probable genetic factors (Campbell et al., 1999; J. Zohar, Kennedy, Hollander, & Koran, 2004) as well as infectious conditions (Giedd et al., 2000; Henry, Perlmutter, & Swedo, 1999), although evidence for either cause is tentative. The compulsive nature of the actions seems to result from a failure to inhibit strongly motivated routines initiated in the striatum, either because striatal networks overrespond to cortical inputs, or because their inhibitory effect on thalamic networks is diminished, or both, leading to ritualization. This picture is consistent with the clinical and pharmacological evidence (Kaplan & Hollander, 2003; J. Zohar et al., 2004). #### 3.2 Cognitive models: General or specific? Cognitive models provide a bridge from neuropsychological findings to the phenomenology of OCD symptoms. A classical cognitive model describes the condition as a disorder of threat-appraisal and cognitive control (Rachman & Shafran, 1998; Salkovskis, 1985). Patients produce a misguided appraisal of intrusive thoughts, exaggerate the threats present in the environment as well as the extent of their own responsibility for what befalls others, and finally fail to appreciate the measure of safety introduced by normal precautions. In this model, OCD differs from other anxiety conditions (general anxiety disorder, panic) only in that the eliciting stimuli are very specific—a series of intrusive thoughts with recurrent themes (Clark, 1999). Obsessions and compulsions might then result from a general failure to appreciate levels of danger, to evaluate one's responsibility in external events, and to form an appropriate picture of one's situation. For instance, ritualized repetition may stem from the patient's failure to realize that he or she has actually accomplished the action (Pitman, 1987). There is indeed evidence (though not conclusive) for general memory problems. OCD patients have the right intuitions in both memory for actions and source monitoring (i.e., whether they performed as opposed to imagined performing an action) but they report less confidence in their own intuition (Hermans, Martens, De Cort, Pieters, & Eelen, 2003). However, there is also definite evidence for domain-specific aspects of OCD. For instance, OCD patients are similar to controls in their recall of neutral objects but are markedly better at recalling dangerous items (Tolin et al., 2001). OCD 'checkers' are impaired in their recall of own actions but less so in recall of other information (Ecker & Engelkamp, 1995). In terms of attention, modified Stroop tasks show that OCD 'washers are more attentive to contamination words than are controls, and OCD patients in general show more interference than controls do from danger-related words (Foa, Ilai, McCarthy, Shoyer, & et al.). #### 3.3 Security-Motivation Most cognitive models of OCD are phrased in domain-general terms. An exception is Abed and de Pauw's evolutionary hypothesis about OCD as a disruption of a specific 'psychological immune system' (Abed & de Pauw, 1998). The hypothesis starts from the observation that the prevalence of OCD would suggest the tail of a phenotypic distribution rather than harmful mutations. According to Abed and de Pauw, obsessional phenomena are an exaggerated version of thought processes selected because they lead to risk-avoidance behavior (in particular through fear or disgust). Central to the hypothesis is the fact that intrusive thoughts, in patients and normal individuals, consist of detailed scenarios of possible danger, an 'Involuntary Risk Scenario Generating System' (Abed & de Pauw, 1998). A similar evolutionary background motivates Szechtman and Woody's interpretation of the condition in terms of a 'securitymotivation' system (Szechtman & Woody, 2004). The model is an attempt to integrate the diverse components of the relevant behaviors (emotion, perception of specific information, typical actions, inhibition or disinhibition of automatic routines) in a motivational system functionally specialized in potential danger. In contrast to general cognitive impairment models, both Abed and de Pauw's and Szechtman and Woody's models provide a parsimonious account for the specificity of OCD intrusions. The security system is present in all normal human beings and monitors external signals of particular kinds of potential danger. The neural circuitry involved in both normal and pathological safety motivation can be broken down into three major functional components with excitatory and inhibitory links. An appraisal system handles information that matches input conditions for environmental cues of potential danger. A security motivation system handles the evaluation of these cues. A set of various evolved security-related programs is engaged, depending on the outcome of this motivation assessment, with specific motor and visceral output (see Fig. 2 ). As a result of engaging security-related motor-programs (this may consist in visual inspection of one's environment, cleaning, ordering, etc.), the security motivation system produces a specific experience of things being 'just right' which feeds back into the danger appraisal system. Szechtman and Woody's identification of the neural correlates of these systems extends beyond the cortico-striatal pathways. The appraisal of potential danger involves perceptual and memory information and feeds Fig. 2. An interpretation of Szechtman and Woody's (2004) model. Rectangles correspond to distinct systems activated, rounded boxes to behavioral results and call-outs to aspects of the processing. Danger clues are evaluated and action-plans selected, resulting in a 'just right feeling' that sends negative feedback to danger appraisal. This loop is absent or impaired in patients, leading to doubts about performance, which themselves result in repetition and rigid action-plans. (Figure by P Boyer, 2006). into both orbital cortex and the cortical-striatal pathways. From there, Szechtman and Woody identify two distinct informational loops. One of them, the affect loop, includes most of the 'indirect pathway' structures, producing a specific anxiety. In parallel, a 'security-related programs' loop, connects striatum to the globus pallidus (internal) and ventral thalamus to elicit the performance of stored motor routines. Finally, the normal inhibition of these two loops is provided by brainstem structures after performance of the elicited motor routines. The model states that OCD is the result of a dysfunction in a satiety signal, plausibly generated in brainstem structures, that connects the performance of security related behaviors as inhibitory feedback to a subsystem that generates and sustains security motivation. #### 3.4 Outstanding Questions In our view, while current models of compulsion have great descriptive and explanatory value, they still provide an incomplete account of various aspects of the obsessive and compulsive spectrum, especially if we include normal as well as pathological manifestations of ritual dispositions. A more complete model should account for the following aspects of ritualized behavior: 1. Why these specific themes? The thoughts patients and others report are clearly not random conceptual associations. They center on a few threats that are particularly disturbing. Even this is much too broad a description. People have intrusive thoughts about causing accidents involving their kin, but not complete strangers; they fear contamination more than bone fracture or inflammation; they fear that they may have left the back door open or the oven on, not that their car will be stolen or the fridge will break down. 2. Why these specific actions? Compulsions seem to focus on a narrow set of possible actions. This is clear for contamination compulsions which result in repeated washing and cleaning. The same applies to checking behaviors, limited to visual cues. Not all actions seem likely to become compulsive. 3. Why combine the actions in that way? Many compulsive rituals organize action in a very specific way. For instance, there are many negative rules in compulsions (avoid treading on the lines on the pavement). Also, there are specific rules about the number of iterations (touch this chair three times) or about the order of actions (tie the right shoe before the left one). 4. Why does ritual provide relief? Most clinicians agree on a temporary lowering of anxiety levels after the performance of rituals. The question points to one possible explanation for the compulsive character of the behavior. Could it be that patients intuitively reproduce behaviors that reduce anxiety? But then, what is it about such organization of action that could reduce anxiety? 5. Why does ritual eventually strengthen obsessions? This too is a feature often noted by clinicians (see, e.g., Salkovskis, 1985). Although rituals provide some relief, this is only temporary and the intrusive thoughts quickly come back. Indeed, it would seem that the more rituals one performs, the more focused and bothersome are the intrusive thoughts. # 4. Ritualized Action: The Core Process What follows is a list of the different points of the model which will be explained in the following sections . In our view, ritualization in young children, in normal adults at particular life-stages, and in patients comprises a series of processes in which specific information is acquired or retrieved and specific behavioral plans are engaged: 1. Security-motivation systems are engaged. This may be because of potential danger cues in the environment (described below), information imparted by other people, self-generated thoughts, or intrusions. In any case, these thoughts focus on cues for potential hazards chosen in a small set that we call the Potential Hazard Repertoire. 2. Safety motivation triggers an arousal state in which non-action is intuitively considered dangerous (something must be done) although there need be no clear representation of why that is the case. 2a. This state triggers a non-deliberate, non-controlled search for action-sequences that appear intuitively appropriate. Some cues make some actions seem apposite although the subject generally has no explanation for the intuition (or may only have ex post facto rationalizations). These actions are selected from what we call an Evolutionary Precaution Repertoire. 2b. The arousal triggers a special attentional state that focuses on low-level properties of own actions. The action-flow is parsed in smaller units than is usually the case. 2c. The arousal state may bias the appraisal system in such a way that 'just right' or 'closure' experience is delayed. This triggers doubts about actual or proper performance and reiteration of action-plans. 3. Performance of the actions with attention to low-level parsing [see 2b above ] may impose a heavy load on working memory-systems, with two consequences: 3a. The intrusive themes are temporarily pushed away from conscious access, resulting in a short-lived reduction in anxiety level. 3b. The intrusive themes are monitored by automatic, not controlled processes, which should result in higher salience (and renewed intrusion) after performance. These different steps are summarized in Figure 3 . In what follows we explain the processes engaged in more detail and provide arguments for their presence in most domains of ritualization. An important point to emphasize is that we do not identify any particular component of the overall process as being exclusively pathological. In our view, most reactions to inferred threats engage all these processes. Whether or not a given action triggers doubt about proper performance, leading to rigid repetition, that is, ritualization of these reactions, may be a matter of degree. Fig. 3. Summary of our Potential Hazard and Precaution model. Boxes denote specific processes with corresponding neural systems. Rounded box describes performance. Dark call-outs describe some of their typical properties. Clues for danger must suggest hazards from the Potential Hazard Repertoire. Appraisal of the clues if modulated by anxiety, leading to activation of plans from Evolutionary Precaution Repertoire and actionmonitoring systems. At the normal end of the spectrum, performance triggers satiety feelings with a negative feedback to danger appraisal systems. At the pathological end of the spectrum, doubts about proper performance lead to repetition and a positive feedback to danger appraisal. (Figure by P Boyer, 2006). # 5. Why These Particular Obsessions and Compulsions? #### 5.1 Logic of Our Evolutionary Approach Intrusions and compulsions are bothersome and time-consuming. Not only do they confer no particular adaptive advantage, they seem to be clearly maladaptive in diverting attention and memory resources from valuable goals. However, note that OCD and other disorders of the frontostriatal circuitry (Tourette's syndrome, ADHD, and schizophrenia) all have some genetic basis, as may be suspected from their prevalence (Bradshaw & Sheppard, 2000) and is tentatively confirmed by geneloci studies (Arnold, Zai, & Richter, 2004; Grados, Walkup, & Walford, 2003). To the extent that a specific kind of motivation is involved in the pathology of ritualization (perhaps also in its normal occurrence), it makes sense to wonder why and how humans are endowed with this special focus on particular kinds of hazards. In particular, are such systems the outcome of the evolutionary history of the species? In this case ultimate explanations would help us make sense of the pathology (Nesse, 1998), a strategy used in physiology (Nesse & Williams, 1996), psychiatry (Baron-Cohen, 1997; Cosmides & Tooby, 1999; Stevens & Price, 2000) and neuropsychology (Duchaine, Cosmides, & Tooby, 2001), and, as mentioned earlier, already outlined in some studies of OCD (Abed & de Pauw, 1998). Providing an evolutionary model requires the following steps: (1) identify the relevant fitness-related problem; (2) identify the knowledge base and computational rules that would be minimally required to solve that problem in ancestral environments; and (Willour et al., 2004) provide experimental evidence for the actual operation of a mental system that meets this computational specification. Once this is accomplished, such a model may allow us to delineate possible pathogenic scenarios that are causally deeper than the vague clusters identified in DSM-IV (Murphy & Stich, 2000). There are some indications that this approach may be appropriate for anxiety disorders and OCD in particular. First, negative emotions like anxiety or persistent low mood should not be considered as dysfunctional. They may consist in evolved warning systems whose negative rewards steer organisms away from fitness-reducing situations (Nesse, 1998). Second, the specific thoughts and actions that compose the symptoms may be linked to evolutionary concerns (Leckman, 2003; Mataix-Cols et al., 2005). Third, some of the conditions associated with fronto-striatal impairment may actually result in adaptive phenotypes (Bradshaw & Sheppard, 2000). #### 5.2 Two Types of Fitness-Threats We know enough of early primate and early human living conditions to identify broad categories of highly salient danger in our evolutionary past: reproductive risk (e.g., for females, mating with un-nurturing or low-fitness males; for males, cuckoldry or choosing unhealthy females); predation (failing to detect or deter predators); contamination from pathogens (bacteria, viruses, toxins); resource scarcity (e.g., failing to anticipate seasonal changes); social harm (e.g., ostracism, but also reduced cooperation). From an evolutionary standpoint, we should expect (1) that such recurrent hazards, not more recent ones, would be the target of specific emotions, and (2) that different kinds of hazard require different decision rules. On the first point, it is clear that specific emotions target hazards of great evolutionary ancestry rather than more recent ones, even though the latter may be much more dangerous. Our danger-avoidance systems do not seem to rely on an unprejudiced tabulation of which features of the environment effectively predict harm or misfortune. If this were the case, we would observe in modern conditions many cases of anxieties, fear, or even phobic aversions to electricity, cars, and cigarettes, which cause vastly more deaths than do spiders and rats. But we observe the opposite. Second, it seems that different kinds of fitnessthreats do activate different inferential rules. Specific principles inform the gender-specific perception of particular mates as more or less of a waste of reproductive potential (Buss, 1989). Predator-prey interaction is governed by early-developed intuitions that do not apply to other interactions (Barrett, 1999). Recurrent features of disgust reactions suggest a pathogen-minimizing system that adapts to local conditions (Fessler, Arguello, Mekdara, & Macias, 2003; Rozin, Haidt, & McCauley, 1993) or to particular individual circumstances such as pregnancy (Fessler & Navarrete, 2003; Profet, 1993). Problems of resource scarcity are handled by specific foraging strategies (Krebs & Inman, 1994) which can override explicit reasoning (Rode, Cosmides, Hell, & Tooby, 1999). Finally, a host of 'social intelligence' principles support the monitoring of social interaction, from the establishment of friendships and coalitions (Harcourt & de Waal, 1992; Kurzban & Leary, 2001; Tooby & Cosmides, 1996), to dominance (Sidanius & Pratto, 1999) and punishment (Boyd & Richerson, 1985; Kurzban & Leary, 2001). At this point we must introduce an important distinction between two types of fitness-threatening situations. First, there are cases of manifest threats, cases in which the organism receives signals about the presence of the source of danger: for example, a predator or enemy attack, or seeing one's infant in danger. Situations of this type are handled by specialized and context-specific fear-mechanisms in humans as in other primates (LeDoux, 2003; Maren & Quirk, 2004) and result in aggression, freezing, or flight routines (Blair, 2001; Payne, 1998). Second, there are inferred threats, when the potential danger is probable given certain clues in the environment. For instance, the strange taste of a particular dish may be evidence of rotting; tracks may betray the recent passage of a dangerous predator; a particular person's attitude may indicate that they will not cooperate. Such circumstances typically engage what Abed and de Pauw called an 'Involuntary Risk Scenario Generation.' Naturally, the distinction is a rough one (many situations involve threats for which there are direct and indirect clues). It is also, obviously, species-specific since some situations are a threat to some organisms but not others. #### 5.3 Potential Danger as a Specific Domain It may seem odd to hypothesize a domain-specific system whose activation is triggered by such disparate potential inputs as a footprint, a disgusting odor, or the fact that one's infant is out of sight for a moment. How specific is the system if it can encompass such physically different stimuli? But this objection assumes that domain-specific inference systems are tied to a physically specified range of stimuli, which is true for some perceptual systems (e.g., 3D vision) but certainly not for most higher-level functional systems. A human mind can parse linguistic input in just the same way on the basis of auditory, visual, or tactile information. Neuro-cognitive systems specialized in assessing the value of potential mates use information from conversations, from comparison of visual information to some ideal template, from observed interactions between the potential mate and other people, and so forth. Indeed, it would be surprising (and maladaptive) if a particular kind of physical input always triggered a unique inference-system. A man is a man is a man, but a father, a brother, an attacker, and a potential mate should activate different mental systems. So the autonomy or specificity of a domain-specific system can be inferred, not from focus on a physically specific range of cues, but from specific processing principles, a specific kind of output, a specific learning logic, and—in some cases—a specific pattern of impairment. These are criteria that seem present in the case of the Hazard-Precaution system. There is indeed some behavioral evidence that humans have specific inference rules for information relative to precautions. Fiddick and colleagues have demonstrated that when considering precautionary rules (e.g., 'if you take oranges on board you will not get scurvy'), subjects pass logical tests for verification of rule-violation that they fail in other contexts (Fiddick, Cosmides, & Tooby, 2000). This is a replication, in another domain, of the performance on rule-verification in the Wason selection task observed when the rules allude to social contracts, however unfamiliar, as opposed to other deontic domains, however familiar (Cosmides, 1989; Fiddick et al., 2000). Although these findings concern explicit judgment more than intuition, they suggest that potential hazard management might require cognitive processing that is quite different from other inferential tasks. #### 5.4 The Limited Range of Obsessions and Compulsions To explain the recurrent features of both intrusions and compulsions, our model stipulates two kinds of databases, called Potential Hazard Repertoire and Precaution Repertoire respectively. Intrusions and compulsions have to do with a specific, narrow range of hazards, which, in our view, are best explained as recurrent threats to fitness in ancestral environments. One reason for defending this hypothesis is that the actions combined in ritual sequences are generally (i) species-specific and (ii) precaution-related. Ritualists do not generally design entirely novel behavioral sequences from scratch. Rather, they combine familiar elements of actions (e.g., washing, cleansing, checking) into novel sequences. This is also manifest in animal models of the condition. The ritualistic behaviors triggered in rats treated with quinpirole (a dopamine agonist) are species-specific, consisting in checking with return to a home-base, similar to those of controls, but stylized, redundant, and time-consuming (Szechtman et al., 1998). Second, these actions are generally relevant ones as a protection against various kinds of fitness-threatening situations (Rapoport & Fiske, 1998). A review of the different dimensions of OCD obsessions but also adult normal intrusions and children's anxieties should illustrate the point. 5.4.1. Contamination. Thoughts about contamination and contagion are too specific to be interpreted as the outcome of a general lowering of the anxiety threshold. They tend to center on invisible agents such as toxins, viruses, and microbes—of obvious evolutionary import. Besides, people's anxious thoughts about contamination focus on modes of contact (touching with the hand, kissing, licking, having sex, sharing food, breathing next to a particular source) that are actually used by pathogen vectors. In patients, the compulsions associated with these thoughts are not arbitrary either. They center on measures such as washing and cleansing, protecting oneself from intrusive material by staying at a distance, avoiding contact, suspending breathing. In ancestral environments, before the discovery of asepsis, these procedures would indeed constitute the only measures to reduce or control contamination. There is behavioral and cross-cultural evidence that a concern with possible contamination triggers specialized inferential circuitry in humans. For instance, Fessler and colleagues have documented the disproportionate representation of meat among the foods that are 'good to taboo' in many cultures. They connect this to the specific challenges of meat consumption caused by protozoa and other pathogens (Fessler & Navarrete, 2003). In the same way, meat seems to be the chief target of early-pregnancy aversions, a period of dangerous immuno-depression (Fessler, 2002). More generally, many sources of disgust are also sources of contamination: decaying corpses but also rotting substances, feces, spit, and so on. 5.4.2. Symmetry and order in one's environment. Many children and adults are concerned with creating an orderly environment. Children align toys in a particular order, ritual participants need to create elaborately ordered displays, and the same is true of many OCD 'checkers.' These behaviors are often construed, especially in the domain of children's rituals, as the expression of a need for reassurance; as the urge to create a recognizable and therefore reassuring environment. However, this 'therefore' is question-begging. What is reassuring about a predictable environment? True, predictability implies a reduction in computational load, but that cannot be the reason, as children and ritualists in general devote great amounts of time and cognitive resources creating their orderly world. So there might be other aspects of order and symmetry that motivate cognitive investment. In our view, ordered environments combine two properties that may explain this motivation. First, alignments and symmetry are such that they make other agents' intrusions clearly visible. Anecdotal (but massive) evidence suggests that children but also various sub-clinical obsessive personality-types get quite upset when 'intruders' such as parents or cleaners disrupt their sequences and alignments. We speculate that the point of the ordering may be precisely to detect such disruptions. Or rather, that the behavior may be a stored action-plan that would have this function in other environments. This is indeed the one explanation of some animals' 'tidying up' routines as an anti-predator strategy (Curio, 1993). So the creation of a non-trivial order that is not immediately detectable by intruders may be a powerful motivation in such compulsions. Note that childhood rituals center on the home environment and in particular on children's own personal space (usually their bedroom). Second, the specific use of symmetry and conceptual order (alternating colors, corresponding shapes) is diagnostic of uniquely human dispositions to alter the environment. Bowerbirds may be among the few exceptions—and seem to resort to similar ways of making a display salient: pure colors, symmetry, and so on. Indeed, people readily detect such specific alterations—which has been used for millennia as a way of advertising human presence. Cairns are improbable pilings of rocks that no species other than human beings would build. Broken twigs, straight paths, and color markings serve as landmarks for the same reason. What makes this possible is the combination of sophisticated symmetry and pattern-detection capacities in humans (Bornstein & Krinsky, 1985; Bornstein & Stiles-Davis, 1984; Fisher, Ferdinandsen, & Bornstein, 1981) and sophisticated tool-making capacities (Wynn, 1993). This is particularly relevant to children's construction of ordered environments, which may consist of a period of systematic training in the construction of such signals of human presence. These are bound to remain speculative as there is, to our knowledge, no systematic research on the cognitive and emotional processes involved in ordered displays, particularly in children's strong motivation to produce such environments. 5.4.3. Social offence. Some of the intrusive thoughts of obsessive people center on possible acts that would offend or harm other people, resulting in social exclusion. These fears also represent, in our view, a domain of evolutionary hazard. Given human dependence on conspecifics for all aspects of survival, it is not surprising to find that possible social strife is seen as extremely dangerous. Life in complex societies makes this dependence diffuse and impersonal. By contrast, in ancestral environments people depended on known members of the group. Conflict in such groups threatens each member's access to resources, cooperation, and information (Tooby & DeVore, 1987). In this domain too, it seems that the precautionary measures taken by obsessives are in fact rather appropriate. For instance, one of the features of OCD patients (especially checkers) is a tendency to monitor actions, in particular the minutiae of one's own behavior, well beyond the 'normal' limits. Another common feature is that people choose to avoid social contact lest they insult or assault others, which again is intuitively appropriate as a precautionary device. 5.4.4. Harm to offspring. Intrusive thoughts reported by adults often focus on possible harm to one's own offspring, accompanied by fears of handling tools and utensils in a dangerous way, smothering or dropping the infant, as well as forgetting about the baby and losing it (particularly in stores and other public places). Again, the danger is one of obvious evolutionary significance, as tools and weapons are part of our ancestral past. Also, shifting attention away from one's infant is risky but unavoidable in humans who need to attend to such tasks as foraging or processing food. Again, the compulsive precautions (hyper vigilance, neglect towards other people and social interactions, etc.) would seem appropriate given these hazards. ### 5.5 The Precaution System Associates Domain-Specific Repertoires Specific reactions to inferred threats suggest a functional system that we called the Precaution System, whose specific input consists in inferences to non-manifest threat and whose output is selective activation of particular precautions. At both ends of its operation, the postulated system is highly specific. The Precaution System does not respond to all or most actually significant signals of potential danger, but to a limited repertoire of cues. As we said above, humans seem to infer fitness threats, with a specific anxiogenic response, from wounds or rotting carcasses, but not from tobacco smoke or electricity. The range of action-plans activated is also restricted to a few possible precautions (washing, avoiding contact, etc.) that may or may not be most appropriate given changing circumstances. Note that this model does not account for some sub-varieties of OCD symptomatology. Hoarding, for instance, does not seem to result in ritualized behavior in the precise sense described here. This may be because the underlying processes are different from other OCD dimensions, as is suggested by neuroimaging studies (Calamari et al., 2004; Saxena et al., 2004). In our model, the specificity of cues and responses maps a set of highly recurrent threats in human evolutionary history. # 6. Why the Complicated Action? #### 6.1 Ordinary Action-Parsing The ritualization process imposes particular constraints on the performance and sequencing of action. This is why the features of ritual should be considered in the context of action representation in general. Human beings attend to each other's behavior and react to it, which means that they must 'parse' other people's and their own behavior in meaningful units (Newtson, 1973). The experimental study of such parsing mechanisms provides a background against which we can understand specific features of ritual. People identify actions as belonging to particular categories (e.g., putting on one's socks) but also as part of larger sequences (putting on one's socks as part of getting dressed). This 'partonomic' structure is general to action sequences in normal contexts. Small units are parts of larger units and the boundaries between large units tend to coincide with a boundary at a lower level. Zacks and colleagues distinguish between three levels of representation: that of simple gestures (sequences of a few seconds), that of behavioral episodes (an order of longer magnitude, actions like 'getting dressed'), and that of a script (series that can span a much longer time, e.g., 'eating out,' 'giving a talk') (Zacks & Tversky, 2001; Zacks, Tversky, & Iyer, 2001). In the absence of specific instructions to the contrary, people spontaneously describe and recall behavior in terms of middle-level behavioral units (Zacks & Tversky, 2001; Zacks, Tversky, et al., 2001), that could be called a 'basic level' for event-taxonomies (Rifkin, 1985). Indeed, people can generate far more categories of events at that middlelevel than either super- or subordinates (Morris & Murphy, 1990). Midlevel breakpoints also correspond to specific neural activity (Speer, Swallow, & Zacks, 2003; Zacks, Braver, et al., 2001). It is certainly not a coincidence that this is also the level of description at which people typically ascribe goals to behavior. While gestures do not readily reveal intention, and scenes include many different intentions, behavioral episodes typically constitute the realization of a particular goal. Actionparsing develops early in infants and seems to focus on the intentional unit level from that early stage (Baldwin & Baird, 1999; Baldwin, Baird, Saylor, & Clark, 2001). #### 6.2 Goal-Demotion in Ritualized Action These studies converge to suggest that spontaneous parsing focuses on middle-level action-units connected to specific goals. It is very difficult for normal humans not to parse action at that level. Indeed, an excessive focus on a low-level, gestural description of behavior, with the attendant imprecision about goals, is characteristic of frontal lobe or schizophrenic patients (Janata & Grafton, 2003; Zalla, Pradat-Diehl, & Sirigu, 2003). Now this focus on low-level gesture analysis of the action-flow is precisely what happens in cultural and individual rituals. People's attention is typically drawn to the details of performance, the particular direction of a gesture, the specific number of times an action should be performed, and so on. Conversely, the description of ritual action in terms of goals is either not available or in any case irrelevant. This is what we call 'goal-demotion.' Although there may be a goal for the overall ritual script, there are no obvious sub-goals for its components. In typical patients' rituals or in developmental rituals, there may be an explicit goal. For instance, producing a particular alignment of twigs in a particular order is supposed to ward off intruders; or a sequence of familiar actions, for example, tying one's shoes in a very specific way, will prevent accidents. But the contribution of each part of the script is not connected to particular sub-goals. For some ritual actions it is impossible for the actor to imagine what contribution they would make as they reverse the results achieved through previous actions (e.g., piling up objects and carefully putting them back in a line before piling them up again). More generally, the actions are considered an indispensable part of the script although the subject has no representation of why he or she should be included in it. This contrasts with the standard parsing of action-flow, where the units identified at all levels of partonomic division correspond to specific goals. Indeed, in a typical example of routinized efficient practice, that of blacksmithing techniques, the correspondence between action-units and goals serves to mobilize different units of knowledge as they become relevant to the sub-task at hand (Keller & Keller, 1996). This is emphatically not the case in ritualized behavior, the performance of which seems to be a 'tunnel' in which each action only points to the following one in the prescribed sequence (Bloch, 1974). #### 6.3 Swamping of Working Memory There is very little study of the attentional effects of the focus on lowlevel features of action, combined with high control and hypersensitivity to possible mistakes, during performance of personal rituals. Our model suggests a specific, temporary effect on working memory which would explain some effects of rituals. Working memory is a specific memory system that holds information for a short time and allows updates and transformations of that information (Baddeley, 2000). In typical working memory tasks subjects are asked to repeat a sequence of letters in the right order, repeat in inverse order, repeat the sequence formed by letters while ignoring digits provided in between, or specify which was the third letter before last in a series that ends unexpectedly. In all such tasks, the subject must consider a certain set of information units or chunks at the same time in order to perform the required operations (Baddeley, 2000). In our view, one of the effects of prescribed, rigidly compulsory action-sequences is a momentary overloading or 'swamping' of working memory, especially if the action sequences are represented at the finegrain parsing level. This is very much what happens to some patients whose spontaneous action-parsing remains at this same low level of description. As Zalla puts it in her description of frontal lobe patients, 'the weakening of the causal connections between the component actions leads to the decomposition and the fragmentation of the action representation. […] The increased amount of fragmented information rapidly overloads subjects' working memory capacity' (Zalla, Verlut, Franck, Puzenat, & Sirigu, 2004). A similar point can be made about fragmentation of action in OCD compulsions (Ursu et al., 2003). Many ritual prescriptions resemble the tasks designed by cognitive psychologists in the study of working memory. They require focused attention on a set of different stimuli and their arrangement. For instance, a requirement to turn round a ritual pole three times clockwise without ever looking down imposes executive control of two tasks at the same time. Also, the frequent combination of a positive prescription ('do x. . .') and a negative one ('. . .while avoiding doing y') would seem to engage working memory and executive control in a way that is not usually present in everyday action flow. #### 6.4 Core Ritualization is the Opposite of Routinization In the model proposed here, ritualized acts are very different from other routines. However often an individual may perform a ritualized action, it does not seem to become automatic. On the contrary, it remains constrained by high-level cognitive control. Ritualized actions as described here require high cognitive control because the rules often apply to familiar actions (e.g., walking, talking, preparing food) and turn them into more difficult tasks (e.g., walking without treading on the line). This clashes with a commonsense notion that rituals only include actions that one performs 'routinely' or 'without thinking.' Indeed, it is essential to our model that the component of rituals that we called Ritualized Behavior cannot be automatic. # 7. Implications of the Model: Individual Ritual #### 7.1 Intrusions as Context-Sensitive Adaptive Algorithms A surprising conclusion from the very few systematic studies of intrusions and mild compulsions in the normal population is that thoughts about potential dangers (contamination, social harm) and some compulsive reactions are not confined to the clinical population. Most normal people seem to experience the same kind of intrusive thoughts as patients do, and to some degree generate the same ritualized action-plans to avoid such dangers (Abramowitz et al., 2003; Rachman & de Silva, 1978). The crucial difference, then, is not in the contents of the thoughts but in their appraisal (Salkovskis, 1985). The evidence available is insufficient to address the fundamental questions of the distribution, themes, intensity, and effects of intrusions in the normal population. Our model implies that intrusions are generally not dysfunctional. They are the outcome of systems geared to protecting the organism against potential dangers by over-interpreting specific inputs, which would suggest this prediction: [P1] The position of an individual along fitness-related life-cycle dimensions (young vs. old, male vs. female, nulliparous vs. multiparous, high vs. low status) should predict the frequency, intensity and contents of intrusive thoughts. So far, we only know that contagion and risk intrusions become highly salient during the perinatal period (Abramowitz et al., 2003; Leckman et al., 2004). This may also be true of other stages in the lifecycle, such as puberty, menarche, and the death of relatives. There is simply no general, population-sample study of thought-intrusions and their correlates. Sampling bias is particularly likely in this domain. Perinatal intrusions get noticed only because pregnancy is a period of higher medical monitoring. #### 7.2 Spontaneous Optimization and Relief Why the strange rules and prescriptions in compulsive action? Also, why should such performances induce temporary relief? Many patients explicitly associate their rituals with specific obsessions, stating that performing the ritual is one way of inhibiting or repressing the unwanted thoughts (Salkovskis, 1985). Clinicians' observations and patients' reports converge in suggesting that the relief from unbearable anxiety, though temporary, is palpable. But there is nothing in current cognitive models to explain the fact. In our view these two questions are related, and the common answer lies in the effects of ritualization on cognitive control and working memory. We suggested earlier that the performance of rituals, accompanied by numerous, specific, attention-demanding prescriptions, has the effect of 'swamping' working memory. We propose that such rituals constitute spontaneous and moderately efficient forms of thought-suppression. The difficulties of thought suppression in everyday life (trying not to recall unpleasant experiences or not to mull over possible future misfortunes) are familiar to everyone. Dan Wegner and colleagues have studied the phenomenon in controlled environments and demonstrated the complex control processes at work in such attempts (Wegner & Erskine, 2003; Wegner & Schneider, 2003). One interesting feature of these experimental studies is that only a few techniques are available to effectively 'push away' unwanted thoughts. They include focusing on emotional information of greater intensity than the target thoughts, or focusing attention on intrinsically difficult tasks like mathematical problems. These are difficult precisely because they recruit working memory to a greater extent than most everyday tasks and cannot be accomplished automatically. Obviously, compulsive rituals are in many ways different from the phenomena observed in such studies. First, Wegner's subjects generally have no intrinsic motivation to avoid the suppressed thoughts, other than compliance with the experimenter's instructions. By contrast, OCD patients are strongly motivated. Second, the intrusions in patients are far stronger (more difficult to push away from consciousness) than a simple neutral theme suggested by an experimenter. Third, patients have a history of thought-intrusion and thought-avoidance, whereas experimental subjects are genuine beginners in the domain. Notwithstanding these differences, we think the studies on thoughtsuppression are important to suggest a possible mechanism for the elaboration and rigidity of ritual prescriptions. In our view, patients with complicated compulsions have spontaneously attained an optimal point in the kind of activity that is so demanding in cognitive control that intrusive thoughts can be, at least for a while, pushed away from consciousness. This 'trick' exploits certain features of the action-parsing systems reviewed (see Section 6.1 ). Given that action-parsing processes are engaged when any behavior is witnessed or produced, there are not many tricks that could force attention to focus on the low-level description of action. Among these features is repetition, which results in goal-demotion. Another such gimmick, obviously, is to borrow a sequence from ordinary scripts and perform it in a context that makes goal-ascription impossible: for example, wash objects without using water, pretend to trace an imaginary line, and so on. What results from these 'tricks' is what we called 'goal-demotion' above. Actions are represented without attaching a goal to each behavioral unit, as would be the case in non-ritual contexts. This has several implications for the organization of compulsive rituals: [P2] Compulsive actions should be such that they mobilize working memory and require high degree of cognitive control. We have suggested that this is precisely what complicated prescriptions achieve, in particular when they result in control of usually automatic actions, such as choosing which shoe to tie first, or whether to push the doorbell button with this or that finger. [P3] Compulsive rituals may be the outcome of a trial-and-error process. This means that patients more or less deliberately (usually not) try various behaviors with various prescriptive rules until they reach an optimum, that is, the maximal occupation of working memory that is compatible with the intrinsic limits of memory itself. This would carry another consequence: [P4] The symptoms should become unstable if the actions become routinized. Working memory is effectively swamped when usually automatic actions are submitted to cognitive control. But even demanding tasks (e.g., tying one's shoes in a particular order that changes with the time of the day) might become partly automatic with time. One would predict that this would result in diminished efficacy and the spontaneous search for different prescriptions, or for more complex sequences. Naturally, this dynamic model does not imply that patients are at any point aware of the effect of prescriptive rules on memory. They may simply come to associate slightly more controlled action to slightly diminished intrusion, which would be enough gradually to lead to the baroque complications of individual rituals. We do not have much comparative clinical evidence concerning the particular contents of obsessive-compulsive rituals, that is, the number of actions, their precise description, their prescribed order, and so on, as opposed to general descriptions such as 'washing' or 'checking.' Nor do we have much in terms of longitudinal studies of ritual elaboration or progression; which is why these remain speculative predictions from the model. #### 7.3 Ironic Outcomes Studying normal subjects instructed not to think about a particular item, Wegner showed that thought suppression typically results in a 'rebound'—in higher salience of the unwanted thoughts (Wegner & Schneider, 2003). This, in Wegner's model, is caused by the combination of two distinct processes engaged in thought suppression. While an explicit process directs and monitors the suppression, implicit processes are engaged that detect material associated with the target item (Wegner & Erskine, 2003). Here again, we do not wish to read too much in the parallel between an experimental paradigm and a long-lasting behavior pattern. However, an ironic outcome would seem to follow from the working-memory swamping scenario: [P5] The precise intrusions that rituals can tone down should become more frequent or more difficult to resist as rituals are frequently practiced. Although it has not been studied in precise quantitative terms, this ironic rebound does seem characteristic of compulsive rituals (Rachman & de Silva, 1978). The patients who perform more rituals are typically more anxious, and also more bothered by their intrusive thoughts. In other words, the long-term effects of ritual performance are the opposite of its short-term results. Indeed, this may be why an effective cognitive and behavioral therapy for OCD, in particular exposure and reaction prevention (ERP), requires that the patient evoke the dangerous thoughts but restrain the compulsive response (Rachman, Hodgson, & Marks, 1971). #### 7.4 Developmental Calibration Our model implies specific claims about the Hazard-Precaution system in children, suggesting that early childhood is a period of calibration of the system. Many cognitive systems require calibration, that is, a change in parameters as a function of specific information picked up in the child's environment (Bjorklund & Pellegrini, 2002). A salient example is the development of food-preferences in young children, with a period of unlimited tolerance followed by 'parameter-setting' when young children reject anything that does not taste familiar (Birch, 1990). Another domain would be predator-prey relations, in which common assumptions are gradually refined in view of local circumstances (Barrett, 1999). We can make a similar point about the Potential Hazard Repertoire. As we said, the system should handle indirect clues and produce inferences about the potential presence of dangerous substances, predators, and conspecifics. But it immediately appears that the number of possible clues is multiplied by the fact that (a) any one of these dangerous situations could be detected using a large number of possible clues and (b) the situations themselves must have changed a great deal, and changed frequently, during human evolution. Indeed, modern humans have adapted to variable conditions of subsistence in primary forests, grasslands, and dry savannas. They also had to adapt to seasonal changes. Most important, cultural evolution led to rapid cultural change, or 'life in the fast lane' (Boyd & Richerson, 1985). Ecological and cultural change means that old predators are gone but new ones are present; that noxious substances are not found in the same plants or animals; and that social interaction is handled in significantly different ways. In this way the security system is a learning system, that appears in infants as a disposition to pick up particular kinds of locally relevant information from the natural and social environment, and changes its parameters as a function of that information. This would explain not just why children perform ritualistic behaviors, but also why the phenomenon appears and subsides at particular stages of development and why its typical manifestations evolve from prepotent fears for which there is clear preparedness, to more complex inferred threats like social harm. The system is designed to address a specific question: How to create a secure environment and to provide a series of contextually relevant solutions like washing, cleaning, checking, or modifying one's interaction with other agents? This implies particular directions for development in the kinds of thoughts and compulsions found in childhood. If the system is in calibration during that period, we should observe the following: [P6] Anxiogenic thoughts should become gradually more specific with development. [P7] Compulsive reaction should become more specific with development. In terms of anxiety, a fear of vaguely defined predatory animals should become a fear of particular animals, a fear of strangers should become a fear of particular strangers, and so forth, as the system picks up information from the environment. This applies to compulsions, too. At an early stage, all recipes should be equiprobable. At a later stage, children should acquire locally relevant associations between a particular potential danger and a particular recipe. This also predicts differences in the rituals of older children from different groups. To the extent that different cultural groups live in different conditions, different kinds of dangers would be relevant and different clues significant: [P8] Fears and compulsions should become more culturally specific as children get older. We already have some fragmentary evidence that developmental trends in children's fears support these predictions. Fantasies and intrusive thoughts change with development, as mentioned earlier (Evans et al.; Leonard et al.). ### 7.5 Cultural Similarities and Differences in Pathological Ritual Our model assumes that there is a Precaution system focused on certain kinds of potential danger. We also suggested that this system undergoes calibration during childhood, given that clues about potential danger change with changing environments. This would imply specific predictions about cross-cultural variations in the condition: [P9] Anxieties and fears that result in compulsion belong to the narrow range of ancestral potential dangers: contamination, intrusion, social offence, and resource-depletion. [P10] We should observe important cultural differences in the relative prevalence of symptom clusters (or 'subtypes'). There is very scant comparative anthropological evidence for anxieties or fears, although it seems to suggest something of this kind. In industrialized countries, the notion of electricity and cars as massive killers is virtually absent from the repertoire of phobic and obsessive patients. Also, the few studies of OCD patients in non-Western environments report the familiar obsessive themes of social offence, contagion, and potential danger (Arrindell, de Vlaming, Eisenhardt, van Berkum, & Kwee, 2002; Barker-Collo, 2003; Bertschy & Ahyi, 1991; Sasson et al., 1997) and the prevalence of OCD as a general category is the same in different places (Weissman et al., 1994). Cultural differences too are suggestive, although there are to date very few (reliable) comparative studies of the condition and most of them only bear on clinical populations (so we have no evidence of what intrusive thoughts are common or exceptional in the population at large). For instance, a study from Bali documents a culture-specific tweaking of the general OCD themes. The patient needs to identify all passers-by in terms of genealogy and status, and reports obsessions about spirits and witches (Lemelson, 2003). Both are culturally specific variants of the social harm and social exposure obsessions, as hierarchy and status are fundamental to social interaction in Balinese society and social strife is expressed through witchcraft accusations (Barth, 1993). In Muslim countries, by contrast, many patients report concerns about pollution and contamination strongly influenced by religious prescriptions on hygiene and purity of thought (Al-Issa, 2000; Mahgoub & Abdel-Hafeiz, 1991; Okasha, Saad, Khalil, el Dawla, & Yehia, 1994). A sample of Bahrain patients showed that the fear of blasphemy was prevalent (about 40% of cases), which may be a local expression of the fear of social harm and potential exclusion (Shooka, Al-Haddad, & Raees, 1998). This would suggest that an important calibrating factor is the range of cultural messages emphasizing potential danger. In particular, further epidemiological studies of the various dimensions of OCD (contagion, social offence, checking) may be correlated to the intensity of precautionary messages available in the environment of development. While Islam includes many descriptions of possibly impure actions or thoughts, Western children are bombarded with insistent warnings about invisible germs. Whether this results in significantly different normal and pathological intrusions is simply not documented yet. # 8. Implications of the Model: Cultural Ritual as Derivative So far, we have not mentioned one of the most salient and socially important manifestations of ritualized behaviors, namely, collective, culturally sanctioned rituals. We consider that the model presented so far can help us understand why rituals are widespread the world over and why they are compelling—an argument summarized here and presented elsewhere in more ethnographic detail (Liénard & Boyer, 2006). #### 8.1 A Capacity for Ritual? We start from the work of Fiske and colleagues. Comparing hundreds of ritual sequences with clinical descriptions of OCD cases, they showed that the same themes recur over and over again in both domains (Dulaney & Fiske, 1994; Fiske & Haslam, 1997). OCD-typical features that also enter into rituals include specific (lucky or unlucky) numbers, use of special colors, repetition of actions, measures to prevent harm, ordering and symmetry, stylized verbal expressions, washing, concern with contagion, and so forth (Fiske & Haslam, 1997). Fiske and colleagues speculate that there may be a human capacity to perform cultural rituals, that is distorted or hyperactive in pathological individual ritual (Fiske & Haslam, 1997). In Fiske's model, rituals are used to channel individual anxiogenic thoughts and make them bearable by providing a broader cultural context in which they can be shared and make better sense. Fiske and Haslam did not pursue the psychological and cultural implications of this hypothesis. It would provide a simple and elegant way of explaining the similarities in themes and actions between pathological and cultural ritual. Moreover, it would do so by connecting both to evolved, species-specific anxiogenic situations. However, we consider that cultural rituals may be better explained in a different way, as partly parasitic on the Hazard-detection and Precaution systems described above. Our main reason for preferring this account is that it is more parsimonious. There is no empirical evidence that humans do have a specific capacity for ritual. There are no evolutionary grounds to consider that such a specific capacity would be adaptive (see our discussion of rituals as possible adaptation in Section 9.1.) So this is a costly hypothesis. By contrast, we have seen that there is solid evidence for systems specialized in responses to potential hazard. So if the disposition to perform cultural rituals is a by-product of these systems, we do not need to posit additional mechanisms. #### 8.2 The Cultural Selection Background The first assumption in our treatment is that cultural rituals, like other forms of cultural behaviors, should be treated as the outcome of cultural selection (Boyd & Richerson, 1985; Durham, 1991; Sperber, 1985). Representations that we call 'cultural' occur with roughly the same content in other minds among people of a particular group. Indefinitely many factors (local or universal, psychological as well as physical) can in principle contribute to the spread of a particular mental representation. One type of factor of great interest to us is the set of general human dispositions that make certain representations, once they are expressed or conveyed by some people, particularly attention-grabbing or memorable or compelling, leading to their cultural transmission (Sperber, 1994). We observe that people seem compelled to perform particular ceremonies at particular junctures, and also that they seem compelled to perform them in (what they judge to be) the prescribed way. This is what we need to explain. Now, one way to explain this would be to posit that there must be a particular urge to perform such ceremonies, or that they may fulfill particular needs of the human mind or of human groups. However, there may be another kind of explanation, based on the fact that people who receive information about particular performances already have sets of mental systems designed to respond to particular classes of stimuli. The question becomes: Which mental systems would be activated, such that performing this ceremony in these circumstances would seem compelling? #### 8.3 Cultural Information, Mimicry, and Cognitive Capture Cognitive systems can be functionally described in terms of their particular input format, their operating principles, and their output. The input formats of cognitive systems are, in some cases, well known. For instance, the auditory stream provides information about pitch and location, which is then routed to different systems (Romanski et al., 1999). The pitch information is divided into linguistic input and nonlinguistic input, transmitted to different parts of the auditory cortex (Liegeois-Chauvel, de Graaf, Laguitton, & Chauvel, 1999). At each step, the transfer from one system to the other depends on the signals' format. This extends to higher cognitive systems. The range of stimuli or internally generated information that meets the input format of a system is its domain. Now it is important to distinguish between an evolutionary or proper domain of stimuli and an actual domain (Sperber, 1994, 1996). The proper domain includes those objects or situations that played a causal role in giving the particular system a selective advantage. The actual domain includes all objects or situations that trigger activation of the system. In most evolved cognitive systems, the actual domain is larger than the proper domain, giving rise to false alarms. The frog snaps at any small objects whizzing by in its visual field, not just to actual edible insects. Any system of this kind is vulnerable to capture and mimicry. The terms describe situations in which the system reacts to an input that matches its input format, and is part of its actual domain, yet is not among the classes of stimuli that the system was designed to process, its proper domain. We reserve the term 'mimicry' for the situations in which a particular behavior or physical trait in an organism gains adaptive value by entering the actual domain of another organism's cognitive system. This is what happens in familiar cases, like that of Viceroy butterflies adopting the genuine poison-warning garb of Monarchs without having to manufacture the poison. A different situation is what we call 'cognitive capture.' Consider a familiar example. Most visual art in humans (from tattooing to painting to architecture) seems strongly biased towards vertically symmetrical displays, while other symmetries are less salient. Vertical symmetry detection capacity appears in infancy (Bornstein & Krinsky, 1985; Fisher et al., 1981), influences pattern recognition in childhood (Bornstein & Stiles-Davis, 1984; Mendelson & Lee, 1981), and has evolved for purposes other than the appreciation of aesthetic displays, most probably for detecting facing predators and healthy mates (Thornhill, 1998). Music too is a good example, as it 'hijacks' certain parts of the auditory cortex and provides auditory super-stimuli (Jerison). Narratives about imagined persons can be, as we say, 'captivating' because they capture our capacities for mind-reading and the explanation of behavior. This is not mimicry since in the cases mentioned here the organism's Type I error does not benefit another organism. The important point about cognitive capture is that a great deal of human culture is acquired and transmitted because of this inevitable propensity of cognitive systems to 'fire' beyond their proper functional range. Most items of 'culture' in the sense of group-specific sets of norms and concepts depend for their transmission on cognitive capture of this kind (Sperber, 1994). #### 8.4 Core Ritualization in Cultural Rituals To understand the cognitive effects of collective rituals, we must describe the kinds of information available to the participants. At first sight, it would seem that most people who participate in most rituals do not have much information at all. People do not generally hold a 'theory' of their own rituals—this is what makes ethnography indispensable and difficult. However, this is not to say that people participate in a ritual on the basis of mere imitation, peering at their cultural elders and simply performing similar gestures. This would be implausible, given that very little human cultural transmission actually involves such mindless imitation (Sperber, 1996). In this particular case, some behavior activates some mental templates in the mind of observers, and triggers non-random inferences about what is accomplished by the behavior. This, we contend, may be sufficient to explain the cultural success of Ritualized Behavior. To make comparisons simpler, we follow in our description the outline of action ritualization processes described earlier. The individual reaction to a particular cultural ritual can be functionally described as consisting in the following elements: People receive specific information about the ritual: a. They are told that a ritual should be performed and are led to infer that nonperformance is a dangerous option. For instance, one is told that because of a particular event (someone's illness, a death or a birth, the change of seasons, a war with another group, possible damnation), it is necessary to go through a particular ritual sequence. b. People also receive information and produce inferences about the kind of danger against which the ritual is supposed to protect the group, for example, 'pollution' by invisible substances, attacks by invisible predators like witches or spirits, threat of disease, possible famine, social strife, and so on. These themes substantially overlap with the Potential Hazard Repertoire. This triggers a (dampened) activation of Hazard-Precaution system. People are instructed to participate in the ritual in particular ways. That is, people are generally not allowed to just add to their ritual whatever action they think fit. They are enjoined, more or less explicitly, to follow a particular script. Information about the script has the following properties: a. Action descriptions include themes that mimic some of the typical outputs of the Hazard-Precaution system: actions such as cleansing, washing, checking. b. Descriptions of prior conditions, particular taboos, substances to avoid, et cetera, reinforce activation of security motivation system. c. There is great emphasis on the details of each action, inducing lowlevel parsing of the action flow during performance, especially because of negative prescriptions. d. Description induces goal-demotion, by insisting on repetition, redundancy, apparently pointless acts, and so forth. Performance enacted in these conditions temporarily swamps working memory because of the attentional demands of the tasks. Performance ironically strengthens the salience of particular themes associated with gestures or situations to avoid during ritual. These various elements and their putative causal relations are outlined in Figure 4. In the next sections we present some evidence for these various claims and for the psychological and cultural effects of the processes. #### 8.5 Cognitive Capture in Cultural Rituals Our model suggests that ritualized actions are culturally successful to the extent that they activate information-processing and motivation systems made manifest in other domains of ritualization. In this sense, cultural rituals result in cognitive capture of the systems described so far, and this is why they can seem attention-demanding and compelling to participants. Many features of collective rituals activate the Hazard-Precaution system by including cues for potential dangers of the Evolutionary Potential Hazard Repertoire. First, occasions for ritual often allude to clues of possible danger that overlap with the Potential Hazard Repertoire: for example, threats to fitness such as famine or illness, invisible germs or miasma, dangerous invisible pollution present in newborn infants, dead bodies and menstruating women (Bloch & Parry, 1982; Metcalf & Huntington, 1991). Second, details of prescribed performance also include many security-related motifs. As we said previously, many collective rituals include such operations as washing and cleaning, checking and re-checking that a particular state of affairs really obtains, as well as creating a symmetrical or otherwise orderly environment (Dulaney & Fiske, 1994; Fiske & Haslam, 1997), so we will not comment on this any further. Fig. 4. A simplified model of action ritualization in cultural rituals. Boxes identity different functional systems in the same way as in Fig. 3 . Participants in rituals are provided with two kinds of information, (a) statements about potential danger and (b) scripted recipes for ritual action, that activate the security-motivation systems. Rules for ritual performance result in both goal-demotion and low-level action-parsing with the resulting swamping of working memory. These processes result in highly attention-demanding and compelling performance of rigidly scripted actions. This in turn makes the associations more salient, which should make subsequent messages about ritual more intuitively compelling. (Figure design by P Boyer, 2006). In our model, precaution systems are activated to the extent that particular themes (e.g., 'this village must be purified') and prescribed actions (e.g., 'wash hands three times in this particular river') trigger activation of evolved Precaution systems. This, however, does not entail that the ritual as a whole should be explicitly and exclusively about these themes. Indeed, there are many ceremonies in which prescribed behavior is only weakly related to these themes, while other themes (e.g., procreation, social exchange, hierarchy) are at the forefront of people's attention. Our claim is only that the ritualization itself is derived from the operation of Precaution and action-parsing systems. #### 8.6 Ceremonies, Ritualized Action, and Routinization This model, in our view, at least provides elements that go some way towards an explanation of why ceremonies that include ritualized actions are found in most human groups and are generally stable within traditions. The model also has some implications that make it diverge from received anthropological usage and common intuitions about ritual. Ritualized actions are not 'rituals.' Ritualized actions as described here are only a subset of what people actually do in what are called 'rituals.' For instance, a ceremony may include a typical example of what we described earlier, such as, a prescription to turn around a cow three times clockwise while avoiding to stare above the horizon and making sure to touch the cow with one's thumb only. But the circumambulation of the cow may be an element of a larger ceremony that also includes singing, dancing, feasting, and all sorts of other behaviors that are not precisely scripted in the sense described here. In other words, ritualized behaviors are certainly not the whole of 'rituals.' Ritualization is not routinization. The model has the slightly counterintuitive implication, that ritualized action is described as quite different from routinized behavior, indeed as its opposite. In most ceremonies we expect to find an alternation between phases of ritualized action (high control, attentional focus, explicit emphasis on proper performance) and routinized action (possible automaticity, low attentional demands, lesser emphasis on proper performance). Cultural ritual is not individual ritual writ large. We said that cultural ritualized actions are 'derivative' and it is important to stress that they are a by-product of the Precaution systems and the action-parsing systems, not of individual ritualized behavior. Given the similarities between individual and cultural forms of ritual, it is of course tempting to take one as a scaled version of the other, as Freud suggested (Freud, 1928). But this is clearly misguided. First, to maintain the parallel, cultural rituals would need to be behaviors that social groups initiate because they perceive certain potential dangers. But groups as a whole do not literally behave or perceive, only their members do. Also, cultural rituals differ from individual ones in the way the information about compelling action is acquired—from other agents and from personal intuition, respectively. Most importantly, what compels performance is entirely different in the two situations. While individual ritualists (especially patients) may feel great anxiety at the prospect of not going through the ritual sequence, participants in a cultural ritual are likely to participate (among other reasons) to the extent that the particular sequence meets a minimal threshold of relevance. The idea of 'scaling' would also predict all sorts of interesting phenomena that are simply not observed; for example, that people who become more religious would tend to become more obsessive, or that OCD patients would tend to be more religious than controls, that children during early childhood should be more interested in religious ritual than at other stages of development, and so on. Although there are connections between certain forms of religious practice and obsessionality (Fallon, Liebowitz, Hollander, Schneier, et al., 1990; Hermesh, Masser-Kavitzky, & Gross-Isseroff, 2003), they fail to support these general conjectures. # 9. Conclusions #### 9.1 Ritualization and Cognitive Adaptations Our models of individual and cultural ritualization take as a starting point a specific connection between obsessive pathology and security motivation (Mataix-Cols et al., 2005; Szechtman & Woody, 2004) but also a more general set of assumptions about the adaptive character of specialized neuro-cognitive function (Cosmides & Tooby 1994; Duchaine et al., 2001). We have assumed that the Hazard-Precaution system was the outcome of selective pressure for gradually finer-grained inferential detection of and appropriate response to recurrent hazards in ancestral environments. This naturally leads to the question, whether action-ritualization might constitute a cognitive adaptation, in the same way as other domain-specific capacities do (Cosmides & Tooby, 1994). The question should be more specific and bear on either individual or cultural rituals, since the cognitive processes involved are so different. Let us consider cultural rituals first. In the anthropological literature, there are various hypothetical models of the ways in which participation in collective ceremonial may have conferred adaptive advantage to individuals (Burkert, 1996; Knight, Power, & Mithen, 1998; Rappaport, 1979; Sosis, 2000; Watanabe & Smuts, 1999). This stems from a long anthropological tradition of construing ritual as crucial to social organization and cohesion (Durkheim, 1947; Hocart, 1970; Smith, 1889). We discuss the various hypotheses in more detail elsewhere (Liénard & Boyer, 2006). Suffice it to say that these different models may well explain a disposition to participate in coordinated social action, but not why these common endeavors should include scripted, goal-demoted, redundant scripting of familiar actions. The question of individual ritualization is more complex. In our model, the activation of the Precaution system normally results in performance of appropriate actions from the Precaution Repertoire and this, in most circumstances, should produce enough of a closure or satiety experience (Szechtman & Woody, 2004) to preclude reiteration. However, the closure experience probably is the outcome of continuous changes in the relevant circuitry, leading to various degrees of repetitiveness and anxiety about proper performance. So, in our model, it is not the ritualized behavior but the Precaution system itself that constitutes a cognitive-motivational adaptation. It has the hallmarks of such adaptations, such as a specific class of inputs, a specific mode of operation, a particular series of fitness-enhancing consequences, a nontrivial functional design—and, in this particular case, a specific neural implementation as well as specific impairment. #### 9.2 Phylogeny: Rituals and Displays What is the connection between human and other animal 'ritual'? We use scare quotes here, as the term is stretched to encompass highly disparate forms of behaviors (Gluckman, 1975). Nevertheless, one should comment on the obvious similarities between human rituals and various forms of animal communication, notably in the context of agonistic and sexual displays where stylized behavior, repetition, and redundancy are clearly present. Is this evidence for the deep phylogenetic ancestry of ritual? In our view, this question suffers from several ambiguities: First, although we may sometimes follow a 'same effects, same causes' rule of thumb, this is rather misguided if it leads us to confuse observable behaviors with the neuro-cognitive systems that support them (Povinelli, Bering, & Giambrone, 2000). Indeed, even in the limited domain of human rituals, apparently similar behaviors (in patients and in cultural ritual participants) actually stem from very different cognitive processes. This should a fortiori be expected when comparing widely different species. Second, the question downplays the extent to which certain features of behavior are constrained. Consider OCD patients for instance. They are not motivated by a positive urge to ritualize. Rather, ritualized behavior happens to constitute an optimal response to the anxiety produced by cognitive impairment. Other forms of behavior would not seem appropriate given the anxious concerns; they would not produce temporary relief. So the redundancy, et cetera, in this case stems from the properties of action-parsing and precaution systems in humans. Now consider animal displays. They are strongly constrained too, in this case by the logic of signaling processes. For instance, signals must be clear and distinct enough to preclude ambiguities, which typically results in redundancy (Rowe, 1999). The evolution of attentive receivers requires that signals maintain a relatively high level of accuracy (Bradbury & Vehrencamp, 2000; Silk, Kaldor, & Boyd, 2000) and that the content of the signals be directly related to the fitness dimensions they advertise (Zahavi & Zahavi, 1997). In other words, in both human rituals and animal displays, features like stylization, redundancy, and repetition are the outcome of external constraints, but these seem to be different in the two cases. This would support the tentative conclusion that the presence of 'ritual' in both cases is a case of behavioral analogy rather than the index of similar capacity and processes. (Obviously, this is not to deny that humans like other animals do engage in stereotypical displays, in situations of courtship or aggression). This is tentative in the sense that we do not know much about the phylogenetic history of ritualization (in the precise sense used here) in the hominin line. The evidence so far simply does not support the notion of a direct evolutionary homology. #### 9.3 Epilogue It is a cognitive and evolutionary puzzle that humans perform rituals, given the waste of time and resources involved. We aimed to solve the puzzle by piecing together the evidence from neuroimaging, neuropsychology, clinical psychology, developmental studies, and evolutionary anthropology. Ritualization may be seen as an occasional by-product of specific precaution systems and action-parsing capacities in humans. This explanation however compels us to discard the common intuition that there is a natural kind of phenomena called 'rituals.' If valid, our model does not explain 'rituals' but a highly specific form of behavior that is found in many of them and occurs for different reasons in the behavior of most normal children and obsessive patients, on the one hand, and in the context of collective rituals, on the other. Discarding misleading categories of behavior (like 'ritual'—but there are many others) may well be the inevitable consequence and benefit of proposing integrated explanations. Our model is an attempt to bring together neural systems, evolutionary background, behavioral manifestations, and developmental trajectory to the understanding of action-ritualization. We consider this indispensable. True, much work remains to be done to understand the phenomenon. For instance, the cognition of children's ritual is still largely unexplored; the connections between ritual performance and anxiety relief in patients need a proper neurophysiological study; the persuasive power of cultural rituals is not properly explained. But we are confident that all these and other puzzles will be solved by the kind of 'general behavioral science' that transcends fields and discipline boundaries. # References taxonomy. Behaviour Research & Therapy, 42, 647–670. https://doi.org/ 10.1016/S0005-7967(03)00173-6 symptoms? Journal of the American Academy of Child and Adolescent Psychiatry, 29, 17–23. https://doi.org/10.1097/00004583-199001000-00004 ——. (1999). Ritual and Religion in the Making of Humanity. Cambridge; New York: Cambridge University Press. are characteristic of obsessive compulsive disorder. Behaviour Research & Therapy, 38, 347–372. https://doi.org/10.1016/s0005-7967(99)00071-6 disorder. The Cross National Collaborative Group. Journal of Clinical Psychiatry, 55, 5–10. # 4. Social Groups and Adapted Minds # Introductory Note Relations between different ethnicities are often fraught with conflict, from mild suspicion to avoidance, discrimination or violent inter-group conflict. In many places, inter-group conflict has consequences for people's well-being, including their health. But why is that the case? In this article, Rengin Firat, Florian van Leeuwen and I tried to propose a general model of inter-group conflict that would explain these welldocumented and sometimes paradoxical public health outcomes. In our view, one cannot properly address questions of inter-group relations without a clear understanding of the ways humans form alliances. In traditional social sciences, people often simply assumed that there are social groups, that people find it self-evident that they 'belong' to a particular collection of individuals, and that people sometimes sacrifice their individual welfare for what they see as the good of the group. Social scientists, like the rest of us, used to find all these phenomena self-evident, simply because they are very familiar. One advantage of taking an evolutionary standpoint is that the familiar cannot be taken for granted, because it happens to be particular to our species. Sustained alliances between genetically unrelated individuals are rare and limited in scope in most animal species (Dugatkin, 1998). But they are ubiquitous among humans, who can form and maintain alliances both stable and extensive, scaling up from a few individuals to several millions. How is this possible? A possible, common and often sterile approach is to catalogue those collections of individuals that people identify as different 'groups' in their society, and to try to classify kinds of groups. One could sort them, for instance, in terms of size (a street gang vs. an army), permanence (crowds vs. nations), or solidarity (a village vs. a group of commuters) and try to produce a taxonomy of groups based on these observable features. Is that really wrong? In a sense, it is not, since documenting varieties of phenomena is the starting point in any scientific study. But that does not by itself provide us with explanations for the observed similarities and differences. Alliances between unrelated individual agents are (at least as a goal) mutually beneficial interactions. They dissolve when members do not see participation as favorable to their welfare. That is why the most promising interpretation of group formation and dynamics came from rational choice models (Elster, 1989; Hechter, 1987). These described the way aggregate individual interests could explain group dynamics the conditions under which each individual may expect to gain from participation in alliances. The one advantage and limitation of these rational choice models is that they assume no complex psychology in the agents, except a set of prior preferences, some perception of the expected benefits from different courses of actions, and of course a motivation to increase their expected benefits. This description of agents, adopted from micro-economics, is often very powerful, especially in the aggregate. But, as many have pointed out, it comes with two limitations. First, it assumes that agents have an accurate perception of the benefits that may result from their behavior, which is a convenient idealization. Second, more important, rational choice models do not (try to) explain why agents have the preferences they have. Now understanding the origin of preferences, and describing the nature and limits of human capacities, are precisely the main goals of evolutionary psychology, in combination with the models and findings of economics, neuroscience and anthropology. In this perspective, we can put forward precise, testable hypotheses about the kinds of preferences that would have been the object of positive selection in human evolution. What makes human alliances and groups possible is a set of evolved mechanisms that allow us to see the benefits of coalitions, to detect what alliances are present in our social environment, to monitor who is and who is not committed to the coalitions we join, to signal our own commitment, and so forth. Over the last thirty years, evolutionary scientists have added considerable detail to our understanding of these capacities. For instance, they demonstrated how people are intuitively suspicious of the status of newcomers in a group (Cimino & Delton, 2010), which neuro-physiological systems support coalitional affiliation and rivalry (De Dreu et al., 2011), how people in some countries readily encode 'racial' identities as coalitional rather than merely perceptual (Kurzban et al., 2001; Pietraszewski et al., 2014), how accent is seen as a cue of alliances (Pietraszewski & Schwartz, 2014), and much more. For general surveys of the field, see Boyer (2018, Chapter 1), Pietraszewski (2016), and Tooby & Cosmides (2010). # References Regulates Categorization by Coalition and Race, but Not Sex. PLoS One, 9, 1–19. https://doi.org/10.1371/journal.pone.0088534 # Safety, Threat, and Stress in Intergroup Relations: A Coalitional Index Model with Rengin Firat & Florian van Leeuwen1 Contacts between people from different groups engage a variety of human competencies and motivations, from high-level representations of social categories to visceral responses when confronted with strangers, from cognitive appraisal of conflict to a desire to exclude or even attack 'others.' There is a correspondingly diverse set of fields and subfields in psychology and the social sciences focusing on such specific topics as racial prejudice, ingroup bias, ethnic identity, xenophobia, and nationalism. In this article, we propose a model that cuts across boundaries between these different fields to describe and explain fundamental aspects of intergroup relations. The psychological literature in this domain comprises a vast number of empirical generalizations without an overarching explanatory perspective. This results in many ambiguities and paradoxes. For instance, belonging to a subordinate or stigmatized group is often described as intrinsically stressful, with negative health effects, but living among one's own stigmatized group sometimes has a positive impact on health (Shaw et al., 2012). Or, racism is commonly found to be associated with conservative or authoritarian values, but the supposedly <sup>1</sup> An earlier version of this chapter was originally published as Boyer, P., Firat, R., & van Leeuwen, F. (2015). Safety, threat and stress in inter-group relations. A coalitional index model, Perspectives in Psychological Science 10(4): 434–450. https://doi. org/10.1177/1745691615583133. Reprinted with permission from Sage Publications. conservative army is the one setting in the United States where people are most satisfied with interrace relations (Bullock, 2013). Or, people are considered to resent immigrants because they threaten the host population's cultural and symbolic supremacy, but when immigrants assimilate and adopt to the majority's cultural symbols, this triggers even stronger resentment in many people (Guimond, De Oliveira, Kamiesjki, & Sidanius, 2010). Many empirical findings are treated as unrelated phenomena, mostly because they are studied in distinct subfields of the social sciences. Finally, a great deal of the social psychological literature in this domain makes no connection to equally salient processes of intergroup relations studied in anthropology, human evolution, history, and economics. We propose that many aspects of intergroup relations should be construed as different manifestations of a coalitional psychology. We describe coalitional psychology as a set of evolved mechanisms designed to garner support from conspecifics, organize and maintain alliances, and increase an alliance's chance of success against rival coalitions. In this perspective, the core psychological mechanisms are the same, independent of whether the alliance in question is formed as ethnic (based on perceived similarity and common origin), racial (based on ethnicity combined with phenotypic similarity), regional, or political, and so forth. The point of the proposed paradigm is not to discard or replace extant models or explanations but to illustrate how they can be integrated into a broader framework, which we hope will give rise to new predictions and hypotheses. Consistent with other research in evolutionary psychology (Kurzban & Neuberg, 2005; Navarrete, McDonald, Molina, & Sidanius, 2010; Neuberg, Kenrick, & Schaller, 2010; Tooby & Cosmides, 2010), we argue that whether the coalitional cognitive system is activated, and what information it processes, may provide a parsimonious causal explanation for many representations, attitudes, and behaviors in intergroup relations. Also, we contend that intergroup relations are strongly influenced by threat-detection mechanisms. Threat detection results in the adjustment of an internal variable, the coalitional safety index, an individual's representation of the safety induced by membership in an alliance. The level of this variable is modulated by cues of coalitional threat and support, for example, cues of decreasing support from one's own group or of increasing menace from rival groups. These threat cues can lead to coalitional stress, with standard physiological stress responses. # 1. Evolved Cognition Background 1.1 Human Coalitional Psychology Stable alliances are rare in most animal species (Harcourt & de Waal, 1992). By contrast, cooperation among non-kin toward a common goal in stable alliances is ubiquitous in human social interaction, suggesting a suite of specialized motivations and capacities that appeared during human evolution. Coalitional processes may be found at many different levels of organization, such as political parties, street gangs, office cliques, academic cabals, and bands of close friends, and can include thousands or millions of individuals when ethnic or national categories are construed as coalitions. Coalitional psychology is a crucial element of the human capacity for collective action, in which a collection of agents cooperate toward a particular (set of) goal(s) that cannot be achieved by any single individual (or only at much greater cost); these agents behave in ways that increase each agent's welfare by making it more likely that the goal is achieved (Hardin, 1982; Myatt & Wallace, 2009). Humans for a long time have required, for their survival and reproduction, extensive support from kin but also from non-kin conspecifics, for example, in hunting (Dubreuil, 2010; Kelly, 1995), parenting (Hrdy, 1999, 2009), trade (Jaeggi & Van Schaik, 2011), and defense against other humans (Gat, 2006; Keeley, 1996). These evolutionary conditions explain why human groups are often stable and competitive. Humans need relatively stable alliances, because many endeavors require a prior assurance that support will be available when needed—warfare is a case in point. Also, human alliances may become rival even in contexts that may not require competition, because social support itself is a rival good. If an alliance builds up offering its members mutual support, it deprives others of that resource, so that one would expect coalitions to emerge as a response to the existence of other coalitions. Collective action, as described by biologists and economists (Dugatkin, 1998; Medina, 2007; Mesterton-Gibbons & Sherratt, 2007), requires that agents engage in highly specific information processing concerning their own and others' behaviors. For instance, (a) payoffs to other members of the group are considered as gains for self (and, obviously, negative payoffs as losses to self); (b) payoffs for rival alliances are assumed to be zero-sum—the rival group's success is our loss, and vice versa; and (c) other members' commitment to the common goal is crucial to one's own welfare. As a consequence, (a) each member monitors other members' levels of commitment, (b) there is a strong motivation to demonstrate one's commitment to the other members, and (c) there is an inclination to make defection less likely, notably by making it costly. Participants in coalitional interactions rarely, if ever, represent these principles explicitly. All they are aware of are intuitive preferences, for instance, a desire to punish a renegade, a motivation to engage in risky behaviors for the good of the cause, an interest in whether and how far a specific person can be trusted or the fact that one's enemies' enemies can be strategic allies. Such motives and cognitions may seem self-evident to both actors and observers, and the necessary complex computations are not available to conscious inspection (Kurzban & Neuberg, 2005). To say that there is a coalitional psychology, distinct from other mental system, does not entail that there is a demarcation between coalitions and non-coalitions in social life. First, coalitional psychology can be activated in relation to very different types of groupings—some may be based on a common category or origin (e.g., gender, ethnicity, nation) and others not (e.g., office cliques). Second, activation of coalitional psychology is in many cases contextual—an agent may treat a certain category as coalitional (e.g., the young against the old, Blacks versus Whites) in some situations but not in others. Third, one may treat a collection of agents as coalitional, while one's partners do not. The coalitional construal is in the eye of the beholder and need not align with others' construals. When one's representations of a social category activate coalitional psychology, one (implicitly or explicitly) assumes that people belonging to that category have a greater stake in each other's welfare than they have in that of outsiders; one also assumes that they are committed to the common goal, that is, prepared to suffer some costs to advance the overall position of the alliance. This background of assumptions may shape people's representations of group interactions in terms of common goals, potential cooperation, and indirect or direct reciprocity—the features that most explicitly influence group-oriented behavior (Balliet, Wu, & De Dreu, 2014). ### 1.2 Coalitional Psychology in Context: Threat-Detection Systems Important aspects of human coalitional psychology should be understood in the context of threat detection. Natural selection results in systems that attend to recurrent danger cues in environments of evolution and guide appropriate responses (Boyer & Bergstrom, 2011; Boyer & Lienard, 2006). Survival and reproductive success require not just avoiding present danger (e.g., a predator present) but also detecting potential fitness threats (e.g., footprints indicating predator presence). Evidence from ethology, neurophysiology, and experimental psychology shows that present and potential hazards elicit different reactions and orchestrate distinct neural circuitry (Blanchard, Griebel, Pobbe, & Blanchard, 2011; Woody & Szechtman, 2011). Research on threat detection has described two features of animal threat-detection systems that are likely relevant for humans' coalitional psychology. First, safety and threat are not two sides of the same coin (Szechtman & Woody, 2004). Threats can be inferred from the actual presence of particular cues in the environment (e.g., the smell of a predator), but the absence of predators is not usually indicated by any perceptible property of the environment. The absence of evidence is not evidence for absence. Indeed, most complex animals do not immediately infer safety from the removal of threat cues (Dielenberg & McGregor, 1999). Rather, animals' return to a baseline level of perceived security seems to be internally generated, mostly through performance of precautionary routines (Woody & Szechtman, 2011). Second, the costs and benefits of inferring safety and threat are often asymmetrical (Haselton & Buss, 2000; Haselton & Funder, 2006; Haselton & Nettle, 2006). Individuals usually face a trade-off between false alarms (e.g., inferring the presence of a predator, when none is present) and misses (e.g., failing to infer the presence of a predator, when one is present), where false alarms are much less costly than misses. Therefore, error management models predict that many features of social psychology are characterized by displaying false alarms—that is, by erring on the side of caution. More generally, such models explain why cues that indicate a potential reduction of safety tend to have a stronger impact on attention and motivation than cues that indicate increased safety (Baumeister, Bratslavsky, Finkenauer, & Vohs, 2001; Rozin & Royzman, 2001). # 2. The Model ### 2.1 The Coalitional Safety Index is an Internal Regulatory Variable We propose that various cues concerning potential social threats and social support are summed up as an internal regulatory variable, a coalitional safety index, the level of which is adjusted in each individual from situation to situation. Formally, this variable is similar to other regulatory variables proposed in the biological psychology and physiology (Tooby, Cosmides, Sell, Lieberman, & Sznycer, 2008), such as indexes for hunger or thirst (Loewenstein, 1996), overall security (McGregor, Adamec, Canteras, Blanchard, & Blanchard, 2005; Szechtman & Woody, 2004), and kinship (Lieberman, Tooby, & Cosmides, 2007). Such an index (a) integrates information from many other cognitive systems and sums them in a single value, which (b) has effects throughout the organism, such as allocating cognitive resources, modifying goal priorities, and triggering emotional and physiological reactions, and (c) predictably affects behavior (Tooby et al., 2008). Given human dependence on social support, we expect human cognitive systems to provide efficient monitoring of the availability of coalitional help. Indeed, the evidence shows that people automatically attend to alliance-relevant information in their social environment. For instance, they look for cues of reliability in potential partners by monitoring their behavior (Bacharach & Gambetta, 2001) or their faces (van't Wout & Sanfey, 2008); they seek information about others, for example, through gossip (Dunbar, 1996; Hess & Hagen, 2006; Wert & Salovey, 2004); they automatically monitor alliances among others, even among outsiders (Pietraszewski, Cosmides, & Tooby, 2014)and they carefully evaluate the status of ongoing friendship ties (DeScioli & Kurzban, 2009; Tooby & Cosmides, 1996) #### 2.2 Coalitional Threat Coalitional psychological systems, as well as delivering a representation of the social environment as composed of competing alliances, also produce inferences of danger (i.e., information likely to activate appropriate emotional systems and engage specific danger-related physiological response). Other alliances can be seen as threats both to the person (e.g., losing one's job, being attacked) and to his or her group (losing influence, power, cultural pre-eminence, and so on; (Rosenstein, 2008). People should be able to detect both within and between-alliance threat cues. Cues suggesting that coalitional support is diminishing or absent should result in reduced levels of coalitional safety in people within an alliance (Pratto & John, 1991). Such cues include information pointing out that one's coalition partners do not consider one an actual member of the alliance, that they do not consider one sufficiently committed and trustworthy, or that they are less committed to the coalition than oneself. In situations that allow for potential physical conflict, we would expect people to be sensitive to other coalitions' number, cohesiveness, and aggressiveness, as each of these factors is relevant to the level of safety provided by one's own group (see, e.g., Schaller & Abeysinghe, 2006). Coalitional threat cues would trigger a strong motivation to engage in a variety of behaviors to avoid the threat and return to a higher level of coalitional safety, for example, by sending clearer commitment signals, by cultivating homogeneity in the group, by avoiding members of other alliances, and by competing with or fighting against members of rival coalitions. Threat-detection systems do not just raise a general alarm level in the face of generic danger. They typically respond in highly specific ways, in social as well as other domains. Other groups may be associated with economic or territorial competition but also with potential physical violence or with pathogen transmission (Schaller, 2006). Neuberg and colleagues have shown that these diverse kinds of threat representations trigger distinct, appropriate emotional responses and precautionary behaviors (Cottrell & Neuberg, 2005; Schaller & Neuberg, 2012). However, on a physiological level, qualitatively different threats may evoke fairly uniform stress responses. #### 2.3 Coalitional Stress Mammals have evolved two neurophysiological responses to direct challenges (Gunnar & Quevedo, 2007). One response is immediate (i.e., within seconds) and involves the fight-or-flight response; the other is a slower, more durable response (i.e., within minutes or hours) that organizes longer-term changes of behavior. The fast reactions are orchestrated by the sympathetic-adrenal medullary system, associated with activation of the sympathetic nervous system, and expressed through release of epinephrine. The slower response involves activation of the hypothalamic-pituitary-adrenal system, is associated with parasympathetic activation, and results in the release of glucocorticoids (cortisol in humans). Repeated activation of these responses results in chronic stress, with important consequences for health and well-being (Sapolsky, 2007). A crucial part of our model is that the detection of coalitional threat cues in one's social environment triggers a stress response. Repeated exposure to such cues may lead to chronic stress, which in turn yields negative health consequences. Therefore, to the extent that many individuals in a specific social category are exposed to similar coalitional threats, we should expect these effects to translate into differences in health outcomes at the level of social groups. #### 2.4 Specific Computations In the model proposed here, many aspects of intergroup psychology are construed as domain specific, geared to the management of coalitions. This stands in contrast to some classical models of social affiliation in terms of broad, domain-general processes, such as stereotyping, preference for familiarity, motives for distinct identity, or desires for self-esteem (see 'Integrating Classical Frameworks' later in this article). We propose that specialized cognitive systems orient attention to specific information relevant for computing coalitional safety and threat. In the course of everyday life, people are constantly sampling their social environment and automatically making inferences about properties of that environment. For instance, perception of the numbers of immigrants in one's country is heavily influenced by the number of visibly 'foreign' individuals encountered (Center, 2006). For the purpose of making inferences about coalitional safety and threat, we expect coalitional psychology to focus on such information as the number of individuals in one's coalitions, the number of individuals in other perceived coalitions, changes in those numbers, the perceived aggressiveness of these coalitions, their cohesiveness, and their respective members' commitment, strength, and so on. The model predicts that these inferences regarding coalitional safety and threat result not in unspecified positive or negative affective states but in domain-specific affective states that motivate a limited set of courses of action, appropriate for coalitional purposes. We summarize the model in Figure 1. Below we survey a number of well-known aspects of intergroup relations and describe how they can be understood in terms of cues that increase or decrease the coalitional safety index. # 3. Intergroup Encounters as Threat Cues We start with the individual impact of intergroup encounters. In the short survey that follows, we emphasize how a coalitional appraisal system integrates various cues and as a result adjusts the coalitional safety index. #### 3.1 Association between 'Outgroups' and Danger The literature on the association between outgroups and danger is vast but essentially convergent, suggesting that this relationship is implicit and largely automatic, resulting in an 'avoidance' rather than 'approach' motivation (Paladino & Castelli, 2008). For example, when primed with faces of Black men, American subjects expect weapons rather than tools (Payne, 2001; Payne, Lambert, & Jacoby, 2002). People categorized as potential enemies seem physically stronger than controls (Fessler & Holbrook, 2013), whereas being in the company of friends Fig. 1. Schematic representation of functional processes involved in the adjustment of the coalitional safety index (left) and examples of such processes (right). The model describes how attention to social information, for instance, about people's behaviors indicating affiliation, leads to inferences of social threat and social support. These inferences modulate the coalitional safety index, which has two main consequences. First, it changes motivations concerning action plans, for instance, an effort to remain within one's group, to avoid others, or to boost solidarity in one's own group. Second, lowering the coalitional safety index triggers a stress response, which can have adverse long-term consequences. (Figure design by P Boyer. 2015). makes potential enemies seem physically smaller (Fessler & Holbrook, 2013). Fear is more easily attributed to out-group than ingroup faces (Navarrete et al., 2009), even when participants were assigned to minimal groups—artificial groups construed for the purpose of the experiment based on an arbitrary criterion (Navarrete et al., 2012). Encounters with outgroups are experienced as uncertain and demanding (Blascovich, Mendes, Hunter, Lickel, & Kowai-Bell, 2001), often create a specific form of 'intergroup anxiety' (Stephan & Stephan, 1985), and are associated with an increase in behaviors like blinking and fidgeting (Fazio, Jackson, Dunton, & Williams, 2008). Neuroimaging studies have also demonstrated specific fear-circuitry activation in response to stimuli depicting outgroups (Hart et al., 2000). Why are outgroups implicitly and often explicitly perceived as potential danger? This association is often explained in terms of shared stereotypes about social categories. In this view, encounters between Blacks and Whites in the United States, for example, are stressful because of a White stereotype of Blacks as violent and a Black expectation of White racism. However, it is not parsimonious to explain each case of difficult intergroup contact in terms of specific cultural stereotypes, as they occur even with minimal groups for which there are no stereotypes and more generally because the phenomenon is ubiquitous and thus demands a general explanation. Indeed, most people in most cultures known to history and anthropology have expected intergroup relations to be fraught with danger or at least some measure of hostility (Gat, 2006; Keeley, 1996). The notion of 'others' as threatening is an essential component of the ethnocentric prejudice generally observed in human societies (LeVine & Campbell, 1972). ### 3.2 Categories Tacitly Construed as Coalitions: Race in the United States In our model, what drives people's intuitions about members of some social category as potential danger is not (just) information about characteristics of that category but the specific inference that members of that category are a coalition, that they are striving to achieve common goals against other alliances, including their own. Consider interracial encounters in the United States. Usually, race is automatically encoded by American participants, regardless of protocols and task demands. However, 'race' is unlikely to be part of our evolved conceptual repertoire, because encounters with people of visibly different ancestry are a recent phenomenon in terms of evolutionary history (Cosmides, Tooby, & Kurzban, 2003). These encounters did not occur regularly before efficient modes of long-range transportation were invented. Excluding a long-evolved adaptation to interracial encounters, one possible interpretation of automatic race encoding is that it is simply a byproduct of general perceptual biases. A more plausible alternative is that race, in the United States, is a proxy for coalitional affiliation. To demonstrate that, Kurzban, Tooby, and Cosmides (2001) used a memory-confusion paradigm in which they presented participants with different target faces, together with text suggesting that two coalitions were involved in a conflict. Each of the suggested alliances crossed racial categories. As predicted, this manipulation resulted in significantly less accurate memory for race than in conditions without such coalitional cues (Kurzban et al., 2001), showing that retrieving coalitional affiliation interfered with race but not with other distinctive features like gender. In other words, current coalitional concerns and external cues can easily interfere with encoding or retrieval of racial categories, because they activate the same cognitive systems (Pietraszewski, 2009). The automatic encoding of racial categories, then, is not a simple matter of perceptual cues but requires a conceptual elaboration of these cues as a proxy for coalitional rivalry, which is a specific consequence of the U.S. social history (Cosmides et al., 2003). This coalitional interpretation also makes sense of other empirical results concerning Black–White encounters. For instance, automatic race encoding is stronger when perceiving lower-class Blacks than when perceiving middle-class Blacks (Weeks & Lupfer, 2004), presumably because lower-class Blacks are seen as more hostile than middle-class Blacks by most research participants. Also, gathering participants into mixed-race minimal groups interferes with the familiar phenomenon of racial ingroup bias (Van Bavel & Cunningham, 2009). #### 3.3 Intergroup Encounters and the Stress Response As outgroups are associated with hazard, encounters with them trigger physiological processes appropriate in the face of potential danger, a process that is crucial to understanding the cognitive effects of intergroup relations (Blascovich et al., 2001; Page-Gould, Mendoza-Denton, & Tropp, 2008). Specific cardiovascular responses may result from unexpected and limited physical contact with an unfamiliar outgroup (Vrana & Rollock, 1998) or from imposed dyadic interaction with outgroup members (Littleford, Wright, & Sayoc-Parial, 2005). Such cardiovascular reactions are modulated by cognitive appraisal of the situation. For instance, White participants' physiological reactions during dyadic interaction are modulated by the self-description of Black interaction partners (confederates) as advantaged or disadvantaged (Mendes, Blascovich, Lickel, & Hunter, 2002). Also, uncertainty about another's attitudes and intentions is a major contributor to the physiological response. In one study, responses of both Black and White participants to negative evaluations depended on the race of the evaluator and triggered threat reactions only when the evaluator was of the same race. Positive evaluations tended not to trigger threat reactions, except when they contradicted stereotypes. Whites may expect positive evaluations from both Whites and Blacks, and Blacks may expect positive evaluations from Blacks. But Black participants likely expected negative evaluations from Whites and responded to 'suspicious' positive evaluations by Whites with an increased threat response (Mendes, Major, McCoy, & Blascovich, 2008). Such responses are also observed as reactions to merely anticipated interracial encounters, as measured in both cardiovascular responses (Sawyer, Major, Casad, Townsend, & Mendes, 2012) and subjective ratings of health (Page‐Gould, Mendoza‐ Denton, & Mendes, 2014). These effects of intergroup contact are best understood in terms of an intuitive appraisal of the resources available to each partner (Blascovich et al., 2001). Cognitive, emotional, and physiological responses to intergroup encounters engage both a primary appraisal (to evaluate potential danger) and a secondary appraisal of one's own resources, leading to a coping versus stress polarity, which in turn results in either engagement or antagonizing behaviors (Trawalter, Richeson, & Shelton, 2009). Stress is a response to situations appraised as incompatible with an organism's goals (Lazarus, 1984). So understanding stress responses requires that we identify the appraisal system involved (Smith & Kirby, 2011). We propose that the coalitional safety index constitutes precisely such an appraisal. Stress responses would make little functional sense if people were confronted only with instances of stereotypes. For instance, the fact that one's partner in an experimental dyad comes from a group reputed to be incompetent should not impair one's own performance or result in a specific cardiovascular response. By contrast, if that encounter is implicitly framed as potential danger, the response is clearly functional. # 4. Coalitional Stress and Health We should expect repeated exposure to stressors, in the coalitional domain as elsewhere, to result in chronic stress with observable physical and mental health consequences. #### 4.1 Minority–Majority Health Disparities The world over, immigrants and minorities suffer from worse health than host or majority populations (D. R. Williams, 2012). In many cases, obviously, immigration is confounded with oppression, poverty, or trauma from exile. However, the pattern also obtains in settled immigrant communities, such as Latinos in the United States (Osypuk, Bates, & Acevedo-Garcia, 2010) or South Asians in Britain (Carpenter & Brockington, 1980). Such negative impact of emigration on health sometimes leads to the 'immigrant paradox' whereby foreign-born members of these groups fare better than those born in the host country (Alegría et al., 2008). Health disparities between immigrant and host populations can be observed in virtually all modern industrial societies with large migrant groups (Bak-Klimek, Karatzias, Elliott, & Maclean, 2014; Noymer & Lee, 2013) A similar disparity is observed between low-status social categories and the rest of the population. For instance, racial-minority members in the United States get sick more often, die at younger ages, and have more hypertension and lower levels of subjective well-being than Whites (Geronimus, Bound, Waidmann, Hillemeier, & Burns, 1996; D. R. Williams, 2012). Though there are exceptions to this pattern (Morales, Lara, Kington, Valdez, & Escarce, 2002), it seems that in general, native ethnic and racial minorities fare worse on various health outcomes and rate their well-being lower than non-minorities. Part of this disparity stems from economic conditions, such as access to nutrition, type of work, and access to healthcare (Lynch, 2000). However, the differences persist even after controlling for these factors, suggesting that discrimination as such has a general deteriorating effect on health (Mays, Cochran, & Barnes, 2007; Pascoe & Smart Richman, 2009). The poor health outcomes for minorities and stigmatized groups may result from a range of social processes, including categorization, hierarchical ranking of groups, and perceived levels of achievement or competence (Major, Mendes, & Dovidio, 2013). Stress is generally recognized as the crucial causal link between discrimination and health (Major et al., 2013; D. R. Williams & Mohammed, 2009). Perceived discrimination tends to elevate physiological stress responses such as blood pressure, cardiovascular reactivity, and heart rate (Brondolo, Rieppi, Kelly, & Gerin, 2003; Guyll, Matthews, & Bromberger, 2001; Utsey & Hook, 2007). Epidemiological studies support this stress-based explanation for Blacks in the United States (Clark, 2000; Clark, Anderson, Clark, & Williams, 1999) and for ethnic minorities more generally (McEwen, 2004; McEwen & Stellar, 1993). Even merely anticipated discrimination can produce stress (Karlsen & Nazroo, 2004). That discrimination causes stress explains why the greatest health disparities between minority and majority groups is found in conditions typically brought about or worsened by chronic stress, such as obesity, heart disease, and hypertension (Geronimus et al., 1996). A similar process may be responsible for deteriorated health among immigrants, as 'acculturation stress' accumulates in individuals confronted with new values or norms (C. L. Williams & Berry, 1991). The connection between acculturation and stress has been observed among Asian immigrants in the United States (Chung & Epstein, 2014), Latino students (Cano, Castillo, Castro, de Dios, & Roncancio, 2014), and older adults (Kwag, Jang, & Chiriboga, 2012). Indeed, newly arrived Latino immigrants in the United States enjoy a health advantage (in terms of adverse effects of chronic stress) over the rest of the Hispanic population, which decreases with each decade spent in the United States (Kaestner, Pearson, Keene, & Geronimus, 2009). In a survey of health disparities, Major and colleagues reviewed a variety of factors (stereotype threat, excessive vigilance, memories of injustice, attributional ambiguity, and many more), all of which are documented as contributing to stress responses (Major et al., 2013). In summary, there is overwhelming evidence that, in many different ways, the experience of minority or immigrant individuals includes a frequent occurrence of stress-inducing episodes, more so than for host or majority populations (Contrada et al., 2000, 2001). #### 4.2 Ethnic Density Effects There is an interesting exception to general health disparities between majority and minority groups: the ethnic density effect. This effect refers to the situation when immigrants or members of minorities who live among other members of their group fare better than those who live among the majority population. This effect is counterintuitive, as immigrant or minority neighborhoods are generally poorer, less pleasant, and afford less access to health resources. Ethnic or group density effects were first observed in the domain of mental health (Bosqui, Hoy, & Shannon, 2014; Halpern, 1993; Shaw et al., 2012). For instance, British Asian immigrants in more homogeneous environments have a lower incidence of psychoses (Boydell et al., 2001; Das-Munshi et al., 2012; Das-Munshi, Becares, Dewey, Stansfeld, & Prince, 2010) and other pathologies like self-harm (Neeleman, Wilson-Jones, & Wessely, 2001). In the United States, the effects of acculturation on depression are modulated by group density among Latinos (Kwag et al., 2012). Ethnic density also influences general health outcomes (Becares & Nazroo, 2013; Pickett & Wilkinson, 2008). For example, birth weight among U.S. Latinos is higher in mostly Latino neighborhoods (Osypuk et al., 2010); Black mortality from cardiovascular conditions is higher in more mixed neighborhoods in New York (Fang, Madhavan, Bosworth, & Alderman, 1998). There is no consensus explanation for such density effects and surprisingly little systematic hypothesis testing about its causes (Shaw et al., 2012). Density may correlate with better social integration—that is, each individual has more and better social ties in homogeneous places (Pickett & Wilkinson, 2008). However, it is not clear that social capital mediates the density effect (Becares & Nazroo, 2013). An alternative is that ethnic density provides 'buffering' against the social psychological effects of discrimination (Becares & Nazroo, 2013). Living in an ethnically homogeneous place may decrease the psychological weight of stigma (Pickett & Wilkinson, 2008). Assuming that this is the case, we still have no precise functional description of the processes whereby stigma or, conversely, protection from stigma would result in specific health outcomes. #### 4.3 Coalitional Interpretation A proper explanation of the effects of intergroup relations on health should account for both the overall disparity between groups and the interaction with ethnic homogeneity. Explanations in terms of societal phenomena like stigmas or shared stereotypes may not provide a sufficiently specific description of the psychological and physiological processes involved. We propose that the coalitional safety index is affected by a variety of threat cues, including the absence of individuals willing to extend support, decreases in number of such individuals, the presence of members of rival groups, their number, an increase in their number, their perceived level of hostility, and their perceived capacity to inflict harm. In other words, the perception of coalitional safety is influenced not just by activated beliefs about one's own and other groups but also by inferring probable states of the world from such features as relative numbers, frequencies of encounters, and tenor of interaction. The daily experiences of minorities or immigrants, on the one hand, and majority or host populations, on the other, diverge on these elementary metrics. First, even assuming an equal level of perceived danger in all intergroup encounters and all else being equal, minority individuals are bound to encounter majority individuals more frequently than vice versa. Second, in these encounters, minority members are more likely than majority individuals to appraise the situation as one of weaker coalitional position. For minority individuals, each encounter with majority members potentially constitutes a threat cue, in that it reminds the minority person that he or she is a member of a less numerous and probably weaker group. So even in terms of low-level properties of the social environment, the natural sampling described above should result in a higher frequency of stressors (i.e., a higher number of situations in which the coalitional safety index is down-regulated, if only momentarily). Such an information-processing account also explains the ethnic density effect. Living in ethnically homogeneous neighborhoods changes the base rates of encounters with same- and rival-coalition members, thus reducing the number of stressors. One would expect that the cumulative, chronic stress effect is therefore smaller for minority individuals living in homogenous neighborhoods. Our interpretation predicts that this beneficial effect of homogeneous neighborhoods may be diminished if an individual does not perceive the frequent own-ethnicity encounters as interactions with coalitional allies. Indeed, people of very low status in their communities do not benefit from ethnic homogeneity (Ayers et al., 2009; Cano et al., 2014; Chae, Park, & Kang, 2014). # 5. Integrating Classical Frameworks Beyond providing explanations for consequences of intergroup contacts, the coalitional model may also help us integrate some standard perspectives on intergroup relations. Specifically, we consider here distance and con-tact approaches, social identity perspectives, and finally social dominance theory. #### 5.1 Intergroup Contact and its Paradoxes We argue that some aspects of intergroup relations should be explained in terms of the psychological processes involved in individual encounters with outgroups. That is also the starting point of the various hypotheses put for-ward in the 'contact' tradition (Dovidio, Gaertner, & Kawakami, 2003), which aims to reduce the prevalence of negative stereotypes and attitudes about outgroups by increasing the frequency and quality of encounters with outgroup members (Thomas F. Pettigrew & Linda R. Tropp, 2006). However, generalizing this association between more contact and more positive relations would be clearly difficult. Places of high outgroup fear and rejection, like the antebellum South in the United States or apartheid South Africa, were also places of intense, daily contact and deep familiarity between dominant and dominated individuals. That is why the contact literature emphasizes that increased intergroup contact diminishes prejudice only if the persons concerned are equal in status, have common goals, are not in competition, and the contact is sanctioned by authority (Pettigrew, 1997; Thomas F Pettigrew & Linda R Tropp, 2006). These conditions for beneficial contact show that the benefits depend on coalitional cooperation, which would raise the coalitional safety index in the individuals in contact. People from different social categories may find that categories matter little when they are equal partners in a joint collective action. A case in point is the U.S. military, which started integrating all its units in 1948. Decades later, U.S. military personnel report levels of satisfaction with intergroup personal relations far above those of civilians (Bullock, 2013). Shortly after the start of the integration process, the units with higher numbers of minority (Black) soldiers reported greater satisfaction than others with interracial relations (Moskos, 1966). This increased satisfaction in heterogeneous units would seem to support the contact hypothesis. But note that the military is a very special social environment, as it constitutes in many ways a situation of coalitional affiliation. Military units are explicitly described as alliances against enemies. In small units like platoons, the specific coalitional dynamic of race is replaced with another one, in which individuals of all categories engage in a high-stakes collective action. Consistent with this interpretation, the beneficial outcomes of army integration change with contexts. Although prejudice is lowest in combat units in times of combat and in dangerous places, it tends to increase in times of peace and in civilian life, when the individuals of different ethnicities are no longer members of the same coalition (Bullock, 2013). Conversely, the coalitional perspective makes sense of the fact that contact does not reduce prejudice or rejection in situations in which individuals from different categories cannot engage in mutually advantageous collective action, because of institutional or other barriers, as was the case for Blacks and Afrikaners in South Africa (Korf & Malan, 2002). More generally, coalitional dynamics explain why, in contrast to the original formulations of contact theories, intense or frequent intergroup contact can be detrimental. As in the context of health outcomes, intergroup encounters are stressors before they are construed as situations of collective action. #### 5.2 Limits of Social Identity Interpretations Safety and threat dimensions of intergroup contact are addressed only indirectly in the framework of social identity theory, self-categorization theory, or what could be called more generally the social identity approach (Hornsey, 2008). Developed on the basis of minimal groups studies in the 1970s and 1980s (Tajfel & Turner, 1986), this approach has been applied to group polarization, group solidarity and cohesiveness, stereotyping, crowd violence and rioting, social influence, conformity, and power. A starting point of this framework is that people are motivated to engage in intergroup competition and other strategies in order to protect and/or promote a positive and secure self-concept (Brewer, 1979). Self-esteem or maintenance of a coherent sense of self are postulated as primary drives, which, combined with comparative assessment, lead people to hold representations of their own and other groups (stereotypes) with associated valence (attitudes) (Hornsey, 2008). One clear limit of social identity approach lies in justifying these general conjectures. That is, even though intergroup attitudes may be connected to self-concepts, it is not clear why maintaining a positive or 'secure' self-concept would be a fundamental human motivation and through what evolutionary process this could have become a general human need. Moreover, the notion of people choosing among a variety of available identities in the service of maintaining a self-concept is clearly confined to some modern mass societies. It would be irrelevant in places where identity is assigned by genealogy, like most societies in human history. Even as a descriptive framework, social identity theory has difficulties integrating some common aspects of intergroup relations. A good example is that of immigrant assimilation. From the standpoint of social identity theory, immigrants' adoption to the host population's cultural norms should be seen by members of the latter population as clearly positive, as it reinforces the assumption that these norms are superior. However, that is far from being the case. Studies carried out in the United States, Sweden, and France observed two divergent paths. Some individuals were hostile to cultural differentiation and therefore to immigrants holding on to cultural and ethnic markers. By contrast, others were hostile to immigrants' assimilation, which they saw as a menace. These attitudes correlate with different personality orientations. High authoritarianism predicts the rejection of cultural differentiation. High preference for hierarchical intergroup relations predicts rejection of assimilation (Guimond et al., 2010; Thomsen, Green, & Sidanius, 2008). These two variables account for the two contrary attitudes to assimilation, and neither of them is influenced by the need for a positive and coherent self-concept. More generally, the connection (in a limited number of modern Western societies) between identity and self-esteem may be more economically interpreted as an effect of fundamental psychological processes. People are motivated to join groups, build them, and maintain them because of the safety and support provided by membership. They are motivated to describe their group as superior because (among other things) this signals to other members their commitment to the group. Safety and signaling motivations are established by independent evidence and have a long evolutionary history. They provide a more parsimonious explanation than self-esteem motives for intergroup dynamics. #### 5.3 Social Dominance Orientation and Coalitional Investment Research on social dominance theory (Sidanius & Pratto, 1999) has anticipated some of the hypotheses presented here. Social dominance theory also starts from the observation that most intergroup relations are competitive and emphasizes that humans readily construe hierarchical intergroup relations on arbitrary bases (i.e., not based on age or sex). Also convergent with the coalitional perspective, social dominance theory implies that stereotypes and attitudes are the effect rather than the cause of discriminatory behaviors. As Guimond et al. put it, '[social dominance] theory conceptualizes prejudice as a form of hierarchyenhancing legitimizing myth, an ideology that justifies intergroup inequality' (Guimond et al., 2010). Such a conceptualization of prejudice is consistent with the notion that stereotypes are explanations rather than descriptions of the social environment (McGarty, Yzerbyt, & Spears, 2002; Yzerbyt, Rocher, & Schadron, 1997). In our perspective, stereotypes are tools used to explicate and communicate to others the contents of one's intuitive expectations about other individuals, for example, that they are in some alliance and constitute a potential danger. Regarding the psychological variables involved, social dominance theory postulates the personality variable of social dominance orientation (SDO), measuring the extent to which people are motivated to preserve and reinforce the subordination of some social groups (Pratto, Sidanius, Stallworth, & Malle, 1994; Sidanius, Pratto, & Bobo, 1994). The SDO measure predicts a number of attitudes and motivations associated with intergroup differentiation and contact (Guimond et al., 2010; Pratto et al., 1994; Sidanius et al., 1994; Thomsen et al., 2008). We propose to interpret SDO as one of the stable personality factors contributing to the coalitional safety index. SDO may be a measure of (a) the extent to which individuals construe a particular category as a collective action they are part of, so that they perceive their welfare as dependent on the welfare of the group; and (b) the extent to which they are willing to invest in defending coalitional interests, which would in turn motivate them to preserve group boundaries. Returning to the example discussed above, this may provide an explanation for the association between high SDO and rejection of assimilation. Immigrants' assimilation constitutes a threat because it dilutes the benefits of membership in a dominant group and because it makes member identification more difficult (uncertainty about affiliation increases transaction costs in collective action and creates opportunities for free riding). Blurring of the boundaries between national categories would be perceived as costly and therefore rejected most strongly by those who have construed national categories as coalitions and have invested heavily in this coalition. # 6. Implications We have argued that the coalitional safety index model provides an integrated and parsimonious understanding of safety, threat, and stress in intergroup relations. The model also suggests directions for further investigation. #### 6.1 Coalitional Cues: Microprocesses of Social Sampling In the social science literature, people are often described as experiencing social phenomena as large societal entities. For instance, immigrants are said to be directly affected by the host population's xenophobia. Models of 'racism as stressor' simply assume that negative stereotypes and attitudes toward one's own group will trigger stress responses (Lewis-Coles & Constantine, 2006). The connections are clear but lack an explanation. The coalitional perspective provides such an explanation, as indices of racism, negative attitudes, and so forth are construed as reminders of one's coalitional vulnerability. So, for example, it is not racism as such that is stressful but the easy inference from putative racism to one's reduced safety. Our model emphasizes the microprocesses involved in computing one's coalitional safety index and suggests specific hypotheses about these processes. A system that computes coalitional safety should attend to various cues of the safety provided by one's own coalition and the threat posed by rival coalitions. As mentioned above, such microprocesses may provide a causal understanding of observed connections between anticipated discrimination and stress, minority status and stress, and ethnic density and relative immunity from stress. We would expect that people's reactions to an immigrant group might be affected by general information about that group's size but also by the frequency of actual encounters with immigrant individuals. The model also predicts that people should attend to the cohesiveness of coalitions, whether members of a coalition act in concert toward a common goal, which may result in lower coalitional safety when one infers one's own coalition to be weaker (or in higher coalitional safety when one infers one's own coalition to be stronger). Cohesiveness cannot really be observed; it must be inferred, for instance, from the similarity (in dress, speech, behavior) of the coalition members. In short, the coalitional perspective suggests that further exploration of intergroup dynamics should pay special attention to the cognitive processes whereby people automatically sample their social environment and infer underlying properties on the basis of that sampling. This research program would benefit from cognitive psychology findings and models concerning intuitive statistics, 'fast and frugal heuristics,' and other aspects of ecological rationality (Gigerenzer, 2007). #### 6.2 Gender Differences Human dispositions and capacities are shaped by what worked toward reproductive success, on average, in evolutionary conditions. This provides a starting point for investigating and explaining gender differences in coalitional psychology. In standard social psychological models, there is little reason to expect, and generally no explicit predictions of, differences between men and women regarding inter-group processes. By contrast, an evolutionary perspective predicts profound sex differences, as already emphasized in social dominance theory (Sidanius et al., 1994). Through most of human evolution, groups were patrilocal, as men stayed and women moved between groups (Pasternak, Ember, & Ember, 1997; Seielstad, Minch, & Cavalli-Sforza, 1998). Women had to establish support networks with non-kin (Taylor et al., 2000), while men needed to bolster alliances between kin groups to compete with other coalitions (Kaplan, Hill, Lancaster, & Hurtado, 2000), most clearly in tribal warfare, an almost exclusively male (Gat, 2006; Keeley, 1996). As predicted, different patterns of socialization can be found cross-culturally from early childhood (Geary, 2003). As a consequence, we may expect men to be more motivated than women to see interindividual relations in terms of rival coalitions and more motivated than women to engage in violent coalitional strife; both men and women should be biased toward representing coalitional enemies as typically male. Some psychological evidence supports these conjectures. For instance, after threat priming, men are more likely than women to activate concepts of groups and coalitions (Bugental & Beaulieu, 2009). Women cooperate within a group regardless of competition with rival groups, while rivalry makes men more cooperative inside the group (van Vugt, Cremer, & Janssen, 2007). Men are implicitly biased to see men more than women as enemies (Plant, Goplen, & Kunstman, 2011). In both genders, the association of anticipated harm with a male's face is more difficult to extinguish than the association with a female face (Navarrete et al., 2009). Sex differences in coalitional psychology may also account for the effects described by Sidanius and colleagues in terms of a subordinate male target hypothesis. According to this hypothesis, which is supported by many empirical studies, adult men of the dominated group are the focus of more intense discrimination than women (Sidanius & Pratto, 1999). One possible explanation for this phenomenon is in terms of the potential reproductive value of subordinate women, which would palliate discriminatory attitudes toward women (Sidanius & Pratto, 1999). Further developments of social dominance models point to a simpler and broader explanation, that men are the target because group rivalry recruits mental systems that evolved in the context of tribal warfare, in which males are more likely than females to be aggressors (McDonald, Navarrete, & Van Vugt, 2012; Navarrete et al., 2010; Yuki & Yokota, 2009). #### 6.3 Coalitional Effects beyond Minorities The literature reviewed above describes the poor health outcomes of subordinate groups (controlling for confounding socioeconomic variables) as an effect of prejudice, stereotype, or discrimination. The coalitional model by contrast emphasizes the number of encounters with individuals of a rival coalition, especially if these rival coalitions are perceived as stronger, more numerous, increasing in number, or more cohesive than one's own. A prejudice model would not predict that members of majorities experience a negative health impact when an ethnic minority in their neighborhood increases in number or visibility. By contrast, the coalitional perspective predicts that increasingly frequent encounters with people of a rival coalition (the minority), especially when the minority is apparently cohesive (e.g., inferred from displays of common markers, a distinct unfamiliar language, and so on), would increase the number of stress responses in majority individuals. Note that such negative effects on majority individuals have already been observed in another domain, that of trust. In studies by Putnam and others, generalized social trust (the extent to which one thinks one can trust others in one's social environment) decreases with greater ethnic diversity (Putnam, 2000, 2007). Further studies have shown that this effect depends on the frequency of encounters at the level of small neighborhoods (Dinesen & Sønderskov, 2012). Our coalitional stress model would predict that this may have effects on health as well. There is some evidence in that direction—for example, Whites who live in more homogeneous neighborhoods have better health in New York (Fang et al., 1998) and fewer psychiatric admissions in Chicago (Halpern, 1993). But the data are really sparse, and only largescale surveys could overcome the obvious confounds created by the overall inequality between majority and minorities, as well as potentially harmful effects of majority individuals' own prejudices. # 7. Conclusion The proposed model stipulates that an internal regulatory variable, the coalitional safety index, corresponds to an individual's perceived coalitional security. The index reflects the extent to which he or she can depend on others in the competition against other alliances. It is down-regulated by specific threat cues of reduced support from one's own coalition or increased menace from a rival coalition, which trigger motivations for appropriate precautionary behaviors. Repeated perceptions of such threat cues may cause chronic stress, with negative health consequences. This perspective allows for the explanation of a great variety of phenomena described in the social psychology of intergroup relations, such as stereotyping, racism, ethnocentrism, stress, and health disparities, in terms of a suite of capacities and motivations shaped by natural selection. The evolved human coalitional psychology is described as a set of universal systems that take as their input specific information about the social environment and activate appropriate motivations to maximize coalitional safety. Interactions between such systems and highly variable social conditions result in culturally and historically specific representations of the social world, which motivate equally specific attitudes and behaviors. # References: ——. (2009). Mothers and Others: The Evolutionary Origins of Mutual Understanding. Cambridge, MA: Belknap Press of Harvard University Press. American Journal of Public Health, 102, 1020–1026. https://doi.org/10.2105/ AJPH.2011.300620 # 5. How People Think about the Economy # Introductory Note People have views about the economy, about such things as unemployment, trade, taxation, etc. Where do these opinions come from? Explaining that would certainly count as an example of 'useful' anthropology or political science, considering that most political programs are based on some particular vision of the way a modern economy works, and how it could be made better. Michael Petersen and I were interested in explaining how people acquire these representations of the economy and, as a result, favor this or that political program. Our aim was to explain economic ideologies, something that economists are not terribly interested in. Economists generally stop at pointing out that these ideologies are often based on erroneous assumptions, e.g., that labor is what creates value, that trade benefits one party at the expense of the other, that regulations have the intended effects, etc. But why would people reason on the basis of these misleading notions? We are often told that this happens because people are uneducated, or cognitively limited, or they just accept what is 'in their culture', or what fits their interests, or what politicians tell them. But none of these explanations are satisfactory, as we explain in the article. We considered the hypothesis that economic ideologies are compelling and persistent in modern societies, because of their 'fit' with our evolved dispositions. How is our genetic evolution relevant to our views on international trade and income taxes? Obviously, such issues were unknown in our environment of evolution, when we were (mostly) living in small bands of nomadic foragers. But that, in a way, is just the point. Our evolutionary heritage includes not just cognitive systems for understanding the natural world, but also capacities for managing life in groups—in particular, for cooperation and collective action, in which we pool efforts to obtain mutually beneficial outcomes. Over the last thirty years, evolutionary biologists, psychologists and economists have proposed and tested ever more refined models of the way cooperation occurs between humans, and of the psychological capacities and motivations that underpin the exceptional level of cooperation among humans—see summaries in André & Baumard (2011), Boyd & Richerson (2006), Cosmides & Tooby (2015). This evolved cooperation psychology is part of our adaptations. It governs our reactions to information we receive, concerning the allocation of resources between partners, when we interact with others, share or trade with them. And—this was our starting point in this article—it may also explain our reactions to messages (from news organizations, political agents) concerning such mass-level phenomena as inflation, trade or unemployment. This should illustrate how evolutionary models and findings are very much relevant to modern, mass-scale societies. A persistent misunderstanding, on the part of those unfamiliar with the field is that such models only apply to technologically simple societies, and that modern patterns of production, consumption, and communication create conditions so special that evolved preferences and capacities become less relevant. But that is just not the case. For instance, Michael Petersen pioneered an evolutionary perspective that promised to account for important features of mass-politics in modern societies (Petersen, 2012a; 2015). Consumption, too, is best understood in terms of evolved motivations (Saad, 2012), and even recent developments of electronic communication, including webpages, social media, etc., illustrate typically human capacities and motivations (Acerbi, 2019). So, our evolved psychology influences the way we think of the immensely complex set of interactions that constitute an economy—and our representations of the economy in turn make particular political programs attractive. Our article only considered the first causal link, from evolved psychology to economic ideologies. Speculating further, one might wonder which political programs would best fit our evolved psychology. If we followed our Stone Age intuitions and preferences, what would we choose as our economic policy? Others have wondered about that, and addressed the question with a great deal of sophistication, in particular Paul Rubin and Peter Singer. Rubin emphasizes that trade, being a cooperative interaction that benefits both parties, is an outgrowth of our cooperation psychology and also notices that much cooperation in humans is based on partnerchoice, on the possibility of selecting good partners and rejecting others. These dispositions would favor the free exchange of goods or services, away from the diktats of a chief, a king or a state (2002). Singer places much more emphasis on our capacities for sharing and mutual help and on the evolutionary basis for fairness and moral intuitions. These would favor generous welfare policies, when modern conditions create disadvantages or inequalities (2000). Both are right, in the sense that our cooperation psychology does respond to these two distinct sets of motivations, for mutually beneficial voluntary trade and for social support as a palliative to misfortune (Boyer, 2018, pp. 163–202). Indeed, Michael Petersen's experimental studies show that, regardless of their political affiliation, people can approve or disapprove of particular policy proposals, depending on which of these cognitive systems the material activates (Petersen, 2012b). # References # Folk-Economic Beliefs: An Evolutionary Cognitive Model1 with Michael Bang Petersen2 Abstract: The domain of 'folk-economics' consists in explicit beliefs about the economy held by laypeople, untrained in economics, about such topics as, for example, the causes of the wealth of nations, the benefits or drawbacks of markets and international trade, the effects of regulation, the origins of inequality, the connection between work and wages, the economic consequences of immigration, or the possible causes of unemployment. These beliefs are crucial in forming people's political beliefs and in shaping their reception of different policies. Yet, they often conflict with elementary principles of economic theory and are often described as the consequences of ignorance, irrationality, or specific biases. As we will argue, these past perspectives fail to predict the particular contents of popular folk-economic beliefs and, as a result, there is no systematic study of the cognitive factors involved in their emergence and cultural success. Here we propose that the cultural success of particular beliefs about the economy is predictable if we consider the influence of specialized, largely automatic inference systems that evolved as adaptations <sup>1</sup> An earlier version of this chapter was originally published as Boyer, P., & Petersen, M. B. (2017). Folk-Economic Beliefs: An Evolutionary Cognitive Model. Behavioral and Brain Sciences, 41, 1–51. https://doi.org/10.1017/S0140525X17001960. Republished with permission from Cambridge University Press. <sup>2</sup> Acknowledgements: We are grateful to Nicolas Baumard, Martin Bisgaard, Timothy Blaine, Thom Scott-Phillips, Don Ross, Paul Rubin, and four anonymous reviewers for thoughtful and detailed comments on a previous version. to ancestral human small-scale sociality. These systems, for which there is independent evidence, include free-rider detection, fairness-based partner choice, ownership intuitions, coalitional psychology, and more. Information about modern mass-market conditions activates these specific inference systems, resulting in particular intuitions, for example, that impersonal transactions are dangerous or that international trade is a zero-sum game. These intuitions in turn make specific policy proposals more likely than others to become intuitively compelling, and, as a consequence, exert a crucial influence on political choices. # 1. The Domain of Folk-Economic Beliefs 1.1 What Folk-Economic Beliefs Are The term folk-economic beliefs denotes a large domain of explicit, widespread beliefs, to do with economic and policy issues, held by individuals without systematic training in economic theory. These beliefs include mental representations of economic topics as diverse as tariffs, rents, prices, unemployment, and welfare or immigration policies, as well as mental models of interactions between different economic processes, for example, inflation and unemployment. Our perspective on the origins and forms of folk-economics is based on two major assumptions. First, we argue that folk-notions of the economy should not be described solely in terms of deviations from normative economic theory. That has, unfortunately, been the common approach to the subject. Folk-views are generally described as the outcome of 'biases,' 'fallacies,' or straightforward ignorance. But describing how human cognition fails to work according to some norm of rationality tells us little about how it actually works. Second, we propose to make sense of folk-economic beliefs by considering the environment in which many, if not most, human cognitive mechanisms evolved. The study of folk-economic beliefs should be distinguished from other domains of investigation. Microeconomics addresses actual choices of agents in conditions of scarcity, independently of whatever mental representations trigger these behaviors in actual individuals, and also of the representations they may form of their behavior upon reflection. Another field, behavioral economics often uses experimental designs as a way to elucidate tacit motivations and capacities that direct economic choices in contexts where experimenters can manipulate incentives and information flow between agents (Plott, 1974). Finally, neuro-economics elucidates the brain systems involved in appraising utility and making economic decisions (Camerer et al., 2007; Loewenstein et al., 2008). The scope of a study of folk-economics is quite different from these three fields (see Figure 1 ). It focuses on people's deliberate, explicit beliefs concerning economic facts and processes, for example, that foreign prosperity is good or bad for one's own nation, that welfare programs are necessary or redundant, that minimal wages help or hurt the poor, and that rent controls make prices go down or up, and so forth. Fig. 1. A summary of the systems and representations involved in forming folkeconomic beliefs. External information about economic matters triggers activation of specific mental systems, which results in both economic behavior and explicit folk-economic beliefs. The latter's effects on behavior cannot be assumed. Different fields, represented as clouds, focus on different parts of these processes. The model presented here is about the causal arrow linking specific mental systems to the occurrence of folkeconomic beliefs in people's minds. (Figure by P Boyer. 2017) One should not assume that folk-economic beliefs (henceforth FEBs) have direct and coherent effects on actual economic behaviors. Many FEBs are about macroeconomic processes—for example, the level of unemployment, or the need for foreign trade, or the need for a nation to balance its budget—that are unrelated to people's everyday transactions. Also, even FEBs that do bear on micro-economic realities, for example, on 'fair' prices or wages, may remain insulated from the psychological processes that drive actual economic behavior, as we explain below, which is why people may recommend specific policy outcomes and behave in ways that contradict that choice (Smith, 2007). Figure 1 summarizes the different domains of thought and behavior and the research programs involved. #### 1.2 Why Folk-Economic Beliefs (FEBs) Matter Understanding FEBs is of crucial importance, even if they do not govern people's economic behavior, because they play a critical role in political choices. Perceptions of macro-economic developments influence how favorably people view the government and how they cast their votes (Nannestad & Paldam, 1994). The translation of inflation, unemployment, and income dynamics into political choices is mediated by people's beliefs about the economy, for example, whether rising unemployment is affected by government policy (Peffley, 1984; Rudolph, 2003a, 2003b). Similarly, economic beliefs underpin people's answers to such questions as: Is it a good idea to increase welfare benefits, impose tariffs on imports, cap rent increases, or institute minimum wages? Folkeconomic beliefs constitute a largely unexplored background against which most information about policy is acquired, processed, and communicated among nonprofessionals (Rubin, 2003). ### 1.3 A Different Approach to the Study of Folk-Economic Beliefs It is a matter of common knowledge that most people, including the educated public in modern democratic societies, do not think like economists (Smith, 2007, pp. 147–166). It is, for instance, a familiar finding that people are overinfluenced by consideration of sunk costs (Magalhães & Geoffrey White, 2016) or fail to consider opportunity costs (Hazlitt, 2010) in evaluating possible courses of action. More important for social and political debates, people often also express views on economic processes that seem misguided, if not downright fallacious, to most professional economists. There is a growing literature documenting this divergence (see, e.g., Blinder & Krueger, 2004; Caplan, 2006; Haferkamp et al., 2009; Hirshleifer, 2008; Rubin, 2003; Sowell, 2011; Wood, 2002; Worstall). However, there is still very little research on why such beliefs appear, and why they are so widespread. We argue that many folk-views on the economy are strongly influenced by the operation of non-conscious inference systems that were shaped by natural selection during our unique evolutionary history, to provide intuitive solutions to such recurrent adaptive problems as maintaining fairness in exchange, cultivating reiterated social interaction, building efficient and stable coalitions, or adjudicating issues of ownership, all within small-scale groups of foragers. The inference systems we describe further on are not specified as ad hoc explanations for folk-economic beliefs. All of these systems have been independently documented by evolutionary biologists, psychologists, and anthropologists who focus on such issues as the evolution of exchange and trade, its form in the small-scale societies in which humans evolved, and its consequences for psychological dispositions and preferences that can be observed in experimental studies on individuals in modern societies; for an overview, see Buss (2015). So, we are not proposing a new description or interpretation of the human evolved psychology of exchange, but rather, using prior findings to illuminate the emergence of folk-economic beliefs in modern contexts. #### 1.4 Models of Folk-Economic Beliefs Are Not Normative The model described here is emphatically not a normative proposal. That is, we do not intend to suggest that there is a right way to consider economic processes, and to evaluate folk-economic beliefs in terms of their validity or coherence. This deserves mention, for two reasons. First, as discussed below, most descriptions of these beliefs, in the literature, were originally motivated by the realization that people do not think like economists, and that they often commit what trained economists would describe as fallacies. By contrast, we argue that this is not a promising way of approaching cultural beliefs in this domain, as the validity (or lack thereof) of these beliefs do not explain their spread. Second, because FEBs are politically consequential, readers may wonder whether studying them is by itself a political project. That would be the case if, for instance, widespread beliefs were contrasted with a supposedly true picture of the economy, and if that picture was associated with a particular kind of political project. But we suspect (and to a certain degree, the evidence confirms) that individuals of all kinds of political persuasions are equally likely to entertain beliefs that are, in some sense, misguided or incoherent. Indeed, one could argue that the epistemic value of FEBs is largely orthogonal to their political import. That is, the economy is not a political end in itself but a political means to ends that are essentially contested. In principle, even completely misguided FEBs might give rise to outcomes that are, by some other standards, 'good' or 'just,' at least as far as some specific social group is concerned. Our more general point is that we believe that the question of whether FEBs are correct or incorrect is orthogonal to the importance of studying them. Few individuals receive formal training in economics and, hence, if they happen to hold correct beliefs, this is as much in need of an explanation as when they generate incorrect ones. # 2. Some Folk-Economic Beliefs and Possible Explanations Evidence for folk-economic beliefs is still scattered and unsystematic. Some FEBs are widespread and well-documented, either through surveys of attitudes such as the General Social Survey (2011), or by more-specific, smaller-scale investigations such as the Kaiser Foundation's 'Survey of Americans and Economists on the Economy' (Kaiser Foundation, 1996). Others are less systematically documented, being inferred from the platforms and common phraseology of political operators, as well as from common journalistic discourse (Wood, 2002; Worstall). #### 2.1 Examples of Folk-Economic Beliefs In the following, we present a few examples of widespread beliefs about the economy, selected for their potential influence on political choice. Given that such beliefs are often expressed in vague or emotional terms (e.g., 'markets are bad for society,' 'trade will make us poorer and others richer'), what we propose here are, by necessity, reconstructions of possible beliefs as implied by people's explicit statements or questionnaire responses. FEB 1. International trade is zero-sum, has negative effects. The notion is expressed in many forms in everyday conversations and in political discourse, and it was also a recurrent theme in early political economy (Hainmueller & Hiscox, 2007). This belief may take many forms. For instance, trade is said to create unemployment at home because foreigners instead of locals are making the things we need (Wood, 2002, pp. 53–55). Also, it is claimed that a nation should always try to export more goods than it imports (Worstall, pp. 29–32). This belief is often associated with the assumption that the wealth of nations is the outcome of a zero-sum game. As a consequence, the assumption that foreigners profit from trade entails that 'we' are losing out. Consistent with this assumption, many people believe (against possible comparative advantage) that trade cannot be beneficial if 'we' import goods that we could manufacture ourselves (Baron & Kemp, 2004, p. 567). After the 2008 recession, many Americans interpreted increased unemployment as an effect of international trade and feared that continued trade would worsen their conditions (Mansfield et al., 2019). FEB 2. Immigrants 'steal' jobs. Beliefs about the negative economic impact of immigration lie at the center of many policy debates. It is a consistent finding among political scientists that immigration, especially of low-skilled immigrants, is viewed as threatening (Hainmueller & Hiscox, 2010), and a common formulation is that immigrants 'take our jobs' (Simon & Lynch, 1999). This view is associated with the assumption that there is a fixed quantity of jobs to share among people (Wood, 2002, p. 23; Worstall, 2014, p. 75). FEB 3. Immigrants abuse the welfare system. Another belief, almost diametrically opposite but equally widespread, is that immigrants are a fiscal burden on the welfare system, using up common resources (Sniderman et al., 2014). So, immigrants are intuitively viewed as freeriding both on the jobs 'we' created and the welfare systems 'we' paid for (Alesina & Glaeser, 2004). Given these beliefs, co-occurrences of immigration and fiscal stress can be viewed as causally linked, with important consequences in terms of both policy opinions and of holding immigration-friendly politicians accountable on Election Day. FEB 4. Necessary social welfare programs are abused by scroungers. Welfare programs, for example, unemployment benefits, are the object of apparently opposing economic beliefs (Aarøe & Petersen, 2014; Alesina & Glaeser, 2004). Experimental studies show the coexistence of those contrary beliefs within individuals. On the one hand, welfare programs are viewed as desirable insurance schemes against unavoidable, essentially random misfortune. On the other hand, unemployment benefits are widely viewed as encouraging laziness and a culture of dependency (Aarøe & Petersen, 2014; Kameda et al., 2002). FEB 5. Markets have a negative social impact. Rubin (2014) coined the term emporiophobia for the generally negative attitude towards markets observed in many modern societies and documented in many surveys. The belief is that markets as such produce negative outcomes for most participants. Surveys offer evidence that many people, against economists, see markets not as the encounter of buyers and sellers who mutually benefit from trade, but as a place of struggle between partners with unequal bargaining power. The anti-market attitude may also contribute to the rejection of market solutions for the allocation of 'sacred' goods, like organs or children in need of adoption. Many people seem to consider more arbitrary allocations (lotteries, first come first served) as not just fairer than auctions, but also probably more efficient (Alan Page Fiske & Tetlock, 1997; Tetlock et al., 2000) FEB 6. The profit motive is detrimental to general welfare. The profit motive is seen as an attempt to extract more from transactions than would be warranted by 'fair' pricing. That is why there is a tendency to see private firms as less 'caring' than non-profits, and therefore more likely to create negative externalities (Bhattacharjee et al., 2017). One version of this belief is that there is a special class of 'excessive' profit that differs from the regular or fair allocation of profit to businesses (Wood, 2002, pp. 10–12). Related to this assumption is the notion that regulation is required to limit the excesses of profit-driven businesses (Hirshleifer, 2008). In general, then, the belief seems to be that if most economic actors act on the basis of maximizing their profits, non-economic social domains will be negatively affected, for example, by externalities such as pollution, or more generally through a decrease in solidarity, social trust, and so forth. Contra Adam Smith, the notion that private selfregard creates general welfare seems to be unintuitive (Rubin, 2003). FEB 7. Labor is the source of value. This is the assumption that the amount of labor necessary to produce a good is an essential (or the only) factor that determines its 'value,' a (generally undefined) quantity that is not necessarily expressed by market price. This assumption is not often expressed in such general terms, but the proposition is implicit in many widespread beliefs about labor and wages (Wood, 2002, pp. 175–178; Worstall, pp. 15–17). It is also present in opinions on the unfairness of low wages for hard or unpleasant jobs, especially those involving hard physical labor. FEB 8. Price-regulation has the intended effects. The belief is that regulation generally does what it is supposed to do, as government policy can direct the economy towards desired results (Hirshleifer, 2008); (Wood, 2002, p. 77). For example, in the United States, many cities imposed rent-control in the 1960s—and such measures were a major item in politicians' platforms (Dreier, 1999)—with the goal of creating an ample supply of cheap housing; see Schipper (2015) for similar processes in Israel. The FEB here is that such regulation efforts will work as intended, for example, that rents will stay low after the imposition of rent-control, or that minimum wages can affect wages without affecting the demand for labor (some people even think that the latter measure could boost employment rates (Haferkamp et al., 2009, p. 533). More broadly, regulation is often seen as an efficient way to protect people against undesirable market dynamics. Chinese respondents, for instance, believe that China was spared the worst effects of the 2008 downturn by its government regulations (Yuen & Greene, 2011). This is only a short list of widespread folk-beliefs about the economy. Because there is very little study of such cultural beliefs as of yet, we have scant evidence for the relative cultural spread of each of these FEBs, and of possible associations between them and various social or cultural variables. The beliefs in question may well vary between social classes, cultures, age-groups, and so on. One aim of this article is to demonstrate the importance and theoretical interest of this domain of cultural beliefs and motivate more detailed empirical research in the domain. #### 2.2 Common Explanations: Ignorance, Self-Interest, Biases There are three main ways of explaining the divergence between laypeople's and economists' views: in terms of ignorance, in terms of self-interest, or as the outcome of specific biases that affect people's perception of economic facts. 2.2.1. Lack of economic knowledge or training. The ignorance hypothesis simply assumes that non-normative views stem from a lack of relevant information, similar to the widespread ignorance in the political domain, long lamented by political scientists (Converse, 1964). It is certainly true that most laypeople are unaware of many fundamental principles of economic analysis. For instance, if people knew some rudiments of price theory, they would not be surprised that useful water is much cheaper than useless diamonds. If they knew about comparative advantage, they might see international trade in a different way (Haferkamp et al., 2009). However, this interpretation has one major defect—it predicts that people's common views will be nonnormative, but it does not predict that they will be non-normative in any particular way. Not knowing about a domain would predict random, vague, or nonexistent opinions, as in popular conceptions of quantum mechanics, rather than the specific set of beliefs observed (Caplan, 2008, pp. 9–11). 2.2.2. Self-interested beliefs. If beliefs are not random, that may be because they are influenced by people's perception of their interests. In this view, people adopt beliefs that would justify more resources being apportioned to them and less to their enemies or competitors (Dahl & Ransom, 1999). One difficulty with this interpretation is that it accounts for only some of the beliefs described above. It can explain, for example, how industrial workers in the United States might feel they will lose out if their jobs move to China, and therefore consider that protectionism is overall a good thing. But beliefs are sometimes less clearly connected to self-interest. For instance, many people feel that markets are bad, even though larger, more competitive markets provide them with cheaper goods, which is clearly in their interest. So, self-interest is at best an incomplete explanation, and in general is not a straightforward predictor of economic beliefs, or indeed of political choices (Caplan, 2008; Green & Shapiro, 1994). It should be noted that one type of interest that does seem to explain some variation in FEBs is partisan interests. During economic downturns, for example, people are much more likely to ascribe the government responsibility if they identify with the opposition party than with the government party (Martin Bisgaard, 2015; M Bisgaard & Slothuus, 2018). However, although partisanship provides a motivation to reach certain conclusions (e.g., 'the government is responsible for this economic downturn' or 'the government is not responsible for this downturn'), the question still remains as to how people generate the particular beliefs about the workings of the economy that allow them to reach their desired conclusion. 2.2.3. Cognitive biases. Finally, another alternative to the knowledge gap is to consider that people's views are the outcome of specific biases. The term denotes tacit patterns of reasoning that orient people towards a limited set of conclusions from the evidence. There is a vast psychological literature for reasoning biases (Gilovich et al., 2002). For example, the 'confirmation bias' is the tendency to notice and remember instances of the hypotheses we hold and to ignore other cases as noise, with the result that prior assumptions seem ever more strongly confirmed. In the domain of beliefs about the economy, Bryan Caplan, for instance, identified an anti-foreign bias (what is good for foreigners is bad for us), an anti-market bias (inability to see how markets would turn private greed into a social good), a make-work bias (if people work more, there will be more wealth), and a pessimistic bias (economies are heading towards less prosperity) (Caplan, 2008). In a similar way, Haferkamp et al. argue that the divergence between economists' and laypeople's views does not reduce to self-interest or ignorance, but rather results from multiple biases, like the well-documented status-quo bias and omission bias (doing something detrimental is worse than not doing something beneficial) (Haferkamp et al., 2009, p. 530). Finally, people's selection of economic beliefs often reflects own-side partisan bias (Martin Bisgaard, 2015). #### 2.3 Proximate and Ultimate Factors Models based on identifying particular cognitive 'biases' have the merit of taking seriously the fact that the emergence of these beliefs may lie in the way information about the economy is processed in human minds, which is certainly the right starting point. However, we propose that the study of folk-economic beliefs should move beyond a description on terms of fallacies and biases. One major problem with bias-oriented accounts of cognitive phenomena is that a bias is often simply a re-description of the empirical phenomenon under investigation (Gigerenzer, 1991; Gigerenzer et al., 1999). For example, when it is observed that people attend more to more recent and vivid information, this is explained by an 'availability heuristic' that simply stipulates that people attend more to more recent information. In a sense, this is fine; after all, science requires the systematization of observations about the world. But explanations require causal models as well. Within the biological sciences, researchers distinguish between 'proximate' and 'ultimate' explanations, where proximate explanations describe how a biological system works and ultimate ones explain why the system exists (Buss et al., 1998; T. C. Scott-Phillips et al., 2011). Bias-based models are largely equivalent to proximate explanations. To develop a scientific understanding of folk-economic beliefs, we need to attend also to the level of ultimate explanations, not just because doing so provides a more complete understanding, but also because we will then be able to develop more precise predictions about the psychology behind folk-economic beliefs. # 3. Our Model: Inference Systems, Beliefs, Cultural Transmission In the model we propose here, the emergence and spread of folkeconomic beliefs is influenced by specific intuitions about interpersonal exchange. These are not the outcome of explicit scholarly training. Nor are they the simple consequence of persuasion from political elites (politicians, journalists, pundits, etc.), or the straightforward absorption of particular cultural values. Rather, because of evolution in the context of small groups with intensive exchange, humans have developed an intuitive psychology of exchange, for which there is independent anthropological and psychological evidence (Cosmides & Tooby, 2015a). This psychology consists of a collection of highly specialized inference systems, each of which is designed to solve one kind of exchange problem recurrent in our ancestral environments. #### 3.1 Properties of Domain-Specific Inference Systems We can describe the mind as consisting of many distinct, specialized systems, each of which corresponds to recurrent adaptive challenges in human evolution, attends to limited domains of available information, is organized along specific inferential principles, orchestrates neural structures in a specific functional manner, and is the outcome of a specific developmental pathway (Boyer & Barrett, 2015; Cosmides & Tooby, 2015b; Hirschfeld & Gelman, 1994). A few examples may help illustrate the relevant functional properties of this broad class of cognitive systems. In the auditory stream, the sound events identified as instances of lexical items are handled by a parsing system that assigns various syntactic roles to the different words (Pickering & van Gompel). In the visual field, some configurations are identified as human faces by a face-recognition system that computes a holistic description of the face, which is then processed by other memory and affective systems (Kanwisher, 2000; Solomon-Harris et al., 2013; Tsao & Livingstone, 2008). Information from multiple modalities is integrated to compute the extent to which a particular person is attractive as a potential mate (Fink & Penton-Voak, 2002; Grammer & Thornhill, 1994). However different the domains, there are some important functional properties common to these systems: 1. Specific input format. The face-identification systems respond to visual displays that include points or lines interpreted as eyes and mouth. Any such elements presented in the appropriate configuration trigger the system, which is why cartoons and other stylized renditions of human faces activate it, whereas displays with scrambled features, or features in the wrong alignment, do not. The parsing system responds only to words in the stream of speech. Other sounds are not processed. Sexual attractiveness computations only consider very narrow aspects of information about a person, for example, the pitch of the voice rather than prosody, skin-reflectance (an index of youth) rather than skintone, facial symmetry rather than facial length, and so on. In general, then, domain-specific inference systems may ignore information that might be relevant to an organism but fails to meet the input conditions. 2. Automatic activation. Specialized inference systems are neither initiated nor stopped by deliberate intentions. Once information with the appropriate input format is detected, the systems proceed to produce the relevant inferences, which are then passed on to other inference systems. 3. Specific inference rules. Each system operates on highly specific inferential rules. The computational principles that assign words to their syntactic roles are found only in that domain, and the same goes for the matching between faces and memories about persons, or the computation of sexual attractiveness. 4. Unconscious computation. The operation and inference rules of each system are generally outside conscious access. Only some outputs of these computational systems can be accessed, such as, for example, the meaning of a sentence or the general attractiveness of an individual. 5. Intuitive output. The output of specialized inference systems, when consciously accessible, consists of intuitions—that is, a description of a particular situation or a motivation to behave in a particular way—that do not include any indication of the computational steps that resulted in that particular description or motivation. #### 3.2 Intuitive Systems Output Can Lead to Reflective Beliefs It is important here to keep in mind the difference between intuitive output on the one hand, and reflective representations on the other (Sperber, 1997). Reflective representations add information to intuitions, explicate them, extend or restrict their scope, offer a comment on the intuitions, or link them to specific sources, as in, for example, 'the reason this sentence is strange is that there is no verb,' or 'this person has the same round face as Humpty Dumpty,' or 'it is sad that this attractive person has a bad personality,' and so forth (Cosmides & Tooby, 2000; Sperber, 1997, 2000). Most of our 'folk-theories' of particular domains consist of explicit, conscious reflective beliefs about our intuitions. That is why we can better understand the diffusion of beliefs in social groups, if we follow closely the interaction between intuitions delivered by specialized inference systems, on the one hand, and their reflective interpretation, on the other. Here, again, examples may be of help. Human minds include an intuitive physics, a set of assumptions that helps us predict the trajectory of objects, expect solid objects to collide when their trajectories intersect, and so forth. These expectations appear early in infancy long before language acquisition (Baillargeon et al., 1995; Spelke et al.). But we can also entertain explicit thoughts that (to some extent) explicate and comment on these intuitions, for example, a belief that heavy objects have more momentum than lighter ones. Some of these reflective beliefs are wrong, others are too vague even to be wrong, and some are in agreement with physical science (Kaiser et al., 1986). In the same way, we have a set of intuitive biological expectations, for example, that all living things come in exclusive, taxonomically ordered categories (Atran, 1995), and that they are propelled by internal energy sources (R. Gelman et al., 1995; Tremoulet & Feldman, 2000). But we also have reflective and explicit beliefs, for example, that each species has unique essential properties that cannot change (S. A. Gelman & Wellman, 1991); that there must be some 'catness' about cats that makes them what they are. Here, the intuitive expectation (all cats share external features, their behavior is highly predictable, etc.) is explained by the reflective belief, which postulates a hidden, undefined essence inside organisms of the same species. Folk-economic beliefs are widespread, culturally transmitted, explicitly held reflective beliefs about economic processes. These are to be distinguished from the intuitive thoughts that emerge as a result of the operation of specialized intuitive systems. We reserve the term 'folk' for beliefs held by layfolk as a result of the interaction between information about the economy, and the operation of some inference systems. (This is in contrast to some parts of the psychological literature, where the term folk has been sometimes, confusingly, used to characterize both the products of intuitive inference systems and the cultural beliefs that emerge as a result of their operation.) ### 3.3 Why We Should Not Expect Consistency or Coherence in FEBs Explicit reflective beliefs may be extremely vague in their implications. One may hold that there must be a special essence present in all cats that makes them different from dogs, without specifying what that essence consists of—in fact, that is the most common form of essentialism (S. A. Gelman, 2004). Also, reflective beliefs may be inconsistent or incoherent, mostly because they come in a meta-representational format. In contrast to the output of intuitive systems, for example, the intuitive belief that 'there is a cat here on the mat,' reflective beliefs consist in comments on intuitions, for example, 'it is true in some sense that "the market is bad."' A meta-representational format allows one to be committed to a belief, without the contents of the belief being processed in detail (Cosmides & Tooby, 2000; Mercier & Sperber, 2009; Sperber, 1997). That is the case for mystical or religious statements, for example, 'the true path is not a path' or 'three persons are one being,' which people can hold to be true, in the form 'the proper interpretation of 'p' is true,' without processing their contents (Mercier & Sperber, 2009; Sperber, 1997). This applies to the domain of folk-economic beliefs as well. A belief that markets are socially negative can be held true, without triggering specific representations about, for example, how markets would decrease social welfare, in what domains of activity, to what extent, through what economic mechanisms, and so forth, as long as it is held in a meta-representational format, for example, 'It is true in some sense that "markets are bad for society."' For the same reason, one can hold that meta-representational belief, and also hold other beliefs that may seem to contradict it, for example, 'It is a good thing that we have many butchers here, so they have to keep prices low.' Finally, if folkeconomic opinions consist of reflective, meta-representational beliefs, then different beliefs can be held in relative isolation from each other without ever being integrated in a general theory of the economy. So, we should not expect precision, consistency, or integration in the domain of reflective folk-economic beliefs. ### 3.4 Proposed Mechanism: Intuitions, Beliefs, Cultural Transmission Folk-economic beliefs are cultural beliefs—which simply means that they are represented in roughly similar ways in the minds of different individuals in a group, as a result of communication between individuals. Folk-economic beliefs are communicated—between laypeople, but also between media and their customers, and between political entrepreneurs and the public. That is why it is important to consider the mechanisms that lead to their cultural spread, that is, the extent to which they are likely to be entertained, in roughly similar ways, by different minds. An essential component of cognitive theories of cultural transmission is that prior psychological assumptions and expectations make certain representations easier to acquire, store, and communicate than others (Boyd & Richerson, 1985; Sperber, 1991). Cognitive dispositions make people transform input in such a way that it is more similar to the types that match these dispositions, an 'attraction' process that results in the spread of highly particular mental representations (Claidière et al., 2014). In Section 4 , we document the existence of various intuitive inference systems dedicated to representing social exchange. We then examine how these different systems make particular views of the economy, in general, particularly easy to acquire and represent, turning them into cultural beliefs. # 4. Relevant Cognitive Systems #### 4.1 Relevant Systems Evolved before and outside Markets Evolutionary theory predicts that cognitive systems are geared towards solving specific, recurrent problems in environments in which humans evolved. Specifically, what evolutionary theorists call the environment of evolutionary adaptedness (or EEA) for a trait is a statistical construct, an aggregate of the conditions under which there was selection for or against that trait, weighted for frequency and time. In that sense, the EEA is not a particular time or place, but a collection of features. As an illustration, we can consider that optimization problems such as hunting, foraging, choosing the best mate, selecting nutritious foods, and garnering social support were present, and relevant to fitness, throughout human evolution. By contrast, urban life, masscommunication, rapid long-distance travel, and mass-market economies only occurred for a small duration and only in some places at first. So it is more plausible that human minds were selected for systems geared to the first kind of adaptive problems, than to the second. One feature that is universally prominent in both modern and ancestral human societies is the exchange of goods (e.g., tools, food) and services (everything from back-up in conflicts to help with hunting, foraging, parenting, or shelter-building) (Brown, 1991). Developmental psychology studies show that children readily engage in exchange in early years (Levitt et al., 1985). Exchange provides significant fitness benefits. It allowed our ancestors, as it allows us, to exploit cooperative positive-sum games, engage in collective action, and buffer against predicaments such as hunger and injury (Gurven, 2004; Sugiyama, 2004). For us and for our ancestors, engaging in exchange requires the existence of distinct, specialized cognitive mechanisms (Cosmides & Tooby, 1992), including mechanisms for estimating costs and benefits of goods and services for the self and other; for comparing them in an abstract format (equivalent to utility in the vocabulary of economics); and for motivating exchange when the benefits of exchange exceed the costs for oneself. The human mind, in other words, contains a rudimentary exchange psychology, evolved by natural selection to help facilitate transactions. Although it evolved within ancestral small-scale hunter-gatherer groups, the cues inherent in modern markets economies (transactions, bargaining, prices, etc.) also bring it online. However, market economies are a novelty at the scale of biological evolution, so we should not expect specific adaptations to their features, as the differences between ancestral exchange and the market are vast (Rubin, 2003). A crucial difference is that economic activity in nonmarket societies, and by extension during most of human evolution, does not and did not take place in isolation from other aspects of social interaction. Indeed, the clear separation between economic exchange and other forms of social interaction is a by-product of market conditions (Polanyi, 2001). Throughout human evolution, most transactions affected not only the agents' welfare, what they gained or lost on the spot, but also their reputation, their social standing, the nature of their relationship to exchange partners, the extent to which they could rely on others, the cohesiveness of the groups they belonged to, and so forth. That is why mechanisms for reasoning about exchange are designed to take in a whole range of social considerations that are not relevant in the impersonal modern market. In the following pages, we examine some of the systems that evolved to facilitate exchange, the evidence for their operating principles, and their potential effects on the perception of modern market phenomena. #### 4.2 Detecting Free-Riders in Collective Action In any exchange, it is crucial to monitor whether the implicit or explicit terms of the exchange are being followed. For example, if two individuals take turns helping each other forage, does one person provide less help than he receives? To solve this problem, human exchange psychology needs to contain specific mechanisms for detecting and responding to free-riders. There is considerable evidence that humans, in general, are attentive to potential cheating in social exchanges, so proximate psychological mechanisms are congruent with the ultimate fitness benefit of detecting and deterring free-riders. Indeed, a situation where some agent has taken a benefit without paying the cost for it is psychologically more salient than the opposite situation of an agent paying some cost but not getting the associated benefit (Cosmides, 1989; Cosmides & Tooby, 2005; Gigerenzer & Hug, 1992; Sugiyama et al., 2002). Also, information that some agent received benefits from cooperation without contributing triggers punitive motivations, as a way of depriving them of the benefits of free-riding (Price et al., 2002). The ultimate rationale for free-riding detection is to preserve cooperation, including in the future. This would suggest that we do not intuitively classify as free-riders those individuals who make honest mistakes or whom accidents bar from cooperating. Indeed, Delton et al. have shown that the intuitive freerider categorization is highly sensitive to intentions, rather than just tallying who contributed what to the collective action (Delton et al., 2012). #### 4.3 Partner-Choice for Exchange To engage in exchange, one needs to choose among available social partners. Given the possibility of choice, human exchange and cooperation from ancestral times have taken place in the context of competition for cooperation (Noë & Hammerstein, 1994), as each agent could advertise a willingness to cooperate (and signal how advantageous cooperation would be), and could choose or reject partners depending on their past and potential future behavior (Barclay, 2016; Delton & Robertson, 2012; Panchanathan & Boyd, 2004). Cases of mutualism between species illustrate the efficiency of partner-choice for stabilizing mutually beneficial cooperation, for example, between cleaner fish and their clients (Bshary & Grutter, 2005). Human communicative abilities allow this kind of mutualism to occur between conspecifics, with reputation as an essential factor in the selection of partners. Agents have access to information about other agents' past interactions as an index of likely future behaviors. In such conditions, there is of course a cost in engaging with free-riders, but also a cost in not cooperating with an honest partner (in terms of potential cooperative positive-sum games) (Krasnow et al., 2012; Milinski et al., 2002). Competition for cooperation has specific consequences on fairness intuitions in the context of collective action. Given that two (or more) partners contribute equal effort to a joint endeavor, and receive benefits from it, an offer to split the benefits equally is likely to emerge as the most frequent strategy anyone faced with a meaner division of spoils will be motivated to seek a more advantageous offer from other partners. So, to the extent that people have partner options, the constraints of partner-choice explain the spontaneous intuition that benefits from collective action must be proportional to each agent's contribution (André, 2010; André & Baumard, 2011; André & Day, 2007). The existence of partner-choice based on shared information and reputation may explain why people select partners, in the context of laboratory economic games, on the basis of criteria that may seem economically irrational, but that happened to be ecologically predictive in our environments of evolution. For instance, people prefer partners who express moral judgments in deontic (i.e., 'moral' and emotional) rather than rational terms (Everett et al., 2016). They also prefer potential partners whose faces suggest productivity, prosocial attitudes, and relatively high social status (Eisenbruch et al., 2016). #### 4.4 Exchange and Assurance by Communal Sharing One important form of social relations is founded on communal sharing, where resources are pooled (Alan P Fiske, 1992). This is found to some variable extent in all human groups, particularly in food provision, and seems crucial to social interaction in small-scale societies, especially in foraging economies similar to those in which humans evolved (Kelly, 1995). That is why this form of apparently unconditional altruism has been the focus of so much research in evolutionary anthropology and psychology (Kaplan et al., 2005). A major result of those observations and models is that communal allocations is not the outcome of an indiscriminate motivation to share with others, but rather follows implicit rules that make sense given the conditions of human evolution. For example, band-wide sharing in hunter-gatherer economies is generally confined to game, especially large game, whereas gathered and extracted foods are mostly shared with close kin. An explanation for this spontaneous preference in allocations lies in the differences in variance in the supply of these goods (Cosmides & Tooby, 1992), as gathering typically produces low-variance resources, in contrast with hit-or-miss hunting expeditions. So communal sharing provides insurance against random bad luck such as the vicissitudes of hunting expeditions (Kaplan & Hill, 1985) or injury that prevents hunters from going on expeditions (Sugiyama, 2004). This is reinforced by the low marginal value of food units when they come in large packages, like big game. Communal sharing, although typically presented as including all group members, is often in fact modulated by past or expected reciprocation. Even where there is a norm of unconditional sharing, those who give more freely also receive more (Gurven, 2004; Gurven et al., 2000). Communal sharing is founded on specific assumptions and principles, distinct from those that govern, for example, direct exchange or authority-based social relations (Alan P Fiske, 1992). The norm of communal sharing is readily acquired by children, and intuitively deployed by adults in the appropriate contexts (Birch & Billman, 1986; Hamann et al., 2011; Rao & Stewart, 1999). In different places, different sets of resources and occasions are designated as proper goods to share. People notice (and are usually shocked by) the application of one type of inference system to the wrong domain according to the local norms, for example, offering to pay your friends for coming to dinner, or asking for a discount as a personal favor at a supermarket checkout. The structure of the psychology for exchange resources through communal sharing implies that if people find that a need is caused by random circumstances beyond their own control, they intuitively represent that need as potentially alleviated through communal sharing. By consequence, they would think it as unfair if others try to profit from this type of need (i.e., turning the exchange into a direct form of exchange rather than communal sharing). #### 4.5 Coalitional Affiliation Humans are special in that they build and maintain highly stable associations bounded by reciprocal and mutual duties and expectations. Such groups—called alliances or coalitions—may be found at many different levels of organization, such as political parties, street gangs, office cliques, academic cabals, and groups of close friends, and can include thousands or millions of individuals when ethnic or national categories are construed as coalitions (Tooby & Cosmides, 2010). The psychology underlying coalitional strategies include the following assumptions: (a) relevant payoffs to other members of the coalition are considered as gains for self (and obviously, negative payoffs as losses to self); (b) payoffs for rival coalitions are assumed to be zerosum—the rival coalition's success is our loss, and vice-versa; and (c) the other members' commitment to the common goal is crucial to one's own welfare (Pietraszewski, 2013, 2016). These assumptions reflect two crucial selection pressures operating on human groups: First, that alliances are competitive and exclusive, because social support is a rival good. Second, that resources, status, and many other goods are zerosum and, hence, the object for rivalry between alliances. As consequence, allied agents spontaneously share the intuition that achieving their goal requires avoiding or overcoming opposition from other, similar alliances and coalitions in a zero-sum fashion (Tooby & Cosmides, 2010). A vast literature in social psychology and behavioral economics documents the proximate psychological mechanisms involved in coalitional situations. For instance, people do indeed consider benefits for the coalition as (presumed) benefits for themselves (Baron, 2001). Second, social psychology studies of in-group favoritism show how very subtle cues of group membership and coalitional rivalry can activate coalitional assumptions. In so-called minimal group paradigms, people favor fellow members of an arbitrarily constructed category (Tajfel, 1970). This occurs when the categories in question are construed by participants as groups within which members can reciprocate favors (Karp et al., 1993; Kiyonari & Yamagishi, 2004). In human coalitions, members monitor each other's level of commitment, are motivated to demonstrate their commitment to the other members, and are also motivated to make defection less likely, notably by making it costly. Monitoring of other people's behavior is frequent, all the more so if the collective action is risky and success is crucially dependent on numbers. Such surveillance is manifest in voluntary groups and associations, and the extent to which monitoring is possible is a predictor of group stability (M Hechter, 1987, pp. 146–156). #### 4.6 Ownership Psychology For exchange to happen over human evolutionary history, our ancestors needed an elaborate psychology of ownership. Who is entitled to enjoy possession of a good, and to exchange it? Ownership is expressed in all human languages (Heine, 1997); in all human cultures, there is a principled distinction between mere possession and ownership; and ownership is associated everywhere with specific emotions and motivations (Brown, 1991). At the same time, explicit norms of ownership and property rights differ from one place or time to another in terms of both scope (who can own things and what things can be owned) and implications (what one may do with specific types of property) (Hann, 1998). Surprisingly, despite a long history of legal and economic reflection on property, there are only recent and relatively sparse experimental studies of our spontaneous intuitions about use, possession, and ownership (Boyer, 2015; DeScioli & Karpoff, 2015; O. Friedman, 2010). We must distinguish between intuitions and reflective representations about ownership. Adults and even very young children have definite intuitions about who owns what particular good, in a specific situation. For instance, they generally assume that ownership applies to rival resources (that is, such that one person's enjoyment of the resource diminishes another person's); that prior possession implies ownership; that extracting a resource from the environment makes one the owner; that transforming an existing resource confers ownership rights; and that ownership can be transferred, but only through codified interactions (O. Friedman et al., 2011). By contrast, people's explicit beliefs about ownership are often vague and sometimes incoherent (Noles & Keil, 2011). Also, these explicit, reflective norms often do not even reflect actual legal practices. In fact, people who live in societies with legal systems generally (and often wrongly) assume that the law must somehow accord with their intuitions—see County and Ellickson (1991) for an illustration in the domain of externalities and tort. In terms of proximate mechanisms, this suggests that the inference system takes as its input information about specific connections between a thing and an agent and outputs an 'owner' tag. In particular, this system is highly sensitive to such cues as first possession (O. Friedman & Neary, 2008), but also to information about an object's history (e.g., past possession, transactions between past and present possessor) (Blake & Harris, 2009; O. Friedman et al., 2011), as well as the work invested in the object by its current possessor; even young children consider that creative work that transforms an object creates, at least presumptively, a claim to ownership (Kanngiesser et al., 2010). # 5. Effects of Intuitive Systems on Folk-Economic Beliefs In our model, folk-economic beliefs are a result of the activation of the intuitive systems for exchange described above (and many others). The processes are illustrated in Figure 2 below. Information about economic processes, from news media, political discourse, from occasional pronouncements by economists, from other individuals, or any other sources, sometimes happens to match the input conditions of some intuitive inference system. As a consequence, the system is activated and produces specific inferences in the form of intuitive representations. These intuitive representations, in some cases, become the object of explicit, deliberate reflections, which may attribute an intuition to a source, put together several intuitive inferences, or compare them, or provide an explanatory context for intuitions, giving rise to folkeconomic beliefs. In this context, it is also worth emphasizing again that a single belief need not be the product of a single, intuitive inference system. The more inference systems that are underlying a particular belief, the more cognitive scaffolding it receives (see Fig. 2 ). In the following sections (5.1. to 5.6), we discuss the possible connections between specific evolved inference systems and specific folk-economic beliefs—that is, how activation of the systems may make a particular belief received from external sources more natural and compelling. The examples that we draw on are meant as mere Fig. 2. Illustration of the sequence of cognitive processes involved in acquisition of economy-related information and generation of folk-economic beliefs. (Figure by P Boyer. 2017) illustrations of many potential connections, providing the first small steps towards an empirical research program. # 5.1 Explaining FEB 1: International Trade as Coalitional Rivalry We begin with what we referred to above as FEB 1, the statement that international trade has negative consequences. This contains several pieces of information likely to activate specific inference systems. Let us consider a news headline like 'China sells more to the U.S. than to Russia.' Selling involves receiving resources and, importantly, resources in this case transfer from one nation to another. In psychological terms, nations are 'imagined communities' (Anderson, 1983) or, with the vocabulary presented above, nations are coalitions to the mind and, hence, mention of nations activates the coalitional psychological machinery (Gat, 2006; Michael Hechter, 1987). Nations are exclusive groups, citizens of a nation are assumed to have common interests, and nations are equipped with armies to fight each other. The activation of this machinery has the downstream consequence, we argue, that Americans will evaluate the transfer of resources to China—and, hence, the headline—negatively. As argued above, one key assumption of the coalitional system, once activated about two categories or groups, is that there is a zero-sum interaction between the mutually exclusive groups. As a consequence, there is a strong prior belief that any advantage to another group is detrimental to one's own (Hiscox, 2006). Any information to the effect that other groups are prosperous, or getting better, is equivalent to a threat-cue, indicating that our group stands to lose out. It is relevant to note how this interpretation of FEB 1 (i.e., the disadvantage of trade) is different from the standard 'fallacy'-oriented interpretation. According to our view, FEB 1 does not occur as a result of any cognitive or intellectual dysfunction. Instead, we argue that the zero-sum assumption is part of the design of coalitional reasoning. The resulting motivations are part of the architecture of this system. To maintain stable and efficient coalitions, humans in many different contexts must have assumed that other groups' advantage was a potential loss. Viewing the 'international trade is bad' belief as supported by coalitional psychology does not just explain the belief but also suggests novel testable predictions. In particular, we should expect the view that trade is bad to be particularly attractive when the trading crosses perceived coalitional boundaries. It is predicted to invariably occur in the context of, precisely, debates about trade between countries. American consumers may find it intuitive that the United States might suffer from Chinese prosperity, but, on this theory, they would find it less compelling that development in Vermont damages the economy of Texas. Similarly, the survival value of the belief might depend on the relationship between the countries. Trading between long-term allies (e.g., trading between Great Britain and the United States) should be viewed as less problematic than trading between rivals (e.g., trading between China and the United States), even if all else were equal. # 5.2 Explaining FEB 2 and FEB 3: Immigration and the Dual Activation of the Psychologies of Coalitions and Cheater-Detection In Section 2 , we outlined two FEBs about immigration. FEB 2 is the belief that immigrants 'steal' jobs and FEB 3 the somewhat contrary belief that 'immigrants abuse welfare systems.' Although these two beliefs seem inconsistent (how can immigrants take both jobs and unemployment benefits?), they do share a key common assumption, a stipulation that immigrants use up valuable resources to which they are not entitled. This assumption, we argue, is what makes either of these ideas resonate with the evolved psychology of social exchange. Specifically, the representation of recipients as not entitled to resources receives support from the interaction of two crucial inference systems: (a) coalitional psychology, and (b) cheater-detection. Immigrants are by definition newcomers to the community. Psychological research has shown that newcomers to groups activate this connection between coalitional cognition and cheater-detection, in particular, in situations where group membership is construed as conferring particular benefits. In such situations, newcomers are typically regarded with great suspicion. Cimino and colleagues interpret this in terms of cheater-detection. When new members join a group, they are in a position to receive some of the benefits of membership (e.g., becoming a Marine makes one a respected member of a prestigious military corps), without having (yet) paid any costs (e.g., risked one's life in action). This combination of features may activate cheater-detection mechanisms, as persons in this situation effectively meet the input criterion of Benefit Received without Cost Paid, which would explain the considerable hostility towards newcomers in many voluntary groups that is sometimes expressed in the form of painful hazing and initiation rituals (Cimino, 2011). Experiments show that there is indeed an implicit concept of NEWCOMER that motivates such aggressive attitude, even when people consider membership in imaginary groups (Cimino & Delton, 2010; Delton & Cimino, 2010). The tight relationship between the concepts of nation and coalition may explain the attractiveness of the statement that immigrants must be free-riders, scrounging on the past efforts of the host community. But, at the same time, the involved psychological systems leave open whether it is on job creation or on the welfare system that immigrants free-ride. This interpretation suggests new research avenues. The argument is that the public's intuitions about the economic effects of immigration does not just reflect diffuse prejudice (Stephan et al., 1999) but is the outcome of very precise psychological mechanisms that work in tandem with beliefs about jobs, the welfare state, and so on, as collectively produced resources. As a consequence, it will be difficult for immigrant populations to behave in ways that increase acceptance by the native population. Any involvement with what is construed as a 'resource' is likely to trigger intuitions of free-riding—see, for example, Guimond et al. (2010). Furthermore, our interpretation suggests that there is an intimate connection between the perceived motivations of immigrants and the presumed economic consequences of immigration. Only in instances where specific immigrant groups are seen as willing to sacrifice self-interest for collective goods—that is, the exact opposite of free-riding motivations—should the public view the economic effects as positive. Finally, this shows that there is no one-to-one mapping from specific systems for social exchange and specific FEBs. When FEBs—sometimes contradictory ones like FEB 2 and FEB 3—become culturally available, their acceptance depends on the degree to which they resonate with human exchange psychology. In this particular case, it is the dual appeal of the FEBs to both coalitional psychology and cheater-detection psychology that ensures their cultural survival in the minds of the public. # 5.3 Explaining FEB 4: Social Welfare and Intuitions about Free Riding and Communal Sharing FEB 4 refers to beliefs about the effects of economic investments in welfare programs. In fact, as laid out in Section 2 , folk-economic beliefs about these effects consist of two separate and diametrically opposed beliefs. One belief is that unemployment benefit programs, for instance, lead to decreased economic activity because welfare programs benefit unproductive individuals. Another belief is that, in the long run, these benefits increase economic activity because they sustain productive individuals during periods of bad luck and, hence, facilitate the transition to new jobs. These opposite beliefs are not randomly distributed. In fact, their distribution demonstrates our key point about the relevance of FEBs: that they are associated with particular political positions. Support for welfare programs is strongly related to the belief that they sustain unfortunate individuals. Opposition to welfare programs is strongly related to the belief that they sustain unproductive—that is, lazy—individuals. In our perspective, beliefs surrounding welfare programs—and, in particular, the link between beliefs about welfare recipients' productivity and support for welfare programs—are a key example of how psychological adaptations designed for social exchange shape economic policy views. What is surprising is not just the existence but also the strength of this link between perceived character of recipients and presumed economic benefits of welfare programs. In one of the most extensive studies of Americans' views on welfare, Gilens (1999) concluded that the perception of welfare recipients as 'undeserving' is the strongest predictor of individual-level opposition to welfare programs. This, we argue, is a consequence of the way in which the cues surrounding welfare programs activates mechanisms designed for cheater-detection. Debates about welfare programs contain a number of cues that should elicit cheater-detection psychology. Welfare recipients are in need, and welfare programs provide benefits to the recipients and does so at a cost for the collective. For a mind designed to scan the social environment for cheaters, this particular set of cues automatically raises the question: Have the recipients paid sufficient costs in order to be entitled to these benefits? (Petersen et al., 2012). Or, more specifically: Are the recipients valuable enough as cooperation partners to be included within the exchange system? This, then, motivates scanning for additional information about the cooperative motivations of welfare recipients, activating either cheater-avoidance motivations (if low) or communal sharing motivations (if high), and, in the end, providing an exceptionally fertile soil for infusing economic opinions with beliefs about whether or not welfare recipients are lazy. This psychological process is one of the more well studied aspects of folk-economics. The most direct test comes from a series of studies utilizing the memory confusion paradigm. They suggest that welfare recipients are mentally represented by activating the exact same psychological categories that people use to represent cheaters and reciprocators in everyday social interaction (Petersen, 2012). The results show that memory processes confuse lazy welfare recipients with everyday cheaters and unfortunate recipients with everyday reciprocators (but not lazy recipients and everyday reciprocators or unfortunate recipients with cheaters). Participants in these studies even forget whether those specific individuals were presented in the context of economically relevant welfare debates or everyday face-toface interaction. This process operates in a similar fashion, regardless of people's political ideology, their level of political engagement, and whether they live in a society with an expansive welfare state (Denmark) or a reduced one (United States). This particular explanation for FEB 4 makes sense of empirical findings concerning the relationship between cultural factors and beliefs about welfare. Individuals with liberal or left-leaning views tend to view social welfare recipients as productive individuals. Individuals with conservative or right-leaning views tend to view welfare recipients as unproductive individuals. Similarly, in social democratic societies, the former belief tends to dominate, whereas the latter belief dominates in societies with minimal welfare states. As consequence, cultural explanations have largely dominated the literature. For example, Americans' perception that many welfare recipients are lazy, and the association with anti-welfare sentiments, has been argued to reflect an 'individualistic' American culture (Gilens, 1999). Similar arguments have been made with regard to right-wing ideology: that it contains an 'ideological script' that binds together perceptions of laziness and welfare opposition in the mind of right-wing individuals (Skitka & Tetlock, 1993). From the evolutionary cognitive perspective, however, this structure is imposed by evolved mechanisms for exchange that are operating flexibly on the available cues. As a consequence, it should be easy to reverse apparently stable cultural patterns in welfare beliefs, if the right cues are provided. Research shows that this is indeed the case. Among a sample of Danish political science majors (who should be able to reason ideologically), ideological differences in opinions completely disappear when the participants form views about the deservingness of recipients cast as either lazy or unfortunate (Petersen et al., 2012). Even more dramatically, cultural differences between Scandinavians and Americans in support for the welfare state completely disappear when participants from these populations have to form views about specific recipients. Two sentences of text that contain evolutionarily relevant cues for cheater-detection are enough to displace 150 years of historical experience with two very different welfare systems (Aarøe & Petersen, 2014). Another insight from the evolutionary cognitive perspective is that people's priority with regard to welfare economy is not so much to ensure a particular overall distribution of resources but more to ensure that resources go to the right individuals. Although the notion that people generally prefer equal to unequal distributions of resources (Fehr & Schmidt, 2006) has been popular, recent research suggests that people are much more concerned with a fair distribution. Unequal distributions are perfectly acceptable, if those who are bypassed are viewed as cheaters (Starmans et al., 2017). ### 5.4 Explaining FEBs 5 and 6: Impersonal Markets and Mechanisms for Partner-Choice A common feature across numerous FEBs is the notion that markets are, in different ways, 'bad' for general welfare. FEB 5 is an expression of what Rubin (2014) called emporiophobia. FEB 6 refers to the more specific notion that transactions on the market are somehow 'unfair.' There is a common thread in these beliefs, the role of perceived social motivations. In most cases, the perceived negative effects of the market are seen as originating from particular sets of social motivations, believed to be pervasive in market transactions. From a cognitive evolutionary perspective, we argue, these beliefs emerge naturally due to the way market interactions differ from the types of social exchanges we evolved to value. Specifically, to explain these FEBs, we need to describe in cognitive terms, in what way market transactions are, as is often claimed, 'impersonal.' This description combines several features of potential relevance to our intuitive systems. First, people in modern conditions do not in principle need information about their exchange partners, beyond knowledge of their positions (seller, buyer), the particular goods they sell or buy, and their price. Second, there is no expectation that considerations other than price and utility should govern people's behaviors in such exchanges. That is, you may be interested in patronizing local stores because that helps keep the town pleasant, but that motivation is clearly extrinsic to the terms of exchange. Third, there is no expectation of reiterated transactions. One can in principle behave in opportunistic ways, patronizing Baker A when his prices are lower and defecting to Baker B when that is more advantageous. These features all constitute advantages of market transactions from an economic standpoint. Yet, for intuitive inference systems designed for established, long-term, cooperative exchange, these same features will be interpreted in a different manner—as threat-cues. First, our partnerchoice system requires that the parties in a transaction be identifiable as specific individuals. In small-scale interactions, the balancing of costs and benefits occurs over reiterated exchanges, and, in order to predict these long-term outcomes, information about the partner's reputation and past exchanges are key. Impersonal transactions, in contrast, are often anonymous, and therefore make it more difficult to track the reputation of one's partners. To a psychology designed for partner-choice, this is likely to trigger an alarm signal, indicating that such a situation should be avoided. Second, strictly impersonal exchange goes against motivations to generate bonds of cooperation with particular individuals, as a form of social insurance. This may reinforce the intuition that impersonal transactions involve, if not danger, at least a missed opportunity. Finally, systems for partner-choice are set up to avoid engaging in exchange relationships with individuals who are much more powerful, in order to avoid exploitation (Petersen, 2013; Trivers, 1971). In modern markets, however, many exchanges take place with corporations or business that seem exceptionally powerful from the perspective of the individual. While these corporations are actually affected by consumer choice, this only occurs at the aggregate level. As a result, each individual can form the perception that powerful corporations set the terms of exchange in potentially exploitative ways. Such intuitive computations would provide the cognitive context in which the mind processes socially transmitted information, for example, to the effect that it seems true that 'markets are cruel and selfish,' or that 'a free market makes wolves free to attack sheep.' In such circumstances, external information provides a context in which some of the intuitions described here receive an explanation or a justification. Conversely, such explicit discourse about the economy is attention-grabbing for people to the extent that it matches some of these intuitions. This perspective on the emergence of emporiophobia is a recent theoretical proposal (Rubin, 2014). There is no specific test of the hypothesis as yet. However, a range of evidence on related phenomena is congruent with this psychological description. Behavioral economics studies show how trust and cooperation are inhibited when social situations are made anonymous (Bohnet, 1999; Hoffman et al., 1996); neuro-economic studies show how monetary rewards elicit greater emotional responses if we experience the source as a human rather than, for example, an impersonal computer—for a review, see Petersen et al. (2009); and management studies show that more impersonal forms of interaction (e.g., e-mail rather than face-to-face interaction) reduce satisfaction with the interaction, in part because of a lack of emotional coordination (Baltes et al., 2002; Hibbing et al., 2013). Future research could test the proposed explanation directly by utilizing an individual differences approach: Do individual differences in attention to cooperative positive-sum games in everyday life predict endorsement of emporiophobia-related beliefs? This would not only provide a test of the link between perceptions of the market and social motivations, but also could illuminate some of the political implications of FEBs. Emporiophobia is more outspoken among liberals than conservatives and, consistent with the proposed explanation, there is evidence that liberals in general are more oriented towards cooperative, positive-sum games, in particular with strangers (Hibbing et al., 2013). In this regard, it is important to note, again, that emporiophobia is a matter of stated, explicit beliefs, which may or may not reflect the intuitive principles that actually guide people's economic behavior. People who say that markets are 'bad' may still behave as roughly rational agents in markets, and they may even detect the advantages of competition in their everyday economic behavior. But, if asked whether a given domain of activity should be left to a market of competitors, or when asked the extent to which markets should be regulated, they readily express the view that market outcomes are socially detrimental. > 5.5 Explaining FEB 7: Wages, Labor, and the Effects of Ownership Intuitions FEB 7 is the belief that labor is the source of 'value.' Experimental studies have carefully documented this effect. For instance, adults and even young children assume that working to transform an object carries a potential claim to ownership such that, for example, the artist, not the owner of the quarry, is the owner of a sculpture. This ownership claim is made stronger by the extent of the transformation (O. Friedman, 2010; O. Friedman & Neary, 2008; O. Friedman et al., 2011). From a cognitive evolutionary perspective, human ownership psychology reflects the features of evolutionarily recurrent environments. Ancestrally, most valued and owned goods were previously unclaimed natural resources that time and effort turned into something useable (whether food, tools, or shelter). In such situations, labor is indeed the exclusive generator of both 'value' and ownership. Features of modern economies that influences ownership and price, such as ownership of capital and consumer demand, were not crucial features of ancestral environments in the context of production. For example, claiming ownership over something processed by an unrelated person ancestrally would instead signal the existence of a clear dominance relationship. Although good evidence exists for the importance of labor for intuitions of ownership and value, future studies should seek to directly test people's intuitions about the relative contributions of labor, as well as capital provision and consumer demands, in determining ownership. The prediction that emerges from the cognitive evolutionary perspective is that labor should be intuitively associated with ownership, while other factors are represented in explicit afterthoughts rather than through automatic intuitions. Studies utilizing measures of explicit and implicit processing could tease such effects apart. This set of folk-economic beliefs (and, in particular, intuitions about value) illustrates an important point: that information that does not meet the input conditions of a system is simply not handled by that system. Here, our ownership inference system takes as its input the fact of original possession, the original state, and the amount of work that transformed a thing. These are the conceptual slots, the place-holders, to be filled by appropriate information. By contrast, the fact that there is, or is not, some demand for the work in question, does not fit any specific conceptual slot in our intuitive ownership system. So, it is simply not processed at all by the relevant intuitive system. These beliefs also illustrate the political importance of FEBs. Intuitions about ownership and value resonate with arguments about large wage differentials between, for example, managers and frontline workers being unfair, and that the latter contribute more 'value' than the former. Such arguments have particular appeal if used to argue in favor of higher taxation or the regulation of business. Historically, Marxian ideologies have also continuously framed owners of capital as exploitive. In this regard, the evolutionary cognitive model entails novel testable predictions: The underlying intuition that owners of companies or factories are exploiting workers may not ultimately stem from observed differences in wealth, or poor conditions for the workers. Instead, an important contribution may lie in the fact that workers are perceived as investing more effort, often in the form of more physically demanding labor. To the evolved mind, this may trigger the intuition that workers are natural owners of products. Future studies could directly test this by examining how different factors such as wealth differences between management and workers, differences in working conditions, and differences in effort, shape the view that particular corporations are exploiting their employees. ### 5.6 Explaining FEB 8: Large-Scale Regulation and Small-Scale Minds FEB 8 is the belief that regulation has the intended economic effects. Specific examples include the belief that rent controls drive down the average rent, that minimum wages increase average income, or that there is a fixed amount of work to be done, so that limiting the working hours will palliate unemployment by distributing that amount (Worstall, p. 75). Economists generally point out that, even in the best scenario, unintended effects occur and, in some cases, reverse the desired outcome. Trust in regulation seems to be based on specific non-economic assumptions (Hirshleifer, 2008) and, in particular, an assumption of stable supply. For example, people expect price-controls to affect market prices but have no effect on quantities supplied. To explain this FEB, we need to take into account the fact that unintended consequences of this kind are second-order effects that occur in large-scale social systems. They reflect aggregate market responses to changes in costs and benefits (e.g., if the price of the good is regulated downwards, the market responds by decreasing quantities supplied). But our psychology of social exchange is designed for smallscale social systems, for personal exchanges between oneself and one or more identified others. The intuitive inference systems that evolved to deal with such situations do not, because of the small-scale nature of the situations, include any conceptual slots for aggregate dynamics such as origins of supply. In this way, FEBs about regulation do not emerge from a single set of intuitive inference systems. Rather, they emerge from the failure of particular pieces of information to be processed by any intuitive inference system. Let us consider the specific example of rent control to illustrate this interpretation in more detail. To the evolved mind, rent control can be intuitively construed as a form of assistance that makes sense from a small-scale perspective, as it seems that resources are transferred from richer landlords to poorer tenants. It is likely that systems designed for cheater-detection provides the motivational impetus to support such policies. The situation can be mentally represented as including a generic landlord who intentionally takes an extra benefit (increasing rent) without incurring an extra cost (providing better housing), thereby meeting the input conditions for the 'cheater' concept. In this context, the regulatory state appears to redress the situation; the rent ideally decreases, so that the situation no longer activates free-riding detection. Economists have pointed out that the adverse consequences of rent controls (i.e., a lower supply of rental units) may offset any positive effects, although there is disagreement over the size of these negative dynamics (Jenkins, 2009). From an evolutionary cognitive perspective, people will fail to consider such aggregate effects, as the activation of evolved categories entails a perception of the situation as small-scale interaction. The cheater-detection system has no slot for information about the origin of supply and takes quantity supplied as a given. Indeed, in the exchange situations typical of our ancestral past, distribution typically had little effect on production. As described above, opportunity costs, insurance expectations, and reputation management made it possible for people to both distribute most of the game they caught and be motivated to hunt again. Since there is no conceptual slot for information about the origin of supply—for example, the incentives that make people offer housing for rent—this information does not enter into computations about regulations, thereby allowing a belief that regulation will have only the intended effects. No existing studies have directly tested this argument, and there is only scant evidence at present concerning the psychological representation of regulation (Hirshleifer, 2008). An initial set of evolutionary cognitive studies on regulation should test (1) whether the presence of evolutionarily recurrent cues (e.g., cues of cheaters) automatically induces the intuition that regulation in the relevant domain (e.g., rent) works, and (2) whether explicit information about second-order dynamics (e.g., decreased supply) are discounted in the face of such cues. # 6. Transmission and Effects of Folk-Economic Beliefs #### 6.1 Intuitive Systems Create Cultural Attractors So far, we have analyzed the ways in which various cognitive systems could affect the relevance of particular pieces of information about the economy, making some views about, for example, unemployment or trade, particularly salient because of their fit with the contents of intuitive assumptions. We can now examine how the agreement or discrepancy between intuitions and some explicit notions of the economy impacts the transmission of information between individuals, thereby creating culturally successful representations. Here we are extending the work of economists who emphasized some particular ways in which individual psychology may influence economic beliefs (Caplan, 2008; D. D. Friedman, 2004; Rubin, 2002). Closest to the kind of model presented here, David Hirshleifer proposed a 'psychological attraction' account of popular opinion on regulation, following which 'certain beliefs […] are especially good at exploiting psychological biases to attract attention and support' (Hirshleifer, 2008, p. 857). Our model extends this form of explanation to most domains of folkeconomic opinion. We predict that information about economic matters will be all the more widespread, easy to acquire, natural, compelling, and so forth, when it matches the input conditions of the inference systems described above, thereby creating widespread folk-economic beliefs. Human communication does not consist in 'downloading' representations from one mind to another. Rather, it consists of inferential processes, whereby a listener makes use of observable cues provided by a speaker to reconstruct that individual's possible communicative intentions (T. Scott-Phillips, 2014; Sperber & Wilson, 1995). Because of this interpretive quality of communication, cultural transmission will often follow unpredictable paths. We should not expect the contents of two minds from the same social group to be similar. And to a large degree, of course, they are not. Among the myriad mental representations created and sustained in individual human minds, only a minuscule fraction are shared with other individuals. Precisely for that reason, these common beliefs require special explanation. Why do people in a social group sometimes hold roughly similar representations? This question stands in contrast to the questions of classical social science, for which social change was the problem, while the continuity of traditions was taken for granted (Morin, 2016). A crucial insight of evolutionary anthropology is that cultural transmission processes are strongly constrained by the structure of human psychology (Sperber, 1985, 1996). The mind is prepared to acquire certain representations more easily than others. As a consequence, these representations are found, in roughly similar forms, in many different minds, becoming what we call cultural beliefs. The combination of expectations from our domain-specific intuitive systems, with communicative input from other members of our group, form what anthropologists call cultural attractors, positions in the space of possible representations where many minds seem to converge (Claidière et al., 2014; Claidière & Sperber, 2007). Cultural transmission creates stable representations, not just because people discard or forget material that is far from the attractors, but also because human minds actively distort fragmentary or deviant material. In other words, transmission is reconstructive rather than just selective (Claidière & Sperber, 2007; Morin, 2013). This perspective on cultural transmission helps make sense of the cultural recurrence of some folk-economic beliefs, explaining for instance why the belief that imports from other countries are a bad thing, or the notion that immigrants are welfare-scroungers, are made more salient by their interaction with intuitions about coalitions and communal sharing. It is important to notice that the effect of intuitive systems on the spread of cultural beliefs are probabilistic. For example, our intuitive free rider detection system, or our evolved set of preferences for partnerchoice, do not by themselves directly generate particular views of the economy. The intuitive systems only provide a context against which external information, provided by mass media, economists, political entrepreneurs, or simply other individuals, is likely to become relevant, attention-grabbing, and therefore susceptible of cultural transmission. Conversely, we are obviously not suggesting that the human mind is condemned to process only mental representations that are relevant to our intuitive systems. There are many circumstances in which humans have acquired and communicated thoughts that are entirely non-intuitive, in the sense that they do not match our evolved inference systems. People can, for instance, learn to think in terms of scientific physics, which often go against our intuitive physics. In the case at hand, people can learn economics and produce reasoning that diverges from the beliefs described here. However, acquisition of such non-intuitive thoughts requires effort, and in most cases institutional support for sustained learning (Boyer, 1998). ### 6.2 Folk-Beliefs Do Not Reveal an Implicit Theory of the Economy Is there an economic system in the mind, a set of processes specially dedicated to economic transactions? It would be tempting, though in our view seriously misleading, to consider the set of folk-economic beliefs as a (spontaneous, popular, perhaps misguided) alternative to economic theory. In this view, FEBs would be the outcome of a particular vision of society and the economy. We resist this interpretation, as there is little evidence for such an integrated, quasi-theoretical picture of the economy among layfolk. In fact, the few studies of lay models clearly suggest the opposite. For instance, Williamson and Wearing interviewed 95 individuals and extracted from this material their implicit views about economic processes. They conclude that 'the outcome was 95 unique cognitive models' (Williamson & Wearing, 1996, p. 3). Indeed, folk-economic beliefs may vary not just between individuals, but also within the same person, at different times or in different contexts. That is, people do not seem to have stable economic beliefs, in long-term memory, that they could pull out on demand. In the field of public opinion, researchers have made a strong case that we should dispense with such 'file-drawer' models of opinion formation (Wilson & Hodges, 1992; Zaller, 1992). People do not build and store stable, organized beliefs about the economy, ready to be made available when surveyed by a pollster. Instead, they make up their attitudes and beliefs 'on the spot,' by retrieving relevant cultural representations, and (in our view) activating the relevant intuitive inference systems. For most individuals in modern mass-societies, there is little monetary incentive to evaluate one's own beliefs about the economy or the political process (in contrast to many other domains in their everyday lives), and there is almost no price to pay for being factually wrong, which would explain why there is relatively little cognitive investment in evaluating their validity (Caplan, 2008). The exceptional range of different understandings of the model identified by Williamson and Wearing (1996) also suggests that, for each individual, the model might be different if surveyed in another context. Indeed, there is evidence for such systematic changes. In an analysis of British voters during the recent economic crisis, Martin Bisgaard (2015) found that people rapidly shift their understanding of how much control the government has over the economy, depending on how the economy is doing and whether or not their favored party is in government. If the economy gets worse and people support the governing party, the government is suddenly no longer viewed as in control. From an evolutionary cognitive perspective, such partisan motivations most likely stem from the operations of coalitional psychology (Haidt, 2012; Petersen, 2015). People signal support to their coalition by construing beliefs that protect it against criticism. Experimental results show that, like national or ethnic identities, partisanship is processed as a coalitional affiliation to the evolved mind (Pietraszewski et al., 2015). Hence, it might matter for people whether they have the 'right' FEBs from a coalitional perspective but not whether they have the 'true' FEBs from an epistemic perspective. The fact that folk-economic beliefs can change rapidly should not be surprising, as most of them are reflective, not intuitive beliefs. To illustrate this reflective nature of FEBs, consider 'emporiophobia.' Information about the fact that market transactions are one-shot interactions can lead to the intuition 'there is danger here,' because our evolved social exchange preferences include reiterated transactions with known individuals. This intuition of danger can then lead to forming, acquiring, or accepting explicit reflective beliefs of the form 'the market is bad.' From an evolutionary standpoint, it should come as no surprise that human minds do not comprise a specific 'economics' module. Decision-making under scarcity, traditionally described as the domain of economic models, is not a unified domain of social interaction, for which evolution would have given us specific inference systems. Instead, the evidence from experimental psychology studies suggests that human evolution resulted in specialized systems for scarcity in food provision (foraging), in mates (sexual preferences), in social support (coalitional psychology), and so on Buss (2015). Even in the domain of social exchange, as described above, we spontaneously activate diverse systems with different principles and potentially inconsistent responses. #### 6.3 Relationship Between FEBs and Economic Behavior The model presented here leaves a gap in our understanding, as concerns the connections (or lack thereof) between folk-economic views on the one hand, and economic behavior on the other. Many people in modern societies have explicit folk-economic views that do not just fly in the face of economic theory, but are also incompatible with their own behavior in markets. For example, people may both have the explicit belief that 'markets produce negative outcomes' and an implicit trust in competition in their search for the best prices. We propose here that economic beliefs are largely constrained by evolved, domain-specific systems concerned with social exchange. So, there might be connections between FEBs and economic behavior, to the extent that these same domain-specific intuitive systems are activated when people engage in actual economic transactions. Unfortunately, this aspect of economic cognition is still very much a terra incognita. We can assume that economic decision-making is governed by a variety of intuitive systems, the aggregate output of which is an intuition that the transactions is desirable or best avoided, and that intuition motivates the eventual decision. Over the last decades, studies within behavioral economics have demonstrated how this intuitive output diverges, in relatively systematic ways, from the subjective utility maximization predictions of standard microeconomics (Plott, 2001; Smith, 2003). However, we still lack a computationally precise and reasonably predictive description of the cognitive processes engaged (Ross, 2005). Indeed, a large part of the behavioral economic literature assumes what could be called a person-level description of economic decisionmaking, in which information about possible strategies is combined and evaluated by a general-purpose, centralized utility-evaluating system the difference from neoclassical models being that considerations of fairness, reputation, and other nonstandard forms of utility are added to the classical homo economicus agent. This notion of utility as considered by a centralized agent corresponds to what Dennett calls the 'intentional stance,' in which we explain behavior in terms of reasons, knowledge, and intentions (Dennett, 1989). This way of explaining behaviors is produced by our intuitive psychology, or 'theory of mind.' It is very successful in explaining and predicting other human beings' behavior. The operation of this intuitive psychology is so natural and invisible that it often seems difficult even to imagine another way of explaining behavior. But there is an alternative, what Dennett called a 'design stance,' in which we consider behavior in terms of the various computational systems involved in acquiring information about the environment and motivating specific behaviors (Dennett, 1989). Approaching economic decision-making in this perspective could make economic theory more congruent with findings and models from the cognitive sciences (Ross, 2005). In that perspective, decision-making in any domain is the outcome of a competition between distinct computational processes and this of course applies to economic decisions as well (Kenrick et al., 2012), a view that is supported by behavioral evidence (Ainslie & Monterosso, 2004) and neurocognitive findings (Glimcher, 2009; Loewenstein et al., 2008). However, it is still difficult to describe how these models and findings could be integrated with classical, and often empirically successful, descriptions of economic behavior in terms of rationality (Ross, 2005) and utility (Burnham, 2013). As a consequence, the actual connections between micro-processes of economic decisionmaking on the one hand, and folk-economic beliefs on the other, remain unexplored. #### 6.4 Political Relevance of Folk-Economic Beliefs In this model, because of the activation of intuitive inference systems, some ways of presenting economic processes are more compelling than others. This would constrain political communication, not just from elites to the rest of the population, but also among layfolk, with important consequences for political debate. Importantly, this would imply that a particular economic issue is often not discussed in the format that provides most information about the causes and consequences of policy, but in the format that is intuitively compelling, even if that obscures a great deal of the relevant information. FEBs are politically important because they act as a set of background assumptions that forms the basis of the formation of political opinions. One important area of opinions relates to political candidates. A wealth of research within political science has shown that incumbent parties or candidates are punished and rewarded for bad and good economic developments, respectively. When unemployment soars, incumbents are more likely to lose. Importantly, however, research also shows that the link between economic circumstances and voting behavior is mediated by the perceived responsibility of the incumbents (Rudolph, 2003a, 2003b). Assignment of responsibility for macro-economic events necessarily relies on FEBs and an interpretation of the relationship between the actions of the candidate and the economic developments. Beliefs about the relationship between economic hardship, on the one hand, and international trade and immigration, on the other hand, could be influential. If the economy is doing badly and the incumbent government has increased immigration and trade, then our analyses suggest that it is more likely that the government will be held accountable on Election Day. Such effects of intuitive systems are also relevant to policy choices. FEBs, and the intuitive systems underlying them, shape political behavior because they make certain ways of organizing the economy more compelling. Importantly, these compelling policies will in some cases be misguided, as the psychological systems were designed for small-scale exchange rather mass markets. For example, in the smallscale environments of our ancestors, helping was a matter of transferring resources to a needy individual. Arguments for welfare policy that are framed that way should be persuasive. In modern markets, however, the effectiveness of any social solutions is also affected by equilibrium considerations. Consider the difference between targeted versus universal welfare programs. From a small-scale perspective, targeted programs should be most effective in helping the needy, because they bring resources specifically to those in need. Yet, in market economies, comparative studies provide compelling evidence that welfare programs are more redistributive, and help the neediest people more, when they are universal rather than targeted. That is due, again, to macro-level dynamics ignored by our intuitive systems. Research shows that it is possible to sustain high levels of benefits from a welfare program, but only when the politically influential middle-class are among those benefiting from that program (Korpi & Palme, 1998; Rothstein, 1998). When they do not benefit, most voters are persuaded that the benefits should be scaled down. Because of this electoral dynamic, universal programs are on balance more redistributive than targeted programs. This net result arises from both their high benefit rates and the fact that higher-income groups contribute more to the program by means of taxation than low-income groups. But, again, evolved exchange intuitions would make people less likely to be persuaded by arguments that touch on such dynamics, compared to arguments that fit our intuitive systems for allocating benefits between individuals. Folk-economic beliefs are politically important because they constrain how politicians can talk about policies to the public. Political scientists have documented the effects of 'framing' on policy views (Chong & Druckman, 2007). The model presented provides a more specific understanding of these processes. In our view, certain policyrelated messages are more compelling or persuasive, not just because they are framed in more 'concrete' or 'simple' or 'vivid' terms, as is often suggested, but also because they meet specific expectations from our intuitive systems. For instance, policies that increase international trade with rival countries or that allow more immigrants to enter the country can be more easily framed as economically problematic than as beneficial, not because the former description is 'simpler' but because of the match it offers between intuitive inference systems and a particular constellation of arguments (Arceneaux, 2012). # 7. Conclusion In 1922, the American journalist Walter Lippmann grasped the characteristic of modern mass societies when he wrote: 'Our opinions cover a bigger space, a longer reach of time, a greater number of things, than we can directly observe' (Lippmann, 1922, p. 42). If this was true in 1922, it is even more true in the twenty-first century. And if it is true about mass societies, in general, it is nowhere else as true as with the market. No citizen can ever observe each of the distant transactions that comprise the market economy. It is not just a matter of practicality. The market mechanism is in principle unobservable. Even if all transactions could be observed, one would still not observe the economy as such such a claim would be a category-mistake in the sense of Ryle (1949). The 'hand' that governs the causal processes of the market is, as already pointed out by Adam Smith, invisible—that is, not just hidden but in principle difficult to detect (Nozick, 1994). As consequence, laypeople, when forming their internal representations of the economy, cannot rely on much, if any, feedback from direct experience. And without external experiences as a reality-check on their beliefs, they are left with what others report and what they themselves can imagine. We proposed a new explanation for the differences between laypeople and economists' views on a number of economic issues. Instead of considering folk-economic views as irrational deviations from normative understandings of economic processes, we explain them as the outcome of principled cognitive systems. These appeared in human evolution as adaptive response to specific challenges, and they are automatically activated whenever a situation meets their input criteria. The intuitions provide support for deliberate, explicit, reflective thoughts, among which are the culturally transmitted folk-economic beliefs considered here. How and why people acquire and stabilize beliefs about the economy is, obviously, crucial to understanding political dynamics. Economic policies are central to the overt choices offered in most liberal democracies, but we are only starting to figure out the effects of intuitive systems, typical of all normal human minds, on the acquisition and transmission of people's explicit beliefs about the economy. # References ——. (2014). Emporiophobia (fear of markets): Cooperation or competition? Southern Economic Journal, 80(4), 875–889. https://doi. org/10.4284/0038-4038-2013.287 # Introductory Note Who do we see as mad, and why? How do people decide that some person (possibly themselves) suffers from a mental disorder? History and anthropology tell us that in all human societies, people readily identify some forms of behavior as evidence for some dysfunction. In modern societies, we delegate final decisions about such matters to medical specialists. But that is of course a recent phenomenon (Porter, 2004). And, even in places with psychiatric experts, an individual must be identified as suffering from some disorder before medicine is involved. All this raises the question, how do people detect mental disorder? I was surprised to find that there was very little description of these criteria in the literature. Anthropologists did describe various local interpretations and explanations of madness, e.g., as the work of spirits, a consequence of witchcraft, an imbalance in humors or elemental components of the person… but what behavior had prompted the initial perception of disorder? In brutal terms, what do you have to do to seem mad? One might imagine that the criteria could be entirely specific to each culture, but that is certainly false. Manifestations of mental disorder are identified in strikingly similar ways in very different places. We can recognize what Horatio describes as Hamlet's 'wild and whirling words' (Hamlet, I–v), as well as the hallucinations, incoherent speech, inappropriate emotional reactions, conversations with non-existent interlocutors, etc. Anthropologists who do fieldwork have little difficulty in perceiving mental disorder in the most exotic (to them) cultural environments. In this article, I proposed that people in different cultures use the same implicit criteria for mental disorder, which derive from our intuitive psychology, sometimes called 'theory of mind' (Leslie et al., 2004). It provides us with interpretations of people's observable behaviors, utterances, and gestures, in terms of things that we could not observe, such as other people's beliefs and intentions. Our intuitive psychology works on particular assumptions about the way minds work, how perception causes beliefs, how beliefs interact with intentions, how intentions explain behavior, etc. Naturally, all these assumptions are implicit. In our everyday interactions we need not be aware of their content or their operation. Intuitive psychology is present in all normally functioning human minds, mostly as a result of natural selection pressures for cooperation (Tomasello, 2009). Humans cannot coordinate their behavior on joint goals unless they mentally represent other agents' intentions and beliefs. This evolutionary context explains why our intuitive psychology generally works smoothly when we interact with typical adults who would have been the cooperation partners that mattered most to our fitness, but is defeated by atypical minds, like those of infants or animals from another species. (Interaction with those two kinds of agents is handled by systems specialized in kin-selection and parenting (Hrdy, 2009) or predator-prey relations (H. C. Barrett, 2005), respectively). This origin in cooperation has the important consequence that intuitive psychology is not just a descriptive mechanism that tells us what happened in other minds. It is also normative—it implies a description of the way a mind ought to work (Stich, 1983). But that also explains why our intuitive psychology alone does not produce any description or explanation of mental disorder. It just produces a 'mind not working' signal when it is defeated, a natural equivalent of the 'syntax error' of computer systems. In this article I describe two consequences of these features of intuitive psychology. First, they allow us to predict which kinds of behaviors will be identified as evidence of underlying mental disorder, and which will remain 'invisible' to our intuitions. Second, the fact that intuitive psychology detects dysfunction but produces no representation of how it occurs creates an explanatory gap that is filled by all manner of culturally transmitted explanations, in many cases imagined agents like witches or spirits. Interestingly, people imagine those mystical agents as endowed with the kinds of minds that our intuitive psychology expects—minds that perceive what happens around them, form beliefs on the basis of perceptions, combine desires and beliefs to form goals, and so forth (J. L. Barrett, 2000; Boyer, 2003). So it seems that in most human societies, people cannot escape intuitive psychology when they want to explain behavior—first, it triggers the intuition of disorder, and second, it is used to explain disorder. Isn't modern psychiatry often engaged in the same operation? Medical detection of mental disorder is of course framed by expectations of intuitive psychology. Patients are confirmed as patients because their behavior deviates from the expectations of intuitive psychology. Indeed, the catalogue of symptoms used by the American psychiatric profession, the Diagnostic and Statistical Manual (American Psychiatric Association, 1995), is mostly a list of deviations from the expectations of normative intuitive psychology. But beyond detection, the ideal of a scientific psychiatry would be to provide explanations, to describe the connections between observed behaviors and reported states of mind, on the one hand, and particular neuro-cognitive mechanisms, on the other (Murphy, 2006). Unfortunately, we are very far from having sufficiently precise computational descriptions of the neuro-cognitive processes that underpin mental dysfunction. Perhaps that is because the initial description, in terms of deviations from our theory of mind expectations, just does not capture the underlying similarities and differences in disorders. That discrepancy between our intuitions and possible underlying mechanisms is particularly clear in extreme cases of delusion, in which the patient seems to hold an irrational belief, e.g., that a part of their body is not actually theirs, that a relative has been replaced with a clone, and so forth. Interpreting delusions constitutes a formidable challenge, precisely because we cannot use any of the inferential tools supplied by our intuitive psychology (McKay, 2012). The most precise models for such delusions necessarily rely on neuroscience models that are completely alien to intuitive psychology (Gerrans, 2014). So intuitive psychology, which is indispensable to human interaction its impairment in autism, for instance, has catastrophic consequences for the social life of patients (Lai et al., 2014)—may also constitute the most formidable obstacle to our understanding of mental disorder. # References # Intuitive Expectations and the Detection of Mental Disorder: A Cognitive Background to Folk-Psychiatries1 Abstract. How is mental dysfunction detected? How do cultural models of mental disorder affect this process of detection? Attempts to answer these questions have not often been made in the research, as they fall between two domains: that of crosscultural psychiatry (which looks at the dysfunction itself) and anthropological ethno-psychiatry (which looks at cultural models of sanity and insanity). In this paper, I set out a model to illustrate this 'missing link' between behavior and cultural models, founded on experiential evidence for intuitive psychology. Typical adult minds contain certain intuitive expectations about mental function and behavior, and these are used to perceive certain sorts of dysfunctional behavior. It appears that there is a 'catalogue' of potential behaviors that activate this intuition, and therefore the symptoms that are present in culturally specific folk-understandings of mental dysfunction are also restricted. It is also suggested that certain mental dysfunctions are 'invisible' to folk-understandings due to their lack of obvious breaches of principles of intuitive psychology. This standpoint helps us to <sup>1</sup> Some of the contents of this chapter have been expressed earlier in Boyer, P. (2010) Intuitive Detection of Mental Disorder: Cognitive Background to Folk-Psychiatries, Philosophical Psychology 23(6): 821–844. https://doi.org/10.1080/09515089.2010.529 049 comprehend the cultural stability and spread of certain views of mental disorder. # 1. Introduction The concept of mental disorder is one which exists across the world people in each and every country have a certain way of classifying and modelling this notion. This leads us to pose several questions: do those in different groups and communities view the concept of mental disorder as consisting of similar aspects? Are there limitless ways to envisage the concept or are our categorizations based on some common underlying principles? If these principles exist, where did they emanate from, and do they have influence over the purportedly scientific models and classifications in psychiatry? These questions were traditionally approached by two disparate disciplines: cross-cultural psychiatry—a branch of mainstream psychiatry — and ethno-psychiatry, stemming from cultural anthropology. This disparity has led to various theoretical problems and uncertainties, which may yet be overcome by the advances in intuitive psychology (or 'theory of mind') and in the psychology of culture. We are now able to set forth a synthetic model which suggests that all normal adult minds across the world contain certain intuitive expectations about normative mental function and behavior, based on common underlying principles. These expectations are constrained by intuitive psychology, which influences both the detection of disorder (through affecting people's recognition of certain kinds of behavior as symptomatic of mental dysfunction) and the explanation of disorder. Thus, it seems that, despite the very different cultural conceptions of sanity and mental dysfunction, the underlying principles are simple and the same. # 2. A Prototypical Scenario and a Question The social interactions that we will consider throughout this paper usually take the following form: 1. An individual experiences some type of mental dysfunction. - a. At a certain point, the individual's behavior subverts the expectations of the other people involved in an interaction; - b. The behavior cannot be easily or logically explained or repaired, and/or: - c. The behavior is repeated, or similar behavior occurs, with similar results (lack of clear or logical explanation). Cross- (or trans-) cultural psychiatry predominantly focuses on step [1], attempting to discover the limits of variation within mental dysfunction, subject to cultural background (Stein, 1993). Inversely, ethno-psychiatry predominantly focuses on steps [3–5], delineating the models or suppositions lying beneath certain kinds of nosography and etiology, as well as contemplating treatment, the potential for specialist care, and the effectiveness of different techniques (Jovanovski, 1995). It is notable that neither of these disciplines mentions the cognitive processes through which mental disorders are recognized (step [2] of the above scenario). Georges Devereux, a founder of modern ethnopsychiatry, comments: The patient appears on scene—on the printed page [in ethnopsychiatric monographs]—as an already recognized and more or less completely diagnosed neurotic or psychotic, with no mention of the manner in which this status—for a status it is!—has been assigned to him. (Devereux, 1980) p. 247 While evidence from history and cultural anthropology has made us aware that steps [3–5] in the above scenario vary around the world, no principled model exists to illustrate the potential for variation in the area of detection ([step 2]), and there is additionally no formal research on the topic. In order for us to understand how mental disorders are detected, we should examine whether common principles that underlie regular cognitive function play a part. In doing so, we extend the existing research program that explores cultural phenomena as bounded variations within limits set by human cognitive capacities (Sperber & Hirschfeld, 2004). Research performed by cognitive scientists and evolutionary anthropologists has found that the underlying principles we develop at a young age lead to suppositions that help us to acquire our specific cultural norms and ideas — for example, in the areas of folkbiology (Atran, 1990, 1998), kinship and ethnic categories (Hirschfeld, 1994a, 1996), racial categories (Kurzban, Tooby, & Cosmides, 2001), religious beliefs (Atran, 2002), social interaction (Cosmides & Tooby, 1992); (Fiske, 1992); (Tooby & Cosmides, 1996). As a result, we would expect that these underlying principles would also have an influence over how we determine what is acceptable versus atypical behavior. # 3. The Background: Intuitive Psychology Expectations The term 'intuitive psychology' straightforwardly describes the collection of cognitive capacities that help us to understand our own and others' behavior as the result of unobservable mental states and processes (Baron-Cohen, 1995), (Leslie, 1987), (Perner, Leekam, & Wimmer, 1987). The majority of these capacities are subconscious in function — we are unaware of the inferential processes that lead us to establish overt explanations of other people's behaviors. The capacities are likely to be situation-specific and distinct from each other, each with their own lower-level neural sub-capacities (C. D. Frith, 1996). We have only gradually begun to be able to (tentatively) describe the capacities involved in normal, adult intuitive psychology—our initial, more detailed descriptions came from instances where the system does not function, such as in autistic children or in animals from other species. In this paper, we can limit our concentration to only the most basic intuitive principles involved in the process. Whilst there are key theoretical disparities between models of intuitive psychology, it is fortunate that its core standards are remarkably uncontroversial, and include: [1] Intentional states; representations of our existing apparent realities. One notion of intuitive psychology is that people's minds have memories, beliefs, and perceptions 'inside' them, which represent or duplicate the conditions in a person's physical reality. Children realize from the age of three that mental representations are not physical objects (Wellmann & Estes, 1986). Beyond that age, we progressively cultivate the idea that our thoughts contain a representation of the circumstances surrounding us—there is a causal link between manifest states of affairs and mental representations (Leslie, Friedman, & German, 2004). Additionally, we use indirect cues — such as the extent to which people's gaze follows objects and other people's gaze—to subconsciously confirm the link between external objects and people's mental states (Friesen & Kingstone, 1998). Broadly speaking, by seeing that an object aligns with and seems to 'attend' to another object, we get a strong indication that it is an intentional agent (Johnson, Slaughter, & Carey, 1998). [2] Agency as internal causation. One of the first assumptions that infants make is that agents, unlike other objects, decide their own behavior from within (Baldwin, Baird, Saylor, & Clark, 2001); (Rochat, Morgan, & Carpenter, 1997). This assumption is founded on the particular psychophysics of animate motion (Michotte, 1963); (Schlottman & Anderson, 1993); (Tremoulet & Feldman, 2000) and on additional cues that reveal internal causation and production of behavior (Gelman, Durgin, & Kaufman, 1995); (Williams, 2000). Intentional agents' actions relate to objects and states in a principled way (Blythe, Todd, & Miller, 1999), and even infants can decode their orientations — for example, by reaching a specific object of interest and avoiding obstacles (Csibra, Gergely, Biro, Koos, & Brockbank, 1999); (Gergely, Nadasdy, Csibra, & Biro, 1995). Attributing more intangible intentions to objects is something that children learn to do at a young age, such as by emulating effective rather than ineffective actions in the handling of tools (Want & Harris, 2002) and using actors' visible emotions to deduce the effectiveness of the action (Phillips, Wellman, & Spelke, 2002). [3] Memory as a store. Throughout the world, the inherent idea of memory as a store is pervasive and meaningful—to the extent that even certain scientific models of memory processes are based upon it (Roediger & Geraci, 2004). While research has not focused on this theme, intuitive psychology also appears to accept the copying of experiences onto memory stores and their later retrieval in the configuration in which they were experienced, which is in conflict with a large amount of psychological research into human memory (Ross & Wilson, 2000); (Rubin, Schrauf, & Geenberg, 2003). [4] Inferential and communicate coherence. In order to communicate, we must make inferences and follow strict, implicit pragmatic principles (Grice, 1975) which we develop at a very young age. These principles influence infants' communicative development (Trevarthen & Aitken, 2001) and acquisition of new vocabulary (Bloom, 2000). We generally cannot interpret others' utterances or communications without comprehending the speaker's intentions—the way in which they are trying to influence the listener's representations (Noveck & Sperber, 2004); (Sperber & Wilson, 1995). During conversations, implicit adaptations and small 'repairs' allow people to sustain comparable representations of the circumstances they are discussing. [5] Emotion, norms and empathy. We build much of our information about other people's mental processes on the inadvertent reading of minute emotional cues — such as facial expressions, voice and gestures—and of the potential reasons behind them. Again, this ability evolves from a young age—at five months old, infants behave differently when faced with demonstrations of different emotions on a familiar face (D'Entremont & Muir, 1997). Different cultural groups develop comparable cues at similar times (Ekman, 1999). To detect and recognize certain kinds of emotions, specific neural circuitry is required (Kesler-West et al., 2001), separate from broader facial identity processing. If cues are lacking, we use our inherent psychological principles to deduce what the emotional repercussions of certain circumstances would be. A substantial database of certainties about social relations is necessary in order to do this—we need to recognize the emotions that influence the different types of relationships, from family to partners, friends to acquaintances, and so on. People tend to assume that those who are part of their cultural group adhere to the cultural norms to an equivalent extent—these norms explicitly prescribe behavior, which is connected to emotion or attitude (Nichols, 2002a). Assumptions about emotions originate from a broad ability to be empathic using simulation—such as internally simulating our own emotions resulting from a situation in order to perceive others (Decety & Sommerville, 2003). Our inherent expectations about emotionally relevant hypotheticals (such as deducing how somebody feels when their family is threatened, or when they are abandoned by their friends) could also be founded on this sort of simulation (Gordon & Olson, 1998). [6] Principled motivation. Intuitive psychology comprises a particular model of how intentions are connected to available information and background preferences (Malle, 2004). It also differentiates desire and intention—desire is seen as a simple preference state, and intention is seen as the fusion of that state with available information to produce a plan of action (Malle & Knobe, 2001). Intentions are presupposed to make the attainment of desires and goals more probable. Children appear to develop their understanding of desire and intention before the age of three (Wellman, Phillips, & Rodriguez, 2000). They view desires as foundational when clarifying behavior, to a greater extent than physiological states (Moses, Coon, & Wusinich, 2000), but even young children appreciate that desires may clash (Bennett & Galpert, 1993). And numerous other principles. This list of inherent principles is merely a summary of research in this area—it does not assert that there is concurrence on the functioning of intuitive psychological capacities in this field. With the completion of more research, we may be able to expand the list, but the key points at present are the generally undisputed findings above. Later in this paper, the restrictions of intuitive psychology will be further discussed, but it is now pertinent to discuss the way it is used to comprehend the perception of mental disorders. # 4. Intuitive Catalogue of Detectable Mental Dysfunction We can now attempt to detail the potential types of evident mental disorder using the broad inherent principles of intuitive psychology. Table 1 below details a list of assumptions and behaviors that defy these assumptions. This list is not exhaustive—it is merely an initial attempt to illustrate that intuitive psychology does tacitly anticipate particular manifestations of mental disorder. Below, the items in the list are discussed in more depth, correlating with the categories introduced in the previous section: [1] Intentional states; representations of our existing apparent realities. Our inherent presuppositions about intentionality are violated when an individual does not react to occurrences (for example, if they are in a vegetative state, hebetude, or coma) or to noteworthy stimuli (for example, if their reflexes are lacking or have declined, they neglect to gaze at moving objects, or display general indifference). Similarly, individuals who do not follow culturally appropriate types of gaze following and direct gaze cause a similar violation. [2] Agency as cause of behavior. In this area, potential indications of atypical functioning comprise involuntary motor behavior (such as Vitus, tics, or alien hand), and circumstances where an individual's intention and action appear not to correspond (for example, if they are surprised by their own actions). Involuntary speech that flouts social norms (such as in Tourette Syndrome) are recognized in the same manner. [3] Memory as a store. This model hypothesizes that memory impediments would be interpreted as leakage. Unlike in scientific models, failing to recall a recent, common occurrence would be viewed as a procedure of deletion, rather than a breakdown in organization or encoding. Table 1. Inherent expectations and corresponding potential violations . Table by P Boyer. [4] Inferential and communicative coherence. An inability to sustain coherence within a conversation would activate the perception of disordered mental processes, as we can see from the clinical accounts of many types of mental disorder—senile dementia and Alzheimer's are often both detected through the manifestation of violations of the principles of pragmatic relevance (Hays, Niven, Godfrey, & Linscott, 2004). As a result, caregivers must slowly modify their expectations of conversations with patients, adapting to their lack of responses, 'repairs', or strategies to diminish ambiguity or misunderstandings (Orange & Zanon, 2006). Likewise, the speech of schizophrenic patients often contains instances of wrong speech-acts, flouting of turn-taking, referential ambiguity (i.e. using pronouns without their referents), lack of repair, referential incoherence—where multiple subjects are covered in the same statement (Corcoran & Frith, 1996); (Meilijson, Kasher, & Elizur, 2004)—and diminished comprehension of figurative speech and proverbs (Brüne & Bodenstein, 2005). [5] Emotion cues. Individuals recognize when another person seems impassive in response to others' emotional cues, or exhibit unintelligible emotional cues themselves — for example, patients with dementia experience diminished control over emotional expression (Smith, 1995). Schizophrenic patients are often impeded in the perception and analysis of emotional cues, and in the same way, so are autistic children, even high-functioning or Asperger patients (Teunisse & Gelder, 2001). Those with autism find it extremely difficult to recognize emotional cues (Adolphs, Sears, & Piven, 2001), which is exacerbated by their struggle to comprehend the potential basis for others' emotions. Any breaches of cultural norms or other emotional expectations should be conspicuous. While breaches of etiquette, for example, will not necessarily be perceived as indicative of atypical mental processes they might instead indicate aggression or poor childhood experiences persistent breaches with no explanation may be seen as confirmation of disorder (see Clement, 1981, #1956 for an account of this in Samoan culture). Breaches of familial expectations (for example, neglect or abuse towards family members) may also be seen as indicators of basal dysfunction. [6] Motivation. Behaviours that seem to conflict with or oppose a person's own preferences would likely be detected as a disturbance in cognitive functioning. Additionally, individuals with low motivation ('indifference') or, in contrast, those who are highly motivated—to the point of mania—would be contenders for detection. For example, those with schizophrenia are often found to have low levels of motivation, just as high-risk children do (Watt, Grubb, & Erlenmeyer-Kimling, 1982). Similarly, Alzheimer's is frequently detected at an early stage via the degeneration of motivation, past what would be expected for those who are ageing (Ready, Ott, Grace, & Cahn-Weiner, 2003). The above table does not constitute a list of potential types of mental disorder—it merely looks at the circumstances or behavioral events that could lead to the perception of atypical mental processes. At this point, we have no presuppositions about whether natural or sound instances of mental disorder can be detected consistently via intuition. # 5. An Illustration: Mohave Cases In order to connect a range of observed behaviours with the intuition of disorder, the dysfunction-detection model is suggested. Cases outlined by George Devereux in 'Mohave ethnopsychiatry and suicide' (1961) are useful demonstrations of the model—Devereux details a number of varying case-histories, in detail, separate from his and others' analyses of the behavior, which is an approach scarcely found in ethnopsychiatric ethnography. This permits us to notice the initial perception of the behavior as extraordinary and the later categorisation of it as an example of culturally specific dysfunction. In Table 2below, all symptoms outlined within case-studies are set out—only behaviors described by participants, excluding the ethnographer and other investigators, are included. We can presuppose that the disclosure of these utterances indicates that individuals thought the accounts of behavior were pertinent and indicative of an intrinsic disorder or condition. It is significant, therefore, that the majority of the utterances describe obvious breaches of inherent psychological expectations (compare Table 2 with Table 1 ). As mentioned previously, psychological contraventions often appear alongside other norm-contraventions, but the list additionally indicates that the psychological contraventions are recognized and acknowledged without fail. For example, a woman is reported to be a nymphomaniac (case-study 14), but for a Mohave onlooker, a salient aspect of the situation is that she was sometimes unaware of what she was doing. Similarly, losing consciousness during epileptic fits (case-study 6) is made significant by the individual not experiencing pain. An anti-social person (case-study 20) is additionally reported to be misled ('he seemed to believe he was going to war'). Naturally, this is not intended as overwhelming proof for the violationdetection model. That would necessitate a detailed examination of casestudies pertaining to a range of cultural environments, beyond what can be achieved in this paper. The intention here is to exemplify that breaches of psychological expectations are core to the perception of putative dysfunction. In fact, out of all the cases detailed by Devereux, only one (case-study 23) describes atypical or improper behavior (beating one's in-laws) that could be interpreted in any way other than psychologically. Table 2. Collation of symptoms outlined in Devereux's case-studies. First column, case-study number; second column, page in Devereux (1961). Third column, quotation from the case-study. Fourth column, principles from Table 1 that may be breached in that case. # 6. Scope and Limits of Intuitive Psychology Principles Intuitive psychology comprises a collection of critical inferential systems that function straightforwardly in general, giving us an analysis of perceived behavior with regard to beliefs, purposes, and emotional states—this is known and accepted. It is suggested here that, in the same way, intuitive psychology helps us to discern that a certain individual's mind is dysfunctional; this is the only clear explanation for the individual's behavior breaching the inherent psychology expectations. This model gives inherent psychological expectations the main causal role — it would thus be useful to define their key characteristics. Are the principles universal? The core question is whether these principles are replicated across the world—do individuals in different cultures possess the same intuitive psychology? It is clear that much of our cognizance of behavior originates from local norms and beliefs about the right way to behave. As a result, individuals' overt interpretations of mind vary significantly (Lillard, 1997; Vinden, 1998), at least in the case of those individuals who attempt to produce an interpretation. However, there is no corroboration of comparable variation within the principles of intuitive psychology themselves—literature from around the world has not found that individuals in different communities decline to orient to the principles above (Table 1 ) or follow completely different principles (Leslie, 1994; Sperber & Hirschfeld, 2004). The majority of applied studies have looked at developmental features of intuitive psychology, discovering a large amount of overlap in early intuitive psychology principles (Astuti, 2001; Avis & Harris, 1991; Tardif & Wellman, 2000; Yazdi, German, Defeyter, & Siegal, 2006), the timescale of their development (Callaghan et al., 2005; Wellman & Fang, 2006), and their relationships to other mental processes (Chasiotis, Kiessling, Hofer, & Campos, 2006). If we observe the profuse amount of verification for early development of brain structures in line with intuitive psychology, this is to be expected (C. D. Frith, 1996; Luo & Baillargeon, 2007). Are intuitive principles a conjecture of rationality? Psychological expectations should not be understood as an assumption of rationality, nor muddled with the broad presupposition that 'people generally behave rationally.' In fact, it seems clear that intuitive psychology is made up of exact, situational expectations and deductions—not an inferential hypothesis of the way individuals behave. Research has shown, for example, that even at an early age, we envision that people's beliefs are shaped according to their observations, rather than their observations being shaped by their beliefs (Leslie et al., 2004), and as a result, we have specific assumptions (e.g. 'X saw a dog here, so X believes there's a dog here'). However, this principle has not been shown to be related to a broader presupposition of people behaving logically the majority of the time—in contrast, we often expect others to behave illogically. Many cultures presuppose that in situations where an individual is angry (and other similar situations), they are likely to act in a way that they will find regrettable afterwards. Intuitive principles are not fixed—they can be both reinforced or dismissed when additional context-specific considerations are taken into account. For example, while small children presuppose that the presence of an object A will be detected by everyone in the vicinity, this presupposition can be overcome if there is an obvious obstruction to other people's perception of object A (Luo & Baillargeon, 2007). Intuitive psychological analysis of behavior is, for the most part, a kind of relevance process, consisting of a detailed succession of presuppositions which lead to an optimal interpretation of the observable behavior (Sperber & Wilson, 1995). There is an unlimited set of circumstances where perceptions and beliefs do not match up—the principles of intuitive psychology are merely preset assumptions, with 'all else being equal' (Leslie, German, & Polizzi, 2005). This should also be true for the discernment of dysfunction that occurs when presuppositions are breached, which would explain the variance of behaviors that precipitate such a discernment across culture and history. For example, in the past, speaking aloud in public when no other conversational partners were present was viewed as indicative of dysfunction in many parts of the world, but this behavior has come to be expected, and it is presupposed that the individual is likely to be on the phone. Mental dysfunction is perceived as a result of a network of illative operations, which may either support or weaken the original intuition. Intuitive principles are not invariably correct. In the field of philosophy, it is still being debated as to whether the constructs that our psychological expectations predicate (such as beliefs, purposes, emotions, etc.) are 'really real'—whether our intuitive psychology has scientific value (Churchland, 1981). However, this discussion is immaterial for our purposes, as we are focusing on the way in which intuitive psychology functions, and not whether it does so correctly or not. This should be noted as we move to discussing our inklings of dysfunction. These inklings that are activated by an individual's behavior in a certain context may be erroneous. Broadly speaking, the set of behaviors that lead to such discernments may be divergent—in the fields of established neuro-psychology or psychiatry, they may be classified separately. Our perceptions of dysfunctional behavior might only have a minor overlap with true dysfunctionality. In fact, many types of dysfunction are likely to go undetected by intuitive psychology. Are presumed dysfunction and broader norm-violation alike? It could be questioned whether breaches of psychological expectations are actually the basis for the perception of dysfunctional behavior—some might argue that they relate back to a more general defiance of social norms. For example, if someone declines to respond to a question, they are not only breaching a psychological expectation, but also flouting the rules of conversation; similarly, a sociopath who lacks remorse after causing pain is not only behaving atypically, but also flouting the social rules that surround violence. However, this straightforward, domain-general analysis is not supported by research findings. Studies in the areas of behavioral and developmental neuropsychology and neuroscience show that intuitive psychology is domain-specific—its inputs and principles are separate from those contained within other mental systems (Blakemore et al., 2001; U. Frith, 2001; Leslie et al., 2004). There is an overwhelming lack of convincing affirmations for the existence of cognitive mechanisms that survey norms (and their violations). Instead, we find evidence for highly specific mechanisms that keep track of violations of moral imperatives as distinct from social conventions (Haidt, Kesebir, Plessner, Betsch, & Betsch, 2008; Turiel, Eisenberg, Damon, & Lerner, 2006); violations of exchange principles and economic fairness (Cosmides & Tooby, 1992; Kurzban, 2001); violations of incest revulsion (Lieberman, Tooby, & Cosmides, 2007); betrayal of the implicit requirements of friendship (Hess & Hagen, 2006), and violations of status and manners (Nichols, 2002b). These kinds of violations all transpire in a certain type of situation, due to certain sorts of purposes. A broad operation that monitors their concurrent features does not seem to exist. These conclusions lead us to suggest that manifest breaches of presuppositions lead to a certain sort of 'dysfunction intuition' that differs from the contravention of other norms. This intuition might be supported, altered, or weakened in reality due to the contravention of other non-psychological expectations. Additionally, we can assume that there are numerous instances where an individual is assessed as atypical due to psychological or non-psychological violations, but neither we nor the person who assesses this are aware of it. # 7. Prediction of 'Invisible' Conditions Our intuitive psychology comprises a group of presuppositions that allow us to comprehend and anticipate other people's behavior in the majority of everyday situations. However, it is not a fully comprehensive account of how and why intentional agents behave. Intuitive psychology wholly focuses on conspecifics. The majority of animals' motivation and conceptions are significantly disparate from our intuitive expectations (for example, see Grandin, 2005 #3976). It happens that, even among conspecifics, our intuitive psychology frequently cannot help us to interpret behavior—for example, the behavior of young children, whose utterances and arrivals at certain conclusions can be perplexing. This can be explained by the assumption that, if intuitive psychology only accounts for a section of mental function, dysfunction within systems which it contains no presuppositions for will be 'invisible'. This assumption appears to be correct, especially when the dysfunction pertains to certain features of cognition, excepting reasoning, planning, and decision-making. The condition of prosopagnosia—the failure to associate the visual stimulus of a person' face with information held in memory about the person (de Renzi, Faglioni, Grossi, & Nichelli, 1991)—serves as a good example. This handicap only applies to the global visual trace of the face, not to face-details or other aspects of an individual (Farah, Levinson, & Klein, 1995). Consequently, patients can overcome it by heeding facial features, voice, gait, and other characteristics in order to interact appropriately. It is improbable that this condition could be detected via intuitive psychology, because its only symptom is a prolonged interval before responding to an individual's presence. This is unlikely to be recognized—if it was, it may be misinterpreted as illustrating some sort of difficulty with vision, memory, or social interaction (i.e. an unwillingness to interact with others). In a similar way, the majority of kinds of visual agnosia—where an individual becomes confused when asked to name and describe common types of objects or animals (Dixon, 2000)—could be missed or misinterpreted. Specific neuro-psychological conditions are generally expected to activate the sense that a dysfunction of some sort is present—however, it is hard for intuitive psychology to define what, exactly, this dysfunction is. For example, Tourette's syndrome causes breaches of etiquette that are detectable even in divergent cultures (Staley, Wand, & Shady, 1997), and these breaches are often not attributed to cognitive control, due to our intuitive psychology's unfamiliarity with the idea of distinct neural procedures and control loops connecting them. Another example is aphasia, which is frequently assumed to be a kind of insanity, due to the lack of meaning that can be derived from their utterances. Because intuitive psychology contains no detailed account of the intricate ways in which thoughts and speech are linked, disruption of speech is often assumed to represent disruption of thought. # 8. From Intuitions of Disorder to Folk-Models The operations that lead to the perception and analysis of mental disorder can be most accurately portrayed by describing causal links between disorder, the resulting behavior, the perception of behaviors controlled by intuitive psychology, and the patterns of acquisition and communication that cause models to be chosen. These links are illustrated in Figure 1 below. #### 8.1 What Makes Folk-Models ''Folk''? Up to this point, we have mainly concentrated on how intuitive psychology sorts the behaviors that are notable for their breaching of expectations from the others. This leads us to question whether this Fig. 1. A reduced model of the detection of mental disorder. Dysfunction sets off behaviors (stars), of which only a few can be perceived as breaches of intuitive psychology (many 'bounce off' intuitive perception). In certain cases, perception may be erroneous (where the origin of the behavior is something other than dysfunction). Perception of dysfunction notifies and limits people's models—only some of these complete the stages of acquisition and communication (ineffective models 'bounce off' transmission). Models which are often triggered may have feedback ('looping') effects both on themselves (transmission biases) and on certain people's behaviors. These are illustrated using dotted lines. (Figure by P Boyer) system of perception induces general cross-cultural ideas of mental disorder. In order to answer this question, we must first discuss contemporary anthropological models that illustrate the way cultural knowledge is transferred. How are certain models straightforwardly and customarily transmitted within a group? This sort of question is handled by presentday anthropological theory through cultural selection frameworks (Boyd & Richerson, 1985; Durham, 1991; Sperber, 1985). A key presupposition is that, similarly to other kinds of human interaction, cultural transmission does not involve 'downloading' notions from one mind to another; instead, deductive processes are necessary, in which individuals notice signals in other's behavior, deduce their communicative purposes, and establish concepts founded on their deductions (Sperber, 1996; Tomasello, Kruger, & Ratner, 1993). Consequently, people are always generating modifications of others' representations. The unpredictability of communication and deductions leads us to seek an explanation for the presence of shared representations or 'cultural' information, where many varying models would be expected (Sperber, 1985). By terming certain representations 'cultural', we focus on the similar representations held by participants in a certain group. This similarity is indicative of certain notions and norms being chosen during the procedure of transmission, while others are altered, abandoned, and forgotten. Cognitive predispositions go some way towards providing an explanation of the repetition of certain notions and norms (Sperber & Hirschfeld, 2004). Research and models originating from experimental and developmental psychology, linguistics, neuro-psychology, and the neurosciences are all unified in the contemporary cognitive anthropology approach, in an attempt to illustrate the power of cognitive predispositions in increasing the probability of specific types of notions and deductions appearing. Certain principles—the majority of which are implicit—accompany and arrange incoming data, which causes certain deductions to follow, regardless of their origin. As a result, we are left with statistical 'attractors' in the population dynamics of cultural transmission (Claidière & Sperber, 2007; Sperber & Hirschfeld, 2004). #### 8.2 A Basis for Dysfunction Intuition The argument here is that a sizable amount of culturally transmitted folk-understandings of mental illness originates from cognitive dispositions, which impact cultural transmission significantly. This indicates that instincts about mental disorder are based on a cognitive network that is predominantly applicable across cultures, as opposed to the conventional presuppositions of ethno-psychiatry. Perceptions of atypical behavior are frequently called intrinsically 'cultural' (Gaines, 1992; Jovanovski, 1995) and, in all likelihood, differ to a large extent as a result. In the word of Anthony Marsella: 'mental disorders cannot be understood apart from the [culturally specific] concept of self, because it is the nature of the self which serves to identify 'reality' for a given cultural group and which dictates the definition of what constitutes a symptom [italics added]' (1981, p. 362; see also Good, 1994 #3892 and Sadowsky, 2003 for similar arguments). It is useful to explore this statement in more detail. Here, the word 'symptom' could be interpreted in two ways—it could mean 'behaviors that people believe to be triggered by the mental dysfunction' or, alternatively, 'behaviors triggered by the mental dysfunction which are overtly built into a model of mental dysfunction'. Marsella appears to have intended the second meaning—in this sense, the statement seems totally justified. Our 'cultural models' of mental dysfunction unquestionably differ according to culture, within certain confines, as we will discuss below. However, we cannot assume that local models govern whether behaviors are or are not perceived as potential proof of mental dysfunction. An assortment of the behaviors recounted above—including breakdowns in appropriate communication or motor control, incomprehensible emotions and self-destructive behaviors) could activate the instinct that an individual may have a mental disorder, no matter whether that can be related back to or clarified by a local cultural model or not. All European or Western individuals, even those who are not familiar with psychiatry, can interpret the motions of a Tourette's patient as indicative of dysfunction, though they often cannot diagnose what this dysfunction might specifically be. This is also true in other cultures—a range of other atypical behaviors are recognizable as symptomatic of mental disorder, even without the ability to carry out further analysis of it. #### 8.3 An Illustration: Haslam's Model of Folk-Psychiatry Certain kinds of behavior are highlighted as representative of mental disorder in the majority of human groups—they correlate across cultures. The steps through which mental disorder is brought about are also hypothesized and locally agreed upon. Conventional ethno-psychiatry details these two stages in comprehending dysfunction (Kleinman, 1988), but may face the topic of culture in an overly 'culturalist' manner, believing culture to be an extrinsic network of representations that is considered from a theoretical point of view, separate from actual cognition (Jovanovski, 1995). Therefore, there is very little structured research into the cognitive procedures incorporated within it. One anomaly is a sequence of conceptual and experiential papers by Haslam and colleagues that propose a psychological description of Western 'folk-psychiatry' (Giosan, Glovsky, & Haslam, 2001; Haslam, 2005; Haslam & Giosan, 2002). Their model details four different ways that perceptions of behaviors may fluctuate: [1] pathologizing, or in other words, the degree to which the behavior is interpreted as atypical due to it being difficult to clarify; [2] moralizing, where the behavior is thought to be governed by the person and having a certain moral valence; [3] medicalizing, where the behavior is thought to be a direct consequence of an implicit natural condition; [4] psychologizing, where the behavior is thought to be produced mentally but not intentionally — it is the consequence of a mental dysfunction, and the cornerstone is its origin, not its reasons, with decreased moral judgment (Haslam, 2005). Haslam and colleagues also recorded significant cultural variation in the comparative significance of these measurements. While US participants are more likely to prefer an 'internal' perspective of mental dysfunction (particularly 'psychologized' internal disputes), Romanian and Brazilian participants highlight external explanations for it (Giosan et al., 2001). This model gives us a good foundation for exploring the cognitive procedures that are the basis of Western folk-understandings of mental disorder. Additionally, it gives us a guide for carrying out further experiential studies that build on conventional ethno-psychiatry—in other words, those that build on the accounts of the cognitive procedures involved in individuals considering mental disorder. In future research, it is important to appreciate that pan-specific aspects of human minds are probable authorities over cultural models. #### 8.4 ''Looping Effects'' from Models to Behaviors Ian Hacking detailed the intricate network of links between pathology, its cultural context of appearance, its typical manifestations within that context, its popular categorization, and its scholarly description in a sequence of inquiries into past 'ways of being mad' (Hacking, 1995b, 1998). A 'looping effect' exists, where certain symptoms that have become core to scientific understanding of a condition guide people towards standard exemplifications of mental dysfunction. An instance of this phenomenon comes from Western psychiatry, when conditions such as female hysteria, long-lasting fugue states, and multiplepersonality disorder were recognized in the research and thereafter spread throughout the culture (Hacking, 1995a). This examination of looping effects adds to the wealth of research focusing on historical and cultural procedures within the displays and models of dysfunction (see, for example, Porter, 1987 #7829 and Porter, 2004). We can assist our comprehension of these feedback loops by looking at feed-forward links between the procedures discussed above. Hacking's statements about ideas of mental dysfunction actually constitute a broad evaluation of 'epidemiological' models of cultural transmission. The reality of a highly diffused representation within a certain group helps us to foresee the ways in which it might be transmitted in the future. For example, there is a 'frequency bias', in which it is probable that individuals will acquire and pass on representations that are already popular (Boyd & Richerson, 1985). Instances of disordered behavior overtly noted by others as being dysfunctional have a high probability all else being equal—of being more salient and more memorable than other atypical behaviors. Just as in other areas of cultural transmission, behaviors that match up with an established pattern are much more likely to be understood and recalled (Bartlett, 1932), where alternative types of atypical or surprising behavior might be brushed off as behavioral 'noise'. Contemporary cognitive anthropology recounts the looping effect of popular ideas and norms in the area of 'race' concepts (Hirschfeld, 1994b), religious and supernatural beliefs (Boyer, 1994), and many more (see e.g. Hirschfeld, 1994 #4583). # 9. Conclusion Our models of behavioral dysfunction originate from culture, in the same way that narratives, scholarship, etiquette, politics, cuisine, musical traditions, and religious rituals do. Mental dispositions that make up a section of our common cognitive architecture govern these cultural formations (Sperber, 1996). This is the foundation of my argument that intuitive psychology should be considered to be the prime point of derivation of implicit presuppositions about other agents' behaviors and, as a result, a prime component in making us discern that an individual may have a mental disorder. While intuitive psychology does not describe why a behavior is atypical or the reasons behind its occurrence, it leaves a gap in which we can place a causal process that gives rise to this particular dysfunction. We may or may not fill the gap with a model of mental disorder that others in our cultural group subscribe to. Since some dysfunctions are invisible, and certain types of causal models are inherently more credible than others, intuitive psychology restricts the manner in which individuals form culturally pervasive ideas of mental disorder in two areas. One objective of this model is to supply the 'missing link' between the incidence of certain behaviors (including those ascribable to mental disorder) and pervasive cultural models of mental disorder. The majority of cross-cultural psychiatry is centered around mental disorder without considering why certain forms of dysfunction are more noticeable than others, or why some recognizable atypicality is salient but some is not, or why certain recognizable atypicality is the focus of culturally transmitted models. In contrast, the majority of ethnopsychiatric research presupposes that the cultural models supply us with a notional grid, anything external to which will not be perceived as atypical or indicative of dysfunction. However, this is not correct—there are many instances where the detection of atypical behavior cannot be traced back to a shared model. The most plausible explanation for this is that inklings of mental disorder come from manifest, repetitive, and unaccountable breaches of implicit psychological expectations. It is as yet unclear whether this proposal can clarify why such a high level of variation in individual and shared interpretations of mental disorder exists. However, it is suggested that a useful approach to further research is to involve intuitive psychology and its well-founded, intricate, early-acquired, implicit principles within our attempts to relate and comprehend the causal links between mental disorder and cultural representations. # References ——. (2002). In Gods We Trust: The Evolutionary Landscape of Religion. Oxford; New York: Oxford University Press. Mapping the Mind: Domain-Specificity in Culture and Cognition (pp. 391–411). New York: Cambridge University Press. Michotte, A. (1963). The Perception of Causality. New York: Basic Books. ——. (1996). Explaining Culture: A Naturalistic Approach. Oxford: Blackwell. # 7. The Ideal of Integrated Social Science # Introductory Note This essay starts with the question of why the discipline of cultural anthropology is marginal in public debates, when it should and might be central. (I provide data that may seem dated, but the trends described here have if anything become stronger.) The diagnosis is that this is a self-inflicted wound—and perhaps more interestingly, I try to describe how some kinds of social science do contribute to public discourse. But this is not intended as a series of recommendations for anthropologists. To understand why, we must keep in mind a simple distinction between disciplines and intellectual projects. Disciplines are associated with university departments, teaching appointments, professional associations, etc. There is a discipline of anthropology, in that sense, in the same way as chemistry or biology. Intellectual projects are about a set of questions and methods. One example of such a project is the idea of explaining the diversity of human cultures in the context of the unity of human motivations and mental capacities. This was a central project for many (not all) professional anthropologists of the twentieth century. But the project of course existed long before that, in the works of Montesquieu or Ibn Khaldun, and many others before and after them. So the idea of explaining cultures in terms of human nature pre-existed the profession of anthropology, and persists, nowadays, largely outside professional anthropology, being pursued by people labeled biologists, linguists or economists, as well as historians in some cases. This evolution is not uncommon. Projects can migrate into or out of disciplines. The idea of constructing mathematical models for genetic evolution was first handled by professional mathematicians like Fisher, and only gradually became central to the discipline of biology. The arrival of some projects and departure of others is the reason why most academic disciplines, like the ship of Theseus, are incrementally modified to such an extent that in some cases nothing remains of the original set of ideas or methods. In this essay, I try to describe the separation between professional anthropology (the discipline) and the goal of explaining the diversity of human cultures in terms of our common human nature (the project). This does not entail that actual anthropologists should abandon their current pursuits and join my favorite project—although I of course wish my tribe will increase and prosper. No, the only negative comment on the discipline of (cultural) anthropology is that it tends to create its own intellectual isolation. What matters, then, are the projects. At the end of the chapter, I sketch a version of a research program that was advocated and implemented by many before me—a cognitive explanation of human cultures that is based on evolutionary principles (Boyd & Richerson, 1985; Sperber, 1985; Tooby & Cosmides, 1992). I described the main achievements of that research program in some detail elsewhere (Boyer, 2018). Just as they crisscross or transcend disciplines, intellectual projects also ignore such common divisions as that between the sciences and the humanities, or Natur- and Geisteswissenschaften, which are descendants of those highly misleading and highly persistent distinctions between nature and culture, innate and acquired traits, etc. These segregation principles do not make much sense, as social sciences continue to become closely integrated, gradually realizing the ideal of consilience described by E.O. Wilson (1998). # References # Modes of Scholarship in the Study of Culture1 Why is it that the majority of cultural anthropology is no longer relevant? The debates within this specific field are generally absent from wider academic conversations, its scholars no longer rank amongst the most renowned and significant intellectuals of their day, and its contribution to non-academic discourse is basically nonexistent. This third aspect is even more alarming, given that the actual subject matter of cultural anthropology situates it at the core of pressing social issues. Although I will qualify this stern appraisal, the aim of the present chapter is to investigate the causes and to propose a possible solution for (rather than to lament) the current status of cultural anthropology. My suggestion is that this condition is in large part self-inflected. Cultural anthropology lacks any function in wider discourse, since many cultural anthropologists have spoken and written themselves out of popular debates. This situation is on the verge of changing, although this change is occurring at the margins (rather than the mainstream) of the discipline. I will begin by emphasizing that there is a considerable amount of reputable, and indeed, brilliant research in cultural anthropology this is hardly in question. What is, however, of concern is a particular academic style (which entered the field of cultural anthropology relatively recently, but has dominated other fields for much longer) that has curtailed the creative vitality and social relevance of the discipline. It is also evident that by no means all anthropology scholarship is <sup>1</sup> Some of the contents of this chapter have been expressed earlier in Boyer, P. (2003). Science, Erudition and Relevant Connections, Journal of Cognition and Culture, 3(4): 344–358. irrelevant: biological anthropology and archaeology are both alive and well. It is also worth noting that evolutionary biologists and economists are currently rejuvenating the established concerns of cultural anthropology in the public consciousness, which indicates the potential for a 'science of culture' field, or some emergent shift towards an integrated discipline of this sort. # 1. Public Decline Let us consider questions of public debate, such as the organization of marriage, gender and familial relations, the formation of social trust and cooperative norms, the outcomes of mass immigration, the impact of global cultural contact, the functions of religious persuasion, the links between civil society and religious institutions, or processes of ethical dispute. A whole range of disciplines—from history to evolutionary biology, and from neuroscience to economics—have much to contribute on all of these topics, but cultural anthropology is, for the most part, too readily introspective and concerned with obscure academic fads. This is not merely an opinion. A brief scan of references to cultural anthropologists and anthropological themes within popular debates corroborates the field's declining relevance. For example, Richard Posner's painstaking study, Public Intellectuals, which lists prominent contributors to public debates (in books, magazines, newspapers, and journals) over the last twenty years in the United States, is instructive (Posner, 2001). Somewhat remarkably, in a list of 416 public intellectuals, only five are anthropologists, and four of these five (Margaret Mead, Ruth Benedict, Claude Lévi-Strauss, Ernest Gellner) are no longer alive. One could be forgiven for assuming that Posner prefers pundits to specialists, and politics to broader social debates, but this would be wrong. The study lists educational psychologists Jerome Bruner and Howard, psychologist and linguist Steven Pinker, literary critic and moral philosopher Tzvetan Todorov, philosopher Robert Nozick, and economist Thomas Sowell. It is worth observing that, save for Mead, the five renowned anthropologists listed are quite detached from the relativist, 'textual' trends of contemporary cultural anthropology. Why this stark lack of influence? It is possible that cultural anthropology's recent propensity for academic fads is responsible for its declining relevance. Treatises on culture as text, postcolonialism, or more arcane and reflexive topics likely are not of much use to those concerned with matters of serious public debate, such as how nontraditional family units will raise children, how mass immigration might result in harmonious co-existence, or how we might overcome religious hatred. 'Mission creep' is the process by which a finite strategic goal snowballs into an excessively ambitious project, and is greatly feared by members of the military and certain politicians. Over the last fifty years, cultural anthropology has encountered the inverse issue, which we might term a dramatic 'mission shrink'. In contrast to its original scope and what is often referred to in textbooks as its 'mission', the focus of cultural anthropology has gradually waned to a few minor problems. Anthropology's official mission over the last century, as emphasized in most textbooks on the subject, has been to understand human nature through the lens of the most challenging and typical features of the species, specifically, the production of vastly different norms, concepts, and social structures. Interestingly, however, nobody working in cultural anthropology pays much attention to these questions, and the majority of cultural anthropologists in fact consider such an approach to be either outdated or audacious. They have for the most part renounced the 'nature' aspect of human nature and cultural diversity. Instead of confronting so-called 'big' questions, the majority of cultural anthropologists gladly confine themselves to geographically specific, narrowly defined analyses. What it worse is that this shift took place at exactly the moment when other fields began to produce many methods and results that could, when paired with cultural anthropological scholarship, revive our understanding of human cultures. Cultural anthropology has, far from embracing such advances, seemingly severed ties with other fields that could aid this progress (even the related fields of biological anthropology and archaeology). It has also doggedly ignored dramatic breakthroughs in the fields of psychology, economics, linguistics, and cognitive science. # 2. Modes of Scholarship—Scientific and Erudite What prompted this shift? I have a provisional diagnosis for this state of affairs that demands us to consider what I term modes of scholarship. These are the means by which we distinguish scholarly works from one another and acknowledge them as legitimate contributions to a given field, or recognize their authors as genuine members of the academy. In the present inquiry, the question is: how do scholars of cultural anthropology reach a decision on whether an individual may be awarded a position as a cultural anthropologist, or on whether their publications constitute valid contributions to the field? The humanities-science binary is far too general and simplistic for a comprehension of the present situation. Instead, there are three different modes of scholarship: science, erudition, and salient connections. #### 2.1 The Science Mode The science mode should not take too long to describe. This is not because scientific authority and authoritativeness are simple matters far from it. Philosophy of science is difficult precisely because it is not easy to explain what this particular mode of scholarship consists of and what really makes it different from (and vastly more successful than) all other ways of gathering knowledge (Klee, 1999). This does not matter for present purposes, however, because the scientific mode, if difficult to explain, is very easy to recognize. You know it when you see it. Here is a short list of the common 'symptoms' by which we recognize a field that employs the science mode of scholarship: a. There is an agreed corpus of knowledge. What has been achieved so far is taken as given by most practitioners. The common corpus also includes a set of recognized methods, and a list of outstanding questions and puzzles to solve. People also tend to agree on which of these questions are important and which only require some puzzle solving and some tidying up of the theoretical landscape. b. The fundamentals of the discipline and its results are explained in textbooks and manuals that are all extraordinarily similar, as the essential points and the way to get there are agreed in the discipline. c. It does not really matter who said what or when. Indeed, many practitioners have a rather hazy picture of the history of their disciplines. Many young biologists would have a hard time explaining what the New Synthesis was, who was involved, and why a synthesis was needed in the first place. Revered figures from the past may be a source of inspiration, demonstrating how to make great discoveries, but they are not a source of truth. Darwin believed in continuous rather than particulate heredity and in some transmission of acquired traits—on both counts we think he was simply wrong, great man though he was (Mayr, 1991). d. People typically publish short contributions. They do not need to establish why the specific problem addressed is a problem or why the methods are appropriate, since that is all part of the agreed background. e. The typical biographical pattern is that the aspiring member of the guild is intensively trained from an early age in the specialized field and makes important contributions after only a few years of training. f. There is a large degree of agreement (because of the various features already mentioned) on whether a given person meets the requirements for being a practitioner of the particular field, and there is also a large agreement on how important each individual's contribution is. Again, let me emphasize that this is by no means a description of science, but only of the scientific mode of scholarship, identified here on the basis of fairly superficial but sufficient criteria. By the same token, I am not claiming that all 'scientists' work in that way (more on that later) or that 'science' only occurs when these features apply. The point of all this is to draw a contrast with other modes of scholarship, where legitimacy and standards are established quite differently. #### 2.2 The Erudition Mode Another mode of scholarship is erudition, understood as the requirement that specialists of the discipline should have detailed knowledge of a particular domain of facts. Consider, for instance, Byzantine numismatics or the history of Late Renaissance painting. We expect specialists of these fields to have knowledge of the corpus of coins or paintings. We turn to them to identify new findings. The erudition mode was essential to (and still plays a great part in) the development of many scientific fields. For instance biology started as natural history and still includes a large part of it. The features of erudition are partly similar and partly different from those of science, as we can see by listing some of erudition's key features: a. There is an agreed corpus of knowledge. There is also a large agreement on what remains to be done. For instance, only a small part of the extant corpus of Mesopotamian tablets has been deciphered. A great number of languages remain to describe. So the remaining tablets or languages are offered to the aspiring specialist as a possible domain of study. b. A great deal of knowledge is not made explicit in manuals. One picks it up by working under the tutelage of more experienced practitioners and immersing oneself in the material for many years. c. The history of the field matters and practitioners generally know it. There are some great masters, whose intuitions matter a lot, although they may have been wrong. For instance, to this day classical scholars know their Bachofen or Straus, religious scholars cite Otto or Eliade. But these are not considered infallible sources. d. People often publish short descriptive contributions, e.g., the first description of a new insect genus or the phonology of a specific language. They also compile monographs that incorporate vast amounts of information about a particular domain (e.g., the comparative morphology of ant species, an encyclopedia of New-Guinean languages, a concordance of Ben Jonson's plays, a catalogue raisonne of Guido Reni). e. Age is a necessary component of competence. Older experts are generally better, because expertise consists in the accumulation of vast amounts of specific facts, also because an expert needs the kind of intuition that is only shaped by long-lasting familiarity with the material. Only a seasoned Renaissance scholar can tell you that this particular painting is from the Venetian not the Milanese school. A younger scholar may be misled by superficial features. f. Within a narrow field, people agree on whether a given individual is competent or not, generally based on that person's knowledge of a monograph-sized subfield. Now, as I said earlier, there is nothing essential about these distinct modes—indeed, as we shall see, they are often found in combination, and this may be an index of 'healthy' disciplines. Also, whether a given field uses more or less of one of these modes can change with time. Technical change can have dramatic effects on the mix of modes. Classics used to be strongly based on erudition in the corpus. Knowing obscure (but relevant) textual sources was a sine qua non, and the outcome of many years of sustained training, the way it still is for, say scholars of Indian philosophy. Now that the entire Greek and Latin canon is available (and searchable) on CD-ROM, this particular form of knowledge cannot be used as a criterion for admission. # 3. How Science and Erudition Combine In healthy empirical disciplines, the science and erudition modes very often co-exist harmoniously. Two illustrative examples are biology and linguistics. Today, molecular biologists principally employ the scientific mode. Conversely, evolutionary biologists often have a defined 'field' of research (for instance social organization amongst wasps, or lekking in antelopes), therefore necessitating a combination of both scientific and erudition modes. The two are not mutually exclusive, and certain fields, such as ecology, often demand scientific knowledge (such as how to apply optimal foraging models, how to run simulations, or awareness of epidemiological techniques) alongside erudite knowledge (such as the ways in which different species interact, the predators or prey of a certain genus, or the minimal density of resources required). Often, a productive information exchange between these two modes can take place. Natural history and evolutionary theory inform one another. E. O. Wilson is simultaneously one of the most significant evolutionary theorists of the last century and one of the world experts on ant behavior, to give just one example (Hölldobler & Wilson, 1990; Wilson, 1975). Linguistics nowadays also combines these two modes in multiple ways, according to the particular sub-field. Whilst certain linguists exclusively work in the science mode (for instance, exploring which formal models might account for linguistic regularity), others employ a more field-oriented approach (for instance investigating Amazonian languages), and many others marry the two approaches. Erudite comparisons of creoles and pidgins have for example inspired certain scientific models of linguistic evolution (Bickerton, 1990). Whilst we may observe these two modes within a single field, or even within the scholarly approach of a single person, their purposes and the manner in which they are applied nonetheless remain distinct. Biologists and linguists rely on empirical evidence for a proposed theory, as well as generating the relevant evidence, through experimentation or selection from a corpus, for example testing the notion that all languages have a noun-verb distinction by analyzing a large number of separate grammar systems. The erudition mode is driven by description, rather than by hypotheses or explanations. The aspirant scholar must catalogue the many forms of a particular genus of orchid, or the various coins found in a certain Byzantine palace because the given genus or collection has not previously been taxonomized. A 'pure' or 'a-theoretical' description does not exist, and particular hypotheses about what is or is not deemed to be relevant are usually established in a given discipline's existing descriptive methods. It is crucial that the distinction between different modes of scholarship should not be conflated with the other (in my opinion) extremely confusing distinction between academic fields belonging to the humanities, the sciences or the social sciences. This institutional distinction operates on a different axis to the modal distinction. Examples of the erudition mode abound in the sciences, and there are also a fair few instances of the scientific mode found in the humanities. In Humanities fields, scholars may for instance be working on a catalogue raisonné of a particular painter, a documentation of Greek coins (erudition), while others study how ecology constrains state formation or how visual perception influences aesthetics (science). In the social sciences, we find projects such as a study of comparative forms of nationalism (erudition) and formal models of cooperation and trade (science). In the so-called STEM fields, one could map the geological formations of England (erudition) while others study the physics of plate-tectonics (science). As I mentioned, erudition and science projects overlap. But the distinction between these styles of scholarship clearly cuts across the familiar humanities/sciences division. # 4. A Third Mode of Scholarship: Salient Connections The third mode of scholarship is the most elusive one, as it has not been systematically described, yet it is also most important to our understanding of many modern disciplines, including cultural anthropology. In this mode, people assess new contributions in terms of the connections they establish between facts or ideas which, by themselves, are not necessarily novel or even interesting. Although this way of judging new work has been around for a long time, it has become characteristic of many academic fields of a recent vintage and of the recent evolution of older disciplines. I call this the 'salient connections' mode. Again, I should provide examples before a model, because this is a phenomenon, we all know when we see it, even if we do not always reflect on the mechanism at work. For instance, a recent book reframes the discourse of love in Shakespeare's plays and sonnets as an expression of the colonial outlook. The lover's loving gaze transparently expresses the conqueror's prospect on a recently discovered, clearly gendered, and mythically virginal New World. A student is planning to work on Indian public executions during the Raj as a form of theater, a ritualized performance that constructs colonial power at the same time as it undermines it by exhibiting the gossamer of its dramatic texture. Another colleague has recently finished a study of gay fathers in the Caribbean in the framework of Benjamin's and Bourdieu's accounts of culture, technology, and late capitalism. Steel drums and strong rum prop up the local habitus of globalized self-empowerment. What is the common thread in these disparate examples? They all seem to offer a new connection between elements that were previously known to everyone in the field and indeed, in many cases, to any educated reader. For instance, all literary scholars presumably know their Shakespeare and educated folk know a little about the conquest of America. But they (supposedly) had never considered Ophelia as American. In the same way, most historians know about the political organization of the Raj and its fondness for state pageantry. They are also cognizant of the 'comedian's paradox' from Diderot or some other source. The author's hope is in the fact that the connection between the two—between state ceremonial and precarious theatrical mimesis—is new. In the same way, most cultural anthropologists have some notion of the Caribbean as a place of contrasting influences and original cultural mixes. They also know a little about the various ways in which homosexuality is construed in different places, as well as cultural variation in fathers' duties or roles. The innovative point is to put all these together, creating salient associations, especially by throwing in Bourdieu and Benjamin—two rather dour, bookish, and strait-laced dead Europeans who would seem far removed from your typical Trinidadian gay dad. One could multiply the examples, but it may be of more help to compare the features of this with the other two modes: a. In salient-connections fields, there is no agreed corpus of knowledge. Indeed, there is no 'knowledge' in the sense of accumulated and organized information, but rather a juxtaposition of different views on different topics. b. There are no manuals, no agreed techniques or methods. Indeed, each contribution constitutes (ideally) a new paradigm or method, each author is an island. c. The history of the field, its self-definition, as well as the reframing of past theories, are crucial. A lot of scholarly activity in salient connections-based fields consists in citing various masters, commenting on their texts, finding some connection between what they said and the issue at hand. In cultural anthropological studies, authors like Walter Benjamin or Pierre Bourdieu or the entire Frankfurt school are part of this Pantheon (a very ephemeral one, with a high turnover rate). The masters are generally invoked as validating authority. That is, the particular fact that one is describing (the gay Caribbean father, etc.) is presented as illustrating the general principle laid down by Benjamin or some other luminary. (Incidentally, these authors are never shown to have been wrong. Indeed, their work is never discussed as having any connection to empirical fact that could make them right or wrong. Benjamin's or Bourdieu's conceptions of culture are not judged in terms of how much they explain). Also, there is a great deal of emphasis on the self-definition of the field, the ideas various practitioners have about what they do and what they ought to do, compared to what others do. Indeed, most important works are supposed to be not just contributions to the field, but also reflections on the field itself. For instance, a study of German post-Expressionist 1960s cinema will be praised, not just because it tells us a lot that we want to know about that specific genre, but also because it reframes our views of the connections between cinema or society. A study of recent rock songs is good because it establishes a new approach to popular culture. d. Books are more important than articles. This, in part, reflects the fact that each contribution should ideally reframe a field as a whole, introduce a new way of looking at issues, and so on, something that cannot be done in a short article. e. There is no specific developmental curve. Some authors produce interesting connections in their first piece of work, others are seasoned specialists of the erudition mode who, at some point, decide to let their hair down, as it were, and let salient connections govern their next project. f. There is no agreement whatsoever on who a competent performer in this mode is, apart from the (generally dead) masters like Bakhtin or Benjamin or Raymond Williams for cultural studies, Derrida or de Man for literary criticism. A consequence is that there are tightly coalitional cliques and exceedingly bitter feuds about who should get what jobs, who is allowed to publish and where, and so on. In the last three decades or so, some fields have dramatically evolved from almost pure erudition mode to the salient-connections mode. Literary criticism is a case in point. In the past, one could not really expatiate on Shakespeare's plays without thorough knowledge of the First Folio and Quartos and other such recondite source criticism. This kind of erudition is still practiced, but it is not the major criterion of a relevant contribution to Elizabethan studies (Garber, 2004). Saying something new about the plays is what matters. One could say that the specialists have (perhaps excessively) taken to heart Forster's dictum. They only connect. There are various accounts of why this happened to literary studies, whether this is a Good Thing or not, and if not, whether it is all the fault of that awful Leavis or of the dreaded French structuralists (Kermode, 1983). I am not enough of an erudite to adjudicate between these normative interpretations of history. I can only comment that polemical narratives generally get in the way of a proper explanation. Neither jeremiad ('No-one knows the Canon anymore!') nor triumphalist epic ('We have overcome! The Canon is dead') is of great help here. # 5. Effects of Salient Connections The particular mode of scholarship I have described above could be explained by some as merely the outcome of a specific framework of ideas. For example, readers of earlier drafts of this essay highlighted similarities to postmodernist thought. This comparison is flawed, since the mode of scholarship I outline extends well beyond a certain intellectual trend (Gellner, 1992). Furthermore, and perhaps more significantly, assuming that an individual's actions (in this instance, the means by which academics validate scholarly contributions or acknowledge new academics) may be adequately accounted for by their own explanation of their rationale (in this instance, a certain intellectual trend). Such trends are no more intelligible than other social tendencies, and consequently we should also seek to explain them. The results of the salient connections mode of scholarship are of greater concern than its sources, and are quite easily observed. For people with the correct grounding, the connections are salient, but they do not translate easily. Imagine explaining to a biochemist that the essays of Walter Benjamin provide an excellent context for a description of gay fathers in Trinidad. There is, understandably, a somewhat limited audience for salient connections, and these often pose a challenge even for scholars of a given field. Ernest Gellner mocked the pitiful Wittgensteinian philosophers propagating the idea that linguistic issues lay at the heart of any epistemological or metaphysical philosophy problems. They generally ended up teaching pupils who had never been particularly interested in philosophical problems per se, whether epistemological or not, and who consequently took on this idea with calm impassivity (Gellner, 1959). David Lodge has also drawn on the ample comic material in such a setup, through fictional professors forced to teach poorly-read students unfamiliar with the concept of the Canon that the margin is text, or the Canon is dead (Lodge, 1988). An arguably more pressing concern is that writing like this does not solve any issues. It does not seek to generate a more accurate explanation of the world, nor even to highlight the boundaries of our knowledge. Salient connections ultimately are not sturdy or flexible knowledge. So what can we do about it? # 6. Integrated Study of Cultures—An Incipient Program Integrated scholarship is the basis of the most promising breakthroughs in comprehending human behavior. By 'integrated', I mean explanatory models that move beyond established oppositions between 'levels' or 'domains' of reality (Bechtel, 1993), so in this instance I have in mind 'culture' rather than human psychology, genetics, or economics. I also have in mind models that are steadfastly adaptable in using any available explanatory tools, irrespective of the specific disciplinary context from which those tools have originated. There is now much greater potential for an integrated study of human culture, thanks to rapid progress on the three fundamental fronts of human cognition, economic models of behavior, and evolutionary biology. Contemporary findings in all three of these arenas are already changing perspectives on the study of culture: One may regard the spread of cultural representations, concepts, and norms as forms whose limits are dictated by human cognitive abilities (Sperber & Hirschfeld, 2004). As evolutionary anthropologists and cognitive scientists have shown, cognitive principles that are developed early on form a framework of expectations that enable the acquisition of specific cultural concepts and norms (Boyer & Barrett, 2005) in a diverse array of domains, from folk-biology (Atran, 1990, 1998), to kinship and ethnic categories (Hirschfeld, 1994, 1996), to racial categories (Kurzban, Tooby, & Cosmides, 2001), to religious beliefs (Atran, 2002), and social interaction (Barkow, Cosmides, & Tooby, 1992; Fiske, 1992; Tooby & Cosmides, 1996). Economic theory gives us the most accurate tools for describing opportunities and predicting options, and these tools are undoubtedly applicable beyond the bounds of exclusively economic issues (Gintis, 2000a). In particular, experimental and behavioral economics have demonstrated how we might move pas strict rationality assumptions (Smith, 2003), and how we might incorporate factors such as reputation (Sperber & Baumard, 2012) punitive feelings (Fehr, Schmidt, Kolm, & Ythier, 2006; Price, Cosmides, & Tooby, 2002) and intuitive standards of fairness (McCabe & Smith, 2001) into economic models. These models are responsible for the dissemination modes of cooperation specific to a given culture (Gintis, 2000b). If we do not situate human culture within an evolutionary context, we cannot provide a thorough account of it. Evolution in humans (and other species) generates decision-making processes that are extremely context-dependent, meaning that environmental and social aspects may dictate the limits of an individual's personal preferences. An evolutionary framework can give a useful explanation of a wide range of cultural phenomena, e.g., reproductive strategies including teenage pregnancies (Ellis et al., 2003; Quinlan, 2003), different responses or uniform objections to cheating in social exchange—in both forager and industrial societies (Sugiyama, Tooby, & Cosmides, 2002); local particularities of 'race' categories (Kurzban et al., 2001; Sidanius & Veniegas, 2000); and many others (Barkow et al., 1992; Buss & Kenrick, 1998). # 7. Back to What Matters A vast domain is open to cultural anthropological investigation, provided that the practitioners accept substantive re-tooling and discard old fetishes. If slogans are needed, an integrated study of culture should proclaim the great values of reductionism, the ambition to understand the causal processes underpinning behaviors; opportunism, the use of whatever tools and findings get us closer to that goal; and revisionism, a deliberate indifference to disciplinary creeds and traditions. The integrated view of human culture—what some may call a 'vertical integration' in the field—will allow cultural anthropology to return to the highly ambitious set of questions it should have addressed all along. For the sake of illustration, here is a far from exhaustive list of such questions: The list is not exhaustive but it is indicative, at least, of the potential scope and diversity of a vertically integrated approach to cultural anthropology. The list should also suggest why an integrated program is a Good Thing: because it finally allows cultural anthropology to talk about things that matter. Cultural anthropology is simply not heard in the public forum, and the simplest explanation is that it is not talking or rather, not talking about anything of great importance. This should change soon. # References # Chapter 3 Fig. 4. A simplified model of action ritualization in cultural rituals. Boxes identity different functional systems in the same way as in Fig. 3 . Participants in rituals are provided with two kinds of information, (a) statements about potential danger and (b) scripted recipes for ritual action, that activate the securitymotivation systems. Rules for ritual performance result in both goal-demotion and low-level action-parsing with the resulting swamping of working memory. These processes result in highly attention-demanding and compelling performance of rigidly scripted actions. This in turn makes the associations more salient, which should make subsequent messages about ritual more intuitively compelling. (Figure design by P Boyer, 2006). 95 # Chapter 4 Fig. 1. Schematic representation of functional processes involved in the adjustment of the coalitional safety index (left) and examples of such processes (right). The model describes how attention to social information, for instance, about people's behaviors indicating affiliation, leads to inferences of social threat and social support. These inferences modulate the coalitional safety index, which has two main consequences. First, it changes motivations concerning action plans, for instance, an effort to remain within one's group, to avoid others, or to boost solidarity in one's own group. Second, lowering the coalitional safety index triggers a stress response, which can have adverse long-term consequences. (Figure design by P Boyer. 2015). 126 # Chapter 5 # Chapter 6 - impact of 257–259 123, 130, 155–157, 159–170, 173–174, game theory 9 health, public 113, 130, 132–133, 135, 141–142 immigration 118, 125, 130–133, 136–137, 139, 159–160, 165, 185–186, 197, 202–203, 258–259 folk beliefs about 118, 125, 186 insanity 2, 217, 221, 232, 233, 238. See also dysfunction, mental cultural models of 221, 241–242, 244 - neo-institutional models of 13, 16, 18–19, 29, 40 naturalism 17 OCD, obsessive-compulsive disorder 49–50, 53–56, 58–59, 61–67, 71, 75–78, 81, 83, 85–86, 88–89, 97, 99, 275 profit, perception of 166 - coalitional 30–32, 118–121, 125, 140, 142, 160, 184–186, 199 - evolutionary 8, 17, 114, 118 - intuitive 170, 200–201, 218–219, 221– 222, 224–225, 227, 234–239, 244, 277 - precautionary 53–54, 74, 77–78, 86, 88–90, 93–98, 121, 124, 142 - effects on health 1, 8, 113, 124, 130–133, 139, 141–142 cultural 51, 53–55, 59–62, 90, 93–97, 99–100, 276 stigmatization 27, 117, 131, 133, 270 effects on health 117, 131–133 stress 117, 119, 124, 126, 128–132, 134, 138–139, 141–142, 165, 276 physiology of 113, 124, 128, 131 threat, coalitional 118, 123–124 # About the Team Alessandra Tosi was the managing editor for this book. Melissa Purkiss performed the copy-editing and proofreading. Anna Gatti designed the cover. The cover was produced in InDesign using the Fontin font. Luca Baffa typeset the book in InDesign and produced the paperback and hardback editions. The text font is Tex Gyre Pagella; the heading font is Californian FB. Luca produced the EPUB, MOBI, PDF, HTML, and XML editions — the conversion is performed with open source software freely available on our GitHub page (https://github.com/ OpenBookPublishers). # This book need not end here… # Share All our books — including the one you have just read — are free to access online so that students, researchers and members of the public who can't afford a printed edition will have access to the same ideas. This title will be accessed online by hundreds of readers each month across the globe: why not share the link so that someone you know is one of them? This book and additional content is available at: https://doi.org/10.11647/OBP.0257 # Customise Personalise your copy of this book or design new books using OBP and thirdparty material. 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Printed and digital editions, together with supplementary digital material, can also be found at http://www.openbookpublishers.com Photo by Marc-Olivier Jodoin on Unsplash at https://unsplash.com/photos/-TQUERQGUZ8 Cover Design by Anna Gatti www.openbookpublishers.com Pascal Boyer Cognitive Anthropology Essays in Evolutionary HUMAN CULTURES THROUGH THE SCIENTIFIC LENS	doab	2025-04-07T03:56:57.561690	5-8-2021 06:01	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "0004666f-919b-4eaa-ac9b-a02a0e3c589b", "url": "https://library.oapen.org/bitstream/20.500.12657/50315/1/9781800642089.pdf", "author": "Boyer, Pascal", "title": "Human Cultures through the Scientific Lens", "publisher": "Open Book Publishers", "isbn": "", "section_idx": 0 }
0004847e-ab80-435d-9a35-50f7f1b13b00.0	Edited by Gorazd Drevenšek The book provides chapters on sex hormones and their modulation in neurodegenerative processes and pathologies, from basic molecular mechanisms, physiology, gender differences, to neuroprotection and clinical aspects for potential novel pharmacotherapy approaches. The book contains 14 chapters written by authors from various biomedical professions, from basic researchers in biology and physiology to medicine and veterinary medicine, pharmacologists, psychiatrist, etc. Chapters sum up the past and current knowledge on sex hormones, representing original new insights into their role in brain functioning, mental disorders and neurodegenerative diseases. The book is written for a broad range of audience, from biomedical students to highly profiled medical specialists and biomedical researchers, helping them to expand their knowledge on sex hormones in neurodegenerative processes and opening new questions for further investigation. ISBN 978-1-78923-014-7 Sex Hormones in Neurodegenerative Processes and Diseases Published in London, UK © 2018 IntechOpen © nnorozoff / iStock	doab	2025-04-07T03:56:57.613510	1-12-2023 17:26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0004847e-ab80-435d-9a35-50f7f1b13b00", "url": "https://mts.intechopen.com/storage/books/5994/authors_book/authors_book.pdf", "author": "", "title": "Sex Hormones in Neurodegenerative Processes and Diseases", "publisher": "IntechOpen", "isbn": "9781789230154", "section_idx": 0 }
0004847e-ab80-435d-9a35-50f7f1b13b00.1	Sex Hormones in Neurodegenerative Processes and Diseases Edited by Gorazd Drevenšek SEX HORMONES IN PROCESSES AND Edited by Gorazd Drevenšek DISEASES NEURODEGENERATIVE	doab	2025-04-07T03:56:57.613732	1-12-2023 17:26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0004847e-ab80-435d-9a35-50f7f1b13b00", "url": "https://mts.intechopen.com/storage/books/5994/authors_book/authors_book.pdf", "author": "", "title": "Sex Hormones in Neurodegenerative Processes and Diseases", "publisher": "IntechOpen", "isbn": "9781789230154", "section_idx": 1 }
0004847e-ab80-435d-9a35-50f7f1b13b00.2	SEX HORMONES IN NEURODEGENERATIVE PROCESSES AND DISEASES Edited by Gorazd Drevenšek #### Sex Hormones in Neurodegenerative Processes and Diseases http://dx.doi.org/10.5772/66585 Edited by Gorazd Drevenšek #### Contributors Dong-Dong Ren, Zi-Yu He, Ramon Sotomayor, Jonathan Martinez, Rodrigo Castillo, Jose Hamilton Nascimento, Fernando A. C. Seara, Rodrigo Soares Fortunato, Denise Pires Carvalho, Slavi Dimitrov Delchev, Katerina Georgieva, Vianey Rodriguez-Lara, Maria Rosa Avila-Costa, Ana Luisa Gutiérrez-Valdez, Verónica Anaya-Martínez, José Luis Ordóñez-Librado, Javier Sanchez-Betancourt, Enrique Montiel-Flores, Leonardo Reynoso-Erazo, Rocio Tron-Alvarez, Patricia Aley-Medina, Jesus Espinosa-Villanueva, Cesar Sanchez-Vazquez Del Mercado, Tracey Quinn, David Walker, Claire Flaherty, Arghavan Sadeghi Zangeneh, Marissa A. Harrison, Sanjana Marikunte, Ksenija Gersak, Ziva Miriam Gersak, Arijana Turčin, Wafik Said Bahnasy, Yasser ElHeneedy, Ehab El- Seidy, Mushap Kuru, Hasan Oral, Abdulsamed Kükürt, Metin Öğün, Tomoya Kataoka, Kazunori Kimura, Katalin Prokai-Tatrai, Manuela Cristina Russu, Alexandra Cristina Antonescu #### © The Editor(s) and the Author(s) 2018 The rights of the editor(s) and the author(s) have been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights to the book as a whole are reserved by INTECHOPEN LIMITED. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECHOPEN LIMITED's written permission. Enquiries concerning the use of the book should be directed to INTECHOPEN LIMITED rights and permissions department ([email protected]). Violations are liable to prosecution under the governing Copyright Law. Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be foundat http://www.intechopen.com/copyright-policy.html. #### Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. First published in London, United Kingdom, 2018 by IntechOpen eBook (PDF) Published by IntechOpen, 2019 IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, The Shard, 25th floor, 32 London Bridge Street London, SE19SG – United Kingdom Printed in Croatia British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Additional hard and PDF copies can be obtained from [email protected] Sex Hormones in Neurodegenerative Processes and Diseases Edited by Gorazd Drevenšek p. cm. Print ISBN 978-1-78923-014-7 Online ISBN 978-1-78923-015-4 eBook (PDF) ISBN 978-1-83881-265-2	doab	2025-04-07T03:56:57.613772	1-12-2023 17:26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0004847e-ab80-435d-9a35-50f7f1b13b00", "url": "https://mts.intechopen.com/storage/books/5994/authors_book/authors_book.pdf", "author": "", "title": "Sex Hormones in Neurodegenerative Processes and Diseases", "publisher": "IntechOpen", "isbn": "9781789230154", "section_idx": 2 }
0004847e-ab80-435d-9a35-50f7f1b13b00.3	We are IntechOpen, the first native scientific publisher of Open Access books 3,400+ Open access books available 109,000+ International authors and editors 115M+ Downloads 151 Countries delivered to Our authors are among the Top 1% most cited scientists 12.2% Contributors from top 500 universities Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI)	doab	2025-04-07T03:56:57.614022	1-12-2023 17:26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0004847e-ab80-435d-9a35-50f7f1b13b00", "url": "https://mts.intechopen.com/storage/books/5994/authors_book/authors_book.pdf", "author": "", "title": "Sex Hormones in Neurodegenerative Processes and Diseases", "publisher": "IntechOpen", "isbn": "9781789230154", "section_idx": 3 }
0004847e-ab80-435d-9a35-50f7f1b13b00.4	Interested in publishing with us? Contact [email protected] Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com	doab	2025-04-07T03:56:57.614084	1-12-2023 17:26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0004847e-ab80-435d-9a35-50f7f1b13b00", "url": "https://mts.intechopen.com/storage/books/5994/authors_book/authors_book.pdf", "author": "", "title": "Sex Hormones in Neurodegenerative Processes and Diseases", "publisher": "IntechOpen", "isbn": "9781789230154", "section_idx": 4 }
0004847e-ab80-435d-9a35-50f7f1b13b00.5	Meet the editor Gorazd Drevenšek holds an MSc degree in Pharmacology and a PhD degree in Medical Sciences, both from the University of Ljubljana, Faculty of Medicine. He started his research in cardiovascular pharmacology, modelling ischemic and reperfusion injuries and atherosclerotic processes. His focus is on pharmacological and toxicological evaluation of natural compounds as potential therapeutic agents with cardio- and neuro-protectant potential. His laboratory and research skills comprise methods used with isolated organs, in vivo animal pharmacology and human studies. His present engagement is with psychopharmacology-oriented research. Dr. Drevenšek teaches Psychopharmacology, Psychopharmacology of Mental Disorders and Molecular Basis of Neurodegenerative Diseases, for students of Biopsychology at the University of Primorska in Koper, Slovenia, where he is involved in neuroscience research, coupling psychopharmacology to EEG and autonomic nervous system-based studies. At the Faculty of Medicine at the University of Ljubljana, he is heading the Laboratory for Cardiovascular Pharmacology. Contents Preface VII Chapter 1 Cellular and Molecular Mechanisms of the Effects of Sex Hormones on the Nervous System 1 Slavi Delchev and Katerina Georgieva Chapter 3 Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Roles in Chapter 4 Gender Differences in Frontotemporal Lobar Degeneration Chapter 5 Reproductive Aging: Perimenopause and Psychopathological Ksenija Gersak, Ziva Miriam Gersak and Arijana Turcin Manuela Cristina Russu and Alexandra Cristina Antonescu Wafik Said Bahnasy, Yasser A. El-Heneedy and Ehab A. El-Seidy Chapter 6 Neuroprotection in Perimenopausal Women 127 Chapter 7 Sex Hormones and Alzheimer's Disease 145 Tracey A. Quinn, Stephen R. Robinson and David Walker (FTLD) Support an Estrogenic Model of Delayed Onset 69 Claire V. Flaherty, Arghavan S. Zangeneh, Marissa A. Harrison and Chapter 2 17β-Estradiol as a Neuroprotective Agent 21 Katalin Prokai-Tatrai and Laszlo Prokai Brain Function and Disease 41 Sanjana Marikunte Symptoms 95	doab	2025-04-07T03:56:57.614117	1-12-2023 17:26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0004847e-ab80-435d-9a35-50f7f1b13b00", "url": "https://mts.intechopen.com/storage/books/5994/authors_book/authors_book.pdf", "author": "", "title": "Sex Hormones in Neurodegenerative Processes and Diseases", "publisher": "IntechOpen", "isbn": "9781789230154", "section_idx": 5 }
0004847e-ab80-435d-9a35-50f7f1b13b00.6	Contents #### Preface XI Chapter 7 Sex Hormones and Alzheimer's Disease 145 Wafik Said Bahnasy, Yasser A. El-Heneedy and Ehab A. El-Seidy Preface very few drugs available. reduce excitotoxicity. leading drug. Neurodegenerative diseases represent one of the main global health burdens and one of the public health priorities in the Western world. Due to population ageing, this health problem around the globe is increasing. Aetiology of neurodegenerative diseases is unclear, and ther‐ apeutic approaches are very limited and individually diverse. Therapy is a subject of con‐ stant modification during progression of these diseases, and pharmacotherapy is limited to Neurodegenerative diseases are diversely affecting men and women during their lifetime. One of the differences between male and female population appears due to sex hormone alterations that affect initiation of neurodegenerative processes. Not enough studies are fo‐ cused on sex hormones in neurodegenerative diseases. Their potential therapeutic role in neurodegenerative processes has been largely neglected in comparison to other therapeutic research. Non-genomic action of sex hormones, their protective role and their modulatory effects on neural tissues are becoming more important with epidemiologic data on the sexrelated differences. Also, sex hormones acting as neuroactive steroids show pleiotropic ef‐ fects that enable neuroprotection, neuroplasticity, neuron survival and regeneration and Potential new therapeutic approaches based on sex hormone effects are described in the present publication. As such, 17-beta-estradiol, DHT, pregnenolone and DHEA can be consid‐ ered as neurosteroids, enabling neuroprotection also against neurodegenerative processes. The book starts with a chapter describing the molecular mechanisms of sex hormones in the nervous system, dividing them into "classical", i.e., intracellular or genomic effects, and non-classical or non-genomic membrane receptor effects. Some steroids are capable of in‐ ducing rapid neurotransmitter-like effects, similar to those of dopamine or glutamate that alter the activity of the neuronal systems via different receptors. Sex hormones according to their pleiotropic effects can be considered neuro-protectants and anti-excitotoxicity agents. As such, 17β-estradiol is a neurosteroid agent in the central nervous system that shows ben‐ eficial effects against many critical steps in neurodegeneration, from initiation to the pro‐ gression of neuronal cell death. 17β-estradiol can protect the vulnerable neurons of the central nervous system as an anti-inflammatory and antioxidant agent. A translational effort that represents its powerful neuroprotective potential is appropriate for clinical setting as a neurotherapeutic drug that ensures therapeutic efficacy and clinical safety. In chapter two, authors studied 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED) as potential therapeutic Chapter 11 Testosterone and Erectile Function: A Review of Evidence from Basic Research 257 Tomoya Kataoka and Kazunori Kimura ## Preface Chapter 8 Differences Between Intact and Ovariectomized Cytological Alterations 171 Addiction 207 VI Contents Sotomayor-Zárate Basic Research 257 Neuroprotection 273 in Ruminants 303 Chapter 14 Sex Hormones and Inner Ear 329 Zi-Yu He and Dong-Dong Ren Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and L-DOPA/Melatonin Coadministration on Motor Behavior and Betancourt, Enrique Montiel-Flores, Leonardo Reynoso-Erazo, Rocio Tron-Alvarez, Patricia Aley-Medina, Jesús Espinosa-Villanueva, Cesar Fernando de Azevedo Cruz Seara, Rodrigo Soares Fortunato, Denise Ana Luisa Gutiérrez-Valdez, Vianey Rodríguez-Lara, Verónica Anaya-Martínez, José Luis Ordóñez-Librado, Javier Sanchez- Sanchez-Vazquez del Mercado and María Rosa Avila-Costa Jonathan Martínez Pinto, Rodrigo L. Castillo and Ramón Chapter 10 Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative Disorders 225 Pires Carvalho and José Hamilton Matheus Nascimento Chapter 11 Testosterone and Erectile Function: A Review of Evidence from Chapter 12 New Insights for Hormone Therapy in Perimenopausal Women Chapter 13 Clinical Use of Progesterone and Its Relation to Oxidative Stress Manuela Cristina Russu and Alexandra Cristina Antonescu Mushap Kuru, Abdulsamed Kükürt, Hasan Oral and Metin Öğün Tomoya Kataoka and Kazunori Kimura Chapter 9 Sex Hormones: Role in Neurodegenerative Diseases and Neurodegenerative diseases represent one of the main global health burdens and one of the public health priorities in the Western world. Due to population ageing, this health problem around the globe is increasing. Aetiology of neurodegenerative diseases is unclear, and ther‐ apeutic approaches are very limited and individually diverse. Therapy is a subject of con‐ stant modification during progression of these diseases, and pharmacotherapy is limited to very few drugs available. Neurodegenerative diseases are diversely affecting men and women during their lifetime. One of the differences between male and female population appears due to sex hormone alterations that affect initiation of neurodegenerative processes. Not enough studies are fo‐ cused on sex hormones in neurodegenerative diseases. Their potential therapeutic role in neurodegenerative processes has been largely neglected in comparison to other therapeutic research. Non-genomic action of sex hormones, their protective role and their modulatory effects on neural tissues are becoming more important with epidemiologic data on the sexrelated differences. Also, sex hormones acting as neuroactive steroids show pleiotropic ef‐ fects that enable neuroprotection, neuroplasticity, neuron survival and regeneration and reduce excitotoxicity. Potential new therapeutic approaches based on sex hormone effects are described in the present publication. As such, 17-beta-estradiol, DHT, pregnenolone and DHEA can be consid‐ ered as neurosteroids, enabling neuroprotection also against neurodegenerative processes. The book starts with a chapter describing the molecular mechanisms of sex hormones in the nervous system, dividing them into "classical", i.e., intracellular or genomic effects, and non-classical or non-genomic membrane receptor effects. Some steroids are capable of in‐ ducing rapid neurotransmitter-like effects, similar to those of dopamine or glutamate that alter the activity of the neuronal systems via different receptors. Sex hormones according to their pleiotropic effects can be considered neuro-protectants and anti-excitotoxicity agents. As such, 17β-estradiol is a neurosteroid agent in the central nervous system that shows ben‐ eficial effects against many critical steps in neurodegeneration, from initiation to the pro‐ gression of neuronal cell death. 17β-estradiol can protect the vulnerable neurons of the central nervous system as an anti-inflammatory and antioxidant agent. A translational effort that represents its powerful neuroprotective potential is appropriate for clinical setting as a neurotherapeutic drug that ensures therapeutic efficacy and clinical safety. In chapter two, authors studied 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED) as potential therapeutic leading drug. In chapter three it is discussed that other potential neuroactive steroids, dehydroepiandros‐ terone (DHEA) and its sulphated metabolite DHEA-sulphate (DHEA(S)), are potent modu‐ lators of neurogenesis, neuronal growth and differentiation, and neuroprotection. Its serum concentrations decrease with age, with lowest concentrations at the time of onset of neuro‐ degenerative processes. Pathology expressed as cognitive decline, age-related neurological disorders and dementia and others may, in part at least, be attributed to decreased secretion of DHEA that acts probably through diminished glutamate-induced excitotoxicity. functional dopaminergic cells, which in turn imply a well-preserved neuropil of a less de‐ Preface IX Brain-modulated body homeostasis and behaviours such as motivation, reward, memory and movement control are complex processes, regulated also by dopaminergic neurons, which can be stimulated by several triggers throughout the life. The homeostasis issue is discussed in detail in chapter nine. For example, early exposure to sex hormones or endo‐ crine disruptors during critical period of neuronal development affects dopaminergic path‐ ways permanently for later life, producing some potential subjugation to mental disorders such as drug addiction. Present knowledge on neurodegeneration in Parkinson's and Alz‐ In chapter ten authors present how despite their therapeutic benefits, anabolic steroid abuse has spread for improvements on physical appearance and performance. The illicit use of anabolic AS has been correlated with several adverse effects, such as cardiovascular, endo‐ crine, reproductive and neurobehavioral dysfunctions. Declines on cognitive and mnemonic performance have been demonstrated due to steroid abuse. These neurological dysfunctions are correlated to neuronal apoptosis in the hippocampus and cortex as well as in induced neurodegeneration. It is considered to be a prognostic factor for neurodegenerative diseases. Pathophysiological mechanisms that have been linked to the anabolic steroid-induced neu‐ rotoxicity are redox imbalance and pro-apoptotic pathways. Abuse of anabolic steroids is Androgens are essential for male physical activity and normal erectile function, a topic pre‐ sented in chapter eleven. Age-related testosterone deficiency is considered a risk factor for erectile dysfunction. Testosterone affects nitric oxide production and phosphodiesterase type 5 (PDE-5) expression that preserves smooth muscle contractility by regulating both contraction and relaxation. Interestingly, testosterone deficiency is related to neurological diseases that lead to erectile dysfunction. Testosterone replacement therapy is widely used to treat patients with testosterone deficiency. PDE-5 inhibitors, L-citrulline and/or resvera‐ trol therapy might be effective therapeutic options for testosterone deficiency-induced erec‐ In the following chapter, chapter twelve, hormone replacement therapy in women that may enable delayed onset of neuro-ageing and neurodegenerative diseases is described. Such therapy during perimenopause is potential protectant against Alzheimer's disease. Oestro‐ gens, progesterone and androgens, with their connections to cholinergic, GABAergic, sero‐ tonergic and glutamatergic signalling, are involved in women's brain functioning through a variety of mechanisms. These agents can modulate/protect plasticity, glucose and ATP me‐ tabolism, ketones, insulin resistance and inflammation of the ageing brain through bloodbrain-barrier disruption, microglial aberrant activation and neural cell survival/loss. Endocrine, neural and metabolic pathways are giving new insights into the sequential view of Aβ in AD pathogenesis. In perimenopause, prevention and treatment can maintain wom‐ Among other sex hormones, progesterone, a steroid hormone secreted by the corpus lu‐ teum and formed after ovulation, is described for its practical use in veterinary medicine in chapter thirteen. Progesterone maintains the continuity of pregnancy. Progestogens can be used also for oestrus synchronization in farm animals, i.e., cows and heifers. Similarly, they are used for oestrus synchronization during the breeding season or outside the breeding heimer's diseases exposed neuroprotective effects of estradiol. also a potential risk factor for the development of Alzheimer's disease. nervated striatum. tile dysfunction. en's neurological health. Chapter four presents how oestrogenic pharmacotherapy can be applied in many neurode‐ generative disorders. Oestrogens delay the onset of frontotemporal dementia (FTLD) in pre‐ menopausal women compared to age-equivalent men and may provide neuroprotection in the early postmenopausal period. Oestrogens possess regulatory role in attenuating the mi‐ croglia hyperactivation in response to cell stress that might trigger an overexpressed inflam‐ matory response. Acting as microglia stabilizer, oestrogens preserve the homeostasis of both the ubiquitin-proteasome degradation system and lysosome-autophagy recycling system. Crucial period for triggering the potential degenerative changes in women is perimeno‐ pause. It is defined by menstrual cycle and endocrine changes in ovarian-pituitary-hypo‐ thalamic feedback relationships, inaccurate oestrogen levels and decreased progesterone levels. These changes in women are most commonly experienced as mild cognitive impair‐ ment, anxiety, irritability, mood swings and depression. In chapter five it is discussed how oestrogens and other shifted hormones influence all these changes including depression and depressive-like behaviour through interactions with neurotrophic factors and through an in‐ fluence on the serotonergic system. Chapter six shows how in perimenopausal period, endocrine and neural degenerative changes overlap, and thus perimenopause is a "critical period" in neuro-ageing, when the neurodegenerative processes may initiate. In Alzheimer's disease, metabolic and inflamma‐ tory changes are characterized, with onset during menopausal transition and early years of menopause. Endocrine, neural and metabolic pathways due to endocrine changes are pre‐ senting new insights into the Aβ-centric AD pathogenesis. The following chapter presents the link between Sex Hormones and Alzheimer's Disease . Alzheimer's disease is one of the most studied neurodegenerative diseases characterized by brain pathological changes, i.e., amyloid plaque accumulation and neurofibrillary tangle deposition, synaptic loss, neuronal death and brain atrophy in later stages. These changes result in progressive memory and cognitive decline. Oestrogens, progesterone and androgens are important for AD pathogen‐ esis, and their effect to the brain results in gender susceptibility to the disease. Sex hormones play an important neuroprotective role against AD development, mild cognitive impair‐ ment and AD progression. Hormonal replacement therapy in AD treatment may represent a new strategy for the development of personalized, gender-specific AD management. In chapter eight authors show that higher incidence of Parkinson's disease is present in post‐ menopausal compared to premenopausal women of similar age, suggesting that oestrogens possess neuroprotective effects. Melatonin alone or in combination with L-DOPA protects nigrostriatal dopaminergic loss induced by 6-OHDA in a rat Parkinson's disease model and improves motor ability and biological alterations, compared with the results of L-DOPA-on‐ ly-treated rats. Rats treated with L-DOPA and melatonin showed decreased dyskinesia and showed better performance at motoric tests. L-DOPA and melatonin co-administration in oestrogen native animals resulted in reduced dyskinesia through the conservation of some functional dopaminergic cells, which in turn imply a well-preserved neuropil of a less de‐ nervated striatum. In chapter three it is discussed that other potential neuroactive steroids, dehydroepiandros‐ terone (DHEA) and its sulphated metabolite DHEA-sulphate (DHEA(S)), are potent modu‐ lators of neurogenesis, neuronal growth and differentiation, and neuroprotection. Its serum concentrations decrease with age, with lowest concentrations at the time of onset of neuro‐ degenerative processes. Pathology expressed as cognitive decline, age-related neurological disorders and dementia and others may, in part at least, be attributed to decreased secretion Chapter four presents how oestrogenic pharmacotherapy can be applied in many neurode‐ generative disorders. Oestrogens delay the onset of frontotemporal dementia (FTLD) in pre‐ menopausal women compared to age-equivalent men and may provide neuroprotection in the early postmenopausal period. Oestrogens possess regulatory role in attenuating the mi‐ croglia hyperactivation in response to cell stress that might trigger an overexpressed inflam‐ matory response. Acting as microglia stabilizer, oestrogens preserve the homeostasis of both the ubiquitin-proteasome degradation system and lysosome-autophagy recycling system. Crucial period for triggering the potential degenerative changes in women is perimeno‐ pause. It is defined by menstrual cycle and endocrine changes in ovarian-pituitary-hypo‐ thalamic feedback relationships, inaccurate oestrogen levels and decreased progesterone levels. These changes in women are most commonly experienced as mild cognitive impair‐ ment, anxiety, irritability, mood swings and depression. In chapter five it is discussed how oestrogens and other shifted hormones influence all these changes including depression and depressive-like behaviour through interactions with neurotrophic factors and through an in‐ Chapter six shows how in perimenopausal period, endocrine and neural degenerative changes overlap, and thus perimenopause is a "critical period" in neuro-ageing, when the neurodegenerative processes may initiate. In Alzheimer's disease, metabolic and inflamma‐ tory changes are characterized, with onset during menopausal transition and early years of menopause. Endocrine, neural and metabolic pathways due to endocrine changes are pre‐ senting new insights into the Aβ-centric AD pathogenesis. The following chapter presents the link between Sex Hormones and Alzheimer's Disease . Alzheimer's disease is one of the most studied neurodegenerative diseases characterized by brain pathological changes, i.e., amyloid plaque accumulation and neurofibrillary tangle deposition, synaptic loss, neuronal death and brain atrophy in later stages. These changes result in progressive memory and cognitive decline. Oestrogens, progesterone and androgens are important for AD pathogen‐ esis, and their effect to the brain results in gender susceptibility to the disease. Sex hormones play an important neuroprotective role against AD development, mild cognitive impair‐ ment and AD progression. Hormonal replacement therapy in AD treatment may represent a new strategy for the development of personalized, gender-specific AD management. In chapter eight authors show that higher incidence of Parkinson's disease is present in post‐ menopausal compared to premenopausal women of similar age, suggesting that oestrogens possess neuroprotective effects. Melatonin alone or in combination with L-DOPA protects nigrostriatal dopaminergic loss induced by 6-OHDA in a rat Parkinson's disease model and improves motor ability and biological alterations, compared with the results of L-DOPA-on‐ ly-treated rats. Rats treated with L-DOPA and melatonin showed decreased dyskinesia and showed better performance at motoric tests. L-DOPA and melatonin co-administration in oestrogen native animals resulted in reduced dyskinesia through the conservation of some of DHEA that acts probably through diminished glutamate-induced excitotoxicity. fluence on the serotonergic system. VIII Preface Brain-modulated body homeostasis and behaviours such as motivation, reward, memory and movement control are complex processes, regulated also by dopaminergic neurons, which can be stimulated by several triggers throughout the life. The homeostasis issue is discussed in detail in chapter nine. For example, early exposure to sex hormones or endo‐ crine disruptors during critical period of neuronal development affects dopaminergic path‐ ways permanently for later life, producing some potential subjugation to mental disorders such as drug addiction. Present knowledge on neurodegeneration in Parkinson's and Alz‐ heimer's diseases exposed neuroprotective effects of estradiol. In chapter ten authors present how despite their therapeutic benefits, anabolic steroid abuse has spread for improvements on physical appearance and performance. The illicit use of anabolic AS has been correlated with several adverse effects, such as cardiovascular, endo‐ crine, reproductive and neurobehavioral dysfunctions. Declines on cognitive and mnemonic performance have been demonstrated due to steroid abuse. These neurological dysfunctions are correlated to neuronal apoptosis in the hippocampus and cortex as well as in induced neurodegeneration. It is considered to be a prognostic factor for neurodegenerative diseases. Pathophysiological mechanisms that have been linked to the anabolic steroid-induced neu‐ rotoxicity are redox imbalance and pro-apoptotic pathways. Abuse of anabolic steroids is also a potential risk factor for the development of Alzheimer's disease. Androgens are essential for male physical activity and normal erectile function, a topic pre‐ sented in chapter eleven. Age-related testosterone deficiency is considered a risk factor for erectile dysfunction. Testosterone affects nitric oxide production and phosphodiesterase type 5 (PDE-5) expression that preserves smooth muscle contractility by regulating both contraction and relaxation. Interestingly, testosterone deficiency is related to neurological diseases that lead to erectile dysfunction. Testosterone replacement therapy is widely used to treat patients with testosterone deficiency. PDE-5 inhibitors, L-citrulline and/or resvera‐ trol therapy might be effective therapeutic options for testosterone deficiency-induced erec‐ tile dysfunction. In the following chapter, chapter twelve, hormone replacement therapy in women that may enable delayed onset of neuro-ageing and neurodegenerative diseases is described. Such therapy during perimenopause is potential protectant against Alzheimer's disease. Oestro‐ gens, progesterone and androgens, with their connections to cholinergic, GABAergic, sero‐ tonergic and glutamatergic signalling, are involved in women's brain functioning through a variety of mechanisms. These agents can modulate/protect plasticity, glucose and ATP me‐ tabolism, ketones, insulin resistance and inflammation of the ageing brain through bloodbrain-barrier disruption, microglial aberrant activation and neural cell survival/loss. Endocrine, neural and metabolic pathways are giving new insights into the sequential view of Aβ in AD pathogenesis. In perimenopause, prevention and treatment can maintain wom‐ en's neurological health. Among other sex hormones, progesterone, a steroid hormone secreted by the corpus lu‐ teum and formed after ovulation, is described for its practical use in veterinary medicine in chapter thirteen. Progesterone maintains the continuity of pregnancy. Progestogens can be used also for oestrus synchronization in farm animals, i.e., cows and heifers. Similarly, they are used for oestrus synchronization during the breeding season or outside the breeding season in small ruminants. Progesterone treatment contributes to the resolution of the anoestrus by rearranging hypothalamic functions in cattle with follicular cysts. In addition, lactation can be successfully induced in cows with a combination of oestrogen and proges‐ terone for 7 or 10 days. The oxidative stress index in the luteal phase, when progesterone is high in ruminants, is higher than in the follicular phase. The last chapter describes the sex hormones modulation in the inner ear. Interactions between sex hormones and the structure and function of the inner ear are important especially in hear‐ ing impairment and balance disorders. Innovative treatments on hearing loss, tinnitus, au‐ tophony and dizziness resulted from the changes in oestrogen and progesterone levels. The presence of oestrogen receptors α and β has earlier been shown in the inner ear of mice and rats, where oestrogen receptor expression correlates with the protection of auditory function. Evidence for the treatment of sex hormone-induced symptoms is principally restricted to case reports and retrospective studies. Recognition and understanding of sex hormone-related in‐ ner ear problems will allow otologists to notice and better manage these patients. The chapters in the book provide an overview of the past and current knowledge on the role of sex hormones in brain functioning, mental disorders and neurodegenerative diseases. The book is interesting for highly profiled clinicians and biomedical researchers, clearly describ‐ ing new knowledge and therapeutic potential for the sex hormones based therapies. The book will help them expand their knowledge on modulation of sex hormones in neurodegenerative processes and diseases, thus opening new questions for readers' further investigations. > Gorazd Drevenšek University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia University of Primorska, FAMNIT, Koper, Slovenia Chapter 1 Provisional chapter Cellular and Molecular Mechanisms of the Effects of The mechanisms of the action of sex steroid hormones on the nervous system are related to both classical, intracellularly mediated effects and non-classical membrane effects due to binding to membrane receptors. Some steroids are capable of inducing rapid neurotransmitter-like effects, similar to those of dopamine or glutamate that alter the activity of neuronal systems via different types of receptors. The neuroactive steroids are endogenous neuromodulators synthesized in the brain and rapidly affecting neuronal excitability. Sex steroids exert many pleiotropic effects in the nervous system: they modulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination, and influence cognitive processes. Estradiol protects neurons from excitotoxic damage and increases neuronal survival. Progesterone stimulates neurological and functional recovery. Androgens also exhibit a wide array of neuroprotective effects in motoneurons, including supporting cell survival, axonal regeneration, and dendritic maintenance. Despite the considerable increase of sex hormones and neurosteroids research in recent years and the ongoing discovery of biochemical mechanisms of action, their role in neurodegenerative processes remains not well determined. neuroprotection, neurodegenerative diseases Keywords: sex hormones, neurosteroids, genomic effects, non-genomic effects, Cellular and Molecular Mechanisms of the Effects of DOI: 10.5772/intechopen.71140 © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. and reproduction in any medium, provided the original work is properly cited. Sex hormones are synthesized from cholesterol mainly in the gonads and adrenal cortex. In the brain, different sex steroids can also be further metabolized to different neurosteroids or be produced anew in neurons and glial cells, with an even more potent effect on the nervous system. The mechanisms of action of the sex steroid hormones on the brain are related to both classical, intracellularly mediated effects and non-classical (non-genomic) membrane effects Sex Hormones on the Nervous System Sex Hormones on the Nervous System Slavi Delchev and Katerina Georgieva Slavi Delchev and Katerina Georgieva http://dx.doi.org/10.5772/intechopen.71140 Abstract 1. Introduction Additional information is available at the end of the chapter Additional information is available at the end of the chapter Provisional chapter #### Cellular and Molecular Mechanisms of the Effects of Sex Hormones on the Nervous System Sex Hormones on the Nervous System Cellular and Molecular Mechanisms of the Effects of DOI: 10.5772/intechopen.71140 Slavi Delchev and Katerina Georgieva Slavi Delchev and Katerina Georgieva Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.71140 #### Abstract season in small ruminants. Progesterone treatment contributes to the resolution of the anoestrus by rearranging hypothalamic functions in cattle with follicular cysts. In addition, lactation can be successfully induced in cows with a combination of oestrogen and proges‐ terone for 7 or 10 days. The oxidative stress index in the luteal phase, when progesterone is The last chapter describes the sex hormones modulation in the inner ear. Interactions between sex hormones and the structure and function of the inner ear are important especially in hear‐ ing impairment and balance disorders. Innovative treatments on hearing loss, tinnitus, au‐ tophony and dizziness resulted from the changes in oestrogen and progesterone levels. The presence of oestrogen receptors α and β has earlier been shown in the inner ear of mice and rats, where oestrogen receptor expression correlates with the protection of auditory function. Evidence for the treatment of sex hormone-induced symptoms is principally restricted to case reports and retrospective studies. Recognition and understanding of sex hormone-related in‐ The chapters in the book provide an overview of the past and current knowledge on the role of sex hormones in brain functioning, mental disorders and neurodegenerative diseases. The book is interesting for highly profiled clinicians and biomedical researchers, clearly describ‐ ing new knowledge and therapeutic potential for the sex hormones based therapies. The book will help them expand their knowledge on modulation of sex hormones in neurodegenerative processes and diseases, thus opening new questions for readers' further investigations. University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia University of Primorska, FAMNIT, Koper, Slovenia Gorazd Drevenšek ner ear problems will allow otologists to notice and better manage these patients. high in ruminants, is higher than in the follicular phase. X Preface The mechanisms of the action of sex steroid hormones on the nervous system are related to both classical, intracellularly mediated effects and non-classical membrane effects due to binding to membrane receptors. Some steroids are capable of inducing rapid neurotransmitter-like effects, similar to those of dopamine or glutamate that alter the activity of neuronal systems via different types of receptors. The neuroactive steroids are endogenous neuromodulators synthesized in the brain and rapidly affecting neuronal excitability. Sex steroids exert many pleiotropic effects in the nervous system: they modulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination, and influence cognitive processes. Estradiol protects neurons from excitotoxic damage and increases neuronal survival. Progesterone stimulates neurological and functional recovery. Androgens also exhibit a wide array of neuroprotective effects in motoneurons, including supporting cell survival, axonal regeneration, and dendritic maintenance. Despite the considerable increase of sex hormones and neurosteroids research in recent years and the ongoing discovery of biochemical mechanisms of action, their role in neurodegenerative processes remains not well determined. Keywords: sex hormones, neurosteroids, genomic effects, non-genomic effects, neuroprotection, neurodegenerative diseases #### 1. Introduction Sex hormones are synthesized from cholesterol mainly in the gonads and adrenal cortex. In the brain, different sex steroids can also be further metabolized to different neurosteroids or be produced anew in neurons and glial cells, with an even more potent effect on the nervous system. The mechanisms of action of the sex steroid hormones on the brain are related to both classical, intracellularly mediated effects and non-classical (non-genomic) membrane effects Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons due to their binding to membrane receptors. Some steroids are capable of inducing rapid neurotransmitter-like effects. Sex steroids exert diverse pleiotropic effects on the nervous system: they modulate major neurotransmitter systems, promote the viability of neurons, play an important role in myelination, and influence cognitive processes. Estradiol increases neuronal survival and recovery. It protects neurons from excitotoxic damage, amyloid β (Aβ) toxicity, oxidative stress, and glucose deprivation. The defense induced by estrogens is mediated by complex mechanisms. Progestins have also been found to exert neuroprotective effects similar to those of estrogens. Androgens exhibit a wide range of neuroprotective effects in motoneurons, including supporting cell survival, axonal regeneration, and dendritic maintenance. The relationship between sex steroids and the brain-derived neurotrophic factor (BDNF) has garnered a growing interest due to the role BDNF plays in the pathogenesis of neurodegenerative diseases. #### 2. Steroidogenesis Sex hormones are steroid compounds synthesized from cholesterol mainly in the testes, ovaries, and adrenal cortex. The male sex hormones (androgens) and female sex hormones (estrogens and gestagens) have a common biosynthetic pathway (Figure 1). The final product of the steroidogenesis of sex hormones depends on whether or not specific metabolizing enzymes are available in the respective cell [1]. The sex steroids in human blood include androgens (testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, and dihydrotestosterone), estrogens (estradiol, estriol, and estrone), and gestagens (progesterone and 17α-hydroxyprogesterone). The major male hormone, testosterone, is produced by the Leydig cells in the testes. Dihydrotestosterone (DHT) is a potent androgen, derived from testosterone by the enzyme 5α-reductase (type 1 and type 2) in some peripheral tissues, mediating some testosterone-induced effects. This enzyme is expressed in the skin, scalp, prostate, epididymis, liver, and nervous system (neocortex, subcortical white matter, and hippocampal tissues) [2]. DHEA, DHEAS, and androstenedione are secreted mainly by the adrenal cortex in the same amounts in both sexes. DHEA and androstenedione are steroids involved in the sex hormones' biosynthesis pathway; both are primary endogenous precursors of testosterone and estrogens. Although they are weak androgens, they are circulating steroids that can be converted into active androgens and estrogens in the peripheral tissues [1, 3]. expressed in steroidogenic tissues, the brain, and nonsteroidogenic tissues, especially fat and bone. Progesterone is the major progestogen and is produced in both theca and granulosa Figure 1. Sex steroid biosynthesis pathway. Enzymes are shown as follows: (1) P450 side-chain cleavage enzyme; (2) 3β-hydroxysteroid dehydrogenase; (3) 17β-hydroxysteroid dehydrogenase; (4) 5α-reductase; (5) aromatase. The dashed arrow indicates poor flux. Not all intermediate steroids, pathways, and enzymes are included (modified from Cellular and Molecular Mechanisms of the Effects of Sex Hormones on the Nervous System http://dx.doi.org/10.5772/intechopen.71140 3 The neuroactive steroids are brain-synthesized endogenous neuromodulators that rapidly alter neuronal excitability. Some of them reach the brain from adrenals and gonads and are further metabolized locally just like the aromatization of testosterone into estradiol [4]. They have been referred to as neurosteroids as they can be derived anew from cholesterol in neurons and the glial cells [5]. The synthesis of neuroactive steroids requires the translocation of cholesterol across the mitochondrial membrane [6]. This process occurs through a molecular complex formed by the translocator protein 18 kDa (TSPO), the steroidogenic acute regulatory protein (StAR), the voltage-dependent anion channel protein (VDAC), and the adenine In the mitochondria, cholesterol is converted into pregnenolone by the P450 side-chain cleavage enzyme (P450scc). Soluble pregnenolone diffuses into the cytosol (the endoplasmic reticulum) where it is further metabolized into various neuroactive steroids such as progesterone, cells, the adrenal gland, and testes [1, 3]. Refs. [1, 55]). nucleotide transporter protein (ANT). Estrogens are produced by aromatization of androgens, including those derived from adrenal steroidogenesis. Although the ovaries produce large amounts of androgens, they secrete little of these into the blood, while the rest are aromatized to estradiol, which is the major estrogen. The theca cells in the ovaries synthesize testosterone and androstenedione, which then diffuse into the granulosa cells of the follicles. There androstenedione is converted into testosterone, which in turn is aromatized to estradiol that enters the blood stream. A portion of the androstenedione is aromatized to estrone, which in turn is converted into estradiol. Androgen aromatization is realized under the influence of the enzyme aromatase, which is Cellular and Molecular Mechanisms of the Effects of Sex Hormones on the Nervous System http://dx.doi.org/10.5772/intechopen.71140 3 due to their binding to membrane receptors. Some steroids are capable of inducing rapid neurotransmitter-like effects. Sex steroids exert diverse pleiotropic effects on the nervous system: they modulate major neurotransmitter systems, promote the viability of neurons, play an important role in myelination, and influence cognitive processes. Estradiol increases neuronal survival and recovery. It protects neurons from excitotoxic damage, amyloid β (Aβ) toxicity, oxidative stress, and glucose deprivation. The defense induced by estrogens is mediated by complex mechanisms. Progestins have also been found to exert neuroprotective effects similar to those of estrogens. Androgens exhibit a wide range of neuroprotective effects in motoneurons, including supporting cell survival, axonal regeneration, and dendritic maintenance. The relationship between sex steroids and the brain-derived neurotrophic factor (BDNF) has garnered a growing interest due to the role BDNF plays in the pathogenesis of neurodegen- Sex hormones are steroid compounds synthesized from cholesterol mainly in the testes, ovaries, and adrenal cortex. The male sex hormones (androgens) and female sex hormones (estro- The final product of the steroidogenesis of sex hormones depends on whether or not specific metabolizing enzymes are available in the respective cell [1]. The sex steroids in human blood include androgens (testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, and dihydrotestosterone), estrogens (estradiol, estriol, and estrone), and gestagens (progesterone and 17α-hydroxyprogesterone). The major male hormone, testosterone, is produced by the Leydig cells in the testes. Dihydrotestosterone (DHT) is a potent androgen, derived from testosterone by the enzyme 5α-reductase (type 1 and type 2) in some peripheral tissues, mediating some testosterone-induced effects. This enzyme is expressed in the skin, scalp, prostate, epididymis, liver, and nervous system (neocortex, subcortical white matter, and hippocampal tissues) [2]. DHEA, DHEAS, and androstenedione are secreted mainly by the adrenal cortex in the same amounts in both sexes. DHEA and androstenedione are steroids involved in the sex hormones' biosynthesis pathway; both are primary endogenous precursors of testosterone and estrogens. Although they are weak androgens, they are circulating steroids that can be converted into active androgens Estrogens are produced by aromatization of androgens, including those derived from adrenal steroidogenesis. Although the ovaries produce large amounts of androgens, they secrete little of these into the blood, while the rest are aromatized to estradiol, which is the major estrogen. The theca cells in the ovaries synthesize testosterone and androstenedione, which then diffuse into the granulosa cells of the follicles. There androstenedione is converted into testosterone, which in turn is aromatized to estradiol that enters the blood stream. A portion of the androstenedione is aromatized to estrone, which in turn is converted into estradiol. Androgen aromatization is realized under the influence of the enzyme aromatase, which is gens and gestagens) have a common biosynthetic pathway (Figure 1). and estrogens in the peripheral tissues [1, 3]. erative diseases. 2. Steroidogenesis 2 Sex Hormones in Neurodegenerative Processes and Diseases Figure 1. Sex steroid biosynthesis pathway. Enzymes are shown as follows: (1) P450 side-chain cleavage enzyme; (2) 3β-hydroxysteroid dehydrogenase; (3) 17β-hydroxysteroid dehydrogenase; (4) 5α-reductase; (5) aromatase. The dashed arrow indicates poor flux. Not all intermediate steroids, pathways, and enzymes are included (modified from Refs. [1, 55]). expressed in steroidogenic tissues, the brain, and nonsteroidogenic tissues, especially fat and bone. Progesterone is the major progestogen and is produced in both theca and granulosa cells, the adrenal gland, and testes [1, 3]. The neuroactive steroids are brain-synthesized endogenous neuromodulators that rapidly alter neuronal excitability. Some of them reach the brain from adrenals and gonads and are further metabolized locally just like the aromatization of testosterone into estradiol [4]. They have been referred to as neurosteroids as they can be derived anew from cholesterol in neurons and the glial cells [5]. The synthesis of neuroactive steroids requires the translocation of cholesterol across the mitochondrial membrane [6]. This process occurs through a molecular complex formed by the translocator protein 18 kDa (TSPO), the steroidogenic acute regulatory protein (StAR), the voltage-dependent anion channel protein (VDAC), and the adenine nucleotide transporter protein (ANT). In the mitochondria, cholesterol is converted into pregnenolone by the P450 side-chain cleavage enzyme (P450scc). Soluble pregnenolone diffuses into the cytosol (the endoplasmic reticulum) where it is further metabolized into various neuroactive steroids such as progesterone, 5α-dihydroprogesterone, DHEA, androstenedione, etc. The enzyme 3β-hydroxysteroid dehydrogenase, required for further conversion of pregnenolone into progesterone, has been found in the brain [7]. The enzyme 17β-hydroxysteroid dehydrogenase type 10 catalyzes the oxidation of neuroactive steroids in mitochondria with NAD<sup>+</sup> as the coenzyme. This enzyme catalyzes most effectively the oxidation of allopregnanolone and allotetrahydrodeoxycorticosterone, which is essential for the homeostasis of these neuroactive steroids [8]. (AR), resulting in conformational change of the receptor and translocation of the androgen/ AR complex from the cytosol to the nucleus. Various AR coregulators can further modulate the transcriptional regulation of target genes [11]. AR receptors are expressed in neurons and glial cells and their expression can be regulated by injury and by circulating testosterone concentration [12–14]. AR mRNA is downregulated post-orchidectomy and after axotomy [12]. AR levels also decrease with aging, especially in the nucleus basalis of Meynert (which Cellular and Molecular Mechanisms of the Effects of Sex Hormones on the Nervous System http://dx.doi.org/10.5772/intechopen.71140 5 The estrogen receptor-α (ERα) was characterized as an intracellular, ligand-regulated transcription factor located primarily in the nucleus [16]. Once bound to estradiol, ERα dimers were shown to regulate gene expression via interaction with estrogen response elements. Following a series of discoveries, a structurally related estrogen receptor-β (ERβ) was identified [17]. Sites of estrogen receptor expression identified in the brain comprised the hypothalamus, pituitary, and preoptic area, among others, which, based on a series of lesion and stimulation studies, were known to affect physiology and behavior related to endocrine function [18]. Apart from the great number of various isoforms, the classic intracellular receptors have also many splice variants that have been studied and characterized. For example, for estrogens besides the ERα and ERβ isoforms, multiple splice variants (e.g., ERαΔ4) can initiate signaling from the membrane [19]. Experiments demonstrated that the same protein is capable of mediating both intracellular and membrane actions of estradiol. For progesterone, a whole new class of progesterone receptors (PRs) has been identified—the membrane PRs localized on the membrane and involved in the reproductive actions of progesterone [20]. The classic genomic mechanism of the action of steroid hormones alone cannot account for all subsequent changes in the target cells; hence, it has been updated to include an additional (non-classic) explanation of the rapid, non-genomic, membrane-initiated action. For decades, steroid hormones have been known to induce acute changes (within minutes) in the physi- Recent research demonstrated that steroids can function in a "neurotransmitter-like" way, being synthesized at precise spatial locations within neural circuits in the brain and acting within minutes as local neuromodulators that rapidly regulate cognitive functions and behavior [24–27]. Some steroids, such as progesterone, are capable of inducing rapid neurotransmitter-like effects, similar to those of dopamine or glutamate, which alter the activity of neuronal systems via multiple types of receptors [19, 25, 28]. Some of these steroid receptors have been classified as extranuclear or membrane receptors, which signal through G-proteins or other second messenger systems [29, 30]. There is recent evidence of these classical steroid receptors binding to response elements on DNA to regulate gene expression, showing that they contain palmitoylation sequences allowing them to be trafficked to the plasma membrane to quickly alter cellular activity [19, 31]. After being trafficked, these nuclear transcription factors interact with other proteins to initiate their signaling at the level of the plasma membrane. From here, intracellular signaling cascades involving effectors (e.g., the mitogen-activated protein kinase (MAPK) and cAMP response element binding protein (CREB)) are initiated via ological functions [21], neuronal activity [22], and behavior [23]. degenerates in Alzheimer's disease (AD)) and the diagonal band of Broca [15]. 3.2. Non-genomic action Although TSPO is highly expressed in microglia and astrocytes and is less abundant in neurons, neurosteroidogenesis occurs primarily in principal neurons of several brain areas that have the necessary set of enzymes to convert cholesterol into neuroactive steroids [9]. #### 3. Mechanisms of action The first thing a hormone does is to bind to specific receptors on the target cell. Cells without receptors for the hormone do not respond to the action. The receptors for certain hormones are localized on the cell membrane, while others are located in the cytoplasm or nucleus. After binding to the specific receptor, the hormone triggers a cascade of cellular responses that become increasingly potent with each successive stage. Thus even small concentrations of the hormone can produce a significant effect [3]. #### 3.1. Genomic action via steroid receptors According to the classic genomic theory of action, sex hormones as steroid hormones bind preferentially to specific protein receptors within the cell rather than to receptors located on the cell membrane. These hormones are fat-soluble and can easily pass through the cell membrane and bind to specific receptors in the cytoplasm. Depending on the steroid and tissue, however, unbound steroid receptors may be located in the nucleus as well. The particular distribution of the receptor between the cytoplasm and nucleus varies. When the cytoplasmic receptors bind to their specific steroid hormone ligands, they translocate to the nucleus. Depending on their mechanism of action and subcellular distribution, nuclear receptors may be classified into at least two groups [10]. Nuclear receptors that bind to steroid hormones are all classified as type I receptors. Only type I receptors have a heat shock protein (HSP) associated with the inactive receptor that will be released when the receptor interacts with the ligand. Type II nuclear receptors have no HSP and in contrast to the classical type I receptor are located in the cell nucleus. The activated hormone-receptor protein complexes then bind to a specific regulatory section of DNA, called hormone response element, by activating or inhibiting the transcription of specific genes and the formation of messenger RNA. Later on, after an extended period of time (usually from a few hours to a few days) counted from the entry of the hormone into the cell, new proteins develop in the cell and alter the cell functions. The complexity of the steroid action can be accounted for by the abundance of identified steroid receptors and their affinity for the hormone. The excess/deficiency of the respective sex steroid regulates the number of the active receptors (downregulation/upregulation) in the target cells. Testosterone and DHT exert their functions via binding to the androgen receptor (AR), resulting in conformational change of the receptor and translocation of the androgen/ AR complex from the cytosol to the nucleus. Various AR coregulators can further modulate the transcriptional regulation of target genes [11]. AR receptors are expressed in neurons and glial cells and their expression can be regulated by injury and by circulating testosterone concentration [12–14]. AR mRNA is downregulated post-orchidectomy and after axotomy [12]. AR levels also decrease with aging, especially in the nucleus basalis of Meynert (which degenerates in Alzheimer's disease (AD)) and the diagonal band of Broca [15]. The estrogen receptor-α (ERα) was characterized as an intracellular, ligand-regulated transcription factor located primarily in the nucleus [16]. Once bound to estradiol, ERα dimers were shown to regulate gene expression via interaction with estrogen response elements. Following a series of discoveries, a structurally related estrogen receptor-β (ERβ) was identified [17]. Sites of estrogen receptor expression identified in the brain comprised the hypothalamus, pituitary, and preoptic area, among others, which, based on a series of lesion and stimulation studies, were known to affect physiology and behavior related to endocrine function [18]. Apart from the great number of various isoforms, the classic intracellular receptors have also many splice variants that have been studied and characterized. For example, for estrogens besides the ERα and ERβ isoforms, multiple splice variants (e.g., ERαΔ4) can initiate signaling from the membrane [19]. Experiments demonstrated that the same protein is capable of mediating both intracellular and membrane actions of estradiol. For progesterone, a whole new class of progesterone receptors (PRs) has been identified—the membrane PRs localized on the membrane and involved in the reproductive actions of progesterone [20]. #### 3.2. Non-genomic action 5α-dihydroprogesterone, DHEA, androstenedione, etc. The enzyme 3β-hydroxysteroid dehydrogenase, required for further conversion of pregnenolone into progesterone, has been found in the brain [7]. The enzyme 17β-hydroxysteroid dehydrogenase type 10 catalyzes the oxi- catalyzes most effectively the oxidation of allopregnanolone and allotetrahydrodeoxycortico- Although TSPO is highly expressed in microglia and astrocytes and is less abundant in neurons, neurosteroidogenesis occurs primarily in principal neurons of several brain areas that The first thing a hormone does is to bind to specific receptors on the target cell. Cells without receptors for the hormone do not respond to the action. The receptors for certain hormones are localized on the cell membrane, while others are located in the cytoplasm or nucleus. After binding to the specific receptor, the hormone triggers a cascade of cellular responses that become increasingly potent with each successive stage. Thus even small concentrations of the According to the classic genomic theory of action, sex hormones as steroid hormones bind preferentially to specific protein receptors within the cell rather than to receptors located on the cell membrane. These hormones are fat-soluble and can easily pass through the cell membrane and bind to specific receptors in the cytoplasm. Depending on the steroid and tissue, however, unbound steroid receptors may be located in the nucleus as well. The particular distribution of the receptor between the cytoplasm and nucleus varies. When the cytoplasmic receptors bind to their specific steroid hormone ligands, they translocate to the nucleus. Depending on their mechanism of action and subcellular distribution, nuclear receptors may be classified into at least two groups [10]. Nuclear receptors that bind to steroid hormones are all classified as type I receptors. Only type I receptors have a heat shock protein (HSP) associated with the inactive receptor that will be released when the receptor interacts with the ligand. Type II nuclear receptors have no HSP and in contrast to the classical type I receptor are located in the cell nucleus. The activated hormone-receptor protein complexes then bind to a specific regulatory section of DNA, called hormone response element, by activating or inhibiting the transcription of specific genes and the formation of messenger RNA. Later on, after an extended period of time (usually from a few hours to a few days) counted from the entry of the hormone into the cell, new proteins develop in the cell and alter the cell functions. The complexity of the steroid action can be accounted for by the abundance of identified steroid receptors and their affinity for the hormone. The excess/deficiency of the respective sex steroid regulates the number of the active receptors (downregulation/upregulation) in the target cells. Testosterone and DHT exert their functions via binding to the androgen receptor have the necessary set of enzymes to convert cholesterol into neuroactive steroids [9]. sterone, which is essential for the homeostasis of these neuroactive steroids [8]. as the coenzyme. This enzyme dation of neuroactive steroids in mitochondria with NAD<sup>+</sup> 4 Sex Hormones in Neurodegenerative Processes and Diseases 3. Mechanisms of action hormone can produce a significant effect [3]. 3.1. Genomic action via steroid receptors The classic genomic mechanism of the action of steroid hormones alone cannot account for all subsequent changes in the target cells; hence, it has been updated to include an additional (non-classic) explanation of the rapid, non-genomic, membrane-initiated action. For decades, steroid hormones have been known to induce acute changes (within minutes) in the physiological functions [21], neuronal activity [22], and behavior [23]. Recent research demonstrated that steroids can function in a "neurotransmitter-like" way, being synthesized at precise spatial locations within neural circuits in the brain and acting within minutes as local neuromodulators that rapidly regulate cognitive functions and behavior [24–27]. Some steroids, such as progesterone, are capable of inducing rapid neurotransmitter-like effects, similar to those of dopamine or glutamate, which alter the activity of neuronal systems via multiple types of receptors [19, 25, 28]. Some of these steroid receptors have been classified as extranuclear or membrane receptors, which signal through G-proteins or other second messenger systems [29, 30]. There is recent evidence of these classical steroid receptors binding to response elements on DNA to regulate gene expression, showing that they contain palmitoylation sequences allowing them to be trafficked to the plasma membrane to quickly alter cellular activity [19, 31]. After being trafficked, these nuclear transcription factors interact with other proteins to initiate their signaling at the level of the plasma membrane. From here, intracellular signaling cascades involving effectors (e.g., the mitogen-activated protein kinase (MAPK) and cAMP response element binding protein (CREB)) are initiated via the transactivation of cell surface–bound receptors, most notably the metabotropic glutamate receptors (mGluRs). Subsequently, estrogen membrane–initiated signaling can in turn activate the regulatory section of DNA and trigger transcription processes. Another mechanism of steroid action takes effect at the level of the microtubules via a proposed receptor microtubule-associated protein of type 2 (MAP2) [40]. Neuronal microtubules play an important role in the growth and maintenance of neurites during neuronal differentiation. They are composed of tubulin and microtubule-associated proteins (MAPs). MAPs determine neuronal shape and control the balance between rigidity and plasticity in neuronal processes. Neurosteroids may be involved in the formation and stabilization of microtubules and thus neuronal plasticity and function [40]. Experimental data demonstrate that progester- Cellular and Molecular Mechanisms of the Effects of Sex Hormones on the Nervous System http://dx.doi.org/10.5772/intechopen.71140 7 Testosterone and its metabolite estradiol induce numerous effects during critical periods of pre- and perinatal brain developments (organizational effects) that are necessary for brain sexual differentiation. Testosterone exposure is an essential requirement for masculinization of the brain. Nuclear volume, neuronal morphology, and astrocyte complexity are examples of the wide range of effects by which testosterone and estradiol can induce permanent changes in the function of neurons [42]. In the developing male rat, testosterone secreted from the testes is not bound by α-fetoprotein and freely enters the brain where it is locally converted into estradiol in specific nuclei. Consequently, neonatal males have more than double the levels of estradiol than females in brain regions subject to sexual differentiation [43]. High levels of the ER are concentrated in the same brain regions and ER is essential for transducing the steroid signal [44]. The gain or loss of function upon developmental estradiol exposure corresponds to the specific cellular morphological changes observed during the critical period, and the dendritic spines and astrocytes seen in each brain region retain that "memory" of early ste- It is generally accepted that estrogen acts as a conditional neuroprotectant with a complex pattern of biological actions, which are modulated by several interacting factors [45]. It has been found that administration of estradiol increases neuronal survival and recovery in adult animals and different lesion models [46, 47]. Estradiol protects neurons from excitotoxic damage due to seizures and stroke, as well as in AD [48]. One of the suggested mechanisms of this effect is the ability of estrogens to enhance neuropeptide Y (NPY) expression and release, as NPY has antiexcitatory effects [49]. In vitro estradiol was found to protect neurons from glutamate toxicity and Aβ peptide toxicity, oxidative stress, and glucose deprivation [50–53]. The defense state induced by estrogen is mediated by complex mechanisms that converge upon regulation of mitochondrial function. Estrogen preserves ATP levels via increased oxidative phosphorylation and reduced ATPase activity, thereby increasing mitochondrial respiration efficiency. Estrogen increases antiapoptotic proteins, Bcl-2 and Bcl-xL, which prevent formation of the permeability transition pores protecting against estrogen-induced increase in mitochondrial Ca2+ sequestration and triggering of apoptotic processes [54]. Therefore, the one treatment attenuated the injury-induced loss of MAP2 [41]. roid exposure [42]. 4.1. Effects of female sex steroids 4. Biological effects of sex hormones on the nervous system The modern understanding of a cell response to a steroid action is that it occurs within the same time frame as that of the G protein–coupled receptors influencing a variety of cellular functions such as gating membrane channels, increasing the intracellular calcium release, activating tyrosine-protein kinase (Src), MAPK, and others [27]. Many studies support a model of integrated signaling that couples signal transduction cascades to transcription in the nucleus, providing an integrated view of hormone signaling in the brain [32]. Recently, extensive research focused on the rapid, non-genomic action of estrogens has raised the question of how rapidly the increase of these steroids can occur in the brain. Of course, estrogens, just like any other steroids, cannot be stored in synaptic vesicles prior to their rapid release, due to their lipophilic nature [4]. It has been suggested, therefore, that the rapid effects of estrogens require a corresponding rapid change of local steroid concentration via rapid changes in their rate of synthesis by androgen conversion [24, 33], which implies changes in aromatase activity. Changes of aromatase activity reflect changes in aromatase protein concentrations. For instance, sex steroids control the hypothalamic aromatase expression in most vertebrates: weak aromatase expression is detected in castrated male animals, while testosterone replacement increases significantly aromatase protein and enzyme activity [34, 35]. There is strong evidence suggesting that aromatase activity can be rapidly modulated via translational modifications, most notably via phosphorylation. The rapid modulation of aromatase activity by phosphorylation is a widespread mechanism present in certain tissues of various species, including humans [4]. The enzymatic changes lead to a rapid local modulation of estrogen availability and consequently to a modification of cellular estrogen-dependent processes that are not mediated by the genomic actions of these steroids. The phosphorylation/ dephosphorylation processes provide a new widespread mechanism by which estrogen concentration could be rapidly altered in the brain and other tissues. Although most of the research on neurotransmitter-like actions of steroid hormones is focused on sex hormones and reproduction, other steroids also induce effects through non-classic mechanisms. As with estrogens and progestins, glucocorticoids can act on the membrane to alter physiology, functioning more like neurotransmitters than classical steroid hormones. Neurosteroids are also capable of interacting with cell surface neurotransmitter receptors to modulate neural cell physiology. Two of the endogenous neurosteroids, pregnenolone sulfate and pregnanolone sulfate, can potentiate or inhibit N-methyl-D-aspartate (NMDA) receptor responses [36]. GABAA receptors represent one of the most elaborate neurotransmitter receptor structures, harboring multiple binding sites for allosteric modulators, neuroactive compounds, and neuroactive steroids [37]. Allopregnanolone has been shown to promote neurogenesis in both rodent and human neuroprogenitor cells, most likely through binding to the GABAA receptor [38]. The modulation of the activity of receptors by neurochemicals such as allopregnanolone has been extensively studied in the context of neurodegenerative disorders [39]. Another mechanism of steroid action takes effect at the level of the microtubules via a proposed receptor microtubule-associated protein of type 2 (MAP2) [40]. Neuronal microtubules play an important role in the growth and maintenance of neurites during neuronal differentiation. They are composed of tubulin and microtubule-associated proteins (MAPs). MAPs determine neuronal shape and control the balance between rigidity and plasticity in neuronal processes. Neurosteroids may be involved in the formation and stabilization of microtubules and thus neuronal plasticity and function [40]. Experimental data demonstrate that progesterone treatment attenuated the injury-induced loss of MAP2 [41]. #### 4. Biological effects of sex hormones on the nervous system Testosterone and its metabolite estradiol induce numerous effects during critical periods of pre- and perinatal brain developments (organizational effects) that are necessary for brain sexual differentiation. Testosterone exposure is an essential requirement for masculinization of the brain. Nuclear volume, neuronal morphology, and astrocyte complexity are examples of the wide range of effects by which testosterone and estradiol can induce permanent changes in the function of neurons [42]. In the developing male rat, testosterone secreted from the testes is not bound by α-fetoprotein and freely enters the brain where it is locally converted into estradiol in specific nuclei. Consequently, neonatal males have more than double the levels of estradiol than females in brain regions subject to sexual differentiation [43]. High levels of the ER are concentrated in the same brain regions and ER is essential for transducing the steroid signal [44]. The gain or loss of function upon developmental estradiol exposure corresponds to the specific cellular morphological changes observed during the critical period, and the dendritic spines and astrocytes seen in each brain region retain that "memory" of early steroid exposure [42]. #### 4.1. Effects of female sex steroids the transactivation of cell surface–bound receptors, most notably the metabotropic glutamate receptors (mGluRs). Subsequently, estrogen membrane–initiated signaling can in turn acti- The modern understanding of a cell response to a steroid action is that it occurs within the same time frame as that of the G protein–coupled receptors influencing a variety of cellular functions such as gating membrane channels, increasing the intracellular calcium release, activating tyrosine-protein kinase (Src), MAPK, and others [27]. Many studies support a model of integrated signaling that couples signal transduction cascades to transcription in the nucleus, Recently, extensive research focused on the rapid, non-genomic action of estrogens has raised the question of how rapidly the increase of these steroids can occur in the brain. Of course, estrogens, just like any other steroids, cannot be stored in synaptic vesicles prior to their rapid release, due to their lipophilic nature [4]. It has been suggested, therefore, that the rapid effects of estrogens require a corresponding rapid change of local steroid concentration via rapid changes in their rate of synthesis by androgen conversion [24, 33], which implies changes in aromatase activity. Changes of aromatase activity reflect changes in aromatase protein concentrations. For instance, sex steroids control the hypothalamic aromatase expression in most vertebrates: weak aromatase expression is detected in castrated male animals, while testosterone replacement increases significantly aromatase protein and enzyme activity [34, 35]. There is strong evidence suggesting that aromatase activity can be rapidly modulated via translational modifications, most notably via phosphorylation. The rapid modulation of aromatase activity by phosphorylation is a widespread mechanism present in certain tissues of various species, including humans [4]. The enzymatic changes lead to a rapid local modulation of estrogen availability and consequently to a modification of cellular estrogen-dependent processes that are not mediated by the genomic actions of these steroids. The phosphorylation/ dephosphorylation processes provide a new widespread mechanism by which estrogen con- Although most of the research on neurotransmitter-like actions of steroid hormones is focused on sex hormones and reproduction, other steroids also induce effects through non-classic mechanisms. As with estrogens and progestins, glucocorticoids can act on the membrane to alter physiology, functioning more like neurotransmitters than classical steroid hormones. Neurosteroids are also capable of interacting with cell surface neurotransmitter receptors to modulate neural cell physiology. Two of the endogenous neurosteroids, pregnenolone sulfate and pregnanolone sulfate, can potentiate or inhibit N-methyl-D-aspartate (NMDA) receptor responses [36]. GABAA receptors represent one of the most elaborate neurotransmitter receptor structures, harboring multiple binding sites for allosteric modulators, neuroactive compounds, and neuroactive steroids [37]. Allopregnanolone has been shown to promote neurogenesis in both rodent and human neuroprogenitor cells, most likely through binding to the GABAA receptor [38]. The modulation of the activity of receptors by neurochemicals such as allopregnanolone has been extensively studied in the context of neurodegenerative vate the regulatory section of DNA and trigger transcription processes. 6 Sex Hormones in Neurodegenerative Processes and Diseases providing an integrated view of hormone signaling in the brain [32]. centration could be rapidly altered in the brain and other tissues. disorders [39]. It is generally accepted that estrogen acts as a conditional neuroprotectant with a complex pattern of biological actions, which are modulated by several interacting factors [45]. It has been found that administration of estradiol increases neuronal survival and recovery in adult animals and different lesion models [46, 47]. Estradiol protects neurons from excitotoxic damage due to seizures and stroke, as well as in AD [48]. One of the suggested mechanisms of this effect is the ability of estrogens to enhance neuropeptide Y (NPY) expression and release, as NPY has antiexcitatory effects [49]. In vitro estradiol was found to protect neurons from glutamate toxicity and Aβ peptide toxicity, oxidative stress, and glucose deprivation [50–53]. The defense state induced by estrogen is mediated by complex mechanisms that converge upon regulation of mitochondrial function. Estrogen preserves ATP levels via increased oxidative phosphorylation and reduced ATPase activity, thereby increasing mitochondrial respiration efficiency. Estrogen increases antiapoptotic proteins, Bcl-2 and Bcl-xL, which prevent formation of the permeability transition pores protecting against estrogen-induced increase in mitochondrial Ca2+ sequestration and triggering of apoptotic processes [54]. Therefore, the decreased levels of estrogen could most likely contribute to the increased risk of developing neurodegenerative diseases, especially in postmenopausal women [52, 55]. Progestins have also been found to exert neuroprotective effects similar to those of estrogens. Progesterone stimulates the neurological and functional recovery after spinal and brain trau- Cellular and Molecular Mechanisms of the Effects of Sex Hormones on the Nervous System http://dx.doi.org/10.5772/intechopen.71140 9 The effects of androgens on the nervous system have been far less characterized than those produced by estrogens and progestins. Androgens also exhibit a wide array of neuroprotective effects in motoneurons, including supporting cell survival, axonal regeneration, and dendritic maintenance [72]. Testosterone influences neuroplastic changes in nuclei of the limbic system, particularly in the amygdala, bed nucleus of the stria terminalis, and the hippocampus [73, 74]; it exerts neuroprotective effects by stimulating neuron survival and regeneration after a nerve injury by actions mediated via the androgen receptor [75, 76]. It has been observed to have a protective effect on apoptosis in cell cultures of human neurons. This effect is mediated directly by androgen receptors, without testosterone aromatization to estradiol [77]. Testosterone replacement in gonadectomized male adult mice reverses the pathological changes in the spine morphology of hippocampal CA1 pyramidal neurons. The dendritic spines are specialized to receive synaptic inputs, and a change in spine morphology is correlated with the strength and maturity of each synapse [78]. Similar data were obtained in experimental motoneuron damage, with the use of DHT reducing the atrophy of adjacent dendrites [79]. Recent findings suggest that one of the mechanisms of the neuroprotective effects of physical training is the increased DHT production in the hippocampus providing evidence for androgenic mediation of neurogenesis by androgen receptors [80]. degradation, thereby decreasing Aβ levels in AD [84]. 4.3. Effects of steroid precursors effects [87]. Androgens may regulate the production and the levels of Aβ, by a classic genomic mechanism and rapid non-genomic signaling or via aromatization to estradiol and activation of estrogen pathways [81, 82]. Testosterone can attenuate the toxicity of Aβ in cultured hippocampal neurons via a rapid, estrogen-independent mechanism [83]. DHT increases Aβ-catabolizing enzyme neprilysin in cultured neurons by an AR-dependent mechanism, which promotes Aβ Precursors of estrogens, progestins, and androgens (pregnenolone and DHEA) also affect neuronal functions. When administered in vivo, pregnenolone reduces histopathological changes, protects neural tissues from secondary lesions, and promotes the recovery of motor functions after spinal cord injury [85, 86]. DHEA is one of the first neurosteroids identified in rat brains. Neuroprotective effects induced by DHEA and its sulfate DHEAS, defined as primary in their biological action, have been documented [87]. Both steroids contribute to the differentiation and survival of neurons in cell cultures [88]; have a protective effect on hippocampal neurons against the toxic effects of glutamate [89]; stimulate the growth of neuritis of the cortical neurons of embryonic rat brains [90]; affect apoptosis, catecholamine synthesis, and secretion; and have exhibited anti-oxidant, anti-inflammatory, and anti-glucocorticoid mas [56, 69] and exerts neuroprotection in cerebral ischemia [70, 71]. 4.2. Effects of androgens It is suggested that in addition to having a direct effect on neurons, estrogens may affect the astrocytes by stimulating them to release protective growth factors and regulate the astrocytes genes and proteins associated with the glutamate level control. Other mechanisms implicated here may include the anti-inflammatory effect associated with suppression of microglia, inflammatory cytokines, and free radicals production, which cause inflammatory damage to the neurons, effects on endothelial cells realized by increasing the mitochondrial efficiency and stimulating angiogenesis, genomic influence on anti-apoptotic protein genes of Bcl family and reduction of apoptotic trends and effect of free radical scavenging. These are the hypothetical models of estrogen neuroprotection in cerebral ischemia and in other neurodegenerative disorders such as Parkinson's disease (PD) and AD [52, 56]. There is growing evidence that estrogen may have a neuroprotective role in PD. Experimental studies have demonstrated that estrogen is neuroprotective in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal lesions, an animal model of idiopathic PD [57, 58]. In these and other studies, 17β-estradiol was used and its effect was shown to be stereospecific. An isomer with weak estrogenic activity, 17α-estradiol, was ineffective with regard to the prevention of MPTP-induced dopamine loss [52]. What is worthy of note is that the receptors ERα and ERβ are sparsely localized in the striatum and substantia nigra of mice, and treatment with MPTP or estrogen does not change the distribution and density of the estrogen receptor. Despite the low availability of ER in these parts of the brain, estrogen has managed to induce a protective effect on the striatum against MPTP-induced loss of dopaminergic neurons [59]. Studies in humans showed that short-term estrogen treatment in postmenopausal women increased dopamine transporter availability in the caudate putamen [60] and that women who had taken postmenopausal estrogen replacement therapy were less likely to develop PD than those who had not [61]. There is evidence of inducing differentiation of human neural stem cells, which develop in the tyrosine hydroxylase (dopaminergic) neurons, and the effect was blocked by application of an estrogen receptor antagonist [62, 63]. As it is supposed that oxidative stress plays an important role in the processes of neuronal degeneration in the PD, it is interesting that estrogens suppress free radical production and protect striatal neurons against oxidative stress, providing another mechanism of estrogen neuroprotection in PD [64, 65]. Recent studies in both animals and humans have provided additional evidence supporting a potentially beneficial protective role for estrogen in AD. The mechanisms of estrogen protection in AD are not clear. At the molecular level, estrogen has been shown to enhance activation of the survival factors, protein kinase B, BDNF [66, 67], while inducing phosphorylation and deactivation of glycogen synthase kinase (GSK3B) and Bcl-2 associated agonist of cell death (BAD), involved in death signaling pathways in neurons [67, 68]. Progestins have also been found to exert neuroprotective effects similar to those of estrogens. Progesterone stimulates the neurological and functional recovery after spinal and brain traumas [56, 69] and exerts neuroprotection in cerebral ischemia [70, 71]. #### 4.2. Effects of androgens decreased levels of estrogen could most likely contribute to the increased risk of developing It is suggested that in addition to having a direct effect on neurons, estrogens may affect the astrocytes by stimulating them to release protective growth factors and regulate the astrocytes genes and proteins associated with the glutamate level control. Other mechanisms implicated here may include the anti-inflammatory effect associated with suppression of microglia, inflammatory cytokines, and free radicals production, which cause inflammatory damage to the neurons, effects on endothelial cells realized by increasing the mitochondrial efficiency and stimulating angiogenesis, genomic influence on anti-apoptotic protein genes of Bcl family and reduction of apoptotic trends and effect of free radical scavenging. These are the hypothetical models of estrogen neuroprotection in cerebral ischemia and in other neuro- There is growing evidence that estrogen may have a neuroprotective role in PD. Experimental studies have demonstrated that estrogen is neuroprotective in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal lesions, an animal model of idiopathic PD [57, 58]. In these and other studies, 17β-estradiol was used and its effect was shown to be stereospecific. An isomer with weak estrogenic activity, 17α-estradiol, was ineffective with regard to the prevention of MPTP-induced dopamine loss [52]. What is worthy of note is that the receptors ERα and ERβ are sparsely localized in the striatum and substantia nigra of mice, and treatment with MPTP or estrogen does not change the distribution and density of the estrogen receptor. Despite the low availability of ER in these parts of the brain, estrogen has managed to induce a protective effect on the striatum against MPTP-induced loss of dopami- Studies in humans showed that short-term estrogen treatment in postmenopausal women increased dopamine transporter availability in the caudate putamen [60] and that women who had taken postmenopausal estrogen replacement therapy were less likely to develop PD There is evidence of inducing differentiation of human neural stem cells, which develop in the tyrosine hydroxylase (dopaminergic) neurons, and the effect was blocked by application of an As it is supposed that oxidative stress plays an important role in the processes of neuronal degeneration in the PD, it is interesting that estrogens suppress free radical production and protect striatal neurons against oxidative stress, providing another mechanism of estrogen Recent studies in both animals and humans have provided additional evidence supporting a potentially beneficial protective role for estrogen in AD. The mechanisms of estrogen protection in AD are not clear. At the molecular level, estrogen has been shown to enhance activation of the survival factors, protein kinase B, BDNF [66, 67], while inducing phosphorylation and deactivation of glycogen synthase kinase (GSK3B) and Bcl-2 associated agonist of cell death (BAD), involved in death signaling pathways in neurons [67, 68]. neurodegenerative diseases, especially in postmenopausal women [52, 55]. 8 Sex Hormones in Neurodegenerative Processes and Diseases degenerative disorders such as Parkinson's disease (PD) and AD [52, 56]. nergic neurons [59]. than those who had not [61]. estrogen receptor antagonist [62, 63]. neuroprotection in PD [64, 65]. The effects of androgens on the nervous system have been far less characterized than those produced by estrogens and progestins. Androgens also exhibit a wide array of neuroprotective effects in motoneurons, including supporting cell survival, axonal regeneration, and dendritic maintenance [72]. Testosterone influences neuroplastic changes in nuclei of the limbic system, particularly in the amygdala, bed nucleus of the stria terminalis, and the hippocampus [73, 74]; it exerts neuroprotective effects by stimulating neuron survival and regeneration after a nerve injury by actions mediated via the androgen receptor [75, 76]. It has been observed to have a protective effect on apoptosis in cell cultures of human neurons. This effect is mediated directly by androgen receptors, without testosterone aromatization to estradiol [77]. Testosterone replacement in gonadectomized male adult mice reverses the pathological changes in the spine morphology of hippocampal CA1 pyramidal neurons. The dendritic spines are specialized to receive synaptic inputs, and a change in spine morphology is correlated with the strength and maturity of each synapse [78]. Similar data were obtained in experimental motoneuron damage, with the use of DHT reducing the atrophy of adjacent dendrites [79]. Recent findings suggest that one of the mechanisms of the neuroprotective effects of physical training is the increased DHT production in the hippocampus providing evidence for androgenic mediation of neurogenesis by androgen receptors [80]. Androgens may regulate the production and the levels of Aβ, by a classic genomic mechanism and rapid non-genomic signaling or via aromatization to estradiol and activation of estrogen pathways [81, 82]. Testosterone can attenuate the toxicity of Aβ in cultured hippocampal neurons via a rapid, estrogen-independent mechanism [83]. DHT increases Aβ-catabolizing enzyme neprilysin in cultured neurons by an AR-dependent mechanism, which promotes Aβ degradation, thereby decreasing Aβ levels in AD [84]. #### 4.3. Effects of steroid precursors Precursors of estrogens, progestins, and androgens (pregnenolone and DHEA) also affect neuronal functions. When administered in vivo, pregnenolone reduces histopathological changes, protects neural tissues from secondary lesions, and promotes the recovery of motor functions after spinal cord injury [85, 86]. DHEA is one of the first neurosteroids identified in rat brains. Neuroprotective effects induced by DHEA and its sulfate DHEAS, defined as primary in their biological action, have been documented [87]. Both steroids contribute to the differentiation and survival of neurons in cell cultures [88]; have a protective effect on hippocampal neurons against the toxic effects of glutamate [89]; stimulate the growth of neuritis of the cortical neurons of embryonic rat brains [90]; affect apoptosis, catecholamine synthesis, and secretion; and have exhibited anti-oxidant, anti-inflammatory, and anti-glucocorticoid effects [87]. Studies suggest that these are different mechanisms for DHEA and DHEAS effects. It is assumed that DHEAS mediates its effects via GABAA receptors, probably by metabolizing DHEAS into a GABAA receptor agonist, such as androsterone or androstanediol [91]. The neuroprotective effect of DHEAS to NMDA receptor-induced cytotoxicity is probably mediated by the σ1 receptor, while DHEA inhibits NMDA-induced nitric oxide (NO) production and NO synthase activity by NMDA receptor, modulating calcium/NO signaling pathway [92]. Concentrations of DHEA and of its sulfate are also important with respect to the final effect. Low concentrations of these steroids may be neuroprotective, while high concentrations of DHEA are ineffective or neurotoxic and lead to the inhibition of complex I of the mitochondrial respiratory chain [93]. Testosterone administration was shown to increase BDNF protein levels in motoneurons of spinal nucleus of the bulbocavernosus of castrated male rats [102]. Gonadectomy induces a significant decrease in the protein levels of BDNF and its downstream target post-synaptic density protein 95 (PSD-95) in the hippocampal CA1 area, which is reversed by testosterone replacement [78]. Knowledge of the interactions between BDNF and sex steroids could be essential for the understanding of the BDNF role in brain development, adaptation during Cellular and Molecular Mechanisms of the Effects of Sex Hormones on the Nervous System http://dx.doi.org/10.5772/intechopen.71140 11 The functions of the sex hormones exceed the limits of reproduction in that they regulate vital neuronal and glial features. The chronic effects of neurosteroids are due to both genomic (classical intracellular steroid receptors) and non-genomic rapid effects (ion channels and Some of the hypothetical models of estrogen neuroprotection include complex mechanisms, which converge upon regulation of mitochondria function–preserved ATP levels via increased oxidative phosphorylation and increased antiapoptotic proteins of Bcl family. Estrogen stimulates the astrocytes to release protective growth factors and has an anti-inflammatory effect associated with suppression of microglia and inflammatory cytokines. It suppresses free radical production and protects striatal neurons against oxidative stress, providing another mechanism for neuroprotection in PD. The female sex steroids promote cell survival via protein kinase B activation and BDNF upregulation; they inactivate GSK3B and BAD, involved in neuronal death signaling pathways in AD. The androgens also have neuroprotective effects in motoneurons, including supporting neuron survival, axonal regeneration, and dendritic maintenance. Despite the growing amount of research on sex hormones and neurosteroids in recent years and the ongoing discovery of biochemical mechanisms of action, their role in neurodegenerative processes remains uncertain. Further elucidation of the cellular and molecular mechanisms responsible for the effects of neurosteroids on the normal function of neuronal and glial cells would provide important insights related to the development of new therapeutic strategies aimed at delaying the onset and slowing the progression of cognitive dysfunctions 1 Department of Anatomy, Histology and Embryology, Faculty of Medicine, Medical 2 Department of Physiology, Faculty of Medicine, Medical University – Plovdiv, Plovdiv, Bulgaria Testosterone can attenuate the toxicity of Aβ and decreases Aβ levels in AD. aging, and the pathogenesis of neurodegenerative diseases. 5. Conclusion membrane receptors) in the brain. and neurodegenerative diseases. \* and Katerina Georgieva<sup>2</sup> \Address all correspondence to: [email protected] University – Plovdiv, Plovdiv, Bulgaria Author details* Slavi Delchev1 #### 4.4. Interaction between steroids and neurotrophins Recently, researchers have studied the relationship between the gonadal steroids, adrenal steroids, and BDNF focusing on intersexual differences and incidence of mental diseases [94]. BDNF belongs to the neurotrophin family and plays an important role in the survival, differentiation, and outgrowth of select peripheral and central neurons during development. BDNF impacts significantly on neuronal survival, acting in the adult brain through a variety of cell types, which include neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells. It is essential for the process of learning and improvement of cognitive function via activation of the TrkB receptor [95]. Our previous data demonstrated that the negative effect of the anticonvulsant lacosamide on the processes of learning and memory is related to suppressed expression of BDNF/TrkB ligand receptor system in the hippocampus of rats [96]. Sex steroid hormones and neurotrophic factors are involved in the neuroendocrine control of reproduction as well as in brain adaptation during reproductive aging. There is a great body of evidence showing the role BDNF plays in the pathogenesis of neurodegenerative diseases. Low post-mortem parietal cortex BDNF levels have been found in patients with mild cognitive impairment [97] and AD [97, 98]. Research shows that BDNF mRNA and protein expression levels in the brain cognitive regions are affected in a region-specific manner when hormone replacement therapy is administered. BDNF mRNA levels have been reported to be significantly reduced in almost all hippocampal layers and the cortex in 28-week ovariectomized rats [99]. Estradiol replacement therapy reverses this effect in the hippocampus, suggesting a regional divergence in ovarian steroid requirements for BDNF expression. After gonadectomy, BDNF mRNA levels are significantly reduced at postnatal day 7 in male rat pups, but after treatment with estradiol benzoate, the levels were similar to those in intact animals. The authors demonstrated that ERα and BDNF were localized in the same cells (pyramidal cells of the CA3 sub-region and to a lesser extent in CA1) within the developing hippocampus [100]. Estrogens have been implicated in the increase of hippocampal BDNF mRNA and protein levels in exercising animals. The exercise effect on BDNF upregulation was reduced after 7 weeks of estrogen deprivation. Exercise in combination with long-term estrogen replacement increased the BDNF protein above the effects of estrogen replacement alone [101]. Androgens also have a bearing on the BDNF expression; some of their effects on the nervous system are most likely to be realized through influencing the production of this neurotrophin. Testosterone administration was shown to increase BDNF protein levels in motoneurons of spinal nucleus of the bulbocavernosus of castrated male rats [102]. Gonadectomy induces a significant decrease in the protein levels of BDNF and its downstream target post-synaptic density protein 95 (PSD-95) in the hippocampal CA1 area, which is reversed by testosterone replacement [78]. Knowledge of the interactions between BDNF and sex steroids could be essential for the understanding of the BDNF role in brain development, adaptation during aging, and the pathogenesis of neurodegenerative diseases. #### 5. Conclusion Studies suggest that these are different mechanisms for DHEA and DHEAS effects. It is assumed that DHEAS mediates its effects via GABAA receptors, probably by metabolizing DHEAS into a GABAA receptor agonist, such as androsterone or androstanediol [91]. The neuroprotective effect of DHEAS to NMDA receptor-induced cytotoxicity is probably mediated by the σ1 receptor, while DHEA inhibits NMDA-induced nitric oxide (NO) production and NO synthase activity by NMDA receptor, modulating calcium/NO signaling pathway [92]. Concentrations of DHEA and of its sulfate are also important with respect to the final effect. Low concentrations of these steroids may be neuroprotective, while high concentrations of DHEA are ineffective or neurotoxic and lead to the inhibition of complex I of the mitochon- Recently, researchers have studied the relationship between the gonadal steroids, adrenal steroids, and BDNF focusing on intersexual differences and incidence of mental diseases [94]. BDNF belongs to the neurotrophin family and plays an important role in the survival, differentiation, and outgrowth of select peripheral and central neurons during development. BDNF impacts significantly on neuronal survival, acting in the adult brain through a variety of cell types, which include neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells. It is essential for the process of learning and improvement of cognitive function via activation of the TrkB receptor [95]. Our previous data demonstrated that the negative effect of the anticonvulsant lacosamide on the processes of learning and memory is related to suppressed expression of BDNF/TrkB ligand receptor system in the hippocampus of rats [96]. Sex steroid hormones and neurotrophic factors are involved in the neuroendocrine control of reproduction as well as in brain adaptation during reproductive aging. There is a great body of evidence showing the role BDNF plays in the pathogenesis of neurodegenerative diseases. Low post-mortem parietal cortex BDNF levels have been found in patients with mild cogni- Research shows that BDNF mRNA and protein expression levels in the brain cognitive regions are affected in a region-specific manner when hormone replacement therapy is administered. BDNF mRNA levels have been reported to be significantly reduced in almost all hippocampal layers and the cortex in 28-week ovariectomized rats [99]. Estradiol replacement therapy reverses this effect in the hippocampus, suggesting a regional divergence in ovarian steroid requirements for BDNF expression. After gonadectomy, BDNF mRNA levels are significantly reduced at postnatal day 7 in male rat pups, but after treatment with estradiol benzoate, the levels were similar to those in intact animals. The authors demonstrated that ERα and BDNF were localized in the same cells (pyramidal cells of the CA3 sub-region and to a lesser extent Estrogens have been implicated in the increase of hippocampal BDNF mRNA and protein levels in exercising animals. The exercise effect on BDNF upregulation was reduced after 7 weeks of estrogen deprivation. Exercise in combination with long-term estrogen replacement increased the BDNF protein above the effects of estrogen replacement alone [101]. Androgens also have a bearing on the BDNF expression; some of their effects on the nervous system are most likely to be realized through influencing the production of this neurotrophin. drial respiratory chain [93]. tive impairment [97] and AD [97, 98]. in CA1) within the developing hippocampus [100]. 4.4. Interaction between steroids and neurotrophins 10 Sex Hormones in Neurodegenerative Processes and Diseases The functions of the sex hormones exceed the limits of reproduction in that they regulate vital neuronal and glial features. The chronic effects of neurosteroids are due to both genomic (classical intracellular steroid receptors) and non-genomic rapid effects (ion channels and membrane receptors) in the brain. Some of the hypothetical models of estrogen neuroprotection include complex mechanisms, which converge upon regulation of mitochondria function–preserved ATP levels via increased oxidative phosphorylation and increased antiapoptotic proteins of Bcl family. Estrogen stimulates the astrocytes to release protective growth factors and has an anti-inflammatory effect associated with suppression of microglia and inflammatory cytokines. It suppresses free radical production and protects striatal neurons against oxidative stress, providing another mechanism for neuroprotection in PD. The female sex steroids promote cell survival via protein kinase B activation and BDNF upregulation; they inactivate GSK3B and BAD, involved in neuronal death signaling pathways in AD. The androgens also have neuroprotective effects in motoneurons, including supporting neuron survival, axonal regeneration, and dendritic maintenance. Testosterone can attenuate the toxicity of Aβ and decreases Aβ levels in AD. Despite the growing amount of research on sex hormones and neurosteroids in recent years and the ongoing discovery of biochemical mechanisms of action, their role in neurodegenerative processes remains uncertain. Further elucidation of the cellular and molecular mechanisms responsible for the effects of neurosteroids on the normal function of neuronal and glial cells would provide important insights related to the development of new therapeutic strategies aimed at delaying the onset and slowing the progression of cognitive dysfunctions and neurodegenerative diseases. ### Author details Slavi Delchev1 \* and Katerina Georgieva<sup>2</sup> \Address all correspondence to: [email protected] 1 Department of Anatomy, Histology and Embryology, Faculty of Medicine, Medical University – Plovdiv, Plovdiv, Bulgaria 2 Department of Physiology, Faculty of Medicine, Medical University – Plovdiv, Plovdiv, Bulgaria #### References* [1] Miller WL, Auchus RJ. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocrine Reviews. 2011;32:81-151 [14] Jones KJ, Coers S, Storer PD, Tanzer L, Kinderman NB. 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Broad-spectrum neuroprotective agents with multiple mechanisms of action rather than a single druggable target are, therefore, highly desirable. The main human estrogen, 17β-estradiol, can also be considered a neurosteroid as it forms de novo in the central nervous system, and it possesses beneficial effects against practically all critical contributors to neurodegeneration to collectively thwart both the initiation and the progression of neuronal cell death. This chapter details the main aspects of the hormone's genomic and non-genomic actions important to protect the highly vulnerably neurons of the central nervous system, as well as translational efforts to successfully realize its powerful neuroprotective potential in clinical setting while ensuring both therapeutic safety and Keywords: antioxidant, brain, bioprecursor prodrug, broad-spectrum neuroprotectant, brain-selective estrogen therapy, cell death, 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED), estrogens, genomic and non-genomic estrogenic actions, neuroprotection, The steroid hormone 17β-estradiol (E2, Figure 1) is the main human estrogen that is not only involved in sexual maturation and reproduction but also has a myriad of important roles throughout the body affecting, for example, the cardiovascular system, lipid metabolism and brain health [1–4]. Therefore, E2 cannot only be considered as just a "female hormone." In humans, the other two endogenously formed estrogens are estrone (E1, Figure 1) and the lesser-known estriol (E3, Figure 1) that is the 16-hydroxy derivate of E2 and formed mostly during gestation by the placenta. E1 becomes the predominant estrogen in women after para-quinol, Prokai antioxidant cycle, stroke, translational research © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. and reproduction in any medium, provided the original work is properly cited. Katalin Prokai-Tatrai and Laszlo Prokai Katalin Prokai-Tatrai and Laszlo Prokai http://dx.doi.org/10.5772/intechopen.72682 Abstract efficacy. 1. Introduction Additional information is available at the end of the chapter Additional information is available at the end of the chapter Provisional chapter ## 17β-Estradiol as a Neuroprotective Agent 17β-Estradiol as a Neuroprotective Agent #### Katalin Prokai-Tatrai and Laszlo Prokai Additional information is available at the end of the chapter Katalin Prokai-Tatrai and Laszlo Prokai Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.72682 Abstract The pathophysiology of neurodegeneration in the central nervous system is complex and multifactorial in nature and yet to be fully understood. Broad-spectrum neuroprotective agents with multiple mechanisms of action rather than a single druggable target are, therefore, highly desirable. The main human estrogen, 17β-estradiol, can also be considered a neurosteroid as it forms de novo in the central nervous system, and it possesses beneficial effects against practically all critical contributors to neurodegeneration to collectively thwart both the initiation and the progression of neuronal cell death. This chapter details the main aspects of the hormone's genomic and non-genomic actions important to protect the highly vulnerably neurons of the central nervous system, as well as translational efforts to successfully realize its powerful neuroprotective potential in clinical setting while ensuring both therapeutic safety and efficacy. DOI: 10.5772/intechopen.72682 Keywords: antioxidant, brain, bioprecursor prodrug, broad-spectrum neuroprotectant, brain-selective estrogen therapy, cell death, 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED), estrogens, genomic and non-genomic estrogenic actions, neuroprotection, para-quinol, Prokai antioxidant cycle, stroke, translational research #### 1. Introduction The steroid hormone 17β-estradiol (E2, Figure 1) is the main human estrogen that is not only involved in sexual maturation and reproduction but also has a myriad of important roles throughout the body affecting, for example, the cardiovascular system, lipid metabolism and brain health [1–4]. Therefore, E2 cannot only be considered as just a "female hormone." In humans, the other two endogenously formed estrogens are estrone (E1, Figure 1) and the lesser-known estriol (E3, Figure 1) that is the 16-hydroxy derivate of E2 and formed mostly during gestation by the placenta. E1 becomes the predominant estrogen in women after Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons 2. Mechanistic overview of neuroprotection by estrogens the CNS, as detailed in the following section. 2.1. Genomic pathways in E2 neuroprotection tion and reproduction [41]. The broad-spectrum protective mechanism of E2 on injured neurons is the end result of wellorchestrated and synergistic combination of genomic and non-genomic actions of the hormone that allows for prevention of both the initiation and progression of neuronal cell death. This implicates a significant translational value for the hormone upon restricting its action to 17β-Estradiol as a Neuroprotective Agent http://dx.doi.org/10.5772/intechopen.72682 23 Estrogen receptors (ERs) are expressed throughout the brain [35, 36] indicating their role in various CNS functions including neuroprotection. ER density is higher in the hypothalamus than in extrahypothalamic areas with overlapping expression of the two isoforms ERα and ERβ [35, 36]. In some brain regions, ERα or ERβ may also be co-localized in cells [37]. However, ERβ is highly expressed in the cortex [38] and hippocampus [39, 40]. Consequently, estrogen impacts the function of extrahypothalamic areas that are not involved in sex matura- The two ERs have similar affinities to endogenous estrogens [42]. Just like many other members of the nuclear receptor superfamily of proteins, they elicit their genomic effect through gene transcription [43]. The sequence of the classical ligand-dependent genomic mechanism of estrogens' neuroprotective action is summarized in Figure 2. After E2 (or in general, an estrogen) distributes into the neuron and reaches the nucleus, it is ligated to its cognate receptor. The ligated ERs form homo- (ERα/ERα or ERβ/ERβ) or heterodimers (ERα/ERβ) that bind to the estrogen-response element (ERE) of the nuclear DNA. Transcriptional activation is enabled by Figure 2. A simplified model for the ligand-dependent genomic mechanism of E2 neuroprotection. After E2 distributes into the neuron (A) and enters the nucleus, it binds to the cognate receptor followed by dimerization of the ligated ER that binds, together with co-regulators, to the nuclear DNA's ERE, which results in the transcription of the corresponding gene (B). E2, ER, co-regulator (Co-r) and the nuclear DNA are symbolized by the filled steroid shape, rounded rectangles and elongated rectangle showing an ERE (shaded area), respectively. Figure 1. Chemical structure of human estrogens: estrone (E1), 17β-estradiol (E2) and estriol (E3). menopause, when it is synthesized largely in subcutaneous fat from androstenedione. The unique structure of estrogens among steroids arises from the presence of the aromatic A-ring (Figure 1). E2 is now also considered as one of the neurosteroids, as its regioselective local formation in the brain has been established [5–8]. Indeed, with today's modern analytical instrumentations and using validated bioassays that are devoid of the limitations of immunoassays [9], brain E2 level even in ovariectomized animals (i.e., in animals without gonadal E2 source) can be measured [10, 11]. It has been hypothesized that this de novo central formation of E2 due to the lack of gonadal E2 sources, for example, as in case of ovariectomy, is essentially a compensatory mechanism to protect the estrogen-deprived brain that cannot receive the hormone from the circulation any more, although plasma estrogen levels do not directly correlate with that of brain [12, 13]. Additionally, de novo synthesis of E2 with a presumed role of neuroprotection in the developing mammalian brain has also been shown [14]. Independent of the loci of estrogen's gonadal or extra-gonadal biosynthesis from cholesterol via a number of enzyme-catalyzed steps, the final process is the oxidation of the 10-methyl group of testosterone, followed by elimination and subsequent aromatization into E2 by aromatase. This step is the one that has been controlled by aromatase inhibitors in clinical practice to prevent the reoccurrence of estrogen-dependent malignancies [15]. Among estrogens and estrogenic compounds, E2 also is the best-known estrogen to be used as a powerful neuroprotective agent in various in vitro and preclinical animal models of neurodegeneration impacting the central nervous system (CNS) [16–20]. It is important to emphasize, though, that E2 has a large array of other beneficial effects in the CNS, including regulating body temperature, enhancing cognition and memory and ameliorating neuropsychiatric conditions in both females and males [7, 11, 21–23]. While the brain is undoubtedly the most frequently studied part of the CNS in the context of neuroprotection [24–26], the utility of E2 in protecting the eye (retina and optic nerve) [27–29] and spinal cord [30–32] have also been explored with promising outcomes. Altogether, extensive basic science investigations brought about convincing data on the plethora of mechanisms by which E2 promotes neuronal survival and protects neurons against a wide variety of stressors addressing, thereby, practically all proposed critical contributors to neurodegeneration such as inflammation, oxidative stress, excitotoxicity and collapse of mitochondrial membrane potential. Clinical and epidemiological observations also suggest that in humans the better outcome after neurotrauma (e.g., traumatic brain and spinal cord injuries) in premenopausal females compared with age-matched men, at least in part, is due to the protective role of endogenous estrogens against neuronal injuries [33, 34]. #### 2. Mechanistic overview of neuroprotection by estrogens The broad-spectrum protective mechanism of E2 on injured neurons is the end result of wellorchestrated and synergistic combination of genomic and non-genomic actions of the hormone that allows for prevention of both the initiation and progression of neuronal cell death. This implicates a significant translational value for the hormone upon restricting its action to the CNS, as detailed in the following section. #### 2.1. Genomic pathways in E2 neuroprotection menopause, when it is synthesized largely in subcutaneous fat from androstenedione. The unique structure of estrogens among steroids arises from the presence of the aromatic A-ring Figure 1. Chemical structure of human estrogens: estrone (E1), 17β-estradiol (E2) and estriol (E3). 22 Sex Hormones in Neurodegenerative Processes and Diseases E2 is now also considered as one of the neurosteroids, as its regioselective local formation in the brain has been established [5–8]. Indeed, with today's modern analytical instrumentations and using validated bioassays that are devoid of the limitations of immunoassays [9], brain E2 level even in ovariectomized animals (i.e., in animals without gonadal E2 source) can be measured [10, 11]. It has been hypothesized that this de novo central formation of E2 due to the lack of gonadal E2 sources, for example, as in case of ovariectomy, is essentially a compensatory mechanism to protect the estrogen-deprived brain that cannot receive the hormone from the circulation any more, although plasma estrogen levels do not directly correlate with that of brain [12, 13]. Additionally, de novo synthesis of E2 with a presumed role of neuroprotection Independent of the loci of estrogen's gonadal or extra-gonadal biosynthesis from cholesterol via a number of enzyme-catalyzed steps, the final process is the oxidation of the 10-methyl group of testosterone, followed by elimination and subsequent aromatization into E2 by aromatase. This step is the one that has been controlled by aromatase inhibitors in clinical prac- Among estrogens and estrogenic compounds, E2 also is the best-known estrogen to be used as a powerful neuroprotective agent in various in vitro and preclinical animal models of neurodegeneration impacting the central nervous system (CNS) [16–20]. It is important to emphasize, though, that E2 has a large array of other beneficial effects in the CNS, including regulating body temperature, enhancing cognition and memory and ameliorating neuropsychiatric conditions in both females and males [7, 11, 21–23]. While the brain is undoubtedly the most frequently studied part of the CNS in the context of neuroprotection [24–26], the utility of E2 in protecting the eye (retina and optic nerve) [27–29] and spinal cord [30–32] have Altogether, extensive basic science investigations brought about convincing data on the plethora of mechanisms by which E2 promotes neuronal survival and protects neurons against a wide variety of stressors addressing, thereby, practically all proposed critical contributors to neurodegeneration such as inflammation, oxidative stress, excitotoxicity and collapse of mitochondrial membrane potential. Clinical and epidemiological observations also suggest that in humans the better outcome after neurotrauma (e.g., traumatic brain and spinal cord injuries) in premenopausal females compared with age-matched men, at least in part, is due to the protective role of endogenous estrogens against neuronal injuries [33, 34]. in the developing mammalian brain has also been shown [14]. also been explored with promising outcomes. tice to prevent the reoccurrence of estrogen-dependent malignancies [15]. (Figure 1). Estrogen receptors (ERs) are expressed throughout the brain [35, 36] indicating their role in various CNS functions including neuroprotection. ER density is higher in the hypothalamus than in extrahypothalamic areas with overlapping expression of the two isoforms ERα and ERβ [35, 36]. In some brain regions, ERα or ERβ may also be co-localized in cells [37]. However, ERβ is highly expressed in the cortex [38] and hippocampus [39, 40]. Consequently, estrogen impacts the function of extrahypothalamic areas that are not involved in sex maturation and reproduction [41]. The two ERs have similar affinities to endogenous estrogens [42]. Just like many other members of the nuclear receptor superfamily of proteins, they elicit their genomic effect through gene transcription [43]. The sequence of the classical ligand-dependent genomic mechanism of estrogens' neuroprotective action is summarized in Figure 2. After E2 (or in general, an estrogen) distributes into the neuron and reaches the nucleus, it is ligated to its cognate receptor. The ligated ERs form homo- (ERα/ERα or ERβ/ERβ) or heterodimers (ERα/ERβ) that bind to the estrogen-response element (ERE) of the nuclear DNA. Transcriptional activation is enabled by Figure 2. A simplified model for the ligand-dependent genomic mechanism of E2 neuroprotection. After E2 distributes into the neuron (A) and enters the nucleus, it binds to the cognate receptor followed by dimerization of the ligated ER that binds, together with co-regulators, to the nuclear DNA's ERE, which results in the transcription of the corresponding gene (B). E2, ER, co-regulator (Co-r) and the nuclear DNA are symbolized by the filled steroid shape, rounded rectangles and elongated rectangle showing an ERE (shaded area), respectively. a constitutively active and a ligand-dependent function located at the amino-terminus and in the carboxy-terminal ligand-binding domain of ERs, respectively [44]. The DNA-bound dimers recruit co-regulator proteins [45], which can participate in and also recruit many enzymatic and structural proteins permitting the modulation of chromatin structure to facilitate or block gene expression [46]. Additionally, ERE-independent mechanisms have also been shown [47]. Neuroprotective target genes for E2 that directly support vital neuronal functions include neurotrophic factors such as the brain-derived neurotrophic factor [48]. Additional target genes are involved in apoptosis to remove unneeded, damaged or potentially deleterious cells [49] playing thereby a central role in development and homeostasis. Through the apoptosis-associated genomic mechanism, E2 has been shown to rescue neurons through the induction of anti-apoptotic proteins such as Bcl-2 [50] or suppression of apoptotic proteins such as the Bcl-2-associated X protein [50, 51]. Induction of several gene products that maintain cellular architecture such as neurofilament, tau and microtubulin-associated proteins and many additional genomic pathways potentially associated with E2's neuroprotection have also been described [52]. #### 2.2. Modulation of intracellular signaling by E2 E2 also rapidly induces numerous cellular responses, which cannot be explained by a delayed genomic effect. ERs have been shown to be present in membrane compartments and in the cytoplasm [53, 54]. Specifically, ERα and ERβ are also found as homo or heterodimers at the cell membrane; they are membrane-associated but not actually embedded in the membrane. In addition, a G protein-coupled estrogen receptor (GPER) is localized mainly to intracellular membranes, including the endoplasmic reticulum and Golgi apparatus, under steady-state conditions [55]. G protein-coupled receptors such as GPER can actually signal from intracellular locations [56] and activation results in intracellular Ca2+-mobilization and synthesis of phosphatidylinositol 3,4,5-triphosphate in the nucleus, which could impact gene transcription indirectly. The mitogen-activated protein kinase (MAPK) cascade [57] and the cyclic-AMP-responsive element-binding protein signaling pathway [58, 59] also respond rapidly to E2 and have been implicated in its neuroprotective effects. maintenance of this organelle's integrity could contribute therefore to the neuroprotective action of E2. Essentially, the hormone could minimize mitochondrial dysfunctions, which accompany neurotrauma, aging and neurodegenerative diseases [69]. However, continued research is needed to fully understand molecular details about the apparently complex interactions between ERs and cellular signaling pathways in the context of neuroprotective mechanisms. Figure 3. An example of non-genomic action of E2 involving interaction with intracellular signaling pathways through ERs. As in Figure 2, E2, ER and the nuclear DNA are represented by the filled steroid shapes, rounded rectangles and elongated rectangle indicating a regulated gene promoter (shaded area), respectively, while the ER-interacting protein 17β-Estradiol as a Neuroprotective Agent http://dx.doi.org/10.5772/intechopen.72682 25 The influence of E2 on neuroinflammation, a process commonly accompanying neurotrauma and neurodegenerative diseases [70–72], has been well established. Direct action on microglia and astrocytes (the cellular component of the neuroimmune system) and response to peripheral blood cells' infiltration to the brain have been implicated as major contributors to the observed anti-inflammatory action of the hormone often impacting the cerebral vasculature [73, 74]. For example, E2 can suppress chemokine-mediated induction of the cyclooxygenase-2 (COX-2) pathway in cerebral blood vessels thereby preventing migration of microglia into the brain after an inflammatory challenge [75]. Although inflammatory processes in the brain are usually associated with microglia and astrocytes, expression of the COX-2 gene in neurons and possible mechanisms by which E2 down-regulates this inflammation-associated gene have been shown recently [76]. Specifically, ERβ contributes to neuronal expression of COX-2, and E2 leads to increased recruitment of histone deacetylase 1 (HDAC1), switch-independent 3A (Sin3A) and a concomitant reduction of nuclear factor-κ B (NF-κ B) p65 occupancy and histone 4 acetylation levels. The hormone also prevents the activation of microglia and the recruitment of peripheral monocytes induced by a toxic stimulus. This effect involves ERα activation and reduces the expression of pro-inflammatory mediators and E2 have also shown to prevent morphological changes occurring in microglia during inflammatory response [77]. Decrease of microglial superoxide production and phagocytic activity by both an ER- and 2.3. Anti-inflammatory action (↔) such as PI3K is shown by a shaded oval. An E2-ER complex can also function through cytoplasmic signaling to provide neuroprotection [60]. For example, ERs have been shown to bind in a ligand-dependent manner to the p85 alpha regulatory subunit of phosphatidylinositol 3-kinase (PI3K) [61, 62]. Therefore, stimulation with E2 increases ER-associated PI3K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase. However, modulation of intracellular pathways may occur though the binding of E2 to ERs, or independently of ligand binding [63, 64]. A representative of the non-genomic mechanism involving the modulation of intracellular signaling through ERs is summarized schematically in Figure 3. E2 has been proposed to influence neurotransmission directly by binding to various transmembrane ion channels [65, 66]. Localization of ERβ to the mitochondria has also been shown [67], implicating E2 in the regulation of mitochondrial structure and function in the brain [68]. In addition to estrogen potentially influencing bioenergetics through long-lasting nuclearassociated processes, rapid mitochondria-intrinsic signaling mechanisms that promote the Figure 3. An example of non-genomic action of E2 involving interaction with intracellular signaling pathways through ERs. As in Figure 2, E2, ER and the nuclear DNA are represented by the filled steroid shapes, rounded rectangles and elongated rectangle indicating a regulated gene promoter (shaded area), respectively, while the ER-interacting protein (↔) such as PI3K is shown by a shaded oval. maintenance of this organelle's integrity could contribute therefore to the neuroprotective action of E2. Essentially, the hormone could minimize mitochondrial dysfunctions, which accompany neurotrauma, aging and neurodegenerative diseases [69]. However, continued research is needed to fully understand molecular details about the apparently complex interactions between ERs and cellular signaling pathways in the context of neuroprotective mechanisms. #### 2.3. Anti-inflammatory action a constitutively active and a ligand-dependent function located at the amino-terminus and in the carboxy-terminal ligand-binding domain of ERs, respectively [44]. The DNA-bound dimers recruit co-regulator proteins [45], which can participate in and also recruit many enzymatic and structural proteins permitting the modulation of chromatin structure to facilitate or block gene expression [46]. Additionally, ERE-independent mechanisms have also been shown [47]. Neuroprotective target genes for E2 that directly support vital neuronal functions include neurotrophic factors such as the brain-derived neurotrophic factor [48]. Additional target genes are involved in apoptosis to remove unneeded, damaged or potentially deleterious cells [49] playing thereby a central role in development and homeostasis. Through the apoptosis-associated genomic mechanism, E2 has been shown to rescue neurons through the induction of anti-apoptotic proteins such as Bcl-2 [50] or suppression of apoptotic proteins such as the Bcl-2-associated X protein [50, 51]. Induction of several gene products that maintain cellular architecture such as neurofilament, tau and microtubulin-associated proteins and many additional genomic path- ways potentially associated with E2's neuroprotection have also been described [52]. E2 also rapidly induces numerous cellular responses, which cannot be explained by a delayed genomic effect. ERs have been shown to be present in membrane compartments and in the cytoplasm [53, 54]. Specifically, ERα and ERβ are also found as homo or heterodimers at the cell membrane; they are membrane-associated but not actually embedded in the membrane. In addition, a G protein-coupled estrogen receptor (GPER) is localized mainly to intracellular membranes, including the endoplasmic reticulum and Golgi apparatus, under steady-state conditions [55]. G protein-coupled receptors such as GPER can actually signal from intracellular locations [56] and activation results in intracellular Ca2+-mobilization and synthesis of phosphatidylinositol 3,4,5-triphosphate in the nucleus, which could impact gene transcription indirectly. The mitogen-activated protein kinase (MAPK) cascade [57] and the cyclic-AMP-responsive element-binding protein signaling pathway [58, 59] also respond rapidly to An E2-ER complex can also function through cytoplasmic signaling to provide neuroprotection [60]. For example, ERs have been shown to bind in a ligand-dependent manner to the p85 alpha regulatory subunit of phosphatidylinositol 3-kinase (PI3K) [61, 62]. Therefore, stimulation with E2 increases ER-associated PI3K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase. However, modulation of intracellular pathways may occur though the binding of E2 to ERs, or independently of ligand binding [63, 64]. A representative of the non-genomic mechanism involving the modulation of intracellular sig- E2 has been proposed to influence neurotransmission directly by binding to various transmembrane ion channels [65, 66]. Localization of ERβ to the mitochondria has also been shown [67], implicating E2 in the regulation of mitochondrial structure and function in the brain [68]. In addition to estrogen potentially influencing bioenergetics through long-lasting nuclearassociated processes, rapid mitochondria-intrinsic signaling mechanisms that promote the 2.2. Modulation of intracellular signaling by E2 24 Sex Hormones in Neurodegenerative Processes and Diseases E2 and have been implicated in its neuroprotective effects. naling through ERs is summarized schematically in Figure 3. The influence of E2 on neuroinflammation, a process commonly accompanying neurotrauma and neurodegenerative diseases [70–72], has been well established. Direct action on microglia and astrocytes (the cellular component of the neuroimmune system) and response to peripheral blood cells' infiltration to the brain have been implicated as major contributors to the observed anti-inflammatory action of the hormone often impacting the cerebral vasculature [73, 74]. For example, E2 can suppress chemokine-mediated induction of the cyclooxygenase-2 (COX-2) pathway in cerebral blood vessels thereby preventing migration of microglia into the brain after an inflammatory challenge [75]. Although inflammatory processes in the brain are usually associated with microglia and astrocytes, expression of the COX-2 gene in neurons and possible mechanisms by which E2 down-regulates this inflammation-associated gene have been shown recently [76]. Specifically, ERβ contributes to neuronal expression of COX-2, and E2 leads to increased recruitment of histone deacetylase 1 (HDAC1), switch-independent 3A (Sin3A) and a concomitant reduction of nuclear factor-κ B (NF-κ B) p65 occupancy and histone 4 acetylation levels. The hormone also prevents the activation of microglia and the recruitment of peripheral monocytes induced by a toxic stimulus. This effect involves ERα activation and reduces the expression of pro-inflammatory mediators and E2 have also shown to prevent morphological changes occurring in microglia during inflammatory response [77]. Decrease of microglial superoxide production and phagocytic activity by both an ER- and MAPK-dependent pathway have also been reported among the anti-inflammatory effects of E2 [78]. In addition, the hormone inhibits pro-inflammatory gene expression by controlling intracellular localization of NF-κ B [79]. #### 2.4. Antioxidant effects Oxidative stress-induced damage has been linked to brain aging [80], neurodegenerative diseases [81] and neurotrauma [25, 26]. From a long time, therapeutic antioxidant interventions have been proposed to reduce the detrimental impact of oxidative stress [82]. E2's ER-independent antioxidant effects are mainly due to its ability to attenuate free-radical reactions [83], although indirect mechanisms such as up regulation of antioxidant enzymes [84, 85] and chelation of redox-active metal ions [86] have been reported. The neuroprotective effect of the hormone through direct oxidative stress reduction has been recognized in part by structure-activity relationship studies [87–89]. Acute E2 neuroprotection in ischemic brain [90] or against damage by ionizing radiation [91] may be largely conferred through antioxidant mechanisms. Our laboratory pioneered in recognizing a complementary novel neuroprotective antioxidant cycle that involves a para-quinol as a molecular intermediate of oxyradical scavenging and, then, NADPH-mediated enzyme-catalyzed reductive aromatization [98–100] to regenerate E2, as shown in Figure 5. We wish to name this previously unrecognized antioxidant cycle for simple phenolic antioxidants as the "Prokai antioxidant cycle." The enzyme activity driving the reductive phase of the cycle is observed predominantly in neuronal tissue [101]. Beyond its mechanistic significance regarding oxidative stress-reducing effect, this discovery has prompted a strategy for brain-selective estrogen therapy using a prodrug approach detailed in the following section. Figure 5. The Prokai antioxidant cycle for E2 through the formation of a para-quinol (10β,17β-dihydroxyestra-1,4-dien-3-one, DHED) as an intermediate, which is reduced to the parent hormone by enzyme-catalyzed reduction involving 17β-Estradiol as a Neuroprotective Agent http://dx.doi.org/10.5772/intechopen.72682 27 The pathophysiology of neurodegeneration in the central nervous system is complex and multifactorial in nature [102]. Therefore, it is not surprising that an agent like E2 can provide robust protection against a myriad of neuronal insults owing to its broad-spectrum activity resulting from well-orchestrated genomic and non-genomic actions, as detailed in Section 2. The need for clinical therapeutic interventions that can be used to target multiple parallel mechanisms of neuronal injury has been repeatedly expressed [101–103]. We argue that, despite profound dichotomy between basic science and clinical studies, E2 is ideally suited to be developed as a broad-spectrum neuroprotectant if its action can be restricted to the CNS, that is, to the site of action to avoid undesirable peripheral hormonal burdens. Since neurotrauma triggers a cascade of biochemical events leading to further damages decreasing thereby the chance of appreciable functional recovery [17, 20, 102, 103], chronic pharmacotherapeutic interventions should be considered in the context of translational research. This, on the other hand, brings about critical considerations for safety and efficacy, which highlights the need for brain-selective (or in general CNS-selective) neurotherapy, considering both a preventative and a curative modality. When estrogen neurotherapy is considered, however, one cannot ignore the (in)famous Women's Health Initiative (WHI) study [104]. This was a placebo-controlled, randomized trial of hormone "replacement" therapy in postmenopausal women that indicated detrimental consequences of estrogen and progesterone supplementations, among others, for brain 3. CNS-selective estrogen neurotherapy NADPH as cofactor. The quintessential feature of estrogens as neuroprotective antioxidants is their phenolic A-ring [83, 92, 93]. Because of its lipophilicity, E2 concentrates in lipid-rich regions of the cell such as cellular membranes [94]. Therefore, it is likely that estrogens act in vivo as a highly localized antioxidant [83]. The mechanism of direct oxyradical-scavenging by E2 functioning as a phenolic antioxidant is shown schematically in Figure 4. The process involves H-atom transfer that causes an interruption of free-radical chain reactions, such as lipid peroxidation (R = LOO, where L represents a lipid). Estrogens, indeed, reduce lipid peroxidation in cells and tissues of the CNS [95]. However, the chain-breaking reaction leaves behind a radical product (phenoxyl radical) whose fate has to be explained in consideration of an efficient antioxidant action observed both in vitro and in vivo. Indeed, phenolic antioxidants can be regenerated from the corresponding phenoxyl radicals by a reaction with ascorbic acid (vitamin C) [96] or through glutathione-dependent free-radical reductase [97]; therefore, a continuous antioxidant cycle is established by E2. Figure 4. E2's effect through the classical phenolic antioxidant mechanism. The solid arrows represent the chainbreaking H-atom transfer, such as lipid peroxidation, while the dashed arrows indicate the conversion of the E2-derived phenoxyl radical back to the phenolic compound by an endogenous reductant (AH) such as ascorbic acid or glutathione. Figure 5. The Prokai antioxidant cycle for E2 through the formation of a para-quinol (10β,17β-dihydroxyestra-1,4-dien-3-one, DHED) as an intermediate, which is reduced to the parent hormone by enzyme-catalyzed reduction involving NADPH as cofactor. Our laboratory pioneered in recognizing a complementary novel neuroprotective antioxidant cycle that involves a para-quinol as a molecular intermediate of oxyradical scavenging and, then, NADPH-mediated enzyme-catalyzed reductive aromatization [98–100] to regenerate E2, as shown in Figure 5. We wish to name this previously unrecognized antioxidant cycle for simple phenolic antioxidants as the "Prokai antioxidant cycle." The enzyme activity driving the reductive phase of the cycle is observed predominantly in neuronal tissue [101]. Beyond its mechanistic significance regarding oxidative stress-reducing effect, this discovery has prompted a strategy for brain-selective estrogen therapy using a prodrug approach detailed in the following section. #### 3. CNS-selective estrogen neurotherapy MAPK-dependent pathway have also been reported among the anti-inflammatory effects of E2 [78]. In addition, the hormone inhibits pro-inflammatory gene expression by controlling Oxidative stress-induced damage has been linked to brain aging [80], neurodegenerative diseases [81] and neurotrauma [25, 26]. From a long time, therapeutic antioxidant interventions have been proposed to reduce the detrimental impact of oxidative stress [82]. E2's ER-independent antioxidant effects are mainly due to its ability to attenuate free-radical reactions [83], although indirect mechanisms such as up regulation of antioxidant enzymes [84, 85] and chelation of redox-active metal ions [86] have been reported. The neuroprotective effect of the hormone through direct oxidative stress reduction has been recognized in part by structure-activity relationship studies [87–89]. Acute E2 neuroprotection in ischemic brain [90] or against damage by ionizing radiation [91] may be largely conferred through antioxi- The quintessential feature of estrogens as neuroprotective antioxidants is their phenolic A-ring [83, 92, 93]. Because of its lipophilicity, E2 concentrates in lipid-rich regions of the cell such as cellular membranes [94]. Therefore, it is likely that estrogens act in vivo as a highly localized antioxidant [83]. The mechanism of direct oxyradical-scavenging by E2 functioning The process involves H-atom transfer that causes an interruption of free-radical chain reactions, such as lipid peroxidation (R = LOO, where L represents a lipid). Estrogens, indeed, reduce lipid peroxidation in cells and tissues of the CNS [95]. However, the chain-breaking reaction leaves behind a radical product (phenoxyl radical) whose fate has to be explained in consideration of an efficient antioxidant action observed both in vitro and in vivo. Indeed, phenolic antioxidants can be regenerated from the corresponding phenoxyl radicals by a reaction with ascorbic acid (vitamin C) [96] or through glutathione-dependent free-radical reductase Figure 4. E2's effect through the classical phenolic antioxidant mechanism. The solid arrows represent the chainbreaking H-atom transfer, such as lipid peroxidation, while the dashed arrows indicate the conversion of the E2-derived phenoxyl radical back to the phenolic compound by an endogenous reductant (AH) such as ascorbic acid or glutathione. as a phenolic antioxidant is shown schematically in Figure 4. [97]; therefore, a continuous antioxidant cycle is established by E2. intracellular localization of NF-κ B [79]. 26 Sex Hormones in Neurodegenerative Processes and Diseases 2.4. Antioxidant effects dant mechanisms. The pathophysiology of neurodegeneration in the central nervous system is complex and multifactorial in nature [102]. Therefore, it is not surprising that an agent like E2 can provide robust protection against a myriad of neuronal insults owing to its broad-spectrum activity resulting from well-orchestrated genomic and non-genomic actions, as detailed in Section 2. The need for clinical therapeutic interventions that can be used to target multiple parallel mechanisms of neuronal injury has been repeatedly expressed [101–103]. We argue that, despite profound dichotomy between basic science and clinical studies, E2 is ideally suited to be developed as a broad-spectrum neuroprotectant if its action can be restricted to the CNS, that is, to the site of action to avoid undesirable peripheral hormonal burdens. Since neurotrauma triggers a cascade of biochemical events leading to further damages decreasing thereby the chance of appreciable functional recovery [17, 20, 102, 103], chronic pharmacotherapeutic interventions should be considered in the context of translational research. This, on the other hand, brings about critical considerations for safety and efficacy, which highlights the need for brain-selective (or in general CNS-selective) neurotherapy, considering both a preventative and a curative modality. When estrogen neurotherapy is considered, however, one cannot ignore the (in)famous Women's Health Initiative (WHI) study [104]. This was a placebo-controlled, randomized trial of hormone "replacement" therapy in postmenopausal women that indicated detrimental consequences of estrogen and progesterone supplementations, among others, for brain health, propagating thereby a dogma that all estrogens (and progestins) are "created equal." The fact is that WHI did not use human hormones and, thus, did not study the effect of hormone replacement per se in aging women. On the contrary, conjugated equine estrogens (CEE) and a synthetic progestin were used for women with intact uterus. CEE is a complex mixture of over 60 different estrogens from pregnant mares' urine, and it only contains a small amount of E2: the main constituents are the sulfate esters of B-ring saturated and unsaturated estrogens [105]. The pharmacokinetic and toxicology profiles of these non-human estrogens are different from those of E2; therefore, direct comparison between E2 and CEE is fundamentally unjustified [106, 107]. Accordingly, the beneficial central effects of E2, including robust neuroprotection based on clinical, epidemiological and basic science observations should not be undermined in view of the confusion brought about by the WHI studies. dehydrogenase/reductase metabolized DHED to E2 (Figure 6), while prodrug activation did not occur in the periphery. This is an unprecedented and distinguishing feature of DHED in the context of translational research [101]. With a series of in vitro and in vivo studies, we showed that with DHED, for the first time, E2's actions can be restricted to the brain independently of the route and duration of DHED administration and, therefore, it can be used (at least in preclinical settings) for the efficacious treatment of estrogen-responsive and centrally regulated maladies and injuries, including neurodegeneration brought about by ischemic Figure 6. Schematic illustration of DHED bioprecursor prodrug's CNS-selective enzymatic metabolism to E2 via an 17β-Estradiol as a Neuroprotective Agent http://dx.doi.org/10.5772/intechopen.72682 29 The transient middle cerebral artery occlusion (tMCAO) model followed by reperfusion is one the most frequently used preclinical animal models for testing an agent for its ability to act as a neuroprotectant, that is, to reduce infarct volume and aid in functional recovery [20, 24, 101, 111]. As Figure 7 shows, a dose-dependent reduction of infarct volumes and neurological deficits was observed in DHED-treated animals. Moreover, about 10-times higher systemic E2 (i.e., the parent drug that is formed in the brain from DHED) dose was needed to achieve the same neuroprotection indicating the profound ability of the bioprecursor prodrug to enter into the brain from the circulation and, then, produce E2 within the brain, and only in Figure 7. Dose-dependent (A) brain infarct volumes and (B) neurological deficit (ND) scores in rats treated with DHED 1 h before tMCAO followed by 24-h reperfusion [101]. The control groups received E2 (200 μg/kg, s.c., approximately representing ED50, equivalent to 50% of the maximum effect) or vehicle alone. ©Reproduced with permission by the American Association for the Advancement of Science. stroke, without hormonal burdens for the rest of the body [101]. NADPH-dependent reductase. Nevertheless, an E2-based neurotherapy cannot be realized in clinical settings until E2's actions are restricted to the site of action assuring therapeutic safety and efficacy. Currently approved E2 dosage forms expose the entire body to the hormone through the circulation, potentially leading to detrimental side-effects including cardiovascular problems and the development of certain type of cancers upon chronic administration that is required for long-term neuroprotection and functional recovery after neurotrauma. Feminization (e.g., gynecomastia) is also a critical negative aspect of estrogen therapy, especially in case of children and males. Early attempts to restrict E2's actions to the brain upon systemic administration included the so-called chemical delivery system, which was conceptually a complex prodrug approach carrying a 1,4-dihydrotrigonellyl promoiety and is capable to usher the hormone through the blood brain barrier (BBB). Once in the brain, the prodrug is oxidized analogously to that of NADP(H) ⇌ NADP<sup>+</sup> , locking thereby the oxidized prodrug into the brain before it releases E2 [108]. This approach does result in significantly increased brain-enhanced delivery of the hormone compared to that of simple prodrugs of E2; however, it still results in sufficient increase in circulating E2 that can produce unwanted peripheral hormonal burdens [10, 109]. Prodrugs are inert precursors of their corresponding biologically active parent drugs and they traditionally carry auxiliary bioreversible "promoiety(ies)" that are removed enzymatically (rarely via chemical reaction, such as pH-dependent hydrolysis) in the body [108]. An important development in achieving a true CNS-selective estrogen therapy has been achieved by our laboratory [101] and was derived from our previous discovery of a novel antioxidant cycle for estrogens we call the Prokai antioxidant cycle for simple phenolic antioxidants [98–100], and detailed in Section 2. We recognized that 10β,17β-dihydroxyestra-1,4 dien-3-one (DHED, Figures 5 and 6), which is chemically a para-quinol (not to be mistaken for quinones involved in E2-induced carcinogenesis through redox cycling [99]), can be reductively rearomatized to the parent E2 and, thus, could serve as a bioprecursor prodrug for E2. The lesser-known bioprecursor prodrugs do not carry auxiliary promoiety(ies) [108] because the bioreversible chemical manipulation is carried out within the drug molecule itself [101, 110]. Therefore, creation of bioprecursor prodrugs, such as DHED, requires significantly greater innovation than that of simple prodrugs; moreover, potential toxicity issues that may arise from the release of the "promoity(ies)" from simple prodrugs is eliminated with bioprecursor prodrugs. Indeed, we have established that CNS-specific and NADPH-dependent health, propagating thereby a dogma that all estrogens (and progestins) are "created equal." The fact is that WHI did not use human hormones and, thus, did not study the effect of hormone replacement per se in aging women. On the contrary, conjugated equine estrogens (CEE) and a synthetic progestin were used for women with intact uterus. CEE is a complex mixture of over 60 different estrogens from pregnant mares' urine, and it only contains a small amount of E2: the main constituents are the sulfate esters of B-ring saturated and unsaturated estrogens [105]. The pharmacokinetic and toxicology profiles of these non-human estrogens are different from those of E2; therefore, direct comparison between E2 and CEE is fundamentally unjustified [106, 107]. Accordingly, the beneficial central effects of E2, including robust neuroprotection based on clinical, epidemiological and basic science observations should not Nevertheless, an E2-based neurotherapy cannot be realized in clinical settings until E2's actions are restricted to the site of action assuring therapeutic safety and efficacy. Currently approved E2 dosage forms expose the entire body to the hormone through the circulation, potentially leading to detrimental side-effects including cardiovascular problems and the development of certain type of cancers upon chronic administration that is required for long-term neuroprotection and functional recovery after neurotrauma. Feminization (e.g., gynecomastia) is also a Early attempts to restrict E2's actions to the brain upon systemic administration included the so-called chemical delivery system, which was conceptually a complex prodrug approach carrying a 1,4-dihydrotrigonellyl promoiety and is capable to usher the hormone through the blood brain barrier (BBB). Once in the brain, the prodrug is oxidized analogously to that of E2 [108]. This approach does result in significantly increased brain-enhanced delivery of the hormone compared to that of simple prodrugs of E2; however, it still results in sufficient increase in circulating E2 that can produce unwanted peripheral hormonal burdens [10, 109]. Prodrugs are inert precursors of their corresponding biologically active parent drugs and they traditionally carry auxiliary bioreversible "promoiety(ies)" that are removed enzymatically An important development in achieving a true CNS-selective estrogen therapy has been achieved by our laboratory [101] and was derived from our previous discovery of a novel antioxidant cycle for estrogens we call the Prokai antioxidant cycle for simple phenolic antioxidants [98–100], and detailed in Section 2. We recognized that 10β,17β-dihydroxyestra-1,4 dien-3-one (DHED, Figures 5 and 6), which is chemically a para-quinol (not to be mistaken for quinones involved in E2-induced carcinogenesis through redox cycling [99]), can be reductively rearomatized to the parent E2 and, thus, could serve as a bioprecursor prodrug for E2. The lesser-known bioprecursor prodrugs do not carry auxiliary promoiety(ies) [108] because the bioreversible chemical manipulation is carried out within the drug molecule itself [101, 110]. Therefore, creation of bioprecursor prodrugs, such as DHED, requires significantly greater innovation than that of simple prodrugs; moreover, potential toxicity issues that may arise from the release of the "promoity(ies)" from simple prodrugs is eliminated with bioprecursor prodrugs. Indeed, we have established that CNS-specific and NADPH-dependent , locking thereby the oxidized prodrug into the brain before it releases critical negative aspect of estrogen therapy, especially in case of children and males. (rarely via chemical reaction, such as pH-dependent hydrolysis) in the body [108]. NADP(H) ⇌ NADP<sup>+</sup> be undermined in view of the confusion brought about by the WHI studies. 28 Sex Hormones in Neurodegenerative Processes and Diseases Figure 6. Schematic illustration of DHED bioprecursor prodrug's CNS-selective enzymatic metabolism to E2 via an NADPH-dependent reductase. dehydrogenase/reductase metabolized DHED to E2 (Figure 6), while prodrug activation did not occur in the periphery. This is an unprecedented and distinguishing feature of DHED in the context of translational research [101]. With a series of in vitro and in vivo studies, we showed that with DHED, for the first time, E2's actions can be restricted to the brain independently of the route and duration of DHED administration and, therefore, it can be used (at least in preclinical settings) for the efficacious treatment of estrogen-responsive and centrally regulated maladies and injuries, including neurodegeneration brought about by ischemic stroke, without hormonal burdens for the rest of the body [101]. The transient middle cerebral artery occlusion (tMCAO) model followed by reperfusion is one the most frequently used preclinical animal models for testing an agent for its ability to act as a neuroprotectant, that is, to reduce infarct volume and aid in functional recovery [20, 24, 101, 111]. As Figure 7 shows, a dose-dependent reduction of infarct volumes and neurological deficits was observed in DHED-treated animals. Moreover, about 10-times higher systemic E2 (i.e., the parent drug that is formed in the brain from DHED) dose was needed to achieve the same neuroprotection indicating the profound ability of the bioprecursor prodrug to enter into the brain from the circulation and, then, produce E2 within the brain, and only in Figure 7. Dose-dependent (A) brain infarct volumes and (B) neurological deficit (ND) scores in rats treated with DHED 1 h before tMCAO followed by 24-h reperfusion [101]. The control groups received E2 (200 μg/kg, s.c., approximately representing ED50, equivalent to 50% of the maximum effect) or vehicle alone. ©Reproduced with permission by the American Association for the Advancement of Science. the brain. In the context of translational research, it is noteworthy that the capacity to generate E2 from DHED is not lost in an injured brain, as neuroprotection was highly preserved poststroke and, again, no hormonal exposure to the rest of the body was observed [101]. References er.2012-1055 1998;23:963-987 molmed.2012.12.007 10.1016/j.steroids.2003.08.012 Neuroscience. 2003;23:8701-8705 srep30721s jsbmb.2005.02.016 Acta. 2013;S7:002. DOI: 10.4172/2153-2435.S7-002 [1] Turgeon JL, Carr MC, Maki PM, Mendelsohn ME, Wise PM. Complex actions of sex steroids in adipose tissue, the cardiovascular system, and brain: Insights from basic science and clinical studies. Endocrine Reviews. 2006;27:575-605. DOI: 10.1210/er.2005-0020 17β-Estradiol as a Neuroprotective Agent http://dx.doi.org/10.5772/intechopen.72682 31 [2] Fink G, Sumner BEH, Rosie R, Grace O, Quinn JP. Estrogen control of central neurotransmission: Effect on mood, mental state, and memory. Cellular and Molecular Neuro- [3] Mauvais-Jarvis F, Clegg DJ, Hevener AL. The role of estrogens in control of energy balance and glucose homeostasis. Endocrine Reviews. 2013;34:309-338. DOI: 10.1210/ [4] Gruber CJ, Tschugguel W, Schneeberger C, Huber JC. Production and actions of estrogens. New England Journal of Medicine. 2002;346:340-352. DOI: 10.1056/NEJMra000471 [5] Baulieu EE. Neurosteroids: A novel function of the brain. Psychoneuroendocrinology. [6] Cui J, Shen Y, Li R. Estrogen synthesis and signaling pathways during aging: From periphery to brain. Trends in Molecular Medicine. 2013;19:197-209. DOI: 10.1016/j. [7] Barakat R, Oakley O, Kim H, Jin J, Ko CJ. Extra-gonadal sites of estrogen biosynthesis and function. BMB Reports. 2016;49:488-496. DOI: 10.5483/BMBRep.2016.49.9.141 [8] Simpson ER. Sources of estrogen and their importance. Journal of Steroid Biochemistry [9] Stanczyk FZ, Cho MM, Endres DB, Morrison JL, Patel S, Paulson RJ. Limitations of direct estradiol and testosterone immunoassay kits. Steroids. 2003;68:1173-1178. DOI: [10] Prokai-Tatrai K, Szarka S, Nguyen V, Sahyouni F, Walker C, White S, Talamantes T, Prokai L. "All in the mind?" brain-targeting chemical delivery system of 17β-estradiol (Estredox) produces significant uterotrophic side effect. Pharmaceutical and Analytical [11] Merchenthaler I, Lane M, Sabnis G, Brodie A, Nguyen V, Prokai L, Prokai-Tatrai K. Treatment with an orally bioavailable prodrug of 17β-estradiol alleviates hot flushes without hormonal effects in the periphery. Scientific Reports. 2016;6:30721. DOI: 10.1038/ [12] Mc Cullough LD, Blizzard K, Simpson ER, Öz OK, Hurn PD. Aromatase cytochrome P450 and extragonadal estrogen play a role in ischemic neuroprotection. Journal of [13] Carswell HV, Dominiczak AF, Garcia-Segura LM, Harada N, Hutchison JB, Macrae IM. Brain aromatase expression after experimental stroke: Topography and time course. The Journal of Steroid Biochemistry and Molecular Biology. 2005;96:89-91. DOI: 10.1016/j. & Molecular Biology. 2003;86:225-230. DOI: 10.1016/S0960-0760(03)00360-1 biology. 1996;16:325-344. DOI: 10.1007/bf02088099 #### 4. Conclusion As neurotrauma and neurodegeneration in the CNS are complex and multifactorial in nature requiring therefore broad-spectrum therapeutic interventions, E2 is an attractive lead agent to address unmet medical needs in this field. The powerful antioxidant action of E2 against oxidative stress owing to its phenolic A-ring is unique among neurosteroids with potential neuroprotective roles; therefore, non-genomic mechanisms contribute significantly to the overall neuroprotection. This chapter presented an overview of our current knowledge on the well-orchestrated genomic and non-genomic events by which E2 could beneficially counteract the initiation and/or progression of neuronal cell death. However, there has been incongruency between basic science and clinical studies in terms of estrogen therapy impacting the brain because most researchers ignore the requirement to confine E2's actions into the CNS upon systemic administration to ensure therapeutic safety and efficacy. We highlighted here a novel and unique bioprecursor prodrug approach our laboratory pioneered for brain-selective delivery of E2 without exposing the rest of the body to unwanted hormonal burden. #### Acknowledgements The project was supported in part by the National Institutes of Health for the authors, in particular grant numbers NS044765, AG031535, MH100700, HD078077 & CA215550 to L.P. and AG031421 & EY027005 to K.P.-T. and by the Robert A. Welch Foundation (endowment BK-0031 to L.P.). #### Conflict of interest The authors are inventors in the patents covering the use of 10β,17β-dihydroxyestra-1,4-dien-3 one (DHED) as a CNS-selective bioprecursor prodrug of 17β-estradiol and are co-founders of AgyPharma LLC with equity in the company that licensed the patents. #### Author details Katalin Prokai-Tatrai\* and Laszlo Prokai \Address all correspondence to: [email protected] Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA #### References* the brain. In the context of translational research, it is noteworthy that the capacity to generate E2 from DHED is not lost in an injured brain, as neuroprotection was highly preserved post- As neurotrauma and neurodegeneration in the CNS are complex and multifactorial in nature requiring therefore broad-spectrum therapeutic interventions, E2 is an attractive lead agent to address unmet medical needs in this field. The powerful antioxidant action of E2 against oxidative stress owing to its phenolic A-ring is unique among neurosteroids with potential neuroprotective roles; therefore, non-genomic mechanisms contribute significantly to the overall neuroprotection. This chapter presented an overview of our current knowledge on the well-orchestrated genomic and non-genomic events by which E2 could beneficially counteract the initiation and/or progression of neuronal cell death. However, there has been incongruency between basic science and clinical studies in terms of estrogen therapy impacting the brain because most researchers ignore the requirement to confine E2's actions into the CNS upon systemic administration to ensure therapeutic safety and efficacy. We highlighted here a novel and unique bioprecursor prodrug approach our laboratory pioneered for brain-selective delivery of E2 without exposing the rest of the body to The project was supported in part by the National Institutes of Health for the authors, in particular grant numbers NS044765, AG031535, MH100700, HD078077 & CA215550 to L.P. and AG031421 & EY027005 to K.P.-T. and by the Robert A. Welch Foundation (endowment The authors are inventors in the patents covering the use of 10β,17β-dihydroxyestra-1,4-dien-3 one (DHED) as a CNS-selective bioprecursor prodrug of 17β-estradiol and are co-founders of Department of Pharmacology and Neuroscience, University of North Texas Health Science AgyPharma LLC with equity in the company that licensed the patents. \Address all correspondence to: [email protected] stroke and, again, no hormonal exposure to the rest of the body was observed [101]. 4. 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DOI: 10.3390/ijms14011443 38 Sex Hormones in Neurodegenerative Processes and Diseases 10.1111/j.1471-4159.2011.07334.x 9230(01)00483-X Chapter 3 Provisional chapter Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Among the neuroactive steroids, dehydroepiandrosterone (3b-hydroxyandrost-5-ene-17-one, [DHEA]) and its sulfated metabolite DHEA sulfate (DHEAS) have been shown to be potent modulators of neural function, including neurogenesis, neuronal growth and differentiation, and neuroprotection. Highlighting the potential health significance of DHEA and DHEAS in humans, serum concentrations decrease steadily with age, with lowest concentrations present at the time many diseases of aging and neurodegeneration become apparent. This temporal association has led to the suggestion that pathology associated with cognitive decline, age-related neurological disorders such as Alzheimer's disease, dementia, amyotrophic lateral sclerosis (ALS), and adult onset schizophrenia may, in part at least, be attributed to decreased secretion of DHEA. Animal studies suggest neuroprotective functions for DHEA and DHEAS through reduction of glutamate-induced excitotoxicity. Reduced myelin loss and reactive gliosis after spinal cord injury by DHEA treatment also suggest a role for DHEA in the treatment of white matter pathologies such as multiple sclerosis. In this chapter, we discuss the physiological roles of DHEA and DHEAS in the central nervous system (CNS), their potential as neuroprotective hormones with reference to documented effects on excitotoxicity and oxidative stress, and their anti-glucocorticoid actions during chronic stress. The potential for metabolic derivatives of DHEA, such as estrogens and testosterone on brain function, and their contribution to neurodevelopment and neurodegenerative conditions are also Dehydroepiandrosterone (DHEA) and DHEA Sulfate: DOI: 10.5772/intechopen.71141 © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. and reproduction in any medium, provided the original work is properly cited. Keywords: adrenal zona reticularis, adrenarche, adrenopause, aging, Alzheimer's disease, amyotrophic lateral sclerosis, androgens, C19 steroids, glucocorticoids, neurocognitive decline, neurogenesis, neuroprotection, estrogens, schizophrenia, Roles in Brain Function and Disease Roles in Brain Function and Disease Tracey A. Quinn, Stephen R. Robinson and Tracey A. Quinn, Stephen R. Robinson and Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.71141 David Walker Abstract discussed. steroid biosynthesis David Walker Provisional chapter #### Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Roles in Brain Function and Disease Roles in Brain Function and Disease Dehydroepiandrosterone (DHEA) and DHEA Sulfate: DOI: 10.5772/intechopen.71141 Tracey A. Quinn, Stephen R. Robinson and David Walker David Walker Additional information is available at the end of the chapter Tracey A. Quinn, Stephen R. Robinson and Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.71141 #### Abstract Among the neuroactive steroids, dehydroepiandrosterone (3b-hydroxyandrost-5-ene-17-one, [DHEA]) and its sulfated metabolite DHEA sulfate (DHEAS) have been shown to be potent modulators of neural function, including neurogenesis, neuronal growth and differentiation, and neuroprotection. Highlighting the potential health significance of DHEA and DHEAS in humans, serum concentrations decrease steadily with age, with lowest concentrations present at the time many diseases of aging and neurodegeneration become apparent. This temporal association has led to the suggestion that pathology associated with cognitive decline, age-related neurological disorders such as Alzheimer's disease, dementia, amyotrophic lateral sclerosis (ALS), and adult onset schizophrenia may, in part at least, be attributed to decreased secretion of DHEA. Animal studies suggest neuroprotective functions for DHEA and DHEAS through reduction of glutamate-induced excitotoxicity. Reduced myelin loss and reactive gliosis after spinal cord injury by DHEA treatment also suggest a role for DHEA in the treatment of white matter pathologies such as multiple sclerosis. In this chapter, we discuss the physiological roles of DHEA and DHEAS in the central nervous system (CNS), their potential as neuroprotective hormones with reference to documented effects on excitotoxicity and oxidative stress, and their anti-glucocorticoid actions during chronic stress. The potential for metabolic derivatives of DHEA, such as estrogens and testosterone on brain function, and their contribution to neurodevelopment and neurodegenerative conditions are also discussed. Keywords: adrenal zona reticularis, adrenarche, adrenopause, aging, Alzheimer's disease, amyotrophic lateral sclerosis, androgens, C19 steroids, glucocorticoids, neurocognitive decline, neurogenesis, neuroprotection, estrogens, schizophrenia, steroid biosynthesis Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons #### 1. Introduction Dehydroepiandrosterone (DHEA) is the principal carbon (C)-19 steroid produced by the adrenal gland in humans and mammals [1]. DHEA and its sulfated derivative DHEAS are multifunctional steroids with actions in a wide variety of physiological systems, with effects on the brain [2], immune systems [3], and somatic growth and development [4, 5]. Although DHEA and DHEAS were identified more than 50 years ago, there remains some uncertainty as to their physiological significance, full mechanisms of action [6–9], and their roles in human disease. 2. The physiology of DHEA 17,20-lyase activity of P450c17 [33–35]. Greaves et al. [31]. In humans, DHEA is one of the most abundant hormones synthesized and secreted by the adrenal cortex. This C19 steroid displays an episodic and diurnal rhythm of synthesis and release that parallels that of cortisol [29, 30]. The major synthetic pathways for DHEA and DHEAS are shown in Figure 1. The de novo synthesis of DHEA from cholesterol depends on the presence and activity of the mitochondrial enzyme steroidogenic acute regulatory protein (StAR), the microsomal enzyme cytochrome P450 enzyme 17α-hydroxylase /17,20 lyase (P450c17), and the accessory hemoprotein cytochrome b5 (Cytb5) [32]. Importantly, P450c17 and Cytb5 need to be colocalized, because the function of Cytb5 is to selectively enhance the Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Roles in Brain Function and Disease http://dx.doi.org/10.5772/intechopen.71141 43 DHEAS is the precursor of approximately 50% of androgens in adult men, 75% of active estrogens in premenopausal women, and almost 100% of active estrogens after menopause [36]. DHEA has a 3- to 10-fold predominance of androgenic over estrogenic activity [37], and although a small portion of the circulating pool of DHEA is of gonadal origin in men and women, the majority of DHEA, and virtually all DHEAS, is produced by the adrenal cortex [1]. However, DHEA is also synthesized in the brain, from cholesterol and other hormonal precursors, primarily by astrocytes and oligodendrocytes; indeed, much higher concentrations of DHEAS are found in the brain than in the serum, suggesting that the DHEAS is primarily synthesized in situ, rather than being transported across the blood-brain barrier [38]. Figure 1. The complete steroid pathway showing the formation of DHEA from pregnenolone and 17OH-pregnenolone, and its reversible sulfation, and disposition via androstenes to estradiol and 5α-dihydroxytestosterone. Steroid metabolites identified in serum and urines are shown in light gray boxes and dark gray boxes, respectively. From In humans, DHEA is a crucial precursor of sex steroid biosynthesis and exerts indirect endocrine and intracrine actions following conversion to androgens and estrogens. In addition, DHEA acts as a neurosteroid via its effects on neurotransmitter receptors in the brain. The potential health significance of DHEA in humans is highlighted by the observation that serum concentrations decrease steadily with age, approaching lowest concentrations around the time at which many diseases of aging, particularly neurocognitive decline, become apparent. The age-related decline in DHEA levels [10] has led to the suggestion that this is associated with a decrease in cognitive function as well as the increased rates of neuronal degeneration and dysfunction that occur during aging [11, 12]. Other studies have reported altered DHEA serum concentrations in patients with conditions such as schizophrenia [13], dementia [14], and Alzheimer's disease (AD) [13, 15–18]. Due to these associations, DHEAS has been widely publicized both in the lay press [19, 20] and in the scientific literature [21, 22] for their putative anti-aging and neuroprotective effects. This has sparked controversial speculation that DHEA treatment might be a remedy for neuropsychiatric and neurodegenerative disorders [7, 23–27] and, even more optimistically, that it is a hormone with the potential to increase the life span [28]. As promising as these speculations may seem, there are many contradictions about the roles of DHEA in normal and degenerative brain function. This is especially evident when comparing preclinical and clinical data. For example, studies in animals show a myriad of neuroprotective and trophic effects of DHEAS in development and disease, while clinical studies show inconsistent, and sometimes highly conflicting, results. Clinical studies of neurodegenerative diseases have variously reported increased or decreased DHEAS concentrations in serum, cerebrospinal fluid, and brain tissue, leading to doubt as to the role of DHEA in the neuropathology of aging. It has been suggested that the incongruity in measured DHEAS concentrations may lie in the methodological differences used to sample DHEAS; however, it is possible that these changes are indicative of a more nuanced and multifaceted role. There is consistent evidence that DHEA is neuroprotective with respect to oxidative stress, neuroinflammation, and excitotoxicity, and thus it is possible that DHEA assists the defense of the brain and has a beneficial effect on cognition in healthy brains. Therefore, it is the aim of this review to briefly discuss the physiology of DHEA and its synthesis and secretion during development and aging and to discuss the relationship between alterations in DHEA concentrations and cognition. We further discuss the possible role of DHEAS in a variety of disease states, including AD, and acute illnesses such as schizophrenia, with focus on the fact that these conditions are characterized by imbalances in oxidative stress, neuroinflammation, and excitotoxicity. ### 2. The physiology of DHEA 1. Introduction 42 Sex Hormones in Neurodegenerative Processes and Diseases Dehydroepiandrosterone (DHEA) is the principal carbon (C)-19 steroid produced by the adrenal gland in humans and mammals [1]. DHEA and its sulfated derivative DHEAS are multifunctional steroids with actions in a wide variety of physiological systems, with effects on the brain [2], immune systems [3], and somatic growth and development [4, 5]. Although DHEA and DHEAS were identified more than 50 years ago, there remains some uncertainty as to their physiological significance, full mechanisms of action [6–9], and their roles in human disease. In humans, DHEA is a crucial precursor of sex steroid biosynthesis and exerts indirect endocrine and intracrine actions following conversion to androgens and estrogens. In addition, DHEA acts as a neurosteroid via its effects on neurotransmitter receptors in the brain. The potential health significance of DHEA in humans is highlighted by the observation that serum concentrations decrease steadily with age, approaching lowest concentrations around the time at which many diseases of aging, particularly neurocognitive decline, become apparent. The age-related decline in DHEA levels [10] has led to the suggestion that this is associated with a decrease in cognitive function as well as the increased rates of neuronal degeneration and dysfunction that occur during aging [11, 12]. Other studies have reported altered DHEA serum concentrations in patients with conditions such as schizophrenia [13], dementia [14], and Alzheimer's disease (AD) [13, 15–18]. Due to these associations, DHEAS has been widely publicized both in the lay press [19, 20] and in the scientific literature [21, 22] for their putative anti-aging and neuroprotective effects. This has sparked controversial speculation that DHEA treatment might be a remedy for neuropsychiatric and neurodegenerative disorders [7, 23–27] and, even more optimistically, that it is a hormone with the potential to increase the life span [28]. As promising as these speculations may seem, there are many contradictions about the roles of DHEA in normal and degenerative brain function. This is especially evident when comparing preclinical and clinical data. For example, studies in animals show a myriad of neuroprotective and trophic effects of DHEAS in development and disease, while clinical studies show inconsistent, and sometimes highly conflicting, results. Clinical studies of neurodegenerative diseases have variously reported increased or decreased DHEAS concentrations in serum, cerebrospinal fluid, and brain tissue, leading to doubt as to the role of DHEA in the neuropathology of aging. It has been suggested that the incongruity in measured DHEAS concentrations may lie in the methodological differences used to sample DHEAS; however, it is possible that these changes are indicative of a more nuanced and multifaceted role. There is consistent evidence that DHEA is neuroprotective with respect to oxidative stress, neuroinflammation, and excitotoxicity, and thus it is possible that DHEA assists the defense of the brain and has a beneficial effect on cognition in healthy brains. Therefore, it is the aim of this review to briefly discuss the physiology of DHEA and its synthesis and secretion during development and aging and to discuss the relationship between alterations in DHEA concentrations and cognition. We further discuss the possible role of DHEAS in a variety of disease states, including AD, and acute illnesses such as schizophrenia, with focus on the fact that these conditions are characterized by imbalances in oxidative stress, neuroinflammation, and excitotoxicity. In humans, DHEA is one of the most abundant hormones synthesized and secreted by the adrenal cortex. This C19 steroid displays an episodic and diurnal rhythm of synthesis and release that parallels that of cortisol [29, 30]. The major synthetic pathways for DHEA and DHEAS are shown in Figure 1. The de novo synthesis of DHEA from cholesterol depends on the presence and activity of the mitochondrial enzyme steroidogenic acute regulatory protein (StAR), the microsomal enzyme cytochrome P450 enzyme 17α-hydroxylase /17,20 lyase (P450c17), and the accessory hemoprotein cytochrome b5 (Cytb5) [32]. Importantly, P450c17 and Cytb5 need to be colocalized, because the function of Cytb5 is to selectively enhance the 17,20-lyase activity of P450c17 [33–35]. DHEAS is the precursor of approximately 50% of androgens in adult men, 75% of active estrogens in premenopausal women, and almost 100% of active estrogens after menopause [36]. DHEA has a 3- to 10-fold predominance of androgenic over estrogenic activity [37], and although a small portion of the circulating pool of DHEA is of gonadal origin in men and women, the majority of DHEA, and virtually all DHEAS, is produced by the adrenal cortex [1]. However, DHEA is also synthesized in the brain, from cholesterol and other hormonal precursors, primarily by astrocytes and oligodendrocytes; indeed, much higher concentrations of DHEAS are found in the brain than in the serum, suggesting that the DHEAS is primarily synthesized in situ, rather than being transported across the blood-brain barrier [38]. Figure 1. The complete steroid pathway showing the formation of DHEA from pregnenolone and 17OH-pregnenolone, and its reversible sulfation, and disposition via androstenes to estradiol and 5α-dihydroxytestosterone. Steroid metabolites identified in serum and urines are shown in light gray boxes and dark gray boxes, respectively. From Greaves et al. [31]. The specific receptors that bind DHEA as a ligand have been of great interest for over 20 years. The biological actions of DHEA and its metabolites are mediated through androgen receptors or estrogen receptors, which belong to the nuclear receptor steroid-receptor subfamily [39]. DHEA has been found to exert both agonistic and antagonistic effects on the androgen receptor, and it acts as an agonist at both the estrogen receptor-α and estrogen receptor-β sites, with a binding preference for estrogen receptor-β [40, 41]. In the brain, DHEA is thought to affect neuronal excitability by modulating the N-methyl-D-aspartate (NMDA) [42–44] and sigma receptors [45], and as a positive allosteric modulator of the Gamma-aminobutyric acid type A (GABAA) receptor [46–49]. In addition to this, DHEA has been shown to be a selective antagonist of the glucocorticoid receptor (GR) [50]. #### 2.1. DHEA and DHEAS synthesis during development and aging In humans, the patterns of DHEA synthesis and secretion change markedly throughout life. In the last months of gestation, the fetal adrenal can synthesize and release considerable amounts of DHEA and DHEAS, which together with estrogen and progesterone produced by the placenta play pivotal roles in the maintenance and endocrine control of pregnancy [51]. Although the plasma concentrations of DHEAS remain high in the newborn, they decrease quickly as the fetal zone of the adrenal gland involutes after birth. From 1 to 6 years of age, the adrenal gland secretes very low concentrations of DHEAS and androstenedione [52]. However at approximately 7–8 years of age, the adrenal zona reticularis increases the production of DHEAS and androstenedione, all of which are C19 steroids that exert androgenic activity in several tissues by converting into potent androgens [36]. This pre-pubertal phenomenon is known as adrenarche, a biochemical, endocrine, and morphological event hypothesized to have evolved only in humans and higher primates. From an evolutionary point of view, adrenarche may be related to the highly coordinated events associated with human growth and organ maturation, particularly of the brain [53–55]. decrease in adrenal androgens might relate to a reduction in the number of DHEA-secreting cells in the zona reticularis itself. Although the underlying mechanisms regarding this change must be further elucidated, the temporal association between falling DHEA concentrations and the onset of age-related diseases has led many investigators to suggest that some age-related neurological disorders such as AD and dementia may be partly attributable to the decrease in systemic DHEA Figure 2. Concentrations of serum DHEAS as a function of age in females and males. Values are high in cord blood and immediately after birth, fall in the first months of life as the fetal adrenal zone involutes, and remain low until the onset of adrenarche at about age 8 years in girls and age 9 years in boys. Peak DHEAS concentrations are usually higher in males than in females. In both sexes, the concentrations of DHEAS decline slowly during the adult years. Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Roles in Brain Function and Disease http://dx.doi.org/10.5772/intechopen.71141 45 The gradual decline in serum concentrations from the peak at 20–30 years of age has led to speculations that low DHEA concentrations could have a negative effect on cognitive function in later life. It has been hypothesized that rise in DHEA concentrations from 6 to 8 years until 20–30 years of age might be associated with the extended period of cortical maturation in humans [55]. While numerous animal studies have shown that DHEA can modulate cognitive performance, the outcomes of such studies in humans are less clear. For example, one study reported that DHEA supplementation improves cognitive performance in young men [64], whereas other studies detected no benefit in an older group who were predominantly male and were HIV-1 seropositive [65]. DHEA supplementation does not appear to improve A study evaluating the cognitive domains of working memory, executive function, and word processing speed in men and women aged between 60 and 88 years with low serum DHEAS concentrations found a positive association between serum DHEAS and working memory [67]. However, the relationship was sex-specific, with a trend toward a better executive function in men only. Other studies in males have shown that increased endogenous androgen concentrations (following cessation of chemical castration in males) resulted in improved performance on the Cambridge Cognitive Examination (part of the Cambridge Examination concentrations [11]. From Miller [56]. 2.2. DHEA and cognition cognition in the elderly [66]. Following the onset of adrenarche, plasma concentrations of DHEAS differ between the sexes, with levels of DHEAS being about 2-fold higher in males than in females (Figure 2). This difference may reflect secretion of these androgens by the testes [10, 57], but it has also been proposed that the higher concentration of DHEAS in men may be attributable to steroid sulfatase, which degrades androgens. The gene for steroid sulfatase is located on the X chromosome, and in having only one copy of the gene, men may have less steroid sulfatase and consequently higher DHEAS concentrations [58]. Maximal plasma concentrations of DHEAS normally occur at 20–30 years of age (Figure 2), followed by a progressive decline in adrenal production in both males and females, until serum concentrations of DHEAS return to pre-adrenarche levels in persons over 80 years of age [59, 60]. The magnitude of this decline is such that serum levels of DHEAS in elderly adults are only around 10–20% of those in young adults [1, 61]. The diminution in adrenal androgens with aging is often termed 'adrenopause.' It has been suggested that adrenopause is associated with a generalized reduction in the 17,20 lyase activity of P450c17 in the zona reticularis of the adrenal gland [62]. Interestingly, it has been shown that the zona reticularis of older men is reduced in size when compared to the adrenals of young men [63], suggesting that at least part of the age-associated Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Roles in Brain Function and Disease http://dx.doi.org/10.5772/intechopen.71141 45 Figure 2. Concentrations of serum DHEAS as a function of age in females and males. Values are high in cord blood and immediately after birth, fall in the first months of life as the fetal adrenal zone involutes, and remain low until the onset of adrenarche at about age 8 years in girls and age 9 years in boys. Peak DHEAS concentrations are usually higher in males than in females. In both sexes, the concentrations of DHEAS decline slowly during the adult years. From Miller [56]. decrease in adrenal androgens might relate to a reduction in the number of DHEA-secreting cells in the zona reticularis itself. Although the underlying mechanisms regarding this change must be further elucidated, the temporal association between falling DHEA concentrations and the onset of age-related diseases has led many investigators to suggest that some age-related neurological disorders such as AD and dementia may be partly attributable to the decrease in systemic DHEA concentrations [11]. #### 2.2. DHEA and cognition The specific receptors that bind DHEA as a ligand have been of great interest for over 20 years. The biological actions of DHEA and its metabolites are mediated through androgen receptors or estrogen receptors, which belong to the nuclear receptor steroid-receptor subfamily [39]. DHEA has been found to exert both agonistic and antagonistic effects on the androgen receptor, and it acts as an agonist at both the estrogen receptor-α and estrogen receptor-β sites, with a binding preference for estrogen receptor-β [40, 41]. In the brain, DHEA is thought to affect neuronal excitability by modulating the N-methyl-D-aspartate (NMDA) [42–44] and sigma receptors [45], and as a positive allosteric modulator of the Gamma-aminobutyric acid type A (GABAA) receptor [46–49]. In addition to this, DHEA has been shown to be a selective In humans, the patterns of DHEA synthesis and secretion change markedly throughout life. In the last months of gestation, the fetal adrenal can synthesize and release considerable amounts of DHEA and DHEAS, which together with estrogen and progesterone produced by the placenta play pivotal roles in the maintenance and endocrine control of pregnancy [51]. Although the plasma concentrations of DHEAS remain high in the newborn, they decrease quickly as the fetal zone of the adrenal gland involutes after birth. From 1 to 6 years of age, the adrenal gland secretes very low concentrations of DHEAS and androstenedione [52]. However at approximately 7–8 years of age, the adrenal zona reticularis increases the production of DHEAS and androstenedione, all of which are C19 steroids that exert androgenic activity in several tissues by converting into potent androgens [36]. This pre-pubertal phenomenon is known as adrenarche, a biochemical, endocrine, and morphological event hypothesized to have evolved only in humans and higher primates. From an evolutionary point of view, adrenarche may be related to the highly coordinated events associated with human growth Following the onset of adrenarche, plasma concentrations of DHEAS differ between the sexes, with levels of DHEAS being about 2-fold higher in males than in females (Figure 2). This difference may reflect secretion of these androgens by the testes [10, 57], but it has also been proposed that the higher concentration of DHEAS in men may be attributable to steroid sulfatase, which degrades androgens. The gene for steroid sulfatase is located on the X chromosome, and in having only one copy of the gene, men may have less steroid sulfatase and conse- Maximal plasma concentrations of DHEAS normally occur at 20–30 years of age (Figure 2), followed by a progressive decline in adrenal production in both males and females, until serum concentrations of DHEAS return to pre-adrenarche levels in persons over 80 years of age [59, 60]. The magnitude of this decline is such that serum levels of DHEAS in elderly adults are only around 10–20% of those in young adults [1, 61]. The diminution in adrenal androgens with aging is often termed 'adrenopause.' It has been suggested that adrenopause is associated with a generalized reduction in the 17,20 lyase activity of P450c17 in the zona reticularis of the adrenal gland [62]. Interestingly, it has been shown that the zona reticularis of older men is reduced in size when compared to the adrenals of young men [63], suggesting that at least part of the age-associated antagonist of the glucocorticoid receptor (GR) [50]. 44 Sex Hormones in Neurodegenerative Processes and Diseases and organ maturation, particularly of the brain [53–55]. quently higher DHEAS concentrations [58]. 2.1. DHEA and DHEAS synthesis during development and aging The gradual decline in serum concentrations from the peak at 20–30 years of age has led to speculations that low DHEA concentrations could have a negative effect on cognitive function in later life. It has been hypothesized that rise in DHEA concentrations from 6 to 8 years until 20–30 years of age might be associated with the extended period of cortical maturation in humans [55]. While numerous animal studies have shown that DHEA can modulate cognitive performance, the outcomes of such studies in humans are less clear. For example, one study reported that DHEA supplementation improves cognitive performance in young men [64], whereas other studies detected no benefit in an older group who were predominantly male and were HIV-1 seropositive [65]. DHEA supplementation does not appear to improve cognition in the elderly [66]. A study evaluating the cognitive domains of working memory, executive function, and word processing speed in men and women aged between 60 and 88 years with low serum DHEAS concentrations found a positive association between serum DHEAS and working memory [67]. However, the relationship was sex-specific, with a trend toward a better executive function in men only. Other studies in males have shown that increased endogenous androgen concentrations (following cessation of chemical castration in males) resulted in improved performance on the Cambridge Cognitive Examination (part of the Cambridge Examination for Mental Disorders of the Elderly, a global measure of cognition and memory) and verbal recall tests [68]. A study in a population of older healthy women (aged 21–77 years) further indicated that women with high serum concentrations of DHEAS had increased performance on a variety of cognitive tests, including better verbal, visual, and spatial abilities; working memory; attention; concentration; and accuracy [69]. In older men and women in an Italian cohort, low DHEAS levels were significant predictors of accelerated decline in Mini-Mental State Examination score during the 3-year follow-up period [70]. Despite these associations, Mazat et al. [71] reported no significant role for serum DHEAS concentrations as a predictor of cognitive decline in an elderly population, while other studies conducted in frail elderly patients and nursing home residents found an inverse relationship between DHEAS levels and cognitive abilities [72, 73]. BDNF is expressed in several areas of the CNS and is necessary for cell proliferation and differentiation [88, 89]. In addition, BDNF plays a vital role in neural plasticity, enhances long-term potentiation, and promotes learning and memory [90, 91]. As such, a mutation or deletion of the BDNF gene in mice results in learning deficits and long-term potentiation impairment [92, 93], as well as decreased learning and memory in behavioral paradigms [90]. In humans, low plasma BDNF is associated with impairments in memory and general cogni- Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Roles in Brain Function and Disease http://dx.doi.org/10.5772/intechopen.71141 47 A recent study investigated the effect of DHEA on cognition and learning in a rat model of vascular dementia [86] and found that DHEA treatment significantly preserved working and reference memory, which was accompanied by a significant increase in the levels of acetylcholine, norepinephrine, and dopamine in the brain. Of note was a significant increase in the hippocampal expression of BDNF after DHEA treatment [86]. In a rodent model, Naert et al., [95] showed that DHEAS treatment can lead to biphasic increases in BDNF in the hippocampus and amygdala, but decreased BDNF concentrations in the hypothalamus. It is interesting to note that glucocorticoids are also involved in BDNF regulation [27, 96], where stress has been found to decrease the expression of BDNF, leading to neuronal atrophy and degeneration in the hippocampus and the cortex, a process that may be common to both development and aging [97, 98]. These findings are important, considering, that BDNF expression is also altered in acute psychiatric disorders such as major depression [99, 100] and schizophrenia [101], as AD is a chronic neurodegenerative disorder characterized by progressive memory loss and cognitive deterioration. It is the most common form of dementia, affecting about 50 million people worldwide [103], with the majority of cases in the elderly population, which presents global health and economic challenges [104]. Currently, there are no disease-modifying therapies available to treat AD [105], and it represents a major unmet need in neurological research and patient management. The neuropathological hallmarks of AD include neurofibrillary tangles, which are formed when the neuronal cytoskeletal protein tau becomes hyperphosphorylated and precipitates, and also amyloid plaques, which are abnormal deposits of extracellular protein that accumulate after cleavage of the β-amyloid precursor protein [106]. Other degenerative changes include cerebral amyloid angiopathy, glial inflammatory responses, and synaptic loss. These processes ultimately lead to neuronal atrophy, white matter loss, and a reduction in the volumes of the entorhinal, temporal, and frontal cortices as well as the hippocampus [107], followed by devastating clinical sequelae and resultant mor- Sporadic AD is the predominant form of the disease, present in more than 95% of patients, and it usually occurs after 65 years of age [109]. The etiology of sporadic AD is multifactorial and may be associated with a number of risk factors including advancing age [110, 111], increased oxidative stress [112, 113], autoimmunity [114], and excess glucocorticoids [115–117]. Although serum DHEA levels decrease with age, the majority of studies have reported that serum DHEAS levels in AD patients are even lower than in age-matched healthy controls. For tive function in aging women [94]. 2.3. DHEA and AD bidity and mortality [108]. well as in neurodegenerative diseases such as AD [102]. While the reasons for the conflicting data on DHEAS and cognition require further investigation, the changes in cognition are likely to be reflective of interactions with both the GABAergic and glutamatergic pathways, and possibly through the mediator brain-derived neurotrophic factor (BDNF). Neurosteroids have contrasting effects on GABAA receptors, which when activated result in chloride entry into the cell, hyperpolarization, and reduced membrane excitability [48]. Reduced metabolites of progesterone and deoxycorticosterone have an agonistic effect on GABAA receptors, resulting in chloride ion movement into the cell. In contrast, DHEAS is a GABAA antagonist and thus increases the likelihood of membrane depolarization [48, 74]. Animal studies have shown that acute exposure to DHEAS may facilitate basal synaptic transmission in the CA1 region of the hippocampus through the noncompetitive potentiation of GABAA receptors [75–77]. In terms of learning and memory, studies have shown that acute administration of DHEAS facilitates primed-burst potentiation, but not the induction of long-term potentiation [78], whereas long-term potentiation is stimulated by the chronic administration of DHEAS [79]. In addition to GABAA receptor modulation, neurosteroids have been found to interact in a structure-specific manner with glutamatergic NMDA receptors. DHEAS potentiates the neuronal response to NMDA in the rat hippocampus [80]. These steroids also act as non-selective sigma-1 receptor antagonists [81], thus suppressing the activity of NMDA receptors, which are central to the process of excitotoxicity [82]. In addition, DHEAS may reduce the cytoplasmic Ca2+-induced loss of mitochondrial membrane potential by preventing Ca2+ influx into the mitochondrial matrix [83]. The neuroprotective effect of DHEA against NMDA-induced excitotoxicity may also involve the calcium/nitric oxide signaling pathway, since DHEA has been shown to inhibit NMDA-induced nitric oxide synthase activity and the production of nitric oxide in primary cultures of hippocampal neurons [84]. The potential of DHEAS to modulate the activity of NMDA receptors through a variety of mechanisms is likely to underpin their capacity to protect neurons from excitotoxicity when high levels of extracellular glutamate are present. Of note, glutamate excitotoxicity has been implicated in AD [85] (discussed further below), where a reduction in neurosteroid production may compromise the intrinsic defense mechanisms of the central nervous system (CNS). Another possible mechanism by which DHEAS could promote neurogenesis and neuronal survival in the CNS is through the mediation of the neurotrophin BDNF [86, 87]. BDNF is expressed in several areas of the CNS and is necessary for cell proliferation and differentiation [88, 89]. In addition, BDNF plays a vital role in neural plasticity, enhances long-term potentiation, and promotes learning and memory [90, 91]. As such, a mutation or deletion of the BDNF gene in mice results in learning deficits and long-term potentiation impairment [92, 93], as well as decreased learning and memory in behavioral paradigms [90]. In humans, low plasma BDNF is associated with impairments in memory and general cognitive function in aging women [94]. A recent study investigated the effect of DHEA on cognition and learning in a rat model of vascular dementia [86] and found that DHEA treatment significantly preserved working and reference memory, which was accompanied by a significant increase in the levels of acetylcholine, norepinephrine, and dopamine in the brain. Of note was a significant increase in the hippocampal expression of BDNF after DHEA treatment [86]. In a rodent model, Naert et al., [95] showed that DHEAS treatment can lead to biphasic increases in BDNF in the hippocampus and amygdala, but decreased BDNF concentrations in the hypothalamus. It is interesting to note that glucocorticoids are also involved in BDNF regulation [27, 96], where stress has been found to decrease the expression of BDNF, leading to neuronal atrophy and degeneration in the hippocampus and the cortex, a process that may be common to both development and aging [97, 98]. These findings are important, considering, that BDNF expression is also altered in acute psychiatric disorders such as major depression [99, 100] and schizophrenia [101], as well as in neurodegenerative diseases such as AD [102]. #### 2.3. DHEA and AD for Mental Disorders of the Elderly, a global measure of cognition and memory) and verbal recall tests [68]. A study in a population of older healthy women (aged 21–77 years) further indicated that women with high serum concentrations of DHEAS had increased performance on a variety of cognitive tests, including better verbal, visual, and spatial abilities; working memory; attention; concentration; and accuracy [69]. In older men and women in an Italian cohort, low DHEAS levels were significant predictors of accelerated decline in Mini-Mental State Examination score during the 3-year follow-up period [70]. Despite these associations, Mazat et al. [71] reported no significant role for serum DHEAS concentrations as a predictor of cognitive decline in an elderly population, while other studies conducted in frail elderly patients and nursing home residents found an inverse relationship between DHEAS levels While the reasons for the conflicting data on DHEAS and cognition require further investigation, the changes in cognition are likely to be reflective of interactions with both the GABAergic and glutamatergic pathways, and possibly through the mediator brain-derived neurotrophic factor (BDNF). Neurosteroids have contrasting effects on GABAA receptors, which when activated result in chloride entry into the cell, hyperpolarization, and reduced membrane excitability [48]. Reduced metabolites of progesterone and deoxycorticosterone have an agonistic effect on GABAA receptors, resulting in chloride ion movement into the cell. In contrast, DHEAS is a GABAA antagonist and thus increases the likelihood of membrane depolarization [48, 74]. Animal studies have shown that acute exposure to DHEAS may facilitate basal synaptic transmission in the CA1 region of the hippocampus through the noncompetitive potentiation of GABAA receptors [75–77]. In terms of learning and memory, studies have shown that acute administration of DHEAS facilitates primed-burst potentiation, but not the induction of long-term potentiation [78], whereas long-term potentiation is stimulated In addition to GABAA receptor modulation, neurosteroids have been found to interact in a structure-specific manner with glutamatergic NMDA receptors. DHEAS potentiates the neuronal response to NMDA in the rat hippocampus [80]. These steroids also act as non-selective sigma-1 receptor antagonists [81], thus suppressing the activity of NMDA receptors, which are central to the process of excitotoxicity [82]. In addition, DHEAS may reduce the cytoplasmic Ca2+-induced loss of mitochondrial membrane potential by preventing Ca2+ influx into the mitochondrial matrix [83]. The neuroprotective effect of DHEA against NMDA-induced excitotoxicity may also involve the calcium/nitric oxide signaling pathway, since DHEA has been shown to inhibit NMDA-induced nitric oxide synthase activity and the production of The potential of DHEAS to modulate the activity of NMDA receptors through a variety of mechanisms is likely to underpin their capacity to protect neurons from excitotoxicity when high levels of extracellular glutamate are present. Of note, glutamate excitotoxicity has been implicated in AD [85] (discussed further below), where a reduction in neurosteroid production may compromise the intrinsic defense mechanisms of the central nervous system (CNS). Another possible mechanism by which DHEAS could promote neurogenesis and neuronal survival in the CNS is through the mediation of the neurotrophin BDNF [86, 87]. and cognitive abilities [72, 73]. 46 Sex Hormones in Neurodegenerative Processes and Diseases by the chronic administration of DHEAS [79]. nitric oxide in primary cultures of hippocampal neurons [84]. AD is a chronic neurodegenerative disorder characterized by progressive memory loss and cognitive deterioration. It is the most common form of dementia, affecting about 50 million people worldwide [103], with the majority of cases in the elderly population, which presents global health and economic challenges [104]. Currently, there are no disease-modifying therapies available to treat AD [105], and it represents a major unmet need in neurological research and patient management. The neuropathological hallmarks of AD include neurofibrillary tangles, which are formed when the neuronal cytoskeletal protein tau becomes hyperphosphorylated and precipitates, and also amyloid plaques, which are abnormal deposits of extracellular protein that accumulate after cleavage of the β-amyloid precursor protein [106]. Other degenerative changes include cerebral amyloid angiopathy, glial inflammatory responses, and synaptic loss. These processes ultimately lead to neuronal atrophy, white matter loss, and a reduction in the volumes of the entorhinal, temporal, and frontal cortices as well as the hippocampus [107], followed by devastating clinical sequelae and resultant morbidity and mortality [108]. Sporadic AD is the predominant form of the disease, present in more than 95% of patients, and it usually occurs after 65 years of age [109]. The etiology of sporadic AD is multifactorial and may be associated with a number of risk factors including advancing age [110, 111], increased oxidative stress [112, 113], autoimmunity [114], and excess glucocorticoids [115–117]. Although serum DHEA levels decrease with age, the majority of studies have reported that serum DHEAS levels in AD patients are even lower than in age-matched healthy controls. For instance, Yanase et al. [18] found that patients with AD or cerebrovascular dementia had lower concentrations of serum DHEAS and a lower DHEAS/DHEA ratio when compared to controls. Several other clinical studies have reported lower serum concentrations of DHEAS in patients with AD [14, 118–120], a reduction paralleled by decreases in the brain and cerebral spinal fluid [121, 122]. For instance, Weill-Engerer and colleagues [108] reported that not only are brain levels of DHEAS significantly lower in AD, but also the lower levels are inversely correlated with the presence of phosphorylated tau and β-amyloid. A few studies have not detected differences in serum DHEAS concentrations between AD patients and controls [120, 123], and there is one report that serum DHEAS levels are increased in mild-moderate AD [124]. The reasons for these differences between studies have not yet been elucidated. likely to develop the disorder than age-matched, nonstressed individuals [117]. Cortisol is the most prominent stress-related glucocorticoid in human serum. Serum cortisol levels are elevated in patients with AD [140], as are the levels of urinary cortisol [141]. It is pertinent that the overactivation of GABAA receptors plays a central role in anxiety disorders and consequently these receptors are the principal targets of anxiolytic drugs for the treatment of affective disorders [142]. Since DHEAS antagonizes GABAA receptors, they are thought to act as endogenous anxiolytics, and hence a reduction in the availability of DHEAS in aging or AD Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Roles in Brain Function and Disease http://dx.doi.org/10.5772/intechopen.71141 49 could contribute to increased anxiety and stimulate the chronic production of cortisol. Animal experiments have shown that excess concentrations of glucocorticoids during prolonged periods of stress can have deleterious effects on the brain, especially in aged animals, and particularly affecting the hippocampus [143]. Glucocorticoids exert several actions on the brain, including the stimulation of glutamatergic neurotransmission via the stimulation of glucocorticoid receptors (GR), which if left unchecked can lead to excitotoxicity. Several studies have shown that DHEA can protect against the effects of glucocorticoid-mediated neurotoxicity [144, 145]. The neuroprotective effects of DHEA have been modeled in vivo where the toxic effects of corticosterone in the dentate gyrus of male rats were suppressed by low concentrations of DHEA [146]. The protection conferred by DHEA may be via downregulating the expression of glucocorticoid receptors [147]. In cultured HT-22 cells, DHEA augmentation suppresses the nuclear localization of the GR in response to glutamate toxicity, as assessed by immunohistochemistry [131]. Thus, inhibition of GR translocation into the nucleus is a possible mechanism of DHEA's anti-glucocorticoid effects. DHEA administration reduces GR expression in hippocampal cells in the mouse [131] and reduces glucocorticoid receptors by 50% in the rat liver [145]. Furthermore, DHEA may act as a GR antagonist and can attenuate the translocation of stress-activated protein kinase-3 in rat hippocampal primary cultures [148]. DHEA may also attenuate the neurotoxic effects of cortisol by reducing the regeneration of active glucocorticoids. The 7α-hydroxylated metabolite of DHEA (7α-hydroxy-DHEA) has antiglucocorticoid effects in target tissues by competition with 11-keto glucocorticoids for access to 11β-hydroxysteroid dehydrogenase-1 [149]. Enzyme kinetic data from yeastexpressed human 11β- hydroxysteroid dehydrogenase imply that 7α-hydroxysteroid substrates are preferred to cortisone by this enzyme [150]. Therefore, in tissues such as the brain, 7α-hydroxy-DHEA may act as an endogenous inhibitor of 11β- hydroxysteroid dehydrogenase, thereby reducing the regeneration of active glucocorticoids [151]. 7α-hydroxy-DHEA may have more potent bioactivity and stronger neuroprotective and antiglucocorticoid effects than DHEA itself [152]. Interestingly, some investigators have hypothesized that the degree of metabolism of DHEA to 7α-hydroxy-DHEA is related to the pathology of AD [122, 151, 153, 154]. This is evident in the study by Yau et al. [151], which found that gene expression for cytochrome P4507b (which converts DHEA into 7α-hydroxy-DHEA) was significantly decreased in hippocampal dentate neurons from patients with AD when compared to controls [151]. Another study found lower plasma 7α-hydroxy-DHEA concentrations in patients with AD Taken together, the preceding observations are generally supportive of the view that DHEAS levels in serum are reduced in AD when compared to those in healthy age-matched controls. when compared to controls [154]. In contrast to the majority of studies, Naylor and colleagues [125] reported that cerebral spinal fluid levels of DHEA are significantly elevated in AD, as are tissue levels in the temporal cortex, with the extent of elevation being correlated with disease severity, as assessed by the burden of β-amyloid plaques. Similarly, Brown and colleagues [126] reported increased DHEA concentrations in the brains and cerebral spinal fluid of patients with AD when compared with controls, even though mean serum concentrations of DHEA did not differ. Interestingly, in this study, DHEA concentrations were highest in the hippocampus of AD patients, a region that does not express P450c17. Brown and colleagues speculated that the higher concentrations of DHEA in the hippocampus may have been produced by an as-yet-unknown pathway that involved the oxidation of an unknown precursor. This speculation has been given support by the finding that the addition of redox-active ferrous iron to serum samples causes a significant increase in the amount of detectable DHEA [127]. It is also supported by the demonstration that oxidative stress associated with the presence of β-amyloid treatment induces DHEA synthesis in human and rodent cells in vitro [126–129]. In this context, it is interesting that the brain regions containing the higher concentrations of DHEA [126] also have higher burdens of neuritic plaques and β-amyloid immunoreactivity, features that are generally associated with AD progression [130]. It may be significant that DHEA protects HT-22 cells (an immortalized mouse hippocampal cell line) against amyloid β protein toxicity in a dose-dependent manner [131]. Another link to the pathogenesis and progression of AD comes from the anti-inflammatory properties of DHEA [132]. Hence, the local production of DHEA in the AD brain may function, at least in part, to reduce the level of inflammation that would otherwise be injurious to neurons if left unchecked. Serum levels of DHEAS have been shown to negatively correlate with serum interleukin-6 (IL-6), to inhibit IL-6 secretion from human mononuclear cells [133], and to inhibit cytokine-stimulated, NF-κB–mediated transcription, partly through an antioxidant property [134]. Interestingly, elevated levels of IL-6 are consistently detected in the brains of AD patients, but not in the brains of non-demented elderly persons [135]. Several studies have suggested that an increase of circulating IL-6 in AD patients indicates immune activation and may be related to the pathophysiology of AD [136–138]. Perhaps the most intriguing link between DHEA and AD comes from its association with systemic stress and glucocorticoid production, which has lead to the hypothesis that chronic stress is an important factor in AD pathogenesis [139]. Epidemiological evidence supports a role for stress in AD because elderly individuals prone to psychological distress are more likely to develop the disorder than age-matched, nonstressed individuals [117]. Cortisol is the most prominent stress-related glucocorticoid in human serum. Serum cortisol levels are elevated in patients with AD [140], as are the levels of urinary cortisol [141]. It is pertinent that the overactivation of GABAA receptors plays a central role in anxiety disorders and consequently these receptors are the principal targets of anxiolytic drugs for the treatment of affective disorders [142]. Since DHEAS antagonizes GABAA receptors, they are thought to act as endogenous anxiolytics, and hence a reduction in the availability of DHEAS in aging or AD could contribute to increased anxiety and stimulate the chronic production of cortisol. instance, Yanase et al. [18] found that patients with AD or cerebrovascular dementia had lower concentrations of serum DHEAS and a lower DHEAS/DHEA ratio when compared to controls. Several other clinical studies have reported lower serum concentrations of DHEAS in patients with AD [14, 118–120], a reduction paralleled by decreases in the brain and cerebral spinal fluid [121, 122]. For instance, Weill-Engerer and colleagues [108] reported that not only are brain levels of DHEAS significantly lower in AD, but also the lower levels are inversely correlated with the presence of phosphorylated tau and β-amyloid. A few studies have not detected differences in serum DHEAS concentrations between AD patients and controls [120, 123], and there is one report that serum DHEAS levels are increased in mild-moderate AD [124]. The reasons for In contrast to the majority of studies, Naylor and colleagues [125] reported that cerebral spinal fluid levels of DHEA are significantly elevated in AD, as are tissue levels in the temporal cortex, with the extent of elevation being correlated with disease severity, as assessed by the burden of β-amyloid plaques. Similarly, Brown and colleagues [126] reported increased DHEA concentrations in the brains and cerebral spinal fluid of patients with AD when compared with controls, even though mean serum concentrations of DHEA did not differ. Interestingly, in this study, DHEA concentrations were highest in the hippocampus of AD patients, a region that does not express P450c17. Brown and colleagues speculated that the higher concentrations of DHEA in the hippocampus may have been produced by an as-yet-unknown pathway that involved the oxidation of an unknown precursor. This speculation has been given support by the finding that the addition of redox-active ferrous iron to serum samples causes a significant increase in the amount of detectable DHEA [127]. It is also supported by the demonstration that oxidative stress associated with the presence of β-amyloid treatment induces DHEA synthesis in human and rodent cells in vitro [126–129]. In this context, it is interesting that the brain regions containing the higher concentrations of DHEA [126] also have higher burdens of neuritic plaques and β-amyloid immunoreactivity, features that are generally associated with AD progression [130]. It may be significant that DHEA protects HT-22 cells (an immortalized mouse hippocampal cell line) against amyloid β protein toxicity in a dose-dependent manner [131]. activation and may be related to the pathophysiology of AD [136–138]. Another link to the pathogenesis and progression of AD comes from the anti-inflammatory properties of DHEA [132]. Hence, the local production of DHEA in the AD brain may function, at least in part, to reduce the level of inflammation that would otherwise be injurious to neurons if left unchecked. Serum levels of DHEAS have been shown to negatively correlate with serum interleukin-6 (IL-6), to inhibit IL-6 secretion from human mononuclear cells [133], and to inhibit cytokine-stimulated, NF-κB–mediated transcription, partly through an antioxidant property [134]. Interestingly, elevated levels of IL-6 are consistently detected in the brains of AD patients, but not in the brains of non-demented elderly persons [135]. Several studies have suggested that an increase of circulating IL-6 in AD patients indicates immune Perhaps the most intriguing link between DHEA and AD comes from its association with systemic stress and glucocorticoid production, which has lead to the hypothesis that chronic stress is an important factor in AD pathogenesis [139]. Epidemiological evidence supports a role for stress in AD because elderly individuals prone to psychological distress are more these differences between studies have not yet been elucidated. 48 Sex Hormones in Neurodegenerative Processes and Diseases Animal experiments have shown that excess concentrations of glucocorticoids during prolonged periods of stress can have deleterious effects on the brain, especially in aged animals, and particularly affecting the hippocampus [143]. Glucocorticoids exert several actions on the brain, including the stimulation of glutamatergic neurotransmission via the stimulation of glucocorticoid receptors (GR), which if left unchecked can lead to excitotoxicity. Several studies have shown that DHEA can protect against the effects of glucocorticoid-mediated neurotoxicity [144, 145]. The neuroprotective effects of DHEA have been modeled in vivo where the toxic effects of corticosterone in the dentate gyrus of male rats were suppressed by low concentrations of DHEA [146]. The protection conferred by DHEA may be via downregulating the expression of glucocorticoid receptors [147]. In cultured HT-22 cells, DHEA augmentation suppresses the nuclear localization of the GR in response to glutamate toxicity, as assessed by immunohistochemistry [131]. Thus, inhibition of GR translocation into the nucleus is a possible mechanism of DHEA's anti-glucocorticoid effects. DHEA administration reduces GR expression in hippocampal cells in the mouse [131] and reduces glucocorticoid receptors by 50% in the rat liver [145]. Furthermore, DHEA may act as a GR antagonist and can attenuate the translocation of stress-activated protein kinase-3 in rat hippocampal primary cultures [148]. DHEA may also attenuate the neurotoxic effects of cortisol by reducing the regeneration of active glucocorticoids. The 7α-hydroxylated metabolite of DHEA (7α-hydroxy-DHEA) has antiglucocorticoid effects in target tissues by competition with 11-keto glucocorticoids for access to 11β-hydroxysteroid dehydrogenase-1 [149]. Enzyme kinetic data from yeastexpressed human 11β- hydroxysteroid dehydrogenase imply that 7α-hydroxysteroid substrates are preferred to cortisone by this enzyme [150]. Therefore, in tissues such as the brain, 7α-hydroxy-DHEA may act as an endogenous inhibitor of 11β- hydroxysteroid dehydrogenase, thereby reducing the regeneration of active glucocorticoids [151]. 7α-hydroxy-DHEA may have more potent bioactivity and stronger neuroprotective and antiglucocorticoid effects than DHEA itself [152]. Interestingly, some investigators have hypothesized that the degree of metabolism of DHEA to 7α-hydroxy-DHEA is related to the pathology of AD [122, 151, 153, 154]. This is evident in the study by Yau et al. [151], which found that gene expression for cytochrome P4507b (which converts DHEA into 7α-hydroxy-DHEA) was significantly decreased in hippocampal dentate neurons from patients with AD when compared to controls [151]. Another study found lower plasma 7α-hydroxy-DHEA concentrations in patients with AD when compared to controls [154]. Taken together, the preceding observations are generally supportive of the view that DHEAS levels in serum are reduced in AD when compared to those in healthy age-matched controls. Given that DHEAS reduces oxidative stress and neuroinflammation, protects against glutamate excitotoxicity, and minimizes the negative effects of cortisol on the brain, the reduced levels of serum DHEAS are likely to increase the vulnerability of the brain to these factors. While limited evidence suggests that the brain may compensate by increasing the local production of DHEAS, this may not be sufficient to slow the pathogenesis of the disease. As noted earlier, DHEA can protect neurons from glutamate excitotoxicity, β-amyloid toxicity, and oxidative stress [49, 131], and furthermore, oxidative stress can lead to increased DHEA formation [84, 178]. Oxidative stressors may therefore stimulate DHEA levels in schizophrenic patients [126], in an adaptive change to other precipitating disease factors. Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Roles in Brain Function and Disease http://dx.doi.org/10.5772/intechopen.71141 51 However, other studies have found no difference in DHEA levels between schizophrenic and control subjects [49], and some studies have reported significantly reduced plasma DHEA concentrations [179–181], particularly in the morning [180, 182, 183], as well as abnormal DHEA diurnal rhythms [184] in schizophrenics compared with matched controls. Furthermore, DHEA augmentation has been found to be effective in the management of depressive and anxiety symptoms of patients with schizophrenia [185], suggesting that higher levels of circulating DHEA in schizophrenic populations may be associated with superior functioning [16]. The inconsistency between studies is understandable in view of the wide clinical polymorphism, variability of psychometric properties (distress and anxiety), drug treatment, and clinical responsiveness of schizophrenia patients to their antipsychotic treatment [169]. It may be difficult to interpret the significance of elevated or decreased DHEA levels in the absence of concentrations of other HPA axis hormones. Dysregulation of the HPA axis described in schizophrenia [13] includes increased basal cortisol levels [186], cortisol nonsuppression on the dexamethasone suppression test [187], increased adrenocorticotropic hormone and cortisol response to the dexamethasone/cortisol releasing hormone challenge test [188], and increases in glucocorticoid receptor mRNA as observed post-mortem [189]. DHEA and cortisol are both cleaved from 17-hydroxypregnenolone and are adrenocorticotropic hormone regulated [190]. It is not clear, therefore, if an elevated DHEA concentration is specific to a particular disease state or due to a generalized overactivation of the HPA axis. This difference is of functional significance as DHEA possesses antiglucocorticoid properties and may protect against some of the deleterious effects of persistently elevated cortisol levels [145]. This can be clarified by determining the cortisol/DHEA ratio, which may be a more appropriate measurement than DHEA alone [191]. If the biological response to stress is impaired among schizophrenia patients, it is possible that the cortisol/DHEA ratio would be elevated There is also evidence for oligodendrocyte and myelin dysfunction in neuropathologies such as schizophrenia and bipolar affective disorder, where alterations in the cortisol/DHEA ratio have been observed [16, 17, 155]. Some key oligodendrocyte and myelination genes (such as proteolipid protein 1 and myelin-associated glycoprotein), and transcription factors that regulate the expression of these genes, are downregulated in brains of schizophrenia and bipolar subjects [193]. Together, these studies indicate that common pathophysiological pathways may govern the disease phenotypes of schizophrenia, as well as other neurodegenerative A significant body of preclinical research investigating the biological actions of DHEA have shown that this steroid, and its sulfated congener DHEAS, has a multifunctional role in a as a result of stress associated with the illness [192]. diseases that specifically involve oligodendrocytes. 3. Conclusion #### 2.4. DHEA in schizophrenia In addition to neurodegenerative diseases, there is evidence that low levels of circulating DHEA with normal levels of glucocorticoids (cortisol) place the developing brain at risk for a range of acute neuropsychiatric disorders, including major depressive disorder, bipolar disorder, and anxiety [155–158]. It is further hypothesized that abnormalities of the hypothalamic-pituitary-adrenal (HPA) axis play a central role in the pathogenesis and etiology of schizophrenia [159–161]. Low ratios of DHEA to cortisol have been noted in patients with schizophrenia and are positively associated with the severity of depression, state and trait anxiety, anger, and hostility [155]. DHEA augmentation in affected patients has been seen to attenuate the severity of some negative symptoms associated with this mental illness, including lack of volition and drive, and social withdrawal [16, 162]. Previous studies have found evidence of abnormal dopaminergic activity [163] and deficits in GABAergic and glutamatergic activity [164] in the brain tissue of patients with schizophrenia. Neuroactive steroids such as DHEA modulate the activity of these neurotransmitter systems, both directly and indirectly, and therefore may contribute to the pathophysiology of the illness [82, 165–168]. A number of studies [169] have reported elevated plasma levels of DHEA and DHEAS in severely psychotic male subjects [170, 171], medicated patients with chronic schizophrenia [172], and nonmedicated first-episode patients [170, 173] compared with controls. Elevated DHEA levels have been detected in the post-mortem brain tissue of schizophrenic patients in both the posterior cingulate and parietal cortex [171]. In addition to this, the levels of allopregnanolone are significantly lower in the schizophrenic parietal cortex when compared with healthy controls, whereas pregnenolone levels are significantly higher [49]. Since both of these neurosteroids are downstream metabolites of DHEA, these data suggest that DHEA is preferentially metabolized to pregnenolone in patients with schizophrenia [49]. As DHEA is a positive modulator of excitatory NMDA receptors, and allopregnanolone is a positive modulator of the inhibitory GABAA receptors, the shift in the ratio of DHEA:allopregnanolone could favor a net increase in neuronal excitation [49], similar to the alterations in brain neurotransmitter systems seen in schizophrenia patients. As a result of the positive modulatory effects of DHEA on NMDA receptors [49], in addition to its capacity to enhance learning and memory in rodent models [174], it may be speculated that an elevation of DHEA levels reflects a compensatory process in the schizophrenic brain. It is possible that subjects with schizophrenia may be physiologically resistant to DHEA action in some manner (potentially resulting in the increased synthesis of this neurosteroid) or that there is dysregulation in a feedback system involving the HPA axis [175]. Specifically, DHEA increases following cortisol-releasing hormone [49] and adrenocorticotropic hormone [176] administration in humans, and persistent DHEA elevations may reflect a prolonged upregulation of this axis [177]. As noted earlier, DHEA can protect neurons from glutamate excitotoxicity, β-amyloid toxicity, and oxidative stress [49, 131], and furthermore, oxidative stress can lead to increased DHEA formation [84, 178]. Oxidative stressors may therefore stimulate DHEA levels in schizophrenic patients [126], in an adaptive change to other precipitating disease factors. However, other studies have found no difference in DHEA levels between schizophrenic and control subjects [49], and some studies have reported significantly reduced plasma DHEA concentrations [179–181], particularly in the morning [180, 182, 183], as well as abnormal DHEA diurnal rhythms [184] in schizophrenics compared with matched controls. Furthermore, DHEA augmentation has been found to be effective in the management of depressive and anxiety symptoms of patients with schizophrenia [185], suggesting that higher levels of circulating DHEA in schizophrenic populations may be associated with superior functioning [16]. The inconsistency between studies is understandable in view of the wide clinical polymorphism, variability of psychometric properties (distress and anxiety), drug treatment, and clinical responsiveness of schizophrenia patients to their antipsychotic treatment [169]. It may be difficult to interpret the significance of elevated or decreased DHEA levels in the absence of concentrations of other HPA axis hormones. Dysregulation of the HPA axis described in schizophrenia [13] includes increased basal cortisol levels [186], cortisol nonsuppression on the dexamethasone suppression test [187], increased adrenocorticotropic hormone and cortisol response to the dexamethasone/cortisol releasing hormone challenge test [188], and increases in glucocorticoid receptor mRNA as observed post-mortem [189]. DHEA and cortisol are both cleaved from 17-hydroxypregnenolone and are adrenocorticotropic hormone regulated [190]. It is not clear, therefore, if an elevated DHEA concentration is specific to a particular disease state or due to a generalized overactivation of the HPA axis. This difference is of functional significance as DHEA possesses antiglucocorticoid properties and may protect against some of the deleterious effects of persistently elevated cortisol levels [145]. This can be clarified by determining the cortisol/DHEA ratio, which may be a more appropriate measurement than DHEA alone [191]. If the biological response to stress is impaired among schizophrenia patients, it is possible that the cortisol/DHEA ratio would be elevated as a result of stress associated with the illness [192]. There is also evidence for oligodendrocyte and myelin dysfunction in neuropathologies such as schizophrenia and bipolar affective disorder, where alterations in the cortisol/DHEA ratio have been observed [16, 17, 155]. Some key oligodendrocyte and myelination genes (such as proteolipid protein 1 and myelin-associated glycoprotein), and transcription factors that regulate the expression of these genes, are downregulated in brains of schizophrenia and bipolar subjects [193]. Together, these studies indicate that common pathophysiological pathways may govern the disease phenotypes of schizophrenia, as well as other neurodegenerative diseases that specifically involve oligodendrocytes. ### 3. Conclusion Given that DHEAS reduces oxidative stress and neuroinflammation, protects against glutamate excitotoxicity, and minimizes the negative effects of cortisol on the brain, the reduced levels of serum DHEAS are likely to increase the vulnerability of the brain to these factors. While limited evidence suggests that the brain may compensate by increasing the local pro- In addition to neurodegenerative diseases, there is evidence that low levels of circulating DHEA with normal levels of glucocorticoids (cortisol) place the developing brain at risk for a range of acute neuropsychiatric disorders, including major depressive disorder, bipolar disorder, and anxiety [155–158]. It is further hypothesized that abnormalities of the hypothalamic-pituitary-adrenal (HPA) axis play a central role in the pathogenesis and etiology of schizophrenia [159–161]. Low ratios of DHEA to cortisol have been noted in patients with schizophrenia and are positively associated with the severity of depression, state and trait anxiety, anger, and hostility [155]. DHEA augmentation in affected patients has been seen to attenuate the severity of some negative symptoms associated with this mental illness, includ- Previous studies have found evidence of abnormal dopaminergic activity [163] and deficits in GABAergic and glutamatergic activity [164] in the brain tissue of patients with schizophrenia. Neuroactive steroids such as DHEA modulate the activity of these neurotransmitter systems, both directly and indirectly, and therefore may contribute to the pathophysiology of the illness [82, 165–168]. A number of studies [169] have reported elevated plasma levels of DHEA and DHEAS in severely psychotic male subjects [170, 171], medicated patients with chronic schizophrenia [172], and nonmedicated first-episode patients [170, 173] compared with controls. Elevated DHEA levels have been detected in the post-mortem brain tissue of schizophrenic patients in both the posterior cingulate and parietal cortex [171]. In addition to this, the levels of allopregnanolone are significantly lower in the schizophrenic parietal cortex when compared with healthy controls, whereas pregnenolone levels are significantly higher [49]. Since both of these neurosteroids are downstream metabolites of DHEA, these data suggest that DHEA is preferentially metabolized to pregnenolone in patients with schizophrenia [49]. As DHEA is a positive modulator of excitatory NMDA receptors, and allopregnanolone is a positive modulator of the inhibitory GABAA receptors, the shift in the ratio of DHEA:allopregnanolone could favor a net increase in neuronal excitation [49], similar to the duction of DHEAS, this may not be sufficient to slow the pathogenesis of the disease. ing lack of volition and drive, and social withdrawal [16, 162]. alterations in brain neurotransmitter systems seen in schizophrenia patients. As a result of the positive modulatory effects of DHEA on NMDA receptors [49], in addition to its capacity to enhance learning and memory in rodent models [174], it may be speculated that an elevation of DHEA levels reflects a compensatory process in the schizophrenic brain. It is possible that subjects with schizophrenia may be physiologically resistant to DHEA action in some manner (potentially resulting in the increased synthesis of this neurosteroid) or that there is dysregulation in a feedback system involving the HPA axis [175]. Specifically, DHEA increases following cortisol-releasing hormone [49] and adrenocorticotropic hormone [176] administration in humans, and persistent DHEA elevations may reflect a prolonged upregu- 2.4. DHEA in schizophrenia 50 Sex Hormones in Neurodegenerative Processes and Diseases lation of this axis [177]. A significant body of preclinical research investigating the biological actions of DHEA have shown that this steroid, and its sulfated congener DHEAS, has a multifunctional role in a variety of physiological systems, including in the developing and aging brain. A summary of the actions of DHEA relevant to the discussion above is shown in Table 1. The present review has highlighted the involvement of DHEAS in glutamatergic and GABAergic neurotransmission, where this neurohormone acts as an important modulator of neuronal excitability. Consequently, perturbations in the level of DHEA can affect cognition and mood. DHEAS has also been shown to respond to stress and to modulate the effects of cortisol on the brain. Reductions in the availability of DHEAS can increase the likelihood of glutamate excitotoxicity as well as exacerbate the deleterious effects of cortisol. Evidence indicates that the brain is not dependent on serum levels of DHEA as it is able to synthesis DHEAS in situ. Indeed, there appears to be a capacity to produce DHEA in direct response to oxidative stress. We have shown that in AD, the levels of DHEA are depleted, and the subsequent loss of protection from glutamate, cortisol, and oxidative stress may contribute to the pathogenesis of the disease. Conversely, in schizophrenia, there appears to be an elevation in the availability of DHEA, and this may act to decrease the influence of the GABAergic inhibitory pathways in favor of excitatory neurotransmission. While these emerging roles for DHEA are exciting, the present review also highlighted the discordant findings in the clinical literature, and it is clear that much remains to be learned about the Dehydroepiandrosterone (DHEA) and DHEA Sulfate: Roles in Brain Function and Disease http://dx.doi.org/10.5772/intechopen.71141 53 The authors are grateful for support and many discussions from Dr. Udani Ratnayake, Dr. Stacey Ellery, Dr. Margie Castillo-Melendez, and Dr. Hayley Dickinson from The Ritchie Centre, Hudson Institute of Medical Research, and from Professor Jonathan Hirst, University of Newcastle, New South Wales, Australia. Tracey Quinn received support from an Australian Post-graduate Award (APA) postgraduate scholarship for some of the studies reported above. Tracey Quinn and David Walker are grateful for funding from National Health & Medical Research Council of Australia and Cerebral Palsy Alliance. We also acknowledge generous support from the Victorian Government Infrastructure Fund to the and David Walker<sup>2</sup> [1] Parker CR. Dehydroepiandrosterone and dehydroepiandrosterone sulfate production in the human adrenal during development and aging. Steroids. 1999;64:640-647 2 School of Health and Biomedical Sciences, RMIT University, Melbourne, Australia \* contribution of DHEAS to brain function in both health and disease. Acknowledgements Author details Tracey A. Quinn<sup>1</sup> References Hudson Institute of Medical Research. , Stephen R. Robinson<sup>2</sup> \Address all correspondence to: [email protected] 1 The Ritchie Centre, Monash University, Melbourne, Australia Abbreviations: AR, androgen receptor; BDNF, brain-derived neurotrophic factor; CNS, central nervous system; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; ER, estrogen receptor; GABA-A, Gammaaminobutyric acid receptor A; GR, glucocorticoid receptor; IL-6, interleukin 6; LTP, long-term potentiation; NMDA, N-methyl-D-aspartate. Table 1.* Summary of functions of DHEA related to development and aging. neurotransmission, where this neurohormone acts as an important modulator of neuronal excitability. Consequently, perturbations in the level of DHEA can affect cognition and mood. DHEAS has also been shown to respond to stress and to modulate the effects of cortisol on the brain. Reductions in the availability of DHEAS can increase the likelihood of glutamate excitotoxicity as well as exacerbate the deleterious effects of cortisol. Evidence indicates that the brain is not dependent on serum levels of DHEA as it is able to synthesis DHEAS in situ. Indeed, there appears to be a capacity to produce DHEA in direct response to oxidative stress. We have shown that in AD, the levels of DHEA are depleted, and the subsequent loss of protection from glutamate, cortisol, and oxidative stress may contribute to the pathogenesis of the disease. Conversely, in schizophrenia, there appears to be an elevation in the availability of DHEA, and this may act to decrease the influence of the GABAergic inhibitory pathways in favor of excitatory neurotransmission. While these emerging roles for DHEA are exciting, the present review also highlighted the discordant findings in the clinical literature, and it is clear that much remains to be learned about the contribution of DHEAS to brain function in both health and disease. #### Acknowledgements variety of physiological systems, including in the developing and aging brain. A summary of the actions of DHEA relevant to the discussion above is shown in Table 1. The present review has highlighted the involvement of DHEAS in glutamatergic and GABAergic Receptor interactions: Agonistic and antagonistic effects on AR, agonist at ERα and ERβ [40, 41] Nonselective sigma-1 receptor antagonist [81] administration of DHEAS stimulates LTP [79] primary cultures of hippocampal neurons [84] hippocampal primary cultures [148] Table 1. Summary of functions of DHEA related to development and aging. preventing Ca2+ influx into the mitochondrial matrix [83] Inhibits IL-6 secretion from human mononuclear cells [133] inhibition of GR translocation into the nucleus [131] Reduces the regeneration of active glucocorticoids [149] Downregulation of the expression of glucocorticoid receptors [147] Abbreviations: AR, androgen receptor; BDNF, brain-derived neurotrophic factor; CNS, central nervous system; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; ER, estrogen receptor; GABA-A, Gammaaminobutyric acid receptor A; GR, glucocorticoid receptor; IL-6, interleukin 6; LTP, long-term potentiation; NMDA, Maintenance and endocrine control of pregnancy [51] Positive allosteric modulator of the GABA-A receptor [46–49] Memory and learning: DHEAS may facilitate basal synaptic transmission in the CA1 region of the hippocampus Associated with human growth and organ maturation, particularly of the brain, during Promotes neurogenesis and neuronal survival in the CNS through the mediation of BDNF Acute DHEAS administration facilitates primed-burst potentiation [78] and chronic Reduces the cytoplasmic Ca2+-induced loss of mitochondrial membrane potential by DHEA treatment significantly preserves working and reference memories and increases acetylcholine, norepinephrine, and dopamine concentrations in the rat brain [86] Inhibits NMDA-induced nitric oxide synthase activity and the production of nitric oxide in Protect neurons from glutamate excitotoxicity, β-amyloid toxicity, and oxidative stress Inhibits cytokine-stimulated, NF-κB–mediated transcription, partly through an antioxidant GR antagonist and can attenuate the translocation of stress-activated protein kinase-3 in rat Suppresses the nuclear localization of the GR in response to glutamate toxicity and Modulates the NMDA receptor [42–44] Selective antagonist of the GR [50] adrenarche [53–55] [86, 87] [75–77] [49, 131] property [134] DHEA Effects/function 52 Sex Hormones in Neurodegenerative Processes and Diseases Development & regeneration: Neuroprotection: Anti-excitatory actions Anti-inflammatory Antiglucocorticoid N-methyl-D-aspartate. actions actions The authors are grateful for support and many discussions from Dr. Udani Ratnayake, Dr. Stacey Ellery, Dr. Margie Castillo-Melendez, and Dr. Hayley Dickinson from The Ritchie Centre, Hudson Institute of Medical Research, and from Professor Jonathan Hirst, University of Newcastle, New South Wales, Australia. Tracey Quinn received support from an Australian Post-graduate Award (APA) postgraduate scholarship for some of the studies reported above. Tracey Quinn and David Walker are grateful for funding from National Health & Medical Research Council of Australia and Cerebral Palsy Alliance. We also acknowledge generous support from the Victorian Government Infrastructure Fund to the Hudson Institute of Medical Research. ### Author details #### References [1] Parker CR. Dehydroepiandrosterone and dehydroepiandrosterone sulfate production in the human adrenal during development and aging. Steroids. 1999;64:640-647 [2] Kroboth PD, Salek FS, Pittenger AL, Fabian TJ, Frye RF. DHEA and DHEA-S: A review. Journal of Clinical Pharmacology. 1999;39:327-348 [16] Strous RD, Maayan R, Lapidus R, Stryjer R, Lustig M, Kotler M, Weizman A. Dehydroepiandrosterone augmentation in the management of negative, depressive, and anxiety symptoms in schizophrenia. 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Medical Hypotheses. 1997;49:85-91 Chapter 4 Provisional chapter Gender Differences in Frontotemporal Lobar Gender Differences in Frontotemporal Lobar Delayed Onset Abstract majority of them. 1. Introduction autophagy, microglia, neuroprotection Delayed Onset Claire V. Flaherty, Arghavan S. Zangeneh, Marissa A. Harrison and Sanjana Marikunte Claire V. Flaherty, Arghavan S. Zangeneh, Marissa A. Harrison and Sanjana Marikunte Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.74158 Degeneration (FTLD) Support an Estrogenic Model of Degeneration (FTLD) Support an Estrogenic Model of Gender differences in frontotemporal lobar degeneration (FTLD) have been reported in the literature but not well characterized or explored. In the present work, we propose that steroid hormone estrogens delay the onset of FTLD in pre-menopausal women compared to age equivalent men, and may provide neuroprotection in the early postmenopausal period. We present a model wherein estrogens serve a regulatory role in attenuating the microglia conversion from the benign to active form in response to cell stress that might otherwise trigger an inflammatory response. Via microglia stabilization, estrogens preserve the homeostasis of both the ubiquitin-proteosome degradation system and lysosome-autophagy recycling system. Both systems have been implicated in the genetic forms of FTLD, with the latter system recognized to be associated with the Keywords: frontotemporal lobar degeneration (FTLD), gender differences, estrogens, FTLD is second only to Alzheimer's disease as a leading cause of primary degenerative dementia in those under age 65 [1, 2]. Researchers estimate that it is responsible for one out of six cases of pre-senile dementia in post-mortem confirmed cases of individuals under age 70 [3]. FTLD symptoms range from motor and language impairment to profound behavioral changes and deficits [4, 5], including severely attenuated initiative to profound impulsivity > © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.5772/intechopen.74158 [193] Tkachev D, Mimmack ML, Ryan MM, Wayland M, Freeman T, Jones PB, Starkey M, Webster MJ, Yolken RH, Bahn S. Oligodendrocyte dysfunction in schizophrenia and bipolar disorder. Lancet (London, England). 2003;362:798-805 #### Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model of Delayed Onset Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model of Delayed Onset DOI: 10.5772/intechopen.74158 Claire V. Flaherty, Arghavan S. Zangeneh, Marissa A. Harrison and Sanjana Marikunte Claire V. Flaherty, Arghavan S. Zangeneh, Marissa A. Harrison and Sanjana Marikunte Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.74158 #### Abstract [192] Hechter O, Grossman A, Chatterton RT. Relationship of dehydroepiandrosterone and [193] Tkachev D, Mimmack ML, Ryan MM, Wayland M, Freeman T, Jones PB, Starkey M, Webster MJ, Yolken RH, Bahn S. Oligodendrocyte dysfunction in schizophrenia and cortisol in disease. Medical Hypotheses. 1997;49:85-91 68 Sex Hormones in Neurodegenerative Processes and Diseases bipolar disorder. Lancet (London, England). 2003;362:798-805 Gender differences in frontotemporal lobar degeneration (FTLD) have been reported in the literature but not well characterized or explored. In the present work, we propose that steroid hormone estrogens delay the onset of FTLD in pre-menopausal women compared to age equivalent men, and may provide neuroprotection in the early postmenopausal period. We present a model wherein estrogens serve a regulatory role in attenuating the microglia conversion from the benign to active form in response to cell stress that might otherwise trigger an inflammatory response. Via microglia stabilization, estrogens preserve the homeostasis of both the ubiquitin-proteosome degradation system and lysosome-autophagy recycling system. Both systems have been implicated in the genetic forms of FTLD, with the latter system recognized to be associated with the majority of them. Keywords: frontotemporal lobar degeneration (FTLD), gender differences, estrogens, autophagy, microglia, neuroprotection #### 1. Introduction FTLD is second only to Alzheimer's disease as a leading cause of primary degenerative dementia in those under age 65 [1, 2]. Researchers estimate that it is responsible for one out of six cases of pre-senile dementia in post-mortem confirmed cases of individuals under age 70 [3]. FTLD symptoms range from motor and language impairment to profound behavioral changes and deficits [4, 5], including severely attenuated initiative to profound impulsivity © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. [6]. FTLD is characterized by three subtypes: behavioral variant frontotemporal dementia (bvFTD), semantic dementia (SD), and primary progressive aphasia agrammatic variant (PPA-agrammatic), although there remains ongoing discussion about their classification [5, 7]. As the most prevalent form of pre-senile dementia after early onset Alzheimer and vascular dementia [8], gender differences in onset and characteristics of FTLD have received little attention in comparison to older onset Alzheimer's dementia. The initial conclusions of the Women's Health Initiative Memory Study (WHIMS) associated long-term estrogen replacement in women ≥65 years of age with increased risk of senile dementia but not mild cognitive impairment (MCI) [9]. MCI, as the Alzheimer's dementia prodrome, is known to remain stable in 1/2 to 2/3 of patients, with stability largely dependent upon the absence of apolipoprotein E e4 (APOE e4) alleles [10]. Consistent with this, estrogen replacement risk of senile dementia has been shown to be dependent upon the presence of the APOE e4 gene. In their longitudinal evaluation of the cognitive status of 12,612 participants of the Nurses' Health Study (≥70 years old) over 4 years, Kang and Grodstein [11] found that participants on hormone replacement since menopause onset, who were carriers of the APOE e4 allele demonstrated a significantly worse rate of cognition decline than nonhormone users. In the absence of APOE e4, the cognitive status of participants was equivalent, regardless of hormone replacement status. While findings did not demonstrate any significant benefit from hormone replacement in this cohort, the authors point out that this was a relatively homogeneous population of highly educated female nurses. Cognitive reserve may have protected nonestrogen users from declines into dementia during the 4 year period of study. All three subtypes of FTLD (bvFTLD, SD, and PPA-agrammatic) are associated with alterations in arousal, characterized by apathy. Our recent findings from a national, multicenter study of nondemented ALS with cognitive impairment and/or behavioral impairment were consistent with this, while evidencing a significantly greater incidence of impulsivity and jocularity in males, as well as personal neglect in females [23]. We contend that this distinction is due to the involvement of ventromedial natural reward and dominance circuitry in the emerging neurodegenerative disease process and the role of estrogen therein (Figures 1 and 2). Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model… http://dx.doi.org/10.5772/intechopen.74158 71 Figure 1. Mesostriatal pathway: natural reward–dominance neural system. With respect to FTD, Ratnavalli and colleagues [12] found that men were four times more likely than women to be affected by bvFTD. They posited that this may have been an artifact of their small sample size, but also referred to older research documenting higher rates of FTLD in men [13] and encouraged further exploration. Johnson and colleagues [14] also reported sex differences. They found that more men had bvFTD and SD, while more women had progressive nonfluent aphasia. They noted that this may be due to sex-specific vulnerability to neurodegeneration for women in the left frontal region and men in right frontal and bilateral temporal regions. Bede and colleagues [15] recently evidenced gender differences in amyotrophic lateral sclerosis (ALS), considered to be a motor variant of FTLD on a clinical continuum [16, 17] with characteristics of FTLD in the absence of dementia in up to 50% of cases [18]. #### 2. The role of estrogen in the brain Beyond their role as reproductive hormones, estrogens, specifically 17β estradiol, exert a neuroprotective role in the brain through estrogen receptors widely distributed in the male and female brain. Multiple estrogen signaling pathways are now recognized in the human brain that are involved in the protection of brain from cognitive decline, emotional dysregulation, and neurodegeneration [19]. Moreover, the neuroendocrine response to stress is genderspecific and associated with the presence of gender-specific gonadal steroids [20]. Estrogen has been shown to be involved in cortical and subcortical hypothalamic-pituitary-adrenal (HPA) function [21], with activation of HPA arousal circuitry evidenced to be regulated in adult women by the hormonal cycle [21, 22]. All three subtypes of FTLD (bvFTLD, SD, and PPA-agrammatic) are associated with alterations in arousal, characterized by apathy. Our recent findings from a national, multicenter study of nondemented ALS with cognitive impairment and/or behavioral impairment were consistent with this, while evidencing a significantly greater incidence of impulsivity and jocularity in males, as well as personal neglect in females [23]. We contend that this distinction is due to the involvement of ventromedial natural reward and dominance circuitry in the emerging neurodegenerative disease process and the role of estrogen therein (Figures 1 and 2). [6]. FTLD is characterized by three subtypes: behavioral variant frontotemporal dementia (bvFTD), semantic dementia (SD), and primary progressive aphasia agrammatic variant (PPA-agrammatic), although there remains ongoing discussion about their classification [5, 7]. As the most prevalent form of pre-senile dementia after early onset Alzheimer and vascular dementia [8], gender differences in onset and characteristics of FTLD have received little attention in comparison to older onset Alzheimer's dementia. The initial conclusions of the Women's Health Initiative Memory Study (WHIMS) associated long-term estrogen replacement in women ≥65 years of age with increased risk of senile dementia but not mild cognitive impairment (MCI) [9]. MCI, as the Alzheimer's dementia prodrome, is known to remain stable in 1/2 to 2/3 of patients, with stability largely dependent upon the absence of apolipoprotein E e4 (APOE e4) alleles [10]. Consistent with this, estrogen replacement risk of senile dementia has been shown to be dependent upon the presence of the APOE e4 gene. In their longitudinal evaluation of the cognitive status of 12,612 participants of the Nurses' Health Study (≥70 years old) over 4 years, Kang and Grodstein [11] found that participants on hormone replacement since menopause onset, who were carriers of the APOE e4 allele demonstrated a significantly worse rate of cognition decline than nonhormone users. In the absence of APOE e4, the cognitive status of participants was equivalent, regardless of hormone replacement status. While findings did not demonstrate any significant benefit from hormone replacement in this cohort, the authors point out that this was a relatively homogeneous population of highly educated female nurses. Cognitive reserve may have protected nonestrogen users With respect to FTD, Ratnavalli and colleagues [12] found that men were four times more likely than women to be affected by bvFTD. They posited that this may have been an artifact of their small sample size, but also referred to older research documenting higher rates of FTLD in men [13] and encouraged further exploration. Johnson and colleagues [14] also reported sex differences. They found that more men had bvFTD and SD, while more women had progressive nonfluent aphasia. They noted that this may be due to sex-specific vulnerability to neurodegeneration for women in the left frontal region and men in right frontal and bilateral temporal regions. Bede and colleagues [15] recently evidenced gender differences in amyotrophic lateral sclerosis (ALS), considered to be a motor variant of FTLD on a clinical continuum [16, 17] with characteristics of FTLD in the absence of dementia in up to 50% of cases [18]. Beyond their role as reproductive hormones, estrogens, specifically 17β estradiol, exert a neuroprotective role in the brain through estrogen receptors widely distributed in the male and female brain. Multiple estrogen signaling pathways are now recognized in the human brain that are involved in the protection of brain from cognitive decline, emotional dysregulation, and neurodegeneration [19]. Moreover, the neuroendocrine response to stress is genderspecific and associated with the presence of gender-specific gonadal steroids [20]. Estrogen has been shown to be involved in cortical and subcortical hypothalamic-pituitary-adrenal (HPA) function [21], with activation of HPA arousal circuitry evidenced to be regulated in from declines into dementia during the 4 year period of study. 2. The role of estrogen in the brain 70 Sex Hormones in Neurodegenerative Processes and Diseases adult women by the hormonal cycle [21, 22]. Figure 1. Mesostriatal pathway: natural reward–dominance neural system. Figure 2. Mesocortical pathway: natural reward–dominance–motivation neural system; PN = paranigral nucleus, PBN = parabrachial pigmented area, PFR = parafasciculus retroflexus area, VTT = ventral tegmental tail, DLPFC = dorsolateral pre-frontal cortex, FCC = frontocingulate cortex, ACC = anterior cingulate cortex, and rACC = rostral ACC. Given that midbrain onset females in our study demonstrated estrogen neuroprotection with respect to cognitive executive functions, we expanded our model of midbrain involvement in FTLD to involve the role of the cerebellum in executive functioning decline, based upon our Figure 3. Cerebellar regulation of the mesocortical pathway: regulation of the natural reward-dominance-motivation Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model… http://dx.doi.org/10.5772/intechopen.74158 73 It has been widely established that ovarian gonadosteroidal hormones provide protection against brain injury and degeneration and provide cognitive maintenance [25–27]. Evidence of estradiol (the primary ovarian estrogen) neuroprotection comes from histopathological studies in rats [28] as well as healthy human females [29]. The release of neuroestradiol from the stalk median eminence (SME) of the hypothalamus in ovarectomized female monkeys has recently been evidenced [30]. Electrical stimulation of the medial basal hypothalamus resulted in release of both gonadotrophin releasing hormone (GnRH) and estrodial. This sug- In recent neuroimaging data, the evidence of estrogen regulation of anterior cingulate cortex (ACC)-associated motivation in healthy human females was evidenced by examining the effects of estrogen on the neural correlates of emotional response inhibition. Applying an gests its vital role as a neurotransmitter involved in regulation of GnRH release. own findings and recent imaging evidence [24] (Figure 3). neural system. More recently, we evidenced greater cognitive and behavioral stability in 78 women from this cohort, based upon both site of disease onset (midbrain vs. spinal cord) and estrogen status (high vs. low). Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model… http://dx.doi.org/10.5772/intechopen.74158 73 Figure 3. Cerebellar regulation of the mesocortical pathway: regulation of the natural reward-dominance-motivation neural system. Given that midbrain onset females in our study demonstrated estrogen neuroprotection with respect to cognitive executive functions, we expanded our model of midbrain involvement in FTLD to involve the role of the cerebellum in executive functioning decline, based upon our own findings and recent imaging evidence [24] (Figure 3). It has been widely established that ovarian gonadosteroidal hormones provide protection against brain injury and degeneration and provide cognitive maintenance [25–27]. Evidence of estradiol (the primary ovarian estrogen) neuroprotection comes from histopathological studies in rats [28] as well as healthy human females [29]. The release of neuroestradiol from the stalk median eminence (SME) of the hypothalamus in ovarectomized female monkeys has recently been evidenced [30]. Electrical stimulation of the medial basal hypothalamus resulted in release of both gonadotrophin releasing hormone (GnRH) and estrodial. This suggests its vital role as a neurotransmitter involved in regulation of GnRH release. In recent neuroimaging data, the evidence of estrogen regulation of anterior cingulate cortex (ACC)-associated motivation in healthy human females was evidenced by examining the effects of estrogen on the neural correlates of emotional response inhibition. Applying an More recently, we evidenced greater cognitive and behavioral stability in 78 women from this cohort, based upon both site of disease onset (midbrain vs. spinal cord) and estrogen status Figure 2. Mesocortical pathway: natural reward–dominance–motivation neural system; PN = paranigral nucleus, PBN = parabrachial pigmented area, PFR = parafasciculus retroflexus area, VTT = ventral tegmental tail, DLPFC = dorsolateral pre-frontal cortex, FCC = frontocingulate cortex, ACC = anterior cingulate cortex, and rACC = rostral ACC. (high vs. low). 72 Sex Hormones in Neurodegenerative Processes and Diseases in-subject design involving 20 right-handed female subjects of average age 25.4 years, Amin and colleagues [31] combined 3.0 T functional magnetic resonance imaging (fMRI) with quantitative analysis of ovarian hormones. All participants evidenced stability of mood across the menstrual cycle. Subjects were scanned during the early follicular phase, when levels of estrogen and progesterone were low, and during the mid-luteal phase, when levels of estrogen and progesterone were high. Subjects were scanned while they were engaged in a verbal go/ no-go task involving positive, negative, and neutral stimuli. This task was chosen because it was already evidenced in the literature to activate the dorsolateral prefrontal cortex (DLPFC) and the ACC. Cycle phase and condition were within-subject independent variables, while mean reaction time and accuracy of response were independent variables. During the follicular phase (low hormones), women exhibited significantly decreased activation in the bilateral ACC and some portions of the left PFC in response to positive distracters, relative to positive targets. During the luteal phase (high hormones), however, women exhibited decreased activation in the ACC in response to negative distracters and increased activation in the DLPFC in response to positive distracters. The investigators noted that the luteal phase findings were consistent with literature associating human female estrogen levels with positive affect. They further noted that their findings of negative correlation between estradiol levels and activation in response to negative distractors, relative to negative targets, were consistent with previous research [21]. This contention is further supported by more recent findings from the KEEPS longitudinal clinical trial focused on the potential for estrogen neuroprotection in 693 younger post-menopausal women of average age 52.6 years old and 1.4 years past their last menstrual period [32]. Following 4½ years, the estrogen replacement subgroups (N = 693) evidenced significant improvements in depression and anxiety in comparison to a placebo subgroup (N = 262). Cognitive status remained stable with monitoring to assess dementia incidence with aging ongoing. #### 3. Estrogen regulation of the stress response as a model of neuroprotection Goldstein and colleagues [21] applied an fMRI paradigm to examine the effect of estrogen on brain regions involved in the stress response by using aversive affective stimuli in a group of 12 right-handed women, ages 36–40. Their imaging data evidenced an association between the early follicular phase (low estrogen) and significantly increased activation to neutral stimuli, relative to negative targets, in the central amygdala nuclei, paraventricular hypothalamic nuclei, peripeduncular nuclei, orbital frontal cortex, and AC gyrus (ACG). In comparison to the early follicular phase, the luteal phase (high estrogen) was associated with decreased activation in the central amygdaloid, ventromedial hypothalamic, orbital frontal, and cingulate nuclei in response to negative vs. neutral stimuli. With respect to FTLD, we propose a model whereby estrogen provides neuroprotection by mediation of the neuroimmunological and neuroendocrinological stress response (i.e., release of anti-inflammatories and stress associated hormones) (Figure 4). Figure 4. Estrogen binding to microglia induces feedback inhibition of the inflammatory cascade, resulting in neuroprotection. Mice models of toxic demyelination evidence estrogen induction of insulin-like growth factor 1 (IGF-1) expression by astrocytes. IGF-1 then promotes proliferation of oligodendrocyte precursors and their differentiation into Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model… http://dx.doi.org/10.5772/intechopen.74158 75 mature remyelinating oligodendrocytes. Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model… http://dx.doi.org/10.5772/intechopen.74158 75 in-subject design involving 20 right-handed female subjects of average age 25.4 years, Amin and colleagues [31] combined 3.0 T functional magnetic resonance imaging (fMRI) with quantitative analysis of ovarian hormones. All participants evidenced stability of mood across the menstrual cycle. Subjects were scanned during the early follicular phase, when levels of estrogen and progesterone were low, and during the mid-luteal phase, when levels of estrogen and progesterone were high. Subjects were scanned while they were engaged in a verbal go/ no-go task involving positive, negative, and neutral stimuli. This task was chosen because it was already evidenced in the literature to activate the dorsolateral prefrontal cortex (DLPFC) and the ACC. Cycle phase and condition were within-subject independent variables, while mean reaction time and accuracy of response were independent variables. During the follicular phase (low hormones), women exhibited significantly decreased activation in the bilateral ACC and some portions of the left PFC in response to positive distracters, relative to positive targets. During the luteal phase (high hormones), however, women exhibited decreased activation in the ACC in response to negative distracters and increased activation in the DLPFC in response to positive distracters. The investigators noted that the luteal phase findings were consistent with literature associating human female estrogen levels with positive affect. They further noted that their findings of negative correlation between estradiol levels and activation in response to negative distractors, relative to negative targets, were consistent with previous research [21]. This contention is further supported by more recent findings from the KEEPS longitudinal clinical trial focused on the potential for estrogen neuroprotection in 693 younger post-menopausal women of average age 52.6 years old and 1.4 years past their last menstrual period [32]. Following 4½ years, the estrogen replacement subgroups (N = 693) evidenced significant improvements in depression and anxiety in comparison to a placebo subgroup (N = 262). Cognitive status remained stable with monitoring to assess dementia incidence with aging ongoing. 74 Sex Hormones in Neurodegenerative Processes and Diseases neuroprotection ated hormones) (Figure 4). 3. Estrogen regulation of the stress response as a model of Goldstein and colleagues [21] applied an fMRI paradigm to examine the effect of estrogen on brain regions involved in the stress response by using aversive affective stimuli in a group of 12 right-handed women, ages 36–40. Their imaging data evidenced an association between the early follicular phase (low estrogen) and significantly increased activation to neutral stimuli, relative to negative targets, in the central amygdala nuclei, paraventricular hypothalamic nuclei, peripeduncular nuclei, orbital frontal cortex, and AC gyrus (ACG). In comparison to the early follicular phase, the luteal phase (high estrogen) was associated with decreased activation in the central amygdaloid, ventromedial hypothalamic, orbital frontal, and cingulate nuclei in response to negative vs. neutral stimuli. With respect to FTLD, we propose a model whereby estrogen provides neuroprotection by mediation of the neuroimmunological and neuroendocrinological stress response (i.e., release of anti-inflammatories and stress associ- Figure 4. Estrogen binding to microglia induces feedback inhibition of the inflammatory cascade, resulting in neuroprotection. Mice models of toxic demyelination evidence estrogen induction of insulin-like growth factor 1 (IGF-1) expression by astrocytes. IGF-1 then promotes proliferation of oligodendrocyte precursors and their differentiation into mature remyelinating oligodendrocytes. ### 4. Lysosome and proteosome homeostasis: estrogen mitigation of the microglia inflammatory response in neurodegeneration regulatory role in the immune system, ERβ is involved in tumor suppression [61]. Thus, in recent years, pharmaceutical companies have generated selective agonists for ERα and ERβ, with ERβ research ongoing for ERβ development as a cancer preventative, as an anti-inflam- Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model… http://dx.doi.org/10.5772/intechopen.74158 77 Microglia are mediators of the innate immune defense, acting as antigens and scavenger cells during brain inflammation, acute central nervous system (CNS) injury, and neurodegeneration in the course of aging [63]. As such, they represent the resident macrophages of the CNS, comprising 5–15% of total cells in the brain [64]. As the brain-resident immunocompetent cells, microglia are critical for proper innate immune responses in the brain [42]. In the steady state, they perform housekeeping chores via autophagy [65] and may participate in postnatal neuronal development, whereas following trauma or pathogenic insult; they initiate the inflammatory response as part of the brain immune defense [66]. In the course of this, they increase their release of several inflammatory-associated substances, including reactive oxygen intermediates, nitric oxide (NO), and (inflammatory) cytokines [interleukin-1 and 6 (IL-1 Microglia cells express a set of classical and non-classical steroidal hormonal receptors, including ERα, ERβ, progesterone receptors (PR), glucocorticoid receptors (GR), and mineralocorticoid receptors (MR) [42, 68]. The central role of ERα and ERβ in the regulation of the microglia inflammatory response, in conjunction with the recent discovery of medial basal hypothalamic neuroestradiol, suggests the potential of estrogens to mitigate the pathogenesis and progression of neuroinflammation and neurodegeneration [69]. In particular, two gonadal steroid hormones, 17α-estradiol and progesterone, provide robust neuroprotection in a variety of experimental brain injury models [43, 54, 70, 71] and under neurodegenerative conditions [72–74]. It is possible that 17α-estradiol is an effective neuroprotective agent due to its actions as an anti-oxidant, anti-inflammatory, and anti-apoptotic steroid hormone [42, 57, 75–77]. Given the role of estrogenic steroids in modulating neurogenesis and the generation of dendritic spines and neuroprotection, Jellinck and colleagues [66] explored the question of whether microglia might serve as a regional source for estrogenic steroids. With respect to neuroprotection, dehydroepiandrosterone (DHEA) is considered to exert its positive influence via conversion to estrogen (estrone and estradiol). However, this conversion is slow and limited in brain cell cultures, with the exception of microglia [66]. These researchers were able to demonstrate the presence of the enzyme necessary for the rate limiting step of this conversion within microglia cells, supporting their contention that microglia are integral to regional regulation of In the healthy CNS, microglia appear in a "resident state" with a ramified morphology (Figure 4). However, microglia are very susceptible to changes in the CNS milieu and become rapidly activated in response to CNS insult. Attracted by endogenous and other chemical messenger factors, microglial cells demonstrate the capacity to migrate toward the site of brain injury. Upon activation, microglia undergo morphological and functional changes such as hypertrophy and up-regulation of major histocompatibility complex (MHC) antigens. Activated cells secrete inflammatory mediators, including cytokines and chemokines [78, 79]. Microglia can matory drug and for the attenuation of neurodegenerative diseases [62]. and IL-6), interferon-g (IFN-g), and tumor necrosis factor-α (TNF-α)] [67]. adult estrogen-dependent brain plasticity and neuroprotection. also produce different types of free superoxide radicals and prostanoids [55]. As disorders of aging, many neurodegenerative diseases demonstrate significant gender differences in their prevalence, symptomatology, and prognosis, implicating gonadal steroidal hormones in their pathophysiology [33, 34]. Parkinson's disease (PD) has for a long time been recognized to be more prevalent in males than in females, with a relative male to female ratio ranging from 1.4 to 3.7. Evidence suggests that levels of estrogens or progesterone or differences in their respective receptor levels could account for this gender difference [35, 36]. Particularly with respect to early onset dementia, emerging with onset of menopause or andropause, endogenous estrogen regulates brain physiology through a concerted action on diverse cell types and molecular targets [37]. Women who undergo surgical removal of the ovaries before menopause clearly demonstrate that oophorectomy is associated with an increased risk of cognitive impairment and dementia [38]. Women's Health Initiative (WHI) data, taking into consideration the time between menopause onset and hormone therapy initiation, showed beneficial effects of estrogens when therapy was initiated early after menopause, with detrimental effects associated with treatment started several years following menopause [39]. This supports the theory of estrogen as a neuroprotective agent while underscoring the limitations of the approach. Estrogen protection has a 'window-in-time': potentially effective if applied around the menopause period and maintained for a 5–10 year period of clinical effectiveness, given the absence of significant cancer or dementia risk factors [40], including the presence of the APOE e4 gene [11]. The neuroendocrine system is a powerful regulator of the inflammatory response in health and disease states [41]. In addition to regulating peripheral immune responses, steroid hormones, including glucocorticoids and gonadal steroids, support anti-inflammatory properties in the brain [42]. Steroid hormone protective actions in the nervous system range from stabilizing the blood brain barrier (BBB), alleviating brain edema, dampening pro-inflammatory/ supporting anti-inflammatory processes, activating anti-apoptotic pathways, stimulating survival-promoting factors, counteracting oxidative stress, promoting respiratory chain function, and reducing glutamate excitotoxicity [43–48]. Estrogens are known to exert their actions through members of the nuclear hormone receptor superfamily, ERα [49], and the more recently identified estrogen receptor–ERβ [50–52]. Estrogen is recognized to exert an inhibitory response to neuroinflammation, with specificity of binding to microglia, resulting in the attenuation of the inflammatory response [53]. 17β-estradiol and progesterone have also been shown to mediate anti-inflammatory activity and improve neuronal survival [44, 54–56]. 17β-estradiol targets many pathways active in secondary injury; including oxidative stress, inflammation, apoptosis, and ischemia [57] (Figure 4). Detecting the expression of the two estrogen receptors ERα and ERβ in cells of the monocyte-macrophage lineage, Vegeto and colleagues first evidenced the role that estrogens play in inflammatory diseases [58] with several laboratories later demonstrating that these gonadal steroid hormones act in a variety of macrophage-like cells to regulate the inflammatory response triggered by diverse inflammatory stimuli [59, 60]. In addition to having a regulatory role in the immune system, ERβ is involved in tumor suppression [61]. Thus, in recent years, pharmaceutical companies have generated selective agonists for ERα and ERβ, with ERβ research ongoing for ERβ development as a cancer preventative, as an anti-inflammatory drug and for the attenuation of neurodegenerative diseases [62]. 4. Lysosome and proteosome homeostasis: estrogen mitigation of the As disorders of aging, many neurodegenerative diseases demonstrate significant gender differences in their prevalence, symptomatology, and prognosis, implicating gonadal steroidal hormones in their pathophysiology [33, 34]. Parkinson's disease (PD) has for a long time been recognized to be more prevalent in males than in females, with a relative male to female ratio ranging from 1.4 to 3.7. Evidence suggests that levels of estrogens or progesterone or differences in their respective receptor levels could account for this gender difference [35, 36]. Particularly with respect to early onset dementia, emerging with onset of menopause or andropause, endogenous estrogen regulates brain physiology through a concerted action on diverse cell types and molecular targets [37]. Women who undergo surgical removal of the ovaries before menopause clearly demonstrate that oophorectomy is associated with an increased risk of cognitive impairment and dementia [38]. Women's Health Initiative (WHI) data, taking into consideration the time between menopause onset and hormone therapy initiation, showed beneficial effects of estrogens when therapy was initiated early after menopause, with detrimental effects associated with treatment started several years following menopause [39]. This supports the theory of estrogen as a neuroprotective agent while underscoring the limitations of the approach. Estrogen protection has a 'window-in-time': potentially effective if applied around the menopause period and maintained for a 5–10 year period of clinical effectiveness, given the absence of significant cancer or dementia risk factors [40], The neuroendocrine system is a powerful regulator of the inflammatory response in health and disease states [41]. In addition to regulating peripheral immune responses, steroid hormones, including glucocorticoids and gonadal steroids, support anti-inflammatory properties in the brain [42]. Steroid hormone protective actions in the nervous system range from stabilizing the blood brain barrier (BBB), alleviating brain edema, dampening pro-inflammatory/ supporting anti-inflammatory processes, activating anti-apoptotic pathways, stimulating survival-promoting factors, counteracting oxidative stress, promoting respiratory chain func- Estrogens are known to exert their actions through members of the nuclear hormone receptor superfamily, ERα [49], and the more recently identified estrogen receptor–ERβ [50–52]. Estrogen is recognized to exert an inhibitory response to neuroinflammation, with specificity of binding to microglia, resulting in the attenuation of the inflammatory response [53]. 17β-estradiol and progesterone have also been shown to mediate anti-inflammatory activity and improve neuronal survival [44, 54–56]. 17β-estradiol targets many pathways active in secondary injury; Detecting the expression of the two estrogen receptors ERα and ERβ in cells of the monocyte-macrophage lineage, Vegeto and colleagues first evidenced the role that estrogens play in inflammatory diseases [58] with several laboratories later demonstrating that these gonadal steroid hormones act in a variety of macrophage-like cells to regulate the inflammatory response triggered by diverse inflammatory stimuli [59, 60]. In addition to having a including oxidative stress, inflammation, apoptosis, and ischemia [57] (Figure 4). microglia inflammatory response in neurodegeneration 76 Sex Hormones in Neurodegenerative Processes and Diseases including the presence of the APOE e4 gene [11]. tion, and reducing glutamate excitotoxicity [43–48]. Microglia are mediators of the innate immune defense, acting as antigens and scavenger cells during brain inflammation, acute central nervous system (CNS) injury, and neurodegeneration in the course of aging [63]. As such, they represent the resident macrophages of the CNS, comprising 5–15% of total cells in the brain [64]. As the brain-resident immunocompetent cells, microglia are critical for proper innate immune responses in the brain [42]. In the steady state, they perform housekeeping chores via autophagy [65] and may participate in postnatal neuronal development, whereas following trauma or pathogenic insult; they initiate the inflammatory response as part of the brain immune defense [66]. In the course of this, they increase their release of several inflammatory-associated substances, including reactive oxygen intermediates, nitric oxide (NO), and (inflammatory) cytokines [interleukin-1 and 6 (IL-1 and IL-6), interferon-g (IFN-g), and tumor necrosis factor-α (TNF-α)] [67]. Microglia cells express a set of classical and non-classical steroidal hormonal receptors, including ERα, ERβ, progesterone receptors (PR), glucocorticoid receptors (GR), and mineralocorticoid receptors (MR) [42, 68]. The central role of ERα and ERβ in the regulation of the microglia inflammatory response, in conjunction with the recent discovery of medial basal hypothalamic neuroestradiol, suggests the potential of estrogens to mitigate the pathogenesis and progression of neuroinflammation and neurodegeneration [69]. In particular, two gonadal steroid hormones, 17α-estradiol and progesterone, provide robust neuroprotection in a variety of experimental brain injury models [43, 54, 70, 71] and under neurodegenerative conditions [72–74]. It is possible that 17α-estradiol is an effective neuroprotective agent due to its actions as an anti-oxidant, anti-inflammatory, and anti-apoptotic steroid hormone [42, 57, 75–77]. Given the role of estrogenic steroids in modulating neurogenesis and the generation of dendritic spines and neuroprotection, Jellinck and colleagues [66] explored the question of whether microglia might serve as a regional source for estrogenic steroids. With respect to neuroprotection, dehydroepiandrosterone (DHEA) is considered to exert its positive influence via conversion to estrogen (estrone and estradiol). However, this conversion is slow and limited in brain cell cultures, with the exception of microglia [66]. These researchers were able to demonstrate the presence of the enzyme necessary for the rate limiting step of this conversion within microglia cells, supporting their contention that microglia are integral to regional regulation of adult estrogen-dependent brain plasticity and neuroprotection. In the healthy CNS, microglia appear in a "resident state" with a ramified morphology (Figure 4). However, microglia are very susceptible to changes in the CNS milieu and become rapidly activated in response to CNS insult. Attracted by endogenous and other chemical messenger factors, microglial cells demonstrate the capacity to migrate toward the site of brain injury. Upon activation, microglia undergo morphological and functional changes such as hypertrophy and up-regulation of major histocompatibility complex (MHC) antigens. Activated cells secrete inflammatory mediators, including cytokines and chemokines [78, 79]. Microglia can also produce different types of free superoxide radicals and prostanoids [55]. As a first line of defense, activated microglia perform phagocytosis of apoptotic neuronal cell bodies via the lysozyme-autophagy recycling system. However, chronic activation of microglia with associated excess pro-inflammatory response in the aftermath of CNS insult may overwhelm this natural recycling system of the cell, resulting in cytotoxicity [80]. In the aftermath of stroke, for example, microglial activation is one of the earliest responses; requiring several hours to fully develop, while persisting for up to several days [81, 82]. The microglia inflammatory response, therefore, needs to be tightly controlled to avoid collateral damage within intact brain tissue. Estrogen appears to provide this level of regulation. opportunity for delaying the onset and progression of early onset neurodegenerative diseases Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model… http://dx.doi.org/10.5772/intechopen.74158 79 Importantly, estradiol does not alter the inflammatory signaling cascade in microglia if it is administered after inflammatory stimuli [94, 97, 101]. Moreover, prolonged hormonal deprivation has been evidenced to affect estrogen protective activity in ischemia; resulting in a null or even pro-inflammatory response following administration of exogenous hormone [105]. Collectively, the experimental evidence indicates that the efficacy of estrogenic molecules as anti-inflammatory agents is confined to a therapeutic window and that their use should be considered only as preventive pharmacological strategies. Applied during the pre-clinical or prodrome stage, estrogen represents a therapeutic opportunity to forestall the onset and mitigate the progression of pre-senile neurodegenerative disease processes, particularly those like FTLD that typically emerge in mid-life, prior to or throughout the course of menopause and andropause. With the advent of personalized medicine, it may eventually be possible to identify genotypically high risk individuals and intervene with hormone replacement therapy Estrogen exerts an indirect effect on microglia through specific interactions with cellular signaling molecules. Nitric oxide synthases, a family of enzymes catalyzing the production of NO from l-arginine, are important cellular signaling molecules. The inducible isoform, inducible nitric oxide synthase (iNOS), serves a number of roles, including involvement in the immune response, with production of NO as an immune defense mechanism, due to its free radical nature. It is the proximate cause of septic shock and may function in autoimmune disease [106, 107]. In rats and microglia cell lines, the expression of iNOS and release of reactive oxygen species is reduced in certain cell types through the action of estrogen, including microglia [53, 76, 94], while expression of endothelial and neuronal subtypes of iNOS are increased [108]. and in unidentified cell types [113]. After immunostimulation by lipopolysaccharides, estrogen but not progesterone has been shown to attenuate microglial superoxide release and phagocytotic activity as well as iNOS expression [94]. These effects are transmitted through an estrogen receptor-dependent activation of the MAP-kinase signaling system. Using a transient focal ischemia animal model, investigators have shown that estrogen and progesterone prevent the hypoxia-induced attraction and activation of local microglia and their morphological transition into an activated phenotype in the cortical penumbra [109]. The reduced stimulation of microglia is considered to result from diminished cytokine and interleukin expression and release in local astroglia, consequent to the close concerted communication between these two glial cell types during tissue stress. Focal ischemic mouse model experiments further evidence diminution of the penumbra of estrogen/progesterone-treated animals, along with reduction in chemokine levels, central microglia, and recruited monocytes. Ischemic mouse model data are confirmed by several other studies, which have demonstrated that these steroid hormones affect local cytokine production during brain inflammation in microglia [53, 76, 110], in astroglia [111, 112], such as FTLD, with replacement offsetting menopause associated declines. while the neurodegenerative disease process remains at the sub-clinical level. 5. Estrogen interaction with cellular signaling molecules Microglia express steroid hormone receptors that include ER-α, with immunoreactivity evidenced by electron microscopy studies [35, 42, 68, 83–86]. Indeed, the neurodegenerative process of several CNS diseases, including amyotrophic lateral sclerosis, frontotemporal lobar degeneration, multiple sclerosis, Alzheimer's disease, and Parkinson's disease are associated with the activation of microglia cells, which drive the resident inflammatory response [17, 87–89]. In addition, a number of pro-inflammatory mediators are elevated in the CNS or cerebrospinal fluid of neurodegenerative disease patients [30]. Given this, it is particularly important to recognize that estrogen is understood to maintain microglia in the benign form, associated with suppression of the inflammatory response, suggesting its protective role in the aging brain against ubiquitin-proteosome mediated degradation. Estrogen signaling is characterized by cell-specificity and dose dependent responsiveness. Divergent effects of estrogens have been reported for T cell activation [90], microglia modulation, and astroglia effects based upon different hormone concentrations [91, 92]. 17β-estradiol inhibits microglial activation following exposure to bacterial lipopolysaccharides [53], with estrogen-induced neuroprotection from autophagy lysis or proteasome degradation related to declines in TNF-α expression and NO production [43, 53]. NADPH oxidase represents one important source of free radicals in activated microglia [92], which catalyzes the reduction of oxygen to superoxide radical [93]. 17β-estradiol has been shown to decrease lipopolysaccharides-induced superoxide production and release in N-9 microglia cell lines [94]. ERα and ERβ are intracellular proteins, which activate a multitude of genomic as well as nongenomic effectors in neural cells [87]. Through the use of an estrogen receptor antagonist [ICI 182780], hormone action in microglia has been attributed to the activation of endogenous ERs, since antagonist binding was able to block the effect of estradiol, suggesting a receptor-mediated effect of the hormone [35, 53, 95, 96]. Using estrogen receptor knock out mutant mice, several investigators have described the selective involvement of ERα in the anti-inflammatory and neuroprotective activity of estradiol against neuroinflammatory and vascular pathologies of the brain [97–100]. In ICI 182780 studies, ERα appeared to be selectively involved in estradiol anti-inflammatory activity in microglia, a finding later confirmed by additional experimentation using primary cultures of microglia as well as cell lines [97, 101]. Estrogen-dependent attenuation of microglia activation has been demonstrated to involve reduced lysosome-phagocytic activity, production of reactive oxygen and nitrogen species and other factors of the inflammatory cascade [35, 94, 102–104]. The inhibitory activity of estrogens on microglia-associated neuroinflammation may prove to be a beneficial therapeutic opportunity for delaying the onset and progression of early onset neurodegenerative diseases such as FTLD, with replacement offsetting menopause associated declines. Importantly, estradiol does not alter the inflammatory signaling cascade in microglia if it is administered after inflammatory stimuli [94, 97, 101]. Moreover, prolonged hormonal deprivation has been evidenced to affect estrogen protective activity in ischemia; resulting in a null or even pro-inflammatory response following administration of exogenous hormone [105]. Collectively, the experimental evidence indicates that the efficacy of estrogenic molecules as anti-inflammatory agents is confined to a therapeutic window and that their use should be considered only as preventive pharmacological strategies. Applied during the pre-clinical or prodrome stage, estrogen represents a therapeutic opportunity to forestall the onset and mitigate the progression of pre-senile neurodegenerative disease processes, particularly those like FTLD that typically emerge in mid-life, prior to or throughout the course of menopause and andropause. With the advent of personalized medicine, it may eventually be possible to identify genotypically high risk individuals and intervene with hormone replacement therapy while the neurodegenerative disease process remains at the sub-clinical level. #### 5. Estrogen interaction with cellular signaling molecules As a first line of defense, activated microglia perform phagocytosis of apoptotic neuronal cell bodies via the lysozyme-autophagy recycling system. However, chronic activation of microglia with associated excess pro-inflammatory response in the aftermath of CNS insult may overwhelm this natural recycling system of the cell, resulting in cytotoxicity [80]. In the aftermath of stroke, for example, microglial activation is one of the earliest responses; requiring several hours to fully develop, while persisting for up to several days [81, 82]. The microglia inflammatory response, therefore, needs to be tightly controlled to avoid collateral damage Microglia express steroid hormone receptors that include ER-α, with immunoreactivity evidenced by electron microscopy studies [35, 42, 68, 83–86]. Indeed, the neurodegenerative process of several CNS diseases, including amyotrophic lateral sclerosis, frontotemporal lobar degeneration, multiple sclerosis, Alzheimer's disease, and Parkinson's disease are associated with the activation of microglia cells, which drive the resident inflammatory response [17, 87–89]. In addition, a number of pro-inflammatory mediators are elevated in the CNS or cerebrospinal fluid of neurodegenerative disease patients [30]. Given this, it is particularly important to recognize that estrogen is understood to maintain microglia in the benign form, associated with suppression of the inflammatory response, suggesting its protective role in the aging Estrogen signaling is characterized by cell-specificity and dose dependent responsiveness. Divergent effects of estrogens have been reported for T cell activation [90], microglia modulation, and astroglia effects based upon different hormone concentrations [91, 92]. 17β-estradiol inhibits microglial activation following exposure to bacterial lipopolysaccharides [53], with estrogen-induced neuroprotection from autophagy lysis or proteasome degradation related to declines in TNF-α expression and NO production [43, 53]. NADPH oxidase represents one important source of free radicals in activated microglia [92], which catalyzes the reduction of oxygen to superoxide radical [93]. 17β-estradiol has been shown to decrease lipopolysaccha- ERα and ERβ are intracellular proteins, which activate a multitude of genomic as well as nongenomic effectors in neural cells [87]. Through the use of an estrogen receptor antagonist [ICI 182780], hormone action in microglia has been attributed to the activation of endogenous ERs, since antagonist binding was able to block the effect of estradiol, suggesting a receptor-mediated effect of the hormone [35, 53, 95, 96]. Using estrogen receptor knock out mutant mice, several investigators have described the selective involvement of ERα in the anti-inflammatory and neuroprotective activity of estradiol against neuroinflammatory and vascular pathologies of the brain [97–100]. In ICI 182780 studies, ERα appeared to be selectively involved in estradiol anti-inflammatory activity in microglia, a finding later confirmed by additional experimentation using primary cultures of microglia as well as cell Estrogen-dependent attenuation of microglia activation has been demonstrated to involve reduced lysosome-phagocytic activity, production of reactive oxygen and nitrogen species and other factors of the inflammatory cascade [35, 94, 102–104]. The inhibitory activity of estrogens on microglia-associated neuroinflammation may prove to be a beneficial therapeutic rides-induced superoxide production and release in N-9 microglia cell lines [94]. within intact brain tissue. Estrogen appears to provide this level of regulation. brain against ubiquitin-proteosome mediated degradation. 78 Sex Hormones in Neurodegenerative Processes and Diseases lines [97, 101]. Estrogen exerts an indirect effect on microglia through specific interactions with cellular signaling molecules. Nitric oxide synthases, a family of enzymes catalyzing the production of NO from l-arginine, are important cellular signaling molecules. The inducible isoform, inducible nitric oxide synthase (iNOS), serves a number of roles, including involvement in the immune response, with production of NO as an immune defense mechanism, due to its free radical nature. It is the proximate cause of septic shock and may function in autoimmune disease [106, 107]. In rats and microglia cell lines, the expression of iNOS and release of reactive oxygen species is reduced in certain cell types through the action of estrogen, including microglia [53, 76, 94], while expression of endothelial and neuronal subtypes of iNOS are increased [108]. After immunostimulation by lipopolysaccharides, estrogen but not progesterone has been shown to attenuate microglial superoxide release and phagocytotic activity as well as iNOS expression [94]. These effects are transmitted through an estrogen receptor-dependent activation of the MAP-kinase signaling system. Using a transient focal ischemia animal model, investigators have shown that estrogen and progesterone prevent the hypoxia-induced attraction and activation of local microglia and their morphological transition into an activated phenotype in the cortical penumbra [109]. The reduced stimulation of microglia is considered to result from diminished cytokine and interleukin expression and release in local astroglia, consequent to the close concerted communication between these two glial cell types during tissue stress. Focal ischemic mouse model experiments further evidence diminution of the penumbra of estrogen/progesterone-treated animals, along with reduction in chemokine levels, central microglia, and recruited monocytes. Ischemic mouse model data are confirmed by several other studies, which have demonstrated that these steroid hormones affect local cytokine production during brain inflammation in microglia [53, 76, 110], in astroglia [111, 112], and in unidentified cell types [113]. Mitogen-activated protein kinases (MAPKs) are involved in directing cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock, and microglia generated pro-inflammatory cytokines (e.g., TNFα, IL-6, and IL-1β) [114]. MAPKs regulate proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis [115]. Several MAPK pathways, including p42/44 MAPKs, are known to be activated by 17β estradiol [116]. Moreover, studies have evidenced the importance of activated p42/44 MAPK pathways in estrogen-mediated neuroprotection and microglia homeostasis [117]. When activated in response to inflammation, microglia release large amounts of oxygen and nitrogen radicals. Proteosomes clear oxidized and damaged proteins from cells, serving as a microglia compensatory response to activation. In the mouse model, the p42/44 MAPK pathway participates in estrogen-mediated proteasome activation, with estrogen up-regulation of proteosome activity considered to be one way estrogen potentially promotes microglial viability [117, 118]. mediating mechanisms [131, 134, 135]. In the healthy adult, sex differences in eating exist, regulated by the hypothalamic-pituitary-gonadal (HPG) axis. Little is known about the direct effect of testosterone on eating, while the effects of 17β-estradiol, the primary estrogen, have been well characterized. Hypothalamic centers are recognized to be intimately involved in the regulation of appetite, with extensive neuronal control reflected in their innervation by axons expressing all the major neurotransmitters [136, 137]. Hypothalamic centers are known to play a vital role in neuronal action of insulin and adipose tissue-secreted leptins. Estrogen binding (i.e., estradiol-triggered calcium influx) results in appetite suppression, with parvabumin potentially serving a protective role in the attenuation of calcium overload-associated Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model… http://dx.doi.org/10.5772/intechopen.74158 81 The posterior hypothalamus contains nuclei that play a critical role in regulating feeding behavior [136, 137]. Recent in vivo structural neuroimaging demonstrated a relationship between deterioration in the posterior hypothalamus and appetite disturbances in FTLD, an early sign of disease onset evident within 2 years of diagnosis [133]. Post-mortem analysis further evidenced sparse TDP-43-immunoreactive neurites within TDP-43 positive cases, with occasional intracytoplasmic inclusions in posterior hypothalamic neurons [133]. In their analysis of the differential effects of peripheral hormones vs. hypothalamic pathology on eating behavior in FTLD, Ahmed and colleagues [131] found higher levels of the hypothalamic derived satiation hormone agouti-related peptide in the serum of bvFTD and SD patients, with both groups having elevated scores on a questionnaire of eating behaviors. Atrophy of the posterior and total hypothlamus was found only for the bvFTD subgroup [131]. Interestingly, gender differences could not be examined, due to the relatively low numbers of females in both the bvFTD (4/15) and SD (8/18) subgroups, in comparison to gender As a bvFTD subtype, apathy is characterized by inertia and loss of volition, as well as apathy, in association with pathology within the dorsolateral convexities of the frontal lobe [139]. As an amotivational syndrome, apathy is also well recognized to be associated with disruptions to the ventral anterior cingulate cortex [139, 140]. In addition, regions known to be associated with Apathy include the medial dorsal nucleus of the thalamus, caudate nucleus, ventral medial striate (nucleus accumbens) and globus pallidus; with the cortical-striatal-thalamiccortical circuit being the circuit most implicated in the Apathy syndrome [141]. Apathy neural circuitry is linked to the richly dopaminergic nonmotor limbic loop of the basal ganglia, with adaptive behaviors requiring a combination of reward evaluation, associative learning, and ability to develop appropriate action plans [142]. Dopamine deficiencies are often characteristic of FTLD. Clinical trials involving the use of dopaminergic psychostimulants have evidenced improvements in symptomology ranging from apathy to disinhibition and risk taking behaviors [143]. With respect to the potential for estrogen derivatives to mitigate apathy in FTLD, the responsiveness of dopamine neurons to estrogens has long been established, with inducement of dopamine synthesis and release, as well as dopamine neuron differentia- neuronal degeneration (see Sinchack and Wagner for a detailed review) [138]. matched controls (12/11). 6.2. Motivation regulation tion [144–147]. Calcium-dependent protease with papain-like activity, or calpain, is a cytoplasmic cysteine protease that is activated by calcium [119]. Calpain is involved in neurodegeneration in a variety of injuries and diseases [120]. Calpain cleaves many substrates, including cytoskeletal proteins, axonal, and myelin proteins, and pro-apoptotic Bax causing mitochondrial cytochrome c release, activation of caspase-3, and activation of microglia [121–123]. Studies indicate that estrogen attenuates Ca2+ influx via modulation of L-type Ca2+ channels and the Na<sup>+</sup> / Ca2+ exchanger [124]. Estrogen has also been shown to reduce calpain expression and activity, resulting in reduced axon degeneration and neuronal apoptosis in vitro [125]. Finally, estrogen is involved in regulation of microglial matrix metalloproteinases [126]. Metalloproteases secreted from microglia mediate inflammation and tissue degradation through processing of pro-inflammatory cytokines and damage to the BBB [127]. Progestins and estrogens affect matrix metalloproteinase enzyme activity in microglial cells, reducing indications of microglial inflammation [97, 126, 128]. #### 6. Evidence of estrogenic effects in FTLD Although few studies known to-date have focused on estrogenic effects in FTLD, indirect evidence comes from physiological studies in both normal and genetically disordered individuals. In this regard, it is notable that FTLD is associated with over a dozen genetic mutations that include the ALS-associated X linked UBQLN2 variant [129]. Neuropathologically, FTLD is primarily represented by two subtypes: one involving aberrant inclusions of microtubule protein tau, and one involving inclusions of TAR DNA binding protein [130]. #### 6.1. Appetite regulation Both bvFTD and SD are associated with changes in appetite and eating habits, with overeating and a preference for sweets and excessive seasonings, including salt [131–133]. Appetite is recognized to be modulated by gonadal steroid hormones, including peripheral and central mediating mechanisms [131, 134, 135]. In the healthy adult, sex differences in eating exist, regulated by the hypothalamic-pituitary-gonadal (HPG) axis. Little is known about the direct effect of testosterone on eating, while the effects of 17β-estradiol, the primary estrogen, have been well characterized. Hypothalamic centers are recognized to be intimately involved in the regulation of appetite, with extensive neuronal control reflected in their innervation by axons expressing all the major neurotransmitters [136, 137]. Hypothalamic centers are known to play a vital role in neuronal action of insulin and adipose tissue-secreted leptins. Estrogen binding (i.e., estradiol-triggered calcium influx) results in appetite suppression, with parvabumin potentially serving a protective role in the attenuation of calcium overload-associated neuronal degeneration (see Sinchack and Wagner for a detailed review) [138]. The posterior hypothalamus contains nuclei that play a critical role in regulating feeding behavior [136, 137]. Recent in vivo structural neuroimaging demonstrated a relationship between deterioration in the posterior hypothalamus and appetite disturbances in FTLD, an early sign of disease onset evident within 2 years of diagnosis [133]. Post-mortem analysis further evidenced sparse TDP-43-immunoreactive neurites within TDP-43 positive cases, with occasional intracytoplasmic inclusions in posterior hypothalamic neurons [133]. In their analysis of the differential effects of peripheral hormones vs. hypothalamic pathology on eating behavior in FTLD, Ahmed and colleagues [131] found higher levels of the hypothalamic derived satiation hormone agouti-related peptide in the serum of bvFTD and SD patients, with both groups having elevated scores on a questionnaire of eating behaviors. Atrophy of the posterior and total hypothlamus was found only for the bvFTD subgroup [131]. Interestingly, gender differences could not be examined, due to the relatively low numbers of females in both the bvFTD (4/15) and SD (8/18) subgroups, in comparison to gender matched controls (12/11). #### 6.2. Motivation regulation / Mitogen-activated protein kinases (MAPKs) are involved in directing cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock, and microglia generated pro-inflammatory cytokines (e.g., TNFα, IL-6, and IL-1β) [114]. MAPKs regulate proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis [115]. Several MAPK pathways, including p42/44 MAPKs, are known to be activated by 17β estradiol [116]. Moreover, studies have evidenced the importance of activated p42/44 MAPK pathways in estrogen-mediated neuroprotection and microglia homeostasis [117]. When activated in response to inflammation, microglia release large amounts of oxygen and nitrogen radicals. Proteosomes clear oxidized and damaged proteins from cells, serving as a microglia compensatory response to activation. In the mouse model, the p42/44 MAPK pathway participates in estrogen-mediated proteasome activation, with estrogen up-regulation of proteosome activity considered to be one way estrogen potentially promotes microglial Calcium-dependent protease with papain-like activity, or calpain, is a cytoplasmic cysteine protease that is activated by calcium [119]. Calpain is involved in neurodegeneration in a variety of injuries and diseases [120]. Calpain cleaves many substrates, including cytoskeletal proteins, axonal, and myelin proteins, and pro-apoptotic Bax causing mitochondrial cytochrome c release, activation of caspase-3, and activation of microglia [121–123]. Studies indicate that estrogen attenuates Ca2+ influx via modulation of L-type Ca2+ channels and the Na<sup>+</sup> Ca2+ exchanger [124]. Estrogen has also been shown to reduce calpain expression and activity, Finally, estrogen is involved in regulation of microglial matrix metalloproteinases [126]. Metalloproteases secreted from microglia mediate inflammation and tissue degradation through processing of pro-inflammatory cytokines and damage to the BBB [127]. Progestins and estrogens affect matrix metalloproteinase enzyme activity in microglial cells, reducing Although few studies known to-date have focused on estrogenic effects in FTLD, indirect evidence comes from physiological studies in both normal and genetically disordered individuals. In this regard, it is notable that FTLD is associated with over a dozen genetic mutations that include the ALS-associated X linked UBQLN2 variant [129]. Neuropathologically, FTLD is primarily represented by two subtypes: one involving aberrant inclusions of microtubule Both bvFTD and SD are associated with changes in appetite and eating habits, with overeating and a preference for sweets and excessive seasonings, including salt [131–133]. Appetite is recognized to be modulated by gonadal steroid hormones, including peripheral and central resulting in reduced axon degeneration and neuronal apoptosis in vitro [125]. protein tau, and one involving inclusions of TAR DNA binding protein [130]. indications of microglial inflammation [97, 126, 128]. 6. Evidence of estrogenic effects in FTLD viability [117, 118]. 80 Sex Hormones in Neurodegenerative Processes and Diseases 6.1. Appetite regulation As a bvFTD subtype, apathy is characterized by inertia and loss of volition, as well as apathy, in association with pathology within the dorsolateral convexities of the frontal lobe [139]. As an amotivational syndrome, apathy is also well recognized to be associated with disruptions to the ventral anterior cingulate cortex [139, 140]. In addition, regions known to be associated with Apathy include the medial dorsal nucleus of the thalamus, caudate nucleus, ventral medial striate (nucleus accumbens) and globus pallidus; with the cortical-striatal-thalamiccortical circuit being the circuit most implicated in the Apathy syndrome [141]. Apathy neural circuitry is linked to the richly dopaminergic nonmotor limbic loop of the basal ganglia, with adaptive behaviors requiring a combination of reward evaluation, associative learning, and ability to develop appropriate action plans [142]. Dopamine deficiencies are often characteristic of FTLD. Clinical trials involving the use of dopaminergic psychostimulants have evidenced improvements in symptomology ranging from apathy to disinhibition and risk taking behaviors [143]. With respect to the potential for estrogen derivatives to mitigate apathy in FTLD, the responsiveness of dopamine neurons to estrogens has long been established, with inducement of dopamine synthesis and release, as well as dopamine neuron differentiation [144–147]. #### 6.3. Turner syndrome The overarching role of gonadal steroidal regulation of brain anatomy and physiology with respect to cognitive and affective executive functioning comes from studies of Turner syndrome, one of the most common sex chromosome-associated genetic disorders. Turner syndrome results in females from the complete or partial loss of one X chromosome, with partial loss involving the distal tip of its short arm [148, 149]. Turner syndrome individuals retain a healthy sense of desire for social interaction, while experiencing disruption in social salience in association with a right hemisphere learning disability. Turner syndrome is typically associated with early loss of ovarian function, leading to gonadal steroid deficiencies that result in pubertal delay and lack of developmental maturation. On a cognitive level, Turner Syndrome results in attentional deficits, disruptions to arithmetic reasoning, visuospatial processing, and executive functions. fMRI studies of Turner Syndrome adults provided with estrogen replacement to allow for physical maturation have implicated the parietal, amygdala and prefrontal areas in this condition, in association with tasks of working memory, as well as interpretation of facial emotional expressions, and mediation of arousal by affective stimuli. Provision of estrogen replacement to stimulate developmental maturation thus has the indirect effect of mitigating many cognitive and behavioral abnormalities also characteristic of cognitive and behavioral declines seen in emerging FTLD in females [23, 24, 150]. Author details Claire V. Flaherty<sup>1</sup> References 145-158 chiatry. 2014;85:866-871 2012.6660613 chiatry. 2002;180(2):140-143 \, Arghavan S. Zangeneh<sup>1</sup> \Address all correspondence to: [email protected] 2 Department of Psychology, Penn State Middletown, Harrisburg, PA, USA international initiative. Lancet Neurology. 2007;6(10):857-868 Alzheimer Disease and Associated Disorders. 2002;16(4):203-212 aphasias. Neurology. 2006;67(10):1752-1756 Review of Clinical Psychology. 2014;10:581-606 Behavioral Neurology. 2017;30(1):8-15 1 Department of Neurology, Penn State Hershey Medical Center, Hershey, PA, USA 3 Department of Biochemistry and Molecular Biology, Penn State University, PA, USA [1] Snowden JS, Neary D, Mann DM. Frontotemporal dementia. British Journal of Psy- [2] Rademakers R, Baker M, Gass J, et al. Phenotypic variability associated with progranulin haploinsufficiency in patients with the common 1477C→ T (Arg493X) mutation: An [3] Barker WW et al. Relative frequencies of Alzheimer disease, Lewy body, vascular and frontotemporal dementia, and hippocampal sclerosis in the State of Florida Brain Bank. [4] Rosen HJ et al. Behavioral features in semantic dementia vs other forms of progressive [5] Rascovsky K, Grossman M. Clinical diagnostic criteria and classification controversies in frontotemporal lobar degeneration. International Review of Psychiatry. 2013;25(2): [6] Levenson RW, Sturm VE, Haase CM. Emotional and behavioral symptoms in neurodegenerative disease: A model for studying the neural bases of psychopathology. Annual [7] Chare L, Hodges JR, Leyton CE, et al. Journal of Neurology, Neurosurgery, and Psy- [8] Vieira R, Caixeta L, Machado S, et al. Epidemiology of early-onset dementia: A review of the literature. Clinical Practice & Epidemiology in Mental Health. 2013;9:88-95 [9] Maki PM, Henderson VW. Hormone therapy, dementia, and cognition: The Woman's Health Initiative ten years on. Climacteric. 2012 June;15(3):256-262. DOI: 10.3109/13697137. [10] Clem MA, Holliday RP, Pandya S, Hynan LS, Lacritz LH, Woon FL. Predictors that a diagnosis of mild cognitive impairment will remain stable 3 years later. Cognitive and , Marissa A. Harrison<sup>2</sup> Gender Differences in Frontotemporal Lobar Degeneration (FTLD) Support an Estrogenic Model… and Sanjana Marikunte<sup>3</sup> 83 http://dx.doi.org/10.5772/intechopen.74158 #### 7. Summary Early researchers found no evidence that FTLD affects men and women differentially [151, 152]. However, recent work has demonstrated hormonal differences in FTLD females not seen in either male FTLD or Alzheimer's patients [153]. Moreover, a multitude of genes have been identified associating FTLD characteristics with ALS, a motor variant of FTLD. ALS is known to have a greater percentage of males among patients who present with disease onset prior to mid-life and to have fMRI evidenced distinct patterns of neurophysiological change with ALS disease progression [15], up to half of whom potentially evidenced signs of FTLD associated cognitive and/or behavioral decline [18]. With respect to concerns about the association between estrogen replacement therapy and increased dementia risk in older women, raised by the results of the Woman's Health Initiative studies of the 1990s, estrogen replacement risk of senile dementia has been shown to be dependent upon the presence of the APOE e4 gene [11]. In the present effort, we present evidence that estrogen, in the absence of the APOE e4 genetic risk factor for Alzheimer's dementia, serves a neuroprotective role in females, including an association between female estrogen levels and cognitive and behavioral stability in emerging FTLD. The potential for estrogen replacement to delay disease onset in females vulnerable to FTLD, a condition typically emerging in midlife, is in need of further exploration. #### Disclosure The authors have no conflicts of interest to disclose. ### Author details 6.3. Turner syndrome 82 Sex Hormones in Neurodegenerative Processes and Diseases 7. Summary Disclosure The authors have no conflicts of interest to disclose. The overarching role of gonadal steroidal regulation of brain anatomy and physiology with respect to cognitive and affective executive functioning comes from studies of Turner syndrome, one of the most common sex chromosome-associated genetic disorders. Turner syndrome results in females from the complete or partial loss of one X chromosome, with partial loss involving the distal tip of its short arm [148, 149]. Turner syndrome individuals retain a healthy sense of desire for social interaction, while experiencing disruption in social salience in association with a right hemisphere learning disability. Turner syndrome is typically associated with early loss of ovarian function, leading to gonadal steroid deficiencies that result in pubertal delay and lack of developmental maturation. On a cognitive level, Turner Syndrome results in attentional deficits, disruptions to arithmetic reasoning, visuospatial processing, and executive functions. fMRI studies of Turner Syndrome adults provided with estrogen replacement to allow for physical maturation have implicated the parietal, amygdala and prefrontal areas in this condition, in association with tasks of working memory, as well as interpretation of facial emotional expressions, and mediation of arousal by affective stimuli. Provision of estrogen replacement to stimulate developmental maturation thus has the indirect effect of mitigating many cognitive and behavioral abnormalities also characteristic of cognitive and behavioral declines seen in emerging FTLD in females [23, 24, 150]. Early researchers found no evidence that FTLD affects men and women differentially [151, 152]. However, recent work has demonstrated hormonal differences in FTLD females not seen in either male FTLD or Alzheimer's patients [153]. Moreover, a multitude of genes have been identified associating FTLD characteristics with ALS, a motor variant of FTLD. ALS is known to have a greater percentage of males among patients who present with disease onset prior to mid-life and to have fMRI evidenced distinct patterns of neurophysiological change with ALS disease progression [15], up to half of whom potentially evidenced signs of FTLD associated cognitive and/or behavioral decline [18]. With respect to concerns about the association between estrogen replacement therapy and increased dementia risk in older women, raised by the results of the Woman's Health Initiative studies of the 1990s, estrogen replacement risk of senile dementia has been shown to be dependent upon the presence of the APOE e4 gene [11]. In the present effort, we present evidence that estrogen, in the absence of the APOE e4 genetic risk factor for Alzheimer's dementia, serves a neuroprotective role in females, including an association between female estrogen levels and cognitive and behavioral stability in emerging FTLD. The potential for estrogen replacement to delay disease onset in females vulnerable to FTLD, a condition typically emerging in midlife, is in need of further exploration. Claire V. Flaherty<sup>1</sup> \, Arghavan S. Zangeneh<sup>1</sup> , Marissa A. 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The basis of reproductive senescence in women is oocyte depletion in the ovary. Perimenopause is defined by menstrual cycle and endocrine changes, such as disturbed ovarian-pituitary-hypothalamic feedback relationships, inaccurate estrogen levels, and decreased progesterone levels. Many psychopathological changes can take place, but most commonly women experience mild cognitive impairment, anxiety, irritability, mood swings, and depression. Estrogens influence depression and depressive-like behavior through interactions with neurotropic factors and through Keywords: reproductive aging, menopausal transition, perimenopause, estrogen, The belief that behavioral disturbances are related to manifestations of the female reproduc- This belief regarding the middle-aged years and the negative outlook of the perimenopause is not completely irrational. There are some events that support impressions such as the completion of the reproductive period, separation from children, care for very old parents and relatives, onset of illness, retirement, or financial insecurity. Perimenopausal changes are not tive system is an ancient one that has persisted to contemporary times [1]. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Psychopathological Symptoms Psychopathological Symptoms Ksenija Gersak, Ziva Miriam Gersak and Ksenija Gersak, Ziva Miriam Gersak and http://dx.doi.org/10.5772/intechopen.74159 Additional information is available at the end of the chapter Additional information is available at the end of the chapter an influence on the serotonergic system. progesterone, neurotransmitters, mental disorders Arijana Turcin Abstract 1. Introduction Arijana Turcin #### Chapter 5 Provisional chapter #### Reproductive Aging: Perimenopause and Psychopathological Symptoms Reproductive Aging: Perimenopause and Psychopathological Symptoms DOI: 10.5772/intechopen.74159 Ksenija Gersak, Ziva Miriam Gersak and Arijana Turcin Ksenija Gersak, Ziva Miriam Gersak and Arijana Turcin Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.74159 #### Abstract The female reproductive axis essentially comprises of the hypothalamic-pituitaryovarian axis and the mullerian-derived structures. The reproductive axis ages to a nonfunctional state (menopause) much earlier than the other organ systems do, at a time when a woman is otherwise healthy. The basis of reproductive senescence in women is oocyte depletion in the ovary. Perimenopause is defined by menstrual cycle and endocrine changes, such as disturbed ovarian-pituitary-hypothalamic feedback relationships, inaccurate estrogen levels, and decreased progesterone levels. Many psychopathological changes can take place, but most commonly women experience mild cognitive impairment, anxiety, irritability, mood swings, and depression. Estrogens influence depression and depressive-like behavior through interactions with neurotropic factors and through an influence on the serotonergic system. Keywords: reproductive aging, menopausal transition, perimenopause, estrogen, progesterone, neurotransmitters, mental disorders #### 1. Introduction The belief that behavioral disturbances are related to manifestations of the female reproductive system is an ancient one that has persisted to contemporary times [1]. This belief regarding the middle-aged years and the negative outlook of the perimenopause is not completely irrational. There are some events that support impressions such as the completion of the reproductive period, separation from children, care for very old parents and relatives, onset of illness, retirement, or financial insecurity. Perimenopausal changes are not © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. symbols of some "ominous changes" but are instead a part of reproductive aging, which appear much earlier in life than do the various other physiological organ system changes due to somatic aging. Reproductive aging is a natural process that begins at birth and proceeds as a continuum. The basis of reproductive senescence is oocyte depletion, a steady-state loss through atresia and ovulations. The decline in reproductive capacity is accompanied by increased risk of psychogenic disturbances, osteoporosis, and cardiovascular and cerebrovascular diseases. There is a lot of evidence from basic science, epidemiological data, and interventional studies to indicate that estrogens are positively influencing mental well-being. Depressive symptoms and even an upsurge in the incidence of some mental disorders have been observed around the menopause, suggesting the direct involvement of instant loss of estrogen activity in mental health. #### 2. Stages of reproductive aging Menstruation is the regular discharge of blood and mucosal tissue from the inner lining of the uterus through the vagina as a result of periodic hormonal changes. Menarche is the first menstruation, and menopause is the point in time when permanent cessation of menstruation occurs following the loss of ovarian activity [1]. The term is derived from the Greek words "men" (month) and "pausis" (cessation). Menopause is confirmed 12 months after the onset of amenorrhea. The years prior to menopause are known as the perimenopausal transitional years. An older, more popular, and less precise term is climacteric, the expression derived from the Greek word for "ladder" and should be used only when talking to patients and in the lay press, not in scientific papers [2]. The 2001 Stages of Reproductive Aging Workshop (STRAW) proposed a new nomenclature and staging system for objectifying ovarian aging, including menstrual and quantitative hormonal criteria to define each stage, and has been reviewed and updated in 2011 [3]. The "STRAW+10" staging system is widely considered as the gold standard for characterizing reproductive aging through menopause. It divides the adult female life into three main phases: The phases include a total of seven stages centered around the final menstrual period (Stage 0). The reproductive phase is divided into stages −5, −4, and −3 (early, peak, and late reproductive phase, respectively). The menopausal transition phase consists of stages −2 (early) and −1 (late), whereas the postmenopausal phase contains stages +1 (early) and +2 (late) (Table 1). Stage Terminology Reproductive phase Early > Duration Principal criteria Menstrual cycle regular Supportive criteria Endocrine Low Variable ↑ Variable ↑ >25 IU/L ↑ Variable Stabilizes Low Very low Low Very low Low Low Low Low Low Low Low FSH AMH Inhibin B Antral follicle Low Low Low Low Very low Very low Reproductive Aging: Perimenopause and Psychopathological Symptoms http://dx.doi.org/10.5772/intechopen.74159 97 count Adapted from Harlow et Table 1. The 2011 STRAW+10 staging system for reproductive aging in women. al. [3]. Variable to Regular Regular Subtle changes in Variable length Interval of amenorrhea flow length (≥ 7 days difference) of ≥60 days Variable Peak Late −5 −4 −3b −3a −2 Menopausal transition Early Perimenopause variable 1–3 years 1 1 3–6 Remaining year year years lifespan Late Early Late −1 +1a +1b Postmenopause +1c +2 symbols of some "ominous changes" but are instead a part of reproductive aging, which appear much earlier in life than do the various other physiological organ system changes due Reproductive aging is a natural process that begins at birth and proceeds as a continuum. The basis of reproductive senescence is oocyte depletion, a steady-state loss through atresia and ovulations. The decline in reproductive capacity is accompanied by increased risk of psycho- There is a lot of evidence from basic science, epidemiological data, and interventional studies to indicate that estrogens are positively influencing mental well-being. Depressive symptoms and even an upsurge in the incidence of some mental disorders have been observed around the menopause, suggesting the direct involvement of instant loss of estrogen activity Menstruation is the regular discharge of blood and mucosal tissue from the inner lining of the Menarche is the first menstruation, and menopause is the point in time when permanent cessation of menstruation occurs following the loss of ovarian activity [1]. The term is derived from the Greek words "men" (month) and "pausis" (cessation). Menopause is confirmed The years prior to menopause are known as the perimenopausal transitional years. An older, more popular, and less precise term is climacteric, the expression derived from the Greek word for "ladder" and should be used only when talking to patients and in the lay press, not The 2001 Stages of Reproductive Aging Workshop (STRAW) proposed a new nomenclature and staging system for objectifying ovarian aging, including menstrual and quantitative hormonal criteria to define each stage, and has been reviewed and updated in 2011 [3]. The "STRAW+10" staging system is widely considered as the gold standard for characterizing The phases include a total of seven stages centered around the final menstrual period (Stage 0). The reproductive phase is divided into stages −5, −4, and −3 (early, peak, and late reproductive phase, respectively). The menopausal transition phase consists of stages −2 (early) and −1 (late), whereas the postmenopausal phase contains stages +1 (early) and +2 (late) (Table 1). uterus through the vagina as a result of periodic hormonal changes. genic disturbances, osteoporosis, and cardiovascular and cerebrovascular diseases. to somatic aging. in mental health. 2. Stages of reproductive aging 96 Sex Hormones in Neurodegenerative Processes and Diseases 12 months after the onset of amenorrhea. reproductive aging through menopause. It divides the adult female life into three main phases: in scientific papers [2]. • Reproductive • Postmenopause. • The menopausal transition Table 1. The 2011 STRAW+10 staging system for reproductive aging in women. The system does not use age as the criterion for determining reproductive staging. The principal criteria are the menstrual cycle patterns [1], which can be described with regularity of menstrual bleeding, frequency of onset, duration of menstrual flow, and heaviness (or volume) of menstrual flow. Regular menstrual cycles are usually the outward manifestation of cyclical ovarian activity and ovulation. 2.5. Late postmenopause recurrent urogenital infections. • Inaccurate estrogen levels • Decreased progesterone levels. remaining ones are to undergo atresia. 3. Reproductive physiology of perimenopause It is characterized by three major hormonal changes [1, 4, 5]: • Disturbed ovarian-pituitary-hypothalamic feedback relationships 3.1. Changes in ovarian-pituitary-hypothalamic feedback controls granulosa cells of antral follicles to produce estradiol and inhibins. ated ovulation, which in turn occurs at a smaller follicle size [8]. As defined by STRAW+10, further changes in reproductive endocrine function (Stage +2) are attributable predominantly to somatic aging. Symptoms such as urogenital atrophy progress during the remaining lifespan. Urogenital atrophy is a cluster of symptoms including vaginal dryness, painful intercourse (dyspareunia), vulvar pruritus, burning, discomfort, as well as Reproductive Aging: Perimenopause and Psychopathological Symptoms http://dx.doi.org/10.5772/intechopen.74159 99 Perimenopause is a period of reproductive aging that includes the early and late menopausal transitions (Stages −2 and −1) and the first year of early postmenopause (Stage +1a). It usually occurs in the late fourth to fifth decade of a woman's life and lasts approximately 15 years. Ovarian control of gonadotropin secretion is normally achieved by feedback control mechanisms, including estradiol, progesterone, and ovarian regulatory proteins. FSH is secreted in pulses by the anterior pituitary under the influence of hypothalamic gonadotropin-releasing hormone, with a direct inhibitory feedback by estradiol and inhibin B, and stimulatory action of activin [1]. Recent research has clarified that a fall in inhibin B is the basis for FSH rise with ovarian aging [5, 6]. With a decreasing follicular pool, inhibin B levels, produced by small Follicular growth in the early follicular phase is under control of FSH, which stimulates the Between days 5 and 7 of the menstrual cycle, selection of a follicle takes place whereby only one dominant follicle is destined to ovulate from the cohort of recruited follicles, and the With dominant follicle selection and a subsequent rapid rise in estradiol, the pituitary responds by releasing a luteinizing hormone (LH) surge, which in turns triggers ovulation [1]. The LH surge occurs 34–36 hours prior to ovulation. In order for the positive feedback effect to trigger the LH release, estradiol levels must be greater than 200 pg/mL for at least 48 hours in a continuous duration [1]. Ovulation occurs approximately 10–12 hours after the LH peak, During menopausal transition, the follicular phase is shortened and associated with acceler- antral follicles, decline and thus allow FSH levels to rise in the early follicular phase. and the dominant follicle is almost always >15 mm in diameter on ultrasound [7]. The supportive criteria include endocrine parameters such as serum concentrations of follicle stimulating hormone (FSH), anti-müllerian hormone (AMH), and inhibin-B. Subjective data, such as menstrual flow changes, are considered too subjective and variable, particularly between ethnic groups, to be included in the criteria. Vasomotor symptoms are the only exception, and have been included in the system only as "descriptive" criteria. The main vasomotor symptoms are hot flashes and cold or night sweats. Hot flash is a sensation of heat, usually involving the face and neck and upper part of the chest. It is caused by a transient dilation of the blood vessels of the skin. Women who have undergone hysterectomy or endometrial ablation cannot be staged by menstrual bleeding criteria. Reproductive stages in these women can only be assessed using the supportive endocrine criteria. STRAW+10 stages are outlined in the following paragraphs. #### 2.1. Late reproductive stage As defined by STRAW+10, in the late reproductive phase (Stage −3a), there are subtle changes in menstrual cycle characteristics. The cycles get shorter, early follicular phase FSH levels increase and become more variable, and the AMH and antral follicle counts get low (Table 1). #### 2.2. Early menopausal transition Stage −2 is marked by increased variability in menstrual cycle length with consecutive cycleto-cycle variation of 7 days or more. Early follicular phase FSH levels are elevated and variable, and AMH and antral follicle counts are low (Table 1). #### 2.3. Late menopausal transition In Stage −1, menstrual cycles are characterized by increased variability in cycle length and the occurrence of amenorrhea lasting 60 days or longer. There are extreme fluctuations in hormonal levels, an increased prevalence of anovulation, and FSH levels are greater than 25 IU/L in a random blood draw. Vasomotor symptoms are likely to occur. This stage is estimated to last, on average, 1–3 years and ends with the last menstrual period (Table 1). #### 2.4. Early postmenopause FSH levels continue to increase while estradiol levels continue to decrease until approximately 2 years after the final menstrual period (stage +1a and +1b) (Table 1). Stage +1c represents the period of stabilization of high FSH levels and low estradiol levels. The entire early postmenopause lasts approximately 5–8 years. #### 2.5. Late postmenopause The system does not use age as the criterion for determining reproductive staging. The principal criteria are the menstrual cycle patterns [1], which can be described with regularity of menstrual bleeding, frequency of onset, duration of menstrual flow, and heaviness (or volume) of menstrual flow. Regular menstrual cycles are usually the outward manifestation of cyclical ovarian The supportive criteria include endocrine parameters such as serum concentrations of follicle stimulating hormone (FSH), anti-müllerian hormone (AMH), and inhibin-B. Subjective data, such as menstrual flow changes, are considered too subjective and variable, particularly between ethnic groups, to be included in the criteria. Vasomotor symptoms are the only The main vasomotor symptoms are hot flashes and cold or night sweats. Hot flash is a sensation of heat, usually involving the face and neck and upper part of the chest. It is caused by a Women who have undergone hysterectomy or endometrial ablation cannot be staged by menstrual bleeding criteria. Reproductive stages in these women can only be assessed using As defined by STRAW+10, in the late reproductive phase (Stage −3a), there are subtle changes in menstrual cycle characteristics. The cycles get shorter, early follicular phase FSH levels increase and become more variable, and the AMH and antral follicle counts get low (Table 1). Stage −2 is marked by increased variability in menstrual cycle length with consecutive cycleto-cycle variation of 7 days or more. Early follicular phase FSH levels are elevated and vari- In Stage −1, menstrual cycles are characterized by increased variability in cycle length and the occurrence of amenorrhea lasting 60 days or longer. There are extreme fluctuations in hormonal levels, an increased prevalence of anovulation, and FSH levels are greater than 25 IU/L in a random blood draw. Vasomotor symptoms are likely to occur. This stage is estimated to FSH levels continue to increase while estradiol levels continue to decrease until approximately 2 years after the final menstrual period (stage +1a and +1b) (Table 1). Stage +1c represents the period of stabilization of high FSH levels and low estradiol levels. The entire early last, on average, 1–3 years and ends with the last menstrual period (Table 1). exception, and have been included in the system only as "descriptive" criteria. transient dilation of the blood vessels of the skin. 98 Sex Hormones in Neurodegenerative Processes and Diseases STRAW+10 stages are outlined in the following paragraphs. able, and AMH and antral follicle counts are low (Table 1). the supportive endocrine criteria. 2.2. Early menopausal transition 2.3. Late menopausal transition 2.4. Early postmenopause postmenopause lasts approximately 5–8 years. 2.1. Late reproductive stage activity and ovulation. As defined by STRAW+10, further changes in reproductive endocrine function (Stage +2) are attributable predominantly to somatic aging. Symptoms such as urogenital atrophy progress during the remaining lifespan. Urogenital atrophy is a cluster of symptoms including vaginal dryness, painful intercourse (dyspareunia), vulvar pruritus, burning, discomfort, as well as recurrent urogenital infections. #### 3. Reproductive physiology of perimenopause Perimenopause is a period of reproductive aging that includes the early and late menopausal transitions (Stages −2 and −1) and the first year of early postmenopause (Stage +1a). It usually occurs in the late fourth to fifth decade of a woman's life and lasts approximately 15 years. It is characterized by three major hormonal changes [1, 4, 5]: #### 3.1. Changes in ovarian-pituitary-hypothalamic feedback controls Ovarian control of gonadotropin secretion is normally achieved by feedback control mechanisms, including estradiol, progesterone, and ovarian regulatory proteins. FSH is secreted in pulses by the anterior pituitary under the influence of hypothalamic gonadotropin-releasing hormone, with a direct inhibitory feedback by estradiol and inhibin B, and stimulatory action of activin [1]. Recent research has clarified that a fall in inhibin B is the basis for FSH rise with ovarian aging [5, 6]. With a decreasing follicular pool, inhibin B levels, produced by small antral follicles, decline and thus allow FSH levels to rise in the early follicular phase. Follicular growth in the early follicular phase is under control of FSH, which stimulates the granulosa cells of antral follicles to produce estradiol and inhibins. Between days 5 and 7 of the menstrual cycle, selection of a follicle takes place whereby only one dominant follicle is destined to ovulate from the cohort of recruited follicles, and the remaining ones are to undergo atresia. With dominant follicle selection and a subsequent rapid rise in estradiol, the pituitary responds by releasing a luteinizing hormone (LH) surge, which in turns triggers ovulation [1]. The LH surge occurs 34–36 hours prior to ovulation. In order for the positive feedback effect to trigger the LH release, estradiol levels must be greater than 200 pg/mL for at least 48 hours in a continuous duration [1]. Ovulation occurs approximately 10–12 hours after the LH peak, and the dominant follicle is almost always >15 mm in diameter on ultrasound [7]. During menopausal transition, the follicular phase is shortened and associated with accelerated ovulation, which in turn occurs at a smaller follicle size [8]. With time, the age-related hypothalamic modifications cause a decrease in estrogen sensitivity and the mid-cycle LH surge becomes more erratic. Furthermore, it is hypothesized that the higher FSH levels might interfere with oocyte release and with progesterone production [9]. all menopausal transition cycles show evidence of these events, with a higher estradiol peak Reproductive Aging: Perimenopause and Psychopathological Symptoms http://dx.doi.org/10.5772/intechopen.74159 101 After menarche, it usually takes several years for regular menstrual cycles to establish. Bleeding that can be defined as a "period" is described according to the following four parameters: The normal frequency of menses is between 24 and 38 days [4]. During the early menopausal transition, menstrual cycles remain regular, with a cycle-to-cycle duration variation of 7 days or more; for example, a cycle length of 24 instead of the previously established years-long regularity of 31 days. Late menopausal transition is characterized by two or more skipped Transitionally higher estradiol levels are associated with heavy monthly blood loss and increased endometrial thickness (hyperplasia). In the Seattle Woman's Midlife Health study, the most common subjective menstrual cycle changes were flow-related and included a heavier menstrual flow in 29% and a longer duration of the flow in 20% [14]. Menstrual blood loss was greater following ovulatory rather than anovulatory cycles [15], especially if the ovulatory cycle followed a prolonged interval of anovulation, during which unopposed high estradiol levels contributed to abnormal excessive proliferative changes in the endometrium [16]. A quantitative study by Hale and coauthors shows that blood loss increases in its absolute values and in its variability across the peak reproductive, late reproductive, and late meno- Perimenopause is the time period bridging the mature fully reproductive and the non-reproductive states. The loss of fertility is the first sign of reproductive aging and precedes the The number of non-growing follicles is determined before birth, when oocytes multiply to a maximum of 6–7 million at mid-gestation. Oocytes are then rapidly lost due to apoptosis, The Wallace-Kelsey model matches the logarithm-adjusted non-growing follicle population from conception to menopause to a five-parameter asymmetric double Gaussian cumulative curve [18]. It is based on the assumption that the peak number of non-growing follicles at 18–22 weeks of gestation defines the age at menopause for every individual woman, and it does not take into account the recent evidence of neo-oogenesis during normal human physiological aging. monotropic increase in FSH levels as well as changes in menstrual regularity [2]. leading to a population of 700,000 at birth and 300,000 at puberty. menstrual bleedings and at least one intermenstrual interval of 60 days or more. following the normal mid-cycle estradiol peak [4, 12]. 3.3. Menstrual cycle during menopausal transition • Heaviness (or volume) of menstrual flow [13]. • Regularity of onset • Frequency of onset • Duration of menstrual flow pausal transition phases [17]. 3.4. Fertility and menopausal transition Additional perimenopausal feedback imbalances that lead to anovulation involve changes in LH and estradiol secretion. Despite the occurrence of a normal estradiol peak, an LH surge does not follow. Finally, despite high follicular phase estradiol levels, cycles may have no evidence of either an estradiol or an LH peak, and hence there is no ovulation [5]. The hypothalamus and/or the pituitary can become insensitive to estradiol feedback resulting in anovulation. It is known that with higher baseline FSH levels, average LH levels may remain normal in the perimenopausal transition. Changes however appear in the dynamics of LH release. Although estradiol levels are not significantly different, cycling perimenopausal women appear to lack the slow-frequency, high-amplitude LH pulsatility characteristic of the luteal phase and resulting in decreased progesterone levels. #### 3.2. Inaccurate estrogen levels Estradiol is the main ovarian estrogen produced by follicular granulosa cells. FSH activates the aromatase enzyme, which converts androgens to estrogen. During the follicular phase, serum estradiol levels rise in parallel to follicle size growth as well as to the increasing number of granulosa cells [1, 10]. In the presence of estradiol, FSH stimulates the formation of luteinizing hormone (LH) receptors on granulosa cells allowing the secretion of small quantities of progesterone, which exerts a positive feedback on the estrogen-dependent pituitary LH release [1]. Recent scientific reviews have shown intermittently high levels of estrogen during the perimenopause. This evidence contradicts the assumption of dropping or overall lower estrogen levels during the perimenopause and invalidates the casual use of the term "estrogen deficiency" as a synonym for perimenopause [5]. Santoro and coauthors were the first to propose the then-radical concept about high estradiol levels [11]. The observation was confirmed by a meta-analysis comparing samples from women of reproductive age to those from perimenopausal women within the same research center. Mean estradiol levels were statistically higher in perimenopausal women [9] (29% in the follicular phase and 22% in the late luteal phase). Higher estradiol levels are a common result of a higher number of recruited estradiol-producing follicles, while that in turn results from the net effect of rising FSH levels. As previously discussed, FSH increases early in the follicular phase due to changes in the feedback control mechanisms: impaired suppression of FSH by higher estradiol levels and lower intraovarian production of inhibins and, on the other hand, by the stimulatory input of both activin and gonadotropin-releasing hormone [4]. FSH changes are also related to the second estradiol peak during the luteal phase in the form of a "luteal out of phase" (LOOP) event. Hale and coauthors estimated that about a third of all menopausal transition cycles show evidence of these events, with a higher estradiol peak following the normal mid-cycle estradiol peak [4, 12]. #### 3.3. Menstrual cycle during menopausal transition After menarche, it usually takes several years for regular menstrual cycles to establish. Bleeding that can be defined as a "period" is described according to the following four parameters: • Regularity of onset With time, the age-related hypothalamic modifications cause a decrease in estrogen sensitivity and the mid-cycle LH surge becomes more erratic. Furthermore, it is hypothesized that the higher FSH levels might interfere with oocyte release and with progesterone production [9]. Additional perimenopausal feedback imbalances that lead to anovulation involve changes in LH and estradiol secretion. Despite the occurrence of a normal estradiol peak, an LH surge Finally, despite high follicular phase estradiol levels, cycles may have no evidence of either an estradiol or an LH peak, and hence there is no ovulation [5]. The hypothalamus and/or the It is known that with higher baseline FSH levels, average LH levels may remain normal in the perimenopausal transition. Changes however appear in the dynamics of LH release. Although estradiol levels are not significantly different, cycling perimenopausal women appear to lack the slow-frequency, high-amplitude LH pulsatility characteristic of the luteal phase and result- Estradiol is the main ovarian estrogen produced by follicular granulosa cells. FSH activates During the follicular phase, serum estradiol levels rise in parallel to follicle size growth as well as to the increasing number of granulosa cells [1, 10]. In the presence of estradiol, FSH stimulates the formation of luteinizing hormone (LH) receptors on granulosa cells allowing the secretion of small quantities of progesterone, which exerts a positive feedback on the Recent scientific reviews have shown intermittently high levels of estrogen during the perimenopause. This evidence contradicts the assumption of dropping or overall lower estrogen levels during the perimenopause and invalidates the casual use of the term "estrogen defi- Santoro and coauthors were the first to propose the then-radical concept about high estradiol levels [11]. The observation was confirmed by a meta-analysis comparing samples from women of reproductive age to those from perimenopausal women within the same research center. Mean estradiol levels were statistically higher in perimenopausal women [9] (29% in Higher estradiol levels are a common result of a higher number of recruited estradiol-producing follicles, while that in turn results from the net effect of rising FSH levels. As previously discussed, FSH increases early in the follicular phase due to changes in the feedback control mechanisms: impaired suppression of FSH by higher estradiol levels and lower intraovarian production of inhibins and, on the other hand, by the stimulatory input of both activin and FSH changes are also related to the second estradiol peak during the luteal phase in the form of a "luteal out of phase" (LOOP) event. Hale and coauthors estimated that about a third of pituitary can become insensitive to estradiol feedback resulting in anovulation. the aromatase enzyme, which converts androgens to estrogen. does not follow. ing in decreased progesterone levels. 100 Sex Hormones in Neurodegenerative Processes and Diseases estrogen-dependent pituitary LH release [1]. ciency" as a synonym for perimenopause [5]. gonadotropin-releasing hormone [4]. the follicular phase and 22% in the late luteal phase). 3.2. Inaccurate estrogen levels The normal frequency of menses is between 24 and 38 days [4]. During the early menopausal transition, menstrual cycles remain regular, with a cycle-to-cycle duration variation of 7 days or more; for example, a cycle length of 24 instead of the previously established years-long regularity of 31 days. Late menopausal transition is characterized by two or more skipped menstrual bleedings and at least one intermenstrual interval of 60 days or more. Transitionally higher estradiol levels are associated with heavy monthly blood loss and increased endometrial thickness (hyperplasia). In the Seattle Woman's Midlife Health study, the most common subjective menstrual cycle changes were flow-related and included a heavier menstrual flow in 29% and a longer duration of the flow in 20% [14]. Menstrual blood loss was greater following ovulatory rather than anovulatory cycles [15], especially if the ovulatory cycle followed a prolonged interval of anovulation, during which unopposed high estradiol levels contributed to abnormal excessive proliferative changes in the endometrium [16]. A quantitative study by Hale and coauthors shows that blood loss increases in its absolute values and in its variability across the peak reproductive, late reproductive, and late menopausal transition phases [17]. #### 3.4. Fertility and menopausal transition Perimenopause is the time period bridging the mature fully reproductive and the non-reproductive states. The loss of fertility is the first sign of reproductive aging and precedes the monotropic increase in FSH levels as well as changes in menstrual regularity [2]. The number of non-growing follicles is determined before birth, when oocytes multiply to a maximum of 6–7 million at mid-gestation. Oocytes are then rapidly lost due to apoptosis, leading to a population of 700,000 at birth and 300,000 at puberty. The Wallace-Kelsey model matches the logarithm-adjusted non-growing follicle population from conception to menopause to a five-parameter asymmetric double Gaussian cumulative curve [18]. It is based on the assumption that the peak number of non-growing follicles at 18–22 weeks of gestation defines the age at menopause for every individual woman, and it does not take into account the recent evidence of neo-oogenesis during normal human physiological aging. Wallace and Kelsey estimated that for 95% of women, only 12% of their maximum pre-birth non-growing follicle population is present by the age of 30 years and by the age of 40 years, only 3% of it remains (Figure 1). When only about 1000 oocytes remain, menopause occurs [19]. cells, combined with the theca-lutein cells and the surrounding stroma, form the corpus luteum [1, 10]. The corpus luteum is a transient endocrine organ that predominantly secretes progesterone. Its primary function is to prepare the estrogen-primed endometrium Reproductive Aging: Perimenopause and Psychopathological Symptoms http://dx.doi.org/10.5772/intechopen.74159 103 Secretion of progesterone and estradiol correlates closely with LH level pulses. Eight to nine Despite erratically oscillating high estradiol levels, perimenopause is characterized by more steadily decreasing levels of progesterone. During menstrual transition ovulatory cycles, the decrease in progesterone levels results from diminished progesterone production, shortened luteal phase lengths, and a rising incidence of ovulation disturbance [5]—ovulation only In addition to menstrual cycle disturbances and reduced fertility, symptoms frequently seen Not all women have them, and those who do, experience these symptoms in different combinations and at different intensities [2]. Quantification of these symptoms is difficult because they are subjective in nature. It has been observed that they vary markedly among ethnic groups, cultures, socioeconomic groups, and climates and that they do not correlate closely Vasomotor symptoms are among the most frequently reported physiological symptoms and the most prominent ones during perimenopause. Their prevalence ranges from 30–75% [23]. A hot flush is a sudden episode of vasodilation in the face and neck, which lasts from a few seconds to several minutes and is accompanied by profuse sweating and an increase in heart rate [1, 23]. The frequency of hot flushes may range from a few per day to one every few minutes. Flushes are more frequent and severe at night or during periods of psychological stress Hot flashes usually start occurring in the late menopausal transition (Stage −1), peaking in the first year after menopause (Stage +1a). Some women (10%) may continue to experience The physiology of the hot flush is still not understood. Studies suggest that these women have a narrower zone of temperature regulation, and therefore smaller changes in core body temperature produce compensatory responses such as vasodilation, sweating, and shivering [23]. The flush is not a release of accumulated body heat but a sudden inappropriate activation of The correlation between the onset of flushes and generally diminishing estrogen levels is confirmed by the effectiveness of estrogen therapy in the prevention of flushes, as well as by the absence of flushes in hypoestrogen states, such as gonadal dysgenesis [1]. and can affect a woman's quality of life, interfering with her work or other activities. for implantation. • bone loss, and days after ovulation, peak production is achieved. occurs in 50% of cycles in women aged 46–50 years [5]. • vasomotor instability such as hot flushes and sweating, with menstrual cycle disturbances or endocrine changes. vasomotor symptoms for up to 15 years after menopause [1]. 3.6. Physiological symptoms of perimenopause and related to decreasing ovarian function are: • adverse changes in the lipid profile. the heat release mechanism. Consistent with the continuing apoptosis, along with the loss of oocytes during the 400– 500 cycles of follicular recruitment in a normal reproductive lifespan, the most replicable and linear endocrine change throughout the menopausal transition is the progressive decline of AMH. It marks the decline in follicular mass and explains why fertility is impaired in women well before any disruption of menstrual cycle can be noticed [20, 21]. In the late reproductive stage, AMH is reduced 2 to 10 times compared to the peak reproductive stage (Table 1). Because of its minimal intra-cycle variation, AMH can be useful in late reproductive stage fertility assessment and in predicting the amount of time to menopause occurrence. In the early menopause transition stage, when a variable length of the menstrual cycle occurs, AMH drops to an almost undetectable level, reflecting the diminishing pool of around 1000 non-growing follicles. Besides apoptosis, qualitative oocyte changes also occur. The accelerated follicular phase and monotropic rise of FSH appear to have an adverse effect, leading to a disorganized meiotic spindle assembly in oocytes of reproductively aged women [22]. #### 3.5. Decreased progesterone levels The luteal phase is 14 days long in most women. After ovulation, the remaining granulosa cells continue to enlarge and become vacuolated in appearance. The luteinized granulosa Figure 1. The hypothetical link between ovarian reserve and the age at menopause. This figure describes the hypothesis that an individual's age at menopause is determined by the peak non-growing follicle population count, established at around 20 weeks post-conception. Adapted from Wallace and Kelsey [18]. cells, combined with the theca-lutein cells and the surrounding stroma, form the corpus luteum [1, 10]. The corpus luteum is a transient endocrine organ that predominantly secretes progesterone. Its primary function is to prepare the estrogen-primed endometrium for implantation. Secretion of progesterone and estradiol correlates closely with LH level pulses. Eight to nine days after ovulation, peak production is achieved. Despite erratically oscillating high estradiol levels, perimenopause is characterized by more steadily decreasing levels of progesterone. During menstrual transition ovulatory cycles, the decrease in progesterone levels results from diminished progesterone production, shortened luteal phase lengths, and a rising incidence of ovulation disturbance [5]—ovulation only occurs in 50% of cycles in women aged 46–50 years [5]. #### 3.6. Physiological symptoms of perimenopause In addition to menstrual cycle disturbances and reduced fertility, symptoms frequently seen and related to decreasing ovarian function are: Wallace and Kelsey estimated that for 95% of women, only 12% of their maximum pre-birth non-growing follicle population is present by the age of 30 years and by the age of 40 years, only 3% of it remains (Figure 1). When only about 1000 oocytes remain, menopause occurs [19]. Consistent with the continuing apoptosis, along with the loss of oocytes during the 400– 500 cycles of follicular recruitment in a normal reproductive lifespan, the most replicable and linear endocrine change throughout the menopausal transition is the progressive decline of AMH. It marks the decline in follicular mass and explains why fertility is impaired in women In the late reproductive stage, AMH is reduced 2 to 10 times compared to the peak reproductive stage (Table 1). Because of its minimal intra-cycle variation, AMH can be useful in late reproductive stage fertility assessment and in predicting the amount of time to menopause occurrence. In the early menopause transition stage, when a variable length of the menstrual cycle occurs, AMH drops to an almost undetectable level, reflecting the diminishing pool of Besides apoptosis, qualitative oocyte changes also occur. The accelerated follicular phase and monotropic rise of FSH appear to have an adverse effect, leading to a disorganized meiotic The luteal phase is 14 days long in most women. After ovulation, the remaining granulosa cells continue to enlarge and become vacuolated in appearance. The luteinized granulosa Figure 1. The hypothetical link between ovarian reserve and the age at menopause. This figure describes the hypothesis that an individual's age at menopause is determined by the peak non-growing follicle population count, established at around 20 weeks post-conception. Adapted from Wallace and Kelsey [18]. well before any disruption of menstrual cycle can be noticed [20, 21]. spindle assembly in oocytes of reproductively aged women [22]. around 1000 non-growing follicles. 102 Sex Hormones in Neurodegenerative Processes and Diseases 3.5. Decreased progesterone levels • adverse changes in the lipid profile. Not all women have them, and those who do, experience these symptoms in different combinations and at different intensities [2]. Quantification of these symptoms is difficult because they are subjective in nature. It has been observed that they vary markedly among ethnic groups, cultures, socioeconomic groups, and climates and that they do not correlate closely with menstrual cycle disturbances or endocrine changes. Vasomotor symptoms are among the most frequently reported physiological symptoms and the most prominent ones during perimenopause. Their prevalence ranges from 30–75% [23]. A hot flush is a sudden episode of vasodilation in the face and neck, which lasts from a few seconds to several minutes and is accompanied by profuse sweating and an increase in heart rate [1, 23]. The frequency of hot flushes may range from a few per day to one every few minutes. Flushes are more frequent and severe at night or during periods of psychological stress and can affect a woman's quality of life, interfering with her work or other activities. Hot flashes usually start occurring in the late menopausal transition (Stage −1), peaking in the first year after menopause (Stage +1a). Some women (10%) may continue to experience vasomotor symptoms for up to 15 years after menopause [1]. The physiology of the hot flush is still not understood. Studies suggest that these women have a narrower zone of temperature regulation, and therefore smaller changes in core body temperature produce compensatory responses such as vasodilation, sweating, and shivering [23]. The flush is not a release of accumulated body heat but a sudden inappropriate activation of the heat release mechanism. The correlation between the onset of flushes and generally diminishing estrogen levels is confirmed by the effectiveness of estrogen therapy in the prevention of flushes, as well as by the absence of flushes in hypoestrogen states, such as gonadal dysgenesis [1]. A common belief still persists that bone loss begins and fractures occur in women with constantly low estrogen levels, during the late postmenopause. However, during perimenopause, an early and accelerated rate of bone loss has been observed, particularly in the lumbar spine [24]. In an Australian study, the estimated average annual rate of bone loss around the time of final menstrual period was 2.5% in the lumbar spine and 1.7% in the femoral neck [24]. by osteoblasts. Moreover, the age-associated vitamin D deficiency and impaired synthesis It is well known that women have a lower incidence of cardiovascular risk factors and cardiovascular diseases than their male peers [34]. Estrogen modifies endothelial function by two primary mechanisms: modulation of NO activity and attenuation of vascular response to injury [35]. Estrogen promotes vasodilation through stimulation of eNOS and reduction of NO-synthase activity. At the level of the mitochondria in the vascular endothelium, estrogen stimulates oxidative phosphorylation and reduces mitochondrial production of ROS [36]. Following menopause, there is active progression of atherosclerotic lesions [37]; women also Subclinical development of vascular diseases manifests itself as increased carotid and femoral artery intima-media thickness and accelerated coronary artery calcium deposition, leading to arterial stiffness, which in turn causes impaired flow-mediated vasodilation [23]. The risk of stroke doubles during the first decade after menopause and ultimately exceeds that of men Estrogens enter the brain through the blood-brain barrier and influence the neural activity by multiple pathways. In the central nervous system, they are involved in different processes including cellular protein production, neuronal growth and survival, neural transmission and Estrogens act through both genomic and non-genomic mechanisms. Intracellular estrogen receptors (ER) are widely distributed, and ER subtypes are located in many of the areas that are associated with depression. During perimenopause, estradiol levels show an increase in oscillations, followed by a gradual decline in levels after early postmenopause. Changes in estradiol levels are correlated with region-specific changes in ER expression [38], and women with a greater amount of hormonal fluctuation during perimenopause are at greater risk for Estrogens can influence depression and depression-like behavior through an influence on the There are multiple pathways through which estrogens impact serotonergic activity. Activation of ERs results in increased serotonin release by decreasing the number of presynaptic 5-HT 1A autoreceptors and 5-HT 1A postsynaptic heteroreceptors. ER activation also increases both preand post-synaptic expression of the serotonin transporter (SERT) and release of brain-derived neurotrophic factor (BDNF) [40]. Furthermore, plasma BDNF levels vary across the menstrual cycle [41], and women who have suffered from postpartum depression and/or premenstrual dysphoric disorders are at a greater risk of major depressive disorders during the transition All of the above may also contribute to the pathology of the brain tissue and subsequently serotonergic system and through interactions with neurotrophic factors. to menopause, referred to as perimenopausal depression [40]. produce psychopathological symptoms. which further contributes to accelerated bone resorption [1, 23, 26]. by the kidneys lead to secondary hyperparathyroidism, Reproductive Aging: Perimenopause and Psychopathological Symptoms http://dx.doi.org/10.5772/intechopen.74159 105 of active 1,25-dihydroxyvitamin-D<sup>3</sup> exhibit increases in blood pressure. function, and also synaptogenesis. developing depression [39]. at older age. 4.1. Mood changes These observations are in concordance with out current understanding of bone physiology: downward swinging estradiol levels release cytokines, especially "receptor activator of nuclear factor kappa-B ligand" (RANKL), that cause increased bone resorption [25, 26]. Also, there is additional compelling data, which suggest that perimenopausal bone loss is associated with high levels of FSH rather than falling levels of estradiol [27, 28]. Proving this is the fact that in perimenopausal women, FSH levels significantly correlate with bone resorption. Still, the role of progesterone and inhibins in bone loss and its maintenance remain unclear. Inhibin B appears to inhibit osteoblastogenesis and osteoclastogenesis while also suppressing osteoblast and osteoclast development. Several longitudinal studies have revealed that adverse changes in the lipid profile occur during the time between early menopausal transition and early postmenopause, such as increased LDL and triglycerides [23]. In spite of not directly influencing the development of insulin resistance or diabetes, data from the Study of Women's Health Across the Nation (SWAN) suggest that perimenopause is associated with the development of metabolic syndrome, including abdominal obesity, dyslipidemia, impaired glucose tolerance, and hypertension [29, 30]. As such, together with the decline in endothelial function that appears to occur around the last menstrual period, perimenopause represents the end of the life period in which estrogen effectively contributed to the prevention of cardiovascular diseases. ### 4. Perimenopause and future health risk Postmenopause is not the main topic of this chapter. Still, we would like to summarize some key changes that occur during this time period [1, 23, 31]: The anatomy and function of the female lower genital tract are estrogen-dependent. With postmenopausal estrogen level decline, tissues lining the vagina, vulva, bladder, and urethra undergo atrophy leading to vaginal dryness, dyspareunia, vulvar pruritus, and other urinary difficulties such as recurrent urogenital infections. Unlike hot flushes and night sweating, which improve over time, symptoms of urogenital atrophy persist throughout the entire postmenopausal period [32, 33]. Postmenopausal low estrogen levels result in bone resorption due to excessive production of the cytokine RANKL and its natural inhibitor cytokine osteoprotegerin (OPG or TNFRS11A), by osteoblasts. Moreover, the age-associated vitamin D deficiency and impaired synthesis of active 1,25-dihydroxyvitamin-D<sup>3</sup> by the kidneys lead to secondary hyperparathyroidism, which further contributes to accelerated bone resorption [1, 23, 26]. It is well known that women have a lower incidence of cardiovascular risk factors and cardiovascular diseases than their male peers [34]. Estrogen modifies endothelial function by two primary mechanisms: modulation of NO activity and attenuation of vascular response to injury [35]. Estrogen promotes vasodilation through stimulation of eNOS and reduction of NO-synthase activity. At the level of the mitochondria in the vascular endothelium, estrogen stimulates oxidative phosphorylation and reduces mitochondrial production of ROS [36]. Following menopause, there is active progression of atherosclerotic lesions [37]; women also exhibit increases in blood pressure. Subclinical development of vascular diseases manifests itself as increased carotid and femoral artery intima-media thickness and accelerated coronary artery calcium deposition, leading to arterial stiffness, which in turn causes impaired flow-mediated vasodilation [23]. The risk of stroke doubles during the first decade after menopause and ultimately exceeds that of men at older age. #### 4.1. Mood changes A common belief still persists that bone loss begins and fractures occur in women with constantly low estrogen levels, during the late postmenopause. However, during perimenopause, an early and accelerated rate of bone loss has been observed, particularly in the lumbar spine [24]. In an Australian study, the estimated average annual rate of bone loss around the time of final menstrual period was 2.5% in the lumbar spine and 1.7% in the femoral neck [24]. These observations are in concordance with out current understanding of bone physiology: downward swinging estradiol levels release cytokines, especially "receptor activator of nuclear factor kappa-B ligand" (RANKL), that cause increased bone resorption [25, 26]. Also, there is additional compelling data, which suggest that perimenopausal bone loss is associated with high levels of FSH rather than falling levels of estradiol [27, 28]. Proving this is the fact Still, the role of progesterone and inhibins in bone loss and its maintenance remain unclear. Inhibin B appears to inhibit osteoblastogenesis and osteoclastogenesis while also suppressing Several longitudinal studies have revealed that adverse changes in the lipid profile occur during the time between early menopausal transition and early postmenopause, such as increased LDL and triglycerides [23]. In spite of not directly influencing the development of insulin resistance or diabetes, data from the Study of Women's Health Across the Nation (SWAN) suggest that perimenopause is associated with the development of metabolic syndrome, including abdominal obesity, dyslipidemia, impaired glucose tolerance, and hypertension [29, 30]. As such, together with the decline in endothelial function that appears to occur around the last menstrual period, perimenopause represents the end of the life period that in perimenopausal women, FSH levels significantly correlate with bone resorption. in which estrogen effectively contributed to the prevention of cardiovascular diseases. Postmenopause is not the main topic of this chapter. Still, we would like to summarize some The anatomy and function of the female lower genital tract are estrogen-dependent. With postmenopausal estrogen level decline, tissues lining the vagina, vulva, bladder, and urethra undergo atrophy leading to vaginal dryness, dyspareunia, vulvar pruritus, and other urinary difficulties such as recurrent urogenital infections. Unlike hot flushes and night sweating, which improve over time, symptoms of urogenital atrophy persist throughout the entire post- Postmenopausal low estrogen levels result in bone resorption due to excessive production of the cytokine RANKL and its natural inhibitor cytokine osteoprotegerin (OPG or TNFRS11A), osteoblast and osteoclast development. 104 Sex Hormones in Neurodegenerative Processes and Diseases 4. Perimenopause and future health risk • Urogenital atrophy • Cardiovascular diseases. menopausal period [32, 33]. • Osteoporosis key changes that occur during this time period [1, 23, 31]: Estrogens enter the brain through the blood-brain barrier and influence the neural activity by multiple pathways. In the central nervous system, they are involved in different processes including cellular protein production, neuronal growth and survival, neural transmission and function, and also synaptogenesis. Estrogens act through both genomic and non-genomic mechanisms. Intracellular estrogen receptors (ER) are widely distributed, and ER subtypes are located in many of the areas that are associated with depression. During perimenopause, estradiol levels show an increase in oscillations, followed by a gradual decline in levels after early postmenopause. Changes in estradiol levels are correlated with region-specific changes in ER expression [38], and women with a greater amount of hormonal fluctuation during perimenopause are at greater risk for developing depression [39]. Estrogens can influence depression and depression-like behavior through an influence on the serotonergic system and through interactions with neurotrophic factors. There are multiple pathways through which estrogens impact serotonergic activity. Activation of ERs results in increased serotonin release by decreasing the number of presynaptic 5-HT 1A autoreceptors and 5-HT 1A postsynaptic heteroreceptors. ER activation also increases both preand post-synaptic expression of the serotonin transporter (SERT) and release of brain-derived neurotrophic factor (BDNF) [40]. Furthermore, plasma BDNF levels vary across the menstrual cycle [41], and women who have suffered from postpartum depression and/or premenstrual dysphoric disorders are at a greater risk of major depressive disorders during the transition to menopause, referred to as perimenopausal depression [40]. All of the above may also contribute to the pathology of the brain tissue and subsequently produce psychopathological symptoms. #### 5. Perimenopause and psychopathological symptoms Perimenopause is primarily viewed as a reproductive transition; however, the symptoms of perimenopause are not just largely neurological in nature, but are also indicative of disruption in multiple estrogen-regulated systems (including thermoregulation, sleep, circadian rhythms, sensory processing, and several domains of cognitive function) [1, 42]. • Feeling a lack of energy ders may develop. decreasing income level [63–67]. through dose tapering and/or medication switching [68]. • Experiencing poor memory [53]. and serotonin regulatory effects of estrogen is shorter [58]. 5.1. Anxiety, anxiety-related substance abuse or dependence, and insomnia Epidemiological studies on women in perimenopause show that the relationships between perimenopausal syndrome and mental disorders are strong and positive, with 1 in 4 women suffering from anxiety and approximately 1 in 7 women suffering from depression [54–56]. Usually, women with a history of poor adaptation to stress or specific personality traits, particularly neuroticism, are predisposed to menopausal syndrome, with more than half of women feeling more stressed due to menopause or approaching menopause, and describing menopause as an unpleasant experience that has had a negative effect on their emotional state [43, 53, 56, 57]. Research also showed that a later age of menarche carries more risk for psychiatric morbidity in perimenopause, because the exposure of women to neuroprotective Reproductive Aging: Perimenopause and Psychopathological Symptoms http://dx.doi.org/10.5772/intechopen.74159 107 Anxiety is a subjectively unpleasant feeling of dread over anticipated events, uneasiness, and worry, accompanied by muscular tension, restlessness, fatigue, or diminished concentration, and is not synonymous with fear, which is a response to a real or perceived immediate threat [59]. If anxiety lasts too long or is too intense in regard to the input stimuli, then anxiety disor- Women are more prone to anxiety than men. On average, the proportion of total anxietyrelated visits to the emergency department is higher among women than men [60]. In perimenopause, every fourth woman experiences higher levels of anxiety, with their anxiety state and trait scores higher in perimenopause than in postmenopause [61]. Furthermore, different personality trait predictors are important in different age subgroups; more specifically, anxious response predisposition might contribute to distress in the early stages of perimeno- Affected patients are usually very impatient to alleviate the symptoms. Despite a high addiction potential, benzodiazepines are among the most prescribed drugs for anxiety and one of the most used drug classes in the world [63, 64]. A quick solution of one mental disorder may produce another mental disorder—dependence. Benzodiazepine dependence develops in 35% of persons who take benzodiazepines regularly for 4 weeks or longer, and the majority of users will develop dependence after 4–6 months of daily use [65]. Women use psychotropic medication consistently more often compared to men and these differences also appear to be contingent on the specific mental disorder [66]. Benzodiazepine use was shown to be higher among women, in older age groups, when burdened with a severe degree of anxiety, and with Upon the abrupt discontinuation of the benzodiazepines, withdrawal symptoms may occur, e.g. sweating, tremor, dizziness, headache, insomnia, rebound anxiety, tachycardia, and elevated blood pressure, all of which can closely resemble perimenopausal symptoms, sometimes making it difficult to diagnose properly. This dependence may be controlled and ended pause, whereas anxiety sensitivity might add to distress closer to menopause [62]. In the perimenopausal brain, there is an increased risk for some women of developing neurodegenerative diseases later in life [42, 43]. Neurodegeneration is the progressive loss of functional activity and trophic degeneration of nerve axons and their terminal arborizations following the destruction of their cells of origin or interruption of their continuity with these cells, and it occurs in Alzheimer's disease, Parkinson's disease, progressive supranuclear paralysis, frontotemporal dementia, corticobasal degeneration, Huntington's disease, prion diseases, amyotrophical lateral sclerosis, spinocerebellar ataxia, and multiple sclerosis [44–47]. Aging women face many challenges in the middle and older adult phases of life, becoming more vulnerable to distress [44, 48]. A gradual decline in functioning on multiple levels takes place, and women must adapt to most of them simultaneously. It is quite challenging to cope with physiological changes that not only involve strict perimenopausal symptoms but also include the occurrence of an array of possible diseases (cardiovascular, pulmonary, endocrine, oncological, etc.) and at the same time have to face many psychosocial changes, such as marital problems, grown-up children leaving home, occupational distress, or possible financial ordeals, to name a few. Therefore, in most cases, before neurodegenerative processes cause clinically relevant symptoms, many psychopathological changes can occur and women in perimenopause most commonly experience anxiety, mood swings, depression, insomnia, and mild cognitive impairment [49]. It is important to understand that perimenopausal estrogen level fluctuations may either alter already diagnosed mental disorders (for the better or for worse) or evoke psychopathological symptoms in otherwise mentally healthy women. Psychiatric diagnosis has a long history of scientific investigation and application, with periods of rapid change, instability, and heated controversy associated with it, and despite efforts of scientists to advance a diagnostic classification system that incorporates neuroscience and genetics, psychiatric disorders cannot yet be fully distinguished by any specific biological markers. Hence, the symptom-based criteria are still used to classify mental illnesses such as psychotic disorders, mood disorders, anxiety and stress-related disorders, behavioral syndromes, personality disorders, mental disorders due to psychoactive substance abuse or due to known physiological conditions, intellectual disabilities, pervasive developmental disorders, and mental disorders with onset in childhood or adolescence [50–52]. The top five symptoms (between 84 and 88%) experienced by middle-aged women with serious mental illness were all problems related to psychological issues: • Feeling a lack of energy 5. Perimenopause and psychopathological symptoms 106 Sex Hormones in Neurodegenerative Processes and Diseases Perimenopause is primarily viewed as a reproductive transition; however, the symptoms of perimenopause are not just largely neurological in nature, but are also indicative of disruption in multiple estrogen-regulated systems (including thermoregulation, sleep, circadian In the perimenopausal brain, there is an increased risk for some women of developing neurodegenerative diseases later in life [42, 43]. Neurodegeneration is the progressive loss of functional activity and trophic degeneration of nerve axons and their terminal arborizations following the destruction of their cells of origin or interruption of their continuity with these cells, and it occurs in Alzheimer's disease, Parkinson's disease, progressive supranuclear paralysis, frontotemporal dementia, corticobasal degeneration, Huntington's disease, prion diseases, amyo- Aging women face many challenges in the middle and older adult phases of life, becoming more vulnerable to distress [44, 48]. A gradual decline in functioning on multiple levels takes place, and women must adapt to most of them simultaneously. It is quite challenging to cope with physiological changes that not only involve strict perimenopausal symptoms but also include the occurrence of an array of possible diseases (cardiovascular, pulmonary, endocrine, oncological, etc.) and at the same time have to face many psychosocial changes, such as marital problems, grown-up children leaving home, occupational distress, or possible financial ordeals, to name a few. Therefore, in most cases, before neurodegenerative processes cause clinically relevant symptoms, many psychopathological changes can occur and women in perimenopause most commonly experience anxiety, mood swings, depression, insomnia, and mild cognitive impairment [49]. It is important to understand that perimenopausal estrogen level fluctuations may either alter already diagnosed mental disorders (for the better or for worse) or evoke psychopathological symptoms in otherwise mentally healthy women. ders, and mental disorders with onset in childhood or adolescence [50–52]. ous mental illness were all problems related to psychological issues: • Feeling depressed • Feeling tired or worn out • Feeling anxious Psychiatric diagnosis has a long history of scientific investigation and application, with periods of rapid change, instability, and heated controversy associated with it, and despite efforts of scientists to advance a diagnostic classification system that incorporates neuroscience and genetics, psychiatric disorders cannot yet be fully distinguished by any specific biological markers. Hence, the symptom-based criteria are still used to classify mental illnesses such as psychotic disorders, mood disorders, anxiety and stress-related disorders, behavioral syndromes, personality disorders, mental disorders due to psychoactive substance abuse or due to known physiological conditions, intellectual disabilities, pervasive developmental disor- The top five symptoms (between 84 and 88%) experienced by middle-aged women with seri- rhythms, sensory processing, and several domains of cognitive function) [1, 42]. trophical lateral sclerosis, spinocerebellar ataxia, and multiple sclerosis [44–47]. • Experiencing poor memory [53]. Epidemiological studies on women in perimenopause show that the relationships between perimenopausal syndrome and mental disorders are strong and positive, with 1 in 4 women suffering from anxiety and approximately 1 in 7 women suffering from depression [54–56]. Usually, women with a history of poor adaptation to stress or specific personality traits, particularly neuroticism, are predisposed to menopausal syndrome, with more than half of women feeling more stressed due to menopause or approaching menopause, and describing menopause as an unpleasant experience that has had a negative effect on their emotional state [43, 53, 56, 57]. Research also showed that a later age of menarche carries more risk for psychiatric morbidity in perimenopause, because the exposure of women to neuroprotective and serotonin regulatory effects of estrogen is shorter [58]. #### 5.1. Anxiety, anxiety-related substance abuse or dependence, and insomnia Anxiety is a subjectively unpleasant feeling of dread over anticipated events, uneasiness, and worry, accompanied by muscular tension, restlessness, fatigue, or diminished concentration, and is not synonymous with fear, which is a response to a real or perceived immediate threat [59]. If anxiety lasts too long or is too intense in regard to the input stimuli, then anxiety disorders may develop. Women are more prone to anxiety than men. On average, the proportion of total anxietyrelated visits to the emergency department is higher among women than men [60]. In perimenopause, every fourth woman experiences higher levels of anxiety, with their anxiety state and trait scores higher in perimenopause than in postmenopause [61]. Furthermore, different personality trait predictors are important in different age subgroups; more specifically, anxious response predisposition might contribute to distress in the early stages of perimenopause, whereas anxiety sensitivity might add to distress closer to menopause [62]. Affected patients are usually very impatient to alleviate the symptoms. Despite a high addiction potential, benzodiazepines are among the most prescribed drugs for anxiety and one of the most used drug classes in the world [63, 64]. A quick solution of one mental disorder may produce another mental disorder—dependence. Benzodiazepine dependence develops in 35% of persons who take benzodiazepines regularly for 4 weeks or longer, and the majority of users will develop dependence after 4–6 months of daily use [65]. Women use psychotropic medication consistently more often compared to men and these differences also appear to be contingent on the specific mental disorder [66]. Benzodiazepine use was shown to be higher among women, in older age groups, when burdened with a severe degree of anxiety, and with decreasing income level [63–67]. Upon the abrupt discontinuation of the benzodiazepines, withdrawal symptoms may occur, e.g. sweating, tremor, dizziness, headache, insomnia, rebound anxiety, tachycardia, and elevated blood pressure, all of which can closely resemble perimenopausal symptoms, sometimes making it difficult to diagnose properly. This dependence may be controlled and ended through dose tapering and/or medication switching [68]. Due to the chronic nature of anxiety, long-term low-dose benzodiazepine treatment may be necessary for some patients, despite that, for example SSRI-antidepressants or second-generation antipsychotics administered in low doses are more suitable as they are not addictive [68]. A phenomenological study explored whether older women who are chronic benzodiazepine users identified themselves as dependent. Canham et al. report that the perceptions of dependence and addiction/abuse influenced benzodiazepine use, as the informants stated to avoid consumption of higher doses of benzodiazepines because of concerns of developing addiction [69]. until puberty, it has been hypothesized that the onset of menstruation in girls, triggered by increases in estrogen and other female gonadal hormones, may be responsible for increased depression and anxiety rates [79]. Paradoxically, sudden decreases in estrogen levels at other times of life, such as postpartum and perimenopause, are also accompanied by increased rates of depression and anxiety, thereby suggesting that it may be hormone ratios or changes, rather than absolute levels, which trigger depression and anxiety in vulnerable women [79]. Depressed mood was found to be associated with the severity of menopausal symptoms (somatic and psychological) [80]. Vasomotor symptoms were reported to be harbingers of oncoming depression and also may signal the presence of dysregulated hormones and neurotransmitters [77]. Psychological aspects of perimenopause, such as loneliness and life satisfaction, were reported to be influenced by personal and partner issues, which seem to play a much more relevant role than biological aspects [81]. Menopausal and affective symptoms, but also partner factors, were related to lower sexual function in middle-aged women [82]. Interestingly, the symptom triad of sleep disturbance, depressed mood, and sexual problems was shown to occur simultaneously in only 5% of perimenopausal women, particularly if they were surgically menopausal or in the late perimenopause [55]. Furthermore, this symptom triad was detected most often among women with fair or poor general health, less education, a lower socioeconomic status, and a greater psychosocial distress [54–56]. Women with perimenopausal depression also report significantly decreased quality of life, lack of social support, poor adjustment, Reproductive Aging: Perimenopause and Psychopathological Symptoms http://dx.doi.org/10.5772/intechopen.74159 109 and increased disability compared to non-depressed perimenopausal women [83]. menopausal depression. manic and depressive symptoms) [88, 89]. 5.3. Bipolar disorder In treating depression in perimenopause, relieving vasomotor symptoms may be a necessary dimension [77]. In milder forms of menopausal mood distress, hormone replacement therapy may be sufficient. However, if depression is severe, antidepressants should be prescribed [84–86]. The selective serotonin reuptake inhibitors (SSRIs) are the most frequently prescribed antidepressant drugs, because they are well tolerated and have no severe side effects. They rapidly block serotonin reuptake, yet the onset of their therapeutic action requires weeks of treatment [87]. Regular exercise is important and helps to strengthen the resilience of women [80]. Also, psychosocial interventions are often necessary to alleviate the symptoms of peri- Perimenopausal women may not suffer only from depression, but may also have had already diagnosed other affective disorders. Bipolar disorder is a common, recurrent, and severe psychiatric disorder that is characterized by extreme mood swings that include emotional highs (mania or hypomania) and lows (depression), or mixed states (simultaneously occurring Studies suggest that women with bipolar disorder are at a higher risk for mood episodes during periods of intense hormonal fluctuation (e.g. premenstrual, postpartum, perimenopause) [90–94]. Estrogen and progesterone were shown to modulate neurotransmitter systems and intracellular signaling pathways that are affected by mood stabilizing agents, and these findings may be relevant to the psychopathological aspects of bipolar disorder in women [90]. A progression in female reproductive stages is associated with bipolar illness exacerbation, partic- Addiction to other substances may also be present in middle-aged women. As the difference between women's and men's drinking rates decrease, the number and impact of older female drinkers is expected to increase, and due to differences in metabolism of alcohol, women are at higher risk for negative physical, medical, social, and psychological consequences associated with higher levels of alcohol consumption [70, 71]. Cannabis users described by Guillem et al. were in a greater percentage female, older, more dependent on marijuana, and with a high prevalence of affective and anxiety disorders [72]. Also, women are at higher risk of abusing opioids through a pathway of initial prescription painkiller use [73]. In clinical practice, sometimes it is challenging to distinguish between anxiety, psychoactive substance withdrawal, and sleep disorders. Insomnia is one of the hallmarks of perimenopause and occurs in approximately 40% of perimenopausal women [57, 58]. Hot flashes, night sweats, and other neurovegetative symptoms disrupt sleep, and insomnia may lead to depression [58]. Personality traits also predispose women to insomnia, and it can be most strongly related to neuroticism and DSM-IV personality disorder diagnoses, especially those of Cluster B (emotional, dramatic, and erratic/inconsistent styles) and Cluster C (anxious, fearful, and obsessive-compulsive styles) [56, 57]. Women with perimenopausal insomnia also have a history of greater sensitivity to severe premenstrual symptoms [57]. #### 5.2. Depression Depression is an affective disorder that causes a persistent feeling of sadness, dysphoria and loss of interest, or anhedonia. It is also called major depressive disorder or clinical depression. It affects feelings, thinking, and behavior and can lead to a variety of emotional and physical problems. Normal day-to-day activities become very troublesome, and sometimes life even seems not worth living. Depression is not a weakness, the patient cannot simply "snap out" of it, and it may require long-term treatment [74, 75]. The majority of findings indicate an increased susceptibility to depression during the perimenopausal transition [76, 77]. Perimenopausal depression is significantly associated with lower education, a rural background, a history of psychiatric illness in the family, a later age of menarche, and the late stage of perimenopause [56, 58]. Marital status, type of family, religion, and occupation seem not to be associated with depression in perimenopause [58]. Also, the timing and number of adverse experiences in the childhood and adolescence differentially impact risk and resilience for major depressive disorder across the female life span and during the menopause transition [78]. Repeated epidemiological studies throughout the world show that depression and anxiety prevalence rates are approximately 2:1 for women to men. As prevalence rates of depression and anxiety are approximately equal in boys and girls until puberty, it has been hypothesized that the onset of menstruation in girls, triggered by increases in estrogen and other female gonadal hormones, may be responsible for increased depression and anxiety rates [79]. Paradoxically, sudden decreases in estrogen levels at other times of life, such as postpartum and perimenopause, are also accompanied by increased rates of depression and anxiety, thereby suggesting that it may be hormone ratios or changes, rather than absolute levels, which trigger depression and anxiety in vulnerable women [79]. Depressed mood was found to be associated with the severity of menopausal symptoms (somatic and psychological) [80]. Vasomotor symptoms were reported to be harbingers of oncoming depression and also may signal the presence of dysregulated hormones and neurotransmitters [77]. Psychological aspects of perimenopause, such as loneliness and life satisfaction, were reported to be influenced by personal and partner issues, which seem to play a much more relevant role than biological aspects [81]. Menopausal and affective symptoms, but also partner factors, were related to lower sexual function in middle-aged women [82]. Interestingly, the symptom triad of sleep disturbance, depressed mood, and sexual problems was shown to occur simultaneously in only 5% of perimenopausal women, particularly if they were surgically menopausal or in the late perimenopause [55]. Furthermore, this symptom triad was detected most often among women with fair or poor general health, less education, a lower socioeconomic status, and a greater psychosocial distress [54–56]. Women with perimenopausal depression also report significantly decreased quality of life, lack of social support, poor adjustment, and increased disability compared to non-depressed perimenopausal women [83]. In treating depression in perimenopause, relieving vasomotor symptoms may be a necessary dimension [77]. In milder forms of menopausal mood distress, hormone replacement therapy may be sufficient. However, if depression is severe, antidepressants should be prescribed [84–86]. The selective serotonin reuptake inhibitors (SSRIs) are the most frequently prescribed antidepressant drugs, because they are well tolerated and have no severe side effects. They rapidly block serotonin reuptake, yet the onset of their therapeutic action requires weeks of treatment [87]. Regular exercise is important and helps to strengthen the resilience of women [80]. Also, psychosocial interventions are often necessary to alleviate the symptoms of perimenopausal depression. #### 5.3. Bipolar disorder Due to the chronic nature of anxiety, long-term low-dose benzodiazepine treatment may be necessary for some patients, despite that, for example SSRI-antidepressants or second-generation antipsychotics administered in low doses are more suitable as they are not addictive [68]. A phenomenological study explored whether older women who are chronic benzodiazepine users identified themselves as dependent. Canham et al. report that the perceptions of dependence and addiction/abuse influenced benzodiazepine use, as the informants stated to avoid consumption Addiction to other substances may also be present in middle-aged women. As the difference between women's and men's drinking rates decrease, the number and impact of older female drinkers is expected to increase, and due to differences in metabolism of alcohol, women are at higher risk for negative physical, medical, social, and psychological consequences associated with higher levels of alcohol consumption [70, 71]. Cannabis users described by Guillem et al. were in a greater percentage female, older, more dependent on marijuana, and with a high prevalence of affective and anxiety disorders [72]. Also, women are at higher risk of In clinical practice, sometimes it is challenging to distinguish between anxiety, psychoactive substance withdrawal, and sleep disorders. Insomnia is one of the hallmarks of perimenopause and occurs in approximately 40% of perimenopausal women [57, 58]. Hot flashes, night sweats, and other neurovegetative symptoms disrupt sleep, and insomnia may lead to depression [58]. Personality traits also predispose women to insomnia, and it can be most strongly related to neuroticism and DSM-IV personality disorder diagnoses, especially those of Cluster B (emotional, dramatic, and erratic/inconsistent styles) and Cluster C (anxious, fearful, and obsessive-compulsive styles) [56, 57]. Women with perimenopausal insomnia Depression is an affective disorder that causes a persistent feeling of sadness, dysphoria and loss of interest, or anhedonia. It is also called major depressive disorder or clinical depression. It affects feelings, thinking, and behavior and can lead to a variety of emotional and physical problems. Normal day-to-day activities become very troublesome, and sometimes life even seems not worth living. Depression is not a weakness, the patient cannot simply "snap out" The majority of findings indicate an increased susceptibility to depression during the perimenopausal transition [76, 77]. Perimenopausal depression is significantly associated with lower education, a rural background, a history of psychiatric illness in the family, a later age of menarche, and the late stage of perimenopause [56, 58]. Marital status, type of family, religion, and occupation seem not to be associated with depression in perimenopause [58]. Also, the timing and number of adverse experiences in the childhood and adolescence differentially impact risk and resilience for major depressive disorder across the female life span and during the menopause transition [78]. Repeated epidemiological studies throughout the world show that depression and anxiety prevalence rates are approximately 2:1 for women to men. As prevalence rates of depression and anxiety are approximately equal in boys and girls of higher doses of benzodiazepines because of concerns of developing addiction [69]. 108 Sex Hormones in Neurodegenerative Processes and Diseases abusing opioids through a pathway of initial prescription painkiller use [73]. also have a history of greater sensitivity to severe premenstrual symptoms [57]. of it, and it may require long-term treatment [74, 75]. 5.2. Depression Perimenopausal women may not suffer only from depression, but may also have had already diagnosed other affective disorders. Bipolar disorder is a common, recurrent, and severe psychiatric disorder that is characterized by extreme mood swings that include emotional highs (mania or hypomania) and lows (depression), or mixed states (simultaneously occurring manic and depressive symptoms) [88, 89]. Studies suggest that women with bipolar disorder are at a higher risk for mood episodes during periods of intense hormonal fluctuation (e.g. premenstrual, postpartum, perimenopause) [90–94]. Estrogen and progesterone were shown to modulate neurotransmitter systems and intracellular signaling pathways that are affected by mood stabilizing agents, and these findings may be relevant to the psychopathological aspects of bipolar disorder in women [90]. A progression in female reproductive stages is associated with bipolar illness exacerbation, particularly with lower mood and depression [91]. Interestingly, the exacerbation of perimenopausal symptoms can be predicted in major depressive disorder, but not in bipolar disorder [93]. 5.5. Psychotic disorders Cognitive impairment, anxiety, and mood swings may also occur or worsen in perimenopausal women with psychotic disorders (e.g. schizophrenia, delusional disorder, transient psychotic disorders, or schizoaffective disorder). Schizophrenia is a complex mental disorder that is characterized by positive symptoms (e.g. abnormal perceptions and beliefs), negative symptoms (e.g. anhedonia and social withdrawal), cognitive deficits, and a decline from a previous level of functioning [103]. Schizophrenia and other psychotic disorders are increasingly thought of as neurodevelopmental disorders, where multiple hits accumulate during critical periods of central nervous system (CNS) development to cause the disorders [104, 105]. Reproductive Aging: Perimenopause and Psychopathological Symptoms http://dx.doi.org/10.5772/intechopen.74159 111 The loss of estrogens may lead to increased vulnerability for psychotic relapse, poor clinical outcome, and a need for increased antipsychotic dose [106, 107]. Furthermore, time since menopause is significantly negatively associated with antipsychotic response in postmenopausal women with schizophrenia, suggesting a decline in antipsychotic response after menopause [107]. Hormone replacement therapy during the perimenopause in women with schizophrenia ameliorates psychotic and cognitive symptoms and may also help affective symptoms [108]. Both hormone replacement therapy and changes in antipsychotic manage- An array of neurotransmitters and neuromodulators is involved in the occurrence of psychopathological symptoms, and although neuroscience has elucidated many psychopathological processes, the complexity of the field makes it impossible to obtain definite explanations just yet. Connectomics has already begun to map out large-scale neural circuit diagrams, including ultrastructural analysis of the human brain [109, 110]. It is noteworthy that neural circuits do not have fixed connective properties and the relative concentrations and mixture of neuromodulators at any given moment can provide yet another layer of dynamic functional Neurotransmitters and neuromodulators generally alter circuit function on the timescale of seconds-to-minutes, and thereby they help fill the 'signaling gap' to a substantially slower processes of gene transcription and protein translation (i.e. on the timescale of hours or days) [109]. The production of steroid hormones within brain circuits can rapidly modulate their functional connectivity, thereby affecting the behavior [109]. Effects of long-term 17-β estradiol (E2) replacement on gene expression in brain nuclei were selective and revealed the greatest number of gene changes in the supraoptic nucleus, with no genes affected in the prefrontal cortex [111]. Estrogen has proven neuroprotective effects and estrogen receptors are particularly plentiful in the brain, especially in the hypothalamus, medulla, and limbic system, and therefore, it is not surprising that sudden changes in estrogen levels may affect mood, anxiety, and cognition [58, 79]. Interventional research on early postmenopausal women suggests that estrogen effects on serotonergic function may actually be a key mechanism relating mood and cognitive ment should be considered for women with schizophrenia at menopause [108]. 5.6. Neurocircuitry and perimenopausal psychopathology connections within a circuit [109]. Estrogen seems to be neuroprotective also in affective disorders. Bipolar disorder patients who were using hormone replacement therapy during perimenopause reported significantly less worsening of mood symptoms than the patients without hormone replacement therapy [95]. This should be considered while adjusting the medication for bipolar disorder in perimenopause. #### 5.4. Cognitive impairment Perimenopausal women can also experience different degrees of cognitive impairment. It is characterized with diminished or impaired mental and/or intellectual function and includes deficits in overall intelligence (e.g. with intellectual disabilities) and specific and restricted deficits in cognitive abilities (e.g. attention, working memory, learning, executive function, etc.), or it may describe drug-induced cognitive malfunction (e.g. benzodiazepines, alcohol, illegal drugs, etc.). In perimenopause, cognitive performance does not decline, but improvement is also absent [96]. In the SWAN study, researchers observed that increased anxiety and depressive symptoms had independent and unfavorable effects on cognitive functioning [96]. Women reported trouble with recall of words and numbers, losing or misplacing items, difficulty concentrating, needing to use memory aids, and forgetting appointments. However, the perceived memory difficulties were predominantly a function of stress and multiple burdens resulting in diminished attention and concentration [96]. Perimenopause may have either just contemporary or long-term effects on cognitive function with women being disproportionally more than men affected with Alzheimer's disease and dementia [96, 97]. Cognitive function does not change linearly across perimenopause, and the decreases in attention/working memory, verbal learning, verbal memory, and fine motor speed may be most evident in the first year after the final menstrual period [98]. A premature menopause, either because of a premature bilateral ovariectomy or a premature ovarian failure, was associated with worse verbal fluency and visual memory in later life and also with a 30% increased risk of decline in psychomotor speed and global cognitive function over 7 years [99]. Hormone replacement therapy at the time of premature menopause appeared only partly beneficial for later-life cognitive functioning, and Ryan et al. warn that this should be considered as a part of risk/benefit ratio when deciding on ovariectomy in younger women [99]. It is noteworthy that women do not arrive at the menopause with equal risk of cognitive impairment or equal susceptibility to the effects of hormone replacement therapy [64]. Hormone replacement therapy can have health risks, such as hormone-dependent cancer or cardiovascular pathology that can also cause cognitive deterioration [100–102]. Therefore, it is very important to take into account as many pro and contra arguments for prescribing hormone replacement therapy, as possible. #### 5.5. Psychotic disorders ularly with lower mood and depression [91]. Interestingly, the exacerbation of perimenopausal symptoms can be predicted in major depressive disorder, but not in bipolar disorder [93]. Estrogen seems to be neuroprotective also in affective disorders. Bipolar disorder patients who were using hormone replacement therapy during perimenopause reported significantly less worsening of mood symptoms than the patients without hormone replacement therapy [95]. This should be considered while adjusting the medication for bipolar disorder in perimenopause. Perimenopausal women can also experience different degrees of cognitive impairment. It is characterized with diminished or impaired mental and/or intellectual function and includes deficits in overall intelligence (e.g. with intellectual disabilities) and specific and restricted deficits in cognitive abilities (e.g. attention, working memory, learning, executive function, etc.), or it may describe drug-induced cognitive malfunction (e.g. benzodiazepines, alcohol, In perimenopause, cognitive performance does not decline, but improvement is also absent [96]. In the SWAN study, researchers observed that increased anxiety and depressive symptoms had independent and unfavorable effects on cognitive functioning [96]. Women reported trouble with recall of words and numbers, losing or misplacing items, difficulty concentrating, needing to use memory aids, and forgetting appointments. However, the perceived memory difficulties were predominantly a function of stress and multiple burdens resulting in Perimenopause may have either just contemporary or long-term effects on cognitive function with women being disproportionally more than men affected with Alzheimer's disease and dementia [96, 97]. Cognitive function does not change linearly across perimenopause, and the decreases in attention/working memory, verbal learning, verbal memory, and fine motor A premature menopause, either because of a premature bilateral ovariectomy or a premature ovarian failure, was associated with worse verbal fluency and visual memory in later life and also with a 30% increased risk of decline in psychomotor speed and global cognitive function over 7 years [99]. Hormone replacement therapy at the time of premature menopause appeared only partly beneficial for later-life cognitive functioning, and Ryan et al. warn that this should be considered as a part of risk/benefit ratio when deciding on ovariectomy in It is noteworthy that women do not arrive at the menopause with equal risk of cognitive impairment or equal susceptibility to the effects of hormone replacement therapy [64]. Hormone replacement therapy can have health risks, such as hormone-dependent cancer or cardiovascular pathology that can also cause cognitive deterioration [100–102]. Therefore, it is very important to take into account as many pro and contra arguments for prescribing hor- speed may be most evident in the first year after the final menstrual period [98]. 5.4. Cognitive impairment 110 Sex Hormones in Neurodegenerative Processes and Diseases diminished attention and concentration [96]. illegal drugs, etc.). younger women [99]. mone replacement therapy, as possible. Cognitive impairment, anxiety, and mood swings may also occur or worsen in perimenopausal women with psychotic disorders (e.g. schizophrenia, delusional disorder, transient psychotic disorders, or schizoaffective disorder). Schizophrenia is a complex mental disorder that is characterized by positive symptoms (e.g. abnormal perceptions and beliefs), negative symptoms (e.g. anhedonia and social withdrawal), cognitive deficits, and a decline from a previous level of functioning [103]. Schizophrenia and other psychotic disorders are increasingly thought of as neurodevelopmental disorders, where multiple hits accumulate during critical periods of central nervous system (CNS) development to cause the disorders [104, 105]. The loss of estrogens may lead to increased vulnerability for psychotic relapse, poor clinical outcome, and a need for increased antipsychotic dose [106, 107]. Furthermore, time since menopause is significantly negatively associated with antipsychotic response in postmenopausal women with schizophrenia, suggesting a decline in antipsychotic response after menopause [107]. Hormone replacement therapy during the perimenopause in women with schizophrenia ameliorates psychotic and cognitive symptoms and may also help affective symptoms [108]. Both hormone replacement therapy and changes in antipsychotic management should be considered for women with schizophrenia at menopause [108]. #### 5.6. Neurocircuitry and perimenopausal psychopathology An array of neurotransmitters and neuromodulators is involved in the occurrence of psychopathological symptoms, and although neuroscience has elucidated many psychopathological processes, the complexity of the field makes it impossible to obtain definite explanations just yet. Connectomics has already begun to map out large-scale neural circuit diagrams, including ultrastructural analysis of the human brain [109, 110]. It is noteworthy that neural circuits do not have fixed connective properties and the relative concentrations and mixture of neuromodulators at any given moment can provide yet another layer of dynamic functional connections within a circuit [109]. Neurotransmitters and neuromodulators generally alter circuit function on the timescale of seconds-to-minutes, and thereby they help fill the 'signaling gap' to a substantially slower processes of gene transcription and protein translation (i.e. on the timescale of hours or days) [109]. The production of steroid hormones within brain circuits can rapidly modulate their functional connectivity, thereby affecting the behavior [109]. Effects of long-term 17-β estradiol (E2) replacement on gene expression in brain nuclei were selective and revealed the greatest number of gene changes in the supraoptic nucleus, with no genes affected in the prefrontal cortex [111]. Estrogen has proven neuroprotective effects and estrogen receptors are particularly plentiful in the brain, especially in the hypothalamus, medulla, and limbic system, and therefore, it is not surprising that sudden changes in estrogen levels may affect mood, anxiety, and cognition [58, 79]. Interventional research on early postmenopausal women suggests that estrogen effects on serotonergic function may actually be a key mechanism relating mood and cognitive symptoms in the menopausal transition [112]. Many biogenic amines like serotonin, dopamine, and norepinephrine can directly shift sensory representations through modulatory actions in the frontal lobe, midbrain, and thalamus [109]. A substantial body of evidence has already linked estrogen and serotonin, as well as estrogen and dopamine in the central nervous system [113, 114]. Serotonergic and dopaminergic pathways in the brain play an important role in the pathogenesis of anxiety, affective, and psychotic disorders, and monoamine oxidase A (MAO-A) is an important brain enzyme that metabolizes these biogenic amines. After estrogen level declines, MAO-A density may be elevated for a month or longer, and its change during perimenopausal age is very similar to its change during major depressive episodes and high-risk states for major depressive episodes, thus, being an interesting target for relieving of perimenopausal symptoms [115]. including hot flashes and night sweats [114, 126]. The nighttime prevalence of hot flashes and night sweats could be a result of the conversion of serotonin to melatonin at night, resulting Female steroid hormones also promote dopaminergic neuron survival and protect them from degeneration, as shown in the E2 modulation of striatal neural pathways [128]. Dopamine functions of dopamine, ranging from voluntary movement and reward to hormonal regulation and hypertension [129]. In the brain, dopamine receptors mediate affect, attention, impulse control, decision-making, motor learning, sleep, reproductive behaviors, and the regulation of food intake [129]. On the basis of their structural, pharmacological, and bio- , D<sup>3</sup> role in the development of psychotic symptoms [129–131]. In the brain of ovariectomized rats, estrogen treatment increased levels of dopamine transporters and lowered dopamine D<sup>2</sup> receptor density in the nucleus accumbens and in the caudate nucleus, but also normalized Current scientific evidence suggests that the path to psychopathology is laid by the adverse interaction of multiple risk genes and environmental factors, a constellation that predisposes individuals to the subtle disturbances in brain neurotransmission that ultimately lead to overt Antidepressants and antipsychotics control psychopathological symptoms due to their predominant antagonistic effect on various combinations of serotonin and dopamine receptors subtypes, and research supports an important role of add-on estrogen in alleviating mood, Adaptation to biological changes of perimenopause is largely affected with the psychosocial context of middle-aged women's lives, and studies show that these circumstances may have a greater effect on symptomatology than any biological changes. Hence, we must be careful about an overly reductionist receptor-based and hormonal approach to mood or cognitive symptoms and have to take into account the evidence that psychosocial factors act via epigenetic mechanisms in the pathogenesis of mental disorders [75, 131]. Epigenetic remodeling takes place throughout adult life, under the influence of environmental factors such as nutrition, drugs, and chemical, physical, and psychosocial factors, and psychotherapies were suggested to be conceptualized as epigenetic "drugs," or at least as therapeutic agents that act Middle adulthood is the period in life characterized by gradually decreased biological and physiological functioning [134]. As previously described, a subgroup of vulnerable women may suffer from the hormonal changes naturally occurring during the perimenopause and coinciding with the manifold psychosocial changes coming together during this phase of , and D4 dopamine receptors. Dopamine D<sup>2</sup> ) are G-protein coupled and mediate all of the physiological Reproductive Aging: Perimenopause and Psychopathological Symptoms receptors play a critical ). All clinically effective antipsychot- http://dx.doi.org/10.5772/intechopen.74159 113 in lower circulating serotonin levels [127]. , and D5 chemical properties, these receptors are classified as either D<sup>1</sup> , D<sup>2</sup> , D<sup>3</sup> , D4 ics possess the ability to block D<sup>2</sup> norepinephrine pathway [132, 133]. emotional and behavioral symptoms [134]. 5.7. Psychosocial context in perimenopause anxiety, and psychotic symptoms in perimenopause. epigenetically very similarly or complementary to drugs [131–133]. receptors (D<sup>1</sup> and D5 ) or D<sup>2</sup> Estradiol (E2) was shown to increase the production of tryptophan hydroxylase (TPH), which represents the rate-limiting step in the synthesis of serotonin from its precursor tryptophan, and furthermore, E2 also inhibits the expression of the gene for the SERT and acts as an antagonist at the SERT, thus, increasing the concentrations of serotonin that remains available in the synapses for a longer period of time [114, 116–118]. E2 also modulates the actions of serotonin because the activation of E2 receptors affects the distribution and state of serotonin receptors [114]. Higher levels of E2 in the presence of progesterone upregulate E2 β receptors (ERβ) and downregulate E2 α receptors (ERα). ERα downregulation directly inhibits function of serotonergic 5-HT1A receptors [114, 119]. Additionally, ERβ upregulation in turn upregulates 5-HT2A receptors [114, 120]. Following 5-HT2A activation of protein kinase C, 5-HT1A receptors become unable to reduce serotonin production through negative feedback and serotonin concentrations increase [114]. Alterations in 5-HT1A, 5-HT1B and 5-HT2A mRNA levels and an increase in synaptic serotonin levels reduce symptoms of anxiety, depression, and possible psychosis [121]. The 5-HT1A and 5-HT1B receptors are both inhibitory transmembrane receptors that are located throughout the brain [82]. 5-HT1A receptor is the most studied for its role in depression, but it also modulates anxiety behavior, bipolar disorder, and post-traumatic stress disorder [122]. Also, several lines of evidence support a stimulatory influence of serotonin on the hypothalamo-pituitary-adrenal axis (HPA) in humans and rodents, mediated, in part, by the 5-HT1A receptor. Evidence suggests that the brain serotonergic system has a higher potential for stimulating the HPA axis in females, and under basal conditions, females express higher levels of serotonin than males in brainstem, limbic forebrain, and cortex [123]. The 5-HT1A receptor not only drives the stimulatory effect of serotonin on the HPA axis but is also a critical determinant of the antidepressant response [123]. 5-HT1B receptor is best known for its role in regulating aggressive and impulsive behavior, but it also modulates depression and it has been implicated in the neural basis of dysregulation of reward processing, thus being associated with drug and alcohol abuse [122]. 5-HT2A antagonists have antidepressant-like, anxiolytic, and antipsychotic effects [122, 124]. There is extensive evidence, from both animal and human studies, that the characteristic effects of hallucinogens are mediated by their agonistic interactions with the 5-HT2A receptor [125]. The loss of estrogen in perimenopause leads to a decreased density of 5-HT2A receptors and a lower activity of serotonin, which could explain aberrant temperature regulation, including hot flashes and night sweats [114, 126]. The nighttime prevalence of hot flashes and night sweats could be a result of the conversion of serotonin to melatonin at night, resulting in lower circulating serotonin levels [127]. Female steroid hormones also promote dopaminergic neuron survival and protect them from degeneration, as shown in the E2 modulation of striatal neural pathways [128]. Dopamine receptors (D<sup>1</sup> , D<sup>2</sup> , D<sup>3</sup> , D4 , and D5 ) are G-protein coupled and mediate all of the physiological functions of dopamine, ranging from voluntary movement and reward to hormonal regulation and hypertension [129]. In the brain, dopamine receptors mediate affect, attention, impulse control, decision-making, motor learning, sleep, reproductive behaviors, and the regulation of food intake [129]. On the basis of their structural, pharmacological, and biochemical properties, these receptors are classified as either D<sup>1</sup> -class dopamine receptors (D<sup>1</sup> and D5 ) or D<sup>2</sup> -class dopamine receptors (D<sup>2</sup> , D<sup>3</sup> , and D4 ). All clinically effective antipsychotics possess the ability to block D<sup>2</sup> dopamine receptors. Dopamine D<sup>2</sup> receptors play a critical role in the development of psychotic symptoms [129–131]. In the brain of ovariectomized rats, estrogen treatment increased levels of dopamine transporters and lowered dopamine D<sup>2</sup> receptor density in the nucleus accumbens and in the caudate nucleus, but also normalized norepinephrine pathway [132, 133]. Current scientific evidence suggests that the path to psychopathology is laid by the adverse interaction of multiple risk genes and environmental factors, a constellation that predisposes individuals to the subtle disturbances in brain neurotransmission that ultimately lead to overt emotional and behavioral symptoms [134]. Antidepressants and antipsychotics control psychopathological symptoms due to their predominant antagonistic effect on various combinations of serotonin and dopamine receptors subtypes, and research supports an important role of add-on estrogen in alleviating mood, anxiety, and psychotic symptoms in perimenopause. #### 5.7. Psychosocial context in perimenopause symptoms in the menopausal transition [112]. Many biogenic amines like serotonin, dopamine, and norepinephrine can directly shift sensory representations through modulatory actions in the frontal lobe, midbrain, and thalamus [109]. A substantial body of evidence has already linked estrogen and serotonin, as well as estrogen and dopamine in the central nervous system [113, 114]. Serotonergic and dopaminergic pathways in the brain play an important role in the pathogenesis of anxiety, affective, and psychotic disorders, and monoamine oxidase A (MAO-A) is an important brain enzyme that metabolizes these biogenic amines. After estrogen level declines, MAO-A density may be elevated for a month or longer, and its change during perimenopausal age is very similar to its change during major depressive episodes and high-risk states for major depressive episodes, thus, being an interesting target for relieving of Estradiol (E2) was shown to increase the production of tryptophan hydroxylase (TPH), which represents the rate-limiting step in the synthesis of serotonin from its precursor tryptophan, and furthermore, E2 also inhibits the expression of the gene for the SERT and acts as an antagonist at the SERT, thus, increasing the concentrations of serotonin that remains available in the synapses for a longer period of time [114, 116–118]. E2 also modulates the actions of serotonin because the activation of E2 receptors affects the distribution and state of serotonin receptors [114]. Higher levels of E2 in the presence of progesterone upregulate E2 β receptors (ERβ) and downregulate E2 α receptors (ERα). ERα downregulation directly inhibits function of serotonergic 5-HT1A receptors [114, 119]. Additionally, ERβ upregulation in turn upregulates 5-HT2A receptors [114, 120]. Following 5-HT2A activation of protein kinase C, 5-HT1A receptors become unable to reduce serotonin production through negative feedback and serotonin concentrations increase [114]. Alterations in 5-HT1A, 5-HT1B and 5-HT2A mRNA levels and an increase in synaptic serotonin levels reduce symptoms of anxiety, depression, and possible psychosis [121]. The 5-HT1A and 5-HT1B receptors are both inhibitory transmembrane receptors that are located throughout the brain [82]. 5-HT1A receptor is the most studied for its role in depression, but it also modulates anxiety behavior, bipolar disorder, and post-traumatic stress disorder [122]. Also, several lines of evidence support a stimulatory influence of serotonin on the hypothalamo-pituitary-adrenal axis (HPA) in humans and rodents, mediated, in part, by the 5-HT1A receptor. Evidence suggests that the brain serotonergic system has a higher potential for stimulating the HPA axis in females, and under basal conditions, females express higher levels of serotonin than males in brainstem, limbic forebrain, and cortex [123]. The 5-HT1A receptor not only drives the stimulatory effect of serotonin on the HPA axis but is also a critical determinant of the antidepressant response [123]. 5-HT1B receptor is best known for its role in regulating aggressive and impulsive behavior, but it also modulates depression and it has been implicated in the neural basis of dysregulation of reward processing, thus being associated 5-HT2A antagonists have antidepressant-like, anxiolytic, and antipsychotic effects [122, 124]. There is extensive evidence, from both animal and human studies, that the characteristic effects of hallucinogens are mediated by their agonistic interactions with the 5-HT2A receptor [125]. The loss of estrogen in perimenopause leads to a decreased density of 5-HT2A receptors and a lower activity of serotonin, which could explain aberrant temperature regulation, perimenopausal symptoms [115]. 112 Sex Hormones in Neurodegenerative Processes and Diseases with drug and alcohol abuse [122]. Adaptation to biological changes of perimenopause is largely affected with the psychosocial context of middle-aged women's lives, and studies show that these circumstances may have a greater effect on symptomatology than any biological changes. Hence, we must be careful about an overly reductionist receptor-based and hormonal approach to mood or cognitive symptoms and have to take into account the evidence that psychosocial factors act via epigenetic mechanisms in the pathogenesis of mental disorders [75, 131]. Epigenetic remodeling takes place throughout adult life, under the influence of environmental factors such as nutrition, drugs, and chemical, physical, and psychosocial factors, and psychotherapies were suggested to be conceptualized as epigenetic "drugs," or at least as therapeutic agents that act epigenetically very similarly or complementary to drugs [131–133]. Middle adulthood is the period in life characterized by gradually decreased biological and physiological functioning [134]. As previously described, a subgroup of vulnerable women may suffer from the hormonal changes naturally occurring during the perimenopause and coinciding with the manifold psychosocial changes coming together during this phase of life [106]. In this midlife transition, an intense reappraisal of all aspects of life takes place, and it may result either in decisions to keep most life structures that were built through decades, such as marriages and careers, or major shifts may be made, such as divorce or a job change, and the latter may represent a true midlife crisis, accompanied by significant emotional turmoil for the individual and others [134]. Another phenomenon described in middle adulthood is an empty nest syndrome, the time when the youngest child is about to leave home. Parents may become depressed, and this is especially true of women whose predominant role in life has been mothering [135]. Author details Ljubljana, Slovenia References 76:874-878 rbmo.2014.02.003 1998;70:56-59 1168. DOI: 10.1210/jc.2011-3362 Ksenija Gersak1,2\, Ziva Miriam Gersak2 \Address all correspondence to: [email protected] 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia delphia: Wolters Kluwer; 2010. 1451 p. ISBN: 978-0-7817-7968-5 ovarian hyperstimulation. Endocrine. 2005;26:297-300 shift. Frontiers in Bioscience (Schol Ed). 2011;3:474-486 Endocrinology and Metabolism. Nov 2003;88(11):5502-5509 and Arijana Turcin3 Reproductive Aging: Perimenopause and Psychopathological Symptoms http://dx.doi.org/10.5772/intechopen.74159 115 1 Department of Obstetrics and Gynaecology, University Medical Centre Ljubljana, 3 Outpatient Psychiatry Centre, University Psychiatric Clinic Ljubljana, Ljubljana, Slovenia [1] Fritz MA, Speroff L. Clinical Gynecologic Endocrinology and Infertility. 8th ed. Phila- [2] Soules MR, Sherman S, Parrott E, Rebar R, Santoro N, Utian W, Woods N. Executive summary: Stages of reproductive aging workshop (STRAW). Fertility and Sterility. 2001; [3] Harlow SD, Gass M, Hall JE, Lobo R, Maki P, Rebar RW, et al. Executive summary of the Stages of Reproductive Aging Workshop+10: Addressing the unfinished agenda of staging reproductive aging. Journal of Clinical Endocrinology and Metabolism 2012;97:1159- [4] Prior JC. Ovarian aging and the perimenopausal transition: The paradox of endogenous [5] Prior JC, Hitchcock CL. The endocrinology of perimenopause: Need for a paradigm [6] Messinis IE, Messini CI, Dafopoulos K. Novel aspects of the endocrinology of the menstrual cycle. Reproductive BioMedicine Online. Jun 2014;28(6):714-722. DOI: 10.1016/j. [7] Cahill DJ, Wardle PG, Harlow CR, Hull MG. Onset of the preovulatory luteinizing hormone surge: Diurnal timing and critical follicular prerequisites. Fertility and Sterility. [8] Santoro N, Isaac B, Neal-Perry G, Adel T, Weingart L, Nussbaum A, et al. Impaired folliculogenesis and ovulation in older reproductive aged women. Journal of Clinical [9] Robertson DM, Hale GE, Jolley D, Fraser IS, Hughes CL, Burger HG.Interrelationships between ovarian and pituitary hormones in ovulatory menstrual cycles across reproductive age. Psychopathological symptoms in perimenopause occur as a result of complex changes on several levels of female functioning, may it be biological or psychosocial. Middle-aged women must adapt to loss of sex hormones, and this transition is sometimes extremely troublesome. If they already suffered from any mental illness while being younger, the perimenopausal transition is even harder. If they also live in unfavorable circumstances, lack the support from significant others, are physically ill and poor, it is reflected in the severity of perimenopausal syndrome and a higher incidence of mental disease during perimenopause. These patients need a special attention from different medical practitioners. It is vitally important to tailor the therapy individually while carefully listening to the minute details of what burdens each woman the most. Psychotherapy, regular exercise, social interventions, and partner counseling are just a few of possible actions that may alleviate the severity of perimenopausal and psychopathological symptoms, while the combination of the prescribed medications must be fine-tuned and supported with the estimation of expected true benefits. Last but not least, women with serious mental disorder have deficits in knowledge regarding menopause [53]. Therefore, educational programmes are necessary and should offer valuable information on natural course of perimenopause and strategies to alleviate perimenopausal symptoms, i.e. teaching the women how to reduce stress, eat healthy, engage in different activities, regularly take the prescribed medication if needed, and also recognize the perimenopausal symptoms and learn how to differentiate them from possible serious diseases that need professional help. Perimenopause cannot be prevented, but many of the perimenopausal symptoms can and should be reduced, and that holds true also for perimenopausal psychopathological symptoms. #### 6. Conclusion Menopause is an event in a woman's life that marks the end of reproductive function. The process of reproductive aging is gradual and begins in the early menopausal transition. The decline in ovarian estrogen production causes physical symptoms, metabolic changes, and influences the mood and cognition. The relationships between perimenopausal syndrome and mental disorders are strong and confirmed with many different studies. Given these findings, in the future, strategies to locally regulate hormone bioavailability may offer greater therapeutic potential in the fight against age-related disease. ### Author details life [106]. In this midlife transition, an intense reappraisal of all aspects of life takes place, and it may result either in decisions to keep most life structures that were built through decades, such as marriages and careers, or major shifts may be made, such as divorce or a job change, and the latter may represent a true midlife crisis, accompanied by significant emotional turmoil for the individual and others [134]. Another phenomenon described in middle adulthood is an empty nest syndrome, the time when the youngest child is about to leave home. Parents may become depressed, and this is especially true of women whose Psychopathological symptoms in perimenopause occur as a result of complex changes on several levels of female functioning, may it be biological or psychosocial. Middle-aged women must adapt to loss of sex hormones, and this transition is sometimes extremely troublesome. If they already suffered from any mental illness while being younger, the perimenopausal transition is even harder. If they also live in unfavorable circumstances, lack the support from significant others, are physically ill and poor, it is reflected in the severity of perimenopausal syndrome and a higher incidence of mental disease during perimenopause. These patients need a special attention from different medical practitioners. It is vitally important to tailor the therapy individually while carefully listening to the minute details of what burdens each woman the most. Psychotherapy, regular exercise, social interventions, and partner counseling are just a few of possible actions that may alleviate the severity of perimenopausal and psychopathological symptoms, while the combination of the prescribed medications must be fine-tuned and supported with the estimation of Last but not least, women with serious mental disorder have deficits in knowledge regarding menopause [53]. Therefore, educational programmes are necessary and should offer valuable information on natural course of perimenopause and strategies to alleviate perimenopausal symptoms, i.e. teaching the women how to reduce stress, eat healthy, engage in different activities, regularly take the prescribed medication if needed, and also recognize the perimenopausal symptoms and learn how to differentiate them from possible serious diseases that need professional help. Perimenopause cannot be prevented, but many of the perimenopausal symptoms can and should be reduced, and that holds true also for perimenopausal Menopause is an event in a woman's life that marks the end of reproductive function. The process of reproductive aging is gradual and begins in the early menopausal transition. The decline in ovarian estrogen production causes physical symptoms, metabolic changes, and influences the mood and cognition. The relationships between perimenopausal syndrome and mental disorders are strong and confirmed with many different studies. Given these findings, in the future, strategies to locally regulate hormone bioavailability may offer greater therapeutic potential in the fight against age-related disease. predominant role in life has been mothering [135]. 114 Sex Hormones in Neurodegenerative Processes and Diseases expected true benefits. psychopathological symptoms. 6. Conclusion Ksenija Gersak1,2\, Ziva Miriam Gersak2 and Arijana Turcin3 \Address all correspondence to: [email protected] 1 Department of Obstetrics and Gynaecology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Outpatient Psychiatry Centre, University Psychiatric Clinic Ljubljana, Ljubljana, Slovenia #### References Journal of Clinical Endocrinology and Metabolism. Jan 2009;94(1):138-144. DOI: 10.1210/ jc.2008-1684 [21] Nelson SM. 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DOI: 10.1186/1472-6874-5-12 Chapter 6 Provisional chapter Neuroprotection in Perimenopausal Women Neuroprotection in Perimenopausal Women DOI: 10.5772/intechopen.74330 Endocrine and neural senescence overlap in time, by intertwined complex feedback loops. Womens' brain is genetically more prone to suffer during life, and perimenopause is a "critical period" in neuroaging, when the degenerative processes begin. Many hypotheses on the multifactorial nature of women's brain aging are elaborated, and tested in high-tech research centers. The most analyzed Alzheimer's disease (AD) is characterized not only by Aβ oligomers and fibrils accumulation, but also by metabolic and inflammatory changes, with the onset during menopausal transition and early years of menopause. Deep analysis of endocrine, neural, and metabolic pathways are giving new insights to the sequential view of Aβ-centric in AD pathogenesis, prevention, and treat- ment from perimenopause, for maintaining women's neurological health. 1. Introduction: sex differences in contemporary neurodegenerative Ovarian aging is very well-known in contemporary women's life, and the jeopardizing menopausal effects of sex steroid hormones deficiency are clinically evident in late-life mental disorders. Endocrine and neural senescence overlap in time, and are mechanistically intertwined In the past century, both life expectancy and the average age of onset of menopause for women in many countries from Western Europe and North America were slightly over 50 years, whereas currently, women can expect to live until the age of 80 years, although the average age of menopause remains in the early 50s. Given the importance of the brain as a target organ Keywords: neuroaging, perimenopause, critical period © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Manuela Cristina Russu and Alexandra Cristina Antonescu Abstract disorders in complex feedback loops. Manuela Cristina Russu and Alexandra Cristina Antonescu http://dx.doi.org/10.5772/intechopen.74330 Additional information is available at the end of the chapter Additional information is available at the end of the chapter #### Chapter 6 Provisional chapter #### Neuroprotection in Perimenopausal Women Neuroprotection in Perimenopausal Women DOI: 10.5772/intechopen.74330 #### Manuela Cristina Russu and Alexandra Cristina Antonescu Manuela Cristina Russu and Alexandra Cristina Antonescu Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.74330 #### Abstract Endocrine and neural senescence overlap in time, by intertwined complex feedback loops. Womens' brain is genetically more prone to suffer during life, and perimenopause is a "critical period" in neuroaging, when the degenerative processes begin. Many hypotheses on the multifactorial nature of women's brain aging are elaborated, and tested in high-tech research centers. The most analyzed Alzheimer's disease (AD) is characterized not only by Aβ oligomers and fibrils accumulation, but also by metabolic and inflammatory changes, with the onset during menopausal transition and early years of menopause. Deep analysis of endocrine, neural, and metabolic pathways are giving new insights to the sequential view of Aβ-centric in AD pathogenesis, prevention, and treatment from perimenopause, for maintaining women's neurological health. Keywords: neuroaging, perimenopause, critical period #### 1. Introduction: sex differences in contemporary neurodegenerative disorders Ovarian aging is very well-known in contemporary women's life, and the jeopardizing menopausal effects of sex steroid hormones deficiency are clinically evident in late-life mental disorders. Endocrine and neural senescence overlap in time, and are mechanistically intertwined in complex feedback loops. In the past century, both life expectancy and the average age of onset of menopause for women in many countries from Western Europe and North America were slightly over 50 years, whereas currently, women can expect to live until the age of 80 years, although the average age of menopause remains in the early 50s. Given the importance of the brain as a target organ © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. for sex steroids, it is not surprising that many of the complaints that prompt women to seek treatment related to menopause are neurological in origin. role of time since menopause at initiation of HT/ET may work in preventing the mental deterioration due to aging [17], and perimenopause can be the "critical window" for opportunity in neuroprotection with steroids. The perimenopausal transition might also represent a "win- Neuroprotection in Perimenopausal Women http://dx.doi.org/10.5772/intechopen.74330 129 The data of this chapter are regarding brain aging in perimenopause—a part in woman's Menopausal transition starts with the variation of cycles duration and ends with the last period (recognized only after 12 months of amenorrhea), natural menopause at the average age 51 years, premature menopause [premature ovarian failure (POF)] before 40 years, and early menopause between 40 and 45 years [21, 22]. Women with POF have been reported to have more anxiety, depression, somatization, sensitivity, hostility, and psychological distress than women with normal ovaries [23]. Perimenopause or "near menopause" starts from the stage −2 of menopausal transition and ends at 12 months after last menstruation, may be of 10–15 years. During this period, there are important variations of sex steroids, summarized by low ovarian inhibin [24], which in turn reduces the restraint on both the hypothalamus and pituitary gland, and results in elevated pituitary gonadotropin FSH, increased also by the hypothalamic gonadotropin-releasing hormone (GnRH). During the late menopause transition and a part of perimenopause, despite occasional episodes of normal cycling, women are exposed to periods of estrogen withdrawal, fewer ovulatory cycles, and prolonged hypogonadism, ultimately leading to the last menstrual period, after which is an elevated level of gonadotrophin secretion (only tonic, not phasic) [25]. During this phase, besides the low ovarian E2 and progesterone, there are productions of androgens and growth factors, which will decline in future years of postmenopause [26]. The ovaries are stimulated during menopausal transition and early postmenopausal years by gonadotropins, but the pulsatile GnRH pattern is different in different species before reproductive failure. It is a decrease of GnRH gene expression in many dow of opportunity" to prevent age-related neurological diseases [10, 18, 19]. 2. Hormonal and genetic data on perimenopausal neuroaging reproductive life, systematized in the stages of reproductive aging (STRAW) [20]. middle-aged rats [27], and an increase in perimenopausal rhesus monkeys [28]. [21, 31–33]. Premature menopause/early menopause can be spontaneous or induced; after medical interventions such as chemotherapy/radiotherapy or surgery. The most common cause of premature/early menopause is bilateral oophorectomy with/without hysterectomy. Primary ovarian failure (POF) may be a cause of early/premature menopause, for ischemic stroke [29], as for all cardiovascular diseases risks [30], and these conditions were first described as a cause of neurological disturbances in different European, North American, and Japanese populations Bilateral oophorectomy at premenopausal ages is inducing drops of E2 and testosterone levels, by 40–50%, and an abruptly rise in FSH levels, the levels of androgen being lower than in natural menopause at ages of 65 years, when women in normal or in premature/early menopause continue to have some levels of androgens [34]. As it is shown in the Table 1, the hazard ratios reached statistical significance in cases with bilateral oophorectomy: at the Dementia with its most severe entities, such as the Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), are the most frequent contemporary neurodegenerative disorders, connected in majority by neural cell loss and neuroinflammation. There are sex/gender differences in cognitive trajectories in clinically normal older adults [1], and women are known to have a higher propensity to develop AD versus men [2, 3], a higher risk of mild cognitive impairment and a lower risk, but poorer outcomes after stroke [4]. Women's risk for AD is considered to be through their organizational effects during developmental sexual differentiation of the fetal brain [5]. The AD is the most prevalent form of old-life mental failure in worldwide humans, being a progressive neurodegenerative disorder, for which a number of genetic, environmental, and lifestyle risk factors have been identified. The estimated prevalence of all-cause of dementia varies from 4.7% in Central Europe to 8.7% in North Africa/Middle East, with North America falling at 6.4%. Currently, over 46 million individuals live with dementia worldwide and the number is projected to increase to 131.5 million by 2050 [6]. The AD is familial with earlyonset and sporadic with late-onset, and the present chapter will discuss the sporadic AD with late-onset in women, the most common form of AD representing more than 95% of the current human AD population [7]. In this very moment, the geroscience research is imperative with aims to forward a better and full understanding of neurodegeneration/neuroprotection of "the sexome" [8], and to prevent or delay by every tool the deleterious effects of brain aging [9]. Estrogen deficiency or estrogen disrupters are associated from menopause transition with episodic memory troubles, a cognitive domain in which impairments are associated with the increased risk of AD, being less known the onset of the other neurodegenerative disorders [10]. AD has an insidious onset and a gradual progression over several years—from 1.5 to 8 or up to 10 years, or a preclinical stage with a subtle loss of cognitive functioning—as verbal memory on new information, that precede several years the AD diagnostic, period considered as a transition period from normal aging brain to AD [11]. During this period, there are discovered several subtypes of mild cognitive impairment (MCI), 10–15% with the risk to future evolution to AD per year [12], or a 12% conversion rate from MIC to dementia yearly [13]. The characteristic deposits of β-amyloid and tau proteins depicted by neuroimaging or at autopsy located in the hippocampus, medial temporal regions, parietal, and frontal cortical regions [14] may be prevented from extension, as other structural degenerative diseases. It is imperious to prevent the intracellular appearance of the amyloid peptide, which induces by its toxicity neuronal apoptosis and cell death, events that can be prevented by sex steroid hormones [15]. The clinical symptoms/signs of MCI are considered by neuroscientists as prodrome to AD [16], and their algorithms for diagnosis may permit to initiate the hormone/estrogen therapy (HT/ET), because their onset moment is coincidental to perimenopause, as considering the North American clinicians [17]. The "timing" theory regarding the reproductive stage and role of time since menopause at initiation of HT/ET may work in preventing the mental deterioration due to aging [17], and perimenopause can be the "critical window" for opportunity in neuroprotection with steroids. The perimenopausal transition might also represent a "window of opportunity" to prevent age-related neurological diseases [10, 18, 19]. #### 2. Hormonal and genetic data on perimenopausal neuroaging for sex steroids, it is not surprising that many of the complaints that prompt women to seek Dementia with its most severe entities, such as the Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), are the most frequent contemporary neurodegenerative disorders, connected in majority by neural cell There are sex/gender differences in cognitive trajectories in clinically normal older adults [1], and women are known to have a higher propensity to develop AD versus men [2, 3], a higher risk of mild cognitive impairment and a lower risk, but poorer outcomes after stroke [4]. Women's risk for AD is considered to be through their organizational effects during develop- The AD is the most prevalent form of old-life mental failure in worldwide humans, being a progressive neurodegenerative disorder, for which a number of genetic, environmental, and lifestyle risk factors have been identified. The estimated prevalence of all-cause of dementia varies from 4.7% in Central Europe to 8.7% in North Africa/Middle East, with North America falling at 6.4%. Currently, over 46 million individuals live with dementia worldwide and the number is projected to increase to 131.5 million by 2050 [6]. The AD is familial with earlyonset and sporadic with late-onset, and the present chapter will discuss the sporadic AD with late-onset in women, the most common form of AD representing more than 95% of the cur- In this very moment, the geroscience research is imperative with aims to forward a better and full understanding of neurodegeneration/neuroprotection of "the sexome" [8], and to prevent or delay by every tool the deleterious effects of brain aging [9]. Estrogen deficiency or estrogen disrupters are associated from menopause transition with episodic memory troubles, a cognitive domain in which impairments are associated with the increased risk of AD, being AD has an insidious onset and a gradual progression over several years—from 1.5 to 8 or up to 10 years, or a preclinical stage with a subtle loss of cognitive functioning—as verbal memory on new information, that precede several years the AD diagnostic, period considered as a transition period from normal aging brain to AD [11]. During this period, there are discovered several subtypes of mild cognitive impairment (MCI), 10–15% with the risk to future evolution to AD per year [12], or a 12% conversion rate from MIC to dementia yearly [13]. The characteristic deposits of β-amyloid and tau proteins depicted by neuroimaging or at autopsy located in the hippocampus, medial temporal regions, parietal, and frontal cortical regions [14] may be prevented from extension, as other structural degenerative diseases. It is imperious to prevent the intracellular appearance of the amyloid peptide, which induces by its toxicity neuronal apoptosis and cell death, events that can be prevented by sex steroid hormones [15]. The clinical symptoms/signs of MCI are considered by neuroscientists as prodrome to AD [16], and their algorithms for diagnosis may permit to initiate the hormone/estrogen therapy (HT/ET), because their onset moment is coincidental to perimenopause, as considering the North American clinicians [17]. The "timing" theory regarding the reproductive stage and less known the onset of the other neurodegenerative disorders [10]. treatment related to menopause are neurological in origin. 128 Sex Hormones in Neurodegenerative Processes and Diseases mental sexual differentiation of the fetal brain [5]. loss and neuroinflammation. rent human AD population [7]. The data of this chapter are regarding brain aging in perimenopause—a part in woman's reproductive life, systematized in the stages of reproductive aging (STRAW) [20]. Menopausal transition starts with the variation of cycles duration and ends with the last period (recognized only after 12 months of amenorrhea), natural menopause at the average age 51 years, premature menopause [premature ovarian failure (POF)] before 40 years, and early menopause between 40 and 45 years [21, 22]. Women with POF have been reported to have more anxiety, depression, somatization, sensitivity, hostility, and psychological distress than women with normal ovaries [23]. Perimenopause or "near menopause" starts from the stage −2 of menopausal transition and ends at 12 months after last menstruation, may be of 10–15 years. During this period, there are important variations of sex steroids, summarized by low ovarian inhibin [24], which in turn reduces the restraint on both the hypothalamus and pituitary gland, and results in elevated pituitary gonadotropin FSH, increased also by the hypothalamic gonadotropin-releasing hormone (GnRH). During the late menopause transition and a part of perimenopause, despite occasional episodes of normal cycling, women are exposed to periods of estrogen withdrawal, fewer ovulatory cycles, and prolonged hypogonadism, ultimately leading to the last menstrual period, after which is an elevated level of gonadotrophin secretion (only tonic, not phasic) [25]. During this phase, besides the low ovarian E2 and progesterone, there are productions of androgens and growth factors, which will decline in future years of postmenopause [26]. The ovaries are stimulated during menopausal transition and early postmenopausal years by gonadotropins, but the pulsatile GnRH pattern is different in different species before reproductive failure. It is a decrease of GnRH gene expression in many middle-aged rats [27], and an increase in perimenopausal rhesus monkeys [28]. Premature menopause/early menopause can be spontaneous or induced; after medical interventions such as chemotherapy/radiotherapy or surgery. The most common cause of premature/early menopause is bilateral oophorectomy with/without hysterectomy. Primary ovarian failure (POF) may be a cause of early/premature menopause, for ischemic stroke [29], as for all cardiovascular diseases risks [30], and these conditions were first described as a cause of neurological disturbances in different European, North American, and Japanese populations [21, 31–33]. Bilateral oophorectomy at premenopausal ages is inducing drops of E2 and testosterone levels, by 40–50%, and an abruptly rise in FSH levels, the levels of androgen being lower than in natural menopause at ages of 65 years, when women in normal or in premature/early menopause continue to have some levels of androgens [34]. As it is shown in the Table 1, the hazard ratios reached statistical significance in cases with bilateral oophorectomy: at the Genetic analyses demonstrated that the menstrual cycles acyclicity is accompanied by a rise in genes required for fatty acid metabolism, a decline of genes required for mitochondrial function, β-amyloid degradation, and neuroinflammation including increased number in microglia population in aging hippocampus [41], plus the shift of microglia activation with predominant production of inflammatory cytokines [42], and a higher basal level of comple- Neuroprotection in Perimenopausal Women http://dx.doi.org/10.5772/intechopen.74330 131 There are neuroimmune modulation differences in normal memory processes and memory dysregulation, in the roles of cytokines, astrocytes, and microglia in females and males [44]. These differences are from early development and differentiation of the brain [5], making women's brain inherently vulnerable to neurodegenerative diseases, to a higher risk of mild cognitive impairment and AD in advanced ages [45] (though not all studies are in agreement on this point, [1]), and non-neurodegenerative cognitive impairments fact that drive to the deleterious/beneficial consequences for estrogen therapy. The metabolic and neuroinflammatory changes are connected via redox regulation during normal brain aging, and may be There are new animal studies on female neuroaging, regarding the microglia involvement in neurogenesis [47], to innate immune system [48], being revealed the microglia sensome by direct RNA sequencing [49]. Molecular studies on mice aging [50] revealed a central role of gender in the transcriptomic response in hippocampal and cortex aging, demonstrating sexually divergent changes of neuroinflammation, mainly an increase of microglia-specific genes, and C1q protein expression of the complement system, in the activation of astrocytes, and in cytokine release and function in aging. C1qa induction is a driver of synapse loss with greater C1qa induction associated with poorer cognitive performance. It is considered that the agerelated changes in inflammatory hippocampal genes amplified in women after estrogen failure may contribute to sex differences in age-related neurological diseases. There are classes of genes in which inductions and reductions in gene expression are acting synergistically in The rise of microglia-specific genes in aging females is interrelated to a significant decrease in the activation of two pro-neurogenesis pathways evident in aging hippocampus: Notch1 and Presenilin 1 and 2 (PSEN1 and PSEN2) regulated genes [51]: Notch1 is necessary for neural stem cell maintenance [52], the PSEN1 expression regulates neuroprogenitor cell differentiation [53], and the defects in PSEN1 expression are associated with the manifestation of AD in old age [54]. Another change of neuroinflammatory genes in aging women is that of Tyrobp known as TREM2, as a causal regulator in microglia-associated changes in AD [55], and its The months/years of perimenopause represent an important moment during women's aging, when steroids and their receptors decline is evident in the hippocampal and cortical neurons, after estrogen exposure during the reproductive years. The estrogens decline is associated/acts proper mechanism in AD etiology is still being determined [56]. 3. Hypothesis on brain aging and neurodegeneration during ment cascade genes and interleukin 1 receptor-like 1 in women versus men [43]. predictive for later-life vulnerability to hypometabolic conditions of AD [46]. female aging hippocampus [50]. perimenopause Cases of cognitive impairment/dementia and Parkinson disease (PD) in women with unilateral (813) and bilateral (676) oophorectomy: For a nonmalignant disease, in Olmsted County, Minnesota (USA) during 1950–1987, followed up the death or the finish of study at 2001–2006 (Rocca et al. [32]). Table 1. Utero-ovarian surgery and neurological disturbances in premenopause. age < 34 years for dementia and <38 years for PD. Perimenopause is a "fragile" period in woman's life, comparable to the fragility of the adolescence, if we speak about "hormonal storms," but the hormonal pyramid is upside down, and more than these, the North American neurologists are considering perimenopause as a neurological transition state [35], because the characteristic symptoms regarding thermoregulation, sleep, circadian rhythms, and sensory processing are of neurological nature, besides the changes of cognitive function [36]. The central and peripheral hormonal changes in menopausal transition and perimenopause were assessed in many research centers from Western Europe [37], Australia [24, 38], North America [25], and besides these, the rat models on gene expression analyses demonstrated that there are two distinct aging programs: chronological and endocrine, regarding bioenergetic gene expression involved in brain metabolism and synaptic plasticity [39]. The endocrine transition marked by changing from regular to irregular menstrual cycles is characterized by the impairment of the energy metabolism, glucose hypometabolism, and chronic oxidative stress, which were demonstrated by gene expression in brain metabolism, mitochondrial function, and long-term potentiation. Rat model analysis on brain energetic metabolism in menopausal transition demonstrated that insulin/insulin-like growth factor 1 and adenosine monophosphate-activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator-1-alpha (AMPK/PGC1α) signaling pathways are upstream regulators [39], and these pathways suggest the critical role of E2 in neuronal survival. E2 stimulates the mitochondrial sequestration of Ca2+ and protects neurons against adverse consequences of excess cytoplasmic Ca2+ and subsequent dysregulation of Ca2+ homeostasis, with concomitant preservation of mitochondrial respiratory capacity [40]. Genetic analyses demonstrated that the menstrual cycles acyclicity is accompanied by a rise in genes required for fatty acid metabolism, a decline of genes required for mitochondrial function, β-amyloid degradation, and neuroinflammation including increased number in microglia population in aging hippocampus [41], plus the shift of microglia activation with predominant production of inflammatory cytokines [42], and a higher basal level of complement cascade genes and interleukin 1 receptor-like 1 in women versus men [43]. There are neuroimmune modulation differences in normal memory processes and memory dysregulation, in the roles of cytokines, astrocytes, and microglia in females and males [44]. These differences are from early development and differentiation of the brain [5], making women's brain inherently vulnerable to neurodegenerative diseases, to a higher risk of mild cognitive impairment and AD in advanced ages [45] (though not all studies are in agreement on this point, [1]), and non-neurodegenerative cognitive impairments fact that drive to the deleterious/beneficial consequences for estrogen therapy. The metabolic and neuroinflammatory changes are connected via redox regulation during normal brain aging, and may be predictive for later-life vulnerability to hypometabolic conditions of AD [46]. There are new animal studies on female neuroaging, regarding the microglia involvement in neurogenesis [47], to innate immune system [48], being revealed the microglia sensome by direct RNA sequencing [49]. Molecular studies on mice aging [50] revealed a central role of gender in the transcriptomic response in hippocampal and cortex aging, demonstrating sexually divergent changes of neuroinflammation, mainly an increase of microglia-specific genes, and C1q protein expression of the complement system, in the activation of astrocytes, and in cytokine release and function in aging. C1qa induction is a driver of synapse loss with greater C1qa induction associated with poorer cognitive performance. It is considered that the agerelated changes in inflammatory hippocampal genes amplified in women after estrogen failure may contribute to sex differences in age-related neurological diseases. There are classes of genes in which inductions and reductions in gene expression are acting synergistically in female aging hippocampus [50]. age < 34 years for dementia and <38 years for PD. Perimenopause is a "fragile" period in woman's life, comparable to the fragility of the adolescence, if we speak about "hormonal storms," but the hormonal pyramid is upside down, and more than these, the North American neurologists are considering perimenopause as a neurological transition state [35], because the characteristic symptoms regarding thermoregulation, sleep, circadian rhythms, and sensory Cases of cognitive impairment/dementia and Parkinson disease (PD) in women with unilateral (813) and bilateral (676) oophorectomy: For a nonmalignant disease, in Olmsted County, Minnesota (USA) during 1950–1987, followed up Age at surgery (years) Hazard ratio CI 95% P value <43 1.74 0.97–3.14 0.06 43–48 1.68 1.06–2.66 0.03 >48 1.09 0.74–1.61 0.66 <34 4.61 2.52–8.43 <0.0001 34–41 1.23 0.67–2.26 0.51 >41 1.50 1.05–2.13 0.03 <38 2.85 1.28–6.35 0.001 38–45 1.38 1.28–6.35 0.42 >45 1.38 0.92–3.03 0.09 The central and peripheral hormonal changes in menopausal transition and perimenopause were assessed in many research centers from Western Europe [37], Australia [24, 38], North America [25], and besides these, the rat models on gene expression analyses demonstrated that there are two distinct aging programs: chronological and endocrine, regarding bioener- The endocrine transition marked by changing from regular to irregular menstrual cycles is characterized by the impairment of the energy metabolism, glucose hypometabolism, and chronic oxidative stress, which were demonstrated by gene expression in brain metabolism, mitochondrial function, and long-term potentiation. Rat model analysis on brain energetic metabolism in menopausal transition demonstrated that insulin/insulin-like growth factor 1 and adenosine monophosphate-activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator-1-alpha (AMPK/PGC1α) signaling pathways are upstream regulators [39], and these pathways suggest the critical role of E2 in neuronal survival. E2 stimulates the mitochondrial sequestration of Ca2+ and protects neurons against adverse consequences of excess cytoplasmic Ca2+ and subsequent dysregulation of Ca2+ homeostasis, with processing are of neurological nature, besides the changes of cognitive function [36]. Adjusted odd ratio for dementia after unilateral oophorectomy 130 Sex Hormones in Neurodegenerative Processes and Diseases Adjusted odd ratio for dementia after bilateral oophorectomy Adjusted odd ratio for PD after bilateral oophorectomy the death or the finish of study at 2001–2006 (Rocca et al. [32]). Table 1. Utero-ovarian surgery and neurological disturbances in premenopause. getic gene expression involved in brain metabolism and synaptic plasticity [39]. concomitant preservation of mitochondrial respiratory capacity [40]. The rise of microglia-specific genes in aging females is interrelated to a significant decrease in the activation of two pro-neurogenesis pathways evident in aging hippocampus: Notch1 and Presenilin 1 and 2 (PSEN1 and PSEN2) regulated genes [51]: Notch1 is necessary for neural stem cell maintenance [52], the PSEN1 expression regulates neuroprogenitor cell differentiation [53], and the defects in PSEN1 expression are associated with the manifestation of AD in old age [54]. Another change of neuroinflammatory genes in aging women is that of Tyrobp known as TREM2, as a causal regulator in microglia-associated changes in AD [55], and its proper mechanism in AD etiology is still being determined [56]. ### 3. Hypothesis on brain aging and neurodegeneration during perimenopause The months/years of perimenopause represent an important moment during women's aging, when steroids and their receptors decline is evident in the hippocampal and cortical neurons, after estrogen exposure during the reproductive years. The estrogens decline is associated/acts synergic to other factors as hypertension, diabetes, hypoxia/obstructive sleep apnea, obesity, vitamin B12/folate deficiency, depression, and traumatic brain injury to promote different pathological mechanisms involved in brain aging, memory impairment, and AD. activation M1 (classical) to M2 (alternative) type [68] or it is a maladaptive microglia activation [69], or a shift from neuroprotection to neurotoxicity, underlining chronic neuroinflammation and parainflammation, which is different in women and men [70, 71]. The shift is connected to proinflammatory cytokines and oxidative-nitrosative stress, which plus elevated levels of complement pathway components and other immune factors plays a key pathophysiological role in promoting cognitive dysfunction by enhancing endothelin, Amyloid-β deposition, cerebral amyloid angiopathy, aberrant synapse elimination in the hippocampus Neuroprotection in Perimenopausal Women http://dx.doi.org/10.5772/intechopen.74330 133 AD is characterized by the loss of neurons and synapses from the cerebral cortex and certain subcortical regions of the temporal and parietal lobes, and parts of the frontal cortex and cingulated gyrus [73], and accumulation of plaque made up of small peptides called β-amiloid (also written as A-beta or Aβ). β-amyloid is a fragment from a larger protein called amyloid precursor protein (APP), a transmembrane protein that penetrates through the neuron's membrane. The Italian studies from Florence have demonstrated that estradiol is restoring in menopause the neuroprotective gene, seladin-1 (for SELective Alzheimer's Disease INdicator-1), or the gene DHCR24, which is downregulated in AD [74]. This gene inhibits the activation of caspase-3, a key modulator of apoptosis, and the gene encodes 3β-hydroxysterol, which catalyzes the conversion of desmosterol into cholesterol, and an appropriate amount of membrane cholesterol plays a pivotal role to protect nerve cells against Aβ toxicity and Microglia, a type of glial cell derived from myeloid precursors in the bone marrow that populate the CNS during development, as well as a brain resident innate immune cell, is the first line of defense in the CNS, as a monitor/sensor of neuronal activity in normal brain [77], protecting the local environment against invading pathogens, helping recovery from injury, and also in synapse pruning and neurodevelopment [78]. It is crucial in clearing debris, apoptotic/ necrotic cells, or products from necrotic cells, infiltration of infectious agents, mediating the brain's inflammatory and repair response to traumatic injury, stroke, or neurodegeneration [79]. It was suggested that age-dependent and senescence-driven impairments of microglia functions and responses play essential roles during onset and progression of neurodegenerative diseases as AD and PD, in which molecular changes on microglia senescence are similar [80]. The unique nature and developmental origin of microglia causing microglial selfrenewal and telomere shortening led to the hypothesis that these CNS-specific innate immune cells become senescent [81]. There are two important characteristics of human brain microglia: their heterogeneity observed in brain regions, and their different sensitivity to aging; the microglia from cortex, basal forebrain, and hippocampus are more sensible [81]. Microglia is activated from its normal state of a functionally "resting" resident immune cell of the CNS, and upon activation, microglia may proliferate and undergo a morphological transformation from a ramified to amoeboid appearance, and movement to sites of injury or stress can occur along with a release soluble immune mediators [82]. The activated microglia are functioning like a phagocyte or macrophage, having toll-like receptors (TLRs), that recognize specific molecular patterns as complement, mannose, scavenger, C-type lectin, nucleotidebinding oligomerization domain-like, and this specific action of microglia is called autophagy. [72], and blood-brain barrier disruption. counteracts the synthesis of Aβ in AD [75, 76]. The Californian and Australian neuroscientists had shown that chronic cerebral hypoperfusion deprives the brain from its two paramount trophic substances, oxygen and glucose, and consequently, the brain suffers from synaptic dysfunction and neuronal degeneration/loss, leading to both gray and white matter atrophy. The magnetic resonance imaging of the head used in the North American studies from Kronos Early Estrogen Prevention Study (KEEPS) showed a brain volume decrease with an average of 0.30–0.35% per year, and an increase of 3.59–3.73% in the ventricular volumes in the first 18 months of menopause [57], with a regional reduction of volume, which is more important in the hippocampus [58]. There are two hypotheses regarding neurodegeneration in brain aging, connected to low energy fuel supply, glucose hypometabolism and its complications for normal functioning [59], and microglia activation with associated secondary effect. In these hypothetical conditions, there are sexually divergent differences in gene expression in aging brain with comparing the number of gene expression changes in both males and females, and separating gene expression profiles based on up or downregulation. The first hypothesis regards to the deficiency in glucose availability and mitochondrial dysfunction well-known as hallmarks of brain aging, which are particularly accentuated in neurodegenerative disorders, and the shift from an aerobic glycolytic to a ketogenic phenotype of bioenergetic metabolism. The model on female rat brain aging revealed that bioenergetic decline is starting from perimenopausal transition, which is followed by the decrease of brain synaptic plasticity [39]. The mouse female transgenic model of familial AD revealed that ovariectomy induces a shift in fuel availability and metabolism in the hippocampus, with an increase of enzymes required for long-chain fatty acid and ketone body metabolism, to obtain brain energy [46, 60]. Glucose hypometabolism associated to cerebral hypoperfusion initiated with perimenopausal atherosclerosis [61], hypercholesterolemia, nitric oxide, and impairment of redox homeostasis is considered as the key pathophysiologic promoter of neurodegeneration [59], and the known differences in regional brain metabolism make some women prone to AD [62]. Posterior cingulated and prefrontal cortex, which closely resembles the hypometabolic profile of AD brains are the postmenopausal women's brain areas with reduced cerebral blood flow, with alteration of brain blood barrier glucose transport, and with significant decline in glucose metabolism [63]. It was demonstrated that brain aging is associated with a decrease of central insulin concentration [64–66], with an impairment of insulin receptor binding ability, resulting in an increase in deterioration of glucose homeostasis in the brain. Brain insulin resistance [67] is associated to peripheral insulin resistance–a typical feature of elder ages, associated to atherogenic dyslipidemia [65], and ET influences insulin resistance in medial prefrontal gyrus metabolism. The second hypothesis is focusing on neuroinflammation specifically after low estrogen levels, connected to the shift of microglia activation, with the changing rate of microglia after activation M1 (classical) to M2 (alternative) type [68] or it is a maladaptive microglia activation [69], or a shift from neuroprotection to neurotoxicity, underlining chronic neuroinflammation and parainflammation, which is different in women and men [70, 71]. The shift is connected to proinflammatory cytokines and oxidative-nitrosative stress, which plus elevated levels of complement pathway components and other immune factors plays a key pathophysiological role in promoting cognitive dysfunction by enhancing endothelin, Amyloid-β deposition, cerebral amyloid angiopathy, aberrant synapse elimination in the hippocampus [72], and blood-brain barrier disruption. synergic to other factors as hypertension, diabetes, hypoxia/obstructive sleep apnea, obesity, vitamin B12/folate deficiency, depression, and traumatic brain injury to promote different The Californian and Australian neuroscientists had shown that chronic cerebral hypoperfusion deprives the brain from its two paramount trophic substances, oxygen and glucose, and consequently, the brain suffers from synaptic dysfunction and neuronal degeneration/loss, leading to both gray and white matter atrophy. The magnetic resonance imaging of the head used in the North American studies from Kronos Early Estrogen Prevention Study (KEEPS) showed a brain volume decrease with an average of 0.30–0.35% per year, and an increase of 3.59–3.73% in the ventricular volumes in the first 18 months of menopause [57], with a There are two hypotheses regarding neurodegeneration in brain aging, connected to low energy fuel supply, glucose hypometabolism and its complications for normal functioning [59], and microglia activation with associated secondary effect. In these hypothetical conditions, there are sexually divergent differences in gene expression in aging brain with comparing the number of gene expression changes in both males and females, and separating gene The first hypothesis regards to the deficiency in glucose availability and mitochondrial dysfunction well-known as hallmarks of brain aging, which are particularly accentuated in neurodegenerative disorders, and the shift from an aerobic glycolytic to a ketogenic phenotype of bioenergetic metabolism. The model on female rat brain aging revealed that bioenergetic decline is starting from perimenopausal transition, which is followed by the decrease of brain synaptic plasticity [39]. The mouse female transgenic model of familial AD revealed that ovariectomy induces a shift in fuel availability and metabolism in the hippocampus, with an increase of enzymes required for long-chain fatty acid and ketone body metabolism, to obtain brain energy [46, 60]. Glucose hypometabolism associated to cerebral hypoperfusion initiated with perimenopausal atherosclerosis [61], hypercholesterolemia, nitric oxide, and impairment of redox homeostasis is considered as the key pathophysiologic promoter of neurodegeneration [59], and the known differences in regional brain metabolism make some Posterior cingulated and prefrontal cortex, which closely resembles the hypometabolic profile of AD brains are the postmenopausal women's brain areas with reduced cerebral blood flow, with alteration of brain blood barrier glucose transport, and with significant decline in It was demonstrated that brain aging is associated with a decrease of central insulin concentration [64–66], with an impairment of insulin receptor binding ability, resulting in an increase in deterioration of glucose homeostasis in the brain. Brain insulin resistance [67] is associated to peripheral insulin resistance–a typical feature of elder ages, associated to atherogenic dyslipidemia [65], and ET influences insulin resistance in medial prefrontal gyrus metabolism. The second hypothesis is focusing on neuroinflammation specifically after low estrogen levels, connected to the shift of microglia activation, with the changing rate of microglia after pathological mechanisms involved in brain aging, memory impairment, and AD. regional reduction of volume, which is more important in the hippocampus [58]. expression profiles based on up or downregulation. 132 Sex Hormones in Neurodegenerative Processes and Diseases women prone to AD [62]. glucose metabolism [63]. AD is characterized by the loss of neurons and synapses from the cerebral cortex and certain subcortical regions of the temporal and parietal lobes, and parts of the frontal cortex and cingulated gyrus [73], and accumulation of plaque made up of small peptides called β-amiloid (also written as A-beta or Aβ). β-amyloid is a fragment from a larger protein called amyloid precursor protein (APP), a transmembrane protein that penetrates through the neuron's membrane. The Italian studies from Florence have demonstrated that estradiol is restoring in menopause the neuroprotective gene, seladin-1 (for SELective Alzheimer's Disease INdicator-1), or the gene DHCR24, which is downregulated in AD [74]. This gene inhibits the activation of caspase-3, a key modulator of apoptosis, and the gene encodes 3β-hydroxysterol, which catalyzes the conversion of desmosterol into cholesterol, and an appropriate amount of membrane cholesterol plays a pivotal role to protect nerve cells against Aβ toxicity and counteracts the synthesis of Aβ in AD [75, 76]. Microglia, a type of glial cell derived from myeloid precursors in the bone marrow that populate the CNS during development, as well as a brain resident innate immune cell, is the first line of defense in the CNS, as a monitor/sensor of neuronal activity in normal brain [77], protecting the local environment against invading pathogens, helping recovery from injury, and also in synapse pruning and neurodevelopment [78]. It is crucial in clearing debris, apoptotic/ necrotic cells, or products from necrotic cells, infiltration of infectious agents, mediating the brain's inflammatory and repair response to traumatic injury, stroke, or neurodegeneration [79]. It was suggested that age-dependent and senescence-driven impairments of microglia functions and responses play essential roles during onset and progression of neurodegenerative diseases as AD and PD, in which molecular changes on microglia senescence are similar [80]. The unique nature and developmental origin of microglia causing microglial selfrenewal and telomere shortening led to the hypothesis that these CNS-specific innate immune cells become senescent [81]. There are two important characteristics of human brain microglia: their heterogeneity observed in brain regions, and their different sensitivity to aging; the microglia from cortex, basal forebrain, and hippocampus are more sensible [81]. Microglia is activated from its normal state of a functionally "resting" resident immune cell of the CNS, and upon activation, microglia may proliferate and undergo a morphological transformation from a ramified to amoeboid appearance, and movement to sites of injury or stress can occur along with a release soluble immune mediators [82]. The activated microglia are functioning like a phagocyte or macrophage, having toll-like receptors (TLRs), that recognize specific molecular patterns as complement, mannose, scavenger, C-type lectin, nucleotidebinding oligomerization domain-like, and this specific action of microglia is called autophagy. The autophagy is crucial for neuronal health and survival, the delivery of toxic molecules and organelles from neuronal apoptotic cells to microglia lysosomes may be acutely and/or chronically dysregulated by senescence, affecting phagocytosis and inflammation-innate immune functions in all age-associated neurodegenerative diseases [83]. There are two phenotypes of activated microglia: M1 (the cells become more cytotoxic by releasing additional pro-inflammatory cytokines- TNF, Il-1β, Il-6, and free radicals [84]), and M2, which becomes more anti-inflammatory, by secreting anti-inflammatory cytokines and neurotrophic factors and helps repair local damage [82]. The mouse model on AD is showing a distinct shift in activated microglia phenotypes, that occurs between the beginning of Aβ pathology (alternative phenotype), and advanced stages (classical phenotype), the latter may cause disease-associated neuron loss. In this context, there are comments/discussions on microglia: if it is a scapegoat, a saboteur, or something else. A multicenter research group has discovered the presence of microglia amylin receptors mediating Aβ inflammation and neurodegeneration on primary cultures of fetal human and rats microglia [84], these receptors being common to neurons and microglia. It was proposed a model of microglia activation for AD, and neuronal death, involving these receptors, microglia, neurons, inflammation, amyloid precursor protein, and Aβ (Figure 1). The amylin receptors are increasing as microglia responds to inflammatory triggers, such as lipopolysaccharide, resulting in microglia activation. The interaction of Aβ with amylin receptors of the activated microglia leads to increased production and release of cytokines, which act directly on neurons to produce cell death, with additionally increased production of Aβ via processing the amyloid precursor protein. The Aβ interacts with neurons and microglia amylin receptors to produce cell death [84]. The microglia activation is via the release of ATP, neurotransmitters, growth factors or cytokines, ion changes, special of Ca+2 in the CNS environment, or loss of inhibitor molecules displayed by healthy neurons, or when microglia cells encounter molecules not normally found in the healthy CNS, as blood clotting factors, intracellular constituents released by necrotic cells (hypomethylated mammalian DNA, RNA), externalized phosphatidylserine on apoptotic cells, immunoglobulin-antigen complexes, opsonizing complement, abnormally folded proteins or pathogen-related structures. When microglia activation occurs, the activation is correlated to the severity degree of the stressor, being recorded the disruptions of microglia functions causing synaptic dysfunction and excess synapse loss early in abnormalities of learning and memory [77]. Being a debate about the initiator from the two hypotheses: first, the bioenergetic hypothesis based on mitochondrial dysfunction, and the second on the microglia activation as the driving force for neuroinflammation, which is "a lesson learned from microglia depletion models" [85], there are multiple evidences that these abnormalities exacerbate each other, and these mechanistic diversities have cellular redox dysregulation as a common denominator and connector [86]. According to these, one may consider a metabolic inflammatory axis during brain aging and in neurodegenerative diseases [42]. In conditions of hypoglycemia, lactate can serve as an auxiliary fuel by metabolism of glycogen stores to generate glucose and subsequently lactate; some studies revealed that glial cells are likely to produce lactate in excess to its utilization by neurons [46]. Neurological symptoms that emerge during perimenopause are indicative of disruption in multiple estrogen-regulated systems, and affect multiple domains of cognitive and memory Figure 1. Model of neurodegeneration in AD proposed by Fu et al. [84]: Through the involvement microglia and neural amylin receptors in mediating the Aβ-induced neurodegeneration. Legend: The expression of amylin receptors of resting microglia, increased in response to inflammatory triggers like LPS, induces microglial cells activation. The interaction of Aβ with amylin receptors of the activated microglia leads to increased production and release of cytokines (TNF, Il-1β, and Il-6), which act directly on neurons to produce cell death and additionally augment the production of Aβ via processing of the amyloid precursor protein (APP). The Aβ, in turn, interacts with neuronal and microglial amylin receptors to produce cell death. Adapted from Fu et al. [84]. Open access to this article is distributed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. The creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data Neuroprotection in Perimenopausal Women http://dx.doi.org/10.5772/intechopen.74330 135 Estrogens (special E2) are appreciated as a master regulator of bioenergetic systems in the The hypothesis on estrogen action or "healthy-cell bias" hypothesis [87, 89] is similar to the understanding of the different cardiovascular protective/harmful effects of estrogens at different women ages—protective before 60 years and harmful after 65 years. The protective effect of E2 is altered in the presence of the APOE4 genotype, which alters the response of microglia functions [87]. body and brain [88]. made available in this article, unless otherwise stated. The autophagy is crucial for neuronal health and survival, the delivery of toxic molecules and organelles from neuronal apoptotic cells to microglia lysosomes may be acutely and/or chronically dysregulated by senescence, affecting phagocytosis and inflammation-innate immune functions in all age-associated neurodegenerative diseases [83]. There are two phenotypes of activated microglia: M1 (the cells become more cytotoxic by releasing additional pro-inflammatory cytokines- TNF, Il-1β, Il-6, and free radicals [84]), and M2, which becomes more anti-inflammatory, by secreting anti-inflammatory cytokines and neurotrophic factors and helps repair local damage [82]. The mouse model on AD is showing a distinct shift in activated microglia phenotypes, that occurs between the beginning of Aβ pathology (alternative phenotype), and advanced stages (classical phenotype), the latter may cause disease-associated neuron loss. In this context, there are comments/discussions on microglia: if it is a scapegoat, a saboteur, or something else. A multicenter research group has discovered the presence of microglia amylin receptors mediating Aβ inflammation and neurodegeneration on primary cultures of fetal human and rats microglia [84], these receptors being common to neurons and microglia. It was proposed a model of microglia activation for AD, and neuronal death, involving these receptors, microglia, neurons, inflammation, amyloid precursor protein, and Aβ (Figure 1). The amylin receptors are increasing as microglia responds to inflammatory triggers, such as lipopolysaccharide, resulting in microglia activation. The interaction of Aβ with amylin receptors of the activated microglia leads to increased production and release of cytokines, which act directly on neurons to produce cell death, with additionally increased production of Aβ via processing the amyloid precursor protein. The Aβ interacts with neurons and microglia The microglia activation is via the release of ATP, neurotransmitters, growth factors or cytokines, ion changes, special of Ca+2 in the CNS environment, or loss of inhibitor molecules displayed by healthy neurons, or when microglia cells encounter molecules not normally found in the healthy CNS, as blood clotting factors, intracellular constituents released by necrotic cells (hypomethylated mammalian DNA, RNA), externalized phosphatidylserine on apoptotic cells, immunoglobulin-antigen complexes, opsonizing complement, abnormally folded proteins or pathogen-related structures. When microglia activation occurs, the activation is correlated to the severity degree of the stressor, being recorded the disruptions of microglia functions causing synaptic dysfunction and excess synapse loss early in abnormalities of Being a debate about the initiator from the two hypotheses: first, the bioenergetic hypothesis based on mitochondrial dysfunction, and the second on the microglia activation as the driving force for neuroinflammation, which is "a lesson learned from microglia depletion models" [85], there are multiple evidences that these abnormalities exacerbate each other, and these mechanistic diversities have cellular redox dysregulation as a common denominator and connector [86]. According to these, one may consider a metabolic inflammatory axis during brain aging and in neurodegenerative diseases [42]. In conditions of hypoglycemia, lactate can serve as an auxiliary fuel by metabolism of glycogen stores to generate glucose and subsequently lactate; some studies revealed that glial cells are likely to produce lactate in amylin receptors to produce cell death [84]. 134 Sex Hormones in Neurodegenerative Processes and Diseases learning and memory [77]. excess to its utilization by neurons [46]. Figure 1. Model of neurodegeneration in AD proposed by Fu et al. [84]: Through the involvement microglia and neural amylin receptors in mediating the Aβ-induced neurodegeneration. Legend: The expression of amylin receptors of resting microglia, increased in response to inflammatory triggers like LPS, induces microglial cells activation. The interaction of Aβ with amylin receptors of the activated microglia leads to increased production and release of cytokines (TNF, Il-1β, and Il-6), which act directly on neurons to produce cell death and additionally augment the production of Aβ via processing of the amyloid precursor protein (APP). The Aβ, in turn, interacts with neuronal and microglial amylin receptors to produce cell death. Adapted from Fu et al. [84]. Open access to this article is distributed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. The creative commons public domain dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Neurological symptoms that emerge during perimenopause are indicative of disruption in multiple estrogen-regulated systems, and affect multiple domains of cognitive and memory functions [87]. Estrogens (special E2) are appreciated as a master regulator of bioenergetic systems in the body and brain [88]. The hypothesis on estrogen action or "healthy-cell bias" hypothesis [87, 89] is similar to the understanding of the different cardiovascular protective/harmful effects of estrogens at different women ages—protective before 60 years and harmful after 65 years. The protective effect of E2 is altered in the presence of the APOE4 genotype, which alters the response of microglia and macrophages to 17β-E2 [90], and this fact may be an explanation of some studies showing better results with ET on memory recall in women aged around 70 and 20 years postmenopausal, if women have not demonstrated memory impairment [91]. [2] Vina J, Lloret A. Why women have more Alzheimer's disease than men: Gender and mitochondrial toxicity of amyloid-beta peptide. Journal of Alzheimer's Disease. 2010; Neuroprotection in Perimenopausal Women http://dx.doi.org/10.5772/intechopen.74330 137 [3] Alzheimer's A. Alzheimer's disease facts and figures. Alzheimer's & Dementia. 2013; [4] Appelros P, Stegmayr B, Terent A. Sex differences in stroke epidemiology: A systematic [5] Pike CJ. Sex and the development of Alzheimer's disease. Journal of Neuroscience [6] Prince M, Wimo A, Prina M, et al. Alzheimer's Disease International. World Alzheimer Report 2015: The Global Impact of Dementia: An Analysis of Prevalence, Incidence, Cost [7] Marin R, Guerra B, Diaz M, et al. Estrogen activates classical and alternative mechanisms to orchestrate neuroprotection. Current Neurovascular Research. 2005 Oct;2(4):287-301 [8] Arnold AP, Lusis AJ. Understanding the sexome: Measuring and reporting sex differ- [9] Baron AM, Pike CJ. Sex hormones, aging, and Alzheimer's disease. Frontiers in Bio- [10] Henderson VW. Cognition and cognitive aging. Climacteric. 2007 Oct;10(Suppl 2):88-91 [11] Blacker D, Lee H, Muzikansky A, et al. Neuropsychological measures in normal individuals that predict subsequent cognitive decline. Archives of Neurology. 2007;64(6): [12] Levey J, Lah J, Goldstein F, Steenland K, Bliwise D. Mild cognitive impairment: An opportunity to identify patients at high risk for progression to Alzheimer's disease. [13] Gauthier S, Reisberg B, Zaudig M, Winblad B, et al. Mild cognitive impairment. Lancet [14] Yan Y, Cheng L, Chen X, Ai J, et al. Estrogen deficiency is associated with hippocampal morphological remodeling of early postmenopausal mice. Oncotarget. 2017 Mar [15] Zhang Y, Champagne N, Beitel KL, Goodyer GC, Trifiro M, LeBlanc A. Estrogen and androgen protection of human neurons against intracellular amyloid toxicity through [16] Kohannim O, Hua X, Hibar DP, et al. Boosting power for clinical trials using classifiers based on multiple biomarkers. Neurobiology of Aging. 2010;31(8):1429-1442 [17] Dye VR, Miller JK, Singer JE, Levine JA. Hormone replacement therapy and risk for neurodegenerative diseases. International Journal of Alzheimer's Disease. 2012:18. Jagannatha Rao KS, editor. Article ID 258454. DOI: doi.org/10.1155/2012/258454 heat shock protein 70. The Journal of Neuroscience. 2004;24(23):5315-5321 ences in gene systems. Endocrinology. 2012;153(6):2551-2555. DOI: 10.1210 20(Suppl 2):S527-S533. DOI: 10.3233 review. Stroke. 2009;40(4):1082-1090. DOI: 10.1161 Research. 2017 Jan 2;95(1-2):671-680. DOI: 10.1002 and Trend. https://www.alz.co.uk/research/world-report-2015 9(2):208-245. DOI:10.1016 science. 2012;4:976-997 2006;367:1262-1270 Clinical Therapeutics. 2006;28(7):991-1001 28;8(13):21892-21902. DOI: 10.18632 862-871 It was demonstrated how during reproductive ages the estrogen-induced signaling pathways in hippocampal and cortical neurons converge upon the mitochondria to enhance aerobic glycolysis coupled to the citric acid cycle, mitochondrial respiration, and ATP generation, and in senescence when estrogens are missing, it is a chronic oxidative stress due to the shift from an aerobic glycolytic to a ketogenic profile/phenotype/ [35, 60], and this shift is preceded by the early, already mentioned decline in glucose transport and metabolism [46]. In mouse model, the mitochondrial bioenergetic deficit precedes AD [92]. The estrogen decline in perimenopause is associated to the decline in mitochondria bioenergetics and together with the shift to ketogenetic profile are steps to Aβ depositions in AD [93, 94]. Hexokinase, the first rate limiting step in glycolysis, interacts with mitochondria and prevents mitochondria-mediated apoptosis and through this mechanism, is promoting survival in neurons and other cell types [95], but AD patients exhibit declined hexokinase activity in the brain, cerebral microvessels, leukocytes, and fibroblasts. Calcium dynamics play a pivotal and mandatory role in the estradiol-inducible cascade that leads to neurotrophic and neuroprotective benefit [89]. Dynamics of Ca2+ homeostasis are tightly regulated in healthy neurons and dysfunctional in degenerating neurons at elder ages. The emergence of glucose hypometabolism, microglia activation, and impaired synaptic function in brain provide plausible mechanisms of neurological symptoms of perimenopause and can be predictive of later-life vulnerability to hypometabolic conditions such as AD. The alteration in the bioenergetic profile of the brain in the months/years of perimenopause may be an explanation for the controversies on estrogen therapy/hormone therapy divergent outcomes, beneficial [18, 19] or harmful (WHI Memory Study) effects on neural health, on memory and cognition [46]. ### Author details Manuela Cristina Russu1 \* and Alexandra Cristina Antonescu2 \Address all correspondence to: manuela\[email protected] 1 "Dr. I. Cantacuzino" Clinic of Obstetrics and Gynecology, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania 2 Intromed Laboratories, Bucharest, Romania #### References* [1] McCarrey AC, An Y, Kitner-Triolo MH, Ferrucci L, Resnick SM. Sex differences in cognitive trajectories in clinically normal older adults. Psychology and Aging. 2016;31(2):166- 175. DOI: 10.1037 [2] Vina J, Lloret A. Why women have more Alzheimer's disease than men: Gender and mitochondrial toxicity of amyloid-beta peptide. Journal of Alzheimer's Disease. 2010; 20(Suppl 2):S527-S533. DOI: 10.3233 and macrophages to 17β-E2 [90], and this fact may be an explanation of some studies showing better results with ET on memory recall in women aged around 70 and 20 years postmeno- It was demonstrated how during reproductive ages the estrogen-induced signaling pathways in hippocampal and cortical neurons converge upon the mitochondria to enhance aerobic glycolysis coupled to the citric acid cycle, mitochondrial respiration, and ATP generation, and in senescence when estrogens are missing, it is a chronic oxidative stress due to the shift from an aerobic glycolytic to a ketogenic profile/phenotype/ [35, 60], and this shift is preceded by the early, already mentioned decline in glucose transport and metabolism [46]. In mouse model, the mitochondrial bioenergetic deficit precedes AD [92]. The estrogen decline in perimenopause is associated to the decline in mitochondria bioenergetics and together with the shift to ketogenetic profile are steps to Aβ depositions in AD [93, 94]. Hexokinase, the first rate limiting step in glycolysis, interacts with mitochondria and prevents mitochondria-mediated apoptosis and through this mechanism, is promoting survival in neurons and other cell types [95], but AD patients exhibit declined Calcium dynamics play a pivotal and mandatory role in the estradiol-inducible cascade that leads to neurotrophic and neuroprotective benefit [89]. Dynamics of Ca2+ homeostasis are tightly regulated in healthy neurons and dysfunctional in degenerating neurons at elder ages. The emergence of glucose hypometabolism, microglia activation, and impaired synaptic function in brain provide plausible mechanisms of neurological symptoms of perimenopause and can be predictive of later-life vulnerability to hypometabolic conditions such as AD. The alteration in the bioenergetic profile of the brain in the months/years of perimenopause may be an explanation for the controversies on estrogen therapy/hormone therapy divergent outcomes, beneficial [18, 19] or harmful (WHI Memory Study) effects on neural health, on memory and \* and Alexandra Cristina Antonescu2 1 "Dr. I. Cantacuzino" Clinic of Obstetrics and Gynecology, "Carol Davila" University of [1] McCarrey AC, An Y, Kitner-Triolo MH, Ferrucci L, Resnick SM. Sex differences in cognitive trajectories in clinically normal older adults. Psychology and Aging. 2016;31(2):166- \Address all correspondence to: manuela\[email protected] Medicine and Pharmacy, Bucharest, Romania 2 Intromed Laboratories, Bucharest, Romania pausal, if women have not demonstrated memory impairment [91]. 136 Sex Hormones in Neurodegenerative Processes and Diseases hexokinase activity in the brain, cerebral microvessels, leukocytes, and fibroblasts. cognition [46]. 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DOI: 10.1186/s12974-017-0972-9 New York Academy of Sciences. 2005;1052:57-74 Chapter 7 Provisional chapter Sex Hormones and Alzheimer's Disease Sex Hormones and Alzheimer's Disease DOI: 10.5772/intechopen.72561 Alzheimer's disease (AD) is the most common type of dementia and the most common neurodegenerative disorder of elderly. It is not an accelerated form of aging but it is characterized by distinct temporospatial brain pathological changes, including amyloid plaques accumulation, neurofibrillary tangles deposition, synaptic loss and neuronal death with gross brain atrophy. These changes result in persistent progressive memory and cognitive decline interfering with the usual daily activities. AD is a multifactorial disorder results from the interaction of genetic, epigenetic, environmental and lifestyle factors. Estrogen, progesterone and androgen effects are important building stones in AD pathogenesis, and their effect in brain modulation and development results in different gender susceptibility to the disease. These sex hormones whether gonadal or neurosteroids (synthesized locally in the brain) play important neuroprotective roles influencing the individual's vulnerability to AD development, rate of mild cognitive impairment (MCI)/AD conversion and speed of AD progression. Despite the little therapeutic implications of hormonal replacement therapy in AD treatment, yet this topic still represents a challenging hopeful way to construct a strategy for the development of personalized, Keywords: Alzheimer's disease, sex hormones, neurosteroids, estrogens, progesterone, Alzheimer's disease (AD) is the most common type of dementia and a key determinant of healthcare costs. It is an age-related neurodegenerative disorder, and due to increased people life expectancy, AD becomes one of the most burdensome threats to public health and a > © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. and reproduction in any medium, provided the original work is properly cited. Wafik Said Bahnasy, Yasser A. El-Heneedy and Wafik Said Bahnasy, Yasser A. El-Heneedy and Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.72561 gender-specific AD management. Ehab A. El-Seidy Abstract androgens 1. Introduction Ehab A. El-Seidy Provisional chapter ## Sex Hormones and Alzheimer's Disease Sex Hormones and Alzheimer's Disease Wafik Said Bahnasy, Yasser A. El-Heneedy and Ehab A. El-Seidy Ehab A. El-Seidy Additional information is available at the end of the chapter Wafik Said Bahnasy, Yasser A. El-Heneedy and Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.72561 #### Abstract Alzheimer's disease (AD) is the most common type of dementia and the most common neurodegenerative disorder of elderly. It is not an accelerated form of aging but it is characterized by distinct temporospatial brain pathological changes, including amyloid plaques accumulation, neurofibrillary tangles deposition, synaptic loss and neuronal death with gross brain atrophy. These changes result in persistent progressive memory and cognitive decline interfering with the usual daily activities. AD is a multifactorial disorder results from the interaction of genetic, epigenetic, environmental and lifestyle factors. Estrogen, progesterone and androgen effects are important building stones in AD pathogenesis, and their effect in brain modulation and development results in different gender susceptibility to the disease. These sex hormones whether gonadal or neurosteroids (synthesized locally in the brain) play important neuroprotective roles influencing the individual's vulnerability to AD development, rate of mild cognitive impairment (MCI)/AD conversion and speed of AD progression. Despite the little therapeutic implications of hormonal replacement therapy in AD treatment, yet this topic still represents a challenging hopeful way to construct a strategy for the development of personalized, gender-specific AD management. DOI: 10.5772/intechopen.72561 Keywords: Alzheimer's disease, sex hormones, neurosteroids, estrogens, progesterone, androgens #### 1. Introduction Alzheimer's disease (AD) is the most common type of dementia and a key determinant of healthcare costs. It is an age-related neurodegenerative disorder, and due to increased people life expectancy, AD becomes one of the most burdensome threats to public health and a Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons grand international research challenges. It is a system-specific brain disease affecting discrete neurons in a nearly consistent temporospatial pattern and is characterized by progressive memory decline and persistent cognitive impairment enough to interfere with the person's performance of the usual daily activities. 1.3.1. The amyloid hypothesis 1.3.2. Neurofibrillary tangles tangles and amyloid plaques. The amyloid cascade theory with the resultant extracellular amyloid plaque aggregation is the leading one for AD pathogenesis. Amyloid plaques are aggregates of amyloid beta (Aβ) peptide derived mainly from the cleavage of a transmembrane protein named amyloid precursor protein (APP) by the sequential action of two aspartyl protease enzymes, β- and γ-secretases (amyloidogenic pathway) in which the APP is firstly cleaved by β-secretase to soluble APP and residual C-terminal segment that is further digested by the γ-secretase to Aβ-40/42 segments. The insoluble Aβ aggregates start to appear 15–25 years prior to the onset of cognitive decline or tau pathology, and their formation is triggered by enhancement of the amyloidogenic pathway with increasing the pool of soluble Aβ production, which in turn aggregate to form monomeric, oligomeric, protofibrils and finally mature insoluble Aβ [10]. Under normal circumstances, the ratio of Aβ-42: Aβ-40 is 1:9 and increase in this ratio due to either aberrant production (increased γ-secretase activity) or clearance (abnormal microglial activities) is the cause of Aβ accumulation as the former has a high tendency to aggregate. The amyloid aggregates spark a sequence of events that lead to AD development including neuronal injury and synaptic loss. It is generally accepted that brain β-amyloid deposition is relatively diffuse, and there is non-linear correlation between the density of mature Aβ aggregate and severity of AD, which denotes that soluble Aβ oligomers per se are neurotoxic and cause synaptic Sex Hormones and Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.72561 147 Tau pathology, including NFTs, neuritic plaques and neuropil threads intraneural deposition is assumed to be the consequence of amyloid accumulation. NFTs are intraneural misfolded twisted paired helical filaments, which accumulate to form intracellular deposits composed of hyperphosphorylated tau protein that concentrates in the inner side of the cell membrane, but when the neurons die, NFT may migrate to other healthy or less affected neurons or may be found extracellular. Tau is essential for NMDA-dependent long-term potentiation and AMPAdependent long-term depression and acts as autophagy regulator by inhibiting histone deacetylase-6 enzyme. Thus, tauopathies result in marked synaptic disturbances and impaired selective autophagic clearance. Tau has more than 25 serine, threonine and tyrosine residue sites, which can be phosphorylated by specific protein kinases and phosphatases [13]. Genetic or acquired Figure 1. Photomicrography of Alzheimer's disease pathology using Bielschowsky stain demonstrating neurofibrillary dysfunction even in the absence of insoluble aggregate [11, 12]. #### 1.1. History The gender difference in the cognitive and neurobehavioral performance had been noticed since ancient time which may be the origin of the popular legend (men are from Mars, women are from Venus and had met here in Earth) [1]. The concept of age-related cognitive decline was well known since antiquity, which progressed through the ages till reached the term dementia. The link between female sex and dementia had also noticed since a very long period and this made Jean Etienne Esquirol put menstrual disorders and sequelae of delivery as direct causes of dementia in his book Des Maladies Mentales [2, 3]. In 25 November 1901, the German neurologist, Dr. Alois Alzheimer admitted a patient presented by recent cognitive decline to the public mental asylum in Frankfurt. Surprisingly, this first description was on a 51-year-old lady named Auguste Deter, who experienced marked memory decline, fear of people and became jealous of her husband in the last year preceded her admission in Frankfurt asylum by Dr. Alois Alzheimer and Dr. Hermann P. Nitsche. Later, the patient developed severe behavioral abnormalities, delusions, disorientation of time and place, hallucinations and severe language difficulties. In 1906, Auguste died and her autopsy revealed gross brain atrophy and microscopically increased silver staining by using Bielschowsky method, which later named amyloid plaques and neurofibrillary tangles (NFTs) [4]. #### 1.2. Epidemiology Dementia affects about 47 million people worldwide, and this number is expected to double every 20 years due to increased life expectancy. AD is the leading cause of dementia accounting for about 30% of early onset cases before the age of 50 years and 60–80% of late onset ones either as pure or mixed form [5]. It is one of the commonest causes of prolonged disability in old age, the sixth cause of death in USA globally and the fifth cause for seniors above the age of 65. Old age is the most important AD risk with estimated prevalence of 3% in people aged 65–74 years, 17% between 75 and 84 years and 32% in those >85 years [6, 7]. AD disproportionately affects both sexes, with females have 2–3 times higher incidence of AD than males of the same age. The age-specific risk of developing AD is almost twofold greater in women than men, 17.2% vs. 9.1% at 65 years and 28.5% vs. 10.2% at 75 years. The incidence of amnestic mild cognitive impairment (MCI) is equal both in male and female, denoting that females take shorter MCI/AD transitional state with rapid conversion to manifest AD [5, 8, 9]. #### 1.3. Pathology AD is not an accelerated form of aging but it is characterized by distinct cellular and molecular pathological changes, including amyloid plaque deposition, NFTs accumulations, synaptic loss and neuronal death with gross brain atrophy. #### 1.3.1. The amyloid hypothesis grand international research challenges. It is a system-specific brain disease affecting discrete neurons in a nearly consistent temporospatial pattern and is characterized by progressive memory decline and persistent cognitive impairment enough to interfere with the person's The gender difference in the cognitive and neurobehavioral performance had been noticed since ancient time which may be the origin of the popular legend (men are from Mars, women are from Venus and had met here in Earth) [1]. The concept of age-related cognitive decline was well known since antiquity, which progressed through the ages till reached the term dementia. The link between female sex and dementia had also noticed since a very long period and this made Jean Etienne Esquirol put menstrual disorders and sequelae of delivery as direct causes of dementia in his book Des Maladies Mentales [2, 3]. In 25 November 1901, the German neurologist, Dr. Alois Alzheimer admitted a patient presented by recent cognitive decline to the public mental asylum in Frankfurt. Surprisingly, this first description was on a 51-year-old lady named Auguste Deter, who experienced marked memory decline, fear of people and became jealous of her husband in the last year preceded her admission in Frankfurt asylum by Dr. Alois Alzheimer and Dr. Hermann P. Nitsche. Later, the patient developed severe behavioral abnormalities, delusions, disorientation of time and place, hallucinations and severe language difficulties. In 1906, Auguste died and her autopsy revealed gross brain atrophy and microscopically increased silver staining by using Bielschowsky method, which later named amyloid plaques and neurofibrillary tangles (NFTs) [4]. 65–74 years, 17% between 75 and 84 years and 32% in those >85 years [6, 7]. loss and neuronal death with gross brain atrophy. Dementia affects about 47 million people worldwide, and this number is expected to double every 20 years due to increased life expectancy. AD is the leading cause of dementia accounting for about 30% of early onset cases before the age of 50 years and 60–80% of late onset ones either as pure or mixed form [5]. It is one of the commonest causes of prolonged disability in old age, the sixth cause of death in USA globally and the fifth cause for seniors above the age of 65. Old age is the most important AD risk with estimated prevalence of 3% in people aged AD disproportionately affects both sexes, with females have 2–3 times higher incidence of AD than males of the same age. The age-specific risk of developing AD is almost twofold greater in women than men, 17.2% vs. 9.1% at 65 years and 28.5% vs. 10.2% at 75 years. The incidence of amnestic mild cognitive impairment (MCI) is equal both in male and female, denoting that females take shorter MCI/AD transitional state with rapid conversion to manifest AD [5, 8, 9]. AD is not an accelerated form of aging but it is characterized by distinct cellular and molecular pathological changes, including amyloid plaque deposition, NFTs accumulations, synaptic performance of the usual daily activities. 146 Sex Hormones in Neurodegenerative Processes and Diseases 1.1. History 1.2. Epidemiology 1.3. Pathology The amyloid cascade theory with the resultant extracellular amyloid plaque aggregation is the leading one for AD pathogenesis. Amyloid plaques are aggregates of amyloid beta (Aβ) peptide derived mainly from the cleavage of a transmembrane protein named amyloid precursor protein (APP) by the sequential action of two aspartyl protease enzymes, β- and γ-secretases (amyloidogenic pathway) in which the APP is firstly cleaved by β-secretase to soluble APP and residual C-terminal segment that is further digested by the γ-secretase to Aβ-40/42 segments. The insoluble Aβ aggregates start to appear 15–25 years prior to the onset of cognitive decline or tau pathology, and their formation is triggered by enhancement of the amyloidogenic pathway with increasing the pool of soluble Aβ production, which in turn aggregate to form monomeric, oligomeric, protofibrils and finally mature insoluble Aβ [10]. Under normal circumstances, the ratio of Aβ-42: Aβ-40 is 1:9 and increase in this ratio due to either aberrant production (increased γ-secretase activity) or clearance (abnormal microglial activities) is the cause of Aβ accumulation as the former has a high tendency to aggregate. The amyloid aggregates spark a sequence of events that lead to AD development including neuronal injury and synaptic loss. It is generally accepted that brain β-amyloid deposition is relatively diffuse, and there is non-linear correlation between the density of mature Aβ aggregate and severity of AD, which denotes that soluble Aβ oligomers per se are neurotoxic and cause synaptic dysfunction even in the absence of insoluble aggregate [11, 12]. #### 1.3.2. Neurofibrillary tangles Tau pathology, including NFTs, neuritic plaques and neuropil threads intraneural deposition is assumed to be the consequence of amyloid accumulation. NFTs are intraneural misfolded twisted paired helical filaments, which accumulate to form intracellular deposits composed of hyperphosphorylated tau protein that concentrates in the inner side of the cell membrane, but when the neurons die, NFT may migrate to other healthy or less affected neurons or may be found extracellular. Tau is essential for NMDA-dependent long-term potentiation and AMPAdependent long-term depression and acts as autophagy regulator by inhibiting histone deacetylase-6 enzyme. Thus, tauopathies result in marked synaptic disturbances and impaired selective autophagic clearance. Tau has more than 25 serine, threonine and tyrosine residue sites, which can be phosphorylated by specific protein kinases and phosphatases [13]. Genetic or acquired Figure 1. Photomicrography of Alzheimer's disease pathology using Bielschowsky stain demonstrating neurofibrillary tangles and amyloid plaques. induced dysfunctions result in tau hyperphosphorylation, misfolding and fibrillar formation ending in NFTs deposition. On the other hand, tau dephosphorylation is regulated by protein phosphatase 2A enzyme, which activity is impaired in AD. NFTs accumulation starts several years after Aβ deposition but still before AD clinical manifestations and its accumulation dense and distribution is directly proportional with the severity of AD cognitive decline. NFTs deposition usually follows a stepwise progression typically starting in the transentorhinal cortex, the entorhinal cortex, hippocampus, medial temporal cortex and lastly other areas of the neocortex [14, 15] (Figure 1). of aging and why AD progression increases after cerebral ischemic events like stroke and transient ischemic attacks. Cerebral amyloid angiopathy (CAA) is present in more than 75% of autopsy confirmed AD brains especially in mixed AD/vascular dementia where the vascular risk factors predominate. Cerebral microvascular compromise is more common among subjects having the APOE4 allele making them at increased risk of AD development. CAA represents imbalanced Aβ production and clearance with consequent deposition within the basement membrane of the leptomeningeal vessels, intracerebral arteries and arterioles, and Sex Hormones and Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.72561 149 Amyloid deposition in and around the blood vessel wall impairs its endothelial integrity and disturbs the BBB leading to Aβ trapping in the CSF and its diminished clearance to the venous circulation. At the same time, CAA disrupts the microvascular homeostasis leading to chronic cerebral parenchymal hypoperfusion with focal ischemia, microinfarcts, release of inflammatory mediators, oxygen-free radicals, loss of nitric oxide bioavailability and mitochondrial dysfunction. The net result is neurotoxicity, reduced neural plasticity, neural apoptosis and synaptic loss [21]. Sex hormone receptors are heavily expressed in the cerebral blood vessels and exert very important actions to keep the vascular integrity and prevent chronic ischemic hypoperfusion through promoting endothelial relaxation by increasing the production/activity of nitric oxide and prostacyclin and at the same time prevent vascular smooth muscle contraction by inhibiting intracellular Ca2+ influx and antagonize the actions of protein kinases [22]. Synaptic failure is an important factor in the cognitive manifestations of AD before manifest neuronal loss takes place. The neurochemical changes in AD include extensive serotonergic denervation in the hippocampus and neocortex, depletion of the cholinergic neurons in the basal forebrain, loss of >70% of noradrenergic locus coeruleus neurons, reduction of dopamine, dopamine metabolites and dopamine receptors, histaminergic tuberomammillary nucleus degeneration and impaired melatonin secretion and action in the pineal body and Glutamate is a non-essential amino acid but it is one of the most important excitatory synaptic neurotransmitter as most of the CNS myelinated axons are glutamatergic. AD patients show aberrant increase in extracellular glutamate, which enhances tau pathology and enhances glutamate receptors expressed oligodendroglia to transport tau from one brain area to another leading to AD spatial progression. At the same time, there is a reciprocal relationship between glutamate and Aβ as soluble amyloid oligomers as well as insoluble Aβ deposits increases the extracellular glutamate concentration resulting in AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) and NMDA (N-methyl-d-aspartate) receptors dysfunction, disturbed synaptic pruning and impaired synaptic plasticity with promotion of long-term synaptic depression and inhibition of long-term synaptic potentiation leading to cognitive decline especially memory domain. At the same time, NMDA receptor inhibition promotes amyloidogenic γ-secretase activities and inhibits non-amyloidogenic α-secretase with the resultant increase in Aβ production and accumulation and vice versa. Many studies revealed protective effects of sex hormones against glutamate-induced neurotoxicity through inhibition of glutamate less frequently in capillaries and veins [20]. 1.3.5. Monoaminergic and cholinergic abnormalities suprachiasmatic hypothalamic nucleus, respectively [23]. #### 1.3.3. Microglia and neuroinflammation It is well known that the density of β-amyloid deposition is not proportional with the severity of AD cognitive decline making the amyloid hypothesis alone is not sufficient to explain the whole AD pathological cascade and in turn the possible role of additional pathogenetic factors, including neuroinflammation and vascular amyloidosis. The neuroinflammatory theory was supposed after identification of activated microglia within the vicinity of the amyloid plaques, which number was proportional with the size of the plaques. At the same time, several microglial expressed genes were associated with AD predisposition, including TREM2, CD33, CR1, CLU, CD2AP, EPHA1, ABCA7 and INPP5D. Under normal circumstances, microglial activities are modulated by several neuroimmune regulatory proteins, including insulin-like growth factor-1, brain-derived neurotrophic factor, transforming growth factor-b and nerve growth factor, which help in slowdown and resolving the inflammatory process [16]. Microglia have no role in Aβ production; however, they act as Aβ scavengers as they play major roles in its clearance either directly through phagocytosis or indirectly via the secretion of several enzymes, including insulin degrading enzyme, neprilysin, matrix metalloproteinase-9 and plasminogen. At the same time, microglia regulate synaptic network remodeling (synaptic pruning) and neural circuit maintenance [17]. In AD, chronic reactivation and excessive proliferation of microglia result in the production of inflammatory mediators, including reactive oxygen species, interleukin-1, interferon-γ and tumor necrosis factor-alpha. This imbalanced microglial function results in aberrant synaptic pruning, pathological synaptic stripping, neuronal loss, enhancing the endothelial response to hypoxia with impaired blood-brain barrier (BBB) stability, disturbed Aβ clearance, increased levels of phosphorylated tau protein, promoting NFTs accumulation and, consequently, cognitive decline. Microglia also transport amyloid and tau from one brain area to another; thus, they play a major role in spatial AD progression. Microglia are candidate for the action of sex hormones, and they express abundant sex hormone receptors. These receptors modulate microglial activities producing potent anti-inflammatory actions that resist AD development and progression [18, 19]. #### 1.3.4. Vascular theory Diabetes, hypertension, smoking and heart diseases are associated with increased risk of AD. This concept resulted in the emergence of the AD vascular theory, which can explain why aging is the major risk of AD as vascular dysfunction is considered as a universal feature of aging and why AD progression increases after cerebral ischemic events like stroke and transient ischemic attacks. Cerebral amyloid angiopathy (CAA) is present in more than 75% of autopsy confirmed AD brains especially in mixed AD/vascular dementia where the vascular risk factors predominate. Cerebral microvascular compromise is more common among subjects having the APOE4 allele making them at increased risk of AD development. CAA represents imbalanced Aβ production and clearance with consequent deposition within the basement membrane of the leptomeningeal vessels, intracerebral arteries and arterioles, and less frequently in capillaries and veins [20]. Amyloid deposition in and around the blood vessel wall impairs its endothelial integrity and disturbs the BBB leading to Aβ trapping in the CSF and its diminished clearance to the venous circulation. At the same time, CAA disrupts the microvascular homeostasis leading to chronic cerebral parenchymal hypoperfusion with focal ischemia, microinfarcts, release of inflammatory mediators, oxygen-free radicals, loss of nitric oxide bioavailability and mitochondrial dysfunction. The net result is neurotoxicity, reduced neural plasticity, neural apoptosis and synaptic loss [21]. Sex hormone receptors are heavily expressed in the cerebral blood vessels and exert very important actions to keep the vascular integrity and prevent chronic ischemic hypoperfusion through promoting endothelial relaxation by increasing the production/activity of nitric oxide and prostacyclin and at the same time prevent vascular smooth muscle contraction by inhibiting intracellular Ca2+ influx and antagonize the actions of protein kinases [22]. #### 1.3.5. Monoaminergic and cholinergic abnormalities induced dysfunctions result in tau hyperphosphorylation, misfolding and fibrillar formation ending in NFTs deposition. On the other hand, tau dephosphorylation is regulated by protein phosphatase 2A enzyme, which activity is impaired in AD. NFTs accumulation starts several years after Aβ deposition but still before AD clinical manifestations and its accumulation dense and distribution is directly proportional with the severity of AD cognitive decline. NFTs deposition usually follows a stepwise progression typically starting in the transentorhinal cortex, the entorhinal cortex, hippocampus, medial temporal cortex and lastly other areas of the neocortex It is well known that the density of β-amyloid deposition is not proportional with the severity of AD cognitive decline making the amyloid hypothesis alone is not sufficient to explain the whole AD pathological cascade and in turn the possible role of additional pathogenetic factors, including neuroinflammation and vascular amyloidosis. The neuroinflammatory theory was supposed after identification of activated microglia within the vicinity of the amyloid plaques, which number was proportional with the size of the plaques. At the same time, several microglial expressed genes were associated with AD predisposition, including TREM2, CD33, CR1, CLU, CD2AP, EPHA1, ABCA7 and INPP5D. Under normal circumstances, microglial activities are modulated by several neuroimmune regulatory proteins, including insulin-like growth factor-1, brain-derived neurotrophic factor, transforming growth factor-b and nerve growth factor, which help in slowdown and resolving the inflammatory process [16]. (synaptic pruning) and neural circuit maintenance [17]. Microglia have no role in Aβ production; however, they act as Aβ scavengers as they play major roles in its clearance either directly through phagocytosis or indirectly via the secretion of several enzymes, including insulin degrading enzyme, neprilysin, matrix metalloproteinase-9 and plasminogen. At the same time, microglia regulate synaptic network remodeling In AD, chronic reactivation and excessive proliferation of microglia result in the production of inflammatory mediators, including reactive oxygen species, interleukin-1, interferon-γ and tumor necrosis factor-alpha. This imbalanced microglial function results in aberrant synaptic pruning, pathological synaptic stripping, neuronal loss, enhancing the endothelial response to hypoxia with impaired blood-brain barrier (BBB) stability, disturbed Aβ clearance, increased levels of phosphorylated tau protein, promoting NFTs accumulation and, consequently, cognitive decline. Microglia also transport amyloid and tau from one brain area to another; thus, they play a major role in spatial AD progression. Microglia are candidate for the action of sex hormones, and they express abundant sex hormone receptors. These receptors modulate microglial activities producing potent anti-inflammatory actions that resist AD development Diabetes, hypertension, smoking and heart diseases are associated with increased risk of AD. This concept resulted in the emergence of the AD vascular theory, which can explain why aging is the major risk of AD as vascular dysfunction is considered as a universal feature [14, 15] (Figure 1). and progression [18, 19]. 1.3.4. Vascular theory 1.3.3. Microglia and neuroinflammation 148 Sex Hormones in Neurodegenerative Processes and Diseases Synaptic failure is an important factor in the cognitive manifestations of AD before manifest neuronal loss takes place. The neurochemical changes in AD include extensive serotonergic denervation in the hippocampus and neocortex, depletion of the cholinergic neurons in the basal forebrain, loss of >70% of noradrenergic locus coeruleus neurons, reduction of dopamine, dopamine metabolites and dopamine receptors, histaminergic tuberomammillary nucleus degeneration and impaired melatonin secretion and action in the pineal body and suprachiasmatic hypothalamic nucleus, respectively [23]. Glutamate is a non-essential amino acid but it is one of the most important excitatory synaptic neurotransmitter as most of the CNS myelinated axons are glutamatergic. AD patients show aberrant increase in extracellular glutamate, which enhances tau pathology and enhances glutamate receptors expressed oligodendroglia to transport tau from one brain area to another leading to AD spatial progression. At the same time, there is a reciprocal relationship between glutamate and Aβ as soluble amyloid oligomers as well as insoluble Aβ deposits increases the extracellular glutamate concentration resulting in AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) and NMDA (N-methyl-d-aspartate) receptors dysfunction, disturbed synaptic pruning and impaired synaptic plasticity with promotion of long-term synaptic depression and inhibition of long-term synaptic potentiation leading to cognitive decline especially memory domain. At the same time, NMDA receptor inhibition promotes amyloidogenic γ-secretase activities and inhibits non-amyloidogenic α-secretase with the resultant increase in Aβ production and accumulation and vice versa. Many studies revealed protective effects of sex hormones against glutamate-induced neurotoxicity through inhibition of glutamate release by reducing the activities of lactate dehydrogenase, inhibiting intracellular Ca2+ influx, exerting antioxidant action and enhancing mitogen-activated protein kinase action [24, 25]. usually followed by other cognitive domains declines which vary according to the pattern of cortical progression, including apathy, loss of interest in hobbies, sleep disturbances, impaired spatial and temporal navigations, inability to solve problems due to executive dysfunction, behavioral changes, difficulty in using common instruments due to apraxia, Sex Hormones and Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.72561 151 The AD cognitive decline is usually preceded by a period of mild cognitive impairment (MCI) in which the individual retains his usual daily activities but has subnormal performance in cognitive neuropsychological testing. Many researchers reported a prodromal stage of subjective cognitive decline in which the person experiences worsening in his memory and/or cognitive performance despite the normal objective performance in standardized cognitive neuropsychological tests [30]. Atypical AD types start by nonamnestic manifestations but have the same pathological hallmarks include logopenic aphasia type, dysexecutive type, parietal dominant and frontal dominant atrophy sub- It is generally accepted that AD pathological changes begin decades before the appearance of dementia symptoms and this leads to the introduction of the term preclinical AD, which is defined as biomarker evidences of AD pathological changes in a cognitively healthy individual. The current challenge is to develop reliable biomarkers for early pre-dementia AD diagnosis to maintain longer patients' independence and prepare the floor for the discovery of disease modifying agents including hormonal replacement therapy before irreversible neu- The ADNI-2 (Alzheimer Disease Neuroimaging Initiative-2) established CSF biomarkers include reduced CSF Aβ-42 and elevated total and phosphorylated tau-181, which are very accurate in prediction of MCI/AD conversion with 85% sensitivity and 90% specificity. Novel but still non-approved CSF biomarkers include high Aβ oligomers and neurogranin Figure 2. Brain MRI of a 56 years old female with early Alzheimer Disease showing medial temporal atrophy in T1 axial section (left), decreased right entorhinal cortex volume (middle) and right hippocampus volume (right) in coronal 3D language difficulties, incontinence and high dependency on others [7, 11]. types [31, 32]. levels [33]. 1.6. Investigations and biomarkers ral damage takes place [11]. spoiled gradient magnetic resonance images. #### 1.4. Genetics and epigenetics of AD Genetic predisposition to AD is very complex although positive family history is a common patients' finding. The rare early onset AD constitutes less than 1% of cases and often transmitted as autosomal dominant and fully penetrant inheritance. Common affected genes include APP (genes encoding γ-secretase complex), presenelin-1 (PSNL1) and presenelin-2 (PSNL2) gene mutation in chromosomes 21, 14 and 1, respectively. Overexpression of these genes results in increased production of the highly hydrophobic fibrillogenic longer Aβ-42 and on the expense of the relatively shorter Aβ-40 [23]. In late onset AD, apolipoprotein E series, especially APOE4, is the major genetic risk as >60% of AD patients harbor at least one APOE4 allele. APOE is a lipid-binding cholesterol transporter protein essential for maintenance of myelin and neuronal membranes, synaptogenesis and dendritic reorganization. Three APOE isoforms exist in humans: APOE2, APOE3 and APOE4. Heterozygous and homozygous APOE4 are at increased risk of significantly lower age of AD onset and higher rate of AD development by about 4 and 15 folds than other allele types. At the same time, males with APOE4 are more liable to develop MCI than others. APOE4 allele expression interacts with the sex hormones leading to increased risk of AD in women than men of the same age. APOE4 expression results in decreased soluble APPα/Aβ ratio, reduced Sirtuin T1 expression (NAD+-dependent deacetylases that attenuate amyloidogenic), triggered tau phosphorylation and induced neuronal apoptosis [26]. Many studies revealed that people with a rare missense mutation (rs75932628-T) in the gene encoding TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) are at increased risk of developing AD 2–3 folds than others possibly due to reduced clearing abilities of their microglia to Aβ and apoptotic cells [10]. At the same time, other studies suggested a possible role of epigenetic changes and aberrantly expressed micro-RNAs (miRNAs) in the pathogenesis of AD through disturbing neurogenesis, synaptic plasticity, synaptogenesis and neuronal network preservation as well as enhancing Aβ production and neuroinflammation [27]. Both epigenetics and miRNAs are influenced by sex hormone receptor activation, and they are potentially versatile and adaptive, which gives a challenging hope for novel therapeutic approaches in AD management. The epigenetics represents alterations in genetic functions without changing DNA sequence and constitutes an interface of genetic/ environmental factors interplay, e.g. DNA methylation, histone modifications, non-coding RNAs regulation and higher order chromatin remodeling [28]. MiRNAs are 18–22 nucleotide long, non-coding RNAs that are involved in post-transcriptional suppression of gene expression [29]. #### 1.5. Clinical signs and symptoms Dementia of Alzheimer's type typically presents by episodic memory impairment, which gradually progresses to interfere with the activities of daily living. Memory impairment is usually followed by other cognitive domains declines which vary according to the pattern of cortical progression, including apathy, loss of interest in hobbies, sleep disturbances, impaired spatial and temporal navigations, inability to solve problems due to executive dysfunction, behavioral changes, difficulty in using common instruments due to apraxia, language difficulties, incontinence and high dependency on others [7, 11]. The AD cognitive decline is usually preceded by a period of mild cognitive impairment (MCI) in which the individual retains his usual daily activities but has subnormal performance in cognitive neuropsychological testing. Many researchers reported a prodromal stage of subjective cognitive decline in which the person experiences worsening in his memory and/or cognitive performance despite the normal objective performance in standardized cognitive neuropsychological tests [30]. Atypical AD types start by nonamnestic manifestations but have the same pathological hallmarks include logopenic aphasia type, dysexecutive type, parietal dominant and frontal dominant atrophy subtypes [31, 32]. #### 1.6. Investigations and biomarkers release by reducing the activities of lactate dehydrogenase, inhibiting intracellular Ca2+ influx, exerting antioxidant action and enhancing mitogen-activated protein kinase action [24, 25]. Genetic predisposition to AD is very complex although positive family history is a common patients' finding. The rare early onset AD constitutes less than 1% of cases and often transmitted as autosomal dominant and fully penetrant inheritance. Common affected genes include APP (genes encoding γ-secretase complex), presenelin-1 (PSNL1) and presenelin-2 (PSNL2) gene mutation in chromosomes 21, 14 and 1, respectively. Overexpression of these genes results in increased production of the highly hydrophobic fibrillogenic longer Aβ-42 and on In late onset AD, apolipoprotein E series, especially APOE4, is the major genetic risk as >60% of AD patients harbor at least one APOE4 allele. APOE is a lipid-binding cholesterol transporter protein essential for maintenance of myelin and neuronal membranes, synaptogenesis and dendritic reorganization. Three APOE isoforms exist in humans: APOE2, APOE3 and APOE4. Heterozygous and homozygous APOE4 are at increased risk of significantly lower age of AD onset and higher rate of AD development by about 4 and 15 folds than other allele types. At the same time, males with APOE4 are more liable to develop MCI than others. APOE4 allele expression interacts with the sex hormones leading to increased risk of AD in women than men of the same age. APOE4 expression results in decreased soluble APPα/Aβ ratio, reduced Sirtuin T1 expression (NAD+-dependent deacetylases that attenuate amyloido- Many studies revealed that people with a rare missense mutation (rs75932628-T) in the gene encoding TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) are at increased risk of developing AD 2–3 folds than others possibly due to reduced clearing abilities of their microglia to Aβ and apoptotic cells [10]. At the same time, other studies suggested a possible role of epigenetic changes and aberrantly expressed micro-RNAs (miRNAs) in the pathogenesis of AD through disturbing neurogenesis, synaptic plasticity, synaptogenesis and neuronal network preservation as well as enhancing Aβ production and neuroinflammation [27]. Both epigenetics and miRNAs are influenced by sex hormone receptor activation, and they are potentially versatile and adaptive, which gives a challenging hope for novel therapeutic approaches in AD management. The epigenetics represents alterations in genetic functions without changing DNA sequence and constitutes an interface of genetic/ environmental factors interplay, e.g. DNA methylation, histone modifications, non-coding RNAs regulation and higher order chromatin remodeling [28]. MiRNAs are 18–22 nucleotide long, non-coding RNAs that are involved in post-transcriptional suppression of gene Dementia of Alzheimer's type typically presents by episodic memory impairment, which gradually progresses to interfere with the activities of daily living. Memory impairment is genic), triggered tau phosphorylation and induced neuronal apoptosis [26]. 1.4. Genetics and epigenetics of AD 150 Sex Hormones in Neurodegenerative Processes and Diseases expression [29]. 1.5. Clinical signs and symptoms the expense of the relatively shorter Aβ-40 [23]. It is generally accepted that AD pathological changes begin decades before the appearance of dementia symptoms and this leads to the introduction of the term preclinical AD, which is defined as biomarker evidences of AD pathological changes in a cognitively healthy individual. The current challenge is to develop reliable biomarkers for early pre-dementia AD diagnosis to maintain longer patients' independence and prepare the floor for the discovery of disease modifying agents including hormonal replacement therapy before irreversible neural damage takes place [11]. The ADNI-2 (Alzheimer Disease Neuroimaging Initiative-2) established CSF biomarkers include reduced CSF Aβ-42 and elevated total and phosphorylated tau-181, which are very accurate in prediction of MCI/AD conversion with 85% sensitivity and 90% specificity. Novel but still non-approved CSF biomarkers include high Aβ oligomers and neurogranin levels [33]. Figure 2. Brain MRI of a 56 years old female with early Alzheimer Disease showing medial temporal atrophy in T1 axial section (left), decreased right entorhinal cortex volume (middle) and right hippocampus volume (right) in coronal 3D spoiled gradient magnetic resonance images. Blood biomarkers include high plasma homocysteine, high serum angiotensin converting enzyme activities and low plasma levels of the obesity-related hormone leptin [20]. Other ongoing research biomarkers include prostate-specific antigen complexed to α1-antichymotrypsin, pancreatic prohormone, clusterin and fetuin B [34]. 2.2. Estrogen actions in normal cognition secreted only during pregnancy) [41]. ation [46]. aspartate (NMDA) receptor-mediated mechanism [43, 44]. Estrogen is the primary female sex hormone, which regulates fundamental physiological processes in both reproductive and nonreproductive organs, including the CNS. The brain can synthesize estrogens (neuroestrogens) either by the aromatization of androgens or via a series of enzymatic steps from the precursor of all steroids, cholesterol. This neuroestrogen plays major roles in sexual differentiation of brain in both male and female. Four natural types of estrogens exist: estrone (E1; a weak estrogen and the main postmenopausal type), estradiol (E2; the most potent endogenous estrogen and the main type during the reproductive age), estriol (E3; very weak estrogen and hardly detected in non-pregnant females) and estetrol (E4; Sex Hormones and Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.72561 153 Estrogens can cross the cell membrane lipid bilayer to bind to the estrogen receptors (ER), which are of two types: nuclear and membrane ER. The nuclear ER are either ERα or ERβ, which are responsible for the estrogen genomic action through regulation of various transcriptional gene expression mechanisms. The brain contains both types of nuclear ER, which are abundant in the hippocampus, pyramidal cells and glial tissue [42]. The membrane estrogen receptors (mERs) are G protein-coupled and ligand-gated ion channels, including GPER1 (previously known as GPR30), ER-X and Gq-mER, which are responsible for the rapid nongenomic actions of estrogen that is initiated within minutes after estrogen administration (estrogen neurotransmitter actions) due to recruitment and activation of kinase-dependent signaling pathways. Membrane ER are abundant in the neocortex and their activation results in increased activity of nitric oxide synthase and Ca2+ influx to the cells through N-methyl-d- The cellular mechanisms underlying estrogen CNS actions are still uncertain due to the different estrogen expression in both sexes and in different brain areas, but it is generally accepted that estrogens usually promote neurogenesis, exert neuroprotective actions and support neuronal survival by antiapoptotic action, stimulating nerve growth factor and brain-derived neurotrophic factor [45]. Estrogens also improve neuronal plasticity especially in the hippocampus, increase cerebral blood flow by enhancing endothelial derived nitric oxide and prostacyclin pathways, regulate neural mitochondrial functions (both types of ER are expressed in the mitochondria) especially in stressful conditions by stimulating anti-apoptotic proteins and decrease free radical production. At the same time, estrogen exerts anti-inflammatory actions by reducing the expression of astrocyte to chemokines, promoting the maturation of oligodendrocyte precursor cells and improve their ability for CNS repair, which enhances the growth and differentiation of axons and dendrites and prevents axonal loss and demyelin- Estrogens also have well-documented direct cognitive and behavioral actions, and their postmenopausal depletion been associated with cognitive decline and increased risk of AD. The rapid estrogen non-genomic actions are important for hippocampal memory consolidation and hippocampal-dependent spatial navigation memory and improve learning performance, novel objects recognition and object placement tasks when administered before the cognitive tests. At the same time, estrogens improve choline acetyltransferase activity, promote serotonergic neuronal function and stimulate dopamine release in the caudate, prefrontal cortex, nucleus accumbens and dorsal raphe nucleus, which in turn enhance age-related learning and memory declines [36, 47]. The imaging biomarkers that can predict MCI/AD conversion are usually directed to measure the neural and synaptic densities in the commonly affected cortical areas. In volumetric MRI, manual and/or automated techniques can detect hippocampus and entorhinal cortex atrophy with concomitant dilatation of the temporal horns of the lateral ventricle. Other early neuroimaging biomarkers include task-free functional MRI (measures network failure quotient), diffusion tensor imaging (DTI) MRI, SPECT, FDG-PET, amyloid PET and tau PET [35] (Figure 2). ### 2. Sex hormones in normal cognition Better keep progestogen as it is a wider term than progesterone and this is clarified in the section of progesterone are synthesized from cholesterol by the action of aromatase enzyme, and they play important roles in shaping the neural functions and behavior throughout all stages of human life. The sex steroid hormones are potent regulators of neuronal survival and function in multiple CNS regions during normal development, aging and in some neurodegenerative disorders including AD. In aging individuals, low levels of gonadal sex hormones are associated with decline in neurogenesis especially in the hippocampus with the resultant age-dependent memory decline and executive function difficulties [36]. #### 2.1. Gender cognitive variability Over a long time, obvious sexual dimorphism in adult human brain was observed with females harbor larger frontal and medial paralimbic cortices, while males exhibit bigger medial frontal cortex, amygdala and hypothalamic volumes. This gender difference is mainly due to variability in sex chromosome and sex hormone neuronal action. Genetic studies revealed that X-chromosome carry genes which expressions enhance visuospatial, executive and/or social cognitive tasks, whereas genes on Y-chromosome are more responsible for behavioral sexual differentiation [37]. At the same time, there is different gender cognitive performance starting during the early neonatal period and persists throughout human survival. This sex difference may be the base of the striking variable susceptibilities to various cognitive disorders in men and women [38]. Under normal circumstances, there is non-significant sex difference in global cognitive performance, but generally, males are better in mathematics and 3D spatial tests, while females are superior in autobiographic, episodic memory and verbal tests. Regarding spatial navigation, males perform better in allocentric strategy (world-centered object-to-object spatial relations) but females excel in egocentric navigation (self-centered subject-to-object spatial relations). This different cognitive performance is universal and evident in humans and animals which weaken the suggestion that environmental factors or gendered socialization are the causes of these gender variations [39, 40]. #### 2.2. Estrogen actions in normal cognition Blood biomarkers include high plasma homocysteine, high serum angiotensin converting enzyme activities and low plasma levels of the obesity-related hormone leptin [20]. Other ongoing research biomarkers include prostate-specific antigen complexed to α1-antichymotrypsin, The imaging biomarkers that can predict MCI/AD conversion are usually directed to measure the neural and synaptic densities in the commonly affected cortical areas. In volumetric MRI, manual and/or automated techniques can detect hippocampus and entorhinal cortex atrophy with concomitant dilatation of the temporal horns of the lateral ventricle. Other early neuroimaging biomarkers include task-free functional MRI (measures network failure quotient), diffusion tensor imaging (DTI) MRI, SPECT, FDG-PET, amyloid PET and tau PET Better keep progestogen as it is a wider term than progesterone and this is clarified in the section of progesterone are synthesized from cholesterol by the action of aromatase enzyme, and they play important roles in shaping the neural functions and behavior throughout all stages of human life. The sex steroid hormones are potent regulators of neuronal survival and function in multiple CNS regions during normal development, aging and in some neurodegenerative disorders including AD. In aging individuals, low levels of gonadal sex hormones are associated with decline in neurogenesis especially in the hippocampus with the resultant Over a long time, obvious sexual dimorphism in adult human brain was observed with females harbor larger frontal and medial paralimbic cortices, while males exhibit bigger medial frontal cortex, amygdala and hypothalamic volumes. This gender difference is mainly due to variability in sex chromosome and sex hormone neuronal action. Genetic studies revealed that X-chromosome carry genes which expressions enhance visuospatial, executive and/or social cognitive tasks, whereas genes on Y-chromosome are more responsible for behavioral sexual At the same time, there is different gender cognitive performance starting during the early neonatal period and persists throughout human survival. This sex difference may be the base of the striking variable susceptibilities to various cognitive disorders in men and women [38]. Under normal circumstances, there is non-significant sex difference in global cognitive performance, but generally, males are better in mathematics and 3D spatial tests, while females are superior in autobiographic, episodic memory and verbal tests. Regarding spatial navigation, males perform better in allocentric strategy (world-centered object-to-object spatial relations) but females excel in egocentric navigation (self-centered subject-to-object spatial relations). This different cognitive performance is universal and evident in humans and animals which weaken the suggestion that environmental factors or gendered socialization are the causes of age-dependent memory decline and executive function difficulties [36]. pancreatic prohormone, clusterin and fetuin B [34]. 152 Sex Hormones in Neurodegenerative Processes and Diseases 2. Sex hormones in normal cognition 2.1. Gender cognitive variability these gender variations [39, 40]. differentiation [37]. [35] (Figure 2). Estrogen is the primary female sex hormone, which regulates fundamental physiological processes in both reproductive and nonreproductive organs, including the CNS. The brain can synthesize estrogens (neuroestrogens) either by the aromatization of androgens or via a series of enzymatic steps from the precursor of all steroids, cholesterol. This neuroestrogen plays major roles in sexual differentiation of brain in both male and female. Four natural types of estrogens exist: estrone (E1; a weak estrogen and the main postmenopausal type), estradiol (E2; the most potent endogenous estrogen and the main type during the reproductive age), estriol (E3; very weak estrogen and hardly detected in non-pregnant females) and estetrol (E4; secreted only during pregnancy) [41]. Estrogens can cross the cell membrane lipid bilayer to bind to the estrogen receptors (ER), which are of two types: nuclear and membrane ER. The nuclear ER are either ERα or ERβ, which are responsible for the estrogen genomic action through regulation of various transcriptional gene expression mechanisms. The brain contains both types of nuclear ER, which are abundant in the hippocampus, pyramidal cells and glial tissue [42]. The membrane estrogen receptors (mERs) are G protein-coupled and ligand-gated ion channels, including GPER1 (previously known as GPR30), ER-X and Gq-mER, which are responsible for the rapid nongenomic actions of estrogen that is initiated within minutes after estrogen administration (estrogen neurotransmitter actions) due to recruitment and activation of kinase-dependent signaling pathways. Membrane ER are abundant in the neocortex and their activation results in increased activity of nitric oxide synthase and Ca2+ influx to the cells through N-methyl-daspartate (NMDA) receptor-mediated mechanism [43, 44]. The cellular mechanisms underlying estrogen CNS actions are still uncertain due to the different estrogen expression in both sexes and in different brain areas, but it is generally accepted that estrogens usually promote neurogenesis, exert neuroprotective actions and support neuronal survival by antiapoptotic action, stimulating nerve growth factor and brain-derived neurotrophic factor [45]. Estrogens also improve neuronal plasticity especially in the hippocampus, increase cerebral blood flow by enhancing endothelial derived nitric oxide and prostacyclin pathways, regulate neural mitochondrial functions (both types of ER are expressed in the mitochondria) especially in stressful conditions by stimulating anti-apoptotic proteins and decrease free radical production. At the same time, estrogen exerts anti-inflammatory actions by reducing the expression of astrocyte to chemokines, promoting the maturation of oligodendrocyte precursor cells and improve their ability for CNS repair, which enhances the growth and differentiation of axons and dendrites and prevents axonal loss and demyelination [46]. Estrogens also have well-documented direct cognitive and behavioral actions, and their postmenopausal depletion been associated with cognitive decline and increased risk of AD. The rapid estrogen non-genomic actions are important for hippocampal memory consolidation and hippocampal-dependent spatial navigation memory and improve learning performance, novel objects recognition and object placement tasks when administered before the cognitive tests. At the same time, estrogens improve choline acetyltransferase activity, promote serotonergic neuronal function and stimulate dopamine release in the caudate, prefrontal cortex, nucleus accumbens and dorsal raphe nucleus, which in turn enhance age-related learning and memory declines [36, 47]. #### 2.3. Progesterone actions in normal cognition Progesterone is a steroid hormone and it is most active natural progestogen, synthesized in the gonads, placenta, adrenal glands and CNS (neurosteroids). It has many reproductive and non-reproductive functions, including regulation of a wide range of brain functions [48]. Progesterone has a lipophilic structure, which can cross the cell membrane to interact with its specific intracellular progesterone receptors (PRs) expressed throughout the brain without sex difference with special higher expression in the hypothalamus, hippocampus, frontal cortex, medial amygdaloid nucleus, norepinephrine neurons of the nucleus tractus solitaries and cerebellum. Progesterone exerts its CNS actions through regulation of gene expression, modulation of neurotransmitter systems and epigenetic actions as well as enhancing estrogen actions [49]. important roles in cognitive function regulation through promoting neuroprotection (anti-glutamate action) and neurogenesis, improving neuronal survival and anti-apoptotic effect (regulating mitochondrial genome activities and suppressing reactive oxygen species), modulating hippocampal synaptic plasticity, enhancing remyelination and exerting anti-inflammatory action by regulating astrocytic and oligodendrocytic activities [58]. Beside the delayed genomic effects of androgens, non-genomic rapid actions are mediated by trans-membrane G-proteincoupled ARs, which stimulations increase the intracellular Ca2+ influx and result in improved Sex Hormones and Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.72561 155 Androgens especially anabolic androgen steroids (AAS) are not always neurobehaviorally beneficial, and their short-term use results in aggressive and manic behaviors, whereas their long-term use is associated with impairment of decision making, behavioral flexibility, cogni- The neurocognitive actions of sex hormones are not simple but several factors may interact to control their beneficial effects including the inter-balance between their levels as well as the age and sex of the individual. In pregnancy, simultaneous increase in progesterone and estrogens results in impaired mood and decreased memory [63]. At the same time, sex hormones seem to play their major neuromodulatory action in early person's prepubertal life with subsequent decrease in the neuronal sensitivity to their actions with advancement of age. Some studies revealed that prepubertal sex hormones have permanent effects in individual's behaviors and cognition including spatial abilities. Early pubertal testosterone administration to gonadectomized male Syrian hamsters resulted in their attaining adult mating behaviors while administration in late puberty did not give the same results denoting that neurons are highly sensitive to the organizational effect of sex hormones at certain age with decreased sensitivity later after passage of this time [64–66]. On the other hand, brain estrogen expression was reduced in adult female mice previously exposed to stress in adolescence but not in early adulthood which concludes that adolescent stresses suppress estrogen activities and interfere with its organizational actions to attain adult mating behavior. From these results, we can conclude that the individual's susceptibility to many neurodegenerative disorders including AD may be attained since early life and we may not be able to reverse it easily later [67, 68]. Alzheimer's disease is a heterogenous disorder with multiple variants and wide variety of manifestations, which result from the interactions between multiple etiological factors, including genetic, epigenetic, environmental and lifestyle factors. Neuronal action of sex hormones represents one of the well-defined AD pathogenetic factors and may represent a hope to understand the biology of sex-dependent variability in AD predisposition and in turn leads inhibitory avoidance task, spatial learning and memory performance [59, 60]. tive control and spatial memory [61, 62]. 3. Brain responses variability to sex hormones 4. Sex hormones in MCI and Alzheimer's disease to the development of personalized, gender-specific AD management. PRs are either nuclear type (PR-A and PR-B), transmembrane PR (7TMPRβ) or membraneassociated 25-Dx PR (PGRMC1). Nuclear PRs are ligand inducible transcription factors that regulate target genes expression and play important roles in sexual brain differentiation, reproductive behavior, neuroprotection, neurogenesis, Schwan cell activities and their myelination programs, proliferation of neural progenitor cells and the release of the brainderived neurotrophic factor important for cell differentiation and survival. Progesterone also has anti-inflammatory actions through regulation of the activities of astrocytes, microglia and oligodendrocytes [50, 51]. Transmembrane PRs are G protein-coupled receptors responsible for the rapid action of progesterone, and when activated, they block the activity of adenylyl cyclase, including enhancement of mitochondrial functions and regulation of cell viability. At the same time, progesterone alters dopaminergic and GABAergic system activities in many brain regions mainly the hippocampus, amygdala and fusiform gyrus enhancing the memory and learning performances [52, 53]. #### 2.4. Androgen actions in normal cognition Androgens are very important sex steroids that exert cognitive functions in both males and females as they not only regulate the CNS development but also help to maintain its proper function from infancy to adulthood [54]. It is generally accepted that androgens play a pivotal role in cognitive performance and their depletion or signaling inhibition (in normal aging or anti-androgen hormonal therapy in cancer prostate) results in dysfunction in androgenresponsive tissues, including the brain and consequently deleterious cognitive impairment. At the same time, discontinuation of anti-androgen in cancer therapy restores cognitive performance especially verbal memory [55]. The CNS action of androgens is mediated either directly through stimulation of androgen receptors (ARs) (nuclear receptors regulating target genes expression at transcriptional level) or indirectly after conversion to estrogen by the action of aromatase enzyme. ARs are highly expressed in the septum pellucidum, stria terminalis, preoptic area, ventromedial hypothalamus and cerebellum where they regulate the sexual reproductive behaviors [56, 57]. Neuroandrogens production had detected in the hippocampus where they modulate the hippocampal structure specifically CA1 and CA3 areas. In the medial amygdala and prefrontal cortex, androgens exert important roles in cognitive function regulation through promoting neuroprotection (anti-glutamate action) and neurogenesis, improving neuronal survival and anti-apoptotic effect (regulating mitochondrial genome activities and suppressing reactive oxygen species), modulating hippocampal synaptic plasticity, enhancing remyelination and exerting anti-inflammatory action by regulating astrocytic and oligodendrocytic activities [58]. Beside the delayed genomic effects of androgens, non-genomic rapid actions are mediated by trans-membrane G-proteincoupled ARs, which stimulations increase the intracellular Ca2+ influx and result in improved inhibitory avoidance task, spatial learning and memory performance [59, 60]. Androgens especially anabolic androgen steroids (AAS) are not always neurobehaviorally beneficial, and their short-term use results in aggressive and manic behaviors, whereas their long-term use is associated with impairment of decision making, behavioral flexibility, cognitive control and spatial memory [61, 62]. ### 3. Brain responses variability to sex hormones 2.3. Progesterone actions in normal cognition 154 Sex Hormones in Neurodegenerative Processes and Diseases oligodendrocytes [50, 51]. performances [52, 53]. 2.4. Androgen actions in normal cognition formance especially verbal memory [55]. Progesterone is a steroid hormone and it is most active natural progestogen, synthesized in the gonads, placenta, adrenal glands and CNS (neurosteroids). It has many reproductive and non-reproductive functions, including regulation of a wide range of brain functions [48]. Progesterone has a lipophilic structure, which can cross the cell membrane to interact with its specific intracellular progesterone receptors (PRs) expressed throughout the brain without sex difference with special higher expression in the hypothalamus, hippocampus, frontal cortex, medial amygdaloid nucleus, norepinephrine neurons of the nucleus tractus solitaries and cerebellum. Progesterone exerts its CNS actions through regulation of gene expression, modulation of neurotransmitter systems and epigenetic actions as well as enhancing estrogen actions [49]. PRs are either nuclear type (PR-A and PR-B), transmembrane PR (7TMPRβ) or membraneassociated 25-Dx PR (PGRMC1). Nuclear PRs are ligand inducible transcription factors that regulate target genes expression and play important roles in sexual brain differentiation, reproductive behavior, neuroprotection, neurogenesis, Schwan cell activities and their myelination programs, proliferation of neural progenitor cells and the release of the brainderived neurotrophic factor important for cell differentiation and survival. Progesterone also has anti-inflammatory actions through regulation of the activities of astrocytes, microglia and Transmembrane PRs are G protein-coupled receptors responsible for the rapid action of progesterone, and when activated, they block the activity of adenylyl cyclase, including enhancement of mitochondrial functions and regulation of cell viability. At the same time, progesterone alters dopaminergic and GABAergic system activities in many brain regions mainly the hippocampus, amygdala and fusiform gyrus enhancing the memory and learning Androgens are very important sex steroids that exert cognitive functions in both males and females as they not only regulate the CNS development but also help to maintain its proper function from infancy to adulthood [54]. It is generally accepted that androgens play a pivotal role in cognitive performance and their depletion or signaling inhibition (in normal aging or anti-androgen hormonal therapy in cancer prostate) results in dysfunction in androgenresponsive tissues, including the brain and consequently deleterious cognitive impairment. At the same time, discontinuation of anti-androgen in cancer therapy restores cognitive per- The CNS action of androgens is mediated either directly through stimulation of androgen receptors (ARs) (nuclear receptors regulating target genes expression at transcriptional level) or indirectly after conversion to estrogen by the action of aromatase enzyme. ARs are highly expressed in the septum pellucidum, stria terminalis, preoptic area, ventromedial hypothalamus and cerebellum where they regulate the sexual reproductive behaviors [56, 57]. Neuroandrogens production had detected in the hippocampus where they modulate the hippocampal structure specifically CA1 and CA3 areas. In the medial amygdala and prefrontal cortex, androgens exert The neurocognitive actions of sex hormones are not simple but several factors may interact to control their beneficial effects including the inter-balance between their levels as well as the age and sex of the individual. In pregnancy, simultaneous increase in progesterone and estrogens results in impaired mood and decreased memory [63]. At the same time, sex hormones seem to play their major neuromodulatory action in early person's prepubertal life with subsequent decrease in the neuronal sensitivity to their actions with advancement of age. Some studies revealed that prepubertal sex hormones have permanent effects in individual's behaviors and cognition including spatial abilities. Early pubertal testosterone administration to gonadectomized male Syrian hamsters resulted in their attaining adult mating behaviors while administration in late puberty did not give the same results denoting that neurons are highly sensitive to the organizational effect of sex hormones at certain age with decreased sensitivity later after passage of this time [64–66]. On the other hand, brain estrogen expression was reduced in adult female mice previously exposed to stress in adolescence but not in early adulthood which concludes that adolescent stresses suppress estrogen activities and interfere with its organizational actions to attain adult mating behavior. From these results, we can conclude that the individual's susceptibility to many neurodegenerative disorders including AD may be attained since early life and we may not be able to reverse it easily later [67, 68]. #### 4. Sex hormones in MCI and Alzheimer's disease Alzheimer's disease is a heterogenous disorder with multiple variants and wide variety of manifestations, which result from the interactions between multiple etiological factors, including genetic, epigenetic, environmental and lifestyle factors. Neuronal action of sex hormones represents one of the well-defined AD pathogenetic factors and may represent a hope to understand the biology of sex-dependent variability in AD predisposition and in turn leads to the development of personalized, gender-specific AD management. #### 4.1. Sex hormones and MCI The concept of MCI had received much attention nowadays for early detection of those candidates to AD conversion which open a gate for future disease modifying agents including hormonal therapy before irreversible neuronal damages take place. MCI is a clinical condition lies between normal aging and dementia in which the cognitive dysfunctions are greater than expected for age but is not severe enough to significantly interfere with the daily life or warrant the diagnosis of dementia [8, 69]. MCI is classified to amnestic (am-MCI) and non-amnestic (nam-MCI) types. In the former, memory impairment is the dominating manifestation, and in the latter, non-memory cognitive domain is the affected one (language, attention, executive function, visual-spatial). Amnestic MCI is termed multiple domain if another cognitive domain is affected, whereas nam-MCI becomes multiple domain if more than one cognitive non-memory domain is affected. People with am-MCI are more liable to develop AD [11, 70]. network [75]. Estrogens also promote Aβ clearance by stimulation of microglial Aβ phagocytosis and enzymes involved in Aβ degradation, including metalloproteases-2 and -9, insulin- Sex Hormones and Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.72561 157 At the same time, estrogens exert anti-AD actions by increasing dendritic spine densities, promoting synaptogenesis, inhibiting the neurotoxic effects of oxidized low-density lipoproteins and glutamate, improving mitochondrial functions and enhancing the hippocampal cholinergic neurotransmitter system. Estrogens also regulate the epigenetic DNA methylation and miRNAs biogenesis especially in the hippocampus and thus master the genes expressions both transcriptionally and post-transcriptionally, which in turn play pivotal roles in enhancement of neuroprotection and prevention of neurodegeneration. Estrogen actions seem to be age dependent with obvious dysregulation in old-aged people. At the same time, estrogens have neuroprotective actions through increasing the expression of antiapoptotic Bcl-xL and Bcl-w and suppressing the expression of proapoptotic Bim, which lead to prevention of neu- Women are at increased risk of AD due to age-related sharp decline in sex steroid hormones and spending a large proportion of their life in the postmenopausal period because of increased their life longevity with the resultant prolonged hypoestrogenic state and its negative neurological consequences. Studies showed that postmenopausal women with AD had lower estradiol (E2) and estrone (E1) levels in both blood and CSF compared to normal controls. Moreover, female CSF-E2 level is positively correlated with CSF-Aβ level and cerebral glucose metabolism in the left hippocampus PET scan, which denotes that they are at increased risk to develop AD. In short-term studies, transdermal estrogen administration is associated with increased attention, verbal and visual memories; however, long-term studies failed to slow down AD progression or adding benefits to rivastigmine therapy in postmenopausal women [47, 81]. The neuroprotective effect of estrogen is not the same in both sexes as brain E1 and E2 levels have no relations with Aβ accumulation in males pointing to different gender expression of sex steroid hormones. At the same time, estrogen administration in male to female cross sex subjects results in significant decreases in the hippocampal volume despite producing neurogenesis, which means that the estrogen AD risk prevention in males is negligible [82, 83] (Figure 3). Studies in progesterone neuroprotective actions against AD predisposition are not so plenty like those on estrogens but despite this, it is generally accepted that progesterone has a direct neuroprotective action while its indirect actions against AD development by regulating the neuroestrogen effects are matter of controversy as in some studies, progesterone enhances estradiol neuroprotective actions and in others antagonizes them beside reducing the cerebral The direct progesterone protecting actions against AD development and/or progression include regulation of β-amyloid metabolism by reducing Aβ production and decreasing the pool of soluble Aβ by enhancement of the non-amyloidogenic α-secretase pathway, decreasing Aβ accumulation through modulation of γ-secretases activities and increasing Aβ degrading enzyme and neprilysin [76]. ronal loss from Aβ toxicity [77–80]. 4.2.2. Progesterone and Alzheimer's disease blood flow [84]. The prevalence of am-MCI is about 8.5–25.9 per 1000 of general population and 10–20% of those above the age of 60 years; 10–15% of am-MCI persons will develop AD compared to 1–2% of nam-MCI people. Several clinical and biochemical markers had been studied to be used as predictors of MCI/AD progression. In general, women have a higher prevalence of nam-MCI but most metanalytic studies showed non-significant gender difference in the prevalence of am-MCI, which means that women take shorter time to convert from am-MCI to manifest AD [71, 72]. #### 4.2. Sex hormones and Alzheimer's disease One of the most common observations associated with AD onset is decreased levels of sex hormones, including estrogens, progesterone and androgens, pointing the potential role of these hormones in AD pathogenesis and the possible benefits of their targeting in AD management strategies. #### 4.2.1. Estrogens and Alzheimer's disease Estrogen neuroprotective actions in AD are well documented by decades of researches showing that women used estrogen supplements or those with late menopause are at significantly decreased risk of AD development. On the other hand, early menopause because of increased sex hormone-binding globulin is associated with higher risk of AD in later life [73]. Estrogens exert anti-AD actions through different mechanisms including inhibition of tau deposition and Aβ accumulation. The former action is exerted through inhibition of tau hyperphosphorylation and promotion of tau dephosphorylation in an estrogen receptor-dependent mode through inhibition of protein kinases and promotion of protein phosphatase 2A enzyme activities, respectively [13, 74]. Estrogens inhibit Aβ accumulation by several mechanisms, one of which is decreasing Aβ production by enhancement of non-amyloidogenic APP pathway through activation of α-secretase enzyme that cleaves APP to soluble APP-α peptides and shorter membrane-attached C-terminal segment. The latter is further digested by γ-secretase to non-toxic P3 and C59 segments. Other estrogen actions include inhibition of β-secretase (amyloidogenic pathway) and stimulation of APP-containing vesicle budding by trans-Golgi network [75]. Estrogens also promote Aβ clearance by stimulation of microglial Aβ phagocytosis and enzymes involved in Aβ degradation, including metalloproteases-2 and -9, insulindegrading enzyme and neprilysin [76]. At the same time, estrogens exert anti-AD actions by increasing dendritic spine densities, promoting synaptogenesis, inhibiting the neurotoxic effects of oxidized low-density lipoproteins and glutamate, improving mitochondrial functions and enhancing the hippocampal cholinergic neurotransmitter system. Estrogens also regulate the epigenetic DNA methylation and miRNAs biogenesis especially in the hippocampus and thus master the genes expressions both transcriptionally and post-transcriptionally, which in turn play pivotal roles in enhancement of neuroprotection and prevention of neurodegeneration. Estrogen actions seem to be age dependent with obvious dysregulation in old-aged people. At the same time, estrogens have neuroprotective actions through increasing the expression of antiapoptotic Bcl-xL and Bcl-w and suppressing the expression of proapoptotic Bim, which lead to prevention of neuronal loss from Aβ toxicity [77–80]. Women are at increased risk of AD due to age-related sharp decline in sex steroid hormones and spending a large proportion of their life in the postmenopausal period because of increased their life longevity with the resultant prolonged hypoestrogenic state and its negative neurological consequences. Studies showed that postmenopausal women with AD had lower estradiol (E2) and estrone (E1) levels in both blood and CSF compared to normal controls. Moreover, female CSF-E2 level is positively correlated with CSF-Aβ level and cerebral glucose metabolism in the left hippocampus PET scan, which denotes that they are at increased risk to develop AD. In short-term studies, transdermal estrogen administration is associated with increased attention, verbal and visual memories; however, long-term studies failed to slow down AD progression or adding benefits to rivastigmine therapy in postmenopausal women [47, 81]. The neuroprotective effect of estrogen is not the same in both sexes as brain E1 and E2 levels have no relations with Aβ accumulation in males pointing to different gender expression of sex steroid hormones. At the same time, estrogen administration in male to female cross sex subjects results in significant decreases in the hippocampal volume despite producing neurogenesis, which means that the estrogen AD risk prevention in males is negligible [82, 83] (Figure 3). #### 4.2.2. Progesterone and Alzheimer's disease 4.1. Sex hormones and MCI 156 Sex Hormones in Neurodegenerative Processes and Diseases manifest AD [71, 72]. agement strategies. 4.2. Sex hormones and Alzheimer's disease 4.2.1. Estrogens and Alzheimer's disease The concept of MCI had received much attention nowadays for early detection of those candidates to AD conversion which open a gate for future disease modifying agents including hormonal therapy before irreversible neuronal damages take place. MCI is a clinical condition lies between normal aging and dementia in which the cognitive dysfunctions are greater than expected for age but is not severe enough to significantly interfere with the daily life or warrant the diagnosis of dementia [8, 69]. MCI is classified to amnestic (am-MCI) and non-amnestic (nam-MCI) types. In the former, memory impairment is the dominating manifestation, and in the latter, non-memory cognitive domain is the affected one (language, attention, executive function, visual-spatial). Amnestic MCI is termed multiple domain if another cognitive domain is affected, whereas nam-MCI becomes multiple domain if more than one cognitive non-memory domain is affected. People with am-MCI are more liable to develop AD [11, 70]. The prevalence of am-MCI is about 8.5–25.9 per 1000 of general population and 10–20% of those above the age of 60 years; 10–15% of am-MCI persons will develop AD compared to 1–2% of nam-MCI people. Several clinical and biochemical markers had been studied to be used as predictors of MCI/AD progression. In general, women have a higher prevalence of nam-MCI but most metanalytic studies showed non-significant gender difference in the prevalence of am-MCI, which means that women take shorter time to convert from am-MCI to One of the most common observations associated with AD onset is decreased levels of sex hormones, including estrogens, progesterone and androgens, pointing the potential role of these hormones in AD pathogenesis and the possible benefits of their targeting in AD man- Estrogen neuroprotective actions in AD are well documented by decades of researches showing that women used estrogen supplements or those with late menopause are at significantly decreased risk of AD development. On the other hand, early menopause because of increased sex hormone-binding globulin is associated with higher risk of AD in later life [73]. Estrogens exert anti-AD actions through different mechanisms including inhibition of tau deposition and Aβ accumulation. The former action is exerted through inhibition of tau hyperphosphorylation and promotion of tau dephosphorylation in an estrogen receptor-dependent mode through inhibition of protein kinases and promotion of protein phosphatase 2A enzyme activities, respectively [13, 74]. Estrogens inhibit Aβ accumulation by several mechanisms, one of which is decreasing Aβ production by enhancement of non-amyloidogenic APP pathway through activation of α-secretase enzyme that cleaves APP to soluble APP-α peptides and shorter membrane-attached C-terminal segment. The latter is further digested by γ-secretase to non-toxic P3 and C59 segments. Other estrogen actions include inhibition of β-secretase (amyloidogenic pathway) and stimulation of APP-containing vesicle budding by trans-Golgi Studies in progesterone neuroprotective actions against AD predisposition are not so plenty like those on estrogens but despite this, it is generally accepted that progesterone has a direct neuroprotective action while its indirect actions against AD development by regulating the neuroestrogen effects are matter of controversy as in some studies, progesterone enhances estradiol neuroprotective actions and in others antagonizes them beside reducing the cerebral blood flow [84]. The direct progesterone protecting actions against AD development and/or progression include regulation of β-amyloid metabolism by reducing Aβ production and decreasing the pool of soluble Aβ by enhancement of the non-amyloidogenic α-secretase pathway, decreasing Aβ accumulation through modulation of γ-secretases activities and increasing Aβ Males are subjected to andropause due to age-dependent high level of sex hormone-binding globulin with subsequent decrease in androgen levels and effects. Sex hormone-binding globulin is significantly higher in AD patients than age- and sex-matched control resulting in functional impairments of androgen-responsive tissues including the brain with consequent increase in AD risk [73]. Male andropause occurs very slowly over a long period of time where total androgen level starts to decline in thirties in a rate of 0.2–1% per year, while free testosterone decreases in a higher rate (2–3% yearly). This slowly gradual andropause relative to the rapid menopause may be one of the explanations of decreased male gender AD risk, Sex Hormones and Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.72561 159 Based on the abundant data supporting the numerous neuroprotective actions of sex hormones in ameliorating many pathological processes occurring in AD, hormonal replacement therapy (HRT) seems to be theoretically beneficial but the translation of this hypothesis to practice met a lot of difficulties which made the use of HRT in AD management still a matter of skepticism [36, 94]. The values of female estrogens and progesterone replacement therapies carry controversial results, which are mainly dependent on the timing, dose and duration of their application to the AD predisposed individual. Promising results were only attained on early HRT initiation at a close menopause temporal proximity and any delayed administration may even give counterproductive bad consequence. This time limit of proper HRT initiation resulted in introduction of the term the critical window of intervention or the window of opportunity which describes the time after which HRT become worthless. HRT has not the same effect in all genotypes but it is found to be more beneficial in people with APOE2 and APOE3 genotypes than APOE4 [96–100]. At the same time, some studies revealed that the protective effect of HRT against AD is only achieved in long-term users (>10 years), while short-term therapy had no AD preventive actions pointing to the need of long-term HRT use to gain significantly beneficial AD protection [101, 102]. The need for long-term use of conventional HRT opens a new obstacle due to the high cardiovascular risks which in some instances may overwhelm the anti-AD cognitive benefits. This makes AD patients in ultimate need for future introduction of new hormonal drugs with little side effects [103, 104]. At the androgenic level and despite their numerous anti-AD neuroprotective actions, the long-term androgens use carries many neurological and extra-neurological risks including decreased dendritic reorganization and spine density in the limbic regions after initial increase due to increased glutamate turnover and neurotoxicity in amygdala structures with functional impaired con- Alzheimer's disease is a complex multifactorial neurodegenerative disorder resulted from dysregulation of many biological processes at multiple levels in a specific neuronal temporospatial pattern. Sex hormones, including estrogens, progesterone and androgens, play crucial delayed male MCI/AD conversion and slower AD cognitive deterioration [78, 95]. 5. Sex hormone therapy trials for Alzheimer's disease nectivity with areas involved in cognitive functions [105, 106]. 6. Conclusions and future prospect Figure 3. Estrogen anti-Alzheimer's neuroprotective actions. clearance by enhancing insulin-degrading enzyme expression and downregulation of β-secretase gene expression [85, 86]. At the same time, progesterone reduces tau hyperphosphorylation and the serum level of endogenous progesterone is inversely correlated with tau accumulation, and at the same time, progesterone administration in transgenic AD mice improved cognitive performance in object recognition and T-maze task [87]. #### 4.2.3. Androgens and Alzheimer's disease Androgens have neuroprotective effects against AD in both males and females. Many studies had detected lower testosterone level in men with AD relative to normal age-matched control both in the blood and CSF. At the same time, APOE4 allele, which is a major risk of late onset AD, is associated with significantly lower level of circulating testosterone [88]. In accordance with these results, the Baltimore Longitudinal Study on Aging had detected significantly lower testosterone level 5–10 years in healthy men prior to their development of clinically manifest AD compared to those who did not develop AD [89]. Short-term testosterone administration improves cognitive functions in MCI and AD patients possibly through non-genomic transmembrane ARs activation [90, 91]. The long-term direct genomic action of androgens results in reduction of Aβ accumulation through enhancement of non-amyloidogenic APP pathway and promoting Aβ clearance by stimulation of Aβ-degrading enzyme action. Postmortem studies had shown that brain levels of testosterone were inversely correlated with cerebral soluble Aβ, which precedes insoluble fibrillar Aβ accumulation. These androgenic anti-amyloid actions are exerted in both sexes [92, 93]. Androgens can also indirectly reduce Aβ accumulation either through enhancing the estrogen pathway or through hypothalamo-hypophyseal-gonadal axis where they inhibit the release of gonadotropin luteinizing hormone secretion by the negative feedback, and it is well known that the latter hormone increases Aβ production by enhancement of APP/β-secretase initiated amyloidogenic pathway [94]. Males are subjected to andropause due to age-dependent high level of sex hormone-binding globulin with subsequent decrease in androgen levels and effects. Sex hormone-binding globulin is significantly higher in AD patients than age- and sex-matched control resulting in functional impairments of androgen-responsive tissues including the brain with consequent increase in AD risk [73]. Male andropause occurs very slowly over a long period of time where total androgen level starts to decline in thirties in a rate of 0.2–1% per year, while free testosterone decreases in a higher rate (2–3% yearly). This slowly gradual andropause relative to the rapid menopause may be one of the explanations of decreased male gender AD risk, delayed male MCI/AD conversion and slower AD cognitive deterioration [78, 95]. #### 5. Sex hormone therapy trials for Alzheimer's disease Based on the abundant data supporting the numerous neuroprotective actions of sex hormones in ameliorating many pathological processes occurring in AD, hormonal replacement therapy (HRT) seems to be theoretically beneficial but the translation of this hypothesis to practice met a lot of difficulties which made the use of HRT in AD management still a matter of skepticism [36, 94]. The values of female estrogens and progesterone replacement therapies carry controversial results, which are mainly dependent on the timing, dose and duration of their application to the AD predisposed individual. Promising results were only attained on early HRT initiation at a close menopause temporal proximity and any delayed administration may even give counterproductive bad consequence. This time limit of proper HRT initiation resulted in introduction of the term the critical window of intervention or the window of opportunity which describes the time after which HRT become worthless. HRT has not the same effect in all genotypes but it is found to be more beneficial in people with APOE2 and APOE3 genotypes than APOE4 [96–100]. At the same time, some studies revealed that the protective effect of HRT against AD is only achieved in long-term users (>10 years), while short-term therapy had no AD preventive actions pointing to the need of long-term HRT use to gain significantly beneficial AD protection [101, 102]. The need for long-term use of conventional HRT opens a new obstacle due to the high cardiovascular risks which in some instances may overwhelm the anti-AD cognitive benefits. This makes AD patients in ultimate need for future introduction of new hormonal drugs with little side effects [103, 104]. At the androgenic level and despite their numerous anti-AD neuroprotective actions, the long-term androgens use carries many neurological and extra-neurological risks including decreased dendritic reorganization and spine density in the limbic regions after initial increase due to increased glutamate turnover and neurotoxicity in amygdala structures with functional impaired connectivity with areas involved in cognitive functions [105, 106]. #### 6. Conclusions and future prospect clearance by enhancing insulin-degrading enzyme expression and downregulation of β-secretase gene expression [85, 86]. At the same time, progesterone reduces tau hyperphosphorylation and the serum level of endogenous progesterone is inversely correlated with tau accumulation, and at the same time, progesterone administration in transgenic AD mice Androgens have neuroprotective effects against AD in both males and females. Many studies had detected lower testosterone level in men with AD relative to normal age-matched control both in the blood and CSF. At the same time, APOE4 allele, which is a major risk of late onset AD, is associated with significantly lower level of circulating testosterone [88]. In accordance with these results, the Baltimore Longitudinal Study on Aging had detected significantly lower testosterone level 5–10 years in healthy men prior to their development of clinically Short-term testosterone administration improves cognitive functions in MCI and AD patients possibly through non-genomic transmembrane ARs activation [90, 91]. The long-term direct genomic action of androgens results in reduction of Aβ accumulation through enhancement of non-amyloidogenic APP pathway and promoting Aβ clearance by stimulation of Aβ-degrading enzyme action. Postmortem studies had shown that brain levels of testosterone were inversely correlated with cerebral soluble Aβ, which precedes insoluble fibrillar Aβ accumulation. These androgenic anti-amyloid actions are exerted in both sexes [92, 93]. Androgens can also indirectly reduce Aβ accumulation either through enhancing the estrogen pathway or through hypothalamo-hypophyseal-gonadal axis where they inhibit the release of gonadotropin luteinizing hormone secretion by the negative feedback, and it is well known that the latter hormone increases Aβ production by enhancement of APP/β-secretase initiated improved cognitive performance in object recognition and T-maze task [87]. manifest AD compared to those who did not develop AD [89]. 4.2.3. Androgens and Alzheimer's disease Figure 3. Estrogen anti-Alzheimer's neuroprotective actions. 158 Sex Hormones in Neurodegenerative Processes and Diseases amyloidogenic pathway [94]. Alzheimer's disease is a complex multifactorial neurodegenerative disorder resulted from dysregulation of many biological processes at multiple levels in a specific neuronal temporospatial pattern. Sex hormones, including estrogens, progesterone and androgens, play crucial CNS modulatory functions and their disturbances result in impairment of neuroprotection, neurogenesis, synaptogenesis, synaptic plasticity and myelination as well as abnormal glial cell activities. The sharp decrease of neurosteroids influences in menopause relative to the slow andropause makes females sex at increased risk of AD development, rapid MCI/AD conversion and rapid course of cognitive deterioration. Trials used sex hormones as a disease modifying neuroprotective anti-AD agents revealed that their possible beneficial effect can be achieved only by early HRT before the beginning of critical window of intervention and the therapy must continue for long time which may put the treated individuals at increased risk of cardiovascular complications. [6] Scheltens P, Blennow K, Breteler MB, de Strooper B, Frisoni GB, Salloway S, Van der Flier WM. Alzheimer's disease. Lancet. 2016;388:505-517. DOI: 10.1016/S0140-6736(15)01124-1 Sex Hormones and Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.72561 161 [7] Ulrich JD, Ulland TK, Colonna M, Holtzman DM. Elucidating the role of TREM2 in Alzheimer's disease. Neuron. 2017;94:237-248. DOI: 10.1016/j.neuron.2017.02.042 [8] Au B, Dale-McGrath S, Tierney MC. Sex differences in the prevalence and incidence of mild cognitive impairment: A meta-analysis. Ageing Research Reviews. 2017;35:176-199. 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DOI: 10.1016/j.mcn.2017.07.002 jalz.2017.04.007 So, what is nowadays considered normal menopausal or andropausal sex hormones' declines may be sufficient triggers irreversible neuropathological changes which latter on progress to AD in susceptible individuals, and it is the time to use these changes as early AD biomarkers in high risk persons and in turn correct them before the onset of the window of opportunity by safe and effective HRT for long-term use and sufficient to produce significant AD prophylaxis. #### Disclosure of interest No conflict of interest was reported. #### Author details Wafik Said Bahnasy\, Yasser A. El-Heneedy and Ehab A. El-Seidy \Address all correspondence to: [email protected] Department of Neurology, Faculty of Medicine, Tanta University, Tanta, Egypt #### References [6] Scheltens P, Blennow K, Breteler MB, de Strooper B, Frisoni GB, Salloway S, Van der Flier WM. Alzheimer's disease. Lancet. 2016;388:505-517. DOI: 10.1016/S0140-6736(15)01124-1 CNS modulatory functions and their disturbances result in impairment of neuroprotection, neurogenesis, synaptogenesis, synaptic plasticity and myelination as well as abnormal glial cell activities. The sharp decrease of neurosteroids influences in menopause relative to the slow andropause makes females sex at increased risk of AD development, rapid MCI/AD conversion and rapid course of cognitive deterioration. Trials used sex hormones as a disease modifying neuroprotective anti-AD agents revealed that their possible beneficial effect can be achieved only by early HRT before the beginning of critical window of intervention and the therapy must continue for long time which may put the treated individuals at increased risk So, what is nowadays considered normal menopausal or andropausal sex hormones' declines may be sufficient triggers irreversible neuropathological changes which latter on progress to AD in susceptible individuals, and it is the time to use these changes as early AD biomarkers in high risk persons and in turn correct them before the onset of the window of opportunity by safe and effective HRT for long-term use and sufficient to produce significant AD prophylaxis. of cardiovascular complications. 160 Sex Hormones in Neurodegenerative Processes and Diseases Disclosure of interest Author details References No conflict of interest was reported. Wafik Said Bahnasy\, Yasser A. 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DOI: 10.1111/j.1471-4159.2010. 06899.x yhbeh.2015.05.010 168 Sex Hormones in Neurodegenerative Processes and Diseases 2012.660613 en.2013-1598 10.1016/j.jsbmb.2016.03.012 jagp.2015.05.009 Chapter 8 Provisional chapter Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, on Motor Behavior and Cytological Alterations on Motor Behavior and Cytological Alterations Ana Luisa Gutiérrez-Valdez, Vianey Rodríguez-Lara, Javier Sanchez-Betancourt, Enrique Montiel-Flores, Leonardo Reynoso-Erazo, Rocio Tron-Alvarez, Patricia Aley-Medina, Jesús Espinosa-Villanueva, Cesar Sanchez-Vazquez del Mercado and Cesar Sanchez-Vazquez del Mercado and Rocio Tron-Alvarez, Patricia Aley-Medina, Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.70898 Verónica Anaya-Martínez, José Luis Ordóñez-Librado, Vianey Rodríguez-Lara, Verónica Anaya-Martínez, José Luis Ordóñez-Librado, Javier Sanchez-Betancourt, Enrique Montiel-Flores, Leonardo Reynoso-Erazo, Ana Luisa Gutiérrez-Valdez, María Rosa Avila-Costa María Rosa Avila-Costa Abstract Jesús Espinosa-Villanueva, Melatonin, and L-DOPA/Melatonin Coadministration Parkinson's disease (PD) higher incidence has been observed in postmenopausal women compared to premenopausal women, suggesting estrogen neuroprotective effect. L-DOPA (LD) chronic treatment causes dyskinesia; evidences indicate that LD increases the preexisting oxidative stress condition. This study determines melatonin ability, alone or in combination with LD (LD/Mel) to protect dopaminergic loss induced by 6-OHDA in a rat PD model in ovariectomized (OVX) and intact (with ovaries (W/OV)) rats on motor behavior and cytological alterations, comparing with LD-only treated rats. LD/Meltreated rats showed dyskinesia decrease (score 5–7.5) and had the best performance in the staircase test (five pellets) throughout all studies. The beam walking time was 20–35 s, showing good coordination (as control group (20–38 s)), dopaminergic cells increase of 22.8% (W/OV rats) and 27.2% (OVX rats) in the contralateral side as well as 100% conservation in the contralateral dendritic spines. Our results suggest that LD/Mel co-administration and estrogen presence result in an efficient treatment to reduce dyskinesia through the conservation of some dopaminergic cells, which imply a well-preserved > © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, Melatonin, and L-DOPA/Melatonin Coadministration DOI: 10.5772/intechopen.70898 Provisional chapter Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and L-DOPA/Melatonin Coadministration on Motor Behavior and Cytological Alterations Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and L-DOPA/Melatonin Coadministration on Motor Behavior and Cytological Alterations DOI: 10.5772/intechopen.70898 Differences Between Intact and Ovariectomized Ana Luisa Gutiérrez-Valdez, María Rosa Avila-Costa Cesar Sanchez-Vazquez del Mercado and Additional information is available at the end of the chapter María Rosa Avila-Costa http://dx.doi.org/10.5772/intechopen.70898 Additional information is available at the end of the chapter #### Abstract Parkinson's disease (PD) higher incidence has been observed in postmenopausal women compared to premenopausal women, suggesting estrogen neuroprotective effect. L-DOPA (LD) chronic treatment causes dyskinesia; evidences indicate that LD increases the preexisting oxidative stress condition. This study determines melatonin ability, alone or in combination with LD (LD/Mel) to protect dopaminergic loss induced by 6-OHDA in a rat PD model in ovariectomized (OVX) and intact (with ovaries (W/OV)) rats on motor behavior and cytological alterations, comparing with LD-only treated rats. LD/Meltreated rats showed dyskinesia decrease (score 5–7.5) and had the best performance in the staircase test (five pellets) throughout all studies. The beam walking time was 20–35 s, showing good coordination (as control group (20–38 s)), dopaminergic cells increase of 22.8% (W/OV rats) and 27.2% (OVX rats) in the contralateral side as well as 100% conservation in the contralateral dendritic spines. Our results suggest that LD/Mel co-administration and estrogen presence result in an efficient treatment to reduce dyskinesia through the conservation of some dopaminergic cells, which imply a well-preserved © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ana Luisa Gutiérrez-Valdez, Vianey Rodríguez-Lara, Vianey Rodríguez-Lara, Verónica Anaya-Martínez, Verónica Anaya-Martínez, José Luis Ordóñez-Librado, José Luis Ordóñez-Librado, Javier Sanchez-Betancourt, neuropil of a less denervated striatum. We assume that these results are because of a synergistic effect between LD, melatonin and estrogens. investigated the reasons for LD long-term problems. Some proposed mechanism that describes LD to induce oxidative damage, perpetuating the cell death [23–25], and it seems that LD produces 6-OHDA in the mouse striatum, generating more ROS formation [26, 27]. It has been proven that dopaminergic nuclei are full with DA following LD acute, subacute, or chronic administration [26], and the augmented DA can stimulate the 6-OHDA production in the brain [27]. Hence, we assumed that parkinsonian neurotoxins that generate free radicals in a DA-enriched milieu would promote oxidative stress production, and it is possible that melatonin might be a free radical scavenger protecting against ROS formation preventing the Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 173 Melatonin is an indoleamine first described in 1993 by Tan et al. [28] as an effective antioxidant. This indoleamine possesses unique benefits. First, its solubility in both water and lipids allows it to be efficiently allocated to the cell. Second, its capacity to cross the blood-brain barrier allows it to reach the central nervous system [29]. There are reports which mention that melatonin protects neurons from neurotoxin-induced damage in a wide range of neuronal culture systems serving as PD experimental models (for review, see [30]). Previous studies have shown that short-term treatment with melatonin does not exert a neuroprotective effect in DA-depleted animals, probably because the levels of this neurohormone are low in the brain [29]; in this sense, it is suggested that melatonin level has to be high and continuously maintained for a long time in the brain to guarantee its neuroprotective effect [30, 31]. It is important to note that in vivo experiments are still uncommon, and most of them have been done in acute models of the disease. These studies show melatonin protective effects in both the striatum dopaminergic terminals [31] and midbrain neurons [32]. However, there are insufficient reports about its effects on the initial stages of On the other hand, it is known that the prevalence of several neurodegenerative diseases, such as PD, correlates with gender [33]. Therefore, PD happens 1.5 times more frequently in men than in women [34–37]. In women, the onset age of PD relates to the fertile life duration It seems that there are several mechanisms of estrogen protection on the nigrostriatal pathway [39]. It has been reported that estrogen has neuroprotective effects in PD animal models utilizing the neurotoxins MPTP [40, 41], 6-OHDA [42, 43], or methamphetamine [44, 45]. The foundations for these sex/gender differences in SNc DA cell death are not known. Nevertheless, the gonadal steroid hormone estrogen seems to be a critical aspect responsible for these differences [39]. In vivo confirmation of the neuroprotective effects of estrogens has been reported since estrogen treatment in female ovariectomized (OVX) rodents protects against neurotoxin-induced depletion of striatal dopamine [46, 47]. However, it is not well known whether this neuroprotective effect prevents SNc dopaminergic cell death. Besides, it is not known whether L-DOPA/melatonin (LD/Mel) cotreatment can influence the neuroprotection degree. Therefore, the present study tries to investigate the capacity of melatonin or LD/Mel to protect striatal dopaminergic denervation induced by 6-OHDA in a hemiparkinsonian rat model, comparing the results with LD-only treated rats. The treatments were administered cell death. neurodegeneration. [38, 39]. Keywords: L-DOPA/melatonin dyskinesia, estrogen, Parkinson's disease experimental model, rat #### 1. Introduction Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopamine-containing neurons in the substantia nigra compacta (SNc) and by Lewy body presence. The subsequent striatal dopamine (DA) deficiency leads to the parkinsonian condition of bradykinesia, rigidity, tremor, and motor and postural instability [1, 2]. Some efficient drugs, including L-DOPA (LD), dopamine agonists, and inhibitors of dopamine-metabolizing enzymes, have been used for the clinical treatment of LD [3]. Unfortunately, chronic LD therapy is compromised by numerous side effects, the most evident LD-induced dyskinesia (LID) that is abnormal involuntary movements (AIMs), which severely compromise patients' lifestyle [4]. LID usually increases when DA reaches the maximum concentration in the brain per LD dose (peak-dose dyskinesias), and dystonia ("off" dystonia) can occur when the level of LD is very low [5]. Risk factors for LID include duration and dose of LD treatment, and consist of asymmetric choreiform movements, athetosis, and dystonia of facial muscles, jaw, tongue, neck, limbs, and toes [6]. Similarly, rats with unilateral 6-hydroxydopamine (6-OHDA) lesion, LD-treatment produces abnormal involuntary movements (AIMs), which are displayed as asymmetric and purposeless movements affecting the limbs, orofacial muscles, and trunk [7]. AIMs evaluation maintains prognostic validity for the preclinical screening of novel antidyskinetic PD treatments [8, 9]. Therefore, the identification of neurochemical features involved in the regulation of motor function may enable the discovery of new potential targets that perform together with LD, improving the effectiveness of these drugs and decreasing the incidence and severity of AIMs and response fluctuations [10]. The etiology of sporadic PD, which is most PD cases, is still unclear. Numerous results have been accumulated from pharmacological and pathological studies on PD and animal or in vitro reports using dopaminergic toxins, which cause Parkinsonism in animals [11]. These reports have revealed that oxidative stress [12], inflammation [13], and mitochondrial dysfunction [2, 14] play essential roles in the progress and pathogenesis of sporadic PD. Nevertheless, the mechanisms of dopaminergic neuron cell loss have not been entirely elucidated. However, some data suggest oxidative stress as the main candidate to mediate in the primary unknown cell death cause. Studies on PD brains have given evidence to support this hypothesis [15–17]. The free radical formation has been confirmed in lipids [18], proteins, and SNc nucleic acids of PD patients [19]. Therefore, the reactive oxygen species (ROS) production induced by oxidative stress, the basal ganglia and SNc lack of antioxidant defenses, is commonly considered [20] the final cause of neuronal death [21, 22]. On the other hand, previous studies have investigated the reasons for LD long-term problems. Some proposed mechanism that describes LD to induce oxidative damage, perpetuating the cell death [23–25], and it seems that LD produces 6-OHDA in the mouse striatum, generating more ROS formation [26, 27]. It has been proven that dopaminergic nuclei are full with DA following LD acute, subacute, or chronic administration [26], and the augmented DA can stimulate the 6-OHDA production in the brain [27]. Hence, we assumed that parkinsonian neurotoxins that generate free radicals in a DA-enriched milieu would promote oxidative stress production, and it is possible that melatonin might be a free radical scavenger protecting against ROS formation preventing the cell death. neuropil of a less denervated striatum. We assume that these results are because of a Keywords: L-DOPA/melatonin dyskinesia, estrogen, Parkinson's disease experimental Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopamine-containing neurons in the substantia nigra compacta (SNc) and by Lewy body presence. The subsequent striatal dopamine (DA) deficiency leads to the parkinsonian condition of bradykinesia, rigidity, tremor, and motor and postural instability [1, 2]. Some efficient drugs, including L-DOPA (LD), dopamine agonists, and inhibitors of dopamine-metabolizing enzymes, have been used for the clinical treatment of LD [3]. Unfortunately, chronic LD therapy is compromised by numerous side effects, the most evident LD-induced dyskinesia (LID) that is abnormal involuntary movements (AIMs), which severely compromise patients' lifestyle [4]. LID usually increases when DA reaches the maximum concentration in the brain per LD dose (peak-dose dyskinesias), and dystonia ("off" dystonia) can occur when the level of LD is very low [5]. Risk factors for LID include duration and dose of LD treatment, and consist of asymmetric choreiform movements, athetosis, and dystonia of facial muscles, jaw, tongue, neck, limbs, and toes [6]. Similarly, rats with unilateral 6-hydroxydopamine (6-OHDA) lesion, LD-treatment produces abnormal involuntary movements (AIMs), which are displayed as asymmetric and purposeless movements affecting the limbs, orofacial muscles, and trunk [7]. AIMs evaluation maintains prognostic validity for the preclinical screening of novel antidyskinetic PD treatments [8, 9]. Therefore, the identification of neurochemical features involved in the regulation of motor function may enable the discovery of new potential targets that perform together with LD, improving the effectiveness of these drugs and decreasing the The etiology of sporadic PD, which is most PD cases, is still unclear. Numerous results have been accumulated from pharmacological and pathological studies on PD and animal or in vitro reports using dopaminergic toxins, which cause Parkinsonism in animals [11]. These reports have revealed that oxidative stress [12], inflammation [13], and mitochondrial dysfunction [2, 14] play essential roles in the progress and pathogenesis of sporadic PD. Nevertheless, the mechanisms of dopaminergic neuron cell loss have not been entirely elucidated. However, some data suggest oxidative stress as the main candidate to mediate in the primary unknown cell death cause. Studies on PD brains have given evidence to support this hypothesis [15–17]. The free radical formation has been confirmed in lipids [18], proteins, and SNc nucleic acids of PD patients [19]. Therefore, the reactive oxygen species (ROS) production induced by oxidative stress, the basal ganglia and SNc lack of antioxidant defenses, is commonly considered [20] the final cause of neuronal death [21, 22]. On the other hand, previous studies have synergistic effect between LD, melatonin and estrogens. 172 Sex Hormones in Neurodegenerative Processes and Diseases incidence and severity of AIMs and response fluctuations [10]. model, rat 1. Introduction Melatonin is an indoleamine first described in 1993 by Tan et al. [28] as an effective antioxidant. This indoleamine possesses unique benefits. First, its solubility in both water and lipids allows it to be efficiently allocated to the cell. Second, its capacity to cross the blood-brain barrier allows it to reach the central nervous system [29]. There are reports which mention that melatonin protects neurons from neurotoxin-induced damage in a wide range of neuronal culture systems serving as PD experimental models (for review, see [30]). Previous studies have shown that short-term treatment with melatonin does not exert a neuroprotective effect in DA-depleted animals, probably because the levels of this neurohormone are low in the brain [29]; in this sense, it is suggested that melatonin level has to be high and continuously maintained for a long time in the brain to guarantee its neuroprotective effect [30, 31]. It is important to note that in vivo experiments are still uncommon, and most of them have been done in acute models of the disease. These studies show melatonin protective effects in both the striatum dopaminergic terminals [31] and midbrain neurons [32]. However, there are insufficient reports about its effects on the initial stages of neurodegeneration. On the other hand, it is known that the prevalence of several neurodegenerative diseases, such as PD, correlates with gender [33]. Therefore, PD happens 1.5 times more frequently in men than in women [34–37]. In women, the onset age of PD relates to the fertile life duration [38, 39]. It seems that there are several mechanisms of estrogen protection on the nigrostriatal pathway [39]. It has been reported that estrogen has neuroprotective effects in PD animal models utilizing the neurotoxins MPTP [40, 41], 6-OHDA [42, 43], or methamphetamine [44, 45]. The foundations for these sex/gender differences in SNc DA cell death are not known. Nevertheless, the gonadal steroid hormone estrogen seems to be a critical aspect responsible for these differences [39]. In vivo confirmation of the neuroprotective effects of estrogens has been reported since estrogen treatment in female ovariectomized (OVX) rodents protects against neurotoxin-induced depletion of striatal dopamine [46, 47]. However, it is not well known whether this neuroprotective effect prevents SNc dopaminergic cell death. Besides, it is not known whether L-DOPA/melatonin (LD/Mel) cotreatment can influence the neuroprotection degree. Therefore, the present study tries to investigate the capacity of melatonin or LD/Mel to protect striatal dopaminergic denervation induced by 6-OHDA in a hemiparkinsonian rat model, comparing the results with LD-only treated rats. The treatments were administered 4 days after lesioning, daily for 6 months at doses suitable to improve motor performance, and their effects were assessed using measures of skilled forelimb use, stepping ability, and AIMs. At the cellular level, the treatment response has been evaluated using tyrosine hydroxylase (TH) immunoreactivity and estimating the number of dendritic spines in the striatal mediumsized spiny neurons, all in female rats, to examine estrogen's presence or absence. #### 2. Experimental procedures The experiments were conducted in 50 female Wistar rats weighing 180 20 g at the start of the study. The animals were individually placed in plastic cages under controlled light conditions (12:12-h light-dark regime) and fed with Purina Rat Chow® and water ad libitum. Body weight was registered daily. The experimental protocol was conducted out in agreement with the National Institutes of Health, Guide for Care and Use of Laboratory Animals certificated by the Secretaria de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA) (NOM-062-ZOO-1999, Mexico) and approved by UNAM institutional animal care committee. All attempts were made to reduce the number of animals used and their suffering. 2.1.2. Beam walking task after that every 15 days. wall under Isoflurane anesthesia (n = 25). 2.2.2. Stereotactic surgery and treatments 2.2. Surgery 2.2.1. Ovariectomy The additional test to measure motor coordination was evaluating the ability of the animals to traverse a narrow beam (12 mm wide) to reach an enclosed safety platform [50]. The rats were trained for 1 week to cross the wooden beam. The beam measured 2 m long and was elevated to a height of 1 m over the floor with wood supports with 15 inclination. Each test session consisted of four trials in which latency to cross the beam was recorded (establishing a maximum range of 120 s; if the animal did not pass at that time, the activity was terminated and assigned the value of 120 s for that evaluation). Five trials were averaged to give a mean latency [51]. The testing was done every week after 6-OHDA lesion during the first month and Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 175 Figure 1. Staircase test used to assess skilled reaching deficits after 6-OHDA lesion. Bilateral ovariectomy (OVX) was performed through two lateral incisions of the abdominal The rats were anesthetized with Isoflurane and placed in a stereotaxic apparatus. The rats (n = 20 OVX and 20 with ovaries (W/OV)) were infused with 4 μl saline solution carrying 8 μg of 6-OHDA (Sigma Chemical, USA) and 0.2 mg of ascorbic acid into the left medial forebrain bundle (MFB) (n = 40), and sham lesion was made with vehicle (n = 10; 5 OVX and 5 W/OV (control group) [7]. The injections were given over a 5-min period with a Hamilton syringe attached to a glass micropipette with a tip diameter of 20–50 μm. The stereotaxic coordinates were as follows: AP = 3 mm anterior of the ear bar; L = 1.6 mm lateral of bregma; V = 8 mm vertical of the Dura (according to [52]). After anesthesia recovery, the animals were returned to their cages. Apomorphine (Sigma Chemical, USA; 0.25 mg/kg i.p.) provoked contralateral rotational behavior was tested 2 days after lesioning. Only those animals displaying more than 200 full turns in a 30-min period were used [53]. Two days after the rotational behavior test, we began the treatments as follows: 5 OVX and 5 W/OV lesioned rats were treated with 7.5 mg/kg #### 2.1. Motor behavior Before ovariectomy and 6-OHDA surgery, all animals were trained for 1 week in the beam walking and in the staircase tasks to evaluate motor performance. Training and testing were performed during the light part/period of the cycle, at the same hour every day. For the staircase test, rats were food-deprived for 24 h. Afterward, they received a restricted diet of ~10-g/kg body weight adjusted to keep their weight constant. Food restriction considered the natural gain in body weight during the training period, which prevented excessive weight reduction. After the 6-OHDA surgery, each rat was tested once a week, a different day for each test. Two observers blind to the rats' condition perform all behavioral assessments. #### 2.1.1. Staircase test Rats were trained in the staircase test, which measures the independent use of forelimbs in skilled reaching and grasping tasks [48]. Briefly, each rat is placed into a clear plexiglass case (length 30 cm, width 6.8 cm, and height 12 cm) in which the rat rests on a central elevated platform with six stairs descending on each side. Each stair contained one food pellet. Food pellets on the left stairs may be retrieved only using the left paw, whereas pellets on the right stairs must be obtained using the right paw (Figure 1). Rats were trained for a week (2/15 min sessions/day) and were excluded from this test if they did not retrieve at least six pellets/side [49]. The last 5 days of training were used to calculate baseline performance. The skilled reaching ability was quantified by recording the number of food pellets retrieved with each paw. The qualitative analysis of this test comprises the appropriate movements to take the pellets: (1) prepared to take food, (2) stretched the forelimb, (3) took the pellet (pronation movement), (4) paw rotation around the wrist (supination), and (5) eat the food [49]. Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 175 Figure 1. Staircase test used to assess skilled reaching deficits after 6-OHDA lesion. #### 2.1.2. Beam walking task 4 days after lesioning, daily for 6 months at doses suitable to improve motor performance, and their effects were assessed using measures of skilled forelimb use, stepping ability, and AIMs. At the cellular level, the treatment response has been evaluated using tyrosine hydroxylase (TH) immunoreactivity and estimating the number of dendritic spines in the striatal medium- The experiments were conducted in 50 female Wistar rats weighing 180 20 g at the start of the study. The animals were individually placed in plastic cages under controlled light conditions (12:12-h light-dark regime) and fed with Purina Rat Chow® and water ad libitum. Body weight was registered daily. The experimental protocol was conducted out in agreement with the National Institutes of Health, Guide for Care and Use of Laboratory Animals certificated by the Secretaria de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA) (NOM-062-ZOO-1999, Mexico) and approved by UNAM institutional animal care committee. All attempts were made to reduce the number of animals used and their Before ovariectomy and 6-OHDA surgery, all animals were trained for 1 week in the beam walking and in the staircase tasks to evaluate motor performance. Training and testing were performed during the light part/period of the cycle, at the same hour every day. For the staircase test, rats were food-deprived for 24 h. Afterward, they received a restricted diet of ~10-g/kg body weight adjusted to keep their weight constant. Food restriction considered the natural gain in body weight during the training period, which prevented excessive weight reduction. After the 6-OHDA surgery, each rat was tested once a week, a different day for each Rats were trained in the staircase test, which measures the independent use of forelimbs in skilled reaching and grasping tasks [48]. Briefly, each rat is placed into a clear plexiglass case (length 30 cm, width 6.8 cm, and height 12 cm) in which the rat rests on a central elevated platform with six stairs descending on each side. Each stair contained one food pellet. Food pellets on the left stairs may be retrieved only using the left paw, whereas pellets on the right stairs must be obtained using the right paw (Figure 1). Rats were trained for a week (2/15 min sessions/day) and were excluded from this test if they did not retrieve at least six pellets/side [49]. The last 5 days of training were used to calculate baseline performance. The skilled reaching ability was quantified by recording the number of food pellets retrieved with each paw. The qualitative analysis of this test comprises the appropriate movements to take the pellets: (1) prepared to take food, (2) stretched the forelimb, (3) took the pellet (pronation test. Two observers blind to the rats' condition perform all behavioral assessments. movement), (4) paw rotation around the wrist (supination), and (5) eat the food [49]. sized spiny neurons, all in female rats, to examine estrogen's presence or absence. 2. Experimental procedures 174 Sex Hormones in Neurodegenerative Processes and Diseases suffering. 2.1. Motor behavior 2.1.1. Staircase test The additional test to measure motor coordination was evaluating the ability of the animals to traverse a narrow beam (12 mm wide) to reach an enclosed safety platform [50]. The rats were trained for 1 week to cross the wooden beam. The beam measured 2 m long and was elevated to a height of 1 m over the floor with wood supports with 15 inclination. Each test session consisted of four trials in which latency to cross the beam was recorded (establishing a maximum range of 120 s; if the animal did not pass at that time, the activity was terminated and assigned the value of 120 s for that evaluation). Five trials were averaged to give a mean latency [51]. The testing was done every week after 6-OHDA lesion during the first month and after that every 15 days. #### 2.2. Surgery #### 2.2.1. Ovariectomy Bilateral ovariectomy (OVX) was performed through two lateral incisions of the abdominal wall under Isoflurane anesthesia (n = 25). #### 2.2.2. Stereotactic surgery and treatments The rats were anesthetized with Isoflurane and placed in a stereotaxic apparatus. The rats (n = 20 OVX and 20 with ovaries (W/OV)) were infused with 4 μl saline solution carrying 8 μg of 6-OHDA (Sigma Chemical, USA) and 0.2 mg of ascorbic acid into the left medial forebrain bundle (MFB) (n = 40), and sham lesion was made with vehicle (n = 10; 5 OVX and 5 W/OV (control group) [7]. The injections were given over a 5-min period with a Hamilton syringe attached to a glass micropipette with a tip diameter of 20–50 μm. The stereotaxic coordinates were as follows: AP = 3 mm anterior of the ear bar; L = 1.6 mm lateral of bregma; V = 8 mm vertical of the Dura (according to [52]). After anesthesia recovery, the animals were returned to their cages. Apomorphine (Sigma Chemical, USA; 0.25 mg/kg i.p.) provoked contralateral rotational behavior was tested 2 days after lesioning. Only those animals displaying more than 200 full turns in a 30-min period were used [53]. Two days after the rotational behavior test, we began the treatments as follows: 5 OVX and 5 W/OV lesioned rats were treated with 7.5 mg/kg LD (Sinemet® (Carbidopa-L-DOPA 25/250)), 5 OVX and 5 W/OV lesioned rats were treated with 10 mg/kg melatonin (Sigma Chemical, USA), and 5 OVX and 5 W/OV lesioned rats were treated with 7.5 mg/kg LD/10 mg/kg Mel. The drugs were dissolved in 10 ml distilled water and given orally with an insulin syringe for 6 months during the light period (at 10:00 AM every day) [7]. The other 10 (5 OVX and 5 W/OV) lesioned rats without treatment, as well as the control animals (5 OVX and 5 W/OV), were kept for the same time. The motor performance was evaluated weekly during the first month and after every 15 days; the rats were tested during the light period at 14:00 h, a different day for each test. #### 2.3. AIMs rating LD-induced AIMs were calculated at day 30 according to a rat dyskinesia scale [54–56]. Rats were placed individually in transparent cages and observed every 20th min, from 20 min before to 180 min after giving the treatments (10 monitoring periods of 1 min each). Four AIM subtypes were classified according to their topographic distribution as locomotive, axial, forelimb, or orolingual (for details, see Figure 2). Signs of otherwise normal behaviors, such as rearing, sniffing, grooming, and gnawing, were not included in the evaluation [57]. AIM severity was assessed using the method of Cenci et al. [55], and Lundblad et al. [9], which designates a score from 0 to 4 to each of the four AIM subtypes mentioned before according to the proportion of time/monitoring period through whichever AIM is observed. Borderline scores, such as 0.5, 1.5, 2.5, and 3.5, were allowed to increase the sensitivity of the evaluation [7, 57]. #### 2.4. Video recording Performance during motor tests and AIM analysis was video-recorded (Panasonic camcorder DR-H80 model). Representative still frames were captured from digital video recordings with the video-editing software Final Cut Pro. Pictures were cropped and adjusted for color and brightness contrast in Adobe Photoshop but were not altered in any other way [57]. #### 2.5. Cytological analysis All animals were perfused under sodium pentobarbital anesthesia immediately after the 6-month treatments via the aorta, with saline solution followed by fixative including 0.2% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were removed and deposited in the fixative solution for 1 h. For the TH Immunocytochemistry, coronal sections (50 μm) were collected on a vibrating microtome through the mesencephalon. Tyrosine hydroxylase (Chemicon International, Inc., CA, USA; 1:1000) immunostaining with the ABC detection method (Vector Lab MI, USA) was conducted for light microscope analysis. The analysis was performed with a computer-assisted system (Image-Pro Plus, Media Cybernetics, L.P. Del Mar, CA, USA) connected to a CCD camera to Optiphot 2 microscope (Nikon, Japan). The number of TH-positive neurons was calculated in 1500 μm<sup>2</sup> from seven SNc sections of each animal [58]. The dendritic spines analysis was performed by the Golgi method. Blocks from the striatum were cut into 90-μm-thick sections and processed for the rapid Golgi method. The analysis consisted in counting the number of dendritic spines in a 10-μm-long section from 5 secondary dendrites to 20 striatal medium-sized spiny Figure 2. Video recording sequences from rats affected by orolingual (A), axial (B), forelimb (C), and locomotive (D) AIMs. Orolingual AIMs (A) include opening and closing of the jaws and tongue protrusion toward the side contralateral to the lesion (arrow). The series in (B) displays a neck and upper trunk torsion action toward the contralateral side to the lesion. Body torsion is maximally critical (>90), causing the rat to lose equilibrium. Forelimb AIMs (C) include purposeless up and down translocation of the Parkinsonian (right) forelimb (arrow). Locomotive AIMs (D) comprise circular movement toward the contralateral side to the lesion. Only locomotive movements involving all four limbs are considered Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 177 Two-way ANOVA was used to analyze the number of TH-immunoreactive cells, the number of dendritic spines, and the behavioral data. Group differences were considered statistically significant at P < 0.05. When appropriate, post hoc comparisons were made with Tukey test. All analyses were conducted with GraphPad Prism 7 for Mac Software. neurons [58]. under this AIM category. 2.6. Statistical analysis Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 177 Figure 2. Video recording sequences from rats affected by orolingual (A), axial (B), forelimb (C), and locomotive (D) AIMs. Orolingual AIMs (A) include opening and closing of the jaws and tongue protrusion toward the side contralateral to the lesion (arrow). The series in (B) displays a neck and upper trunk torsion action toward the contralateral side to the lesion. Body torsion is maximally critical (>90), causing the rat to lose equilibrium. Forelimb AIMs (C) include purposeless up and down translocation of the Parkinsonian (right) forelimb (arrow). Locomotive AIMs (D) comprise circular movement toward the contralateral side to the lesion. Only locomotive movements involving all four limbs are considered under this AIM category. for the rapid Golgi method. The analysis consisted in counting the number of dendritic spines in a 10-μm-long section from 5 secondary dendrites to 20 striatal medium-sized spiny neurons [58]. #### 2.6. Statistical analysis LD (Sinemet® (Carbidopa-L-DOPA 25/250)), 5 OVX and 5 W/OV lesioned rats were treated with 10 mg/kg melatonin (Sigma Chemical, USA), and 5 OVX and 5 W/OV lesioned rats were treated with 7.5 mg/kg LD/10 mg/kg Mel. The drugs were dissolved in 10 ml distilled water and given orally with an insulin syringe for 6 months during the light period (at 10:00 AM every day) [7]. The other 10 (5 OVX and 5 W/OV) lesioned rats without treatment, as well as the control animals (5 OVX and 5 W/OV), were kept for the same time. The motor performance was evaluated weekly during the first month and after every 15 days; the rats were tested LD-induced AIMs were calculated at day 30 according to a rat dyskinesia scale [54–56]. Rats were placed individually in transparent cages and observed every 20th min, from 20 min before to 180 min after giving the treatments (10 monitoring periods of 1 min each). Four AIM subtypes were classified according to their topographic distribution as locomotive, axial, forelimb, or orolingual (for details, see Figure 2). Signs of otherwise normal behaviors, such as rearing, sniffing, grooming, and gnawing, were not included in the evaluation [57]. AIM severity was assessed using the method of Cenci et al. [55], and Lundblad et al. [9], which designates a score from 0 to 4 to each of the four AIM subtypes mentioned before according to the proportion of time/monitoring period through whichever AIM is observed. Borderline scores, such as 0.5, 1.5, 2.5, and 3.5, were allowed to increase the sensitivity of the evaluation Performance during motor tests and AIM analysis was video-recorded (Panasonic camcorder DR-H80 model). Representative still frames were captured from digital video recordings with the video-editing software Final Cut Pro. Pictures were cropped and adjusted for color and All animals were perfused under sodium pentobarbital anesthesia immediately after the 6-month treatments via the aorta, with saline solution followed by fixative including 0.2% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were removed and deposited in the fixative solution for 1 h. For the TH Immunocytochemistry, coronal sections (50 μm) were collected on a vibrating microtome through the mesencephalon. Tyrosine hydroxylase (Chemicon International, Inc., CA, USA; 1:1000) immunostaining with the ABC detection method (Vector Lab MI, USA) was conducted for light microscope analysis. The analysis was performed with a computer-assisted system (Image-Pro Plus, Media Cybernetics, L.P. Del Mar, CA, USA) connected to a CCD camera to Optiphot 2 microscope (Nikon, Japan). The number of TH-positive neurons was calculated in 1500 μm<sup>2</sup> from seven SNc sections of each animal [58]. The dendritic spines analysis was performed by the Golgi method. Blocks from the striatum were cut into 90-μm-thick sections and processed brightness contrast in Adobe Photoshop but were not altered in any other way [57]. during the light period at 14:00 h, a different day for each test. 176 Sex Hormones in Neurodegenerative Processes and Diseases 2.3. AIMs rating [7, 57]. 2.4. Video recording 2.5. Cytological analysis Two-way ANOVA was used to analyze the number of TH-immunoreactive cells, the number of dendritic spines, and the behavioral data. Group differences were considered statistically significant at P < 0.05. When appropriate, post hoc comparisons were made with Tukey test. All analyses were conducted with GraphPad Prism 7 for Mac Software. #### 3. Results After 6 months, neither clinical alterations nor significant weight changes were detected in the experimental animals compared to controls. #### 3.1. Staircase test It is well known that motor behavior is crossed; We lesioned the left MFB (ipsilateral) affecting the right side (contralateral), so we only show the contralateral paw data. For the treatment's effect analysis, data from OVX and W/OV rats were plotted separately (Figure 3A and B, respectively). Those graphs show that all control rats maintained the same number of reaching success through all the study (5.2 0.20–6) comparing to the baseline. In contrast, the 6-OHDA-lesioned rats showed significant motor alterations during the whole study showing a drastic decrease in the number of pellets reached (1.5 0.28–2.2 0.750). 6-OHDA + LD treatment animals presented motor behavior recovery until 21–28 days after treatment, and then, the rats failed in the task (1.8 0.12–2.4 0.47 pellets), behaving very similar to the untreated lesioned animals (Figure 3A and B). Unlike the 6-OHDA + melatonin rat's motor performance, initially they had similar values to untreated 6-OHDA animals (2.2 0.47) and subsequently had a gradual recovery of reaching values (5.8 0.10) as control animals (5.2 0.2). The 6-OHDA + LD/Mel rats showed improvement in the performance from the start, lasting this effect until the end of the study; the number of successes (4.2 0.25–5 0.40) was similar to control animals (5.4 0.24–5.8 0.20) (Figure 3A and B). To compare estrogen protection data from W/OV and OVX, rats were plotted by treatment (Figure 4). We can observe that 6-OHDA + LD OVX rats at 21 days decrease the reaching values (3 0.49) similar to 6-OHDA animals (2.22 0.27), unlike 6-OHDA + LD W/OV rats presented motor impairment until 42 days (2.2 0.026 pellets), this group exhibited delayed deterioration (Figure 4A). W/OV 6-OHDA + melatonin rats, reaching values (5.8 0.10), were Figure 3. Contralateral forelimb staircase test results. The number of reaching successes recorded in W/OV (A) and OVX (B), with the different treatments. \* = P < 0.05 experimental groups vs. control groups; # = P < 0.05 treatments vs. untreated 6-OHDA; & = P < 0.05 6-OHDA + LD vs. 6-OHDA + melatonin; @ = P < 0.05 6-OHDA + LD and 6-OHDA + melatonin vs. 6-OHDA + LD/Mel. similar to the control animals (5.8 0.20) after 42 days of treatment, unlike OVX rats who Figure 4. Estrogen protection in the staircase test contralateral forelimb. The number of reaching successes recorded in W/OV and OVX. 6-OHDA + LD (A), 6-OHDA+ melatonin (B) and 6-OHDA + LD/Mel (C). \* = P < 0.05 experimental groups Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 179 All animals receiving 6-OHDA + LD/Mel perform similarly to control group animals during the 6 months of treatment and displayed no statistically significant differences between them reached control group values (4.25 0.27) at 84 days of treatment (Figure 4B). vs. control groups; # = P < 0.05 treatments vs. untreated 6-OHDA; @ = P < 0.05 W/OV vs. OVX rats. (Figure 4C). Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 179 3. Results 3.1. Staircase test 6-OHDA + LD/Mel. experimental animals compared to controls. 178 Sex Hormones in Neurodegenerative Processes and Diseases After 6 months, neither clinical alterations nor significant weight changes were detected in the It is well known that motor behavior is crossed; We lesioned the left MFB (ipsilateral) affecting the right side (contralateral), so we only show the contralateral paw data. For the treatment's effect analysis, data from OVX and W/OV rats were plotted separately (Figure 3A and B, respectively). Those graphs show that all control rats maintained the same number of reaching success through all the study (5.2 0.20–6) comparing to the baseline. In contrast, the 6-OHDA-lesioned rats showed significant motor alterations during the whole study showing a drastic decrease in the number of pellets reached (1.5 0.28–2.2 0.750). 6-OHDA + LD treatment animals presented motor behavior recovery until 21–28 days after treatment, and then, the rats failed in the task (1.8 0.12–2.4 0.47 pellets), behaving very similar to the untreated lesioned animals (Figure 3A and B). Unlike the 6-OHDA + melatonin rat's motor performance, initially they had similar values to untreated 6-OHDA animals (2.2 0.47) and subsequently had a gradual recovery of reaching values (5.8 0.10) as control animals (5.2 0.2). The 6-OHDA + LD/Mel rats showed improvement in the performance from the start, lasting this effect until the end of the study; the number of successes (4.2 0.25–5 0.40) To compare estrogen protection data from W/OV and OVX, rats were plotted by treatment (Figure 4). We can observe that 6-OHDA + LD OVX rats at 21 days decrease the reaching values (3 0.49) similar to 6-OHDA animals (2.22 0.27), unlike 6-OHDA + LD W/OV rats presented motor impairment until 42 days (2.2 0.026 pellets), this group exhibited delayed deterioration (Figure 4A). W/OV 6-OHDA + melatonin rats, reaching values (5.8 0.10), were Figure 3. Contralateral forelimb staircase test results. The number of reaching successes recorded in W/OV (A) and OVX (B), with the different treatments. \* = P < 0.05 experimental groups vs. control groups; # = P < 0.05 treatments vs. untreated 6-OHDA; & = P < 0.05 6-OHDA + LD vs. 6-OHDA + melatonin; @ = P < 0.05 6-OHDA + LD and 6-OHDA + melatonin vs. was similar to control animals (5.4 0.24–5.8 0.20) (Figure 3A and B). Figure 4. Estrogen protection in the staircase test contralateral forelimb. The number of reaching successes recorded in W/OV and OVX. 6-OHDA + LD (A), 6-OHDA+ melatonin (B) and 6-OHDA + LD/Mel (C). \* = P < 0.05 experimental groups vs. control groups; # = P < 0.05 treatments vs. untreated 6-OHDA; @ = P < 0.05 W/OV vs. OVX rats. similar to the control animals (5.8 0.20) after 42 days of treatment, unlike OVX rats who reached control group values (4.25 0.27) at 84 days of treatment (Figure 4B). All animals receiving 6-OHDA + LD/Mel perform similarly to control group animals during the 6 months of treatment and displayed no statistically significant differences between them (Figure 4C). #### 3.2. Beam walking test Figure 5A and B illustrates the mean numbers of total time to cross the beam, and the treatments' effect in W/OV and OVX rats. 6-OHDA animals significantly increased the time (120 s) compared to control animals (30 s 2.71–40 s 1.3), remaining these values throughout the study. The 6-OHDA + LD group showed statistically significant improvement for about 21–28 days (25 4.26 and 38.5 2.7), displaying scores like the control group (24.8 1.31 and 20.4 2.24 s). Afterward, these rats increased the time (62 6.1), behaving similarly to untreated 6-OHDA group (100 6.7). 6-OHDA + melatonin rats, at the beginning of the treatment, showed increased time to cross the beam (72.7 4.71) to approximately 28 days, with values like 6-OHDA-untreated animals (112.8 7.2), and then, at 42 days, the animals improved their motor activity (46.6 1.83 s), reaching values of control animals (25.6 2.48 s). 6-OHDA + LD/Mel rats presented values (30.40 2.71 to 40.4 1.37 s) similar to control animals during the entire study (Figure 5A and B). Regarding the comparison between estrogen status, we observed that OVX 6-OHDA + LD decreased the time to cross the beam. They have reached values (62 6.1 s) similar to 6-OHDA untreated group (100 6.78) to day 42, and unlike 6-OHDA + LD W/OV showed similar (67 2.14 s) values to 6-OHDA untreated animals (104.5 9.17) from 126 days of treatment, again, we observed that W/OV rats showed delayed motor impairment compared to 6-OHDA + LD OVX rats (Figure 6A). OVX 6-OHDA + melatonin rats increased the time to cross the beam (107.8 2.2 s) as 6-OHDA untreated animals (105.2 9.82 s) until 21 days of treatment; unlike 6-OHDA + melatonin W/OV rats increased the time (60.83 3.95 s); subsequently, after 28 days of treatment, 6-OHDA + melatonin W/OV animals had similar values (46.66 1.86 s) to the control group (25.60 2.48 s), while OVX rats reached values (50 5.6 s) as control animals (30.2 6.55 s) until 70 days of treatment. W/OV 6-OHDA + melatonin rats recovered faster compared to OVX rats (Figure 6B). It is important to note that all 6-OHDA + LD/Mel animals displayed similar (values 19.8 0.97 to 38.75 1.03) to the control animals (30.4 2.71 to 40.4 3.37) over the 6 months of treatment and no statistically Figure 6. Estrogen protection on the beam walking test. The time to cross the beam recorded in W/OV and OVX rats. 6-OHDA + LD (A), 6-OHDA+ melatonin (B) and 6-OHDA + LD/Mel (C). \* = P < 0.05 experimental groups vs. control Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 181 To get an overview of the development of dyskinesia in the different groups, we carried out the summation of all AIMs subtypes (axial + locomotive + limb + orolingual). As shown in significant differences between groups (Figure 6C). groups; # = P < 0.05 treatments vs. untreated 6-OHDA; @ = P < 0.05 W/OV vs. OVX. 3.3. Abnormal involuntary movements 3.3.1. Time course and overall incidence AIMs Figure 5. Beam walking test evaluation W/OV (A) and OVX (B) rats, with the different treatments. \* = P < 0.05 experimental groups vs. control groups; # = P < 0.05 treatments vs. untreated 6-OHDA; & = P < 0.05 6-OHDA + LD vs. 6-OHDA + melatonin; @ = P < 0.05 6-OHDA + LD and 6-OHDA + melatonin vs. 6-OHDA + LD/Mel. Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 181 Figure 6. Estrogen protection on the beam walking test. The time to cross the beam recorded in W/OV and OVX rats. 6-OHDA + LD (A), 6-OHDA+ melatonin (B) and 6-OHDA + LD/Mel (C). \* = P < 0.05 experimental groups vs. control groups; # = P < 0.05 treatments vs. untreated 6-OHDA; @ = P < 0.05 W/OV vs. OVX. recovered faster compared to OVX rats (Figure 6B). It is important to note that all 6-OHDA + LD/Mel animals displayed similar (values 19.8 0.97 to 38.75 1.03) to the control animals (30.4 2.71 to 40.4 3.37) over the 6 months of treatment and no statistically significant differences between groups (Figure 6C). #### 3.3. Abnormal involuntary movements 3.2. Beam walking test 180 Sex Hormones in Neurodegenerative Processes and Diseases animals during the entire study (Figure 5A and B). Figure 5A and B illustrates the mean numbers of total time to cross the beam, and the treatments' effect in W/OV and OVX rats. 6-OHDA animals significantly increased the time (120 s) compared to control animals (30 s 2.71–40 s 1.3), remaining these values throughout the study. The 6-OHDA + LD group showed statistically significant improvement for about 21–28 days (25 4.26 and 38.5 2.7), displaying scores like the control group (24.8 1.31 and 20.4 2.24 s). Afterward, these rats increased the time (62 6.1), behaving similarly to untreated 6-OHDA group (100 6.7). 6-OHDA + melatonin rats, at the beginning of the treatment, showed increased time to cross the beam (72.7 4.71) to approximately 28 days, with values like 6-OHDA-untreated animals (112.8 7.2), and then, at 42 days, the animals improved their motor activity (46.6 1.83 s), reaching values of control animals (25.6 2.48 s). 6-OHDA + LD/Mel rats presented values (30.40 2.71 to 40.4 1.37 s) similar to control Regarding the comparison between estrogen status, we observed that OVX 6-OHDA + LD decreased the time to cross the beam. They have reached values (62 6.1 s) similar to 6-OHDA untreated group (100 6.78) to day 42, and unlike 6-OHDA + LD W/OV showed similar (67 2.14 s) values to 6-OHDA untreated animals (104.5 9.17) from 126 days of treatment, again, we observed that W/OV rats showed delayed motor impairment compared to 6-OHDA + LD OVX rats (Figure 6A). OVX 6-OHDA + melatonin rats increased the time to cross the beam (107.8 2.2 s) as 6-OHDA untreated animals (105.2 9.82 s) until 21 days of treatment; unlike 6-OHDA + melatonin W/OV rats increased the time (60.83 3.95 s); subsequently, after 28 days of treatment, 6-OHDA + melatonin W/OV animals had similar values (46.66 1.86 s) to the control group (25.60 2.48 s), while OVX rats reached values (50 5.6 s) as control animals (30.2 6.55 s) until 70 days of treatment. W/OV 6-OHDA + melatonin rats Figure 5. Beam walking test evaluation W/OV (A) and OVX (B) rats, with the different treatments. \* = P < 0.05 experimental groups vs. control groups; # = P < 0.05 treatments vs. untreated 6-OHDA; & = P < 0.05 6-OHDA + LD vs. 6-OHDA + melatonin; @ = P < 0.05 6-OHDA + LD and 6-OHDA + melatonin vs. 6-OHDA + LD/Mel. #### 3.3.1. Time course and overall incidence AIMs To get an overview of the development of dyskinesia in the different groups, we carried out the summation of all AIMs subtypes (axial + locomotive + limb + orolingual). As shown in Figure 7. Total AIMs (orolingual, axial, forelimb, and locomotive) within 6 months of treatment of W/OV (A) and OVX (B) rats with the different treatments. \* 6-OHDA + LD vs. untreated 6-OHDA and 6-OHDA + melatonin; # 6-OHDA + LD vs. 6-OHDA + LD/Mel. P < 0.05. Figure 7 A and B, repeated-measures ANOVA revealed significant overall differences between untreated 6-OHDA-lesioned (2.5 0.80 to 2.75 0.14) and melatonin-treated groups (5.3 0.30 to 3.16 0.17) comparing to LD-treated groups. 6-OHDA + LD rats from the first month of the evaluation showed high values (12.30 2.068) of MIAs (Figure 7A and B). Remarkably, all 6-OHDA animals receiving LD/Mel coadministration developed MIAs scores (4.80 0.25 to 5.83 1.20) similar to untreated 6-OHDA (2.5 0.80 to 2.75 0.14) and 6-OHDA + melatonin animals (5.3 0.30 to 3.16 0.17) (Figure 7A and B). Concerning total LIDs and the comparison between estrogen status, we observed that OVX 6-OHDA+ LD began to develop LIDs after 3 months of treatment unlike W/OV 6-OHDA + LD rats, which showed LIDs from the first month, and the OVX 6-OHDA + LD group showed delay in LIDs development, the two groups subsequently, had similar scores (18 2.40 for W/OV 6-OHDA + LD rats and 13.5 0.28 OVX 6-OHDA+ LD), and showed no statistically significant differences between them (Figure 8A). W/OV and OVX 6-OHDA + melatonin rats developed low MIAs scores (5.3 0.30–3.16 0.16 and 4.5 0.1–3.25 0.25, respectively) like the untreated 6-OHDA animals (2.5 0.80–2.70 0.14) and showed no statistically significant differences (Figure 8B). It is important to note that all 6-OHDA + LD/Mel rats, since the first evaluation, showed small LID scores (4.80 0.25–5.83 1.20) and no statistically significant differences (Figure 8C). and display no statistically significant differences between groups. In Figure 9A, it can be observed a drastic dopaminergic neuronal loss in the ipsilateral SNc, W/OV and OVX 6-OHDA-lesioned rats had neuronal survival of 3.97% and 6.14%, respectively, like W/OV and OVX 6-OHDA + LD (2.2% and 3.46%) and 6-OHDA + melatonin (3.45% and 5.9%). Note that both W/OV and OVX rats who received 6-OHD + LD/Mel had a higher percentage of cells 7.67% and 10.46%, respectively; however, we found no statistically significant differences Figure 8. W/OV and OVX rats' comparison in total AIMs during 6 months of treatment. 6-OHDA + LD (A), 6-OHDA + melatonin (B), 6-OHDA + LD/Mel (C). \6-OHDA + LD vs. untreated 6-OHDA; @ OVX vs. W/OV rats, P < 0.05. Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 183 Regarding contralateral SNc, Figure 9B shows that W/OV and OVX 6-OHDA-lesioned groups and all animals with 6-OHDA + LD showed a decline of approximately 20% neuronal loss, compared to control groups, and showed no statistically significant differences between between groups. #### 3.4. TH immunocytochemistry Our results show that W/OV and OVX control rats had similar values in the number of THimmunopositive cells, in both ipsilateral and contralateral sides (Figures 9A and B and 10), Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 183 Figure 7 A and B, repeated-measures ANOVA revealed significant overall differences between untreated 6-OHDA-lesioned (2.5 0.80 to 2.75 0.14) and melatonin-treated groups (5.3 0.30 to 3.16 0.17) comparing to LD-treated groups. 6-OHDA + LD rats from the first month of the evaluation showed high values (12.30 2.068) of MIAs (Figure 7A and B). Figure 7. Total AIMs (orolingual, axial, forelimb, and locomotive) within 6 months of treatment of W/OV (A) and OVX (B) rats with the different treatments. \ 6-OHDA + LD vs. untreated 6-OHDA and 6-OHDA + melatonin; # 6-OHDA + LD Remarkably, all 6-OHDA animals receiving LD/Mel coadministration developed MIAs scores (4.80 0.25 to 5.83 1.20) similar to untreated 6-OHDA (2.5 0.80 to 2.75 0.14) and Concerning total LIDs and the comparison between estrogen status, we observed that OVX 6-OHDA+ LD began to develop LIDs after 3 months of treatment unlike W/OV 6-OHDA + LD rats, which showed LIDs from the first month, and the OVX 6-OHDA + LD group showed delay in LIDs development, the two groups subsequently, had similar scores (18 2.40 for W/OV 6-OHDA + LD rats and 13.5 0.28 OVX 6-OHDA+ LD), and showed no statistically significant differences between them (Figure 8A). W/OV and OVX 6-OHDA + melatonin rats developed low MIAs scores (5.3 0.30–3.16 0.16 and 4.5 0.1–3.25 0.25, respectively) like the untreated 6-OHDA animals (2.5 0.80–2.70 0.14) and showed no statistically significant differences (Figure 8B). It is important to note that all 6-OHDA + LD/Mel rats, since the first evaluation, showed small LID scores (4.80 0.25–5.83 1.20) and no statistically significant Our results show that W/OV and OVX control rats had similar values in the number of THimmunopositive cells, in both ipsilateral and contralateral sides (Figures 9A and B and 10), 6-OHDA + melatonin animals (5.3 0.30 to 3.16 0.17) (Figure 7A and B). differences (Figure 8C). vs. 6-OHDA + LD/Mel. P < 0.05. 182 Sex Hormones in Neurodegenerative Processes and Diseases 3.4. TH immunocytochemistry Figure 8. W/OV and OVX rats' comparison in total AIMs during 6 months of treatment. 6-OHDA + LD (A), 6-OHDA + melatonin (B), 6-OHDA + LD/Mel (C). \6-OHDA + LD vs. untreated 6-OHDA; @ OVX vs. W/OV rats, P < 0.05. and display no statistically significant differences between groups. In Figure 9A, it can be observed a drastic dopaminergic neuronal loss in the ipsilateral SNc, W/OV and OVX 6-OHDA-lesioned rats had neuronal survival of 3.97% and 6.14%, respectively, like W/OV and OVX 6-OHDA + LD (2.2% and 3.46%) and 6-OHDA + melatonin (3.45% and 5.9%). Note that both W/OV and OVX rats who received 6-OHD + LD/Mel had a higher percentage of cells 7.67% and 10.46%, respectively; however, we found no statistically significant differences between groups. Regarding contralateral SNc, Figure 9B shows that W/OV and OVX 6-OHDA-lesioned groups and all animals with 6-OHDA + LD showed a decline of approximately 20% neuronal loss, compared to control groups, and showed no statistically significant differences between Figure 9. TH-immunoreactive cell percentages from the ipsilateral (A) and contralateral (B) SNc in the control and experimental groups. The data are depicted as mean SEM. \ Experimental vs. control; # 6-OHDA + melatonin and 6-OHDA + LD/Mel vs. untreated 6-OHDA and 6-OHDA + LD; & 6-OHDA + melatonin vs. 6-OHDA + LD/Mel; P < 0.05. groups. W/OV and OVX rats 6-OHDA + melatonin-treated had values (values to 102% and 111%, respectively) similar to the control group. Surprisingly, the W/OV and OVX 6- OHDA + LD/Mel rats showed a higher percentage of TH-immunopositive cells (22.8% and 27.2%, respectively) compared to control group and no statistically significant differences. #### 3.5. Dendritic spines When performing dendritic spines counting, we observed that control W/OV rats displayed a mean of 7.94 3.23 in the ipsilateral striatum and 7.97 3.47 in the contralateral side; these values were taken as 100%. OVX rats showed a decreased of 21.3% dendritic spines in the ipsilateral striatum and 20.94% in the contralateral side compared to control W/OV (Figure 11A and B), Figure 10. Representative tyrosine hydroxylase immunostained from coronal sections containing the SNc of control, 6-OHDA-untreated, 6-OHDA + LD, 6-OHDA + melatonin and 6-OHDA + LD/Mel-treated rats. Note the significant cell loss in the ipsilateral SNc in the four experimental groups (arrows), being more evident in the untreated 6-OHDA and LD treated ones; also, the contralateral SNc of melatonin and LD/Mel-treated rats lost fewer neurons than the other two Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 185 experimental groups, and LD/Mel-treated rats had more neurons than control rats (magnification 4). Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 185 Figure 10. Representative tyrosine hydroxylase immunostained from coronal sections containing the SNc of control, 6-OHDA-untreated, 6-OHDA + LD, 6-OHDA + melatonin and 6-OHDA + LD/Mel-treated rats. Note the significant cell loss in the ipsilateral SNc in the four experimental groups (arrows), being more evident in the untreated 6-OHDA and LD treated ones; also, the contralateral SNc of melatonin and LD/Mel-treated rats lost fewer neurons than the other two experimental groups, and LD/Mel-treated rats had more neurons than control rats (magnification 4). groups. W/OV and OVX rats 6-OHDA + melatonin-treated had values (values to 102% and 111%, respectively) similar to the control group. Surprisingly, the W/OV and OVX 6- OHDA + LD/Mel rats showed a higher percentage of TH-immunopositive cells (22.8% and 27.2%, respectively) compared to control group and no statistically significant differences. Figure 9. TH-immunoreactive cell percentages from the ipsilateral (A) and contralateral (B) SNc in the control and experimental groups. The data are depicted as mean SEM. \* Experimental vs. control; # 6-OHDA + melatonin and 6-OHDA + LD/Mel vs. untreated 6-OHDA and 6-OHDA + LD; & 6-OHDA + melatonin vs. 6-OHDA + LD/Mel; P < 0.05. When performing dendritic spines counting, we observed that control W/OV rats displayed a mean of 7.94 3.23 in the ipsilateral striatum and 7.97 3.47 in the contralateral side; these values were taken as 100%. OVX rats showed a decreased of 21.3% dendritic spines in the ipsilateral striatum and 20.94% in the contralateral side compared to control W/OV (Figure 11A and B), 3.5. Dendritic spines 184 Sex Hormones in Neurodegenerative Processes and Diseases Figure 11. Striatal medium-sized spiny neurons dendritic spine percentage ipsilateral (A) and contralateral (B). \* Experimental vs. control; # 6-OHDA + melatonin and 6-OHDA + LD/Mel vs. untreated 6-OHDA and 6-OHDA + LD; @ untreated 6-OHDA OVX vs. untreated 6-OHDA W/OV, P < 0.05. In the ipsilateral striatum, W/OV and OVX 6-OHDA-lesioned rats and 6-OHDA + LD-treated rats presented severe dendritic spines loss (50, 44, 49, and 51%, respectively), unlike 6-OHDA + melatonin-treated (72 and 73%) and 6-OHDA + LD/Mel coadministration rats (77 and 73%), which showed a greater number of dendritic spines and showed no significant differences between groups (Figures 11A and 12). Regarding contralateral striatum, we observed that OVX untreated 6-OHDA rats displayed higher dendritic spines loss (41%) compared to W/OV 6-OHDA untreated animals (36%), showing statistically significant differences. W/OV and OVX all 6-OHDA + LD groups showed significant dendritic spines loss (35% and 34%), showing similar values with W/OV and OVX untreated 6-OHDA rats (36% and 41%), with no statistically significant differences between groups. W/OV 6-OHDA + melatonin (95%) and W/OV 6-OHDA + LD/Mel (99%) rats had similar values for the number of dendritic spines to control group. OVX 6-OHDA + melatonin (92%) and OVX 6-OHDA + LD/Mel-treated rats (97%) had a higher percentage of dendritic spines compared to control group (76%), showing increased number of dendritic spines similar to control W/OV group (Figures 11B and 12). 4. Discussion 4.1. Staircase test melatonin, and the estrogen presence. Our data show that the LD/Mel coadministration and the estrogen presence appear to be a very effective combination to reduce AIMs through the conservation of some functional SNc dopaminergic cells, which in turn imply a well-preserved neuropil of a less denervated striatum. We assume that these results are probably because of a synergistic effect between LD, Figure 12. Representative micrographs of Golgi-stained medium-sized spiny neurons of the ipsilateral striatum with an illustrative box of dendritic spine densities from the control group (A), untreated 6-OHDA group (B), 6-OHDA + LD (C), 6-OHDA + melatonin (D), and 6OHDA + LD/Mel (E). Both untreated 6-OHDA and LD-treated induced a marked decrease in the total number of spines mainly in the ipsilateral striatum. In contrast, melatonin and LD/Mel-treated Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 187 groups showed a well-preserved dendritic spine density (magnification, 40 and 100). It has been reported that PD patients have poor manual skills that worsen as the disease progresses, and patients have difficulty performing tasks that require sequential movements, Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 187 Figure 12. Representative micrographs of Golgi-stained medium-sized spiny neurons of the ipsilateral striatum with an illustrative box of dendritic spine densities from the control group (A), untreated 6-OHDA group (B), 6-OHDA + LD (C), 6-OHDA + melatonin (D), and 6OHDA + LD/Mel (E). Both untreated 6-OHDA and LD-treated induced a marked decrease in the total number of spines mainly in the ipsilateral striatum. In contrast, melatonin and LD/Mel-treated groups showed a well-preserved dendritic spine density (magnification, 40 and 100). #### 4. Discussion In the ipsilateral striatum, W/OV and OVX 6-OHDA-lesioned rats and 6-OHDA + LD-treated rats presented severe dendritic spines loss (50, 44, 49, and 51%, respectively), unlike 6-OHDA + melatonin-treated (72 and 73%) and 6-OHDA + LD/Mel coadministration rats (77 and 73%), which showed a greater number of dendritic spines and showed no significant differences between groups (Figures 11A and 12). Regarding contralateral striatum, we observed that OVX untreated 6-OHDA rats displayed higher dendritic spines loss (41%) compared to W/OV 6-OHDA untreated animals (36%), showing statistically significant differences. W/OV and OVX all 6-OHDA + LD groups showed significant dendritic spines loss (35% and 34%), showing similar values with W/OV and OVX untreated 6-OHDA rats (36% and 41%), with no statistically significant differences between groups. W/OV 6-OHDA + melatonin (95%) and W/OV 6-OHDA + LD/Mel (99%) rats had similar values for the number of dendritic spines to control group. OVX 6-OHDA + melatonin (92%) and OVX 6-OHDA + LD/Mel-treated rats (97%) had a higher percentage of dendritic spines compared to control group (76%), showing increased number of dendritic spines similar to control W/OV group (Figures 11B and 12). Figure 11. Striatal medium-sized spiny neurons dendritic spine percentage ipsilateral (A) and contralateral (B). \* Experimental vs. control; # 6-OHDA + melatonin and 6-OHDA + LD/Mel vs. untreated 6-OHDA and 6-OHDA + LD; @ untreated 6-OHDA OVX vs. untreated 6-OHDA W/OV, P < 0.05. 186 Sex Hormones in Neurodegenerative Processes and Diseases Our data show that the LD/Mel coadministration and the estrogen presence appear to be a very effective combination to reduce AIMs through the conservation of some functional SNc dopaminergic cells, which in turn imply a well-preserved neuropil of a less denervated striatum. We assume that these results are probably because of a synergistic effect between LD, melatonin, and the estrogen presence. #### 4.1. Staircase test It has been reported that PD patients have poor manual skills that worsen as the disease progresses, and patients have difficulty performing tasks that require sequential movements, for example, when performing repetitive movements of forearm pronation and supination, openness and closing hand and reaching for objects [59]. The rats' movements in the staircase test are very like humans, so that test allows evaluating DA deficiency and treatments effectiveness [48, 59]. The rats' movement in the staircase test by using the forelimb to keep the pellet and eat it is a complex and anomalous activity for animals with 6-OHDA unilateral lesion. According to our results, all untreated 6-OHDA animals showed severe motor damage mainly affecting the contralateral side, expressed by the drastic reduction in the number of pellets eaten, which is consistent with other authors [60, 61]; the performance of this activity was abnormal, and although sometimes the rats obtained the food pellet successfully, supination and pronation movements were limited compared to control animals. Some rats also use compensatory strategies such as increasing their digit pressure and frequently used tongue and teeth to achieve the pellet [59]. In this respect, it is known that motor alterations in the staircase test depend on the striatonigral dopaminergic system integrity [60, 62, 63]. Besides, several authors have reported that animals with this motor impairments display severe SNc TH-immunopositive neuronal loss and fewer DA fibers in the striatum [60, 64]. enzymes, which act on the 6-OHDA-free radicals; in addition, it is known that these enzymes are diminished in the DA-depleted brain [74, 75]. Previous studies have shown that short-term melatonin treatment does not exert a neuroprotective effect in DA-depleted animals [76, 77], probably due to the fact that this neurohormone levels are low in the brain [76]. In this sense, it is suggested that melatonin level has to be high and continuously maintained for a long time in Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 189 Remarkably, as shown in our results, all animals treated with 6-OHDA + LD/Mel coadministration showed improvement in their motor performance in the staircase test from the beginning of treatment. We also observed that these animals improved the digit contraction and projection movements, pronation, and supination, in comparison with the other groups. The neuroprotective effect we observed is probably due to the melatonin's characteristic as an antioxidant, avoiding LD autoxidation and the consequent ROS formation, thus restoring LD This test evaluates stereotyped movements, coordination, and motor alterations characteristic of PD in this animal model [80]. The device we used implied greater difficulty in its execution due to the thickness of the beam (12 mm). Besides, when placed diagonally to 15 to the floor, it required more effort to maintain a stable position. In humans, balance deterioration occurs when the loss of dopaminergic neurons is >70% [81]. Bracha et al. [82] report that PD patients, when tested showed asymmetry toward the hemisphere containing less dopaminergic activity, decreased movement initiation (akinesia), and walking was slow and presented postural changes. So that it is suggested that these changes may be similar in hemiparkinsonian rats, The data obtained from the 6-OHDA + LD animals are consistent with data previously reported in our laboratory, where 6-OHDA LD-treated rats show motor activity recovery in the first days of treatment, but after 28 days dramatically increased the time to pass the beam [57]. PD patients' studies treated with LD showed a significant increase in walking speed and balance [83]. In our study, we observed that the animals frequently interrupted their ascent and slipped due to the low digit clamping force produced by the lesion, which is not reversed by LD treatment [68]. The SNc degeneration produced by 6-OHDA lesion considerably decreases LD therapeutic benefit [71] probably because this drug produces oxidative stress and therefore increases the neurodegeneration of the remaining dopaminergic cells [84]. In addition, when animals attempted to 6-OHDA + melatonin-treated animals, after 42 days, showed gradual motor activity recovery, suggesting that somehow melatonin contributed to the improvement motor coordination [57]; which may contribute to motor deficit observed in the beam walking test [50]. traverse the beam, they stopped because they had axial and limb-type AIMs. the brain to guarantee its neuroprotective effect [76, 78, 79]. levels and increasing striatal DA bioavailability [26, 27]. 4.4. LD/Mel treatment 4.5. Beam walking test 4.6. LD treatment 4.7. Melatonin treatment #### 4.2. LD treatment As our results show, all 6-OHDA + LD animals showed recovery since the first day to 21– 28 days of treatment. Subsequently, they displayed notorious motor alterations. Thus, our results are consistent with previous studies where it has been observed that PD-experimental animals LD-treatment therapeutic benefit in rodents are approximately 3 weeks [57, 65]. In this respect, it has also been reported that LD-treated PD patients improve the motor response in tests that include taking objects on a surface, display greater coordination, and recover the movement initiation [66, 67]. However, when LD treatment is chronically administered (6– 13 months), patients do not improve and show alteration in elbow flexion, supination, pronation, and bradykinesia [68]. It is suggested that, after a while, LD treatment is no longer effective [66, 67, 69, 70]. In our results, we also observed 6-OHDA + LD animals when they used the contralateral forelimb, the movement was limited, and the limb tended to remain flexed, which are clear signs of hypokinesia and rigidity. It is important to note that with chronic LD treatment, the animals showed mainly orolingual, axial, and limb-type dyskinesia at the time they were evaluated in the staircase test. Therefore, the pellets were harder to take, corresponding with Winkler et al. [71] results. Besides, it is considered that the motivation that leads the animal to get the food pellets is the food restriction [48], generating anxiety and promoting the realization of quick and inaccurate limb movements [72]. #### 4.3. Melatonin treatment All 6-OHDA + melatonin-treated animals behaved very similarly to untreated 6-OHDA animals at the beginning of the treatment; later at approximately 21 days, they showed gradual improvement. In a study conducted by Singh et al. [73], they show that 35 days of melatonin treatment in 6-OHDA-lesioned animals, they display improvement in posture and ability to take the food pellets in the staircase test with the contralateral forelimb, coinciding with our data, since we found improvement in the animals between days 28 and 42. These authors propose that melatonin neuroprotective effect is due to its ability to stimulate antioxidant enzymes, which act on the 6-OHDA-free radicals; in addition, it is known that these enzymes are diminished in the DA-depleted brain [74, 75]. Previous studies have shown that short-term melatonin treatment does not exert a neuroprotective effect in DA-depleted animals [76, 77], probably due to the fact that this neurohormone levels are low in the brain [76]. In this sense, it is suggested that melatonin level has to be high and continuously maintained for a long time in the brain to guarantee its neuroprotective effect [76, 78, 79]. #### 4.4. LD/Mel treatment for example, when performing repetitive movements of forearm pronation and supination, openness and closing hand and reaching for objects [59]. The rats' movements in the staircase test are very like humans, so that test allows evaluating DA deficiency and treatments effectiveness [48, 59]. The rats' movement in the staircase test by using the forelimb to keep the pellet and eat it is a complex and anomalous activity for animals with 6-OHDA unilateral lesion. According to our results, all untreated 6-OHDA animals showed severe motor damage mainly affecting the contralateral side, expressed by the drastic reduction in the number of pellets eaten, which is consistent with other authors [60, 61]; the performance of this activity was abnormal, and although sometimes the rats obtained the food pellet successfully, supination and pronation movements were limited compared to control animals. Some rats also use compensatory strategies such as increasing their digit pressure and frequently used tongue and teeth to achieve the pellet [59]. In this respect, it is known that motor alterations in the staircase test depend on the striatonigral dopaminergic system integrity [60, 62, 63]. Besides, several authors have reported that animals with this motor impairments display severe SNc TH-immunopositive neuronal loss and fewer DA fibers in the striatum [60, 64]. promoting the realization of quick and inaccurate limb movements [72]. As our results show, all 6-OHDA + LD animals showed recovery since the first day to 21– 28 days of treatment. Subsequently, they displayed notorious motor alterations. Thus, our results are consistent with previous studies where it has been observed that PD-experimental animals LD-treatment therapeutic benefit in rodents are approximately 3 weeks [57, 65]. In this respect, it has also been reported that LD-treated PD patients improve the motor response in tests that include taking objects on a surface, display greater coordination, and recover the movement initiation [66, 67]. However, when LD treatment is chronically administered (6– 13 months), patients do not improve and show alteration in elbow flexion, supination, pronation, and bradykinesia [68]. It is suggested that, after a while, LD treatment is no longer effective [66, 67, 69, 70]. In our results, we also observed 6-OHDA + LD animals when they used the contralateral forelimb, the movement was limited, and the limb tended to remain flexed, which are clear signs of hypokinesia and rigidity. It is important to note that with chronic LD treatment, the animals showed mainly orolingual, axial, and limb-type dyskinesia at the time they were evaluated in the staircase test. Therefore, the pellets were harder to take, corresponding with Winkler et al. [71] results. Besides, it is considered that the motivation that leads the animal to get the food pellets is the food restriction [48], generating anxiety and All 6-OHDA + melatonin-treated animals behaved very similarly to untreated 6-OHDA animals at the beginning of the treatment; later at approximately 21 days, they showed gradual improvement. In a study conducted by Singh et al. [73], they show that 35 days of melatonin treatment in 6-OHDA-lesioned animals, they display improvement in posture and ability to take the food pellets in the staircase test with the contralateral forelimb, coinciding with our data, since we found improvement in the animals between days 28 and 42. These authors propose that melatonin neuroprotective effect is due to its ability to stimulate antioxidant 4.2. LD treatment 188 Sex Hormones in Neurodegenerative Processes and Diseases 4.3. Melatonin treatment Remarkably, as shown in our results, all animals treated with 6-OHDA + LD/Mel coadministration showed improvement in their motor performance in the staircase test from the beginning of treatment. We also observed that these animals improved the digit contraction and projection movements, pronation, and supination, in comparison with the other groups. The neuroprotective effect we observed is probably due to the melatonin's characteristic as an antioxidant, avoiding LD autoxidation and the consequent ROS formation, thus restoring LD levels and increasing striatal DA bioavailability [26, 27]. #### 4.5. Beam walking test This test evaluates stereotyped movements, coordination, and motor alterations characteristic of PD in this animal model [80]. The device we used implied greater difficulty in its execution due to the thickness of the beam (12 mm). Besides, when placed diagonally to 15 to the floor, it required more effort to maintain a stable position. In humans, balance deterioration occurs when the loss of dopaminergic neurons is >70% [81]. Bracha et al. [82] report that PD patients, when tested showed asymmetry toward the hemisphere containing less dopaminergic activity, decreased movement initiation (akinesia), and walking was slow and presented postural changes. So that it is suggested that these changes may be similar in hemiparkinsonian rats, which may contribute to motor deficit observed in the beam walking test [50]. #### 4.6. LD treatment The data obtained from the 6-OHDA + LD animals are consistent with data previously reported in our laboratory, where 6-OHDA LD-treated rats show motor activity recovery in the first days of treatment, but after 28 days dramatically increased the time to pass the beam [57]. PD patients' studies treated with LD showed a significant increase in walking speed and balance [83]. In our study, we observed that the animals frequently interrupted their ascent and slipped due to the low digit clamping force produced by the lesion, which is not reversed by LD treatment [68]. The SNc degeneration produced by 6-OHDA lesion considerably decreases LD therapeutic benefit [71] probably because this drug produces oxidative stress and therefore increases the neurodegeneration of the remaining dopaminergic cells [84]. In addition, when animals attempted to traverse the beam, they stopped because they had axial and limb-type AIMs. #### 4.7. Melatonin treatment 6-OHDA + melatonin-treated animals, after 42 days, showed gradual motor activity recovery, suggesting that somehow melatonin contributed to the improvement motor coordination [57]; so the animals were probably able to make optimal postural adjustments to maintain the balance and move on the beams. Patki and Lau [85] performed a study on DA-depleted animals, which were continuously melatonin-treated for 18 weeks, and when evaluating the animals in the beam walking test, they observed improvement in motor coordination compared to animals that did not receive treatment. In addition, chronic melatonin treatment increased striatal DA levels, so the authors conclude that long-term melatonin treatment has a neuroprotective potential to preserve nigrostriatal dopaminergic function. Probably because during the treatment, high and constant melatonin levels were maintained in the brain [79]. On the other hand, 6-OHDA + LD/Mel animals displayed low AIM scores compared to those receiving exclusively LD, showing that somehow melatonin has some influence on LIDs. It is important to stand out that there are no studies on the effect of LD/Mel coadministration on LIDs in PD. However, several authors suggest that melatonin may have a beneficial effect on LIDs because of its antioxidant properties [27, 98] and its ability to stimulate antioxidant enzymes [99]. Rocchitta et al. [100] reported that LD/Mel coadministration inhibits LD autoxidation, thereby increasing striatal DA bioavailability, and then, melatonin appears to be the most suitable antioxidant drug to be used as LD adjuvant to avoid LD and DA nonenzymatic autoxidation. According to these studies, it is feasible to think that with LD/Mel coadmini- Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 191 As expected, MFB 6-OHDA lesion drastically reduced the number of TH-immunopositive neurons in the SNc, coinciding with previous works in PD patients [101] and 6-OHDA model [80, 102–105]. Thus, it is suggested that this model simulates PD advanced stages. The precise 6- OHDA cytotoxicity molecular mechanism remains under discussion. Several hypotheses have been proposed. One of which is related to free radical formation, in addition to decreasing mitochondrial complex I activity with the consequent ATP decrease and cell death [106], which has also been reported in PD postmortem studies [107]. Moreover, LD-treated animals showed a dramatic loss of TH-immunopositive cells and both, the ipsilateral and contralateral SNc, similar to untreated 6-OHDA-lesioned rats, features are also reported by Smith et al. [108] and by our group [57]. In vivo and in vitro studies confirm that LD-treatment decreases TH-immunopositive cells; these results suggest that LD induces cell death due primarily to the ROS generation [12, 25, 27], which may increase oxidative stress in the nigrostriatal pathway [109, 110]. In addition, previous studies in our laboratory showed that hemiparkinsonian LD-treated animals displayed increased levels of lipid peroxidation, which is the principal oxidative stress characteristic [57]. Melatonin treatment favored SNc dopaminergic neuron preservation compared to untreated rats, consistent with previous studies [57, 85]. It is proposed that melatonin protection may be by direct antioxidant action [57, 111, 112] or by indirect stimulating antioxidant enzymes [112, 113]. LD/Mel-treated animals had lower SNc TH-immunopositive cell death compared to the other groups, although no significant differences; so, it is feasible to think that this small percentage of cells could be involved in improving motor tests and decreased dyskinesia. Surprisingly, on the contralateral SNc, the animals showed dopaminergic neurons increase, probably trying to compensate ipsilateral SNc damage. In this regard, it has been reported that DA is essential for neurogenesis, which was evidenced in DA-depleted animals [114, 115], and this effect was reversed when given LD [116]. Apparently, the neurogenic effect is modulated Our results show that dopaminergic denervation produced by 6-OHDA results in loss of striatal neuron dendritic spines. PD patients' postmortem studies have shown 30% decrease, and this loss can reach 50% in dendritic spine density, and the reduction in the size of dendritic stration the DA concentration fluctuations are avoided, thus reducing LIDs. 4.10. TH immunocytochemistry by activation of DA receptors [115]. 4.11. Dendritic spines #### 4.8. LD/Mel treatment Animals receiving chronic LD/Mel coadministration showed recovery of motor coordination; the animals cross the beam alternating the limbs, which made the movement faster and better so that they presented similar times to pass the beam to the control group animals throughout the experiment. In this regard, recent studies show that melatonin, given in conjunction with LD in MPTP mice, reverses akinesia by restoring the number of dendritic spines in mediumsized spiny neurons and attenuating striatal DA loss. Proposing that melatonin could be an ideal LD adjuvant in PD therapy [77]. In this sense, our data also showed that animals receiving LD/Mel treatment had preservation of dendritic spines and more dopaminergic neurons on the contralateral SNc, so it is feasible to think that maintaining the nigrostriatal connections would allow the animals to make optimal adjustments in their movements to maintain the balance and move better over the beam. #### 4.9. Abnormal involuntary movements As shown in Figures 7 and 8, untreated DA-depleted animals had small AIM scores compared to those receiving LD treatment, which is consistent with results of other authors [71, 86]. Also, animals receiving melatonin treatment showed similar behavior, corroborating these data with those previously reported by our group [57]. These groups of animals are primarily characterized by having contralateral and orolingual AIMs (considered as resting tremor [71]). Previous studies suggest that rat AIMs, regarding severity and topographical distribution, are related to striatal dopaminergic denervation [71], and this can be explained by the somatotopic organization of this structure. According to this, the dorsolateral striatum controls jaw and limb movements. Abnormal function of this region is correlated with the presence of orolingual AIMs [87, 88]. Some studies have shown that the response to LD changes with the progression of the disease. Deogaonkar and Subramanian [89] demonstrated that LD minimal dose produces dyskinesias in PD patients in advanced stages compared to patients in early stages, suggesting that the LD therapeutic window is lost in advanced stages of the disease. The DA fluctuations are closely related to the development of LIDs [90]. Furthermore, LD treatment triggers LIDs via signaling pathways in striatonigral neurons, probably by D1 and D2 receptors' stimulation [91]. On the other hand, there are data which sustained that dopaminergic depletion can generate changes in the postsynaptic neurons, which involve modifications in the neuronal morphology and striatal dendritic spines loss, which would result in a decrease in synaptic connections [92–97]. On the other hand, 6-OHDA + LD/Mel animals displayed low AIM scores compared to those receiving exclusively LD, showing that somehow melatonin has some influence on LIDs. It is important to stand out that there are no studies on the effect of LD/Mel coadministration on LIDs in PD. However, several authors suggest that melatonin may have a beneficial effect on LIDs because of its antioxidant properties [27, 98] and its ability to stimulate antioxidant enzymes [99]. Rocchitta et al. [100] reported that LD/Mel coadministration inhibits LD autoxidation, thereby increasing striatal DA bioavailability, and then, melatonin appears to be the most suitable antioxidant drug to be used as LD adjuvant to avoid LD and DA nonenzymatic autoxidation. According to these studies, it is feasible to think that with LD/Mel coadministration the DA concentration fluctuations are avoided, thus reducing LIDs. #### 4.10. TH immunocytochemistry so the animals were probably able to make optimal postural adjustments to maintain the balance and move on the beams. Patki and Lau [85] performed a study on DA-depleted animals, which were continuously melatonin-treated for 18 weeks, and when evaluating the animals in the beam walking test, they observed improvement in motor coordination compared to animals that did not receive treatment. In addition, chronic melatonin treatment increased striatal DA levels, so the authors conclude that long-term melatonin treatment has a neuroprotective potential to preserve nigrostriatal dopaminergic function. Probably because during the treatment, high and constant melatonin levels were maintained in the brain [79]. Animals receiving chronic LD/Mel coadministration showed recovery of motor coordination; the animals cross the beam alternating the limbs, which made the movement faster and better so that they presented similar times to pass the beam to the control group animals throughout the experiment. In this regard, recent studies show that melatonin, given in conjunction with LD in MPTP mice, reverses akinesia by restoring the number of dendritic spines in mediumsized spiny neurons and attenuating striatal DA loss. Proposing that melatonin could be an ideal LD adjuvant in PD therapy [77]. In this sense, our data also showed that animals receiving LD/Mel treatment had preservation of dendritic spines and more dopaminergic neurons on the contralateral SNc, so it is feasible to think that maintaining the nigrostriatal connections would allow the animals to make optimal adjustments in their movements to As shown in Figures 7 and 8, untreated DA-depleted animals had small AIM scores compared to those receiving LD treatment, which is consistent with results of other authors [71, 86]. Also, animals receiving melatonin treatment showed similar behavior, corroborating these data with those previously reported by our group [57]. These groups of animals are primarily characterized by having contralateral and orolingual AIMs (considered as resting tremor [71]). Previous studies suggest that rat AIMs, regarding severity and topographical distribution, are related to striatal dopaminergic denervation [71], and this can be explained by the somatotopic organization of this structure. According to this, the dorsolateral striatum controls jaw and limb movements. Abnormal function of this region is correlated with the presence of orolingual AIMs [87, 88]. Some studies have shown that the response to LD changes with the progression of the disease. Deogaonkar and Subramanian [89] demonstrated that LD minimal dose produces dyskinesias in PD patients in advanced stages compared to patients in early stages, suggesting that the LD therapeutic window is lost in advanced stages of the disease. The DA fluctuations are closely related to the development of LIDs [90]. Furthermore, LD treatment triggers LIDs via signaling pathways in striatonigral neurons, probably by D1 and D2 receptors' stimulation [91]. On the other hand, there are data which sustained that dopaminergic depletion can generate changes in the postsynaptic neurons, which involve modifications in the neuronal morphology and striatal dendritic spines loss, which would result in a decrease in 4.8. LD/Mel treatment 190 Sex Hormones in Neurodegenerative Processes and Diseases maintain the balance and move better over the beam. 4.9. Abnormal involuntary movements synaptic connections [92–97]. As expected, MFB 6-OHDA lesion drastically reduced the number of TH-immunopositive neurons in the SNc, coinciding with previous works in PD patients [101] and 6-OHDA model [80, 102–105]. Thus, it is suggested that this model simulates PD advanced stages. The precise 6- OHDA cytotoxicity molecular mechanism remains under discussion. Several hypotheses have been proposed. One of which is related to free radical formation, in addition to decreasing mitochondrial complex I activity with the consequent ATP decrease and cell death [106], which has also been reported in PD postmortem studies [107]. Moreover, LD-treated animals showed a dramatic loss of TH-immunopositive cells and both, the ipsilateral and contralateral SNc, similar to untreated 6-OHDA-lesioned rats, features are also reported by Smith et al. [108] and by our group [57]. In vivo and in vitro studies confirm that LD-treatment decreases TH-immunopositive cells; these results suggest that LD induces cell death due primarily to the ROS generation [12, 25, 27], which may increase oxidative stress in the nigrostriatal pathway [109, 110]. In addition, previous studies in our laboratory showed that hemiparkinsonian LD-treated animals displayed increased levels of lipid peroxidation, which is the principal oxidative stress characteristic [57]. Melatonin treatment favored SNc dopaminergic neuron preservation compared to untreated rats, consistent with previous studies [57, 85]. It is proposed that melatonin protection may be by direct antioxidant action [57, 111, 112] or by indirect stimulating antioxidant enzymes [112, 113]. LD/Mel-treated animals had lower SNc TH-immunopositive cell death compared to the other groups, although no significant differences; so, it is feasible to think that this small percentage of cells could be involved in improving motor tests and decreased dyskinesia. Surprisingly, on the contralateral SNc, the animals showed dopaminergic neurons increase, probably trying to compensate ipsilateral SNc damage. In this regard, it has been reported that DA is essential for neurogenesis, which was evidenced in DA-depleted animals [114, 115], and this effect was reversed when given LD [116]. Apparently, the neurogenic effect is modulated by activation of DA receptors [115]. #### 4.11. Dendritic spines Our results show that dopaminergic denervation produced by 6-OHDA results in loss of striatal neuron dendritic spines. PD patients' postmortem studies have shown 30% decrease, and this loss can reach 50% in dendritic spine density, and the reduction in the size of dendritic trees [92, 117]. Similarly, MPTP nonhuman primates and 6-OHDA-lesioned rodents show drastic loss of these structures [7, 57], suggesting nigrostriatal system importance in morphological regulation and plasticity of dendritic spines in the striatum [104]. We have observed that LD chronic treatment does not restore striatal dendritic spine density, which is consistent with previous PD postmortem studies that show that the loss of dendritic spines was present even though all patients were treated with LD for several years [117]. Deutch et al. [92] propose that LD may be ineffective in PD advanced stages, probably due to dendritic spine loss. In rodents with different models of PD LD-treated, the number of dendritic spines [7, 77, 92] is not restored. We also observed that melatonin treatment helped the conservation of dendritic spines. In this regard, it is reported that melatonin prevents cytoskeletal damage by reducing oxidative stress [118]. Interestingly, our results also show that there is a difference between the estrogen condition regarding dyskinesias and motor behavior, noting that W/OV 6-OHDA + LD have motor impairment delayed but are more likely to develop dyskinesias compared to OVX 6-OHDA + LD, which is consistent with previous studies, which shows that there are sex differences in LD treatment, showing that women performed better in the UPDRS test (unified Parkinson's disease Rating Scale) and presented longer "on"-LD state compared to men. However, women had a higher prevalence to develop dyskinesias. It is still uncertain why women are more prone than men to develop dyskinesias, but it is suggested that estrogen may be the basis of this susceptibility [123, 126, 127]. One possible explanation for such proneness is the fact that humans and rats have similar expression characteristics of the catechol-o-methyl transferase (COMT) [128], which is a catecholamine degrading enzyme, and that women have 20–30% decrease in COMT activity compared to men [129]. In this regard, it has been demonstrated that estrogen can decrease the regulation of COMT gene [130]. Therefore, if estrogens decline, the COMT system could have a pharmacological potential to increase the LD striatal availabil- Differences Between Intact and Ovariectomized Hemiparkinsonian Rats in Response to L-DOPA, Melatonin, and… http://dx.doi.org/10.5772/intechopen.70898 193 On the other hand, W/OV melatonin-treated rats recover faster in behavioral tests compared to OVX rats, and in the last month of treatment, all animals had similar control values; cytologically W/OV rats exhibited contralateral SNc dopaminergic cell protection and dendritic spine recovery of both ipsilateral and contralateral striatum. Studies of melatonin and estrogen therapy in neurodegenerative models are few, so this work provides new knowledge about it. In a study of W/OV rats that were subjected to a stroke model and receiving melatonin, it was observed that estrogen and melatonin exhibit synergistic effect to decrease the levels of lipid peroxidation, increasing the activity of free radical scavenger and the number of surviving neurons in the cortex, and improve sensorimotor behaviors [131]. According to these studies, we can expect that after 6-OHDA injection melatonin treatment and estrogen presence in W/ OV rats work together to activate different signaling pathways to reduce oxidative stress and thus protect the dopaminergic neurons (at least the contralateral SNc) and the number of striatal dendritic spines and thus improve motor capacity; this could be a possible explanation of why W/OV rats tend to recover faster in motor performance compared to OVX rats. and cell survival [77, 131], and motor disorders such as dyskinesias. diseases, so careful and controlled administration of estrogens is required. Interestingly, animals receiving LD/Mel treatment showed behavioral recovery from the start of the treatment, increase in the number of dopaminergic neurons in the contralateral SNc and striatal ipsi and contralateral dendritic spine protection; these results were estrogen independent. So, we suggest that LD/Mel cotreatment could improve the LD efficacy by increasing striatal DA levels [77]. Also, it has been suggested that these drugs could act synergistically to exert a modulatory role in nigrostriatal transmission pathway, which may be responsible for many of the beneficial effects, such as biochemical alterations, regulation of dendritic spines Finally, it is important to mention that estrogens act as neuroprotectors in neurodegenerative diseases such as PD and Alzheimer disease, improving women quality of life [126]. But it should be noted that the use of estrogen also involves risks. Women who take hormone replacement treatment are more likely to suffer cancer [132] if there is a family history of these ity and prolong LD-"on" state as well as dyskinesias [130]. LD/Mel coadministration significantly restored the dendritic spine density of both ipsilateral and contralateral striatum. Recent studies show that the presence of dopaminergic neurons enhances dendritic spine formation in medium spiny neurons in culture. So it is possible that dopaminergic neurons have neurotrophic effect [119]. In this context, it is feasible to think that as LD/Mel coadministration increases the number of dopaminergic neurons in contralateral SNc and exerts a neurotrophic effect, promoting the formation of new dendritic spines. Our results are also consistent with those described by Naskar et al. [77], who show that MPTPexposed rodents and LD/Mel-treated for 2 days have restored the morphology and density of dendritic spines of medium-sized spiny neurons, suggesting that melatonin primarily regulates this effect due to its characteristics of reducing excessive calcium flow. #### 4.12. W/OV and OVX comparison In our study, we show that W/OV rats, which were 6-OHDA-lesioned and received different treatments, have greater neuroprotection compared to OVX females, confirming the estrogen protection, besides the neurodegeneration delay difference, suggesting beneficial estrogen effect in the development and progression of the disease. It has been observed that estrogen has a neuroprotective effect on the nigrostriatal system. Recent studies suggest that PD women tend to have a delay in the appearance of certain motor symptoms compared with men [33, 120]. Furthermore, PD postmenopausal women treated with estrogen showed improvement in their motor performance [121, 122], suggesting estrogen symptomatic role [123]. As our results indicate, 6-OHDA-untreated W/OV and OVX rats showed no significant difference from both ipsilateral and contralateral SNc dopaminergic cells. This could be because the neurotoxin is very aggressive, which somehow does not allow the cell survival. It is also proposed that estrogens protect dopaminergic neurons surviving for a short time, but subsequently the cells could die as a result of neurotoxin action [124]. Previous studies showed the effect of replacement estrogen therapy in physiological doses in OVX rats after 6-OHDA injection in the nigrostriatal pathway, reporting that estrogen treatment showed no effect on survival of TH-immunopositive cells. Nonetheless, estrogen attenuated the striaral DA loss; the authors suggest that estrogens can somehow promote an adaptive DA mechanism synthesis, release, and metabolism in the surviving cells, so probably the females may be able to resist the onset and progression of neurodegenerative lesions compared with males [125]. Interestingly, our results also show that there is a difference between the estrogen condition regarding dyskinesias and motor behavior, noting that W/OV 6-OHDA + LD have motor impairment delayed but are more likely to develop dyskinesias compared to OVX 6-OHDA + LD, which is consistent with previous studies, which shows that there are sex differences in LD treatment, showing that women performed better in the UPDRS test (unified Parkinson's disease Rating Scale) and presented longer "on"-LD state compared to men. However, women had a higher prevalence to develop dyskinesias. It is still uncertain why women are more prone than men to develop dyskinesias, but it is suggested that estrogen may be the basis of this susceptibility [123, 126, 127]. One possible explanation for such proneness is the fact that humans and rats have similar expression characteristics of the catechol-o-methyl transferase (COMT) [128], which is a catecholamine degrading enzyme, and that women have 20–30% decrease in COMT activity compared to men [129]. In this regard, it has been demonstrated that estrogen can decrease the regulation of COMT gene [130]. Therefore, if estrogens decline, the COMT system could have a pharmacological potential to increase the LD striatal availability and prolong LD-"on" state as well as dyskinesias [130]. trees [92, 117]. Similarly, MPTP nonhuman primates and 6-OHDA-lesioned rodents show drastic loss of these structures [7, 57], suggesting nigrostriatal system importance in morphological regulation and plasticity of dendritic spines in the striatum [104]. We have observed that LD chronic treatment does not restore striatal dendritic spine density, which is consistent with previous PD postmortem studies that show that the loss of dendritic spines was present even though all patients were treated with LD for several years [117]. Deutch et al. [92] propose that LD may be ineffective in PD advanced stages, probably due to dendritic spine loss. In rodents with different models of PD LD-treated, the number of dendritic spines [7, 77, 92] is not restored. We also observed that melatonin treatment helped the conservation of dendritic spines. In this regard, it is reported that melatonin prevents cytoskeletal damage by reducing LD/Mel coadministration significantly restored the dendritic spine density of both ipsilateral and contralateral striatum. Recent studies show that the presence of dopaminergic neurons enhances dendritic spine formation in medium spiny neurons in culture. So it is possible that dopaminergic neurons have neurotrophic effect [119]. In this context, it is feasible to think that as LD/Mel coadministration increases the number of dopaminergic neurons in contralateral SNc and exerts a neurotrophic effect, promoting the formation of new dendritic spines. Our results are also consistent with those described by Naskar et al. [77], who show that MPTPexposed rodents and LD/Mel-treated for 2 days have restored the morphology and density of dendritic spines of medium-sized spiny neurons, suggesting that melatonin primarily regu- In our study, we show that W/OV rats, which were 6-OHDA-lesioned and received different treatments, have greater neuroprotection compared to OVX females, confirming the estrogen protection, besides the neurodegeneration delay difference, suggesting beneficial estrogen effect in the development and progression of the disease. It has been observed that estrogen has a neuroprotective effect on the nigrostriatal system. Recent studies suggest that PD women tend to have a delay in the appearance of certain motor symptoms compared with men [33, 120]. Furthermore, PD postmenopausal women treated with estrogen showed improvement in their motor performance [121, 122], suggesting estrogen symptomatic role [123]. As our results indicate, 6-OHDA-untreated W/OV and OVX rats showed no significant difference from both ipsilateral and contralateral SNc dopaminergic cells. This could be because the neurotoxin is very aggressive, which somehow does not allow the cell survival. It is also proposed that estrogens protect dopaminergic neurons surviving for a short time, but subsequently the cells could die as a result of neurotoxin action [124]. Previous studies showed the effect of replacement estrogen therapy in physiological doses in OVX rats after 6-OHDA injection in the nigrostriatal pathway, reporting that estrogen treatment showed no effect on survival of TH-immunopositive cells. Nonetheless, estrogen attenuated the striaral DA loss; the authors suggest that estrogens can somehow promote an adaptive DA mechanism synthesis, release, and metabolism in the surviving cells, so probably the females may be able to resist the onset and progression of neurodegenerative lesions compared with males [125]. lates this effect due to its characteristics of reducing excessive calcium flow. oxidative stress [118]. 192 Sex Hormones in Neurodegenerative Processes and Diseases 4.12. W/OV and OVX comparison On the other hand, W/OV melatonin-treated rats recover faster in behavioral tests compared to OVX rats, and in the last month of treatment, all animals had similar control values; cytologically W/OV rats exhibited contralateral SNc dopaminergic cell protection and dendritic spine recovery of both ipsilateral and contralateral striatum. Studies of melatonin and estrogen therapy in neurodegenerative models are few, so this work provides new knowledge about it. In a study of W/OV rats that were subjected to a stroke model and receiving melatonin, it was observed that estrogen and melatonin exhibit synergistic effect to decrease the levels of lipid peroxidation, increasing the activity of free radical scavenger and the number of surviving neurons in the cortex, and improve sensorimotor behaviors [131]. According to these studies, we can expect that after 6-OHDA injection melatonin treatment and estrogen presence in W/ OV rats work together to activate different signaling pathways to reduce oxidative stress and thus protect the dopaminergic neurons (at least the contralateral SNc) and the number of striatal dendritic spines and thus improve motor capacity; this could be a possible explanation of why W/OV rats tend to recover faster in motor performance compared to OVX rats. Interestingly, animals receiving LD/Mel treatment showed behavioral recovery from the start of the treatment, increase in the number of dopaminergic neurons in the contralateral SNc and striatal ipsi and contralateral dendritic spine protection; these results were estrogen independent. So, we suggest that LD/Mel cotreatment could improve the LD efficacy by increasing striatal DA levels [77]. Also, it has been suggested that these drugs could act synergistically to exert a modulatory role in nigrostriatal transmission pathway, which may be responsible for many of the beneficial effects, such as biochemical alterations, regulation of dendritic spines and cell survival [77, 131], and motor disorders such as dyskinesias. Finally, it is important to mention that estrogens act as neuroprotectors in neurodegenerative diseases such as PD and Alzheimer disease, improving women quality of life [126]. But it should be noted that the use of estrogen also involves risks. Women who take hormone replacement treatment are more likely to suffer cancer [132] if there is a family history of these diseases, so careful and controlled administration of estrogens is required. #### 5. Conclusion According to our results, we can conclude that regardless of the estrogen situation, LD/Mel coadministration was the most effective in reducing motor alterations. So, it is feasible to think that the combination of these drugs exerts a modulatory role in the nigrostriatal transmission involving motor activity and dyskinesias by protecting dendritic spines and dopaminergic neurons. Therefore, we consider that the LD/Mel coadministration may be a possible candidate for PD treatment. References 10.1016/0022-510X(73)90175-5 DOI: 10.1016/j.bbadis.2008.11.007 213-246. DOI: 10.1038/npp.2011 458. DOI: 10.1038/npp.2011.212 673. DOI: 10.1136/jnnp.34.6.668 DOI: 10.1097/FBP.0000000000000004 10.1097/FBP.0000000000000004 631-645. 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[9] Lundblad M, Andersson M, Winkler C, Kirik D, Wierup N, Cenci MA. Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson's disease. The European Journal of Neuroscience. 2002;15:120-132. DOI: [10] Perez-Rial S, Garcia-Gutierrez MS, Molina JA, Perez-Nievas BG, Ledent C, Leiva C, et al. Increased vulnerability to 6-hydroxydopamine lesion and reduced development of dyskinesias in mice lacking CB1 cannabinoid receptors. Neurobiology of Aging. 2011;32: Neurology. 2010;9:1106-1117. DOI: 10.1016/S1474-4422(10)70218-0 Furthermore, our data show that W/OV rats have a better response to LD or melatonin treatment, being less motor and cytological damage than in OVX rats, suggesting that estrogens have a beneficial effect on the development and progression of the disease. Those facts could lead us to think about the importance of taking into consideration the estrogens-based therapies for PD as a possible adjunct in women. So it is suggested to study the effect that could have estrogen in males in subsequent studies. The study of estrogens mechanisms of action in the basal ganglia and their role in movement disorders will become stronger. It is recognized that estrogen may have neuroprotective effects in many neurodegenerative processes, including PD. The neurodegenerative diseases field is in great need of therapies that can prevent or slow the disease progression. Thus, the introduction of Melatonin combined with LD treatment is a promising therapeutic strategy. So, we suggest the use of such drug combination plus the estrogen replacement therapy as useful PD treatments. #### Acknowledgements FESI-DIP-PAPCA 2016-13 and PAPIIT-DGAPA IN219617 supported this work. #### Author details Ana Luisa Gutiérrez-Valdez1 , Vianey Rodríguez-Lara<sup>3</sup> , Verónica Anaya-Martínez<sup>1</sup> , José Luis Ordóñez-Librado<sup>1</sup> , Javier Sanchez-Betancourt<sup>1</sup> , Enrique Montiel-Flores<sup>1</sup> , Leonardo Reynoso-Erazo<sup>2</sup> , Rocio Tron-Alvarez2 , Patricia Aley-Medina<sup>1</sup> , Jesús Espinosa-Villanueva1 , Cesar Sanchez-Vazquez del Mercado<sup>1</sup> and María Rosa Avila-Costa<sup>1</sup> \* \Address all correspondence to: [email protected] 1 Department of Neuroscience, National University of Mexico (UNAM) Iztacala, Mexico, Mexico 2 Health Education Project, Facultad de Estudios Superiores Iztacala, UNAM, Tlalnepantla, Mexico 3 Department of Cell Biology, Facultad de Medicina, Nacional University of Mexico (UNAM), Mexico City, Mexico #### References 5. Conclusion Acknowledgements Ana Luisa Gutiérrez-Valdez1 José Luis Ordóñez-Librado<sup>1</sup> Leonardo Reynoso-Erazo<sup>2</sup> Jesús Espinosa-Villanueva1 \ \Address all correspondence to: [email protected] María Rosa Avila-Costa<sup>1</sup> Mexico City, Mexico Mexico Mexico Author details could have estrogen in males in subsequent studies. 194 Sex Hormones in Neurodegenerative Processes and Diseases According to our results, we can conclude that regardless of the estrogen situation, LD/Mel coadministration was the most effective in reducing motor alterations. So, it is feasible to think that the combination of these drugs exerts a modulatory role in the nigrostriatal transmission involving motor activity and dyskinesias by protecting dendritic spines and dopaminergic neurons. Therefore, we consider that the LD/Mel coadministration may be a possible candidate for PD treatment. Furthermore, our data show that W/OV rats have a better response to LD or melatonin treatment, being less motor and cytological damage than in OVX rats, suggesting that estrogens have a beneficial effect on the development and progression of the disease. Those facts could lead us to think about the importance of taking into consideration the estrogens-based therapies for PD as a possible adjunct in women. So it is suggested to study the effect that The study of estrogens mechanisms of action in the basal ganglia and their role in movement disorders will become stronger. It is recognized that estrogen may have neuroprotective effects in many neurodegenerative processes, including PD. The neurodegenerative diseases field is in great need of therapies that can prevent or slow the disease progression. Thus, the introduction of Melatonin combined with LD treatment is a promising therapeutic strategy. So, we suggest the use of such drug combination plus the estrogen replacement therapy as useful PD treatments. FESI-DIP-PAPCA 2016-13 and PAPIIT-DGAPA IN219617 supported this work. , Vianey Rodríguez-Lara<sup>3</sup> , Javier Sanchez-Betancourt<sup>1</sup> 1 Department of Neuroscience, National University of Mexico (UNAM) Iztacala, Mexico, 2 Health Education Project, Facultad de Estudios Superiores Iztacala, UNAM, Tlalnepantla, 3 Department of Cell Biology, Facultad de Medicina, Nacional University of Mexico (UNAM), , Cesar Sanchez-Vazquez del Mercado<sup>1</sup> and , Rocio Tron-Alvarez2 , Verónica Anaya-Martínez<sup>1</sup> , Enrique Montiel-Flores<sup>1</sup> , , Patricia Aley-Medina<sup>1</sup> , , [11] Bezard E, Yue Z, Kirik D, Spillantini MG. Animal models of Parkinson's disease: Limits and relevance to neuroprotection studies. Movement Disorders. 2013;28:61-70. DOI: 10.1002/mds.25108 [24] Olanow CW, Obeso JA. Levodopa toxicity and Parkinson disease: Still a need for equipoise. 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[125] Gillies GE, Murray HE, Dexter D, McArthur S. Sex dimorphisms in the neuroprotective effects of estrogen in an animal model of Parkinson's disease. Pharmacology, Biochem- [126] Brann DW, Dhandapani K, Wakade C, Mahesh VB, Khan MM. Neurotrophic and neuroprotective actions of estrogen: Basic mechanisms and clinical implications. Ste- [127] Solla P, Cannas A, Ibba FC, Loi F, Corona M, Orofino G, et al. Gender differences in motor and non-motor symptoms among Sardinian patients with Parkinson's disease. Journal of the Neurological Sciences. 2012;323:33-39. DOI: 10.1016/j.jns.2012.07.026 [128] Tenhunen J. Characterization of the rat catechol-O-methyltransferase gene proximal promoter: Identification of a nuclear protein-DNA interaction that contributes to the tissue-specific regulation. DNA and Cell Biology. 1996;15:461-473. DOI: 10.1089/ [129] Martinelli P, Contin M, Scaglione C, Riva R, Albani F, Baruzzi A. Levodopa pharmacokinetics and dyskinesias: Are there sex-related differences? Neurological Sciences. [130] Xie T, Ho SL, Ramsden D. Characterization and implications of estrogenic downregulation of human catechol-O-methyltransferase gene transcription. Molecular Phar- [131] Tai S-H, Hung Y-C, Lee E-J, Lee A-C, Chen T-Y, Shen C-C, et al. Melatonin protects against transient focal cerebral ischemia in both reproductively active and estrogendeficient female rats: The impact of circulating estrogen on its hormetic dose-response. Journal of Pineal Research. 2011;50:292-303. DOI: 10.1111/j.1600-079X.2010.00839.x [132] Folkerd E, Dowsett M. Sex hormones and breast cancer risk and prognosis. Breast. Brain Research. 2003;986:200-205. DOI: 10.1016/S0006-8993(03)03198-6 istry, and Behavior. 2004;78:513-522. DOI: 10.1016/j.pbb.2004.04.022 roids. 2007;72:381-405. DOI: 10.1016/j.steroids.2007.02.003 2003;24(3):192. DOI: 10.1007/s10072-003-0125-z macology. 1999;56:31-38. 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The Journal of Comparative Neurology. 2014;522:2336- [117] Zaja-Milatovic S, Milatovic D, Schantz AM, Zhang J, Montine KS, Samii A, et al. Dendritic degeneration in neostriatal medium spiny neurons in Parkinson disease. Neurol- [118] Reiter R, Benitez-King G. Melatonin reduces neuronal loss and cytoskeletal deterioration: Implications for psychiatry. Salud Mental. 2009;32:3-11. ISSN 0185-3325 [119] Fasano C, Bourque M-J, Lapointe G, Leo D, Thibault D, Haber M, et al. Dopamine facilitates dendritic spine formation by cultured striatal medium spiny neurons through both D1 and D2 dopamine receptors. Neuropharmacology. 2013;67:432-443. DOI: [120] Alves G, Muller B, Herlofson K, HogenEsch I, Telstad W, Aarsland D, et al. Incidence of Parkinson's disease in Norway: The Norwegian ParkWest study. Journal of Neurology, Neurosurgery, and Psychiatry. 2009;80:851-857. DOI: 10.1136/jnnp.2008.168211 Neuroscience. 2004;20:575-579. DOI: 10.1111/j.1460-9568.2004.03486.x ogy. 2005;64:545-547. DOI: 10.1212/01.WNL.0000150591.33787.A4 2012;226:281-292. DOI: 10.1016/j.bbr.2011.09.025 204 Sex Hormones in Neurodegenerative Processes and Diseases ogy. 1995;45:S6-S12. DOI: 10.1212/WNL.45.3\_Suppl\_3.S6 Investigation. 1995;95:2458-2464. DOI: 10.1172/JCI117946 Sciences. 2008;53:119-129. DOI: 10.2478/v10039-008-0032-x parkreldis.2008.07.008 2348. DOI: 10.1002/cne.23537 10.1016/j.neuropharm.2012.11.030 06.2006 Chapter 9* Provisional chapter Sex Hormones: Role in Neurodegenerative Diseases The brain is a complex organ in charge of regulating the homeostasis of our body and behaviors such as motivation, reward, memory, and movement control, between others. These behaviors are regulated by dopaminergic neurons, which can be modulated by several stimuli throughout the life of an individual. For example, early exposure to sex hormones or endocrine disruptors during critical period of neuronal development affects dopaminergic pathways permanently, producing some disorders such as drug addiction. On the other hand, current knowledge regarding neurodegeneration in Parkinson and Alzheimer diseases pointed out the neuroprotection that estradiol can exert, but contradictory information can also be found in the literature. To know the underlying mechanisms that are related to the above mentioned diseases will help to Keywords: sex hormones, neonatal programming, dopaminergic circuit, neuroprotection, In the current world, humans are exposed to different compounds that can exert deleterious modifications in their bodies, taking special attention of the short- and long-term effects of endocrine disruptor chemicals, which mimic or block hormonal activity. Endocrine disruptor chemicals are natural or synthetic molecules that can alter the endocrine homeostasis, especially if exposure to these molecules is during critical developmental windows [1]. These compounds are used in plastic industries, chemical, and pharmaceutical industries, and for different events that are bioavailable in the environment affecting animals and humans. Endocrine disruptors exert their action through different pathways that converge on the molecular targets such as hormone receptors, enzymatic pathways involved in biosynthesis and metabolism of endobiotics > © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, Sex Hormones: Role in Neurodegenerative Diseases and DOI: 10.5772/intechopen.71380 Jonathan Martínez Pinto, Rodrigo L. Castillo and improve health policies and treatments development. drug addiction, Alzheimer, Parkinson disease Additional information is available at the end of the chapter Additional information is available at the end of the chapter Jonathan Martínez Pinto, Rodrigo L. Castillo and Addiction Abstract 1. Introduction Addiction Ramón Sotomayor-Zárate and Ramón Sotomayor-Zárate http://dx.doi.org/10.5772/intechopen.71380 #### Sex Hormones: Role in Neurodegenerative Diseases and Addiction Sex Hormones: Role in Neurodegenerative Diseases and Addiction DOI: 10.5772/intechopen.71380 Jonathan Martínez Pinto, Rodrigo L. Castillo and Ramón Sotomayor-Zárate Jonathan Martínez Pinto, Rodrigo L. Castillo and Ramón Sotomayor-Zárate Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.71380 #### Abstract The brain is a complex organ in charge of regulating the homeostasis of our body and behaviors such as motivation, reward, memory, and movement control, between others. These behaviors are regulated by dopaminergic neurons, which can be modulated by several stimuli throughout the life of an individual. For example, early exposure to sex hormones or endocrine disruptors during critical period of neuronal development affects dopaminergic pathways permanently, producing some disorders such as drug addiction. On the other hand, current knowledge regarding neurodegeneration in Parkinson and Alzheimer diseases pointed out the neuroprotection that estradiol can exert, but contradictory information can also be found in the literature. To know the underlying mechanisms that are related to the above mentioned diseases will help to improve health policies and treatments development. Keywords: sex hormones, neonatal programming, dopaminergic circuit, neuroprotection, drug addiction, Alzheimer, Parkinson disease #### 1. Introduction In the current world, humans are exposed to different compounds that can exert deleterious modifications in their bodies, taking special attention of the short- and long-term effects of endocrine disruptor chemicals, which mimic or block hormonal activity. Endocrine disruptor chemicals are natural or synthetic molecules that can alter the endocrine homeostasis, especially if exposure to these molecules is during critical developmental windows [1]. These compounds are used in plastic industries, chemical, and pharmaceutical industries, and for different events that are bioavailable in the environment affecting animals and humans. Endocrine disruptors exert their action through different pathways that converge on the molecular targets such as hormone receptors, enzymatic pathways involved in biosynthesis and metabolism of endobiotics © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. in endocrine, reproductive, and nervous system. In this sense, different brain areas are sensible to the action of endocrine disruptors and sex hormones due to the presence of its receptors that can modulate the synaptic transmission and neuronal survival. In this regard, nuclear receptors for sex hormones are ligand-activated transcriptional factors that regulate different neural process such as neurodevelopment and behaviors. Alterations in hormonal homeostasis (e.g., aging) or signaling (e.g., exposure to agonists or antagonists of sex hormone receptors) may induce the onset of diseases before mentioned, affecting lifespan, quality of life, and high medical costs. have shown that neonatal administration of testosterone reduces spatial memory and TH positive terminals in prefrontal cortex in an animal model of attention deficit disorder with Sex Hormones: Role in Neurodegenerative Diseases and Addiction http://dx.doi.org/10.5772/intechopen.71380 209 In recent years, it has been shown that environmental pollutants (being most of them chemical disruptors) produce a myriad of effects in the brain [12]. For example, in rats, neonatal and postnatal administration of bisphenol A produce an increase of spontaneous locomotion behavior associated with the decreased immunoreactivity for TH in SN and decreased expres- Sex hormone levels affect cortical and subcortical brain areas, especially in sensitive periods of development in childhood and adolescence [14]. In this regard, dopaminergic brain areas such as SN, VTA, and hypothalamus are sensitive to the effects of sex hormones because they It has been demonstrated that exposure to a single dose of sex hormones during the neonatal period can change the profile expression of DA [8]; in fact, when female rats are exposed to a single dose of estradiol during the neonatal period, DA levels are increased in the ventromedial hypothalamus–arcuate nucleus, but not the exposure to testosterone, during adult life [8]. In addition, when male rats are exposed to estradiol or testosterone, DA levels and TH expression are increased in substantia nigra-ventral tegmental in addition to increased dopamine release in nucleus accumbens. This effect is not seen when rats are exposed to a nonaromatizable androgen, dihydrotestosterone, suggesting an estrogenic mechanism involving increased TH expression, either by direct estrogenic action or by aromatization of testos- The programming is exerted through epigenetic modifications, which comprised DNA methylation and post-translational histone modifications, interacting with regulatory proteins and noncoding RNA to reorganize the chromatin in active or inactive domains (euchromatin or heterochromatin), being possible to be inherited from one generation to another without subsequent exposure to the endocrine disruptor [17]. The normal development of mammals involves the activity of DNA methyl transferase (DNMTs) to determine the de novo methylation, where DNMT3A and DNMT3B are involved, and to maintain the methylation pattern in the genome, where DNMT1 is involved. The expression levels of these enzymes are highly regulated during specific stages of life [18], and therefore, the impact of the exposure to In this view, in humans the social alcohol drinking during periconceptional or pregnancy period may induce changes in the promoter methylation of DAT in mothers and their babies. Specifically, using peripheral blood from mothers or cord blood from newborns, they found that alcohol intake decreases the methylation level of the locus-specific DAT promoter region of the parents and newborns [19]. However, these findings are controversial, since also found a decrease of DAT mRNA expression in drug addicts (to opioid drugs) compared to control subjects, but not in the methylation pattern of DAT promoter [20]. One of the methodological sion of dopamine transporter (DAT) in midbrain nuclei [13]. terone to estradiol in substantia nigra-ventral tegmental area [10]. 2.1. Long-term epigenetic programming of the dopaminergic circuit endocrine disruptors and the consequences over the offspring are alarming. express estrogens and androgens receptors [7, 15, 16]. hyperactivity [11]. Worldwide, drug abuse has increased dramatically, especially in susceptible populations such as youth. However, human and animal studies show that not all drug consumers become addicts. In addition, it has determined sex differences in behaviors related to motivation, reward, and cognition, among others. Clinical observation has shown that children who have developed precocious puberty show an increase in risky behaviors such as drugs abuse, sexual risk, and anti-social behaviors in adolescence. Also, when Parkinson (PD) and Alzheimer disease (AD) is analyzed, it observed a sex difference in terms of prevalence, which draws the attention to the possible role of sex hormones in the onset of these pathologies. In this term, meta-analysis has shown that males have augmented prevalence of PD than women, overall in the age range of 50–59 years (134 per 100,000) compared to women (41 per 100,000) [2]. However, the prevalence of AD is greater in women compared to men, considering different age range and ethnicity [3, 4]. In this chapter, we will discuss about the exposure to abnormal levels of sex hormones, due to metabolic alterations or endocrine disruptor chemicals, during critical period of neurodevelopment; and based on clinical evidence and current scientific knowledge, we will discuss the mechanisms involved in the development of drug addiction, Alzheimer and Parkinson disease, and the sex differences observed between patients. #### 2. Programming: early exposure to sex hormones Programming concept was defined by Lucas as the physiological redirection of a tissue or organ by a deleterious stimulus in a sensitive period of development produces adverse functional changes in adulthood [5]. Currently, research in programming has been focused in the study of stimuli that affects sensitive periods of development such as prenatal and neonatal stages. In that sense, experiments are carried out in female rats, where precocious puberty is induced by neonatal exposure to estradiol valerate, is accompanied by increased catecholamine content in the adrenal gland, noradrenaline content in the ovary and reproductive alterations in the adulthood [6]. Using the same model of neonatal administration of estradiol valerate [7], it observed an increase in dopamine (DA) and noradrenaline content in dopaminergic neurons of tuberoinfundibular [8], nigrostriatal, and mesolimbic pathways [9] of the adult. Indeed, neonatal administration of estradiol valerate and testosterone propionate increases DA content and tyrosine hydroxylase (TH), (rate limiting enzyme of dopamine synthesis) expression in substantia nigra (SN), and ventral tegmental area (VTA) of adult male rats [10]. Others works have shown that neonatal administration of testosterone reduces spatial memory and TH positive terminals in prefrontal cortex in an animal model of attention deficit disorder with hyperactivity [11]. in endocrine, reproductive, and nervous system. In this sense, different brain areas are sensible to the action of endocrine disruptors and sex hormones due to the presence of its receptors that can modulate the synaptic transmission and neuronal survival. In this regard, nuclear receptors for sex hormones are ligand-activated transcriptional factors that regulate different neural process such as neurodevelopment and behaviors. Alterations in hormonal homeostasis (e.g., aging) or signaling (e.g., exposure to agonists or antagonists of sex hormone receptors) may induce the onset of diseases before mentioned, affecting lifespan, quality of life, and high medical costs. Worldwide, drug abuse has increased dramatically, especially in susceptible populations such as youth. However, human and animal studies show that not all drug consumers become addicts. In addition, it has determined sex differences in behaviors related to motivation, reward, and cognition, among others. Clinical observation has shown that children who have developed precocious puberty show an increase in risky behaviors such as drugs abuse, sexual Also, when Parkinson (PD) and Alzheimer disease (AD) is analyzed, it observed a sex difference in terms of prevalence, which draws the attention to the possible role of sex hormones in the onset of these pathologies. In this term, meta-analysis has shown that males have augmented prevalence of PD than women, overall in the age range of 50–59 years (134 per 100,000) compared to women (41 per 100,000) [2]. However, the prevalence of AD is greater in women In this chapter, we will discuss about the exposure to abnormal levels of sex hormones, due to metabolic alterations or endocrine disruptor chemicals, during critical period of neurodevelopment; and based on clinical evidence and current scientific knowledge, we will discuss the mechanisms involved in the development of drug addiction, Alzheimer and Parkinson dis- Programming concept was defined by Lucas as the physiological redirection of a tissue or organ by a deleterious stimulus in a sensitive period of development produces adverse functional changes in adulthood [5]. Currently, research in programming has been focused in the study of stimuli that affects sensitive periods of development such as prenatal and neonatal In that sense, experiments are carried out in female rats, where precocious puberty is induced by neonatal exposure to estradiol valerate, is accompanied by increased catecholamine content in the adrenal gland, noradrenaline content in the ovary and reproductive alterations in the adulthood [6]. Using the same model of neonatal administration of estradiol valerate [7], it observed an increase in dopamine (DA) and noradrenaline content in dopaminergic neurons of tuberoinfundibular [8], nigrostriatal, and mesolimbic pathways [9] of the adult. Indeed, neonatal administration of estradiol valerate and testosterone propionate increases DA content and tyrosine hydroxylase (TH), (rate limiting enzyme of dopamine synthesis) expression in substantia nigra (SN), and ventral tegmental area (VTA) of adult male rats [10]. Others works compared to men, considering different age range and ethnicity [3, 4]. ease, and the sex differences observed between patients. stages. 2. Programming: early exposure to sex hormones risk, and anti-social behaviors in adolescence. 208 Sex Hormones in Neurodegenerative Processes and Diseases In recent years, it has been shown that environmental pollutants (being most of them chemical disruptors) produce a myriad of effects in the brain [12]. For example, in rats, neonatal and postnatal administration of bisphenol A produce an increase of spontaneous locomotion behavior associated with the decreased immunoreactivity for TH in SN and decreased expression of dopamine transporter (DAT) in midbrain nuclei [13]. Sex hormone levels affect cortical and subcortical brain areas, especially in sensitive periods of development in childhood and adolescence [14]. In this regard, dopaminergic brain areas such as SN, VTA, and hypothalamus are sensitive to the effects of sex hormones because they express estrogens and androgens receptors [7, 15, 16]. It has been demonstrated that exposure to a single dose of sex hormones during the neonatal period can change the profile expression of DA [8]; in fact, when female rats are exposed to a single dose of estradiol during the neonatal period, DA levels are increased in the ventromedial hypothalamus–arcuate nucleus, but not the exposure to testosterone, during adult life [8]. In addition, when male rats are exposed to estradiol or testosterone, DA levels and TH expression are increased in substantia nigra-ventral tegmental in addition to increased dopamine release in nucleus accumbens. This effect is not seen when rats are exposed to a nonaromatizable androgen, dihydrotestosterone, suggesting an estrogenic mechanism involving increased TH expression, either by direct estrogenic action or by aromatization of testosterone to estradiol in substantia nigra-ventral tegmental area [10]. #### 2.1. Long-term epigenetic programming of the dopaminergic circuit The programming is exerted through epigenetic modifications, which comprised DNA methylation and post-translational histone modifications, interacting with regulatory proteins and noncoding RNA to reorganize the chromatin in active or inactive domains (euchromatin or heterochromatin), being possible to be inherited from one generation to another without subsequent exposure to the endocrine disruptor [17]. The normal development of mammals involves the activity of DNA methyl transferase (DNMTs) to determine the de novo methylation, where DNMT3A and DNMT3B are involved, and to maintain the methylation pattern in the genome, where DNMT1 is involved. The expression levels of these enzymes are highly regulated during specific stages of life [18], and therefore, the impact of the exposure to endocrine disruptors and the consequences over the offspring are alarming. In this view, in humans the social alcohol drinking during periconceptional or pregnancy period may induce changes in the promoter methylation of DAT in mothers and their babies. Specifically, using peripheral blood from mothers or cord blood from newborns, they found that alcohol intake decreases the methylation level of the locus-specific DAT promoter region of the parents and newborns [19]. However, these findings are controversial, since also found a decrease of DAT mRNA expression in drug addicts (to opioid drugs) compared to control subjects, but not in the methylation pattern of DAT promoter [20]. One of the methodological factors that could determine this difference is from where the samples are obtained, in the case of the peripheral blood, it is not a direct measurement of DAT expression in brain. and function in response to injury. Therefore, the neurochemical interaction and environmental aspects can modulate the pathophysiological processes that determine the development of Sex Hormones: Role in Neurodegenerative Diseases and Addiction http://dx.doi.org/10.5772/intechopen.71380 211 The mesocorticolimbic system comprises the midbrain dopaminergic projection from the VTA to the nucleus accumbens (NAcc) [23, 37] and prefrontal and orbitofrontal cortexes [38]. One of the most important neurotransmitters in mesocorticolimbic system is DA, which is released in response to natural rewarding stimuli as food [39] or sex [40]. Drugs of abuse produce an increase in DA release in NAcc and striatum [41]; however, the magnitude and duration of this effect are much greater than with natural reinforces [42]. This acute supraphysiological DA release induced by drugs of abuse in the NAcc exherte its actions through the activation of the DA receptor type 1 (D1 receptor), leading to early gene products induction (e.g., cFos) [43]. In situ hybridization studies have demonstrated the expression of estrogen receptors (ESR1, ESR2) and androgen receptors in SN-VTA [16, 44]. Using immunohistochemistry, it has been shown that ESR2 is expressed in high proportion in TH positive neurons of the VTA, whereas Figure 1. Schematic representation of the influence of neuroactive compounds like estradiol, testosterone, DHT, genistein, and bisphenol A on brain areas. These compounds can cross the blood–brain barrier, reaching brain areas that are related to cognition and learning (prefrontal cortex and hippocampus), movement (striatum), reward (VTA), social emotional (amygdala), the endocrine system (hypothalamus). Abbreviations: E2, estradiol; T, testosterone; DHT, dihydrotestoster- one; PFC, prefrontal cortex; VTA, ventral tegmental area. neurodegenerative events [36] (Figure 1). #### 3. Sex hormones, dopaminergic neurotransmission, and addiction Worldwide, drugs of abuse have increased dramatically, especially in susceptible populations such as youth. However, human and animal studies show that not all drug consumers become addicts [21, 22]. Lately, it has been determined differences in behaviors related to motivation reward and cognition between women and men (for review see 23). Accordingly, sex hormone levels affect cortical and subcortical brain areas, especially in sensitive periods of development in childhood and adolescence [14]. In this regard, dopaminergic brain areas such as SN, VTA, and hypothalamus are sensitive to the effects of sex hormones because they express estrogens and androgens receptors [7, 15, 16]. Interestingly, sex hormones induce opposite effects between female and males. While estrogens increase the expression of tyrosine hydroxylase in SN and VTA of adult female rats [24], androgens such as testosterone and dihydrotestosterone reduce TH expression in the same brain areas in adult male rats [25]. However, in adolescent male rats, androgens increase TH expression in SN [26]. In humans, it has been observed that an excess of testosterone levels during prenatal stage is related with the development and maintenance of alcohol dependence during adolescence and adulthood [27, 28]. Children who have developed precocious puberty (an early activation of the reproductive axis leading to the onset of puberty closer to 8, 9 years in girls and boys, respectively) shows an increase in risky behaviors such as drugs abuse, sexual risk, and anti-social behaviors in adolescence [29]. Many of these behaviors and the neuroendocrine pathways that regulate them are sexually dimorphic. These sex dimorphisms reflect adaptive differences for behavioral strategies in coping as a result of sexual selection. Disruptions in these behaviors may lead to reduced social adaptation and impaired responsiveness to environmental demands [30]. On the other hand, the exposure to several environmental pollutants, with neuroendocrine activity, has been associated with behavioral effects. For example, genistein (a phytoestrogen produced by legumes and present in soy bean-based food) increases locomotor activity in males, ethinylestradiol (a synthetic estrogen used as contraceptive) affects response to reward in females and bisphenol A, an endocrine disruptor, increases anxiety and sexual behavior in males (for review see [12]). In summary, our brain is modulated by sex hormones (or exogenous compounds) and depending on the stage of development, this interaction could affect the organization and activation of neural systems (for review see [31–33]). The alteration of sexually-dimorphic behaviors may be relevant for concerns regarding the increased developmental, cognitive, and⁄or emotional disabilities reported over the past 30 years [34]. Some studies of enrichment and deprivation of sensory inputs to the brain have provided information regarding the role of experience on the development of the brain. These studies suggest widespread effects of experience on the complexity and function of the developing system, while the deprivation studies document the capacity for neural reorganization within particular sensory systems [35]. These studies suggest that plasticity in developing neural systems can modulate the capacity to develop fundamentally different patterns of organization and function in response to injury. Therefore, the neurochemical interaction and environmental aspects can modulate the pathophysiological processes that determine the development of neurodegenerative events [36] (Figure 1). factors that could determine this difference is from where the samples are obtained, in the case Worldwide, drugs of abuse have increased dramatically, especially in susceptible populations such as youth. However, human and animal studies show that not all drug consumers become addicts [21, 22]. Lately, it has been determined differences in behaviors related to motivation reward and cognition between women and men (for review see 23). Accordingly, sex hormone levels affect cortical and subcortical brain areas, especially in sensitive periods of development in childhood and adolescence [14]. In this regard, dopaminergic brain areas such as SN, VTA, and hypothalamus are sensitive to the effects of sex hormones because they express estrogens and androgens receptors [7, 15, 16]. Interestingly, sex hormones induce opposite effects between female and males. While estrogens increase the expression of tyrosine hydroxylase in SN and VTA of adult female rats [24], androgens such as testosterone and dihydrotestosterone reduce TH expression in the same brain areas in adult male rats [25]. However, in In humans, it has been observed that an excess of testosterone levels during prenatal stage is related with the development and maintenance of alcohol dependence during adolescence and adulthood [27, 28]. Children who have developed precocious puberty (an early activation of the reproductive axis leading to the onset of puberty closer to 8, 9 years in girls and boys, respectively) shows an increase in risky behaviors such as drugs abuse, sexual risk, and anti-social behaviors in adolescence [29]. Many of these behaviors and the neuroendocrine pathways that regulate them are sexually dimorphic. These sex dimorphisms reflect adaptive differences for behavioral strategies in coping as a result of sexual selection. Disruptions in these behaviors may lead to reduced social adaptation and impaired responsiveness to environmental demands [30]. On the other hand, the exposure to several environmental pollutants, with neuroendocrine activity, has been associated with behavioral effects. For example, genistein (a phytoestrogen produced by legumes and present in soy bean-based food) increases locomotor activity in males, ethinylestradiol (a synthetic estrogen used as contraceptive) affects response to reward in females and bisphenol A, an endocrine disruptor, increases anxiety and sexual behavior in males (for review see [12]). In summary, our brain is modulated by sex hormones (or exogenous compounds) and depending on the stage of development, this interaction could affect the organization and activation of neural systems (for review see [31–33]). The alteration of sexually-dimorphic behaviors may be relevant for concerns regarding the increased devel- opmental, cognitive, and⁄or emotional disabilities reported over the past 30 years [34]. Some studies of enrichment and deprivation of sensory inputs to the brain have provided information regarding the role of experience on the development of the brain. These studies suggest widespread effects of experience on the complexity and function of the developing system, while the deprivation studies document the capacity for neural reorganization within particular sensory systems [35]. These studies suggest that plasticity in developing neural systems can modulate the capacity to develop fundamentally different patterns of organization of the peripheral blood, it is not a direct measurement of DAT expression in brain. 210 Sex Hormones in Neurodegenerative Processes and Diseases 3. Sex hormones, dopaminergic neurotransmission, and addiction adolescent male rats, androgens increase TH expression in SN [26]. The mesocorticolimbic system comprises the midbrain dopaminergic projection from the VTA to the nucleus accumbens (NAcc) [23, 37] and prefrontal and orbitofrontal cortexes [38]. One of the most important neurotransmitters in mesocorticolimbic system is DA, which is released in response to natural rewarding stimuli as food [39] or sex [40]. Drugs of abuse produce an increase in DA release in NAcc and striatum [41]; however, the magnitude and duration of this effect are much greater than with natural reinforces [42]. This acute supraphysiological DA release induced by drugs of abuse in the NAcc exherte its actions through the activation of the DA receptor type 1 (D1 receptor), leading to early gene products induction (e.g., cFos) [43]. In situ hybridization studies have demonstrated the expression of estrogen receptors (ESR1, ESR2) and androgen receptors in SN-VTA [16, 44]. Using immunohistochemistry, it has been shown that ESR2 is expressed in high proportion in TH positive neurons of the VTA, whereas Figure 1. Schematic representation of the influence of neuroactive compounds like estradiol, testosterone, DHT, genistein, and bisphenol A on brain areas. These compounds can cross the blood–brain barrier, reaching brain areas that are related to cognition and learning (prefrontal cortex and hippocampus), movement (striatum), reward (VTA), social emotional (amygdala), the endocrine system (hypothalamus). Abbreviations: E2, estradiol; T, testosterone; DHT, dihydrotestosterone; PFC, prefrontal cortex; VTA, ventral tegmental area. androgen receptor is expressed in high proportion in TH positive neurons of NAcc [15]. Thus, sex hormones can regulate the expression of Tyrosine hydroxylase; specifically, estrogens can increase the expression of TH in SN and VTA of adult female rats [24], while androgens reduce TH expression in the same brain areas in adult male rats [25]. Noteworthy, in adolescent male rats, androgens increase TH expression in SN [26], suggesting a mechanism that depends on the physiological/hormonal context. The effects of sex hormones are mediated by the activation of specific receptors expressed in cell bodies of midbrain dopaminergic neurons and its limbic projections. The dopamine transporter, DAT, is a protein that mediates the active reuptake of dopamine from the synapse and is a principal regulator of dopaminergic neurotransmission, dopamine receptor 1 and 2 (D1 and D2, respectively) are modulated by 17βestradiol and testosterone. Experiments using ovariectomized adult rats have shown a significant reduction of DAT levels in the NAcc and Striatum, which is restored to normal levels after E2 replacement or the use of diarylpropionitrile (a selective ERβ agonist) and tamoxifen (selective estrogen receptor modulator) [45–48]. In the same model, levels of D1 in mPOA are decreased after E2 replacement [49]. Noteworthy, immunoreaction to D2 is not affected by E2 replacement, when is measured using immunohistochemistry. However, when western blot is used, levels of D2 are apparently increased in mPOA and PLC [49]. In NAcc, D2 levels are significantly increased in the NAcc and striatum of ovariectomized rats and E2 replacement reduced D2 receptors to lower levels than in controls rats [48]. (SHR) (an animal model of attention-deficit hyperactivity disorder [ADHD]) decreases cognitive function and TH immunoreactivity in prefrontal cortex [11]. In this work, the authors implanted at postnatal day 1 pellets of testosterone in SHR rats, observing at postnatal day 45, through the Morris water maze test, an increased latency to find the platform. The authors conclude that the administration of androgens in neonatal period may predispose to ADHD- Sex Hormones: Role in Neurodegenerative Diseases and Addiction http://dx.doi.org/10.5772/intechopen.71380 213 With regard to pharmacological therapies of ADHD, animal studies and case reports have suggested that methylphenidate exerts adverse effects on gonadal hormones. In this case, methylphenidate could be altering testosterone levels in children with attention-deficit/hyperactivity disorder through the comparison of those with or without methylphenidate treatment [56]. Recently, prospective study conducted in Taiwan that included 203 ADHD patients with a mean age of 8.7 years (boys: 75.8%). After the initial recruitment, 137 received daily methylphenidate treatment and 66 were assessed through naturalistic observation (nonmedicated group). During the study period, salivary testosterone levels did not significantly change in the treated group (P = 0.389) or in the nontreated group (P = 0.488). After the correction for potential confounding effects of age and sex, salivary testosterone levels still remained unchanged in the treated and nontreated groups during the 4-week follow-up [57]. Findings suggest that the short-term treatment with methylphenidate at usual doses does not significantly alter salivary testosterone levels in attention-deficit/hyperactivity disorder patients. Future studies should clarify whether long-term methylphenidate treatment disrupts testos- In summary, these evidences indicate that sex hormones play an important role modulating the mesocorticolimbic system and behavioral, neurochemical, and neuroplastic effects of drugs During the menopause in women or andropause in men, there is a normal decrease in sexual hormones, due to the loss of ovarian sex hormones (estrone, estradiol, and progesterone) or to a decrease in testosterone levels, correspondingly. Noteworthy, postmortem analysis has shown lower brain levels of estrogens in women with AD, and lower levels of androgens in men with AD compared to nonAD patients. Specifically, studies performed in caucasic female subjects with neuropathological diagnosis of AD, according to Braak stages V–VI, with the absence of other neuropathologies (ranging in age from 61 to 90 years old) show a decrease of two times in estrone levels (midfrontal gyrus samples) when compared to controls, but not in estradiol, testosterone, or dihydrotestosterone (DHT) levels [58]. However, when male subjects with neuropathological diagnosis of AD, according to Braak stages V–VI, are analyzed (ranging in age from 50 to 97 years old) estradiol or estrone levels are not different between AD subjects and controls, but there is a decrease in androgen levels in AD patients, compared to controls [58]. Thus, it is proposed that the sex hormone decrease observed in brain samples from AD patients is not just related to the normal decrease in gonadal synthesis, but also to a terone production as well as the function of the reproductive system. 4. Sex hormones and Alzheimer disease decrease in local brain steroidogenesis [58, 59]. like behaviors in the adulthood. of abuse. It has been shown that circulating levels of female and male sex hormones modulate the mesocorticolimbic system, regulating the addictive behavior. Women in reproductive age who are users of drugs of abuse show greater rate of escalation of drug use than men [50], leading to the establishment of the addictive behavior quickly [51]. On the other hand, depending on circulating levels of sex hormones in menstrual cycle, the reward effects of psychostimulant drugs such as amphetamine are more potent in follicular phase when estradiol levels are higher than luteal phase, when progesterone levels are higher [52, 53]. Exposure to hormone disruptors has shown to produce effects on the behavior of animals. Thus, the prenatal administration of bisphenol A to pregnant mice and postnatal administration to offspring until postnatal day 15 produces anxiolytic behavior in elevated plus-maze and open field tests [54]. Interestingly, this behavior has been related to a significant decrease of DAT in striatum and NMDA receptor in frontal cortex [54]. Silverman and Koenig [55] showed the involvement of ESR2 in the reinforcement induced by low doses of amphetamine in female rats. In this work, ovariectomized female rats do not show conditional place preference to amphetamine compared with intact female rats. The replacement with estradiol or estradiol plus progesterone reestablishes the conditioned place preference induced by amphetamine in ovariectomized rats [55]. Interestingly, the authors found that conditioning with amphetamine was significant in the ovariectomized groups that were administered with estradiol or the ESR2-specific ligand DPN. These results provide new evidence of the specific requirement of ESR2 in response to drugs of abuse [55]. #### 3.1. Attention-deficit/hyperactivity disorder (ADHD) Regarding the behavioral effects produced by the administration of androgens, it has been observed that the neonatal administration of testosterone in spontaneously hypertensive rats (SHR) (an animal model of attention-deficit hyperactivity disorder [ADHD]) decreases cognitive function and TH immunoreactivity in prefrontal cortex [11]. In this work, the authors implanted at postnatal day 1 pellets of testosterone in SHR rats, observing at postnatal day 45, through the Morris water maze test, an increased latency to find the platform. The authors conclude that the administration of androgens in neonatal period may predispose to ADHDlike behaviors in the adulthood. With regard to pharmacological therapies of ADHD, animal studies and case reports have suggested that methylphenidate exerts adverse effects on gonadal hormones. In this case, methylphenidate could be altering testosterone levels in children with attention-deficit/hyperactivity disorder through the comparison of those with or without methylphenidate treatment [56]. Recently, prospective study conducted in Taiwan that included 203 ADHD patients with a mean age of 8.7 years (boys: 75.8%). After the initial recruitment, 137 received daily methylphenidate treatment and 66 were assessed through naturalistic observation (nonmedicated group). During the study period, salivary testosterone levels did not significantly change in the treated group (P = 0.389) or in the nontreated group (P = 0.488). After the correction for potential confounding effects of age and sex, salivary testosterone levels still remained unchanged in the treated and nontreated groups during the 4-week follow-up [57]. Findings suggest that the short-term treatment with methylphenidate at usual doses does not significantly alter salivary testosterone levels in attention-deficit/hyperactivity disorder patients. Future studies should clarify whether long-term methylphenidate treatment disrupts testosterone production as well as the function of the reproductive system. In summary, these evidences indicate that sex hormones play an important role modulating the mesocorticolimbic system and behavioral, neurochemical, and neuroplastic effects of drugs of abuse. #### 4. Sex hormones and Alzheimer disease androgen receptor is expressed in high proportion in TH positive neurons of NAcc [15]. Thus, sex hormones can regulate the expression of Tyrosine hydroxylase; specifically, estrogens can increase the expression of TH in SN and VTA of adult female rats [24], while androgens reduce TH expression in the same brain areas in adult male rats [25]. Noteworthy, in adolescent male rats, androgens increase TH expression in SN [26], suggesting a mechanism that depends on the physiological/hormonal context. The effects of sex hormones are mediated by the activation of specific receptors expressed in cell bodies of midbrain dopaminergic neurons and its limbic projections. The dopamine transporter, DAT, is a protein that mediates the active reuptake of dopamine from the synapse and is a principal regulator of dopaminergic neurotransmission, dopamine receptor 1 and 2 (D1 and D2, respectively) are modulated by 17βestradiol and testosterone. Experiments using ovariectomized adult rats have shown a significant reduction of DAT levels in the NAcc and Striatum, which is restored to normal levels after E2 replacement or the use of diarylpropionitrile (a selective ERβ agonist) and tamoxifen (selective estrogen receptor modulator) [45–48]. In the same model, levels of D1 in mPOA are decreased after E2 replacement [49]. Noteworthy, immunoreaction to D2 is not affected by E2 replacement, when is measured using immunohistochemistry. However, when western blot is used, levels of D2 are apparently increased in mPOA and PLC [49]. In NAcc, D2 levels are significantly increased in the NAcc and striatum of ovariectomized rats and E2 replacement It has been shown that circulating levels of female and male sex hormones modulate the mesocorticolimbic system, regulating the addictive behavior. Women in reproductive age who are users of drugs of abuse show greater rate of escalation of drug use than men [50], leading to the establishment of the addictive behavior quickly [51]. On the other hand, depending on circulating levels of sex hormones in menstrual cycle, the reward effects of psychostimulant drugs such as amphetamine are more potent in follicular phase when estra- Exposure to hormone disruptors has shown to produce effects on the behavior of animals. Thus, the prenatal administration of bisphenol A to pregnant mice and postnatal administration to offspring until postnatal day 15 produces anxiolytic behavior in elevated plus-maze and open field tests [54]. Interestingly, this behavior has been related to a significant decrease of DAT in striatum and NMDA receptor in frontal cortex [54]. Silverman and Koenig [55] showed the involvement of ESR2 in the reinforcement induced by low doses of amphetamine in female rats. In this work, ovariectomized female rats do not show conditional place preference to amphetamine compared with intact female rats. The replacement with estradiol or estradiol plus progesterone reestablishes the conditioned place preference induced by amphetamine in ovariectomized rats [55]. Interestingly, the authors found that conditioning with amphetamine was significant in the ovariectomized groups that were administered with estradiol or the ESR2-specific ligand DPN. These results provide new evidence of the specific Regarding the behavioral effects produced by the administration of androgens, it has been observed that the neonatal administration of testosterone in spontaneously hypertensive rats diol levels are higher than luteal phase, when progesterone levels are higher [52, 53]. reduced D2 receptors to lower levels than in controls rats [48]. 212 Sex Hormones in Neurodegenerative Processes and Diseases requirement of ESR2 in response to drugs of abuse [55]. 3.1. Attention-deficit/hyperactivity disorder (ADHD) During the menopause in women or andropause in men, there is a normal decrease in sexual hormones, due to the loss of ovarian sex hormones (estrone, estradiol, and progesterone) or to a decrease in testosterone levels, correspondingly. Noteworthy, postmortem analysis has shown lower brain levels of estrogens in women with AD, and lower levels of androgens in men with AD compared to nonAD patients. Specifically, studies performed in caucasic female subjects with neuropathological diagnosis of AD, according to Braak stages V–VI, with the absence of other neuropathologies (ranging in age from 61 to 90 years old) show a decrease of two times in estrone levels (midfrontal gyrus samples) when compared to controls, but not in estradiol, testosterone, or dihydrotestosterone (DHT) levels [58]. However, when male subjects with neuropathological diagnosis of AD, according to Braak stages V–VI, are analyzed (ranging in age from 50 to 97 years old) estradiol or estrone levels are not different between AD subjects and controls, but there is a decrease in androgen levels in AD patients, compared to controls [58]. Thus, it is proposed that the sex hormone decrease observed in brain samples from AD patients is not just related to the normal decrease in gonadal synthesis, but also to a decrease in local brain steroidogenesis [58, 59]. Supporting that, the premenopausal bilateral oophorectomy (surgical menopause), which induces early menopause through an abrupt decrease in circulating estrogen levels in young women, has shown lights about the role of sex hormones, its decline during the menopause and correct timing of hormonal replace therapy [60, 61]. Thus, in a study where 1884 women were followed longitudinally for upto 18 years (natural menopause n = 1277, surgical menopause n = 607) relating the onset of menopause (natural or surgical) to cognitive decline and AD. According to the study, surgical menopause at earlier age was associated with the decline in cognition (decline in episodic and semantic memory) as well as a greater level of Alzheimer disease in women who survived free of dementia to a mean age of 78 years [61]. Noteworthy, when the use of hormone therapy was considered in the study, a protective role was found when the treatment was administrated within 5-year perimenopausal period for at least 10 years: less decline in visuospatial ability, episodic, and semantic memory, but no influence over the onset of AD [61]. formation of Aβ plaques can be induced by a reduction in sensitivity to estrogens or androgens due to long periods of low steroid hormones synthesis from gonads, modifying the mechanism of Sex Hormones: Role in Neurodegenerative Diseases and Addiction http://dx.doi.org/10.5772/intechopen.71380 215 Clinical approach involved the estradiol and its role in maintaining brain architecture and metabolism, and chronically low levels of estradiol associated to anovulation may impair brain health. In women with functional hypothalamic amenorrhea, alterations in the thyroid axis Parkinson disease is a progressive neurodegenerative, multisystemic disorder characterized by a combination of motor symptoms like resting tremors, rigidity, bradykinesia, and postural abnormalities [69]. In addition, there are cognitive, neuropsychiatric, sleep, autonomic, and sensory disturbances associated to PD, which are related to the degeneration of serotoninergic neurons of the raphe nucleus, noradrenergic neurons of the locus coeruleus or cholinergic neurons of the nucleus basalis of Meynert [70]. PD is associated to degeneration of dopamine neurons in substantia nigra pars compacta, being PD the most common disease of dopamine It has been reported that higher incidence rates of PD in men are compared to women [72, 73], and special interest has been put on sex hormones, due to its role on the regulation of dopamine synthesis, being estradiol the main regulator of this synthesis. In addition, studies performed to oophorectomized women, have revealed a higher risk of PD in this patients, suggesting that the abnormal decrease in estradiol prior to the menopause can be related to the onset of PD condition [74]. On other hand, increased exposure to endogenous estrogen can be associated with a late onset of PD and less sever motor impairment according to a study where 579 female patients were analyzed according to menarche age, menopause age, and PD onset age; also, delayed exposure to estrogens, through an increased age at menarche, is associated The synthesis of estrogen differs between reproductive and nonreproductive women, being the extragonadal tissues, like kidney, adipose tissue, skin, and brain, the main source of estrogen in nonreproductive women. In reproductive women, the main sources are ovaries, As was mentioned for Alzheimer, sex hormones levels are crucial to maintain the proper functioning of brain circuits. Regarding to that, the normal decrease of estrogen levels in women, or testosterone in men, has been related to the onset of Parkinson. Many studies have shown that hormonal replacement therapy can reduce the risk of PD is applied during what is called a "window of opportunity", which is immediately after menopause. Using the same treatment after that period, the beneficial effects could be lost, due to a long-term hormone corpus luteum, and placenta. In men, the main source of testosterone is the testis. amyloid precursor protein elimination that is regulated by estradiol. impair neurogenesis and synaptic connectivity [68]. 5. Sex hormones and Parkinson disease dysfunction [71]. with older age at PD onset [75]. deprivation reviewed by [76] (Figure 2). In women, it has been determined that the hormonal treatment with estradiol has more protective effects than the treatment with conjugated equine estrogen. Noteworthy, many studies have pointed out that the hormonal treatment needs to start during what is called a "window of opportunity", since the protective effects of estradiol treatment depend on when hormonal treatment is started. In particular, hormonal treatment needs to start during the perimenopause period (i.e., under age of 65 years). During this period is necessary to maintain a constant treatment (not withdraw), since doing so could decrease the memory improvement obtained by the hormonal treatment [62]. Verbal memory is enhanced with the treatment with E2. Although there are many studies supporting the benefits of hormonal treatment, there are other studies that are against this statement. The window of efficient therapy depends on the capacity of the brain to respond to sexual hormones, and the presence of receptors in brain areas related to memory. This decrease is related to the normal decrease in sexual hormones due to aging, in man and woman. In that term, HT is focused on to keep the hormonal levels constant, so the brain cannot lose its responsiveness to hormones. Basic studies have shown that the role of estrogens or molecules like tamoxifen, a selective estrogen receptor modulator used as hormone therapy, may induce/modulate the dopamine system, inducing neuroprotection. In that term, the use of tamoxifen in murine AD model has shown an increment in dopamine content in striatum, and an improvement in memory tasks [63]. Recently, seven prospective cohort studies with a total of 5251 elderly men and 240 cases of Alzheimer's disease were included into the meta-analysis of AD follow-up. Meta-analysis using random effect model showed that low plasma testosterone level was significantly associated with an increased risk of Alzheimer's disease in elderly men (RR = 1.48, 95% CI 1.12-1.96, P = 0.006) [64]. This decrease is in direct relation with appearance of Aβ plaques in the brain, since androgen and estrogens can regulate the amount of Aβ through the modulation of signal transduction or enzymes related to the clearance of Aβ, like insulin-degrading enzyme, neprilysin, endothelinconverting enzymes 1 and 2, and angiotensin-converting enzyme. In that term, animal models of gonadectomy, to reduce the sexual hormones, have shown a direct relationship between the hormonal decrease and the increased amount of Aβ in the brain [65–67]. Also, in this type of models, the hormone therapy reduces the levels of Aβ and improves the memory. Thus, the formation of Aβ plaques can be induced by a reduction in sensitivity to estrogens or androgens due to long periods of low steroid hormones synthesis from gonads, modifying the mechanism of amyloid precursor protein elimination that is regulated by estradiol. Clinical approach involved the estradiol and its role in maintaining brain architecture and metabolism, and chronically low levels of estradiol associated to anovulation may impair brain health. In women with functional hypothalamic amenorrhea, alterations in the thyroid axis impair neurogenesis and synaptic connectivity [68]. #### 5. Sex hormones and Parkinson disease Supporting that, the premenopausal bilateral oophorectomy (surgical menopause), which induces early menopause through an abrupt decrease in circulating estrogen levels in young women, has shown lights about the role of sex hormones, its decline during the menopause and correct timing of hormonal replace therapy [60, 61]. Thus, in a study where 1884 women were followed longitudinally for upto 18 years (natural menopause n = 1277, surgical menopause n = 607) relating the onset of menopause (natural or surgical) to cognitive decline and AD. According to the study, surgical menopause at earlier age was associated with the decline in cognition (decline in episodic and semantic memory) as well as a greater level of Alzheimer disease in women who survived free of dementia to a mean age of 78 years [61]. Noteworthy, when the use of hormone therapy was considered in the study, a protective role was found when the treatment was administrated within 5-year perimenopausal period for at least 10 years: less decline in visuospatial ability, episodic, and semantic memory, but no influence In women, it has been determined that the hormonal treatment with estradiol has more protective effects than the treatment with conjugated equine estrogen. Noteworthy, many studies have pointed out that the hormonal treatment needs to start during what is called a "window of opportunity", since the protective effects of estradiol treatment depend on when hormonal treatment is started. In particular, hormonal treatment needs to start during the perimenopause period (i.e., under age of 65 years). During this period is necessary to maintain a constant treatment (not withdraw), since doing so could decrease the memory improvement obtained by the hormonal treatment [62]. Verbal memory is enhanced with the treatment with E2. Although there are many studies supporting the benefits of hormonal treatment, there are other studies that are against this statement. The window of efficient therapy depends on the capacity of the brain to respond to sexual hormones, and the presence of receptors in brain areas related to memory. This decrease is related to the normal decrease in sexual hormones due to aging, in man and woman. In that term, HT is focused on to keep the hormonal levels constant, so the brain cannot lose its responsiveness to hormones. Basic studies have shown that the role of estrogens or molecules like tamoxifen, a selective estrogen receptor modulator used as hormone therapy, may induce/modulate the dopamine system, inducing neuroprotection. In that term, the use of tamoxifen in murine AD model has shown an increment in dopamine content in striatum, and an improvement in memory tasks [63]. Recently, seven prospective cohort studies with a total of 5251 elderly men and 240 cases of Alzheimer's disease were included into the meta-analysis of AD follow-up. Meta-analysis using random effect model showed that low plasma testosterone level was significantly associated with an increased risk of Alzheimer's disease in elderly men (RR = 1.48, 95% CI 1.12-1.96, P = 0.006) [64]. This decrease is in direct relation with appearance of Aβ plaques in the brain, since androgen and estrogens can regulate the amount of Aβ through the modulation of signal transduction or enzymes related to the clearance of Aβ, like insulin-degrading enzyme, neprilysin, endothelinconverting enzymes 1 and 2, and angiotensin-converting enzyme. In that term, animal models of gonadectomy, to reduce the sexual hormones, have shown a direct relationship between the hormonal decrease and the increased amount of Aβ in the brain [65–67]. Also, in this type of models, the hormone therapy reduces the levels of Aβ and improves the memory. Thus, the over the onset of AD [61]. 214 Sex Hormones in Neurodegenerative Processes and Diseases Parkinson disease is a progressive neurodegenerative, multisystemic disorder characterized by a combination of motor symptoms like resting tremors, rigidity, bradykinesia, and postural abnormalities [69]. In addition, there are cognitive, neuropsychiatric, sleep, autonomic, and sensory disturbances associated to PD, which are related to the degeneration of serotoninergic neurons of the raphe nucleus, noradrenergic neurons of the locus coeruleus or cholinergic neurons of the nucleus basalis of Meynert [70]. PD is associated to degeneration of dopamine neurons in substantia nigra pars compacta, being PD the most common disease of dopamine dysfunction [71]. It has been reported that higher incidence rates of PD in men are compared to women [72, 73], and special interest has been put on sex hormones, due to its role on the regulation of dopamine synthesis, being estradiol the main regulator of this synthesis. In addition, studies performed to oophorectomized women, have revealed a higher risk of PD in this patients, suggesting that the abnormal decrease in estradiol prior to the menopause can be related to the onset of PD condition [74]. On other hand, increased exposure to endogenous estrogen can be associated with a late onset of PD and less sever motor impairment according to a study where 579 female patients were analyzed according to menarche age, menopause age, and PD onset age; also, delayed exposure to estrogens, through an increased age at menarche, is associated with older age at PD onset [75]. The synthesis of estrogen differs between reproductive and nonreproductive women, being the extragonadal tissues, like kidney, adipose tissue, skin, and brain, the main source of estrogen in nonreproductive women. In reproductive women, the main sources are ovaries, corpus luteum, and placenta. In men, the main source of testosterone is the testis. As was mentioned for Alzheimer, sex hormones levels are crucial to maintain the proper functioning of brain circuits. Regarding to that, the normal decrease of estrogen levels in women, or testosterone in men, has been related to the onset of Parkinson. Many studies have shown that hormonal replacement therapy can reduce the risk of PD is applied during what is called a "window of opportunity", which is immediately after menopause. Using the same treatment after that period, the beneficial effects could be lost, due to a long-term hormone deprivation reviewed by [76] (Figure 2). Figure 2. Effects of estradiol and testosterone on dopamine synthesis. Neonatal exposure to estradiol or testosterone increases TH expression in midbrain dopaminergic neurons. Abbreviations: E2, estradiol; T, testosterone. #### 5.1. Clinical aspects in Parkinson disease Male patients with Parkinson disease have less testosterone and estradiol than healthy males. In a recent study, it was determined if dopaminergic therapy using levodopa and dopamine agonist influenced testosterone levels. In this study, a cohort of 32 consecutive male patients from the INSPECT trial were used. INSPECT is a multi-center, prospective study that primarily examined the effects of short-term treatment with pramipexole or levodopa on cohort of PD patients [77]. There were statistically significant differences in the change in free testosterone level, increased in both the levodopa group and pramipexole group but decreased in the untreated group at 12-weeks post-treatment. These preliminary data support the premise that dopaminergic medications do not reduce testosterone levels in early PD patients. In a clinical study, where male subjects were analyzed (36 PD patients and 69 age-matched controls): prolactin levels were higher in PD subjects, compared to healthy ones. Also, concentrations of estradiol and testosterone in the control group were higher than those found in patients. In addition, the level of sex hormones was positively correlated with better mood and quality of life in patients affected with PD; prolactin levels correlated negatively with sex steroid concentrations [78, 79]. Therefore, it is extremely necessary to determine the level of hormones that may influence patients' cognition, mood, and quality of life of PD patients. The more important clinical trials that show the relationship between sex hormones and neurodegenerative disorders are shown in Table 1. 6. Concluding remarks ADHD Phase III trial (n = 203) > Phase II trial (n = 240) Prospective brain imaging study (n = 54) INSPECT trial–A multi-center Phase III trial (n = 32) Phase II trial (n = 36) Cognitive Impairment in postmenopausal women (n = 4532) Phase II trial Functional hypothalamic amenorrhea (n = 60) Alzheimer disease Parkinson Disease Hormone decrease disorders. Here, we review how sex hormones (i.e., neuroactive modulators) can differentially modulate neuronal neurodegeneration in animal and clinical models. Specifically, we provide an overview of the effects of sex hormones, stress hormones, and metabolic hormones on structural Table 1. More important clinical trials that show the relationship between sex hormones and neurodegenerative Pathology Trial (N) Primary end point Treatment/Results (R) Reference 2-year prospective brain imaging study and randomized trial of HT continuation or discontinuation in a sample of middle-aged postmenopausal women (aged 49-69 years). Testosterone levels and motor The plasma levels of oestradiol, testosterone, prolactin and sex hormone-binding protein were examined in 36 patients affected with Parkinson's disease and in 69 age-matched control subjects, using chemiluminescent scores reactions. Salivary testosterone levels 137 received daily methylphenidate Plasma testosterone Meta-analysis using random effect model elderly men treatment and 66 were assessed through Sex Hormones: Role in Neurodegenerative Diseases and Addiction http://dx.doi.org/10.5772/intechopen.71380 showed that low plasma testosterone level was significantly associated with an increased risk of Alzheimer's disease in Continuation of HT use appears to protect cognition in women with heightened risk for AD when initiated There were statistically significant differences in the change in free testosterone level, increased in both the levodopa group and pramipexole group but decreased in the untreated group at 12-weeks post-treatment The level of sex hormones was positively correlated with better mood and quality with Parkinson's disease; prolactin levels correlated negatively with sex steroid Participants received either one daily tablet of 0.625 mg of conjugated equine therapy or observation for 20 weeks close to menopause onset of life in patients affected estrogen plus 2.5 mg of medroxyprogesterone acetate or a matching placebo. Results demonstrate that estrogen plus progestin therapy increases older women's risk for probable dementia, including Alzheimer concentrations. Ovarian function (ovulating) Randomized to Cognitive behavioral [57] 217 [64] [62] [77] [78] [80] [68] naturalistic observation Table 1. More important clinical trials that show the relationship between sex hormones and neurodegenerative disorders. #### 6. Concluding remarks 5.1. Clinical aspects in Parkinson disease 216 Sex Hormones in Neurodegenerative Processes and Diseases disorders are shown in Table 1. Male patients with Parkinson disease have less testosterone and estradiol than healthy males. In a recent study, it was determined if dopaminergic therapy using levodopa and dopamine agonist influenced testosterone levels. In this study, a cohort of 32 consecutive male patients from the INSPECT trial were used. INSPECT is a multi-center, prospective study that primarily examined the effects of short-term treatment with pramipexole or levodopa on cohort of PD patients [77]. There were statistically significant differences in the change in free testosterone level, increased in both the levodopa group and pramipexole group but decreased in the untreated group at 12-weeks post-treatment. These preliminary data support the premise that dopaminergic medications do not reduce testosterone levels in early PD patients. In a clinical study, where male subjects were analyzed (36 PD patients and 69 age-matched controls): prolactin levels were higher in PD subjects, compared to healthy ones. Also, concentrations of estradiol and testosterone in the control group were higher than those found in patients. In addition, the level of sex hormones was positively correlated with better mood and quality of life in patients affected with PD; prolactin levels correlated negatively with sex steroid concentrations [78, 79]. Therefore, it is extremely necessary to determine the level of hormones that may influence patients' cognition, mood, and quality of life of PD patients. The more important clinical trials that show the relationship between sex hormones and neurodegenerative Figure 2. Effects of estradiol and testosterone on dopamine synthesis. Neonatal exposure to estradiol or testosterone increases TH expression in midbrain dopaminergic neurons. Abbreviations: E2, estradiol; T, testosterone. Here, we review how sex hormones (i.e., neuroactive modulators) can differentially modulate neuronal neurodegeneration in animal and clinical models. Specifically, we provide an overview of the effects of sex hormones, stress hormones, and metabolic hormones on structural plasticity and some pharmacological targets. In addition, we also discuss how sex hormones such as estrogen and testosterone can be affected by variables such as duration and intensity of motor and cognitive impairment. Understanding the neurobiological mechanisms underlying the modulation of neuronal structural plasticity by intrinsic and extrinsic factors will impact the design of new therapeutic approaches aimed at restoring physiological state and determine some pharmacological therapies. This approach is very important for the design of phase III clinical trial (randomized clinical trial) in the clinical practical conditions. [4] Baum LW. Sex, hormones, and Alzheimer's disease. The Journals of Gerontology: Series A. Sex Hormones: Role in Neurodegenerative Diseases and Addiction http://dx.doi.org/10.5772/intechopen.71380 219 [5] Lucas A. Programming by early nutrition in man. Ciba Foundation Symposium. 1991; [6] Sotomayor-Zarate R, Dorfman M, Paredes A, Lara HE. Neonatal exposure to estradiol valerate programs ovarian sympathetic innervation and follicular development in the [7] Perez SE, Chen EY, Mufson EJ. Distribution of estrogen receptor alpha and beta immunoreactive profiles in the postnatal rat brain. Brain research. Developmental Brain [8] Sotomayor-Zarate R, Tiszavari M, Cruz G, Lara HE. Neonatal exposure to single doses of estradiol or testosterone programs ovarian follicular development-modified hypothalamic neurotransmitters and causes polycystic ovary during adulthood in the rat. Fertility [9] Cruz G, Riquelme R, Espinosa P, Jara P, Dagnino-Subiabre A, Renard GM, et al. Neonatal exposure to estradiol valerate increases dopamine content in nigrostriatal pathway during adulthood in the rat. 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Epub 01-01-1991 Research. 2003;145(1):117-139 and Sterility. 2011;96(6):1490-1496 ticity. 2016;2016:4569785 11-08-2007 #### Acknowledgements This work was supported by FONDECYT Grant N 116-0398 and DIUV-CI Grant 01/2006 to Ramón Sotomayor-Zárate. #### Conflict of interest The authors of this work declare that they have no conflicts of interest. ### Author details Jonathan Martínez Pinto1 , Rodrigo L. Castillo<sup>2</sup> and Ramón Sotomayor-Zárate<sup>1</sup> \* \Address all correspondence to: [email protected] 1 Laboratory of Neurochemistry and Neuropharmacology, Center of Neurobiology and Brain plasticity, Institute of Physiology, Faculty of Sciences, Universidad de Valparaíso, Valparaíso, Chile 2 Program of Pathophysiology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile #### References [4] Baum LW. Sex, hormones, and Alzheimer's disease. The Journals of Gerontology: Series A. 2005;60(6):736-743 plasticity and some pharmacological targets. In addition, we also discuss how sex hormones such as estrogen and testosterone can be affected by variables such as duration and intensity of motor and cognitive impairment. Understanding the neurobiological mechanisms underlying the modulation of neuronal structural plasticity by intrinsic and extrinsic factors will impact the design of new therapeutic approaches aimed at restoring physiological state and determine some pharmacological therapies. This approach is very important for the design of phase III This work was supported by FONDECYT Grant N 116-0398 and DIUV-CI Grant 01/2006 to , Rodrigo L. Castillo<sup>2</sup> and Ramón Sotomayor-Zárate<sup>1</sup> 1 Laboratory of Neurochemistry and Neuropharmacology, Center of Neurobiology and Brain plasticity, Institute of Physiology, Faculty of Sciences, Universidad de Valparaíso, Valparaíso, [1] Diamanti-Kandarakis E, Bourguignon J-P, Giudice LC, Hauser R, Prins GS, Soto AM, et al. Endocrine-disrupting chemicals: An Endocrine Society scientific statement. Endo- [2] Pringsheim T, Jette N, Frolkis A, Steeves TD. The prevalence of Parkinson's disease: A systematic review and meta-analysis. Movement Disorders. 2014;29(13):1583-1590. Epub [3] Seshadri S, Wolf PA, Beiser A, Au R, McNulty K, White R, et al. Lifetime risk of dementia and Alzheimer's disease. The impact of mortality on risk estimates in the Framingham study. Neurology. 1997;49(6):1498-1504. Epub 31-12-1997 2 Program of Pathophysiology, Institute of Biomedical Sciences, Faculty of Medicine, \ clinical trial (randomized clinical trial) in the clinical practical conditions. 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Epub 27-06-2009 Chapter 10* Provisional chapter Neurophysiological Repercussions of Anabolic Steroid Neurophysiological Repercussions of Anabolic Steroid Since its discovery, several chemical modifications in the testosterone molecule have been done by pharmaceutical industry in order to improve its pharmacological effects, resulting in the creation of anabolic steroids (AS). Despite the therapeutic benefits, AS abuse has spread among elite and recreational athletes in the search for improvements on physical appearance and physical performance. Illicit use of anabolic AS has been correlated with several adverse effects, such as cardiovascular, endocrine, reproductive, and neurobehavioral dysfunctions. Recently, declines on cognitive and mnemonic performance have been demonstrated clinically and experimentally. Experimental studies have demonstrated that these neurological dysfunctions are correlated to spread neuronal apoptosis throughout important areas of the central nervous system (CNS), such as hippocampus and cortex. Several pathophysiological mechanisms have been linked to the AS-induced neurotoxicity, including redox imbalance and recruitment of pro-apoptotic downstream pathways. Furthermore, exposure to AS has arisen as a potential risk factor to the development of Alzheimer's disease. Altogether, these evidences imply that AS abuse per se induces neurodegeneration and can aggravate the DOI: 10.5772/intechopen.70475 © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. and reproduction in any medium, provided the original work is properly cited. The history of anabolic steroids (ASs) inevitably passes through the discovery of endogenous androgens. Based on previous evidences of several renowned scientists, such as Arnold Adolf Keywords: testosterone, anabolic steroids, neurotoxicity, neurodegeneration Abuse: A Road into Neurodegenerative Disorders Abuse: A Road into Neurodegenerative Disorders Fernando de Azevedo Cruz Seara, José Hamilton Matheus Nascimento José Hamilton Matheus Nascimento http://dx.doi.org/10.5772/intechopen.70475 Additional information is available at the end of the chapter prognosis of neurodegenerative diseases. Additional information is available at the end of the chapter Fernando de Azevedo Cruz Seara, Rodrigo Soares Fortunato, Denise Pires Carvalho and Rodrigo Soares Fortunato, Denise Pires Carvalho and Abstract 1. Introduction #### Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative Disorders Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative Disorders DOI: 10.5772/intechopen.70475 Fernando de Azevedo Cruz Seara, Rodrigo Soares Fortunato, Denise Pires Carvalho and José Hamilton Matheus Nascimento Fernando de Azevedo Cruz Seara, Rodrigo Soares Fortunato, Denise Pires Carvalho and José Hamilton Matheus Nascimento Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.70475 #### Abstract [69] Stacy M. Medical treatment of Parkinson disease. Neurologic Clinics. 2009;27(3):605-631, [70] Lang AE. The progression of Parkinson disease: A hypothesis. Neurology. 2007;68(12): [71] Smith KM, Dahodwala N. Sex differences in Parkinson's disease and other movement [72] Wooten GF, Currie LJ, Bovbjerg VE, Lee JK, Patrie J. Are men at greater risk for Parkinson's disease than women? Journal of Neurology, Neurosurgery & Psychiatry. [73] Baldereschi M, Di Carlo A, Rocca WA, Vanni P, Maggi S, Perissinotto E, et al. Parkinson's disease and parkinsonism in a longitudinal study: Two-fold higher incidence in men. [74] Rocca WA, Bower JH, Maraganore DM, Ahlskog JE, Grossardt BR, de Andrade M, et al. Increased risk of parkinsonism in women who underwent oophorectomy before meno- [75] Cereda E, Barichella M, Cassani E, Caccialanza R, Pezzoli G. Reproductive factors and clinical features of Parkinson's disease. Parkinsonism & Related Disorders. 2013;19(12): [76] Litim N, Morissette M, Di Paolo T. Neuroactive gonadal drugs for neuroprotection in male and female models of Parkinson's disease. Neuroscience and Biobehavioral Reviews. [77] Okun MS, Wu SS, Jennings D, Marek K, Rodriguez RL, Fernandez HH. Testosterone level and the effect of levodopa and agonists in early Parkinson disease: Results from the INSPECT cohort. Journal of Clinical Movement Disorders. 2014;1:8. Epub 01-01-2014 [78] Nitkowska M, Tomasiuk R, Czyzyk M, Friedman A. Prolactin and sex hormones levels in males with Parkinson's disease. Acta Neurologica Scandinavica. 2015;131(6):411-416. [79] Kenangil G, Orken DN, Ur E, Forta H, Celik M. The relation of testosterone levels with fatigue and apathy in Parkinson's disease. Clinical Neurology and Neurosurgery. 2009; [80] Shumaker SA, Legault C, Rapp SR, Thal L, Wallace RB, Ockene JK, et al. Estrogen plus progestin and the incidence of dementia and mild cognitive impairment in postmenopausal women: The Women's Health Initiative memory study: A randomized controlled trial. Journal of the American Medical Association. 2003;289(20):2651-2662. Epub 29-05-2003 disorders. Experimental Neurology. 2014;259:44-56. Epub 01-04-2014 v. Epub 27-06-2009 2004;75(4):637-639 1094-1099 Epub 18-11-2014 Neurology. 2000;55(9):1358-1363 2016;67:79-88. Epub 29-12-2015 111(5):412-414. Epub 10-01-2009 pause. Neurology 2008;70(3):200-209 948-952. Epub 21-03-2007 224 Sex Hormones in Neurodegenerative Processes and Diseases Since its discovery, several chemical modifications in the testosterone molecule have been done by pharmaceutical industry in order to improve its pharmacological effects, resulting in the creation of anabolic steroids (AS). Despite the therapeutic benefits, AS abuse has spread among elite and recreational athletes in the search for improvements on physical appearance and physical performance. Illicit use of anabolic AS has been correlated with several adverse effects, such as cardiovascular, endocrine, reproductive, and neurobehavioral dysfunctions. Recently, declines on cognitive and mnemonic performance have been demonstrated clinically and experimentally. Experimental studies have demonstrated that these neurological dysfunctions are correlated to spread neuronal apoptosis throughout important areas of the central nervous system (CNS), such as hippocampus and cortex. Several pathophysiological mechanisms have been linked to the AS-induced neurotoxicity, including redox imbalance and recruitment of pro-apoptotic downstream pathways. Furthermore, exposure to AS has arisen as a potential risk factor to the development of Alzheimer's disease. Altogether, these evidences imply that AS abuse per se induces neurodegeneration and can aggravate the prognosis of neurodegenerative diseases. Keywords: testosterone, anabolic steroids, neurotoxicity, neurodegeneration #### 1. Introduction The history of anabolic steroids (ASs) inevitably passes through the discovery of endogenous androgens. Based on previous evidences of several renowned scientists, such as Arnold Adolf © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Berthold, Charles Edouard Brown-Séquard, and Fred C. Koch, the group of the pharmacologist Ernst Laqueur purified and described the chemical properties of the testicular-derived substance called testosterone. Subsequently, de novo synthesis of testosterone from cholesterol was described by two different groups leaded by Adolf Butendandt and Leopold Ruzicka. Thus, testosterone became not only the first hormone to be described, but also the first drug to be genuinely synthesized in vitro, since predecessors were plant extracts, fungi, and other sources. Conceptually, ASs are synthetic testosterone derivatives that share a common molecular structure characterized by four aromatic rings of cyclopentanoperhydrophenanthrene with 19 carbon atoms [1]. Given the structural similarities, AS can bind to androgenic receptor (AR) and exert testosterone-like physiological effects. target tissues, such as the blood-brain-barrier. Furthermore, from the cellular perspective, the high hydrophobicity gives AS the capacity to cross plasma membranes without necessarily Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... http://dx.doi.org/10.5772/intechopen.70475 227 Within target tissues, AS can undergo three different pathways. First, biological active compounds can bind directly to the target receptors, promoting their physiological effects. Second, AS can undergo 5α-reduction, by the enzyme 5α-reductase, resulting in specific metabolites [3]. This enzymatic reaction can substantially affect the physiological effects induced by the AS and must be taken into account in both therapeutic and toxicological conditions. For example, the α-reduced nandrolone-derived metabolite, 5α-dihydro-19-nor-testosterone, has a significant decreased binding affinity for the AR when compared to nandrolone, which results in decreased androgenic effects. On the other hand, dihydrotestosterone (DHT), a α-reduced testosterone-derived metabolite, has a binding affinity for the AR approximately 10-fold higher than testosterone and, thus, impose profound androgenic effects. Third, AS can be converted into estrogen by aromatase enzyme, a reaction especially observed in AS from Classically, AS can exert their effects by binding to AR. This receptor is a member of nuclear receptors family, which also includes estrogen, glucocorticoid, mineralocorticoid, progesterone, thyroid hormones, and retinoic acid receptors. In general, ARs have four distinct molecular domains: ligand-binding domain (LBD), which presents a canonic molecular structure among nuclear receptors; N-terminal transactivation domain, which per se confers the capacity of ligand-independent activity in the case of estrogen receptors; DNA-binding domain (DBD); and hinge region [5]. In the absence of agonist, the AR remains in the cytosol due to its bindings to specific chaperone proteins, such as the heat-shock-protein 90 (HSP90). These interactions are thought to be necessary for the stabilization of the receptor in an appropriate conformation that enables the steroid molecule to bind with high affinity to the LBD. Furthermore, the interaction with HSP90 prevents the AR to dimerize and bind to When AS binds to the LBD, the interaction between AR and HSP90 is lost. In such a condition, active AR dimerizes, resulting in the formation of a homodimer, which is translocated into the nucleus by cytoskeleton myofilaments [6]. The interaction between the homodimer and the chromatin occurs due to the binding of zinc fingers located in the DBD at the level of androgen-responsive elements, a complex process that involves the recruitment of a cluster of co-regulators to this site [7]. Co-regulators include co-activators and co-repressors which are crucial to the transcriptional activity of the complex AR steroid. It is generally accepted that the recruitment of co-activators results in increased transcription of a target gene [8]. Besides the bioavailability within target tissues, the extent of the physiological effects induced by each AS is also correlated to the binding affinity to the AR consonant with this view; previous studies have shown that nandrolone binding affinity to the AR is 2- to 3-fold higher than testosterone-related binding affinity for the same receptor [9]. As a result, nandrolone has a more potent anabolic effect in skeletal muscle compared to testosterone [9]. Further experimental studies compared the binding affinity of nandrolone, oxymetholone, stanozolol, 17α-methyltestosterone, methenolone, methandienone, mesterolone, fluoxymesterone, binding to membrane-associated proteins. class I [4]. co-regulators. #### 1.1. General pharmacological aspects of AS Despite the molecular similarities, ASs exhibit different chemical characteristics within their structure in comparison to testosterone, which determines the differences in the pharmacological properties and physiological effects between distinct compounds. So far, three classes of AS have been described. The first class includes injectable AS with esterification of the 17β-hydroxyl group on testosterone molecule, such as testosterone propionate. This chemical modification in the structure of testosterone down-regulates the rates of absorption and degradations, resulting in substantial prolongation of the biological effects [2]. Within bloodstream, the ester bonds are rapidly hydrolyzed by blood esterases, releasing the active compound. Like the first class, the second class of AS is composed by injectable steroids, although the biologically active compound is the 19-nor-testosterone, instead of testosterone. Furthermore, the side chain is significantly longer when compared to AS of the class I. In addition, class II ASs have a methyl group at C19 position, instead of a hydrogen atom [2]. Altogether, these chemical modifications prolong even more the rates of absorption and degradation when compared to the AS of class I. Class II includes mainly nandrolone esters, such as nandrolone decanoate and undecanoate. Basically, the longer the side chain, the prolonger the biological effect. Similarly, the esterification is rapidly hydrolyzed by blood esterases, releasing 19-nor-testosterone into the bloodstream. The third class includes C17-alkylated AS, such as 17α-methyltestosterone, oxymetholone, methandrostenolone, and stanozolol. Given that these drugs can be orally administered, the alkylation is especially important to decrease the first-pass effect and, thus, hepatic metabolism, which could result in decreased absorption [2]. The C-1 group can also be methylated and, thus, present oral activity, but the effects induced by these drugs are relatively weaker compared to C-17-alkylated compounds. AS compounds can be carried throughout the bloodstream by plasma proteins, such as albumin and sexual hormone-binding globulin (SHBG), or they can circulate without being conjugated. Free AS can reach target tissues and, thus, promote physiological effects. The molecular structure of AS, rich in hydrocarbons, confers to them apolar characteristics and the capacity to easily cross hydrophobic layers composed by lipids. From the systemic point of view, this characteristic allows AS to permeate physiological barriers between capillaries and target tissues, such as the blood-brain-barrier. Furthermore, from the cellular perspective, the high hydrophobicity gives AS the capacity to cross plasma membranes without necessarily binding to membrane-associated proteins. Berthold, Charles Edouard Brown-Séquard, and Fred C. Koch, the group of the pharmacologist Ernst Laqueur purified and described the chemical properties of the testicular-derived substance called testosterone. Subsequently, de novo synthesis of testosterone from cholesterol was described by two different groups leaded by Adolf Butendandt and Leopold Ruzicka. Thus, testosterone became not only the first hormone to be described, but also the first drug to be genuinely synthesized in vitro, since predecessors were plant extracts, fungi, and other sources. Conceptually, ASs are synthetic testosterone derivatives that share a common molecular structure characterized by four aromatic rings of cyclopentanoperhydrophenanthrene with 19 carbon atoms [1]. Given the structural similarities, AS can bind to androgenic receptor (AR) and Despite the molecular similarities, ASs exhibit different chemical characteristics within their structure in comparison to testosterone, which determines the differences in the pharmacological properties and physiological effects between distinct compounds. So far, three classes of AS have been described. The first class includes injectable AS with esterification of the 17β-hydroxyl group on testosterone molecule, such as testosterone propionate. This chemical modification in the structure of testosterone down-regulates the rates of absorption and degradations, resulting in substantial prolongation of the biological effects [2]. Within bloodstream, the ester bonds are rapidly hydrolyzed by blood esterases, releasing the active Like the first class, the second class of AS is composed by injectable steroids, although the biologically active compound is the 19-nor-testosterone, instead of testosterone. Furthermore, the side chain is significantly longer when compared to AS of the class I. In addition, class II ASs have a methyl group at C19 position, instead of a hydrogen atom [2]. Altogether, these chemical modifications prolong even more the rates of absorption and degradation when compared to the AS of class I. Class II includes mainly nandrolone esters, such as nandrolone decanoate and undecanoate. Basically, the longer the side chain, the prolonger the biological effect. Similarly, the esterification is rapidly hydrolyzed by blood esterases, releasing 19-nor-testosterone into The third class includes C17-alkylated AS, such as 17α-methyltestosterone, oxymetholone, methandrostenolone, and stanozolol. Given that these drugs can be orally administered, the alkylation is especially important to decrease the first-pass effect and, thus, hepatic metabolism, which could result in decreased absorption [2]. The C-1 group can also be methylated and, thus, present oral activity, but the effects induced by these drugs are relatively weaker AS compounds can be carried throughout the bloodstream by plasma proteins, such as albumin and sexual hormone-binding globulin (SHBG), or they can circulate without being conjugated. Free AS can reach target tissues and, thus, promote physiological effects. The molecular structure of AS, rich in hydrocarbons, confers to them apolar characteristics and the capacity to easily cross hydrophobic layers composed by lipids. From the systemic point of view, this characteristic allows AS to permeate physiological barriers between capillaries and exert testosterone-like physiological effects. 226 Sex Hormones in Neurodegenerative Processes and Diseases 1.1. General pharmacological aspects of AS compound. the bloodstream. compared to C-17-alkylated compounds. Within target tissues, AS can undergo three different pathways. First, biological active compounds can bind directly to the target receptors, promoting their physiological effects. Second, AS can undergo 5α-reduction, by the enzyme 5α-reductase, resulting in specific metabolites [3]. This enzymatic reaction can substantially affect the physiological effects induced by the AS and must be taken into account in both therapeutic and toxicological conditions. For example, the α-reduced nandrolone-derived metabolite, 5α-dihydro-19-nor-testosterone, has a significant decreased binding affinity for the AR when compared to nandrolone, which results in decreased androgenic effects. On the other hand, dihydrotestosterone (DHT), a α-reduced testosterone-derived metabolite, has a binding affinity for the AR approximately 10-fold higher than testosterone and, thus, impose profound androgenic effects. Third, AS can be converted into estrogen by aromatase enzyme, a reaction especially observed in AS from class I [4]. Classically, AS can exert their effects by binding to AR. This receptor is a member of nuclear receptors family, which also includes estrogen, glucocorticoid, mineralocorticoid, progesterone, thyroid hormones, and retinoic acid receptors. In general, ARs have four distinct molecular domains: ligand-binding domain (LBD), which presents a canonic molecular structure among nuclear receptors; N-terminal transactivation domain, which per se confers the capacity of ligand-independent activity in the case of estrogen receptors; DNA-binding domain (DBD); and hinge region [5]. In the absence of agonist, the AR remains in the cytosol due to its bindings to specific chaperone proteins, such as the heat-shock-protein 90 (HSP90). These interactions are thought to be necessary for the stabilization of the receptor in an appropriate conformation that enables the steroid molecule to bind with high affinity to the LBD. Furthermore, the interaction with HSP90 prevents the AR to dimerize and bind to co-regulators. When AS binds to the LBD, the interaction between AR and HSP90 is lost. In such a condition, active AR dimerizes, resulting in the formation of a homodimer, which is translocated into the nucleus by cytoskeleton myofilaments [6]. The interaction between the homodimer and the chromatin occurs due to the binding of zinc fingers located in the DBD at the level of androgen-responsive elements, a complex process that involves the recruitment of a cluster of co-regulators to this site [7]. Co-regulators include co-activators and co-repressors which are crucial to the transcriptional activity of the complex AR steroid. It is generally accepted that the recruitment of co-activators results in increased transcription of a target gene [8]. Besides the bioavailability within target tissues, the extent of the physiological effects induced by each AS is also correlated to the binding affinity to the AR consonant with this view; previous studies have shown that nandrolone binding affinity to the AR is 2- to 3-fold higher than testosterone-related binding affinity for the same receptor [9]. As a result, nandrolone has a more potent anabolic effect in skeletal muscle compared to testosterone [9]. Further experimental studies compared the binding affinity of nandrolone, oxymetholone, stanozolol, 17α-methyltestosterone, methenolone, methandienone, mesterolone, fluoxymesterone, and ethyl estrenol for the AR in both skeletal muscle and prostate tissues of rodents [10]. Among these steroids, nandrolone has shown the highest binding affinity to the AR, followed by methenolone > testosterone > mesterolone. Interestingly, although the binding affinity of stanozolol, fluoxymesterone, and methandienone is thought to be significantly decreased compared with the above-mentioned steroids, the cell-based AR transactivation is comparable [11]. This evidence suggests that the degree of activation of AR does not seem to be strictly dependent on the binding affinity, despite the clear influence exerted by the latest. Indeed, gene expression can be affected in different degree by structurally distinct AS. In this context, the different conformational changes induced by distinct AS in the AR and the subsequent impact in the recruitment of co-regulators might develop a more important role in the dimension of gene expression and biological effects, although more studies are necessary to demonstrate these pharmacodynamics aspects [12]. AS-induced anabolism has been observed in both experimental and clinical studies. These effects have been widely described in several target tissues and are basically related to cellular hypertrophy and hyperplasia. AS significantly potentiates nitrogen retention and protein synthesis, resulting in increased muscle mass, strength, and muscle healing, perhaps the most prominent effects aimed among bodybuilders, elite, and recreational athletes [17]. Skeletal tissue is also affected by AS, in which they stimulate osteoblasts and chondrocytes maturation, leading to epiphyseal fusion, whereas the stimulation of osteocytes promotes increases in bone formation and density. In the bone marrow, ASs stimulate the proliferation of progenitor hematopoietic cells and their maturation directly. Besides the classical androgenic/anabolic effects, AS can induce a broad spectrum of physiological effects that have been widely Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... http://dx.doi.org/10.5772/intechopen.70475 229 ASs were rapidly adopted as the primary therapeutic approach to treat low-circulating testosterone conditions, such as hypogonadism and andropause. Moreover, AS-related therapeutic benefits were also observed in women with endocrine dysfunctions secondary to oophorectomy and menopause. The hematopoietic effect in the bone marrow is frequently explored in the treatment of aplastic anemia and myelofibrosis. In addition, ASs are also indicated for the treatment of catabolic diseases, such as cachexia, sarcopenia, and osteoporosis correlated with ASs are considered controlled medical substances and must be used just for medical purposes and under supervision of physicians. Due to their anabolic properties, the International Olympic Committee Medical Commission banned AS from the list of substances allowed in sports competitions [18]. Currently, the rules and technical documents regarding the use of AS in sports field are under regulation of the World Anti-Doping Association. Furthermore, commercialization and consumption of AS are illegal, such as in Brazil, in the United States of Despite the beneficial therapeutic effects, illicit use of AS by individuals aiming improvements in their physical performance and esthetics has been increasingly reported during the last century. Illicit use of AS is characterized by administration of doses 10 to 1000 times higher than the doses prescribed to treat medical conditions, such as hypogonadism [19]. Dose regimens are mainly characterized by cycling, i.e., intercalation between period of time of administration and withdrawal, stacking, i.e., the combination of different types of AS, especially oral and injectable AS, and pyramiding, i.e., progressive increase of dose and frequency at a peak followed by a progressive decrease on both. Among AS users, these dose regimens are thought to supposedly reduce adverse effects associated with AS abuse, although so far, no scientific evidence supporting this hypothesis has been demonstrated. described and reviewed elsewhere. 1.2. Therapeutic applicability of AS malnutrition, HIV, and cancer. America (USA), and in Great Britain. 2. Epidemiological aspects of AS abuse The classical mechanism of action of AS is the AR-mediated genomic effects; however, rapid, nongenomic effects were also demonstrated for several target organs. Nongenomic effects are generally thought to request faster responses, mainly in the range of seconds to minutes, besides activation of membrane protein–mediated signaling cascades and lack of direct transcriptional/translational activation [13]. Given that rapid AS-induced responses have been observed in cell types that do not express the AR or in the presence of AR antagonists, it is reasonable to hypothesize that these effects might be triggered by mechanisms other than AR mediated. In keeping with this, experimental evidences have demonstrated that the complex AS-SHBG can bind to membrane receptors and induce increases in intracellular levels of second messengers, such as cyclic-adenosine monophosphate and inositol 1,4,5-triphosphate (IP<sup>3</sup> ), resulting in rapid cellular effects. Other studies hypothesized that AS can bind directly to noncharacterized G-protein-coupled-receptors or to nonreceptors tyrosine kinase c-SRC, which pharmacological aspects remain unclear. Furthermore, the recruitment of collateral signaling cascades by the AR activation, aside of the classical genomic mechanism, must also be taken into account [14]. Despite these divergences, evidences that AS can promote rapid changes in intracellular ion concentrations have been widely demonstrated elsewhere. Exposure of neuroblastoma cells to testosterone resulted in concentration-dependent increase of intracellular calcium in a time range of 50–100 sec [15]. Interestingly, knock down or blockade of endoplasmic reticulum IP3 receptor (InsP<sup>3</sup> R) abolished the testosterone-induced increase on intracellular calcium concentration, suggesting that InsP<sup>3</sup> R-mediated testosterone effect [15]. Similarly, it has been shown that the incubation of primary hippocampal neurons with DHT-increased baseline calcium concentration [16]. Testosterone and its synthetic metabolites can modulate several physiological aspects in a wide range of cell types, and their effects can be didactically divided in androgenic and anabolic. Androgenic effects include the development of primary and secondary male sexual characteristics, the initiation and maintenance of spermatogenesis, and the maintenance of sexual behavior, such as the libido and spontaneous erections. However, as previously stated, several testosterone synthetic derivatives have lower androgenic capacity, especially those from class II. AS-induced anabolism has been observed in both experimental and clinical studies. These effects have been widely described in several target tissues and are basically related to cellular hypertrophy and hyperplasia. AS significantly potentiates nitrogen retention and protein synthesis, resulting in increased muscle mass, strength, and muscle healing, perhaps the most prominent effects aimed among bodybuilders, elite, and recreational athletes [17]. Skeletal tissue is also affected by AS, in which they stimulate osteoblasts and chondrocytes maturation, leading to epiphyseal fusion, whereas the stimulation of osteocytes promotes increases in bone formation and density. In the bone marrow, ASs stimulate the proliferation of progenitor hematopoietic cells and their maturation directly. Besides the classical androgenic/anabolic effects, AS can induce a broad spectrum of physiological effects that have been widely described and reviewed elsewhere. #### 1.2. Therapeutic applicability of AS and ethyl estrenol for the AR in both skeletal muscle and prostate tissues of rodents [10]. Among these steroids, nandrolone has shown the highest binding affinity to the AR, followed by methenolone > testosterone > mesterolone. Interestingly, although the binding affinity of stanozolol, fluoxymesterone, and methandienone is thought to be significantly decreased compared with the above-mentioned steroids, the cell-based AR transactivation is comparable [11]. This evidence suggests that the degree of activation of AR does not seem to be strictly dependent on the binding affinity, despite the clear influence exerted by the latest. Indeed, gene expression can be affected in different degree by structurally distinct AS. In this context, the different conformational changes induced by distinct AS in the AR and the subsequent impact in the recruitment of co-regulators might develop a more important role in the dimension of gene expression and biological effects, although more studies are necessary to The classical mechanism of action of AS is the AR-mediated genomic effects; however, rapid, nongenomic effects were also demonstrated for several target organs. Nongenomic effects are generally thought to request faster responses, mainly in the range of seconds to minutes, besides activation of membrane protein–mediated signaling cascades and lack of direct transcriptional/translational activation [13]. Given that rapid AS-induced responses have been observed in cell types that do not express the AR or in the presence of AR antagonists, it is reasonable to hypothesize that these effects might be triggered by mechanisms other than AR mediated. In keeping with this, experimental evidences have demonstrated that the complex AS-SHBG can bind to membrane receptors and induce increases in intracellular levels of second messengers, such as cyclic-adenosine monophosphate and inositol 1,4,5-triphosphate ), resulting in rapid cellular effects. Other studies hypothesized that AS can bind directly to noncharacterized G-protein-coupled-receptors or to nonreceptors tyrosine kinase c-SRC, which pharmacological aspects remain unclear. Furthermore, the recruitment of collateral signaling cascades by the AR activation, aside of the classical genomic mechanism, must also Despite these divergences, evidences that AS can promote rapid changes in intracellular ion concentrations have been widely demonstrated elsewhere. Exposure of neuroblastoma cells to testosterone resulted in concentration-dependent increase of intracellular calcium in a time range of 50–100 sec [15]. Interestingly, knock down or blockade of endoplasmic reticulum shown that the incubation of primary hippocampal neurons with DHT-increased baseline Testosterone and its synthetic metabolites can modulate several physiological aspects in a wide range of cell types, and their effects can be didactically divided in androgenic and anabolic. Androgenic effects include the development of primary and secondary male sexual characteristics, the initiation and maintenance of spermatogenesis, and the maintenance of sexual behavior, such as the libido and spontaneous erections. However, as previously stated, several testosterone synthetic derivatives have lower androgenic capacity, especially those from class II. R) abolished the testosterone-induced increase on intracellular calcium R-mediated testosterone effect [15]. Similarly, it has been demonstrate these pharmacodynamics aspects [12]. 228 Sex Hormones in Neurodegenerative Processes and Diseases (IP<sup>3</sup> IP3 be taken into account [14]. receptor (InsP<sup>3</sup> calcium concentration [16]. concentration, suggesting that InsP<sup>3</sup> ASs were rapidly adopted as the primary therapeutic approach to treat low-circulating testosterone conditions, such as hypogonadism and andropause. Moreover, AS-related therapeutic benefits were also observed in women with endocrine dysfunctions secondary to oophorectomy and menopause. The hematopoietic effect in the bone marrow is frequently explored in the treatment of aplastic anemia and myelofibrosis. In addition, ASs are also indicated for the treatment of catabolic diseases, such as cachexia, sarcopenia, and osteoporosis correlated with malnutrition, HIV, and cancer. ASs are considered controlled medical substances and must be used just for medical purposes and under supervision of physicians. Due to their anabolic properties, the International Olympic Committee Medical Commission banned AS from the list of substances allowed in sports competitions [18]. Currently, the rules and technical documents regarding the use of AS in sports field are under regulation of the World Anti-Doping Association. Furthermore, commercialization and consumption of AS are illegal, such as in Brazil, in the United States of America (USA), and in Great Britain. ### 2. Epidemiological aspects of AS abuse Despite the beneficial therapeutic effects, illicit use of AS by individuals aiming improvements in their physical performance and esthetics has been increasingly reported during the last century. Illicit use of AS is characterized by administration of doses 10 to 1000 times higher than the doses prescribed to treat medical conditions, such as hypogonadism [19]. Dose regimens are mainly characterized by cycling, i.e., intercalation between period of time of administration and withdrawal, stacking, i.e., the combination of different types of AS, especially oral and injectable AS, and pyramiding, i.e., progressive increase of dose and frequency at a peak followed by a progressive decrease on both. Among AS users, these dose regimens are thought to supposedly reduce adverse effects associated with AS abuse, although so far, no scientific evidence supporting this hypothesis has been demonstrated. The misuse of testosterone was firstly reported during the World War II by Nazi German army with the purpose of enhancing soldier's aggressiveness, and there has also been uncertain reports about the administration of AS in Nazi athletes during the Olympic games of 1936 [20]. During the 1940s, the use of synthetic testosterone for medical purposes spread, especially to increase sexual libido and to treat mood disorders, menorrhagia, dysmenorrhea, hypogonadism, and breast cancer [21–25]. Concomitantly, AS use was correlated with a sense of well-being and boosting of physical performance among AS users. approximately one million individuals reported AS use [38]. Furthermore, AS use was positively correlated to the use of other illicit drugs, cigarettes, and alcohol [38]. Interestingly, the perception of AS abuse-induced effects among college athletes has been inversely correlated with the academic performance. In retaliation to this practice, several countries sanctioned Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... http://dx.doi.org/10.5772/intechopen.70475 231 Despite the classical and still frequent AS abuse among elite athletes, recent reports have suggested that the biggest group of AS users are recreational athletes and individuals aiming a supposed improvement on their esthetic appearance [39]. In the USA, epidemiological studies estimated that approximately 2.9–4.0 million Americans have used AS over their lives, a significant rise compared with data from early 1990s. Several epidemiological studies on the consumption of psychotropic drugs in Brazil have revealed that 0.9% of Brazilians have used AS, surpassing the prevalence of use of crack and heroin. Recently, it has been estimated that worldwide prevalence of AS use is approximately 3.3%, although the rate among males can reach 6.4% [40]. Middle East exhibits the highest rate of AS consumption at 21.7%, followed by South America, 4.8%; Europe, 3.8%; North America, 3.0%; Oceania, 2.6%; Africa, 2.4%; and Asia, 0.2% [40]. However, recent reports suggest that the real epidemiological extent of AS abuse might be overshadowed by the high rate of omission among Unsurprisingly, AS abuse can impose harmful adverse effects. The prevalence of these effects among AS abusers remains unclear, and recent reports have demonstrated that approximately 56% of AS users had never reported this practice to any physician, which turns the correlation between AS abuse and the adverse effects elicited by them underreported [41]. In sum, AS-induced adverse effects include reproductive, endocrinological, hepatic, cardiovascular, dermatological, and neurological dysfunctions, as demonstrated by several clinical and experimental studies. Furthermore, many effects can be persistent or even irreversible after Among adverse effects, the most common are dysfunctions in the reproductive system. Given the substantial similarity between AS and endogenous androgens, chronic use of AS results in down-regulation of both follicle-stimulating hormone and luteinizing hormone and overall suppression of the hypothalamus-pituitary-testicular (HPT) axis. Consequently, endogenous production of testosterone can be dramatically reduced, which consists in the main cause of hypogonadism in former AS users [42]. Secondary hypogonadism recovers relatively rapidly after the interruption of AS abuse, although recent reports suggest that it can last for more than a year [42]. Altogether, these abnormalities underlie the significant dysfunction in the spermatic production in AS users. When aromatizable ASs are used, secondary effects linked interruption of AS use, whereas other effects arise only after AS withdrawal. to increased estrogen levels can be seen, such as gynecomastia [43]. laws prohibiting nonmedical use of AS, such as the Anabolic Steroid Act, in the USA. AS abusers [41]. 3. Adverse effects of AS abuse 3.1. Endocrine and reproductive dysfunctions The reports about the relationship between the use of testosterone or its metabolites and the increase in muscle mass and strength resulted in great interest for these substances by elite athletes. It has been suggested that the first report of AS abuse by athletes was during a weightlifting championship in Vienna, 1954, by Russian weightlifters [26]. During the 1950s and 1960s, AS abuse skyrocketed among elite athletes of different countries in several categories, and obviously, it was followed by a rapid and significant increase in the athletic performance in sports competition, such as shot, hammer, and high jump [27, 28]. Interestingly, some reports suggest that popular media supported the supplementation with AS, especially with methandrostenolone, with allegations that it had no side effects [29]. Probably, the most notorious case about AS abuse by athletes involved the State Plan 14.25 of the German Democratic Republic (East German). Although AS abuse had turned into a common practice among elite athletes worldwide, East German government together with both medical and scientific communities organized massive efforts to stimulate AS administration in young and adult athletes, in order to improve their performance in Olympic games [30]. A similar sort of "governmental program" happened in the former Soviet Union between 1960s and 1970s, where it has been believed that athletes as young as 8 years were included [31]. In general, 1950s, 1960s, and 1970s decades were marked by the spread of AS administration among elite athletes. Finally, in 1974, the International Committee banned the use of testosterone and its derivatives in the Olympic games. The first reports about AS use among bodybuilders occurred in the late 1960s and 1970s, but it was in the late 1970s and especially in the 1980s that this practice spread in this class [32]. Interestingly, this delay in comparison to elite athletes was mainly due to a current thinking that AS did not potentiate the gain of muscle mass [26]. Concomitantly, the recreational use of AS rapidly increased throughout the general population, especially in gyms. This scenario was further aggravated by the rising cult for a muscularized body shape among the general population, which was boosted by popular media [33]. Furthermore, given that this body shape paradigm has been even more complex in adolescents, AS abuse also reached highschool students [34]. In this context, underground guidelines containing information about the ways to obtain and use AS have arisen and quickly gained popularity by pseudo-scientific reports [35]. Consequently, AS abuse became a major concern to public health organizations given the severe adverse consequences that frequently follow this practice, and first, epidemiological studies have been conducted during the late 1980s and beginning 1990s showing that approximately 6.6% of 12th grade students reported AS use, and two thirds admitted its use when they were aged 16 years or less [34]. Among male Canadian adolescents, the average AS use was estimated in 5.5%, mostly stimulated by their coaches [36, 37]. In the USA, approximately one million individuals reported AS use [38]. Furthermore, AS use was positively correlated to the use of other illicit drugs, cigarettes, and alcohol [38]. Interestingly, the perception of AS abuse-induced effects among college athletes has been inversely correlated with the academic performance. In retaliation to this practice, several countries sanctioned laws prohibiting nonmedical use of AS, such as the Anabolic Steroid Act, in the USA. Despite the classical and still frequent AS abuse among elite athletes, recent reports have suggested that the biggest group of AS users are recreational athletes and individuals aiming a supposed improvement on their esthetic appearance [39]. In the USA, epidemiological studies estimated that approximately 2.9–4.0 million Americans have used AS over their lives, a significant rise compared with data from early 1990s. Several epidemiological studies on the consumption of psychotropic drugs in Brazil have revealed that 0.9% of Brazilians have used AS, surpassing the prevalence of use of crack and heroin. Recently, it has been estimated that worldwide prevalence of AS use is approximately 3.3%, although the rate among males can reach 6.4% [40]. Middle East exhibits the highest rate of AS consumption at 21.7%, followed by South America, 4.8%; Europe, 3.8%; North America, 3.0%; Oceania, 2.6%; Africa, 2.4%; and Asia, 0.2% [40]. However, recent reports suggest that the real epidemiological extent of AS abuse might be overshadowed by the high rate of omission among AS abusers [41]. ### 3. Adverse effects of AS abuse The misuse of testosterone was firstly reported during the World War II by Nazi German army with the purpose of enhancing soldier's aggressiveness, and there has also been uncertain reports about the administration of AS in Nazi athletes during the Olympic games of 1936 [20]. During the 1940s, the use of synthetic testosterone for medical purposes spread, especially to increase sexual libido and to treat mood disorders, menorrhagia, dysmenorrhea, hypogonadism, and breast cancer [21–25]. Concomitantly, AS use was correlated with a sense The reports about the relationship between the use of testosterone or its metabolites and the increase in muscle mass and strength resulted in great interest for these substances by elite athletes. It has been suggested that the first report of AS abuse by athletes was during a weightlifting championship in Vienna, 1954, by Russian weightlifters [26]. During the 1950s and 1960s, AS abuse skyrocketed among elite athletes of different countries in several categories, and obviously, it was followed by a rapid and significant increase in the athletic performance in sports competition, such as shot, hammer, and high jump [27, 28]. Interestingly, some reports suggest that popular media supported the supplementation with AS, especially with methandrostenolone, with allegations that it had no side effects [29]. Probably, the most notorious case about AS abuse by athletes involved the State Plan 14.25 of the German Democratic Republic (East German). Although AS abuse had turned into a common practice among elite athletes worldwide, East German government together with both medical and scientific communities organized massive efforts to stimulate AS administration in young and adult athletes, in order to improve their performance in Olympic games [30]. A similar sort of "governmental program" happened in the former Soviet Union between 1960s and 1970s, where it has been believed that athletes as young as 8 years were included [31]. In general, 1950s, 1960s, and 1970s decades were marked by the spread of AS administration among elite athletes. Finally, in 1974, the International Committee banned the use of testosterone and its The first reports about AS use among bodybuilders occurred in the late 1960s and 1970s, but it was in the late 1970s and especially in the 1980s that this practice spread in this class [32]. Interestingly, this delay in comparison to elite athletes was mainly due to a current thinking that AS did not potentiate the gain of muscle mass [26]. Concomitantly, the recreational use of AS rapidly increased throughout the general population, especially in gyms. This scenario was further aggravated by the rising cult for a muscularized body shape among the general population, which was boosted by popular media [33]. Furthermore, given that this body shape paradigm has been even more complex in adolescents, AS abuse also reached highschool students [34]. In this context, underground guidelines containing information about the ways to obtain and use AS have arisen and quickly gained popularity by pseudo-scientific reports [35]. Consequently, AS abuse became a major concern to public health organizations given the severe adverse consequences that frequently follow this practice, and first, epidemiological studies have been conducted during the late 1980s and beginning 1990s showing that approximately 6.6% of 12th grade students reported AS use, and two thirds admitted its use when they were aged 16 years or less [34]. Among male Canadian adolescents, the average AS use was estimated in 5.5%, mostly stimulated by their coaches [36, 37]. In the USA, of well-being and boosting of physical performance among AS users. 230 Sex Hormones in Neurodegenerative Processes and Diseases derivatives in the Olympic games. Unsurprisingly, AS abuse can impose harmful adverse effects. The prevalence of these effects among AS abusers remains unclear, and recent reports have demonstrated that approximately 56% of AS users had never reported this practice to any physician, which turns the correlation between AS abuse and the adverse effects elicited by them underreported [41]. In sum, AS-induced adverse effects include reproductive, endocrinological, hepatic, cardiovascular, dermatological, and neurological dysfunctions, as demonstrated by several clinical and experimental studies. Furthermore, many effects can be persistent or even irreversible after interruption of AS use, whereas other effects arise only after AS withdrawal. #### 3.1. Endocrine and reproductive dysfunctions Among adverse effects, the most common are dysfunctions in the reproductive system. Given the substantial similarity between AS and endogenous androgens, chronic use of AS results in down-regulation of both follicle-stimulating hormone and luteinizing hormone and overall suppression of the hypothalamus-pituitary-testicular (HPT) axis. Consequently, endogenous production of testosterone can be dramatically reduced, which consists in the main cause of hypogonadism in former AS users [42]. Secondary hypogonadism recovers relatively rapidly after the interruption of AS abuse, although recent reports suggest that it can last for more than a year [42]. Altogether, these abnormalities underlie the significant dysfunction in the spermatic production in AS users. When aromatizable ASs are used, secondary effects linked to increased estrogen levels can be seen, such as gynecomastia [43]. Important, but still poorly explored endocrine effects of AS abuse are those related to metabolism. In this context, abnormalities in the glucose metabolism have been reported during AS abuse, as evidenced by decreased glucose tolerance in powerlifters under AS abuse [44]. Even so, post-glucose insulin levels were increased in this condition, which suggests that AS can significantly reduce insulin sensitivity [44]. In addition, serum leptin can be significantly increased in AS abusers, without considerable changes in the adipose tissue content [45]. Interestingly, administration of nandrolone decanoate in rats can induce a significant decrease in proopiomelacortin (POMC) expression in the arcuate nucleus, despite the increased levels of leptin and insulin found in AS abusers evaluated in this study [46]. Given that anorexigenic POMC neurons can be directly activated by both insulin and leptin, these findings suggest that POMC neurons might become insensitive to these hormones, which is a common dysfunction observed in obesity and metabolic syndrome. 3.3. General neurological consequences vation of estrogenic receptors. 4. Neurodegenerative diseases abusers. Neurological effects of AS abuse include a broad spectrum of neurobehavioral disturbances. Increased aggressiveness and violence and abnormal sexual behavior have been widely described in AS abusers, whereas anxiety and depression have been observed after AS withdrawal [64]. The behavioral abnormalities found in AS abusers seem to be correlated to profound changes in the neurochemical profile of important limbic regions, such as amygdala, hippocampal, cortical, and hypothalamic regions. These changes are probably promoted by direct bindings to the AR, which is widely expressed throughout the central nervous system, allosteric modulation of neurotransmitter receptors, or by conversion into estrogen and acti- Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... http://dx.doi.org/10.5772/intechopen.70475 233 Experimental studies have demonstrated that the levels of serotonin and catecholamines are closely associated with mood phenotype, motivation, anhedonia, and attention. Specifically, down-regulation of these neurotransmitters throughout limbic regions can increase the susceptibility to depression and anxiety. Interestingly, chronic exposure to AS elicited significant decrease of serotonin levels in the hippocampus, hypothalamus, cortex, and amygdala of rats [65], whereas norepinephrine and dopamine levels are up-regulated in these regions [66, 67]. In the amygdala and hypothalamus, ASs modulate the main excitatory and inhibitory neurotransmitters, namely glutamate and GABA, respectively. ASs have been shown to potentiate glutamate signaling, increasing its excitatory potential in these regions, whereas GABAergic signaling is mainly down-regulated [68, 69]. These limbic regions are associated to a broad spectrum of neurobehavioral functions that include the process of environmental information and memories, as well as the elaboration of a behavioral phenotype in response to these inputs. Therefore, the set of neurochemical alterations elicited by AS within these regions can impose remarkable neurobehavioral manifestations frequently observed in AS Besides the neurobehavioral disturbances, AS abuse has been recently linked to loss of cognition and mnemonic performance. These evidences have been widely demonstrated in animal models of AS abuse, but so far, cognitive performance in human AS abusers remains poorly investigated. It has been reported that decline of cognition is the major consequence of the neurotoxic effect of AS, and consequently, neuronal loss in pivotal areas, such as cortex and hippocampus. Altogether, the changes in the neurophysiology elicited by supraphysiological doses of AS can substantially increase the susceptibility to neurodegenerative diseases. Neurodegenerative diseases are a heterogeneous group of disorders that affect the nervous system, being primarily characterized by degeneration and dysfunction of several neural structures. Despite the efforts to develop therapeutic approaches and the significant number of studies published in this area, such findings have not resulted into development of an effective treatment so far. This lack of success is mainly attributed to the unclear AS abuse has been correlated with overall down-regulation of HPT axis activity. In particular, decreased serum concentration of thyroid-stimulating hormone (TSH), thyroxine (T4), triiodothyronine (T3), free thyroxine, and thyroid-binding globulin have been found in AS abusers [47, 48]. In addition, the stimulatory effect induced by parenteral thyrotropin-release hormone (TRH) injection in the secretion of T3 can be significantly decreased by AS administration, despite the increased level of TSH observed after TRH bolus, suggesting that secondary hypothyroidism can be a prominent consequence of indiscriminate AS use [49]. On the other hand, the level of T3 can be significantly increased after AS withdrawal [47]. Experimentally, rats chronically exposed to nandrolone decanoate can also present significantly decreased serum TSH, T3, and free-T4, besides reduced hepatic deiodinase type 1 activity, followed by secondary thyroid hypertrophy [50]. #### 3.2. Cardiovascular effects AS abuse and cardiovascular adverse effects have long been correlated and reviewed [51]. Cardiovascular effects are marked by dyslipidemia, higher serum low-density-lipoprotein, interstitial fibrosis, cardiac hypertrophy, increased thrombogenesis, arterial hypertension, dysautonomia, and cardiac arrhythmias, as evidenced by clinical and experimental studies [52–58]. Importantly, clinical evidences suggest that some of these abnormalities, such as hypertension and dyslipidemia, are reversible after AS interruption, but others can persist for long periods or are likely irreversible. Notwithstanding in increasing the susceptibility to myocardial infarction and stroke by the above-mentioned abnormalities, chronic administration of AS has been shown to increase the damage induced by myocardial ischemia and reperfusion, which per se can aggravate the post-infarction prognosis [59–61]. Furthermore, recent evidences have demonstrated that therapeutic efficacy of cardioprotective maneuvers against the myocardial ischemia/reperfusion injury can be abolished by chronic exposure to AS [62]. Biochemical and molecular analyses revealed that supraphysiological doses of AS are related to redox imbalance, increased proinflammatory signaling, and overactivation of renin-angiotensin system in the heart, which seems to be closely correlated to the loss of cardioprotection after myocardial ischemia/reperfusion injury [55, 60, 61, 63]. #### 3.3. General neurological consequences Important, but still poorly explored endocrine effects of AS abuse are those related to metabolism. In this context, abnormalities in the glucose metabolism have been reported during AS abuse, as evidenced by decreased glucose tolerance in powerlifters under AS abuse [44]. Even so, post-glucose insulin levels were increased in this condition, which suggests that AS can significantly reduce insulin sensitivity [44]. In addition, serum leptin can be significantly increased in AS abusers, without considerable changes in the adipose tissue content [45]. Interestingly, administration of nandrolone decanoate in rats can induce a significant decrease in proopiomelacortin (POMC) expression in the arcuate nucleus, despite the increased levels of leptin and insulin found in AS abusers evaluated in this study [46]. Given that anorexigenic POMC neurons can be directly activated by both insulin and leptin, these findings suggest that POMC neurons might become insensitive to these hormones, which is a common dys- AS abuse has been correlated with overall down-regulation of HPT axis activity. In particular, decreased serum concentration of thyroid-stimulating hormone (TSH), thyroxine (T4), triiodothyronine (T3), free thyroxine, and thyroid-binding globulin have been found in AS abusers [47, 48]. In addition, the stimulatory effect induced by parenteral thyrotropin-release hormone (TRH) injection in the secretion of T3 can be significantly decreased by AS administration, despite the increased level of TSH observed after TRH bolus, suggesting that secondary hypothyroidism can be a prominent consequence of indiscriminate AS use [49]. On the other hand, the level of T3 can be significantly increased after AS withdrawal [47]. Experimentally, rats chronically exposed to nandrolone decanoate can also present significantly decreased serum TSH, T3, and free-T4, besides reduced hepatic deiodinase type 1 activity, followed by AS abuse and cardiovascular adverse effects have long been correlated and reviewed [51]. Cardiovascular effects are marked by dyslipidemia, higher serum low-density-lipoprotein, interstitial fibrosis, cardiac hypertrophy, increased thrombogenesis, arterial hypertension, dysautonomia, and cardiac arrhythmias, as evidenced by clinical and experimental studies [52–58]. Importantly, clinical evidences suggest that some of these abnormalities, such as hypertension and dyslipidemia, are reversible after AS interruption, but others can persist for long periods or are likely irreversible. Notwithstanding in increasing the susceptibility to myocardial infarction and stroke by the above-mentioned abnormalities, chronic administration of AS has been shown to increase the damage induced by myocardial ischemia and reperfusion, which per se can aggravate the post-infarction prognosis [59–61]. Furthermore, recent evidences have demonstrated that therapeutic efficacy of cardioprotective maneuvers against the myocardial ischemia/reperfusion injury can be abolished by chronic exposure to AS [62]. Biochemical and molecular analyses revealed that supraphysiological doses of AS are related to redox imbalance, increased proinflammatory signaling, and overactivation of renin-angiotensin system in the heart, which seems to be closely correlated to the loss of cardioprotection after myocardial ischemia/reperfusion function observed in obesity and metabolic syndrome. 232 Sex Hormones in Neurodegenerative Processes and Diseases secondary thyroid hypertrophy [50]. 3.2. Cardiovascular effects injury [55, 60, 61, 63]. Neurological effects of AS abuse include a broad spectrum of neurobehavioral disturbances. Increased aggressiveness and violence and abnormal sexual behavior have been widely described in AS abusers, whereas anxiety and depression have been observed after AS withdrawal [64]. The behavioral abnormalities found in AS abusers seem to be correlated to profound changes in the neurochemical profile of important limbic regions, such as amygdala, hippocampal, cortical, and hypothalamic regions. These changes are probably promoted by direct bindings to the AR, which is widely expressed throughout the central nervous system, allosteric modulation of neurotransmitter receptors, or by conversion into estrogen and activation of estrogenic receptors. Experimental studies have demonstrated that the levels of serotonin and catecholamines are closely associated with mood phenotype, motivation, anhedonia, and attention. Specifically, down-regulation of these neurotransmitters throughout limbic regions can increase the susceptibility to depression and anxiety. Interestingly, chronic exposure to AS elicited significant decrease of serotonin levels in the hippocampus, hypothalamus, cortex, and amygdala of rats [65], whereas norepinephrine and dopamine levels are up-regulated in these regions [66, 67]. In the amygdala and hypothalamus, ASs modulate the main excitatory and inhibitory neurotransmitters, namely glutamate and GABA, respectively. ASs have been shown to potentiate glutamate signaling, increasing its excitatory potential in these regions, whereas GABAergic signaling is mainly down-regulated [68, 69]. These limbic regions are associated to a broad spectrum of neurobehavioral functions that include the process of environmental information and memories, as well as the elaboration of a behavioral phenotype in response to these inputs. Therefore, the set of neurochemical alterations elicited by AS within these regions can impose remarkable neurobehavioral manifestations frequently observed in AS abusers. Besides the neurobehavioral disturbances, AS abuse has been recently linked to loss of cognition and mnemonic performance. These evidences have been widely demonstrated in animal models of AS abuse, but so far, cognitive performance in human AS abusers remains poorly investigated. It has been reported that decline of cognition is the major consequence of the neurotoxic effect of AS, and consequently, neuronal loss in pivotal areas, such as cortex and hippocampus. Altogether, the changes in the neurophysiology elicited by supraphysiological doses of AS can substantially increase the susceptibility to neurodegenerative diseases. #### 4. Neurodegenerative diseases Neurodegenerative diseases are a heterogeneous group of disorders that affect the nervous system, being primarily characterized by degeneration and dysfunction of several neural structures. Despite the efforts to develop therapeutic approaches and the significant number of studies published in this area, such findings have not resulted into development of an effective treatment so far. This lack of success is mainly attributed to the unclear pathogenesis underlying neurodegenerative diseases, despite the well-known pathophysiological aspects, such as redox imbalance, autophagy, inflammation, and accumulation of neurotoxic substances. excitotoxicity. As a result, depressive patients generally have loss of hippocampal mass and mnemonic deficit. Furthermore, the extent of dendritic arborization, the density of dendritic spines, and the process of synaptogenesis are crucial aspects in the consolidation of synaptic transmission [75]. During the progression of major depression, increased levels of glucocorticoids elicit a decrease on these events in hippocampus, which contribute to the previously mentioned decline on learning and mnemonic capacities. When neurotransmitter bioavailability, receptor expression and extent of dendritic arborization are chronically up-regulated, synaptic transmission is substantially facilitated. This process is called long-term potentiation (LTP), which is thought to exert a pivotal role in the process of memory consolidation [75]. On the other hand, chronic down-regulation of these properties can elicit a process denominated Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... http://dx.doi.org/10.5772/intechopen.70475 235 It is important to note that all the synaptic abnormalities mentioned above can develop slowly and progressively, being frequently asymptomatic. Overtime, the spread damage culminates in functional deficit. Unfortunately, the lack of sensitive biomarkers to diagnose and to estimate the extent of these changes makes the early diagnostic of neurodegenerative diseases very difficult. As a result, this set of functional abnormalities is most commonly noted only in elderly individuals, in which the prevalence of neurodegenerative diseases is higher. Furthermore, several environmental factors can progressively increase the susceptibility to neurodegenerative diseases over lifetime, such as chronic stress and drug abuse. In this context, recent clinical and experimental findings suggest that long-term AS abuse can induce neurotoxic effects that might increase the susceptibility to loss of cognitive capacity and neu- Studies performed in animal models and cell cultures have demonstrated a broad spectrum of pathophysiological mechanisms underlying the neurotoxicity induced by AS, and all of them seem to culminate in cell apoptosis. Apoptosis is a programmed cell death in which cell volume is progressively decreased, chromatin is condensed, and cell nucleus is fragmented [76]. Generally, apoptosis can be triggered by several distinct intracellular and extracellular stimuli, such as DNA damage, redox imbalance, calcium overload, and excitotoxicity. Naturally occurring apoptosis has been thought to exert a pivotal role in the development of multicellular organisms; moreover, it is considered a defense mechanism in several conditions, such as metabolic imbalance, infections, and neoplasia [77]. However, unbalanced apoptotic process can induce harmful effects into target tissues. In the case of cancer, for instance, the decreased apoptotic rate among neoplastic cells results in growing and spread of tumors. Conversely, increased cell death in cases of neurodegenerative diseases has been attributed to the uncontrollable apoptotic process among neuronal cells [77]. Apoptosis is triggered by two main pathways—the extrinsic, also called death receptor pathway, and the intrinsic, namely mitochondrial pathway. The extrinsic pathway is stimulated by activation of death receptors family, which includes the tumor necrosis factor (TNF)–related apoptosisinducing ligand (TRAIL-R1 and TRAIL-R2), FAS and TNF receptors TNF)-related apoptosisinducing ligand TNF)-related apoptosis-inducing ligand. The activation of these receptors long-term depression, culminating in long-term cognitive impairment. 4.1. Pathophysiological mechanisms associated to AS-induced neurotoxicity rodegenerative diseases. In sum, it has been proposed that genetic and/or environmental factors can trigger early pathophysiological changes, such as aggregation of amyloid-β (Aβ)-protein, a common feature throughout the progression of Alzheimer's disease, resulting in primary neural damage. Subsequently, these early events can evoke secondary damages, due to inflammation, redox imbalance, and endoplasmic reticulum stress, leading to synapse dysfunctions, which has generally been accepted as a reversible process, and culminating in neuronal death and irreversible neuronal damage. Consistent evidences have implied that the accumulation of Aβ correlates—temporally and pathophysiologically—with decreased synaptic function early in the progression of Alzheimer's disease [70]. The occurrence of these events in the hippocampus has been correlated with impaired hippocampal-dependent memory consolidation. Thus, synapse dysfunction and the consequent neuronal death are the cornerstones during the progression of neurodegenerative diseases. Conceptually, synaptic function can be modulated by distinct but correlated mechanisms. Changes in the physiological regulation of these mechanisms can lead to marked neurological effects, including cognitive impairments. First, the bioavailability of neurotransmitters is crucial to an effective synaptic function, thereby neurochemical imbalance can lead to impairment of synaptic transmission. Furthermore, post-synaptic expression of neurotransmitter receptors is equally important in the maintenance of synaptic transmission. For example, down-regulation of acetylcholine signaling in the hippocampal and cortical neuronal networks results in cognitive deficit, as observed during the progression of schizophrenia and Alzheimer's disease [71, 72]. In Parkinson's disease, loss of dopaminergic neurons located in the substantia nigra, and the consequent interruption of neural transmission in the nigrostriatal pathway results in a substantial drop on dopamine bioavailability in the dorsal striatum (i.e., the caudate nucleus and putamen), and decreased activity of GABAergic neurons located in the striatum [73]. Taken together, these disturbances culminate with loss of locomotor control, the most prominent characteristic of Parkinson's disease. Moreover, the pathophysiological development of neurobehavioral dysfunctions and loss of cognitive performance found in major depression are related to a decrease on the bioavailability of noradrenaline, dopamine, and 5-hydroxytryptamine [74]. Thus, drugs that enhance the bioavailability of these neurotransmitters are the first-line therapeutic approach to treat this condition. All the above-mentioned conditions are examples of how down-regulation of synaptic transmission can induce severe degeneration in cognitive, locomotor, and behavioral control. However, synaptic function is not only negatively affected by down-regulation of neurotransmitters but also by an up-regulation of those molecules and their signaling. In some conditions characterized by increased circulating levels of glucocorticoids, such as stress and major depression, glutamatergic signaling can be significantly potentiated in hippocampal neuronal networks by cortisol and corticosterone [75]. Overactivation of glutamate receptors promote a significant increase in intracellular calcium concentration, resulting in recruitment of several downstream signaling that culminate in neuronal death, an event known as excitotoxicity. As a result, depressive patients generally have loss of hippocampal mass and mnemonic deficit. Furthermore, the extent of dendritic arborization, the density of dendritic spines, and the process of synaptogenesis are crucial aspects in the consolidation of synaptic transmission [75]. During the progression of major depression, increased levels of glucocorticoids elicit a decrease on these events in hippocampus, which contribute to the previously mentioned decline on learning and mnemonic capacities. When neurotransmitter bioavailability, receptor expression and extent of dendritic arborization are chronically up-regulated, synaptic transmission is substantially facilitated. This process is called long-term potentiation (LTP), which is thought to exert a pivotal role in the process of memory consolidation [75]. On the other hand, chronic down-regulation of these properties can elicit a process denominated long-term depression, culminating in long-term cognitive impairment. pathogenesis underlying neurodegenerative diseases, despite the well-known pathophysiological aspects, such as redox imbalance, autophagy, inflammation, and accumulation of In sum, it has been proposed that genetic and/or environmental factors can trigger early pathophysiological changes, such as aggregation of amyloid-β (Aβ)-protein, a common feature throughout the progression of Alzheimer's disease, resulting in primary neural damage. Subsequently, these early events can evoke secondary damages, due to inflammation, redox imbalance, and endoplasmic reticulum stress, leading to synapse dysfunctions, which has generally been accepted as a reversible process, and culminating in neuronal death and irreversible neuronal damage. Consistent evidences have implied that the accumulation of Aβ correlates—temporally and pathophysiologically—with decreased synaptic function early in the progression of Alzheimer's disease [70]. The occurrence of these events in the hippocampus has been correlated with impaired hippocampal-dependent memory consolidation. Thus, synapse dysfunction and the consequent neuronal death are the cornerstones during the pro- Conceptually, synaptic function can be modulated by distinct but correlated mechanisms. Changes in the physiological regulation of these mechanisms can lead to marked neurological effects, including cognitive impairments. First, the bioavailability of neurotransmitters is crucial to an effective synaptic function, thereby neurochemical imbalance can lead to impairment of synaptic transmission. Furthermore, post-synaptic expression of neurotransmitter receptors is equally important in the maintenance of synaptic transmission. For example, down-regulation of acetylcholine signaling in the hippocampal and cortical neuronal networks results in cognitive deficit, as observed during the progression of schizophrenia and Alzheimer's disease [71, 72]. In Parkinson's disease, loss of dopaminergic neurons located in the substantia nigra, and the consequent interruption of neural transmission in the nigrostriatal pathway results in a substantial drop on dopamine bioavailability in the dorsal striatum (i.e., the caudate nucleus and putamen), and decreased activity of GABAergic neurons located in the striatum [73]. Taken together, these disturbances culminate with loss of locomotor control, the most prominent characteristic of Parkinson's disease. Moreover, the pathophysiological development of neurobehavioral dysfunctions and loss of cognitive performance found in major depression are related to a decrease on the bioavailability of noradrenaline, dopamine, and 5-hydroxytryptamine [74]. Thus, drugs that enhance the bioavailability of these neurotransmitters are the first-line therapeutic approach to treat this condition. All the above-mentioned conditions are examples of how down-regulation of synaptic transmission can induce severe degeneration in cognitive, locomotor, and behavioral control. However, synaptic function is not only negatively affected by down-regulation of neurotransmitters but also by an up-regulation of those molecules and their signaling. In some conditions characterized by increased circulating levels of glucocorticoids, such as stress and major depression, glutamatergic signaling can be significantly potentiated in hippocampal neuronal networks by cortisol and corticosterone [75]. Overactivation of glutamate receptors promote a significant increase in intracellular calcium concentration, resulting in recruitment of several downstream signaling that culminate in neuronal death, an event known as neurotoxic substances. gression of neurodegenerative diseases. 234 Sex Hormones in Neurodegenerative Processes and Diseases It is important to note that all the synaptic abnormalities mentioned above can develop slowly and progressively, being frequently asymptomatic. Overtime, the spread damage culminates in functional deficit. Unfortunately, the lack of sensitive biomarkers to diagnose and to estimate the extent of these changes makes the early diagnostic of neurodegenerative diseases very difficult. As a result, this set of functional abnormalities is most commonly noted only in elderly individuals, in which the prevalence of neurodegenerative diseases is higher. Furthermore, several environmental factors can progressively increase the susceptibility to neurodegenerative diseases over lifetime, such as chronic stress and drug abuse. In this context, recent clinical and experimental findings suggest that long-term AS abuse can induce neurotoxic effects that might increase the susceptibility to loss of cognitive capacity and neurodegenerative diseases. #### 4.1. Pathophysiological mechanisms associated to AS-induced neurotoxicity Studies performed in animal models and cell cultures have demonstrated a broad spectrum of pathophysiological mechanisms underlying the neurotoxicity induced by AS, and all of them seem to culminate in cell apoptosis. Apoptosis is a programmed cell death in which cell volume is progressively decreased, chromatin is condensed, and cell nucleus is fragmented [76]. Generally, apoptosis can be triggered by several distinct intracellular and extracellular stimuli, such as DNA damage, redox imbalance, calcium overload, and excitotoxicity. Naturally occurring apoptosis has been thought to exert a pivotal role in the development of multicellular organisms; moreover, it is considered a defense mechanism in several conditions, such as metabolic imbalance, infections, and neoplasia [77]. However, unbalanced apoptotic process can induce harmful effects into target tissues. In the case of cancer, for instance, the decreased apoptotic rate among neoplastic cells results in growing and spread of tumors. Conversely, increased cell death in cases of neurodegenerative diseases has been attributed to the uncontrollable apoptotic process among neuronal cells [77]. Apoptosis is triggered by two main pathways—the extrinsic, also called death receptor pathway, and the intrinsic, namely mitochondrial pathway. The extrinsic pathway is stimulated by activation of death receptors family, which includes the tumor necrosis factor (TNF)–related apoptosisinducing ligand (TRAIL-R1 and TRAIL-R2), FAS and TNF receptors TNF)-related apoptosisinducing ligand TNF)-related apoptosis-inducing ligand. The activation of these receptors results in recruitment of pro-apoptotic proteins, such as BAX, BID, BAK, BAD, besides downregulation of anti-apoptotic proteins, like Bcl-2, and culminates in activation of caspases, a family of cysteine protease enzymes, leading to cleavage of caspase substrates and cell death [77, 78]. The intrinsic apoptotic pathway is mainly triggered by intracellular stimuli, such as DNA damage, endoplasmic reticulum stress and redox imbalance. Irrespective of the central cause, these events lead to mitochondrial inner-membrane permeabilization, mainly through opening of mitochondrial permeability transition pore, culminating in mitochondrial swelling and release of apoptosis-triggering factors, such as cytochrome c [78]. that PKCδ might have a pathophysiological role in AS-induced neuronal apoptosis. In contrast, the activation of ERK and Akt, two key proteins involved in the recruitment of cellular pathways linked to cell survival can be considerably decreased in neurons exposed to AS [81]. Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... http://dx.doi.org/10.5772/intechopen.70475 237 Redox imbalance has long been reported as a prominent mechanism underlying the apoptotic process in several pathophysiological conditions. At low concentrations, reactive oxygen species (ROS) can act as second messengers, especially hydrogen peroxide. In the thyroid gland, ROSs have been shown to have a crucial role in thyroid hormones synthesis and overall thyroid homeostasis [90]. However, in high concentration, ROS can induce oxidative damage of Redox homeostasis is characterized by cellular antioxidant activity, such as the enzymes superoxide dismutase, catalase, thioperoxidases and glutathione complex, and ROS production by the mitochondria, nicotinamide dinucleotide phosphate oxidases (NOX), and xanthine oxidases. Thus, redox imbalance can arise in conditions of down-regulation of cellular antioxidant defense and/or ROS overproduction [90]. In the context of neurodegenerative diseases, redox imbalance has been shown to have a pivotal role in synaptic dysfunction and neuronal loss, which is observed in the brain during the development and progression of neurodegenerative diseases [92, 93]. Moreover, the hallmarks of apoptosis, including caspase activation, DNA damage, and binding of pro-apoptotic transcription factors, and cytoskeletal Redox imbalance has been reported as a prominent mechanism underlying the AS-induced cell damage and apoptosis. Experimental studies have demonstrated that chronic administration of AS can up-regulate the activity of NOX in several cell types, resulting in increased ROS production, whereas antioxidant activity seems to be substantially decreased in this condition [63, 94]. In the CNS, chronic administration of nandrolone decanoate in rats has been shown to decrease glutathione peroxidase (Gpx) activity in the hippocampus and pre-frontal cortex [79]. Gpx catalyzes the oxidation of two monomeric glutathione molecules by hydrogen peroxide. Thus, down-regulation of Gpx activity by AS increases hydrogen peroxide bioavailability, which is correlated to increased lipoperoxidation and reduced thiol residues induced Interestingly, pretreatment of neuronal dopaminergic cell lines with testosterone has also been shown to protect them against oxidative damage induced by hydrogen peroxide [95]. The neuroprotective effect was correlated with a slight increase in the calcium-induced mitochondrial ROS production. In contrast, in conditions of sustained redox imbalance, post-exposure of dopaminergic neurons to testosterone can further increase the oxidative damage and decrease cell viability by mitochondrial calcium overload, an effect mediated by membrane-attached receptor [95]. Furthermore, activation of membrane-attached receptors has also been shown to be involved, as co-exposure with flutamide did prevent neither mitochondrial calcium over- O and glutathione disulfide, thus reducing the concentration of hydrogen several cellular structures, culminating in cell death by apoptosis or necrosis [91]. 4.1.1. Redox imbalance alterations can be strictly affected by ROS. by AS exposure in the brain [79, 95]. load nor decreased cell viability [95]. peroxide into H<sup>2</sup> Experimental studies have demonstrated that exposure to high concentrations of AS can elicit both extrinsic and intrinsic apoptosis. Long-term administration of nandrolone decanoate results in increased activation of caspase-3 and apoptosis throughout hippocampal and cortical structures [79]. Several in vitro studies have also demonstrated that exposure of neuroblastoma cells, primary hippocampal cells, and pheochromocytoma cells to AS can result in increased activation of caspase-3 [15, 80, 81]. Caspase-3 can be activated in both extrinsic and intrinsic apoptosis pathways and exerts a pivotal role in the execution of the apoptotic process by proteolytic cleavage of several proteins and chromatin condensation, resulting in DNA fragmentation and other changes throughout the apoptotic process. In the context of neurodegenerative diseases, caspase-3 has been shown to have a prominent role in the proteolytic cleavage of amyloid-β precursor protein and neuronal death during the progression of Alzheimer's disease [82]. Interestingly, in neuroblastoma cell culture, exposure to testosterone-induced concentrationdependent sustained increase in intracellular calcium concentration that involved up-regulation of inositol-triphosphate receptor (InsP<sup>3</sup> R) type I-induced calcium release [15]. As demonstrated elsewhere, prolonged calcium overload can trigger apoptosis in several cell types [83]. Indeed, exposure to testosterone can induce caspase-3 activation in these cells, an event that can be prevented by pharmacological inhibition or knock down of InsP<sup>3</sup> R [15]. The increased activation of caspase-3 by testosterone and its synthetic metabolites induces the cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP), a nuclear protein involved in DNA repair signaling [81]. In response to single-strand DNA breaks induced by cellular stressful conditions, PARP initiates the synthesis of polymeric adenosine diphosphate-ribose and leads to recruitment of DNA-repairing enzymes, such as DNA ligase and DNA polymerase [84]. As a result, cleavage of PARP by caspases can substantially increase DNA damage and apoptosis. Conversely, co-exposure with flutamide prevented the activation of caspase-3 and proteolytic cleavage of PARP, demonstrating that activation of AR is crucial to the process of DNA damage and apoptosis after exposure of neuronal cells to high concentrations of AS [81]. Furthermore, testosterone-induced activation of caspase-3 can induce proteolytic cleavage and activation of protein-kinase Cδ (PKCδ) in different cell types, including neuronal dopaminergic cell line [85]. Although the precise role of PKCδ remains controversial and experimental studies have shown both protective and pro-apoptotic effects, testosterone-induced coronary smooth muscle cell apoptosis was prevented by PKCδ and caspase-3 inhibition [86]. In addition, PKCδ has been shown to have a prominent role in the aging-related decline on hippocampal and mnemonic performance, as well as in the apoptosis of dopaminergic neurons in experimental models of Parkinson's disease [87–89]. Thus, it seems reasonable to hypothesize that PKCδ might have a pathophysiological role in AS-induced neuronal apoptosis. In contrast, the activation of ERK and Akt, two key proteins involved in the recruitment of cellular pathways linked to cell survival can be considerably decreased in neurons exposed to AS [81]. #### 4.1.1. Redox imbalance results in recruitment of pro-apoptotic proteins, such as BAX, BID, BAK, BAD, besides downregulation of anti-apoptotic proteins, like Bcl-2, and culminates in activation of caspases, a family of cysteine protease enzymes, leading to cleavage of caspase substrates and cell death [77, 78]. The intrinsic apoptotic pathway is mainly triggered by intracellular stimuli, such as DNA damage, endoplasmic reticulum stress and redox imbalance. Irrespective of the central cause, these events lead to mitochondrial inner-membrane permeabilization, mainly through opening of mitochondrial permeability transition pore, culminating in mitochondrial swell- Experimental studies have demonstrated that exposure to high concentrations of AS can elicit both extrinsic and intrinsic apoptosis. Long-term administration of nandrolone decanoate results in increased activation of caspase-3 and apoptosis throughout hippocampal and cortical structures [79]. Several in vitro studies have also demonstrated that exposure of neuroblastoma cells, primary hippocampal cells, and pheochromocytoma cells to AS can result in increased activation of caspase-3 [15, 80, 81]. Caspase-3 can be activated in both extrinsic and intrinsic apoptosis pathways and exerts a pivotal role in the execution of the apoptotic process by proteolytic cleavage of several proteins and chromatin condensation, resulting in DNA fragmentation and other changes throughout the apoptotic process. In the context of neurodegenerative diseases, caspase-3 has been shown to have a prominent role in the proteolytic cleavage of amyloid-β precursor protein and neuronal death during the progression of Alzheimer's disease [82]. Interestingly, in neuroblastoma cell culture, exposure to testosterone-induced concentrationdependent sustained increase in intracellular calcium concentration that involved up-regulation elsewhere, prolonged calcium overload can trigger apoptosis in several cell types [83]. Indeed, exposure to testosterone can induce caspase-3 activation in these cells, an event that can be tion of caspase-3 by testosterone and its synthetic metabolites induces the cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP), a nuclear protein involved in DNA repair signaling [81]. In response to single-strand DNA breaks induced by cellular stressful conditions, PARP initiates the synthesis of polymeric adenosine diphosphate-ribose and leads to recruitment of DNA-repairing enzymes, such as DNA ligase and DNA polymerase [84]. As a result, cleavage of PARP by caspases can substantially increase DNA damage and apoptosis. Conversely, co-exposure with flutamide prevented the activation of caspase-3 and proteolytic cleavage of PARP, demonstrating that activation of AR is crucial to the process of DNA dam- Furthermore, testosterone-induced activation of caspase-3 can induce proteolytic cleavage and activation of protein-kinase Cδ (PKCδ) in different cell types, including neuronal dopaminergic cell line [85]. Although the precise role of PKCδ remains controversial and experimental studies have shown both protective and pro-apoptotic effects, testosterone-induced coronary smooth muscle cell apoptosis was prevented by PKCδ and caspase-3 inhibition [86]. In addition, PKCδ has been shown to have a prominent role in the aging-related decline on hippocampal and mnemonic performance, as well as in the apoptosis of dopaminergic neurons in experimental models of Parkinson's disease [87–89]. Thus, it seems reasonable to hypothesize age and apoptosis after exposure of neuronal cells to high concentrations of AS [81]. R) type I-induced calcium release [15]. As demonstrated R [15]. The increased activa- ing and release of apoptosis-triggering factors, such as cytochrome c [78]. of inositol-triphosphate receptor (InsP<sup>3</sup> 236 Sex Hormones in Neurodegenerative Processes and Diseases prevented by pharmacological inhibition or knock down of InsP<sup>3</sup> Redox imbalance has long been reported as a prominent mechanism underlying the apoptotic process in several pathophysiological conditions. At low concentrations, reactive oxygen species (ROS) can act as second messengers, especially hydrogen peroxide. In the thyroid gland, ROSs have been shown to have a crucial role in thyroid hormones synthesis and overall thyroid homeostasis [90]. However, in high concentration, ROS can induce oxidative damage of several cellular structures, culminating in cell death by apoptosis or necrosis [91]. Redox homeostasis is characterized by cellular antioxidant activity, such as the enzymes superoxide dismutase, catalase, thioperoxidases and glutathione complex, and ROS production by the mitochondria, nicotinamide dinucleotide phosphate oxidases (NOX), and xanthine oxidases. Thus, redox imbalance can arise in conditions of down-regulation of cellular antioxidant defense and/or ROS overproduction [90]. In the context of neurodegenerative diseases, redox imbalance has been shown to have a pivotal role in synaptic dysfunction and neuronal loss, which is observed in the brain during the development and progression of neurodegenerative diseases [92, 93]. Moreover, the hallmarks of apoptosis, including caspase activation, DNA damage, and binding of pro-apoptotic transcription factors, and cytoskeletal alterations can be strictly affected by ROS. Redox imbalance has been reported as a prominent mechanism underlying the AS-induced cell damage and apoptosis. Experimental studies have demonstrated that chronic administration of AS can up-regulate the activity of NOX in several cell types, resulting in increased ROS production, whereas antioxidant activity seems to be substantially decreased in this condition [63, 94]. In the CNS, chronic administration of nandrolone decanoate in rats has been shown to decrease glutathione peroxidase (Gpx) activity in the hippocampus and pre-frontal cortex [79]. Gpx catalyzes the oxidation of two monomeric glutathione molecules by hydrogen peroxide into H<sup>2</sup> O and glutathione disulfide, thus reducing the concentration of hydrogen peroxide. Thus, down-regulation of Gpx activity by AS increases hydrogen peroxide bioavailability, which is correlated to increased lipoperoxidation and reduced thiol residues induced by AS exposure in the brain [79, 95]. Interestingly, pretreatment of neuronal dopaminergic cell lines with testosterone has also been shown to protect them against oxidative damage induced by hydrogen peroxide [95]. The neuroprotective effect was correlated with a slight increase in the calcium-induced mitochondrial ROS production. In contrast, in conditions of sustained redox imbalance, post-exposure of dopaminergic neurons to testosterone can further increase the oxidative damage and decrease cell viability by mitochondrial calcium overload, an effect mediated by membrane-attached receptor [95]. Furthermore, activation of membrane-attached receptors has also been shown to be involved, as co-exposure with flutamide did prevent neither mitochondrial calcium overload nor decreased cell viability [95]. Altogether, these evidences imply that ROS might be a "switch" in the neuronal effects induced by AS, as they can determine whether exposure of neuronal cells to AS can result in neurotoxic or neuroprotective effects. In physiological concentrations, testosterone and AS can slightly increase the concentration of ROS, which might induce neuronal preconditioning, protecting these cells against further increases in ROS concentration [96, 97]. However, in supraphysiological concentrations, AS can significantly increase the ROS bioavailability by mitochondrial and nonmitochondrial mechanisms, resulting in oxidative damage and neuronal apoptosis. Interestingly, the increased susceptibility of dopaminergic neurons to AS-induced redox imbalance and oxidative damage suggests that administration of supraphysiological doses of these drugs might also increase the susceptibility to Parkinson's disease. These effects can be modulated by both membrane and cytosolic receptors, although the precise contribution of each one remains unclear. Furthermore, it remains unclear, though, whether AS exposure can induce long-term increased neuronal susceptibility to redox imbalance, as evidenced in cardiac cells [59]. Given that neurodegenerative diseases occur more frequently in elderly people, the elucidation of this aspect can develop an important role in the diagnosis and prognosis of neurodegenerative diseases in former AS abusers. synaptic cleft. Both changes dramatically increase the susceptibility of glutamate storm and glutamate-induced excitotoxicity. Noteworthy, the increased expression of VGLT2 can persist until 3 weeks after interruption of AS administration, implying that these effects might be Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... http://dx.doi.org/10.5772/intechopen.70475 239 Exposure of mixed cultures of mouse cortical cells to testosterone induced concentrationdependent increase in NMDA-induced neurotoxicity, as demonstrated by increases on trypan blue-labeling and the release of lactate-dehydrogenase [99]. This effect was further increased when aromatase inhibitors were co-administered to the culture medium, corroborating the hypothesis that testosterone-induced neuroprotection was at least partially mediated by further conversion into estrogen. In addition, co-administration of flutamide significantly attenuated the increased excitotoxicity induced by high concentrations of testosterone, highlighting the role exerted by the overactivation of AR in this regard [99]. In keeping with this, exposure of neuronal cells to nor-testosterone (i.e., nandrolone) and stanozolol increased NDMAinduced neurotoxicity in a concentration-dependent and aromatase-independent way, given that inhibition of aromatase did not attenuate this effect [99]. As a result, AS-induced potentiation in the glutamate signaling substantially increased the peak of calcium concentration induced by glutamate, whereas the return to calcium baseline levels was prolonged [16]. Conversely, inhibition of AR with flutamide completely abolished this effect. These evidences imply that ASs potentiate excitotoxicity induced by overactivation of glutamate receptor Besides the potentiation of glutamate-induced excitotoxicity, experimental studies have shown that exposure of neuronal cells to AS can also modulate the neurotoxic effects of Aβ. These oligomers are physiologically generated by cleavage of amyloid precursor protein (APP) by β- and γ-secretases, and the β-site APP-cleaving enzyme 1 (BACE1) is the most prominent β-secretase throughout the brain. The most common isoforms are Aβ40 and Aβ42, where the shorter (i.e., Aβ40) is produced in the trans-Golgi apparatus and is the most prominent, whereas the longer is produced in the endoplasmic reticulum and has the most notorious fibrillogenic capacity. The clearance of Aβ is performed by several pathways, including activation of degrading enzymes and receptor-mediated cellular and vascular clearance. Under unclear circumstances, though, Aβ generation and clearance can be unbalanced, resulting in accumulation and aggregation of Aβ [102]. In this context, presenilin 1 (PS1) and presenilin 2 (PS2) regulate the proteolytic function of γ-secretases, and recent studies have demonstrated that mutations in both protein can result in accumulation of Aβ42, which is the hallmark of Alzheimer's disease [103]. Aggregated Aβ can induce neurotoxic effects by several mechanisms, including induction of calcium overload and redox imbalance, culminating in synaptic deterioration and neuronal apoptosis [102]. Furthermore, soluble Aβ, also known to induce neurotoxicity, is increased in the cerebrospinal fluid of patients with Alzheimer's disease [104, 105]. Noteworthy, Aβ can bind to NMDA and AMPA receptors, and these inter- Recently, it has been demonstrated that Aβ levels can be substantially increased in the whole brain and cerebrospinal fluid, but especially in the hippocampus, after short-term exposure to 17β-trenbolone in male rats [80]. Similarly, exposure of primary hippocampal neurons actions can further increase excitotoxicity induced by glutamate [106]. induced by genomic mechanisms [101]. exclusively via classic AR pathway. #### 4.1.2. Excitotoxicity The neurotoxicity induced by AS can be further complicated by the induction of excitotoxicity effect. This phenomenon occurs after a massive release of glutamate, an event called glutamate storm, or exogenous compounds, such as N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA). In such a condition, glutamate ionotropic receptors (i.e., NMDA and AMPA receptors) are overstimulated. Physiologically, activation of these receptors triggers calcium influx and plasma membrane depolarization and exerts a pivotal role in the neurotransmission and LTP process. However, overactivation of NMDA and AMPA receptors results in calcium overload, mitochondrial dysfunction, redox imbalance, and recruitment of pro-apoptotic pathways [98]. Excitotoxicity is thought to develop a prominent role in the cellular damage and tissue injury during the progression of neurodegenerative diseases, as well as major depression-associated loss of neuronal viability and decline on cognitive capacity [98]. Within physiological ranges, testosterone exhibited neuroprotective effects against neurotoxicity induced by kainic acid, an agonist of AMPA receptors and its conversion into estrogen by aromatase is thought to have a key role in this protection [99]. However, as previously stated, most of ASs are poorly converted into estrogen by aromatase, especially class II and III AS. Furthermore, the abusive characteristic of AS illicit consumption can dramatically increase the concentration of testosterone and its metabolites within neural tissue. In keeping with this, it was shown that one administration of nandrolone decanoate in rats can increase the phosphorylation of NMDA receptor subunits NR2A and NR2B in the hippocampus, suggesting that acute exposure to AS is enough to increase the activity of NMDA receptor [100]. In addition, chronic administration of AS in rats can increase the expression of vesicular glutamate transporter 2 (VGLT2) [101]. VGLT2 exerts an important role in the uptake of glutamate into synaptic vesicles, suggesting that exposure to high concentration of AS can increase not only the activity of glutamate receptors but also increase the release of glutamate into synaptic cleft. Both changes dramatically increase the susceptibility of glutamate storm and glutamate-induced excitotoxicity. Noteworthy, the increased expression of VGLT2 can persist until 3 weeks after interruption of AS administration, implying that these effects might be induced by genomic mechanisms [101]. Altogether, these evidences imply that ROS might be a "switch" in the neuronal effects induced by AS, as they can determine whether exposure of neuronal cells to AS can result in neurotoxic or neuroprotective effects. In physiological concentrations, testosterone and AS can slightly increase the concentration of ROS, which might induce neuronal preconditioning, protecting these cells against further increases in ROS concentration [96, 97]. However, in supraphysiological concentrations, AS can significantly increase the ROS bioavailability by mitochondrial and nonmitochondrial mechanisms, resulting in oxidative damage and neuronal apoptosis. Interestingly, the increased susceptibility of dopaminergic neurons to AS-induced redox imbalance and oxidative damage suggests that administration of supraphysiological doses of these drugs might also increase the susceptibility to Parkinson's disease. These effects can be modulated by both membrane and cytosolic receptors, although the precise contribution of each one remains unclear. Furthermore, it remains unclear, though, whether AS exposure can induce long-term increased neuronal susceptibility to redox imbalance, as evidenced in cardiac cells [59]. Given that neurodegenerative diseases occur more frequently in elderly people, the elucidation of this aspect can develop an important role in the diagnosis The neurotoxicity induced by AS can be further complicated by the induction of excitotoxicity effect. This phenomenon occurs after a massive release of glutamate, an event called glutamate storm, or exogenous compounds, such as N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA). In such a condition, glutamate ionotropic receptors (i.e., NMDA and AMPA receptors) are overstimulated. Physiologically, activation of these receptors triggers calcium influx and plasma membrane depolarization and exerts a pivotal role in the neurotransmission and LTP process. However, overactivation of NMDA and AMPA receptors results in calcium overload, mitochondrial dysfunction, redox imbalance, and recruitment of pro-apoptotic pathways [98]. Excitotoxicity is thought to develop a prominent role in the cellular damage and tissue injury during the progression of neurodegenerative diseases, as well as major depression-associated loss of neuronal viability Within physiological ranges, testosterone exhibited neuroprotective effects against neurotoxicity induced by kainic acid, an agonist of AMPA receptors and its conversion into estrogen by aromatase is thought to have a key role in this protection [99]. However, as previously stated, most of ASs are poorly converted into estrogen by aromatase, especially class II and III AS. Furthermore, the abusive characteristic of AS illicit consumption can dramatically increase the concentration of testosterone and its metabolites within neural tissue. In keeping with this, it was shown that one administration of nandrolone decanoate in rats can increase the phosphorylation of NMDA receptor subunits NR2A and NR2B in the hippocampus, suggesting that acute exposure to AS is enough to increase the activity of NMDA receptor [100]. In addition, chronic administration of AS in rats can increase the expression of vesicular glutamate transporter 2 (VGLT2) [101]. VGLT2 exerts an important role in the uptake of glutamate into synaptic vesicles, suggesting that exposure to high concentration of AS can increase not only the activity of glutamate receptors but also increase the release of glutamate into and prognosis of neurodegenerative diseases in former AS abusers. 4.1.2. Excitotoxicity and decline on cognitive capacity [98]. 238 Sex Hormones in Neurodegenerative Processes and Diseases Exposure of mixed cultures of mouse cortical cells to testosterone induced concentrationdependent increase in NMDA-induced neurotoxicity, as demonstrated by increases on trypan blue-labeling and the release of lactate-dehydrogenase [99]. This effect was further increased when aromatase inhibitors were co-administered to the culture medium, corroborating the hypothesis that testosterone-induced neuroprotection was at least partially mediated by further conversion into estrogen. In addition, co-administration of flutamide significantly attenuated the increased excitotoxicity induced by high concentrations of testosterone, highlighting the role exerted by the overactivation of AR in this regard [99]. In keeping with this, exposure of neuronal cells to nor-testosterone (i.e., nandrolone) and stanozolol increased NDMAinduced neurotoxicity in a concentration-dependent and aromatase-independent way, given that inhibition of aromatase did not attenuate this effect [99]. As a result, AS-induced potentiation in the glutamate signaling substantially increased the peak of calcium concentration induced by glutamate, whereas the return to calcium baseline levels was prolonged [16]. Conversely, inhibition of AR with flutamide completely abolished this effect. These evidences imply that ASs potentiate excitotoxicity induced by overactivation of glutamate receptor exclusively via classic AR pathway. Besides the potentiation of glutamate-induced excitotoxicity, experimental studies have shown that exposure of neuronal cells to AS can also modulate the neurotoxic effects of Aβ. These oligomers are physiologically generated by cleavage of amyloid precursor protein (APP) by β- and γ-secretases, and the β-site APP-cleaving enzyme 1 (BACE1) is the most prominent β-secretase throughout the brain. The most common isoforms are Aβ40 and Aβ42, where the shorter (i.e., Aβ40) is produced in the trans-Golgi apparatus and is the most prominent, whereas the longer is produced in the endoplasmic reticulum and has the most notorious fibrillogenic capacity. The clearance of Aβ is performed by several pathways, including activation of degrading enzymes and receptor-mediated cellular and vascular clearance. Under unclear circumstances, though, Aβ generation and clearance can be unbalanced, resulting in accumulation and aggregation of Aβ [102]. In this context, presenilin 1 (PS1) and presenilin 2 (PS2) regulate the proteolytic function of γ-secretases, and recent studies have demonstrated that mutations in both protein can result in accumulation of Aβ42, which is the hallmark of Alzheimer's disease [103]. Aggregated Aβ can induce neurotoxic effects by several mechanisms, including induction of calcium overload and redox imbalance, culminating in synaptic deterioration and neuronal apoptosis [102]. Furthermore, soluble Aβ, also known to induce neurotoxicity, is increased in the cerebrospinal fluid of patients with Alzheimer's disease [104, 105]. Noteworthy, Aβ can bind to NMDA and AMPA receptors, and these interactions can further increase excitotoxicity induced by glutamate [106]. Recently, it has been demonstrated that Aβ levels can be substantially increased in the whole brain and cerebrospinal fluid, but especially in the hippocampus, after short-term exposure to 17β-trenbolone in male rats [80]. Similarly, exposure of primary hippocampal neurons to 17β-trenbolone, but not to DHT, can significantly elevate the levels of Aβ42. Interestingly, this effect was seen only in male rats. Even so, administration of 17β-trenbolone in pregnant female rats resulted in accumulation of Aβ42 in the fetus brain [80]. In keeping with these findings, exposure to 17β-trenbolone also resulted in a concentration-dependent down-regulation of PS1 levels in primary hippocampal neurons [80]. These evidences have demonstrated that AS abuse can induce accumulation of Aβ42 in the hippocampus, which can induce long-term susceptibility to Alzheimer's disease, especially in the offspring of female AS abusers. these abnormalities [109, 110]. Despite these evidences regarding endogenous testosterone levels, consistent data have shown that long-term administration of supraphysiological doses of AS can significantly impair the cognitive capacity. Evaluation of cognitive capacity involves mainly standard tests including attention and psychomotor tests, functional executive tests, Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... http://dx.doi.org/10.5772/intechopen.70475 241 In recent studies, long-term AS users submitted to cognitive analysis have performed significantly worse in visuospatial memory tests and learning capacity compared to nonusers [111, 112]. Basically, AS users made more mistakes in the attempt to recognize visual patterns they had seen immediately before the test. The capacity to distinguish between new and already seen visual pattern was also impaired in AS users when compared to nonusers. In addition, AS users showed a tendency to make more mistakes in the attempt to memorize verbal patterns 30 minutes after the presentation. Interestingly, cognitive impairments were significantly correlated to the total lifetime of AS dose consumption [112]. In another recent study, AS users had worse results in tests evaluating attention and inhibitory control skills [111]. Taken together, these findings evidence that AS abuse can elicit substantial cognitive loss and raise the question of whether the effects of long-term consumption can be even more remarkable with aging. Noteworthy, adolescents have exhibited more sensitivity to AS-induced cognitive impairments when compared to adult AS users, which suggest that chronic AS abuse during pubertal and pre-pubertal phases might induce more severe neurological impairments [111]. Importantly, these individuals might be more susceptible to aging-associated loss of cognitive capacity when compared to individuals that started AS abuse in adulthood. Studies focused in animal models of AS abuse have shown conflicting findings, depending on the test and dose regimen used. In the passive avoidance test, rodents undergo fear-motivated analyses of short-term and long-term memory, as they learn to avoid their innate tendency for preference dark environments, instead of bright areas, by exposure to aversive stimulus (i.e., electric shock) in the dark area. Long-term administration of nandrolone decanoate (4 mg/week) for 10 weeks significantly increased the extinction of learned responses (i.e., avoidance of the learned aversive stimulus) when compared to vehicle-treated rats, suggesting that nandroloneimpaired mnemonic performance, whereas rats administered with testosterone enanthate performed better than control rats [113]. In contrast, one injection of nandrolone decanoate (1–6 mg/ rat) or testosterone enanthate (5–30 mg/rat) significantly improved the mnemonic performance of rats in the passive avoidance test [114]. These evidences suggest that long-term exposure to poorly aromatizable AS, such as nandrolone, can considerably impair learning and memory consolidation, whereas treatment with aromatizable AS might improve these aspects, probably In the Morris water maze test, visuospatial memory is evaluated by repeated presentations of rats to the maze in daily training trials, in which rats must find the target platform. In the day of the test, the latency time spent to find the central platform, the time spent within the target platform, and the time spent in the surrounding areas of the maze are compared between experimental groups. In this context, administration of AS cocktail (2 mg/kg testosterone cypionate, 2 mg/kg nandrolone decanoate, and 1 mg/kg boldenone undecylenate) or 0.375 mg/kg memory tests, and emotional/social cognition tests. by increasing local production of estrogen in the CNS. Notwithstanding the effects with respect to the synthesis of Aβ, AS can modulate the toxicity induced by these oligomers. In this context, testosterone per se can induce neuroprotective effect against the toxicity induced by Aβ, an effect observed in mixed cortical neuronal cell cultures [107]. This effect was completely abolished by co-exposure with aromatase, suggesting that aromatization and the local generation of estrogen can underlie the testosteroneinduced neuroprotection. Interestingly, co-exposure with the AR antagonist flutamide can also attenuate the neuroprotection induced by testosterone against Aβ-induced toxicity, implying that activation of classical AR can attenuate the toxicity induced by Aβ oligomers [107]. In addition, the exposure of neuronal cells to nandrolone, a poorly aromatizable AS, did not affect the neurotoxicity induced by Aβ, whereas it was significantly potentiated by exposure to methandrostenolone [107]. However, nandrolone-BSA (bovine serum albumin) conjugate significantly potentiated the Aβ-induced neurotoxicity, whereas the conjugation of BSA further increased the neurotoxic potentiation induced by methandrostenolone per se. Taken together, these evidences have demonstrated that AS not only increases the generation of Aβ oligomers in crucial areas of CNS associated with the cognitive and mnemonic capacities but also increases the neurotoxic effect of these molecules, which can substantially increase the susceptibility to Alzheimer's disease. The mechanism underlying the elevated level of Aβ remains unclear but might involve disturbances in the organelles, where Aβ is produced (i.e., endoplasmic reticulum and Golgi apparatus); moreover, the role of endoplasmic reticulum stress must be investigated in this regard. Furthermore, the downstream signaling underlying the AS-induced potentiation in the neurotoxic effect promoted by Aβ seems to involve membrane receptors instead of the classical cytosolic AR. Thus, this effect might be more pronounced in drugs that exhibit increased binding affinity for the membrane receptor and that are poorly converted into estrogen by aromatase. #### 4.2. Long-term AS abuse and cognitive impairments Testosterone and other endogenous androgens per se have been shown to exert a pivotal role during the development of CNS. In keeping with this, recent evidences have demonstrated that the development of central nervous system exhibits sexual dimorphic differences, including the size of cortical and sub-cortical structures. In addition, cognitive and mnemonic performances can be strikingly influenced by the levels of testosterone and estrogen within CNS. Indeed, decreased levels of testosterone have been correlated to a poor performance in cognitive tests, increased levels of Aβ throughout the brain, and increased susceptibility to Alzheimer's disease [108]. Conversely, testosterone replacement can significantly restore these abnormalities [109, 110]. Despite these evidences regarding endogenous testosterone levels, consistent data have shown that long-term administration of supraphysiological doses of AS can significantly impair the cognitive capacity. Evaluation of cognitive capacity involves mainly standard tests including attention and psychomotor tests, functional executive tests, memory tests, and emotional/social cognition tests. to 17β-trenbolone, but not to DHT, can significantly elevate the levels of Aβ42. Interestingly, this effect was seen only in male rats. Even so, administration of 17β-trenbolone in pregnant female rats resulted in accumulation of Aβ42 in the fetus brain [80]. In keeping with these findings, exposure to 17β-trenbolone also resulted in a concentration-dependent down-regulation of PS1 levels in primary hippocampal neurons [80]. These evidences have demonstrated that AS abuse can induce accumulation of Aβ42 in the hippocampus, which can induce long-term Notwithstanding the effects with respect to the synthesis of Aβ, AS can modulate the toxicity induced by these oligomers. In this context, testosterone per se can induce neuroprotective effect against the toxicity induced by Aβ, an effect observed in mixed cortical neuronal cell cultures [107]. This effect was completely abolished by co-exposure with aromatase, suggesting that aromatization and the local generation of estrogen can underlie the testosteroneinduced neuroprotection. Interestingly, co-exposure with the AR antagonist flutamide can also attenuate the neuroprotection induced by testosterone against Aβ-induced toxicity, implying that activation of classical AR can attenuate the toxicity induced by Aβ oligomers [107]. In addition, the exposure of neuronal cells to nandrolone, a poorly aromatizable AS, did not affect the neurotoxicity induced by Aβ, whereas it was significantly potentiated by exposure to methandrostenolone [107]. However, nandrolone-BSA (bovine serum albumin) conjugate significantly potentiated the Aβ-induced neurotoxicity, whereas the conjugation of BSA further increased the neurotoxic potentiation induced by methandrostenolone per se. Taken together, these evidences have demonstrated that AS not only increases the generation of Aβ oligomers in crucial areas of CNS associated with the cognitive and mnemonic capacities but also increases the neurotoxic effect of these molecules, which can substantially increase the susceptibility to Alzheimer's disease. The mechanism underlying the elevated level of Aβ remains unclear but might involve disturbances in the organelles, where Aβ is produced (i.e., endoplasmic reticulum and Golgi apparatus); moreover, the role of endoplasmic reticulum stress must be investigated in this regard. Furthermore, the downstream signaling underlying the AS-induced potentiation in the neurotoxic effect promoted by Aβ seems to involve membrane receptors instead of the classical cytosolic AR. Thus, this effect might be more pronounced in drugs that exhibit increased binding affinity for the membrane susceptibility to Alzheimer's disease, especially in the offspring of female AS abusers. 240 Sex Hormones in Neurodegenerative Processes and Diseases receptor and that are poorly converted into estrogen by aromatase. Testosterone and other endogenous androgens per se have been shown to exert a pivotal role during the development of CNS. In keeping with this, recent evidences have demonstrated that the development of central nervous system exhibits sexual dimorphic differences, including the size of cortical and sub-cortical structures. In addition, cognitive and mnemonic performances can be strikingly influenced by the levels of testosterone and estrogen within CNS. Indeed, decreased levels of testosterone have been correlated to a poor performance in cognitive tests, increased levels of Aβ throughout the brain, and increased susceptibility to Alzheimer's disease [108]. Conversely, testosterone replacement can significantly restore 4.2. Long-term AS abuse and cognitive impairments In recent studies, long-term AS users submitted to cognitive analysis have performed significantly worse in visuospatial memory tests and learning capacity compared to nonusers [111, 112]. Basically, AS users made more mistakes in the attempt to recognize visual patterns they had seen immediately before the test. The capacity to distinguish between new and already seen visual pattern was also impaired in AS users when compared to nonusers. In addition, AS users showed a tendency to make more mistakes in the attempt to memorize verbal patterns 30 minutes after the presentation. Interestingly, cognitive impairments were significantly correlated to the total lifetime of AS dose consumption [112]. In another recent study, AS users had worse results in tests evaluating attention and inhibitory control skills [111]. Taken together, these findings evidence that AS abuse can elicit substantial cognitive loss and raise the question of whether the effects of long-term consumption can be even more remarkable with aging. Noteworthy, adolescents have exhibited more sensitivity to AS-induced cognitive impairments when compared to adult AS users, which suggest that chronic AS abuse during pubertal and pre-pubertal phases might induce more severe neurological impairments [111]. Importantly, these individuals might be more susceptible to aging-associated loss of cognitive capacity when compared to individuals that started AS abuse in adulthood. Studies focused in animal models of AS abuse have shown conflicting findings, depending on the test and dose regimen used. In the passive avoidance test, rodents undergo fear-motivated analyses of short-term and long-term memory, as they learn to avoid their innate tendency for preference dark environments, instead of bright areas, by exposure to aversive stimulus (i.e., electric shock) in the dark area. Long-term administration of nandrolone decanoate (4 mg/week) for 10 weeks significantly increased the extinction of learned responses (i.e., avoidance of the learned aversive stimulus) when compared to vehicle-treated rats, suggesting that nandroloneimpaired mnemonic performance, whereas rats administered with testosterone enanthate performed better than control rats [113]. In contrast, one injection of nandrolone decanoate (1–6 mg/ rat) or testosterone enanthate (5–30 mg/rat) significantly improved the mnemonic performance of rats in the passive avoidance test [114]. These evidences suggest that long-term exposure to poorly aromatizable AS, such as nandrolone, can considerably impair learning and memory consolidation, whereas treatment with aromatizable AS might improve these aspects, probably by increasing local production of estrogen in the CNS. In the Morris water maze test, visuospatial memory is evaluated by repeated presentations of rats to the maze in daily training trials, in which rats must find the target platform. In the day of the test, the latency time spent to find the central platform, the time spent within the target platform, and the time spent in the surrounding areas of the maze are compared between experimental groups. In this context, administration of AS cocktail (2 mg/kg testosterone cypionate, 2 mg/kg nandrolone decanoate, and 1 mg/kg boldenone undecylenate) or 0.375 mg/kg methandrostenolone for 10 weeks did not affect the performance, as the latency time to find the target platform was statistically equivalent to the level of vehicle-treated rats. In contrast, after 4 training trials, rats chronically treated with nandrolone decanoate (15 mg/kg each third day, for 14 days) exhibited increased latency time to reach the target platform, whereas the time spent within the target platform was significantly decreased when compared to the control group [115]. Interestingly, long-term AS administration per se not only impaired learning and mnemonic performances but also abrogated the well-known improvements in these skills elicited by chronic treadmill exercise. In sum, treadmill exercised rats chronically administered with nandrolone decanoate spent significantly more time to find the target platform in the Morris water maze when compared to exercised control rats, whereas the time spent in the target platform was significantly decreased [116]. II AS, in adult males and females, and pregnant female rats, resulted in accumulation of AS Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... http://dx.doi.org/10.5772/intechopen.70475 243 Hippocampus is a sub-cortical region that develops a pivotal role in the consolidation of new memories and spatial cognition. Bilateral destruction of hippocampus impairs the formation of new episodic memories and induces anterograde and retrograde amnesia in epileptic patients [118]. Hippocampus has also been correlated to the consolidation of episodic and declarative memories through the process of LTP [119]. Interestingly, experimental studies have demonstrated that specific neuronal clusters within the hippocampus are activated when rats and monkeys pass through particular locations, which suggest that there is a "neuronal mapping" associated with distinct environments [119]. Noteworthy, studies have demonstrated that in several conditions characterized by cognition and memory decline, such as Alzheimer's disease and other forms of dementia, the hippocampus is one of the earliest The density of AR in the hippocampus is the highest of CNS; thus, it is particularly sensitive to oscillation in circulating levels of testosterone [121, 122]. Exposure of neuroblastoma cell culture to different testosterone concentrations induced a concentration-dependent decrease on cell viability [15]. This effect was also observed in primary culture of hippocampal neurons, in which the incubation for 48 hours with 17β-trenbolone significantly decreased cell viability [80]. Furthermore, administration of nandrolone decanoate (15 mg/kg, daily) for 5 days in adult males, females, and pregnant female rat (embryonic day 15) resulted in a significant decrease of BrdU-labeled cells in the dentate gyrus of the hippocampus, indicating that AS The dentate gyrus is a hippocampal area at the interface of entorhinal cortex and CA3 region of hippocampus [124]. Excitatory inputs from the layer II of the entorhinal cortex project to the dentate gyrus, which send neuronal projections to the CA3 region via mossy fibers. This trisynaptic circuit exerts a particular role in the process of spatial memory and cognition. In keeping with this, experimental studies have demonstrated that neuronal death in the dentate gyrus granule cells resulted in significantly decreased performance on hippocampal-sensitive memory tests, such as the Morris water maze, acquisition of reference, and working memory tests [125, 126]. Worth of noting long-term administration of nandrolone decanoate (10 mg/kg/week, for 8 weeks) in rats significantly decreased neuronal density not only in the dentate gyrus but also throughout Noteworthy, the dentate gyrus is one of the few regions of the adult brain to exhibit neurogenesis and acute nandrolone administration decreased the number of newly born neurons within dentate gyrus of adult rats in approximately 75%, implying that short-term administration of AS is enough to significantly impair neurogenic processes [123]. Furthermore, neuronal loss and impaired neurogenesis in the hippocampal and cortical structures have been correlated to the development of Alzheimer's disease-related cognitive decline, as well as to increased number of Aβ plaques in this region [127]. Conversely, experimental evidences have demonstrated that both aerobic and anaerobic exercises can significantly increase neuronal throughout the brain and cerebrospinal fluid, but especially in the hippocampus [80]. structures to exhibit synaptic dysfunctions [120]. overdose decreased cell proliferation [123]. CA1, CA2, CA3, pre-frontal cortex, and parietal cortex [79]. Besides the impairment of spatial memory, exposure to nandrolone decanoate also reduces the social memory capacity of rats [117]. In social memory capacity evaluation, adult rats are allowed to investigate and recognize juvenile rats for 5 minutes. During this period of time, adult rats frequently demonstrate investigatory-like behavior, such as head and body sniffing, anogenital exploration, grooming, close pursuing, touching the flanks with the snout, and manipulation with the forepaws. After an interval of time, the same juvenile rats are reintroduced to the adult rat. In this context, long-term administration of nandrolone decanoate (15 mg/kg, daily, for 6 weeks) significantly increased the recognition time in the second exposure, when compared to control rats [117]. Importantly, this effect was completely abolished when flutamide, an AR antagonist, was co-administered with nandrolone. These findings suggest that long-term exposure to nandrolone impaired the mnemonic capacity by stimulation of AR within CNS. Taken together, these evidences corroborate the findings in AS abusers that the loss on mnemonic capacity might be proportional to the dose and time of AS exposure, as well as the compound administered (i.e. aromatizable or nonaromatizable). More studies are necessary to elucidate whether longer exposure to AS and more cycles can further impair cognitive capacity in experimental models. Furthermore, the impact of chronic administration of AS in aging rats should be investigated, given that the majority of oldest AS abusers in general population (i.e., that started to use AS in the 1970s and 1980s) are entering the age of risk of ND now [112]. Despite the evidences about decline of cognitive and mnemonic capacities after AS administration, the underlying mechanisms are complex and remain unclear so far. Experimental studies have shown that high concentrations of AS can elicit apoptosis of several cell types, including cardiomyocytes, endothelial, and skeletal muscles cells. Even so, the overall consequences of AS exposure on neural cells viability remain poorly explored in in vivo studies. In vitro studies have demonstrated decreased cell viability in neural cell cultures exposed to AS, suggesting that neuronal loss might be the central event in the cognitive decline during supraphysiological AS intake. These neuronal adverse effects are especially critical in the case of AS abuse, given the capacity of these drugs to cross the blood-brain barrier and accumulate in the neural tissue. In keeping with this, short-term administration of 17β-trenbolone, a class II AS, in adult males and females, and pregnant female rats, resulted in accumulation of AS throughout the brain and cerebrospinal fluid, but especially in the hippocampus [80]. methandrostenolone for 10 weeks did not affect the performance, as the latency time to find the target platform was statistically equivalent to the level of vehicle-treated rats. In contrast, after 4 training trials, rats chronically treated with nandrolone decanoate (15 mg/kg each third day, for 14 days) exhibited increased latency time to reach the target platform, whereas the time spent within the target platform was significantly decreased when compared to the control group [115]. Interestingly, long-term AS administration per se not only impaired learning and mnemonic performances but also abrogated the well-known improvements in these skills elicited by chronic treadmill exercise. In sum, treadmill exercised rats chronically administered with nandrolone decanoate spent significantly more time to find the target platform in the Morris water maze when compared to exercised control rats, whereas the time spent in the Besides the impairment of spatial memory, exposure to nandrolone decanoate also reduces the social memory capacity of rats [117]. In social memory capacity evaluation, adult rats are allowed to investigate and recognize juvenile rats for 5 minutes. During this period of time, adult rats frequently demonstrate investigatory-like behavior, such as head and body sniffing, anogenital exploration, grooming, close pursuing, touching the flanks with the snout, and manipulation with the forepaws. After an interval of time, the same juvenile rats are reintroduced to the adult rat. In this context, long-term administration of nandrolone decanoate (15 mg/kg, daily, for 6 weeks) significantly increased the recognition time in the second exposure, when compared to control rats [117]. Importantly, this effect was completely abolished when flutamide, an AR antagonist, was co-administered with nandrolone. These findings suggest that long-term exposure to nandrolone impaired the mnemonic capacity by Taken together, these evidences corroborate the findings in AS abusers that the loss on mnemonic capacity might be proportional to the dose and time of AS exposure, as well as the compound administered (i.e. aromatizable or nonaromatizable). More studies are necessary to elucidate whether longer exposure to AS and more cycles can further impair cognitive capacity in experimental models. Furthermore, the impact of chronic administration of AS in aging rats should be investigated, given that the majority of oldest AS abusers in general population (i.e., that started to use AS in the 1970s and 1980s) are entering the age of risk of ND now [112]. Despite the evidences about decline of cognitive and mnemonic capacities after AS administration, the underlying mechanisms are complex and remain unclear so far. Experimental studies have shown that high concentrations of AS can elicit apoptosis of several cell types, including cardiomyocytes, endothelial, and skeletal muscles cells. Even so, the overall consequences of AS exposure on neural cells viability remain poorly explored in in vivo studies. In vitro studies have demonstrated decreased cell viability in neural cell cultures exposed to AS, suggesting that neuronal loss might be the central event in the cognitive decline during supraphysiological AS intake. These neuronal adverse effects are especially critical in the case of AS abuse, given the capacity of these drugs to cross the blood-brain barrier and accumulate in the neural tissue. In keeping with this, short-term administration of 17β-trenbolone, a class target platform was significantly decreased [116]. 242 Sex Hormones in Neurodegenerative Processes and Diseases stimulation of AR within CNS. Hippocampus is a sub-cortical region that develops a pivotal role in the consolidation of new memories and spatial cognition. Bilateral destruction of hippocampus impairs the formation of new episodic memories and induces anterograde and retrograde amnesia in epileptic patients [118]. Hippocampus has also been correlated to the consolidation of episodic and declarative memories through the process of LTP [119]. Interestingly, experimental studies have demonstrated that specific neuronal clusters within the hippocampus are activated when rats and monkeys pass through particular locations, which suggest that there is a "neuronal mapping" associated with distinct environments [119]. Noteworthy, studies have demonstrated that in several conditions characterized by cognition and memory decline, such as Alzheimer's disease and other forms of dementia, the hippocampus is one of the earliest structures to exhibit synaptic dysfunctions [120]. The density of AR in the hippocampus is the highest of CNS; thus, it is particularly sensitive to oscillation in circulating levels of testosterone [121, 122]. Exposure of neuroblastoma cell culture to different testosterone concentrations induced a concentration-dependent decrease on cell viability [15]. This effect was also observed in primary culture of hippocampal neurons, in which the incubation for 48 hours with 17β-trenbolone significantly decreased cell viability [80]. Furthermore, administration of nandrolone decanoate (15 mg/kg, daily) for 5 days in adult males, females, and pregnant female rat (embryonic day 15) resulted in a significant decrease of BrdU-labeled cells in the dentate gyrus of the hippocampus, indicating that AS overdose decreased cell proliferation [123]. The dentate gyrus is a hippocampal area at the interface of entorhinal cortex and CA3 region of hippocampus [124]. Excitatory inputs from the layer II of the entorhinal cortex project to the dentate gyrus, which send neuronal projections to the CA3 region via mossy fibers. This trisynaptic circuit exerts a particular role in the process of spatial memory and cognition. In keeping with this, experimental studies have demonstrated that neuronal death in the dentate gyrus granule cells resulted in significantly decreased performance on hippocampal-sensitive memory tests, such as the Morris water maze, acquisition of reference, and working memory tests [125, 126]. Worth of noting long-term administration of nandrolone decanoate (10 mg/kg/week, for 8 weeks) in rats significantly decreased neuronal density not only in the dentate gyrus but also throughout CA1, CA2, CA3, pre-frontal cortex, and parietal cortex [79]. Noteworthy, the dentate gyrus is one of the few regions of the adult brain to exhibit neurogenesis and acute nandrolone administration decreased the number of newly born neurons within dentate gyrus of adult rats in approximately 75%, implying that short-term administration of AS is enough to significantly impair neurogenic processes [123]. Furthermore, neuronal loss and impaired neurogenesis in the hippocampal and cortical structures have been correlated to the development of Alzheimer's disease-related cognitive decline, as well as to increased number of Aβ plaques in this region [127]. Conversely, experimental evidences have demonstrated that both aerobic and anaerobic exercises can significantly increase neuronal proliferation in these regions, an effect that has been correlated to the improved mnemonic capacity and neuronal survival [128, 129]. In this context, rats submitted to strength exercise in a vertical ladder showed up-regulation of Ki-67 throughout the dentate gyrus, which is considered a marker of neurogenesis [130]. However, chronic administration of nandrolone decanoate abolished this effect, suggesting that AS abuse can decrease the neurogenic effect induced by exercise. Acknowledgements Author details References WHO; 1992 Fernando de Azevedo Cruz Seara<sup>1</sup> José Hamilton Matheus Nascimento1 \Address all correspondence to: [email protected] University of Rio de Janeiro, Rio de Janeiro, Brazil 5117. DOI: 10.1210/jcem.86.11.7983 DOI: 10.1210/jcem-68-3-578 endo.142.11.8547 Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Federal University of Rio de Janeiro, Rio de Janeiro, Brazil This study was supported by the Brazilian Council for Scientific and Technological Development (CNPq), the Rio de Janeiro State Research Foundation (FAPERJ) and the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES). JHMN, DPC and RSF Neurophysiological Repercussions of Anabolic Steroid Abuse: A Road into Neurodegenerative... , Rodrigo Soares Fortunato<sup>2</sup> 1 Laboratory of Cardiac Electrophysiology, Carlos Chagas Filho Institute of Biophysics, 2 Laboratory of Molecular Radiobiology, Carlos Chagas Filho Institute of Biophysics, 3 Laboratory of Endocrine Physiology, Carlos Chagas Filho Institute of Biophysics, Federal [1] World Health organization. Guidelines for the Use of Androgens in Men. Geneva: [2] Basaria S, Wahlstrom JT, Dobs AS. Anabolic-androgenic steroid therapy in the treatment of chronic diseases. Journal of Clinical Endocrinology and Metabolism. 2001;86:5108- [3] Grino PB, Isidro-Gutierrez RF, Griffin JE, Wilson JD. Androgen resistance associated with a qualitative abnormality of the androgen receptor and responsive to high dose androgen therapy. Journal of Clinical Endocrinology and Metabolism. 1989;68:578-584. [4] Simpson ER, Davis SR. Minireview: Aromatase and the regulation of estrogen biosynthesis – some new perspectives. Endocrinology. 2001;142:4589-4594. DOI: 10.1210/ [5] Shaffer PL, Jivan A, Dollins DE, Claessens F, Gewirth DT. Structural basis of androgen receptor binding to selective androgen response elements. Proceedings of National , Denise Pires Carvalho<sup>3</sup> http://dx.doi.org/10.5772/intechopen.70475 and 245 are research fellows from the CNPq and FACS receive a fellowship from the CNPq. \ In cortical neuronal and astrocytic cultures, 48 hours of exposure to 10 mM of testosterone, nandrolone, or methandrostenolone significantly increased neuronal death [107]. Interestingly, when these ASs were conjugated to BSA, which impedes the AS to cross the plasma membrane and to bind to the cytosolic AR, the neurotoxicity was further increased. Even so, co-exposure of flutamide prevented both testosterone- and nandrolone-induced neurotoxicity, suggesting that the membrane-attached AR shares pharmacological similarities with the cytosolic receptor [107]. Furthermore, these evidences suggest that activation of membrane-attached AR can recruit distinct downstream signaling pathways that culminate in enhanced cell death, when compared to the cytosolic AR. Taken together, these clinical and experimental evidences imply that chronic exposure to supraphysiological doses of AS can severely impair cognitive and mnemonic capacities. This paradigm might be worsened by aging-related neurophysiological effects, which can increase the susceptibility to neurodegenerative diseases, besides the well-described neurobehavioral effects. #### 5. Conclusions The growing misuse of AS is a major concern worldwide due to its harmful effects, including cardiovascular, endocrine, reproductive, behavioral, and neurological abnormalities. Unfortunately, there are several unclear aspects regarding the consequences of AS abuse, such as the prevalence of adverse effects, the repercussions in aging-related dysfunctions, such as neurodegenerative diseases, and if these effects are reversible. Even so, experimental studies have provided consistent evidences that the short-term and long-term exposure to AS can induce neuronal apoptosis throughout important neural regions, such as hippocampus and pre-frontal cortex. As a result, this phenomenon can severely impair cognitive and mnemonic capacities, as evidenced by clinical studies with AS abuses. In addition, exposure to AS can significantly increase the susceptibility to Alzheimer's disease. Taken together, these evidences support the hypothesis that administration of supraphysiological doses of AS is an important risk factor to the development of neurodegenerative diseases, and that the prognosis of these conditions might be worsened by AS abuse. Given the rising misuse of AS among elite athletes and recreational users, these neurological consequences should not be underestimated by physicians and researchers. The understanding of these aspects is particularly important to provide the diagnostic and prognostic of neurological diseases in active and former AS abusers. ### Acknowledgements proliferation in these regions, an effect that has been correlated to the improved mnemonic capacity and neuronal survival [128, 129]. In this context, rats submitted to strength exercise in a vertical ladder showed up-regulation of Ki-67 throughout the dentate gyrus, which is considered a marker of neurogenesis [130]. However, chronic administration of nandrolone decanoate abolished this effect, suggesting that AS abuse can decrease the neurogenic effect In cortical neuronal and astrocytic cultures, 48 hours of exposure to 10 mM of testosterone, nandrolone, or methandrostenolone significantly increased neuronal death [107]. Interestingly, when these ASs were conjugated to BSA, which impedes the AS to cross the plasma membrane and to bind to the cytosolic AR, the neurotoxicity was further increased. Even so, co-exposure of flutamide prevented both testosterone- and nandrolone-induced neurotoxicity, suggesting that the membrane-attached AR shares pharmacological similarities with the cytosolic receptor [107]. Furthermore, these evidences suggest that activation of membrane-attached AR can recruit distinct downstream signaling pathways that culminate in enhanced cell death, when Taken together, these clinical and experimental evidences imply that chronic exposure to supraphysiological doses of AS can severely impair cognitive and mnemonic capacities. This paradigm might be worsened by aging-related neurophysiological effects, which can increase the susceptibility to neurodegenerative diseases, besides the well-described neurobehavioral The growing misuse of AS is a major concern worldwide due to its harmful effects, including cardiovascular, endocrine, reproductive, behavioral, and neurological abnormalities. Unfortunately, there are several unclear aspects regarding the consequences of AS abuse, such as the prevalence of adverse effects, the repercussions in aging-related dysfunctions, such as neurodegenerative diseases, and if these effects are reversible. Even so, experimental studies have provided consistent evidences that the short-term and long-term exposure to AS can induce neuronal apoptosis throughout important neural regions, such as hippocampus and pre-frontal cortex. As a result, this phenomenon can severely impair cognitive and mnemonic capacities, as evidenced by clinical studies with AS abuses. In addition, exposure to AS can significantly increase the susceptibility to Alzheimer's disease. Taken together, these evidences support the hypothesis that administration of supraphysiological doses of AS is an important risk factor to the development of neurodegenerative diseases, and that the prognosis of these conditions might be worsened by AS abuse. Given the rising misuse of AS among elite athletes and recreational users, these neurological consequences should not be underestimated by physicians and researchers. The understanding of these aspects is particularly important to provide the diagnostic and prognostic of neurological diseases in active and induced by exercise. 244 Sex Hormones in Neurodegenerative Processes and Diseases compared to the cytosolic AR. effects. 5. Conclusions former AS abusers. This study was supported by the Brazilian Council for Scientific and Technological Development (CNPq), the Rio de Janeiro State Research Foundation (FAPERJ) and the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES). JHMN, DPC and RSF are research fellows from the CNPq and FACS receive a fellowship from the CNPq. ### Author details Fernando de Azevedo Cruz Seara<sup>1</sup> , Rodrigo Soares Fortunato<sup>2</sup> , Denise Pires Carvalho<sup>3</sup> and José Hamilton Matheus Nascimento1 \* \Address all correspondence to: [email protected] 1 Laboratory of Cardiac Electrophysiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 2 Laboratory of Molecular Radiobiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 3 Laboratory of Endocrine Physiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil #### References* Academy of Sciences United States of America. 2004;101:4758-4763. DOI: 10.1073/ pnas.0401123101 [17] Bhasin S, Storer T, Berman N, Callegari C, Clevenger B, Phillips J, et al. 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Hippocampus. 1999;9:668-681. DOI: 10.1002/(SICI)1098-1063(1999)9:6<668::AID-HIPO8>3.0.CO;2-9 Chapter 11 Provisional chapter Testosterone and Erectile Function: A Review of Testosterone and Erectile Function: A Review of DOI: 10.5772/intechopen.72935 Androgens are essential for male physical activity and normal erectile function. Hence, age-related testosterone deficiency, known as late-onset hypogonadism (LOH), is considered a risk factor for erectile dysfunction (ED). This chapter summarizes relevant basic research reports examining the effects of testosterone on erectile function. Testosterone affects several organs and is especially active on the erectile tissue. The mechanism of testosterone deficiency effects on erectile function and the results of testosterone replacement therapy (TRT) have been well studied. Testosterone affects nitric oxide (NO) production and phosphodiesterase type 5 (PDE-5) expression in the corpus cavernosum through molecular pathways, preserves smooth muscle contractility by regulating both contraction and relaxation, and maintains the structure of the corpus cavernosum. Interestingly, testosterone deficiency has relationship to neurological diseases, which leads to ED. Testosterone replacement therapy is widely used to treat patients with testosterone deficiency; however, this treatment might also induce some problems. Basic research suggests that PDE-5 inhibitors, L-citrulline, and/or resveratrol therapy might be effective therapeutic options for testosterone deficiency-induced ED. Future research should confirm these findings through more specific experiments using molecular tools and may shed more light on endocrine-related ED and its possible treatments. Keywords: testosterone, erectile dysfunction, endothelial function, testosterone > © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. and reproduction in any medium, provided the original work is properly cited. Androgens are essential for male physical activity and normal erectile function [1–5]. Thus, agerelated androgen deficiency, known as late-onset hypogonadism (LOH), is a risk factor for erectile dysfunction (ED) [1, 2]. Several studies have reported that androgen replacement therapy mitigates the symptoms of LOH and ED. In this context, bioidentical or synthetic testosterone Evidence from Basic Research Evidence from Basic Research Tomoya Kataoka and Kazunori Kimura Tomoya Kataoka and Kazunori Kimura http://dx.doi.org/10.5772/intechopen.72935 replacement therapy, basic science Abstract 1. Introduction Additional information is available at the end of the chapter Additional information is available at the end of the chapter Provisional chapter #### Testosterone and Erectile Function: A Review of Evidence from Basic Research Evidence from Basic Research Testosterone and Erectile Function: A Review of DOI: 10.5772/intechopen.72935 Tomoya Kataoka and Kazunori Kimura Tomoya Kataoka and Kazunori Kimura Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.72935 #### Abstract [126] Xavier GF, Oliveira-Filho FJB, Santos AMG. Dentate gyrus-selective colchicine lesion and disruption of performance in spatial tasks: Difficulties in "place strategy" because of a lack of flexibility in the use of environmental cues? Hippocampus. 1999;9:668-681. [127] Rodríguez JJ, Jones VC, Tabuchi M, Allan SM, Knight EM, LaFerla FM, Oddo S, Verkhratsky A. Impaired adult neurogenesis in the dentate gyrus of a triple transgenic mouse model of Alzheimer's disease. PLoS One. 2008;3:e2935. DOI: 10.1371/journal. [128] Chun XL, Jiang J, Qi GZ, Xin JZ, Wang W, Zhi JZ, Han X, Dong YZ. Voluntary exercise-induced neurogenesis in the postischemic dentate gyrus is associated with spatial memory recovery from stroke. Journal of Neuroscience Research. 2007;85:1637-1646. [129] Pereira AC, Huddleston DE, Brickman AM, Sosunov AA, Hen R, McKhann GM, Sloan R, Gage FH, Brown TR, Small AS. An in vivo correlate of exercise-induced neurogenesis in the adult dentate gyrus. Proceedings of National Academy of Sciences USA. [130] Novaes Gomes FG, Fernandes J, Vannucci Campos D, Cassilhas RC, Viana GM, D'Almeida V, de Moraes Rêgo MK, Buainain PI, Cavalheiro EA, Arida RM, Gomes F, Fernandes J, Campos D. The beneficial effects of strength exercise on hippocampal cell proliferation and apoptotic signaling is impaired by anabolic androgenic steroids, Psychoneuroendocrinology. 2014;50:106-117. DOI: 10.1016/j.psyneuen.2014.08.009 DOI: 10.1002/(SICI)1098-1063(1999)9:6<668::AID-HIPO8>3.0.CO;2-9 pone.0002935 DOI: 10.1002/jnr.21317 256 Sex Hormones in Neurodegenerative Processes and Diseases 2007;104:5638-5643. DOI: 10.1073/pnas.0611721104 Androgens are essential for male physical activity and normal erectile function. Hence, age-related testosterone deficiency, known as late-onset hypogonadism (LOH), is considered a risk factor for erectile dysfunction (ED). This chapter summarizes relevant basic research reports examining the effects of testosterone on erectile function. Testosterone affects several organs and is especially active on the erectile tissue. The mechanism of testosterone deficiency effects on erectile function and the results of testosterone replacement therapy (TRT) have been well studied. Testosterone affects nitric oxide (NO) production and phosphodiesterase type 5 (PDE-5) expression in the corpus cavernosum through molecular pathways, preserves smooth muscle contractility by regulating both contraction and relaxation, and maintains the structure of the corpus cavernosum. Interestingly, testosterone deficiency has relationship to neurological diseases, which leads to ED. Testosterone replacement therapy is widely used to treat patients with testosterone deficiency; however, this treatment might also induce some problems. Basic research suggests that PDE-5 inhibitors, L-citrulline, and/or resveratrol therapy might be effective therapeutic options for testosterone deficiency-induced ED. Future research should confirm these findings through more specific experiments using molecular tools and may shed more light on endocrine-related ED and its possible treatments. Keywords: testosterone, erectile dysfunction, endothelial function, testosterone replacement therapy, basic science #### 1. Introduction Androgens are essential for male physical activity and normal erectile function [1–5]. Thus, agerelated androgen deficiency, known as late-onset hypogonadism (LOH), is a risk factor for erectile dysfunction (ED) [1, 2]. Several studies have reported that androgen replacement therapy mitigates the symptoms of LOH and ED. In this context, bioidentical or synthetic testosterone Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons facilitates erectile function by maintaining an adequate supply of nitric oxide (NO), penile structure, and the endothelial functioning of the corpus cavernosum. Thus, reduced NO bioavailability is believed to be the main cause of ED in individuals with testosterone deficiency [6]; however, the pathophysiological mechanisms underlying this process remain unclear and require further study. This chapter summarizes relevant basic research reports examining the effects of testosterone on erectile function. results suggest that testosterone is essential not only for regulating eNOS activity but also for regulating PDE-5 activity. Traish et al. also suggested that while these actions may seem paradoxical, in which androgens are upregulating both signal initiators (NOS) and signal terminators (PDE-5), they may be interpreted to be part of a homeostatic mechanism that maintains a relatively constant ratio of critical pathway enzymes [3]. They also postulated that PDE-5 expression may be controlled by NO. Androgen-mediated upregulation of NOS may lead to increased NO synthesis, which may then upregulate PDE-5 expression and activity. Conversely, androgen deprivation-mediated NOS downregulation also results in the downregulation of PDE-5 expression and activity. More studies are needed to define this delicate Testosterone and Erectile Function: A Review of Evidence from Basic Research http://dx.doi.org/10.5772/intechopen.72935 259 Testosterone also affects the smooth muscle of the corpus cavernosum. Reilly et al. showed that castration reduced the number of α-adrenergic-1 receptors on smooth fascia [24]. They also showed that testosterone modulated the adrenergic response of the corpus cavernosum vascular smooth muscle [25]. Their results indicate that when testosterone levels decrease, smooth muscle contractility also decreases. On the other hand, Wingard et al. showed that castration increased the levels of Rho-A and Rho-kinase proteins in rats. RhoA, a small monomeric GTPase, activates the Rho-associated protein kinase, a serine/threonine kinase, which phosphorylates the myosin-binding subunit of myosin light chain phosphatase, thereby deactivating it and promoting contraction [26]. Their results indicate that when testosterone levels decline, smooth muscle contractility increases, leading not only to the development of ED but also to the hypertension. Thus, although testosterone deficiency might increase contraction, additional research is required to fully elucidate its impact on smooth muscle contraction. Interestingly, testosterone also directly affects smooth muscle relaxation. Yue et al., using an isometric tension study, showed that testosterone relaxed the smooth muscle of rabbit coronary arteries and aortas [27]. Others also showed that testosterone induces the relaxation of isolated human corpora cavernosa strips by activating smooth muscle ATP-sensitive K<sup>+</sup> channels [28]. These findings suggest that testosterone, in addition to its known endothelial action, might regulate erectile function locally by acting on human corpus cavernosum smooth muscle. These results indicate that testosterone might affect both the genomic and Some studies demonstrated that testosterone also impacts the structure of the penis. One group showed that castrated rats show smooth muscle loss and fibrosis [29], and another group reported that castration increases the collagen content of the internal pudendal arteries and decreases α-actin expression [30]. These testosterone effects suggest that testosterone deprivation results in programmed trabecular smooth muscle cell death (apoptosis) and increased development of extracellular matrix [22]. Traish et al. also proposed that testosterone deprivation is associated with the accumulation of fat-containing cells (fibroblasts or preadipocyte-like cells), especially in the subtunical region of the corpus cavernosum, contributing to impaired veno-occlusion [31]. Interestingly, Wang et al. showed that castration attenuates erectile function and induces corporeal fibrosis by inhibiting autophagy and promoting apoptosis of the corpus cavernosum smooth muscle cells in rats [32]. Their study has limitations, but they highlighted the important role of androgens in maintaining the structural integrity and functioning of the corpus cavernosum. This resulted from androgens mediating and crucial mechanism of testosterone action. nongenomic actions of erectile function. #### 2. Testosterone and erectile function Androgens are well established as being essential for erectile function, and their deficiency is considered a risk factor for ED. LOH is a result of the normal aging process and is responsible for androgen deficiency [7, 8]. In recent years, epidemiologic studies have suggested that metabolic syndrome and diabetes mellitus are also associated with the development of androgen deficiency [9–12]. Erectile function is regulated by complex mechanisms [13]. When sexual stimulation occurs, NO is released in the penis, causing corporal smooth muscle relaxation through the activation of the cGMP/protein kinase G signaling cascade. ED results when the relaxant system is weakened; therefore, many studies have focused on smooth muscle relaxation. In contrast, in the flaccid state, corporal smooth muscle contraction is controlled by constrictors such as noradrenaline. Recent studies have indicated that the balance between smooth muscle relaxation and contraction is disturbed by abnormal activation of the RhoA/Rho-kinase signaling pathway. In some syndromes causing ED, such as diabetes mellitus or metabolic syndrome, the RhoA/Rho-kinase signaling pathway is enhanced [14–16]. Additionally, enhancement of the RhoA/Rho-kinase signaling pathway is known to occur in aged individuals, and a Rho-kinase inhibitor (Y-27632) has been shown to improve erectile function in aged rats [17, 18]. As contractility may play a significant role in erectile function, its role in ED should be considered along with contraction. Thus, the balance between smooth muscle contraction and relaxation is important for normal erectile function. #### 3. Testosterone deficiency and ED Most animal studies have shown that castration causes ED by reducing arterial inflow [19]. Further, endothelial nitric oxide synthase (eNOS) and neuronal NOS (nNOS) are important in erectile functioning. In castrated animals, testosterone administration restores the erectile response and NOS expression in the penis [20]. Li et al. showed that testosterone deficiency decreases eNOS activity (phosphor-eNOS/eNOS ratio) by upregulating reactive oxygen species production [21], and the decreased eNOS activity decreases cGMP levels in the penis. Some studies found that testosterone changes phosphodiesterase type 5 (PDE-5) expression in the penis. Traish et al. showed that castration decreased PDE-5 activity in rabbit penises [22], whereas Zhang et al. showed that testosterone deficiency decreased PDE-5 expression in the rat penis and that testosterone administration increased PDE-5 expression [23]. These results suggest that testosterone is essential not only for regulating eNOS activity but also for regulating PDE-5 activity. Traish et al. also suggested that while these actions may seem paradoxical, in which androgens are upregulating both signal initiators (NOS) and signal terminators (PDE-5), they may be interpreted to be part of a homeostatic mechanism that maintains a relatively constant ratio of critical pathway enzymes [3]. They also postulated that PDE-5 expression may be controlled by NO. Androgen-mediated upregulation of NOS may lead to increased NO synthesis, which may then upregulate PDE-5 expression and activity. Conversely, androgen deprivation-mediated NOS downregulation also results in the downregulation of PDE-5 expression and activity. More studies are needed to define this delicate and crucial mechanism of testosterone action. facilitates erectile function by maintaining an adequate supply of nitric oxide (NO), penile structure, and the endothelial functioning of the corpus cavernosum. Thus, reduced NO bioavailability is believed to be the main cause of ED in individuals with testosterone deficiency [6]; however, the pathophysiological mechanisms underlying this process remain unclear and require further study. This chapter summarizes relevant basic research reports examining the effects of testoster- Androgens are well established as being essential for erectile function, and their deficiency is considered a risk factor for ED. LOH is a result of the normal aging process and is responsible for androgen deficiency [7, 8]. In recent years, epidemiologic studies have suggested that metabolic syndrome and diabetes mellitus are also associated with the development of Erectile function is regulated by complex mechanisms [13]. When sexual stimulation occurs, NO is released in the penis, causing corporal smooth muscle relaxation through the activation of the cGMP/protein kinase G signaling cascade. ED results when the relaxant system is weakened; therefore, many studies have focused on smooth muscle relaxation. In contrast, in the flaccid state, corporal smooth muscle contraction is controlled by constrictors such as noradrenaline. Recent studies have indicated that the balance between smooth muscle relaxation and contraction is disturbed by abnormal activation of the RhoA/Rho-kinase signaling pathway. In some syndromes causing ED, such as diabetes mellitus or metabolic syndrome, the RhoA/Rho-kinase signaling pathway is enhanced [14–16]. Additionally, enhancement of the RhoA/Rho-kinase signaling pathway is known to occur in aged individuals, and a Rho-kinase inhibitor (Y-27632) has been shown to improve erectile function in aged rats [17, 18]. As contractility may play a significant role in erectile function, its role in ED should be considered along with contraction. Thus, the balance between smooth muscle contraction and relaxation Most animal studies have shown that castration causes ED by reducing arterial inflow [19]. Further, endothelial nitric oxide synthase (eNOS) and neuronal NOS (nNOS) are important in erectile functioning. In castrated animals, testosterone administration restores the erectile response and NOS expression in the penis [20]. Li et al. showed that testosterone deficiency decreases eNOS activity (phosphor-eNOS/eNOS ratio) by upregulating reactive oxygen species production [21], and the decreased eNOS activity decreases cGMP levels in the penis. Some studies found that testosterone changes phosphodiesterase type 5 (PDE-5) expression in the penis. Traish et al. showed that castration decreased PDE-5 activity in rabbit penises [22], whereas Zhang et al. showed that testosterone deficiency decreased PDE-5 expression in the rat penis and that testosterone administration increased PDE-5 expression [23]. These one on erectile function. androgen deficiency [9–12]. 2. Testosterone and erectile function 258 Sex Hormones in Neurodegenerative Processes and Diseases is important for normal erectile function. 3. Testosterone deficiency and ED Testosterone also affects the smooth muscle of the corpus cavernosum. Reilly et al. showed that castration reduced the number of α-adrenergic-1 receptors on smooth fascia [24]. They also showed that testosterone modulated the adrenergic response of the corpus cavernosum vascular smooth muscle [25]. Their results indicate that when testosterone levels decrease, smooth muscle contractility also decreases. On the other hand, Wingard et al. showed that castration increased the levels of Rho-A and Rho-kinase proteins in rats. RhoA, a small monomeric GTPase, activates the Rho-associated protein kinase, a serine/threonine kinase, which phosphorylates the myosin-binding subunit of myosin light chain phosphatase, thereby deactivating it and promoting contraction [26]. Their results indicate that when testosterone levels decline, smooth muscle contractility increases, leading not only to the development of ED but also to the hypertension. Thus, although testosterone deficiency might increase contraction, additional research is required to fully elucidate its impact on smooth muscle contraction. Interestingly, testosterone also directly affects smooth muscle relaxation. Yue et al., using an isometric tension study, showed that testosterone relaxed the smooth muscle of rabbit coronary arteries and aortas [27]. Others also showed that testosterone induces the relaxation of isolated human corpora cavernosa strips by activating smooth muscle ATP-sensitive K<sup>+</sup> channels [28]. These findings suggest that testosterone, in addition to its known endothelial action, might regulate erectile function locally by acting on human corpus cavernosum smooth muscle. These results indicate that testosterone might affect both the genomic and nongenomic actions of erectile function. Some studies demonstrated that testosterone also impacts the structure of the penis. One group showed that castrated rats show smooth muscle loss and fibrosis [29], and another group reported that castration increases the collagen content of the internal pudendal arteries and decreases α-actin expression [30]. These testosterone effects suggest that testosterone deprivation results in programmed trabecular smooth muscle cell death (apoptosis) and increased development of extracellular matrix [22]. Traish et al. also proposed that testosterone deprivation is associated with the accumulation of fat-containing cells (fibroblasts or preadipocyte-like cells), especially in the subtunical region of the corpus cavernosum, contributing to impaired veno-occlusion [31]. Interestingly, Wang et al. showed that castration attenuates erectile function and induces corporeal fibrosis by inhibiting autophagy and promoting apoptosis of the corpus cavernosum smooth muscle cells in rats [32]. Their study has limitations, but they highlighted the important role of androgens in maintaining the structural integrity and functioning of the corpus cavernosum. This resulted from androgens mediating the counter-regulation of autophagy and apoptosis through regulation of the BECN 1-Bcl-2 (key dual regulators of autophagy and apoptosis) interaction [33, 34]. (T2DM) [1, 48, 49]. Obesity is also considered a strong risk factor for ED [4, 5]. In men, visceral adipose tissue causes arteriosclerosis and vessel endothelial dysfunction [12]. Therefore, men Testosterone and Erectile Function: A Review of Evidence from Basic Research http://dx.doi.org/10.5772/intechopen.72935 261 In recent years, epidemiologic studies have suggested that obesity is also associated with multiple alterations in the gonadal endocrine system, including low testosterone levels [1, 48, 52, 53]. Low testosterone levels have also been reported in animals with T2DM, including two seminal research papers that reported testosterone replacement therapy (TRT) in such animal models [54, 55]. Davis et al. administered TRT to obese Zucker rats, resulting in improved cholesterol parameters and insulin sensitivity [54]. On the other hand, others administered TRT to rabbits with high-fat diet-associated hypogonadotropic hypogonadism [55]. TRT partially ameliorated the animals' blood glucose levels and improved CC sensitivity to acetyl- We also reported that T2DM increased inflammatory biomarker (inducible NO synthase, interleukin-6, and tumor necrosis factor alpha) mRNA expression levels in the CC, but TRT decreased them [56]. Ota et al. reported an in vitro study that demonstrated testosterone prevented inflammation caused by hydrogen peroxide in blood vessel cells by upregulating the sirtuin-1 (Sirt1)/eNOS pathway [57, 58]. In one of our studies, testosterone administration upregulated Sirt1 and eNOS mRNA transcription, possibly preventing CC inflammation in T2DM rats (Figures 1 and 2). Interestingly, serum asymmetric dimethylarginine (ADMA) levels were also increased in T2DM rats, and rats receiving TRT were observed to have decreased ADMA levels. ADMA is an endogenous arginine compound that rises in individuals demonstrating some disease states [59]; in particular, several reports have suggested a potential relationship between ADMA levels and ED [60, 61]. ADMA has NOS inhibitory activity, and the elevation of ADMA levels contributes to decreased NO bioactivity and decreased endothelial functioning of vessel tissues. Zhang with T2DM have a high incidence of ED [5, 48, 50, 51]. Figure 1. The mechanism of erectile dysfunction caused by T2DM. choline and eNOS. #### 4. Testosterone and neurogenic factors ED has relationships between not only cardiovascular diseases but also neurological diseases. Yang et al. found the hazard risk for Alzheimer's disease and non-Alzheimer dementia to be greater in patients with ED [35]. They also found that log-rank test revealed that patients with ED had significantly higher cumulative incidence rates of dementia than those without. Yang et al. found the incidence density rate of Parkinson's disease (PD) was higher in the ED cohort than in the non-ED cohort [36]. Balsamo et al. reported that men with multiple sclerosis had high risk of ED [37]. Interestingly, testosterone deficiency is often observed in these neurological disease patients relative to age-matched controls [38–40]. In basic study, there are some reports on the relationship between testosterone deficiency and neurogenic factors. Baba et al. reported the mean number of NOS-containing nerve fibers in the corpora cavernosa and in both dorsal nerves of castrated rats [41]. Others also showed that castration decreased nNOS protein expression in the corpus cavernosum [32]. However, reports regarding nNOS responses differ significantly; some studies show increased activity but no change in protein expression [42] in rats, whereas others report no effects in rabbits [43]. Thus, more research into the relationship between nNOS and testosterone is required. On the other hand, Suzuki et al. measured the ICP during electrical stimulation of the preoptic area and cavernous nerve in castrated male rats with and without testosterone replacement [44]. They showed the actions of testosterone and its metabolites on both the central and peripheral neural pathways are crucial for maintaining and restoring erectile capacity. Syme et al. reported that castration resulted in a decreased erectile response to electrostimulation following nerve grafting due to decreased graft neuronal nitric oxide synthase-positive axonal regeneration [45]. Armagan et al. indicated that testosterone had a neuroprotective role in the nerve fibers of the dorsal nerve and testosterone deficiency led to different forms of nerve degeneration resulting in anatomic alterations [46]. Baba et al. also reported that castration decreased the number of nicotinamide adenine dinucleotide phosphate diaphorasestaining nerve fibers not only in corpus cavernosum but also in dorsal nerve [47]. These results indicate that testosterone deficiency would cause neurogenic dysfunction of erectile tissues; however, future study needs to unravel the mechanism of testosterone action to the nerve systems. #### 5. Testosterone and metabolic syndrome Obesity has become a major public health issue that is associated with increased mortality primarily due to increased risks of cardiovascular disease and type 2 diabetes mellitus (T2DM) [1, 48, 49]. Obesity is also considered a strong risk factor for ED [4, 5]. In men, visceral adipose tissue causes arteriosclerosis and vessel endothelial dysfunction [12]. Therefore, men with T2DM have a high incidence of ED [5, 48, 50, 51]. the counter-regulation of autophagy and apoptosis through regulation of the BECN 1-Bcl-2 ED has relationships between not only cardiovascular diseases but also neurological diseases. Yang et al. found the hazard risk for Alzheimer's disease and non-Alzheimer dementia to be greater in patients with ED [35]. They also found that log-rank test revealed that patients with ED had significantly higher cumulative incidence rates of dementia than those without. Yang et al. found the incidence density rate of Parkinson's disease (PD) was higher in the ED cohort than in the non-ED cohort [36]. Balsamo et al. reported that men with multiple sclerosis had high risk of ED [37]. Interestingly, testosterone deficiency is often observed in these neuro- In basic study, there are some reports on the relationship between testosterone deficiency and neurogenic factors. Baba et al. reported the mean number of NOS-containing nerve fibers in the corpora cavernosa and in both dorsal nerves of castrated rats [41]. Others also showed that castration decreased nNOS protein expression in the corpus cavernosum [32]. However, reports regarding nNOS responses differ significantly; some studies show increased activity but no change in protein expression [42] in rats, whereas others report no effects in rabbits [43]. Thus, more research into the relationship between nNOS and testosterone is required. On the other hand, Suzuki et al. measured the ICP during electrical stimulation of the preoptic area and cavernous nerve in castrated male rats with and without testosterone replacement [44]. They showed the actions of testosterone and its metabolites on both the central and peripheral neural pathways are crucial for maintaining and restoring erectile capacity. Syme et al. reported that castration resulted in a decreased erectile response to electrostimulation following nerve grafting due to decreased graft neuronal nitric oxide synthase-positive axonal regeneration [45]. Armagan et al. indicated that testosterone had a neuroprotective role in the nerve fibers of the dorsal nerve and testosterone deficiency led to different forms of nerve degeneration resulting in anatomic alterations [46]. Baba et al. also reported that castration decreased the number of nicotinamide adenine dinucleotide phosphate diaphorasestaining nerve fibers not only in corpus cavernosum but also in dorsal nerve [47]. These results indicate that testosterone deficiency would cause neurogenic dysfunction of erectile tissues; however, future study needs to unravel the mechanism of testosterone action to the Obesity has become a major public health issue that is associated with increased mortality primarily due to increased risks of cardiovascular disease and type 2 diabetes mellitus (key dual regulators of autophagy and apoptosis) interaction [33, 34]. logical disease patients relative to age-matched controls [38–40]. 4. Testosterone and neurogenic factors 260 Sex Hormones in Neurodegenerative Processes and Diseases nerve systems. 5. Testosterone and metabolic syndrome In recent years, epidemiologic studies have suggested that obesity is also associated with multiple alterations in the gonadal endocrine system, including low testosterone levels [1, 48, 52, 53]. Low testosterone levels have also been reported in animals with T2DM, including two seminal research papers that reported testosterone replacement therapy (TRT) in such animal models [54, 55]. Davis et al. administered TRT to obese Zucker rats, resulting in improved cholesterol parameters and insulin sensitivity [54]. On the other hand, others administered TRT to rabbits with high-fat diet-associated hypogonadotropic hypogonadism [55]. TRT partially ameliorated the animals' blood glucose levels and improved CC sensitivity to acetylcholine and eNOS. We also reported that T2DM increased inflammatory biomarker (inducible NO synthase, interleukin-6, and tumor necrosis factor alpha) mRNA expression levels in the CC, but TRT decreased them [56]. Ota et al. reported an in vitro study that demonstrated testosterone prevented inflammation caused by hydrogen peroxide in blood vessel cells by upregulating the sirtuin-1 (Sirt1)/eNOS pathway [57, 58]. In one of our studies, testosterone administration upregulated Sirt1 and eNOS mRNA transcription, possibly preventing CC inflammation in T2DM rats (Figures 1 and 2). Interestingly, serum asymmetric dimethylarginine (ADMA) levels were also increased in T2DM rats, and rats receiving TRT were observed to have decreased ADMA levels. ADMA is an endogenous arginine compound that rises in individuals demonstrating some disease states [59]; in particular, several reports have suggested a potential relationship between ADMA levels and ED [60, 61]. ADMA has NOS inhibitory activity, and the elevation of ADMA levels contributes to decreased NO bioactivity and decreased endothelial functioning of vessel tissues. Zhang Figure 1. The mechanism of erectile dysfunction caused by T2DM. Figure 2. The mechanism of ART for T2DM. et al. also reported that testosterone treatment improved nNOS activity using streptozotocin-induced diabetic rat [62]. > the medication, and there are no basic science reports addressing its use. These results demonstrate the need to investigate differences between various testosterone administration Testosterone and Erectile Function: A Review of Evidence from Basic Research http://dx.doi.org/10.5772/intechopen.72935 263 Erectile functioning is a complex process, with an underlying mechanism that is affected by several factors [4, 67–69]. Recent studies have suggested that one of these factors may be endogenous estrogen levels [70–77]. For example, Baser et al. suggested that serum estrogen levels are correlated with aging in men and that estrogen may, therefore, play an important role in the expression of the symptoms of aging [70]. Further, Greco et al. reported that tadalafil treatment suppresses estrogen levels in some obese men and improved their erectile function domain scores [71]. Another group reported high estrogen levels in elderly patients with ED and sexual disinterest; therefore, they suggested that pathophysiological estrogentestosterone imbalance is involved in these conditions among elderly men [72, 73]. In a basic study, Goyal et al. reported that estrogen caused developmental disorders of the rat penis and that it decreased penile testosterone levels [74, 75]. Others reported that estrogen caused pathophysiological changes in the corpus cavernosum and a decline in erectile function in rats [76]. These authors also reported that estrogen induction enhanced corpus cavernosum smooth muscle contraction and decreased smooth muscle relaxation in rabbits [77]. We reported the use of TRT in a rat model of testosterone deficiency induced by estrogen injections. Interestingly, TRT is not an effective ED treatment in the high-estrogen level model [78]. Thus, attention needs to be given not only to the testosterone levels but also to the levels of methods. Figure 3. Testosterone levels after testosterone injection in rat. other hormones. #### 6. TRT limitations TRT is widely used to effectively treat patients with testosterone deficiencies. It has also been applied to animal models for investigating the mechanisms of testosterone action. However, Burns-Cox et al. pointed out that testosterone (testosterone enanthate) injections cause extremely high levels of testosterone after a few days [63]. Similarly, we injected testosterone enanthate into rats, and the animals demonstrated serum testosterone level increases that rose in a dose-dependent manner (Figure 3). Amano et al. reported that the therapeutic administration of testosterone ointment to patients with LOH successfully kept testosterone at normal levels [64]. We administered low-dose testosterone (similar to applying testosterone ointment) to rats, as previous report [65], 4 weeks after castration. Interestingly, this TRT did not improve erectile functioning over the first 4 weeks of administration. However, after 8 weeks of TRT, partial ED improvements were observed (Figure 4). Baba et al. reported that delayed TRT improved ED, in rats, for 4 weeks [41]. However, they used high-dose testosterone administrations and the testosterone levels were ≥10 times normal. These results suggest that low-dose testosterone treatments may require longer treatment periods to overcome testosterone deficiency. Currently, testosterone undecanoate, a drug that is applied over a long period (about 3 months), is widely used in European countries. The medication has been shown to be a safe and effective treatment for patients with testosterone deficiencies [66]. However, some countries have not approved Figure 3. Testosterone levels after testosterone injection in rat. et al. also reported that testosterone treatment improved nNOS activity using streptozoto- TRT is widely used to effectively treat patients with testosterone deficiencies. It has also been applied to animal models for investigating the mechanisms of testosterone action. However, Burns-Cox et al. pointed out that testosterone (testosterone enanthate) injections cause extremely high levels of testosterone after a few days [63]. Similarly, we injected testosterone enanthate into rats, and the animals demonstrated serum testosterone level increases that rose Amano et al. reported that the therapeutic administration of testosterone ointment to patients with LOH successfully kept testosterone at normal levels [64]. We administered low-dose testosterone (similar to applying testosterone ointment) to rats, as previous report [65], 4 weeks after castration. Interestingly, this TRT did not improve erectile functioning over the first 4 weeks of administration. However, after 8 weeks of TRT, partial ED improvements were observed (Figure 4). Baba et al. reported that delayed TRT improved ED, in rats, for 4 weeks [41]. However, they used high-dose testosterone administrations and the testosterone levels were ≥10 times normal. These results suggest that low-dose testosterone treatments may require longer treatment periods to overcome testosterone deficiency. Currently, testosterone undecanoate, a drug that is applied over a long period (about 3 months), is widely used in European countries. The medication has been shown to be a safe and effective treatment for patients with testosterone deficiencies [66]. However, some countries have not approved cin-induced diabetic rat [62]. Figure 2. The mechanism of ART for T2DM. 262 Sex Hormones in Neurodegenerative Processes and Diseases in a dose-dependent manner (Figure 3). 6. TRT limitations the medication, and there are no basic science reports addressing its use. These results demonstrate the need to investigate differences between various testosterone administration methods. Erectile functioning is a complex process, with an underlying mechanism that is affected by several factors [4, 67–69]. Recent studies have suggested that one of these factors may be endogenous estrogen levels [70–77]. For example, Baser et al. suggested that serum estrogen levels are correlated with aging in men and that estrogen may, therefore, play an important role in the expression of the symptoms of aging [70]. Further, Greco et al. reported that tadalafil treatment suppresses estrogen levels in some obese men and improved their erectile function domain scores [71]. Another group reported high estrogen levels in elderly patients with ED and sexual disinterest; therefore, they suggested that pathophysiological estrogentestosterone imbalance is involved in these conditions among elderly men [72, 73]. In a basic study, Goyal et al. reported that estrogen caused developmental disorders of the rat penis and that it decreased penile testosterone levels [74, 75]. Others reported that estrogen caused pathophysiological changes in the corpus cavernosum and a decline in erectile function in rats [76]. These authors also reported that estrogen induction enhanced corpus cavernosum smooth muscle contraction and decreased smooth muscle relaxation in rabbits [77]. We reported the use of TRT in a rat model of testosterone deficiency induced by estrogen injections. Interestingly, TRT is not an effective ED treatment in the high-estrogen level model [78]. Thus, attention needs to be given not only to the testosterone levels but also to the levels of other hormones. gut and liver [3]. However, oral L-citrulline administrations were shown to avoid such metabolism [85]. Accordingly, oral L-citrulline supplementation was reported to increase L-arginine levels more efficiently than oral L-arginine administration; L-citrulline also increased NO production [86]. In addition, we conducted a similar study using an acute arteriogenic ED model [87]. In that study, oral L-citrulline supplementation improved erectile function and increased Testosterone and Erectile Function: A Review of Evidence from Basic Research http://dx.doi.org/10.5772/intechopen.72935 265 Fukuhara et al. showed that resveratrol and vardenafil improved erectile responses in rats with streptozotocin-induced diabetes [88]. Recently, Dalaklioglu et al. also reported that resveratrol improved sildenafil-induced corpus cavernosum relaxation in both diabetic and nondiabetic aged rats, probably by potentiating NOS activity [89]. Oral supplementation might improve the vasculogenic condition, considering our previous study, though this is just spec- Testosterone levels affect several organs, including the functioning of male erectile tissue. Many studies have described the mechanism of testosterone deficiency effects on erectile function as well as the impact of TRT. Testosterone affects NO production and PDE-5 expression in the corpus cavernosum through molecular pathways. It also preserves smooth muscle contractility by regulating both contraction and relaxation. Further, testosterone maintains the structure of the corpus cavernosum. TRT is widely used to treat patients with testosterone deficiencies; however, the present discussion has also documented some problems associated with this therapeutic approach. Basic research has also identified other potentially effective therapeutic methods for treating testosterone deficiency. Among these, PDE-5 inhibitors, L-citrulline, and resveratrol might be options for treating testosterone deficiency-induced ED. Future research should confirm these findings in more specific experiments that use molecular tools. Such additional research may shed more light on possible treatments for endocrine-mediated ED and its treatment. NO production, without side effects (e.g., decreased mean arterial pressure) [87]. ulation and needs to be examined. 8. Conclusions Conflict of interest Author details Tomoya Kataoka<sup>1</sup> University, Japan City University, Japan The authors declare no conflict of interest. and Kazunori Kimura1,2\* \Address all correspondence to: [email protected] 1 Department of Clinical Pharmaceutics, Graduate School of Medical Sciences, Nagoya City 2 Department of Hospital Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya Figure 4.* Different effects of TRT periods for delayed treatment. #### 7. New approaches for testosterone deficiency treatment Although TRT is an effective treatment for testosterone deficiency, some reports have reported a new treatment approach based on the use of testosterone deficiency models that consider the mechanism of testosterone action on erectile function. PDE-5 inhibitors are the first choice for ED patients, but they are not always effective in patients with testosterone deficiencies [79, 80]. One of the reasons for the lack of efficacy might be the PDE-5 expression changes induced by testosterone. A combination therapy involving both testosterone and PDE-5 inhibitors is one choice, but it is the one that is being vigorously debated, with strong reasons being presented for and against its use. PDE-5 inhibitors are also effective, but regardless of their pharmacokinetics or the regimen used, none has been shown to cure ED [2]. Moody et al. showed that L-arginine administration also improves ED in castrated rats [81]. Similarly, we demonstrated that L-citrulline supplementation improves erectile function and penile structure in castrated rats [82]. L-arginine and L-citrulline are amino acids present in free form in the human body. When L-citrulline is orally administered, it is converted to L-argininosuccinate and, subsequently, to L-arginine by renal argininosuccinate lyase [83]. L-arginine is then converted to NO and L-citrulline by NOS [84]. Orally administered L-arginine is known to be extensively metabolized by autochthonous gut bacteria and by arginases in the gut and liver [3]. However, oral L-citrulline administrations were shown to avoid such metabolism [85]. Accordingly, oral L-citrulline supplementation was reported to increase L-arginine levels more efficiently than oral L-arginine administration; L-citrulline also increased NO production [86]. In addition, we conducted a similar study using an acute arteriogenic ED model [87]. In that study, oral L-citrulline supplementation improved erectile function and increased NO production, without side effects (e.g., decreased mean arterial pressure) [87]. Fukuhara et al. showed that resveratrol and vardenafil improved erectile responses in rats with streptozotocin-induced diabetes [88]. Recently, Dalaklioglu et al. also reported that resveratrol improved sildenafil-induced corpus cavernosum relaxation in both diabetic and nondiabetic aged rats, probably by potentiating NOS activity [89]. Oral supplementation might improve the vasculogenic condition, considering our previous study, though this is just speculation and needs to be examined. ### 8. Conclusions Testosterone levels affect several organs, including the functioning of male erectile tissue. Many studies have described the mechanism of testosterone deficiency effects on erectile function as well as the impact of TRT. Testosterone affects NO production and PDE-5 expression in the corpus cavernosum through molecular pathways. It also preserves smooth muscle contractility by regulating both contraction and relaxation. Further, testosterone maintains the structure of the corpus cavernosum. TRT is widely used to treat patients with testosterone deficiencies; however, the present discussion has also documented some problems associated with this therapeutic approach. Basic research has also identified other potentially effective therapeutic methods for treating testosterone deficiency. Among these, PDE-5 inhibitors, L-citrulline, and resveratrol might be options for treating testosterone deficiency-induced ED. Future research should confirm these findings in more specific experiments that use molecular tools. Such additional research may shed more light on possible treatments for endocrine-mediated ED and its treatment. #### Conflict of interest 7. New approaches for testosterone deficiency treatment Figure 4. Different effects of TRT periods for delayed treatment. 264 Sex Hormones in Neurodegenerative Processes and Diseases Although TRT is an effective treatment for testosterone deficiency, some reports have reported a new treatment approach based on the use of testosterone deficiency models that consider the mechanism of testosterone action on erectile function. PDE-5 inhibitors are the first choice for ED patients, but they are not always effective in patients with testosterone deficiencies [79, 80]. One of the reasons for the lack of efficacy might be the PDE-5 expression changes induced by testosterone. A combination therapy involving both testosterone and PDE-5 inhibitors is one choice, but it is the one that is being vigorously debated, with strong reasons being presented for and against its use. PDE-5 inhibitors are also effective, but regardless of Moody et al. showed that L-arginine administration also improves ED in castrated rats [81]. Similarly, we demonstrated that L-citrulline supplementation improves erectile function and penile structure in castrated rats [82]. L-arginine and L-citrulline are amino acids present in free form in the human body. When L-citrulline is orally administered, it is converted to L-argininosuccinate and, subsequently, to L-arginine by renal argininosuccinate lyase [83]. L-arginine is then converted to NO and L-citrulline by NOS [84]. Orally administered L-arginine is known to be extensively metabolized by autochthonous gut bacteria and by arginases in the their pharmacokinetics or the regimen used, none has been shown to cure ED [2]. The authors declare no conflict of interest. #### Author details Tomoya Kataoka<sup>1</sup> and Kazunori Kimura1,2\* \Address all correspondence to: [email protected] 1 Department of Clinical Pharmaceutics, Graduate School of Medical Sciences, Nagoya City University, Japan 2 Department of Hospital Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Japan #### References* [1] Meldrum DR, Gambone JC, Morris MA, Esposito K, Giugliano D, Ignarro LJ. Lifestyle and metabolic approaches to maximizing erectile and vascular health. International Journal of Impotence Research. 2012;24:61-68 [15] Morelli A, Chavalmane AK, Filippi S, Fibbi B, Silvestrini E, Sarchielli E, Zhang XH, Vignozzi L, Vannelli GB, Forti G, Maggi M. 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The Journal of Sexual Medicine. 2009;6:3274-3288 Protective role of eNOS and SIRT1. PLoS One. 2012;7:e29598 function. International Journal of Andrology. 2012;35:660-667 Oxford: Isis Medical Media; 1999. pp. 327-344 therapy. Pharmacological Reports. 2006;58:159-178 European Urology. 2011;59:948-955 270 Sex Hormones in Neurodegenerative Processes and Diseases 2006;3:2535-2545 [80] Mäkinen JI, Huhtaniemi I. Androgen replacement therapy in late-onset hypogonadism: Current concepts and controversies—A mini-review. Gerontology. 2011;57:193-202 Chapter 12 Provisional chapter New Insights for Hormone Therapy in Perimenopausal DOI: 10.5772/intechopen.74332 Perimenopause is a mandatory period in women's life, when the medical staff may initiate hormone therapy with sex steroids for the delay of brain aging and neurodegenerative diseases, during the so-called "window of opportunity." Animals' models are helpful to sustain the still controversial results of human clinical observational and/or randomized controlled studies. Estrogens, progesterone, and androgens, with their nuclear and membrane receptors, genes, and epigenetics, with their connections to cholinergic, GABAergic, serotoninergic, and glutamatergic systems are involved in women's normal brain or in brain's pathology. The sex steroids are active through direct and/or indirect mechanisms to modulate and/or to protect brain plasticity, and vessels network, fuel metabolism—glucose, ketones, ATP, to reduce insulin resistance, and inflammation of the aging brain through blood-brain barrier disruption, microglial aberrant activation, and neural cell survival/loss. Keywords: perimenopause, "window" of opportunity, neuroprotection, sex steroid The months/years of perimenopause represent an important moment during women's aging, when sex steroids and their receptors decline are evident in the hippocampal and cortical neurons, after estrogen exposure during the reproductive years. The sex steroid hormones decline is associated/acts synergic to other factors as hypertension, diabetes, hypoxia/obstructive sleep apnea, obesity, vitamin B12/folate deficiency, depression, and traumatic brain injury to promote > © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. diverse pathological mechanisms involved in brain aging, memory impairment, and AD. New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection Women Neuroprotection http://dx.doi.org/10.5772/intechopen.74332 Abstract hormones 1. Introduction Additional information is available at the end of the chapter Additional information is available at the end of the chapter Manuela Cristina Russu and Alexandra Cristina Antonescu Manuela Cristina Russu and Alexandra Cristina Antonescu #### New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection DOI: 10.5772/intechopen.74332 Manuela Cristina Russu and Alexandra Cristina Antonescu Manuela Cristina Russu and Alexandra Cristina Antonescu Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.74332 #### Abstract [80] Mäkinen JI, Huhtaniemi I. Androgen replacement therapy in late-onset hypogonadism: Current concepts and controversies—A mini-review. Gerontology. 2011;57:193-202 [81] Moody JA, Vernet D, Laidlaw S, Rajfer J, Gonzalez-Cadavid NF. Effects of long-term oral administration of L-arginine on the rat erectile response. The Journal of Urology [82] Hotta Y, Shiota A, Kataoka T, Motonari M, Maeda Y, Morita M, Kimura K. Oral L-citrulline supplementation improves erectile function and penile structure in castrated [83] Curis E, Nicolis I, Moinard C, Osowska S, Zerrouk N, Bénazeth S, Cynober L. Almost all [84] Morris SM Jr. Enzymes of arginine metabolism. The Journal of Nutrition. 2004;134: [85] Schwedhelm E, Maas R, Freese R, Jung D, Lukacs Z, Jambrecina A, Spickler W, Schulze F, Böger RH. Pharmacokinetic and pharmacodynamic properties of oral l-citrulline and l-arginine: Impact on nitric oxide metabolism. British Journal of Clinical Pharmacology. [86] Wijnands KA, Vink H, Briedé JJ, van Faassen EE, Lamers WH, Buurman WA, Poeze M. Citrulline a more suitable substrate than arginine to restore NO production and the [87] Shiota A, Hotta Y, Kataoka T, Morita M, Maeda Y, Kimura K. Oral l-citrulline supplementation improves erectile function in rats with acute arteriogenic erectile dysfunction. [88] Fukuhara S, Tsujimura A, Okuda H, Yamamoto K, Takao T, Miyagawa Y, Nonomura N, Okuyama A. Vardenafil and resveratrol synergistically enhance the nitric oxide/cyclic guanosine monophosphate pathway in corpus cavernosal smooth muscle cells and its therapeutic potential for erectile dysfunction in the streptozotocin-induced diabetic rat: [89] Dalaklioglu S, Bayram Z, Tasatargil A, Ozdem S. Resveratrol reverses diabetes-related decrement in sildenafil-induced relaxation of corpus cavernosum in aged rats. Aging Preliminary findings. The Journal of Sexual Medicine. 2011;8:1061-1071 rats. International Journal of Urology. 2014;21:608-612 about citrulline in mammals. Amino Acids. 2005;29:177-205 microcirculation during endotoxemia. PLoS One 2012;7:e37439 The Journal of Sexual Medicine. 2013;10:2423-2429 Clinical and Experimental Research. 2017;29:345-351 1997;158:942-947 272 Sex Hormones in Neurodegenerative Processes and Diseases 2743S-2747S 2008;65:51-59 Perimenopause is a mandatory period in women's life, when the medical staff may initiate hormone therapy with sex steroids for the delay of brain aging and neurodegenerative diseases, during the so-called "window of opportunity." Animals' models are helpful to sustain the still controversial results of human clinical observational and/or randomized controlled studies. Estrogens, progesterone, and androgens, with their nuclear and membrane receptors, genes, and epigenetics, with their connections to cholinergic, GABAergic, serotoninergic, and glutamatergic systems are involved in women's normal brain or in brain's pathology. The sex steroids are active through direct and/or indirect mechanisms to modulate and/or to protect brain plasticity, and vessels network, fuel metabolism—glucose, ketones, ATP, to reduce insulin resistance, and inflammation of the aging brain through blood-brain barrier disruption, microglial aberrant activation, and neural cell survival/loss. Keywords: perimenopause, "window" of opportunity, neuroprotection, sex steroid hormones #### 1. Introduction The months/years of perimenopause represent an important moment during women's aging, when sex steroids and their receptors decline are evident in the hippocampal and cortical neurons, after estrogen exposure during the reproductive years. The sex steroid hormones decline is associated/acts synergic to other factors as hypertension, diabetes, hypoxia/obstructive sleep apnea, obesity, vitamin B12/folate deficiency, depression, and traumatic brain injury to promote diverse pathological mechanisms involved in brain aging, memory impairment, and AD. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ### 2. Sex steroid hormones involvement in aging brain. The perimenopausal "window" of opportunity for neuroprotection Perimenopause represents the critical period during women's brain aging when it is possible to use the "window" of opportunity to delay/postpone the proved deleterious effects of sex steroids decline. The complex/complicated feedback loops between non-reproductive brain regions—prefrontal neocortex, hippocampus, amigdala, and brainstem, thalamus and hypothalamus, and the ovaries are made by sex steroid hormones, their network receptors through the entire brain, enzymes involved in their metabolism, their metabolites, neurotransmitters, cytokines, chemokines, and many other proteins/peptides. A multitude of studies are demonstrating the effects of estrogens (estradiol-E2, estetrol-E4, and in a less measure estrone-E1), progesterone, and androgens in brain during reproductive years, and have preventive functions to cognitive and memory performances, being involved in brain bioenergetic control by regulation of glucose transport, aerobic glycolysis coupled to the citric acid cycle, and mitochondrial respiration to generate ATP [1], and also in anti-inflammatory actions in the biology of neuroaging and neurodegenerative diseases. These effects of sex steroids are covering the two hypothesis of neurodegeneration discussed in the previous chapter. Beside involvement in reproduction, sex steroids exert regulatory actions on the receptors in non-reproductive brain regions, including (but not limited to) prefrontal cortex and hippocampus, amigdala, thalamus, and brainstem, which occur via neural circuitry linking the hypothalamus to other CNS systems. The steroid hormones are acting by indirect mechanisms on coagulation, metabolisms to prevent atherosclerosis, and to vasodilation of cerebral vessels, increasing the blood flow to the hippocampus and left superior temporal gyrus [2], and through direct cellular mechanisms on different types of neurons, microglia, and astroglia, on their synapses particularly in the brain regions that show preclinical abnormalities in individuals who are at risk for AD (Table 1). Starting from basic science, preclinical and clinical studies, experimental, observational, and controlled trials are linked to estrogen-inducible neuroprotective, neurotrophic, and neurogenic actions. There are animal (rats, mice, non-human primates) and human evidences on cellular mechanisms of estrogen-regulated functions/systems, which are presented in Table 2. #### 2.1. Estrogens' involvement in brain aging All types of studies have demonstrated that the neuroprotective effects of estrogens—mainly 17 β—and 17α-estradiol, progesterone, and androgens (DHEA mainly) are on the vessels, neurons, microglia, and astroglia. The vast majority of gynecological, endocrinological, and/ or neurological research studies are about the thrombotic and ischemic stroke risks after the age of 50 years (median age of menopause). users vs. 158/968 [16.3%] nonusers; 0.40 [95% Cl 0.22–0.85], p < 0.01), and for a longer duration than 1 year, with no effect observed for the age of menopause. At that moment, the researchers considered the necessity of prospective studies for the establishment of the dose and duration of ET to provide this benefit and to assess the safety in elderly postmenopausal women. Legend: E2: estradiol; Ethinyl-E2: ethinylestradiol; MPA: medroxyprogesterone acetate; CEE: conjugated equine estro- Author(s)/year Type of Maki and Resnick [2] McEwen et al. [79] Adams et al. [37] Zhang et al., [34] Norbury et al. [140] Persad et al. [141] Harburger et al. [64] Silverman et al. [142] Tskitishvili et al. [46] Zhao et al. [52] Experimental E2 + CyP4 Tang et al. [138] Prospective study observational (1–5 yrs. follow-up) Prospective (2 yrs. follow-up) Experimental Experimental E2 E2 Experimental 17β E2, testosterone or RCT Ethinyl-E2 and progestin > vs. E2 + CoP4 Table 1. Actions/effects of sex steroid hormones on brain aging. methyl-testosterone Steroid Action/Effect E2 Estrogen promotes the growth and survival of cholinergic which may delay the onset or prevent AD. New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection E2 Increasing the blood flow to the hippocampus and left superior temporal gyrus tau-related changes resonance imaging RCT E2 Higher metabolism in language processing and auditory CEE + MPA) Yao et al. [143] Experimental 17β-E2 Early correction of bioenergetics deficits, and mitochondrial Experimental E4 Antioxidative actions, gens; RCT: randomized control trial; CyP4: progesterone cylic; CoP4: progesterone continuous Experimental E2 + progesterone E2 alone and combined E2 + P4 may influence ERK activation RCT E2 Maintenance of the cholinergic system in the hippocampus and frontal cortex Xu et al. [139] Basic study E2 Provides protection against β-amyloid-induced damage and neurons, and could decrease cerebral Aβ deposition, both of http://dx.doi.org/10.5772/intechopen.74332 275 Estrogen synapses formation in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat E2 did not induced the increased synapses number in aged rats Estrogen & Androgen Protection of Human Neurons against Intracellular Amyloid Toxicity through Heat Shock Protein 70 Increased activation in brain regions associated with the left middle/superior frontal cortex, and left inferior parietal cortex during verbal memory encoding tasks on functional magnetic in different time frames or enhance memory of objects β-amyloid deposition when ovarian hormones decline Differential regulation of hippocampal gene expression neurogenesis and possibly promyelinating activities association areas compared to other HRT regimens (CEE or recognition in young ovariectomized mice according to P4 supplementation to E2 The "healthy-cell bias" hypothesis demonstrated the E2 neural different effects at different ages, and at different stages of Aβ presence, making the explanations of different results Observational studies on large numbers of cases from North America as the Baltimore Longitudinal Study of Aging [3], the Manhattan Study of Aging [4] have associated HT/ET to significant prevention or delay onset of AD, or reduction risk of AD (9/156 [5.8%] estrogen Legend: E2: estradiol; Ethinyl-E2: ethinylestradiol; MPA: medroxyprogesterone acetate; CEE: conjugated equine estrogens; RCT: randomized control trial; CyP4: progesterone cylic; CoP4: progesterone continuous Table 1. Actions/effects of sex steroid hormones on brain aging. 2. Sex steroid hormones involvement in aging brain. The perimenopausal Perimenopause represents the critical period during women's brain aging when it is possible to use the "window" of opportunity to delay/postpone the proved deleterious effects of sex steroids decline. The complex/complicated feedback loops between non-reproductive brain regions—prefrontal neocortex, hippocampus, amigdala, and brainstem, thalamus and hypothalamus, and the ovaries are made by sex steroid hormones, their network receptors through the entire brain, enzymes involved in their metabolism, their metabolites, neurotransmitters, cytokines, chemokines, and many other proteins/peptides. A multitude of studies are demonstrating the effects of estrogens (estradiol-E2, estetrol-E4, and in a less measure estrone-E1), progesterone, and androgens in brain during reproductive years, and have preventive functions to cognitive and memory performances, being involved in brain bioenergetic control by regulation of glucose transport, aerobic glycolysis coupled to the citric acid cycle, and mitochondrial respiration to generate ATP [1], and also in anti-inflammatory actions in the biology of neuroaging and neurodegenerative diseases. These effects of sex steroids are covering the Beside involvement in reproduction, sex steroids exert regulatory actions on the receptors in non-reproductive brain regions, including (but not limited to) prefrontal cortex and hippocampus, amigdala, thalamus, and brainstem, which occur via neural circuitry linking the hypothal- The steroid hormones are acting by indirect mechanisms on coagulation, metabolisms to prevent atherosclerosis, and to vasodilation of cerebral vessels, increasing the blood flow to the hippocampus and left superior temporal gyrus [2], and through direct cellular mechanisms on different types of neurons, microglia, and astroglia, on their synapses particularly in the brain regions Starting from basic science, preclinical and clinical studies, experimental, observational, and controlled trials are linked to estrogen-inducible neuroprotective, neurotrophic, and neurogenic actions. There are animal (rats, mice, non-human primates) and human evidences on cellular mechanisms of estrogen-regulated functions/systems, which are presented in Table 2. All types of studies have demonstrated that the neuroprotective effects of estrogens—mainly 17 β—and 17α-estradiol, progesterone, and androgens (DHEA mainly) are on the vessels, neurons, microglia, and astroglia. The vast majority of gynecological, endocrinological, and/ or neurological research studies are about the thrombotic and ischemic stroke risks after the Observational studies on large numbers of cases from North America as the Baltimore Longitudinal Study of Aging [3], the Manhattan Study of Aging [4] have associated HT/ET to significant prevention or delay onset of AD, or reduction risk of AD (9/156 [5.8%] estrogen that show preclinical abnormalities in individuals who are at risk for AD (Table 1). "window" of opportunity for neuroprotection 274 Sex Hormones in Neurodegenerative Processes and Diseases two hypothesis of neurodegeneration discussed in the previous chapter. amus to other CNS systems. 2.1. Estrogens' involvement in brain aging age of 50 years (median age of menopause). users vs. 158/968 [16.3%] nonusers; 0.40 [95% Cl 0.22–0.85], p < 0.01), and for a longer duration than 1 year, with no effect observed for the age of menopause. At that moment, the researchers considered the necessity of prospective studies for the establishment of the dose and duration of ET to provide this benefit and to assess the safety in elderly postmenopausal women. The "healthy-cell bias" hypothesis demonstrated the E2 neural different effects at different ages, and at different stages of Aβ presence, making the explanations of different results symptoms [7, 14]. The actions of sex steroid hormones are bidirectional to the body periphery and to the brain, and today, there are current evidences that estrogen and progesterone may have beneficial, neutral, or detrimental effects on the brain, depending on age at therapy initiation, type of menopause (natural vs. induced), or stage of menopause, specificity of administered medication, mainly the association of E2 to progesterone (P4) (exogenous), active New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection http://dx.doi.org/10.5772/intechopen.74332 277 There are characteristics that contribute to several discrepancies in the results: age, stage of reproductive aging, duration of hypogonadism, and symptoms presence. A better understanding of the nature of these discrepancies will be base for future studies of clinical relevance of The Canadian gynecologists [16] were the first who described neurological and psychological disturbances after oophorectomy, than the Italian gynecologists [17]. One may consider that these surgical circumstances are an abrupt, deep decline/withdrawal of steroids, different from the gradual decline in natural menopause, and the cognitive decline in surgical/chemical menopause is more severe [18]. In natural menopause, either premature or early or at the median age of 51 years, the hormonal decline is on a slow slope for E2 and E1, and the androgens (testosterone, androstenedione, and dehydroepiandrosterone) [19] are still present Professional organizations including the British Menopause Society [21], the International Menopause Society [22], and North American Menopause Society [23] recommend estrogen replacement therapy for women with premature menopause or premature ovarian failure. There are evidences, although not from RCT that restoring pathologically low estrogen levels will reduce the later development of cardiovascular disease, osteoporosis, and possibly dementia. This leads to the general recommendation that estrogen be continued in women who experience premature menopause or early menopause until at least around the median age of natural menopause (approximate age 51 years), effects which are evident up to 60 years for women in natural menopause treated for menopausal symptoms [10]. Different to this category of age, the initiation of ET alone or in combination with a progestin in the late postmenopausal stage (ages 65–79 years) induced an increased risk of dementia and cognitive decline regardless of the type of menopause ([10], citing WHIMS), as the "continuum of neurological health progresses from healthy to unhealthy so to do the benefits of ET/ Experimental evidences support the favorable estrogen effect in neurons, on spinogenesis and synaptogenesis, and the rationale option for the prevention/reduction of the risk or the delay of the onset of AD in postmenopausal women. The North American experiments of McEwen at the Laboratory of Neuroendocrinology from The Rockefeller University, New York, on rats' brain in the years of 1990s' were a surprising discovery on the modulation of hippocampal structural plasticity done by estrogens, and it was considered as a "whole new field in the science" [24]. In the animal models, there are differences between species, regarding reproductive and brain aging scenarios, both changes precede natural reproductive failure [7]. There are some similarities between humans and rodents, and differences between rats and mice. on cognition through its 5α-reduced metabolite, allopregnanolone [15]. ovarian steroids and hormone therapies in women. up to the age of 65 [20]. HT" [4]. 2.1.1. Animal models Table 2. Evidences on cellular mechanisms of estrogen-regulated functions/systems. between experimental, observational studies and large RCT, that E2 is beneficial on rats' healthy hippocampal neurons before Aβ exposure [4–6]. More than this hypothesis on "healthy-cell bias," there are discussions and comments about the discrepancies between basic science and/or observational studies supporting estrogen neuroprotective effects and RCT results, focusing the attention to age, stage of reproductive aging, duration of hypogonadism, and the presence of symptoms of cognition or memory impairment, in the efforts for reconciliations [7] between HT and risk for AD [17]. If neurons are healthy at the time of estrogen exposure, their response to estrogen is beneficial for both neuronal survival and neurological function. In contrast, if neurological health is compromised, estrogen exposure over time exacerbates neurological demise. These last two statements represent the opinion of Brinton [8] from the Department of Pharmacology and Pharmaceutical Sciences of California University (USA). In concordance to these facts, the North American and Chinese researchers had elaborated, and tried to confirm the "hypothesis of critical period of estrogen neuroprotection," which appreciates the risk of duration of estrogen deprivation [9]. It was suggested as a "critical period" or a "critical window of opportunity" for the beneficial protective effect of E2 on the human brain [10, 11], and that estrogens have to be administered at perimenopause or earlier to observe a beneficial effect on the neural system [12, 13], as it is for the cardiovascular system. A consensus began to emerge (although not without controversies) that HT/ET at the time of the menopause transition and afterward could have beneficial effects on several neurological symptoms [7, 14]. The actions of sex steroid hormones are bidirectional to the body periphery and to the brain, and today, there are current evidences that estrogen and progesterone may have beneficial, neutral, or detrimental effects on the brain, depending on age at therapy initiation, type of menopause (natural vs. induced), or stage of menopause, specificity of administered medication, mainly the association of E2 to progesterone (P4) (exogenous), active on cognition through its 5α-reduced metabolite, allopregnanolone [15]. There are characteristics that contribute to several discrepancies in the results: age, stage of reproductive aging, duration of hypogonadism, and symptoms presence. A better understanding of the nature of these discrepancies will be base for future studies of clinical relevance of ovarian steroids and hormone therapies in women. The Canadian gynecologists [16] were the first who described neurological and psychological disturbances after oophorectomy, than the Italian gynecologists [17]. One may consider that these surgical circumstances are an abrupt, deep decline/withdrawal of steroids, different from the gradual decline in natural menopause, and the cognitive decline in surgical/chemical menopause is more severe [18]. In natural menopause, either premature or early or at the median age of 51 years, the hormonal decline is on a slow slope for E2 and E1, and the androgens (testosterone, androstenedione, and dehydroepiandrosterone) [19] are still present up to the age of 65 [20]. Professional organizations including the British Menopause Society [21], the International Menopause Society [22], and North American Menopause Society [23] recommend estrogen replacement therapy for women with premature menopause or premature ovarian failure. There are evidences, although not from RCT that restoring pathologically low estrogen levels will reduce the later development of cardiovascular disease, osteoporosis, and possibly dementia. This leads to the general recommendation that estrogen be continued in women who experience premature menopause or early menopause until at least around the median age of natural menopause (approximate age 51 years), effects which are evident up to 60 years for women in natural menopause treated for menopausal symptoms [10]. Different to this category of age, the initiation of ET alone or in combination with a progestin in the late postmenopausal stage (ages 65–79 years) induced an increased risk of dementia and cognitive decline regardless of the type of menopause ([10], citing WHIMS), as the "continuum of neurological health progresses from healthy to unhealthy so to do the benefits of ET/ HT" [4]. #### 2.1.1. Animal models between experimental, observational studies and large RCT, that E2 is beneficial on rats' healthy hippocampal neurons before Aβ exposure [4–6]. More than this hypothesis on "healthy-cell bias," there are discussions and comments about the discrepancies between basic science and/or observational studies supporting estrogen neuroprotective effects and RCT results, focusing the attention to age, stage of reproductive aging, duration of hypogonadism, and the presence of symptoms of cognition or memory impairment, in the efforts for reconciliations [7] between HT and risk for AD [17]. If neurons are healthy at the time of estrogen exposure, their response to estrogen is beneficial for both neuronal survival and neurological function. In contrast, if neurological health is compromised, estrogen exposure over time exacerbates neurological demise. These last two statements represent the opinion of Brinton [8] from the Department of Pharmacology and Pharmaceutical Sciences of California University (USA). In concordance to these facts, the North American and Chinese researchers had elaborated, and tried to confirm the "hypothesis of critical period of estrogen neuroprotection," which appreciates the risk of duration of estrogen deprivation [9]. It was suggested as a "critical period" or a "critical window of opportunity" for the beneficial protective effect of E2 on the human brain [10, 11], and that estrogens have to be administered at perimenopause or earlier to observe a beneficial effect on • Estrogens augment the glutamatergic impact on hippocampal function Zhao et al. [153]; Estrogen-regulated functions/systems Reference(s) • Estrogens prevent apoptotic death cascades and neuronal death Lebesgue et al. [40] • Estradiol rapidly stimulates signaling cascades: as the mitogen-activated protein (MAP) kinase family and the phosphatidylinositol 3-kinase (PI3K), pathway leading to the phosphorylation of Akt (a key signaling molecule), and Akt can promote local protein synthesis • Estrogens increase the dendritic spine and synaptic density by 30% on CA1 pyramidal cells • Estrogens provide potential to protect or have the capacity to alter synaptic and postsynap- • Estradiol promotes mitochondrial respiration and hence ATP generation and antioxidant enzymes that offset the increase in free radical generation induced by increased respiration • Estrogen-induced calcium signaling pathways both promote neuronal function and can • Estrogens exerts on the GABAergic and cholinergic systems in the hippocampus and frontal Table 2. Evidences on cellular mechanisms of estrogen-regulated functions/systems. related to the formation of new spines through a non-genomic mechanism • Estradiol increases phosphorylated Akt (pAkt) present in CA1 dendrites, spines, and syn- tic circuitry in hypothalamus, hippocampus, and neocortex 276 Sex Hormones in Neurodegenerative Processes and Diseases • Estrogen- induced mitochondrial functions in brain bioenergetics • Estradiol significantly reduces mitochondrial lipid peroxidation exacerbate neuronal demise in neurodegenerative disease states. apses cortex in the hippocampus Etgen et al. [144] Inagaki et al. [42]; Etgen and Inagaki Cordey et al. [145] Znamensky et al. [90] Zhao et al. [146]; Mannella and Brinton [86]; Gould et al. [36]; Adams et al. [147] Choi et al. [148]. Nilsen and Brinton Nilsen et al. [150]. Simpkins et al. [151] Brewer et al. [152] Rudick et al. [154] Tinkler et al. [155]. [149]. [41] A consensus began to emerge (although not without controversies) that HT/ET at the time of the menopause transition and afterward could have beneficial effects on several neurological the neural system [12, 13], as it is for the cardiovascular system. Experimental evidences support the favorable estrogen effect in neurons, on spinogenesis and synaptogenesis, and the rationale option for the prevention/reduction of the risk or the delay of the onset of AD in postmenopausal women. The North American experiments of McEwen at the Laboratory of Neuroendocrinology from The Rockefeller University, New York, on rats' brain in the years of 1990s' were a surprising discovery on the modulation of hippocampal structural plasticity done by estrogens, and it was considered as a "whole new field in the science" [24]. In the animal models, there are differences between species, regarding reproductive and brain aging scenarios, both changes precede natural reproductive failure [7]. There are some similarities between humans and rodents, and differences between rats and mice. Estrogens influence the process of adult neurogenesis. E2 promotes the migration of newly generated neurons toward the damaged brain regions, facilitating brain remodeling, and repair after ischemic stroke injury [25]. exposed to Aβ, that is possible to prevent neurodegeneration when E2 is administered before or during Aβ exposure, the strongest effect being on continuous administration, and the effects are worsened up to neuronal death when are large doses or when Aβ is already present. These results were obtained after previous studies regarding estrogen critical different effects on synaptic system at different rats' ages. Whereas, young rats displayed a 30% increase in axospinous synapse density in CA1 [36], fact which is absent in aged rats [37], as is mentioned in Table 2. There are findings in rodents and monkeys providing evidences that the hippocampus (in rats) and the frontal cortex (in monkeys) remain responsive to E2 administered either in vivo or in vitro even after prolonged periods of hormone withdrawal [38, 39]. The physiological concentrations of E2 exert profound neuroprotective action on apoptotic death cascades and neuronal death from focal and global ischemia causing selective, delayed death of hippocampal CA1 neurons and associated cognitive deficits after a single injection in acute ischemia [40]. E2 administered at physiological levels for 2 weeks before ischemia rescues neurons destined to die in the hippocampal CA1, and ameliorates ischemia-induced cognitive deficits in ovariectomized female rats [41]. An acute post-ischemic infusion of E2 into the brain ventricles is neuroprotective in aged rats after 6 months of hormone deprivation, and E2 enhances synaptic transmission in CA1 pyramidal neurons of aged long-term hormone deprived females [42]. There are evidences about distinct estrogens effects on different cognitive aspects, anxiety-like, and depressive-like behaviors. There are comparisons regarding the subregion-specific effects on tryptophan hydroxylase-2 (TpH2, the brain-specific, rate-limiting enzyme for 5-HT biosynthesis, a serotonin precursor). The comparison of CEE and E2 treatments on behavior and TpH2 mRNA on female ovariectomized Sprague Dawley rats [43]. Both CEE and E2 exert beneficial behavioral effects, although efficacy depended on the distinct behavior and for cognition, on the task difficulty. Compared to CEE, E2 generally had more robust anxiolytic and antidepressant effects. E2 increased TpH2 mRNA in the caudal and mid dorsal raphe nucleus. The recent Chinese study on adult male Sprague Dawley rats [44] demonstrated that low dose E2 administered for the first 3-months after bilateral common carotid artery occlusion (BCCAO) exerted long-lasting beneficial effects, including significant neuroprotection of hippocampal CA1 neurons and preservation of hippocampal-dependent cognitive function New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection http://dx.doi.org/10.5772/intechopen.74332 279 Recent evidences demonstrate a de novo estradiol synthesis within the hippocampus and other brain regions, which seems highly likely that activity-dependent estradiol signaling can play an essential role in the modulation of discrete signaling units within individual cells, affording Animals and in vitro studies are demonstrating the role of estetrol (E4) on nervous system. The antioxidative actions of E4 mostly depend on ER-α and ER-β, whereas neurogenesis and possibly promyelinating activities might be realized through ER-β, and the membrane GRP30 receptor for estrogens and progesterone is less important for LDH activity and cell survival in The animal experiments reconcile the discordance between studies showing favorable steroids/estrogen effects in neurons to the results from former randomized trials, as the largest randomized clinical trial of HT ever conducted—Women's Health Initiative Memory Study when examined at 6 months after BCCAO. E4 actions [46]. "fine- tuned" control of neuronal excitability [45]. E2 induces neuronal plasticity underlying cognitive function. Acute E2 treatment promotes hippocampal neurogenesis in the female rat [26], which has been linked to hippocampaldependent learning and memory [27]. It was revealed on rats experiments [28] that different forms of estrogens modulate neuroplasticity and cognition in complex and intriguing ways. Estrogens specifically up-regulate adult hippocampal neurogenesis (via cell proliferation) and synaptic protein levels in the hippocampus in a time- and dose-dependent manner [29]. Low levels of E2 facilitate spatial working memory and contextual fear conditioning, while high levels of estradiol impair spatial working, spatial reference memory and contextual fear conditioning, and estrone (E1) impairs contextual fear conditioning. The rats' experiments show that only 17β-E2 and not E1 is increasing the survival and activation of new neurons in the hippocampus in response to spatial memory compared to controls [30]. The Chinese experiments on menopausal mice had identified morphological changes in the hippocampus mitochondrial damage, lipofuscin deposition and microtubule degradation, which were possible to be partially restored: mitochondrial damage and lipofuscin increase, not the microtubules degradation, and only in early postmenopausal stages [31]. Regarding the first hypothesis on bioenergetics failure in females' brain aging, animal models demonstrated estrogens effects on mitochondria energetic metabolism. E2 is regulating mitochondrial proteome, being a key metabolic control of enzymes including pyruvate dehydrogenase, aconitase, and ATP-synthetase, and so it is a high respiratory control ratio, elevated cytochrome-c oxidase activity and expression, and it is reduced brain free radical generation [32], according to the knowledge that in aging brain is a high lactate level [30]. E2 is able to mitigate negative effects of glucocorticoids, as animal and human researches indicate: E2-related mitigation of glucocorticoid damage and interference is one benefit of E2 supplementation during perimenopause or soon after menopause. The evidence for E2-related protection against glucocorticoids suggests that maintaining E2 levels in postmenopausal women could protect them from stress-induced declines in neural and cognitive integrity [33]. Physiological doses of 17β-E2, testosterone or methyl-testosterone reduce induced cell death by 50% in neurons treated after the injection and by 80–90% in neurons treated 1 h before the injection [34]. The effect is mediated by genomic mechanisms proved by the blockage of ERs and ARs, and by a proteomic mechanism—the increasing levels of heat shock protein 70 (Hsp70), and the hormones role is to protect from the development and toxicity of the intracellular Aβ (iA1–42), which induces neuronal apoptosis and death, being known that AD starts with intraneuronal iA1–42 accumulation in human brain [35]. The "estrogen action hypothesis" known as "healthy-cell bias" elaborated in the North American Laboratories for Neuroscience Research [4, 5, 8] tries to explain the differences between the effects of estrogens on normal/healthy neurons and aged/damaged neurons. It was demonstrated with different doses (low dose of 10 ng/ml and large dose of 200 ng/ml) and on different schedules (acute vs. continuous vs. intermittent) in experiments on rats' hippocampal neurons exposed to Aβ, that is possible to prevent neurodegeneration when E2 is administered before or during Aβ exposure, the strongest effect being on continuous administration, and the effects are worsened up to neuronal death when are large doses or when Aβ is already present. These results were obtained after previous studies regarding estrogen critical different effects on synaptic system at different rats' ages. Whereas, young rats displayed a 30% increase in axospinous synapse density in CA1 [36], fact which is absent in aged rats [37], as is mentioned in Table 2. Estrogens influence the process of adult neurogenesis. E2 promotes the migration of newly generated neurons toward the damaged brain regions, facilitating brain remodeling, and E2 induces neuronal plasticity underlying cognitive function. Acute E2 treatment promotes hippocampal neurogenesis in the female rat [26], which has been linked to hippocampaldependent learning and memory [27]. It was revealed on rats experiments [28] that different forms of estrogens modulate neuroplasticity and cognition in complex and intriguing ways. Estrogens specifically up-regulate adult hippocampal neurogenesis (via cell proliferation) and synaptic protein levels in the hippocampus in a time- and dose-dependent manner [29]. Low levels of E2 facilitate spatial working memory and contextual fear conditioning, while high levels of estradiol impair spatial working, spatial reference memory and contextual fear conditioning, and estrone (E1) impairs contextual fear conditioning. The rats' experiments show that only 17β-E2 and not E1 is increasing the survival and activation of new neurons in the The Chinese experiments on menopausal mice had identified morphological changes in the hippocampus mitochondrial damage, lipofuscin deposition and microtubule degradation, which were possible to be partially restored: mitochondrial damage and lipofuscin increase, Regarding the first hypothesis on bioenergetics failure in females' brain aging, animal models demonstrated estrogens effects on mitochondria energetic metabolism. E2 is regulating mitochondrial proteome, being a key metabolic control of enzymes including pyruvate dehydrogenase, aconitase, and ATP-synthetase, and so it is a high respiratory control ratio, elevated cytochrome-c oxidase activity and expression, and it is reduced brain free radical generation E2 is able to mitigate negative effects of glucocorticoids, as animal and human researches indicate: E2-related mitigation of glucocorticoid damage and interference is one benefit of E2 supplementation during perimenopause or soon after menopause. The evidence for E2-related protection against glucocorticoids suggests that maintaining E2 levels in postmenopausal women could protect them from stress-induced declines in neural and cognitive integrity [33]. Physiological doses of 17β-E2, testosterone or methyl-testosterone reduce induced cell death by 50% in neurons treated after the injection and by 80–90% in neurons treated 1 h before the injection [34]. The effect is mediated by genomic mechanisms proved by the blockage of ERs and ARs, and by a proteomic mechanism—the increasing levels of heat shock protein 70 (Hsp70), and the hormones role is to protect from the development and toxicity of the intracellular Aβ (iA1–42), which induces neuronal apoptosis and death, being known that AD starts The "estrogen action hypothesis" known as "healthy-cell bias" elaborated in the North American Laboratories for Neuroscience Research [4, 5, 8] tries to explain the differences between the effects of estrogens on normal/healthy neurons and aged/damaged neurons. It was demonstrated with different doses (low dose of 10 ng/ml and large dose of 200 ng/ml) and on different schedules (acute vs. continuous vs. intermittent) in experiments on rats' hippocampal neurons hippocampus in response to spatial memory compared to controls [30]. not the microtubules degradation, and only in early postmenopausal stages [31]. [32], according to the knowledge that in aging brain is a high lactate level [30]. with intraneuronal iA1–42 accumulation in human brain [35]. repair after ischemic stroke injury [25]. 278 Sex Hormones in Neurodegenerative Processes and Diseases There are findings in rodents and monkeys providing evidences that the hippocampus (in rats) and the frontal cortex (in monkeys) remain responsive to E2 administered either in vivo or in vitro even after prolonged periods of hormone withdrawal [38, 39]. The physiological concentrations of E2 exert profound neuroprotective action on apoptotic death cascades and neuronal death from focal and global ischemia causing selective, delayed death of hippocampal CA1 neurons and associated cognitive deficits after a single injection in acute ischemia [40]. E2 administered at physiological levels for 2 weeks before ischemia rescues neurons destined to die in the hippocampal CA1, and ameliorates ischemia-induced cognitive deficits in ovariectomized female rats [41]. An acute post-ischemic infusion of E2 into the brain ventricles is neuroprotective in aged rats after 6 months of hormone deprivation, and E2 enhances synaptic transmission in CA1 pyramidal neurons of aged long-term hormone deprived females [42]. There are evidences about distinct estrogens effects on different cognitive aspects, anxiety-like, and depressive-like behaviors. There are comparisons regarding the subregion-specific effects on tryptophan hydroxylase-2 (TpH2, the brain-specific, rate-limiting enzyme for 5-HT biosynthesis, a serotonin precursor). The comparison of CEE and E2 treatments on behavior and TpH2 mRNA on female ovariectomized Sprague Dawley rats [43]. Both CEE and E2 exert beneficial behavioral effects, although efficacy depended on the distinct behavior and for cognition, on the task difficulty. Compared to CEE, E2 generally had more robust anxiolytic and antidepressant effects. E2 increased TpH2 mRNA in the caudal and mid dorsal raphe nucleus. The recent Chinese study on adult male Sprague Dawley rats [44] demonstrated that low dose E2 administered for the first 3-months after bilateral common carotid artery occlusion (BCCAO) exerted long-lasting beneficial effects, including significant neuroprotection of hippocampal CA1 neurons and preservation of hippocampal-dependent cognitive function when examined at 6 months after BCCAO. Recent evidences demonstrate a de novo estradiol synthesis within the hippocampus and other brain regions, which seems highly likely that activity-dependent estradiol signaling can play an essential role in the modulation of discrete signaling units within individual cells, affording "fine- tuned" control of neuronal excitability [45]. Animals and in vitro studies are demonstrating the role of estetrol (E4) on nervous system. The antioxidative actions of E4 mostly depend on ER-α and ER-β, whereas neurogenesis and possibly promyelinating activities might be realized through ER-β, and the membrane GRP30 receptor for estrogens and progesterone is less important for LDH activity and cell survival in E4 actions [46]. The animal experiments reconcile the discordance between studies showing favorable steroids/estrogen effects in neurons to the results from former randomized trials, as the largest randomized clinical trial of HT ever conducted—Women's Health Initiative Memory Study (WHIMS), which showed that women who initiated estrogen therapy alone or in combination with the progestin MPA after the age of 60 years had a twofold greater risk to develop dementia [47] or are affected regarding mean cognitive performance over periods of time ranging up to 5 years [10], or estrogen-containing hormone therapy initiated in the late postmenopausal stage (ages 65–79 years) is followed by an increased risk of dementia and cognitive decline regardless the type of menopause—naturally, medically, or surgically induced [48]. on cognition through its 5α-reduced metabolite, allopregnanolone [15, 54], a fact that differentiates P4 from the progestin MPA, which proved as a jeopardizing drug for elder postmenopausal women. P4 has neuroprotective effects mediated by various mechanisms such as reduction of neuronal vulnerability to neurotoxic molecules, reduction of cell loss, inhibition of lipid peroxidation, and expression of pro-inflammatory genes [55–57]. P4 can exert protective effects through its metabolites—allopregnanolone or 3α, 5α-tetrahydroprogesterone, the best known, which can interact with membrane-associated receptors coupled to ion-channels, such as the GABAA receptor system. P4 and allopregnanolone, exert various effects on both cognitive and non-mnemonic functions in females. Allopregnanolone may also elicit its protective effects through its actions on the mitochondria [58]. Allopregnanolone is enhancing cognitive performances and placement memory in mice, by inducing higher levels of brain-derived neurotrophic factor (BDNF) in the prefrontal cortex and hippocampus, an effect that is contrary to the lowest levels among mice administered MPA [15]. MPA—the progestin used to balance CEE in WHIMS —was proved by in vitro studies to be the best antagonist to neurotrophic and neuroprotective estrogen actions in neurons, fact that makes it completely different to P4 which alone is neuroprotective [59], and acts synergistic with estradiol [60]. MPA (Provera®) metabolic involvement is also divergent from P4, regarding the action on nuclear mitogen-activated protein kinase signaling [61], and on the exacerbation of neuroexcitotoxicity of glutamate [62]. The well-known object recognition task is a valuable experimental paradigm that can be used to determine the effects and mechanisms of progestogens for mnemonic effects across the lifespan. Improvements in object recognition performance of rodents are often associated with higher hormone levels in the hippocampus and prefrontal cortex during natural cycles, with progesterone replacement New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection http://dx.doi.org/10.5772/intechopen.74332 281 The estrogens neuroprotective actions are modulated by progesterone. It was demonstrated [63] in young ovariectomized mice that E2 enhances object memory consolidation, which depends on dorsal hippocampal activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway, and the questions were if the E2 actions need progesterone adding, which was latter demonstrated, and more than this the effect is E2 dose-dependent [64]. It was suggested that E2 alone, and combined with P4, may influence ERK activation in different time frames or enhance memory through different mechanisms. E2 alone significantly increased phospho-p42 ERK protein levels in the dorsal hippocampus relative to vehicle controls. In contrast, no combination of E2 and P4 affected dorsal hippocampal phospho-ERK levels. Recent studies on normal age-related testosterone and its androgen metabolite dihydrotestosterone (DHT) loss in plasma and brain in men are emerging AD risk, and the protective role of endogenous testosterone/DHT is not only to increase the neuronal resilience against ADrelated insults, but also to reduce intracellular Aβ accumulation [34, 36, 52], testosterone actions are similar, but also cumulative to those of estrogens in perimenopausal women. In perimenopause, estrogens and androgens are still in physiological levels in plasma and brain, and their presence is considered to prevent the accumulation of intracellular amyloid 1-42 following ovariectomy in young animals, or with aging [54]. 2.3. Androgens neuroprotective role in women's aging #### 2.1.2. Human studies The Mayo Clinic Cohort Studies of Oophorectomy and Aging—unilateral or bilateral oophorectomy [48–50] and estrogens, before the age of menopause. The risk of cognitive impairment/ dementia is increased after either unilateral or bilateral oophorectomy compared to referent women (Hazard ratio [HR] of 1.46; 95% CI 1.13 to 1.90; adjusted for education, type of interview, and history of depression). These associations were similar regardless of oophorectomy indication, and for women who underwent unilateral or bilateral oophorectomy were considered separately. The risk increased with younger age at oophorectomy (test for linear trend; adjusted p < 0.0001). The same study group from the Mayo Clinic showed that women who underwent bilateral oophorectomy before menopause were at increased risk of Parkinsonism, and the risk increased with younger age at time of oophorectomy [49, 50]. Their conclusion was a sizeable neuroprotective effect of estrogen before the age of 50 years. Some studies are sustaining that estrogen neuroprotective actions are modulated by progesterone/progestogens. Specifically, continuous progestogen exposure is associated with inhibition of estrogen actions, whereas cyclic delivery of progestogens may enhance neural benefits of estrogen [51]. In the next subchapter, more evidences on these findings are discussed. The North American study [52] provides evidence at the molecular level that different regimens of HT can induce disparate gene expression profiles in brain. From a translational perspective, confirmation of these results in a model of natural menopause would imply that the common regimen of continuous combined HT may have adverse consequences, whereas a cyclic combined regimen, which is more physiological, could be an effective strategy to maintain neurological health and function. It has to be remembered that different factors may determine the efficacy of ER/HT as age, menopausal status, route of administration and dose, the starting cognitive function, and the presence of pre-existing risk factors (smoking, apolipoprotein E genotype) [53]. #### 2.2. Progesterone neuroprotective role in women's aging During menopausal transition on assists at lowering progesterone (P4) values by luteal defect, and afterward in perimenopause P4 which is absent, and at the beginning of the history of HT recommendations, P4 administration was mandatory for women with intact uterus, fact that continues to be actual. There were intensive efforts to develop progesterone neurobiology in the hippocampus and cortex, and current discoveries are sustaining P4 administration for more than uterine protection from endometrial hyperplasia and cancer, but for brain aging protection, besides the much analyzed "therapeutic window" of progesterone in brain trauma. P4 is active on cognition through its 5α-reduced metabolite, allopregnanolone [15, 54], a fact that differentiates P4 from the progestin MPA, which proved as a jeopardizing drug for elder postmenopausal women. P4 has neuroprotective effects mediated by various mechanisms such as reduction of neuronal vulnerability to neurotoxic molecules, reduction of cell loss, inhibition of lipid peroxidation, and expression of pro-inflammatory genes [55–57]. P4 can exert protective effects through its metabolites—allopregnanolone or 3α, 5α-tetrahydroprogesterone, the best known, which can interact with membrane-associated receptors coupled to ion-channels, such as the GABAA receptor system. P4 and allopregnanolone, exert various effects on both cognitive and non-mnemonic functions in females. Allopregnanolone may also elicit its protective effects through its actions on the mitochondria [58]. Allopregnanolone is enhancing cognitive performances and placement memory in mice, by inducing higher levels of brain-derived neurotrophic factor (BDNF) in the prefrontal cortex and hippocampus, an effect that is contrary to the lowest levels among mice administered MPA [15]. MPA—the progestin used to balance CEE in WHIMS —was proved by in vitro studies to be the best antagonist to neurotrophic and neuroprotective estrogen actions in neurons, fact that makes it completely different to P4 which alone is neuroprotective [59], and acts synergistic with estradiol [60]. MPA (Provera®) metabolic involvement is also divergent from P4, regarding the action on nuclear mitogen-activated protein kinase signaling [61], and on the exacerbation of neuroexcitotoxicity of glutamate [62]. The well-known object recognition task is a valuable experimental paradigm that can be used to determine the effects and mechanisms of progestogens for mnemonic effects across the lifespan. Improvements in object recognition performance of rodents are often associated with higher hormone levels in the hippocampus and prefrontal cortex during natural cycles, with progesterone replacement following ovariectomy in young animals, or with aging [54]. The estrogens neuroprotective actions are modulated by progesterone. It was demonstrated [63] in young ovariectomized mice that E2 enhances object memory consolidation, which depends on dorsal hippocampal activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway, and the questions were if the E2 actions need progesterone adding, which was latter demonstrated, and more than this the effect is E2 dose-dependent [64]. It was suggested that E2 alone, and combined with P4, may influence ERK activation in different time frames or enhance memory through different mechanisms. E2 alone significantly increased phospho-p42 ERK protein levels in the dorsal hippocampus relative to vehicle controls. In contrast, no combination of E2 and P4 affected dorsal hippocampal phospho-ERK levels. #### 2.3. Androgens neuroprotective role in women's aging (WHIMS), which showed that women who initiated estrogen therapy alone or in combination with the progestin MPA after the age of 60 years had a twofold greater risk to develop dementia [47] or are affected regarding mean cognitive performance over periods of time ranging up to 5 years [10], or estrogen-containing hormone therapy initiated in the late postmenopausal stage (ages 65–79 years) is followed by an increased risk of dementia and cognitive decline regardless The Mayo Clinic Cohort Studies of Oophorectomy and Aging—unilateral or bilateral oophorectomy [48–50] and estrogens, before the age of menopause. The risk of cognitive impairment/ dementia is increased after either unilateral or bilateral oophorectomy compared to referent women (Hazard ratio [HR] of 1.46; 95% CI 1.13 to 1.90; adjusted for education, type of interview, and history of depression). These associations were similar regardless of oophorectomy indication, and for women who underwent unilateral or bilateral oophorectomy were considered separately. The risk increased with younger age at oophorectomy (test for linear trend; adjusted p < 0.0001). The same study group from the Mayo Clinic showed that women who underwent bilateral oophorectomy before menopause were at increased risk of Parkinsonism, and the risk increased with younger age at time of oophorectomy [49, 50]. Their conclusion was a sizeable neuroprotective effect of estrogen before the age of 50 years. the presence of pre-existing risk factors (smoking, apolipoprotein E genotype) [53]. During menopausal transition on assists at lowering progesterone (P4) values by luteal defect, and afterward in perimenopause P4 which is absent, and at the beginning of the history of HT recommendations, P4 administration was mandatory for women with intact uterus, fact that continues to be actual. There were intensive efforts to develop progesterone neurobiology in the hippocampus and cortex, and current discoveries are sustaining P4 administration for more than uterine protection from endometrial hyperplasia and cancer, but for brain aging protection, besides the much analyzed "therapeutic window" of progesterone in brain trauma. P4 is active 2.2. Progesterone neuroprotective role in women's aging Some studies are sustaining that estrogen neuroprotective actions are modulated by progesterone/progestogens. Specifically, continuous progestogen exposure is associated with inhibition of estrogen actions, whereas cyclic delivery of progestogens may enhance neural benefits of estrogen [51]. In the next subchapter, more evidences on these findings are discussed. The North American study [52] provides evidence at the molecular level that different regimens of HT can induce disparate gene expression profiles in brain. From a translational perspective, confirmation of these results in a model of natural menopause would imply that the common regimen of continuous combined HT may have adverse consequences, whereas a cyclic combined regimen, which is more physiological, could be an effective strategy to maintain neurological health and function. It has to be remembered that different factors may determine the efficacy of ER/HT as age, menopausal status, route of administration and dose, the starting cognitive function, and the type of menopause—naturally, medically, or surgically induced [48]. 2.1.2. Human studies 280 Sex Hormones in Neurodegenerative Processes and Diseases Recent studies on normal age-related testosterone and its androgen metabolite dihydrotestosterone (DHT) loss in plasma and brain in men are emerging AD risk, and the protective role of endogenous testosterone/DHT is not only to increase the neuronal resilience against ADrelated insults, but also to reduce intracellular Aβ accumulation [34, 36, 52], testosterone actions are similar, but also cumulative to those of estrogens in perimenopausal women. In perimenopause, estrogens and androgens are still in physiological levels in plasma and brain, and their presence is considered to prevent the accumulation of intracellular amyloid 1-42 (iA1-42) in the hippocampus and the entorhinal cortex neurons, preceding amyloid plaque formation, and further induction of neuronal death [65]. Proteomic analyses are demonstrating increased levels of Hsp70 in testosterone- and estrogen-treated human neurons [15], which is a sign of Aβ toxicity inhibition. membrane receptors [73]. In addition, estrogens can affect metabotropic glutamate receptors, and the second messenger systems, including calcium mobilization, and a plethora of kinases to alter cell signaling. This subchapter considers the current knowledge of rapid membraneinitiated and intracellular signaling by steroids in the brain, the nature of receptors involved, New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection http://dx.doi.org/10.5772/intechopen.74332 283 The protective role of estrogens in the brain is sure, and the missing preventive effects revealed by RCT is connected to the age-related changes of ERs, as it is in the endometrium/uterus [74], suggesting that several key players in the local synaptic response to E2 are compromised in aging females. The brain has one of the most complex and complicated ERs network of the body, which is changing life-long. In addition to its well-documented hormonal action, E2 is In the last 10 years, molecular and biochemical animal studies are demonstrating that the mechanisms used by estrogens are greatly influenced by brain cell type, ER type, and metabotropic glutamate receptors (mGluRs) independent of glutamate, and/or region of the brain-cortex and/or hippocampus, all these leading to differential regulation of neuronal circuitry in each area [45, 76]. The hippocampus cognitive performance is directly connected to ER-α, other ERs such as ER-β and GPR30 [8]. The ERs have similar distribution in female and male brains, but may differ in relative expression [77]. ER-α and ER-β expression patterns generally overlap, where ER-α is associated with reproductive behavior, whereas ER-β is associated with non-reproductive behaviors such as learning and memory [78] and anxiety-related behaviors. In hippocampus and cortical neurons, the estrogens—mainly E2 and other estrogenic ligands bind to membrane—associated and mitochondrial-associated G protein-coupled receptor (GPR 30), and activates the classical/ canonical nuclear and extranuclear or intra-cytoplasmatic ER isoforms—α and β—functioning as transcription factors [79–81], and a new type of nuclear ER, the orphan estrogenrelated receptor γ (ERR γ), which regulates dopaminergic neuronal phenotype [82], and 3.1. Estrogen receptors (ERs). Genetic polymorphism and epigenetics of ERs IGF-1 receptor, which was recently recognized as a receptor for estrogens. The nongenomic or alternative signaling pathways mechanism involving extranuclear ERs respond to physiological concentration of estrogens to elicit neuroprotection, resulting in the "fine tuning" of neuronal circuitry [45]. Often, rapid activation of intracellular signalers such as mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) underlie alternative estrogen-induced neuroprotection upon activation of specific binding sites at the plasma membrane. The plasma membrane ER (mER) originates from, or is related to canonical nuclear ERs, and GPR30 mimics short latency E2 facilitation of synaptic transmission in the hippocampus, to enhance memory and cognition [83]. The activation of GPR30 by G-1 (its specific ligand) is associated with a mobilization of calcium in dissociated and cultured rat hypothalamic neurons [80, 84, 85]. There were elaborated cellular models of Aβ toxicity where classical and alternative pathways activated by estrogens seem to coexist to orchestrate neuroprotection, fact that is a unique signaling profile of estrogen neuroprotection, dependent and how they contribute to homeostatic functions. considered as a neurotransmitter in the brain [75]. upon activation of the MAPK signaling [86]. Cell cultures are bringing strong evidences that both androgens and estrogens are neuroprotective, and many studies analyzed the different pathways for neural cells protection from Aβ toxicity. Testosterone is involved in regulation of spine synapse density in the CA1 region of hippocampus [66]. A special analysis is to be made on DHEA(S)—the "youth" hormone—for which human body does not have receptors, but it is a source of intracrinology, with different enzymes for steroidforming and/or for steroid-inactivating, permitting each cell/tissue to synthesize a small amount of androgens and/or estrogens in order to meet the local physiological needs without affecting the other tissues of the organism [67]. Blood concentrations are not different from those observed in normal postmenopausal women having high serum DHEA concentrations, when DHES is supplemented to maintain serum estrogen level at sub-threshold or biologically inactive concentrations. On the other hand, all androgens are made intracellularly from DHEA by the mechanisms of intracrinology, and are always maintained at very low levels in the blood in pre- and postmenopausal women [67]. According to this conceptus, it was proposed a short-term DHEA supplementation (5 mg/day 7 days) in perimenopausal female rhesus macaques [68]. The comparison of serum and hippocampal levels in treated and controls of the same age revealed that despite lower concentrations of the estrogens in the serum of elder animals, their concentrations in the hippocampus did not show any obvious differences due to age or to DHEA supplementation. The results suggest that de novo estrogen synthesis in the brain may compensate for the perimenopausal loss of estrogens in the circulation even without supplemental DHEA. ### 3. Receptors mediators of sex steroid hormone signaling mechanisms of action in neuroprotection The sex steroids can protect through the activation of transcriptional activity in the genomic mechanism or via signaling of neurons survival pathways [69–71] or via non-genomic mechanism through membrane receptors. More and more studies/trials are presenting new insights of sex steroids involvement in hypothalamic, hippocampal, and other brain neurons, their actions being partially common to other organs/tissues effects, but with important peculiarities. The well-known mediation via intracellular receptor/transcription factors that interact with steroid response elements on target genes, regarding the genomic mechanism, is doubled or tripled in the speed of alterations of the neuronal activity within seconds, indicating that some cellular effects can occur via membrane delimited events. Sex steroid hormone ligands bind to membrane-associated G protein-coupled receptor (GPR 30) [72], and caveolin proteins have an essential role for membrane receptors [73]. In addition, estrogens can affect metabotropic glutamate receptors, and the second messenger systems, including calcium mobilization, and a plethora of kinases to alter cell signaling. This subchapter considers the current knowledge of rapid membraneinitiated and intracellular signaling by steroids in the brain, the nature of receptors involved, and how they contribute to homeostatic functions. #### 3.1. Estrogen receptors (ERs). Genetic polymorphism and epigenetics of ERs (iA1-42) in the hippocampus and the entorhinal cortex neurons, preceding amyloid plaque formation, and further induction of neuronal death [65]. Proteomic analyses are demonstrating increased levels of Hsp70 in testosterone- and estrogen-treated human neurons [15], which is a Cell cultures are bringing strong evidences that both androgens and estrogens are neuroprotective, and many studies analyzed the different pathways for neural cells protection from Aβ toxicity. Testosterone is involved in regulation of spine synapse density in the CA1 region of hippocampus A special analysis is to be made on DHEA(S)—the "youth" hormone—for which human body does not have receptors, but it is a source of intracrinology, with different enzymes for steroidforming and/or for steroid-inactivating, permitting each cell/tissue to synthesize a small amount of androgens and/or estrogens in order to meet the local physiological needs without affecting the other tissues of the organism [67]. Blood concentrations are not different from those observed in normal postmenopausal women having high serum DHEA concentrations, when DHES is supplemented to maintain serum estrogen level at sub-threshold or biologically On the other hand, all androgens are made intracellularly from DHEA by the mechanisms of intracrinology, and are always maintained at very low levels in the blood in pre- and postmenopausal women [67]. According to this conceptus, it was proposed a short-term DHEA supplementation (5 mg/day 7 days) in perimenopausal female rhesus macaques [68]. The comparison of serum and hippocampal levels in treated and controls of the same age revealed that despite lower concentrations of the estrogens in the serum of elder animals, their concentrations in the hippocampus did not show any obvious differences due to age or to DHEA supplementation. The results suggest that de novo estrogen synthesis in the brain may compensate for the perimen- The sex steroids can protect through the activation of transcriptional activity in the genomic mechanism or via signaling of neurons survival pathways [69–71] or via non-genomic mecha- More and more studies/trials are presenting new insights of sex steroids involvement in hypothalamic, hippocampal, and other brain neurons, their actions being partially common to other organs/tissues effects, but with important peculiarities. The well-known mediation via intracellular receptor/transcription factors that interact with steroid response elements on target genes, regarding the genomic mechanism, is doubled or tripled in the speed of alterations of the neuronal activity within seconds, indicating that some cellular effects can occur via membrane delimited events. Sex steroid hormone ligands bind to membrane-associated G protein-coupled receptor (GPR 30) [72], and caveolin proteins have an essential role for opausal loss of estrogens in the circulation even without supplemental DHEA. 3. Receptors mediators of sex steroid hormone signaling mechanisms of action in neuroprotection nism through membrane receptors. sign of Aβ toxicity inhibition. 282 Sex Hormones in Neurodegenerative Processes and Diseases inactive concentrations. [66]. The protective role of estrogens in the brain is sure, and the missing preventive effects revealed by RCT is connected to the age-related changes of ERs, as it is in the endometrium/uterus [74], suggesting that several key players in the local synaptic response to E2 are compromised in aging females. The brain has one of the most complex and complicated ERs network of the body, which is changing life-long. In addition to its well-documented hormonal action, E2 is considered as a neurotransmitter in the brain [75]. In the last 10 years, molecular and biochemical animal studies are demonstrating that the mechanisms used by estrogens are greatly influenced by brain cell type, ER type, and metabotropic glutamate receptors (mGluRs) independent of glutamate, and/or region of the brain-cortex and/or hippocampus, all these leading to differential regulation of neuronal circuitry in each area [45, 76]. The hippocampus cognitive performance is directly connected to ER-α, other ERs such as ER-β and GPR30 [8]. The ERs have similar distribution in female and male brains, but may differ in relative expression [77]. ER-α and ER-β expression patterns generally overlap, where ER-α is associated with reproductive behavior, whereas ER-β is associated with non-reproductive behaviors such as learning and memory [78] and anxiety-related behaviors. In hippocampus and cortical neurons, the estrogens—mainly E2 and other estrogenic ligands bind to membrane—associated and mitochondrial-associated G protein-coupled receptor (GPR 30), and activates the classical/ canonical nuclear and extranuclear or intra-cytoplasmatic ER isoforms—α and β—functioning as transcription factors [79–81], and a new type of nuclear ER, the orphan estrogenrelated receptor γ (ERR γ), which regulates dopaminergic neuronal phenotype [82], and IGF-1 receptor, which was recently recognized as a receptor for estrogens. The nongenomic or alternative signaling pathways mechanism involving extranuclear ERs respond to physiological concentration of estrogens to elicit neuroprotection, resulting in the "fine tuning" of neuronal circuitry [45]. Often, rapid activation of intracellular signalers such as mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) underlie alternative estrogen-induced neuroprotection upon activation of specific binding sites at the plasma membrane. The plasma membrane ER (mER) originates from, or is related to canonical nuclear ERs, and GPR30 mimics short latency E2 facilitation of synaptic transmission in the hippocampus, to enhance memory and cognition [83]. The activation of GPR30 by G-1 (its specific ligand) is associated with a mobilization of calcium in dissociated and cultured rat hypothalamic neurons [80, 84, 85]. There were elaborated cellular models of Aβ toxicity where classical and alternative pathways activated by estrogens seem to coexist to orchestrate neuroprotection, fact that is a unique signaling profile of estrogen neuroprotection, dependent upon activation of the MAPK signaling [86]. ER-α and ER-β mediate the effects of E2 on both intracellular signaling and gene transcription, sharing similar domain organization, and using almost identical DNA-binding elements, coregulators, and transcription machinery. There are differences between ER-labeled regarding each female, species (rats, non-human primates, human), age (young, old), estrogen levels, brain reference area, vulnerability of spines/synapses based on size (large or small), or presynaptic/postsynaptic location of ER. Both, ER-α and ER-β are located predominantly at extranuclear sites; ER-α are found in dendritic spines, many originating from pyramidal cells, axons, terminals, astrocytes, and microglia [81], in symmetric and asymmetric synapses. ER-β is detected on or near the plasma membrane of somata and dendritic shafts and spines in hippocampal neurons [81], in axons and axon terminals and both in the cytoplasm and on endomembranes near mitochondria in vivo [81], and within mitochondria in vitro, in pyramidal cells, in newly generated cells in a few interneurons and in glia [81]. Changes in ER-β expression occur in the presynaptic membrane, cleft, and postsynaptic membranes, where neurotransmitter release and postsynaptic receptor binding occurs. Conversely, ER-α changes are detected presynaptically in synaptic vesicles and postsynaptically in plasmalemmal and cytoplasmic regions of spine heads where protein translation occurs. In aged animals, it was demonstrated for the first time [78] that the window for E2-mediated benefits on cognition and hippocampal E2 responsiveness can be reinstated by increased expression of ER-α. estrogen receptor-α in conditions of rats' global ischemia, or of aging hippocampal CA1 region [9]. Natural aging is associated with increased ER-α and ER-β CHIP binding and ubiquitination, and with decreased ER-α and ER-β levels in the hippocampal CA1 region, fact that is different from the aging uterus in the rat model of long-term E2 deprivation (LTED) or after ovariectomy, where the level of ER via the ubiquitin-proteasome degradation pathway is increasing after New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection http://dx.doi.org/10.5772/intechopen.74332 285 The studies on rats and monkeys [39] from Mount Sinai School of Medicine (New York, USA) have emerged three key findings: (1) synaptic ER-α is present in axospinous synapses in monkeys dorsolateral prefrontal cortex, in area 46; (2) it is stable across treatment and age groups (which is not the case in rat hippocampus); and (3) the abundance and distribution of synaptic ER-α is a key correlate of individual variation in cognitive performance in certain age Another interesting and important findings about rats cortex are that ER-α can modulate synaptic function and behavior even in the absence of circulating gonadal E2, in response to E2 synthesized within neurons [94], and that ER-α may initiates the non-genomic signaling mechanisms modulating synaptic plasticity in the hippocampus in response to either circulating or locally synthesized E2 [39, 93]. These findings are considered of great importance for the The detected levels of ERs in postmortem brain tissue of AD patients is related to the severity of cognitive impairment assessed proximate to death, and only the reduction of ER-α from frontal cortex is correlated to Mini-Mental State Examination score, not the ER-β [95]. The spectrometry, immunohistochemistry, and quantitative real-time PCR of the autopsied Japanese AD patients compared to controls [96] have revealed a glial nuclear ER-β expression significantly lower in white matter in the AD group vs. controls, without any significant differences in estrogen concentrations, and the conclusion was that estrogens have effects on glias and neurons in the etiology of AD, and the correlation between BMI and estrogen concentrations in the frontal lobe suggests the importance of non-brain sources of estrogens Long-term E2 treatment initiated 14 days prior to global ischemia in ovariectomized female rats demonstrated that E2 at near physiological concentrations acts via the IGF-1 receptor to protect the functional integrity of hippocampal CA1 neurons and synapses in conditions of global ischemia, but not the classical ER-α or β. [97]. The transactivation of IGF-1 receptors and stimulation of ERK/MAPK signaling pathway maintains CREB activity in the ischemic CA1. All three types of ERs cooperate in neuroprotection against AD [98], in association to intracellular calcium signaling cascade, which is very important, and the ER-α is the central key, for maintaining channel inactivation and may be relevant in neuronal preservation against Aβ injury. It was demonstrated that combination of ERm and caveolin in caveolae, and ER-αmediated inhibition of Death domain-associated protein translocation may protect neurons against Aβ injury. ER-α and IGF-IR co-activation may mediate neuroprotection, and many other growth factors and intracellular signaling responses triggered by ER-α may play important roles in the process [98].These data are crucial for contemporary societies, with high risks for diabetes mellitus, in all populations. A very recent study from the University of Missouri, design of HT strategies for both surgically and naturally menopausal women. estrogen exposure [9]. and treatment groups. particularly in controls. Studies have determined that membrane-localized ER-α and ER-β are capable of activating multiple metabotropic glutamate receptors (mGluRs) independent of glutamate, leading to downstream second messenger signaling [73, 76]. The expression of ER-α and ER-β mRNA in the hippocampus is limited [87], and GPR30 may be the major receptor subtype by which estrogen produces its enhancing effects. The physiological consequence of activation of GPR30 can regulate local synthesis pathways in a novel direction of our understanding of rapid estrogen signaling within the brain and its ability to induce the "fine-tuning" of neuronal circuits [45]. E2 works as a neuroprotector by membrane receptors coupled to E2 induction of intracellular Ca(2+) influx via the L-type channels, connected to memory mechanisms, and through Src/ERK/cyclic AMP response element-binding protein activation in single hippocampal neurons [88]. The presence of the L-type Ca (2+) channel inhibitor, nifedipine (10 microM), partially inhibits 17 β E2 [89]. It was discovered an aging decrease of about 50% of ER-α-labeled synapses [37, 45], with alteration in the ratio of ER-β to ER-α, fact that contributes to age-related decreases in the capacity to form additional spines and synapses in response to E2 in rats. In addition, synaptic pAkt thought is activated by ER-α [90], which is also decreased dramatically in aged CA1 axospinous synapses [91], as is ER-β [92], suggesting that several key players in the local synaptic response to E2 are compromised with age in female rats. It was reported for the first time in Mont Sinai University (New-York, USA) [93] that in the monkey's neocortex 46, approximately 50% of the axospinous synapses contains ER-α, with axon terminals more likely to have ER-α than spines, and that presynaptic ER-α was often associated with vesicles, whereas postsynaptic ER-α was widely distributed in the PSD, adjacent to the PSD, and in the spine core. The duration of brain's estrogen deprivation is connected to C terminus of heat shock cognate protein (Hsc) 70-interacting protein (CHIP)-mediated proteasomal degradation of hippocampal estrogen receptor-α in conditions of rats' global ischemia, or of aging hippocampal CA1 region [9]. Natural aging is associated with increased ER-α and ER-β CHIP binding and ubiquitination, and with decreased ER-α and ER-β levels in the hippocampal CA1 region, fact that is different from the aging uterus in the rat model of long-term E2 deprivation (LTED) or after ovariectomy, where the level of ER via the ubiquitin-proteasome degradation pathway is increasing after estrogen exposure [9]. ER-α and ER-β mediate the effects of E2 on both intracellular signaling and gene transcription, sharing similar domain organization, and using almost identical DNA-binding elements, coregulators, and transcription machinery. There are differences between ER-labeled regarding each female, species (rats, non-human primates, human), age (young, old), estrogen levels, brain reference area, vulnerability of spines/synapses based on size (large or small), or presynaptic/postsynaptic location of ER. Both, ER-α and ER-β are located predominantly at extranuclear sites; ER-α are found in dendritic spines, many originating from pyramidal cells, axons, terminals, astrocytes, and microglia [81], in symmetric and asymmetric synapses. ER-β is detected on or near the plasma membrane of somata and dendritic shafts and spines in hippocampal neurons [81], in axons and axon terminals and both in the cytoplasm and on endomembranes near mitochondria in vivo [81], and within mitochondria in vitro, in pyramidal cells, in newly generated cells in a few interneurons and in glia [81]. Changes in ER-β expression occur in the presynaptic membrane, cleft, and postsynaptic membranes, where neurotransmitter release and postsynaptic receptor binding occurs. Conversely, ER-α changes are detected presynaptically in synaptic vesicles and postsynaptically in plasmalemmal and cytoplasmic regions of spine heads where protein translation occurs. In aged animals, it was demonstrated for the first time [78] that the window for E2-mediated benefits on cognition and hippocampal E2 responsiveness can be reinstated by increased expression of ER-α. Studies have determined that membrane-localized ER-α and ER-β are capable of activating multiple metabotropic glutamate receptors (mGluRs) independent of glutamate, leading to downstream second messenger signaling [73, 76]. The expression of ER-α and ER-β mRNA in the hippocampus is limited [87], and GPR30 may be the major receptor subtype by which estrogen produces its enhancing effects. The physiological consequence of activation of GPR30 can regulate local synthesis pathways in a novel direction of our understanding of rapid estrogen signaling within the brain and its ability to induce the "fine-tuning" of neuronal circuits [45]. E2 works as a neuroprotector by membrane receptors coupled to E2 induction of intracellular Ca(2+) influx via the L-type channels, connected to memory mechanisms, and through Src/ERK/cyclic AMP response element-binding protein activation in single hippocampal neurons [88]. The presence of the L-type Ca (2+) channel inhibitor, nifedipine (10 microM), It was discovered an aging decrease of about 50% of ER-α-labeled synapses [37, 45], with alteration in the ratio of ER-β to ER-α, fact that contributes to age-related decreases in the capacity to form additional spines and synapses in response to E2 in rats. In addition, synaptic pAkt thought is activated by ER-α [90], which is also decreased dramatically in aged CA1 axospinous synapses [91], as is ER-β [92], suggesting that several key players in It was reported for the first time in Mont Sinai University (New-York, USA) [93] that in the monkey's neocortex 46, approximately 50% of the axospinous synapses contains ER-α, with axon terminals more likely to have ER-α than spines, and that presynaptic ER-α was often associated with vesicles, whereas postsynaptic ER-α was widely distributed in the PSD, adja- The duration of brain's estrogen deprivation is connected to C terminus of heat shock cognate protein (Hsc) 70-interacting protein (CHIP)-mediated proteasomal degradation of hippocampal the local synaptic response to E2 are compromised with age in female rats. partially inhibits 17 β E2 [89]. 284 Sex Hormones in Neurodegenerative Processes and Diseases cent to the PSD, and in the spine core. The studies on rats and monkeys [39] from Mount Sinai School of Medicine (New York, USA) have emerged three key findings: (1) synaptic ER-α is present in axospinous synapses in monkeys dorsolateral prefrontal cortex, in area 46; (2) it is stable across treatment and age groups (which is not the case in rat hippocampus); and (3) the abundance and distribution of synaptic ER-α is a key correlate of individual variation in cognitive performance in certain age and treatment groups. Another interesting and important findings about rats cortex are that ER-α can modulate synaptic function and behavior even in the absence of circulating gonadal E2, in response to E2 synthesized within neurons [94], and that ER-α may initiates the non-genomic signaling mechanisms modulating synaptic plasticity in the hippocampus in response to either circulating or locally synthesized E2 [39, 93]. These findings are considered of great importance for the design of HT strategies for both surgically and naturally menopausal women. The detected levels of ERs in postmortem brain tissue of AD patients is related to the severity of cognitive impairment assessed proximate to death, and only the reduction of ER-α from frontal cortex is correlated to Mini-Mental State Examination score, not the ER-β [95]. The spectrometry, immunohistochemistry, and quantitative real-time PCR of the autopsied Japanese AD patients compared to controls [96] have revealed a glial nuclear ER-β expression significantly lower in white matter in the AD group vs. controls, without any significant differences in estrogen concentrations, and the conclusion was that estrogens have effects on glias and neurons in the etiology of AD, and the correlation between BMI and estrogen concentrations in the frontal lobe suggests the importance of non-brain sources of estrogens particularly in controls. Long-term E2 treatment initiated 14 days prior to global ischemia in ovariectomized female rats demonstrated that E2 at near physiological concentrations acts via the IGF-1 receptor to protect the functional integrity of hippocampal CA1 neurons and synapses in conditions of global ischemia, but not the classical ER-α or β. [97]. The transactivation of IGF-1 receptors and stimulation of ERK/MAPK signaling pathway maintains CREB activity in the ischemic CA1. All three types of ERs cooperate in neuroprotection against AD [98], in association to intracellular calcium signaling cascade, which is very important, and the ER-α is the central key, for maintaining channel inactivation and may be relevant in neuronal preservation against Aβ injury. It was demonstrated that combination of ERm and caveolin in caveolae, and ER-αmediated inhibition of Death domain-associated protein translocation may protect neurons against Aβ injury. ER-α and IGF-IR co-activation may mediate neuroprotection, and many other growth factors and intracellular signaling responses triggered by ER-α may play important roles in the process [98].These data are crucial for contemporary societies, with high risks for diabetes mellitus, in all populations. A very recent study from the University of Missouri, St. Louis (USA) [99] demonstrated the neuroprotection of the coupling of IGF1 to estrogens and/androgens. It is considered that both steroids are involved in many neuroprotective processes that operate on similar signaling cascades [100]. are broadly expressed throughout the brain, inclusive the hippocampus, and can be detected in every neural cell type [113]. There are known the classic nuclear PR-A (the N-terminally truncated form of PR-B) and PR-B receptors, and splice variants of each, explaining the P4 genomic mechanism of action through specific progesterone response elements (PRE) within the promoter region of target genes to regulate transcription of the genes [114], and the two types of cell surface-associated proteins [membrane PRs (mPRs) and the progesterone membrane receptor component (PGMRC)].These PRs induce classic regulation of gene expression while also transducing signaling cascades that originate at the cell membrane and ultimately activate transcription factors. As for estrogens the genomic and non-genomic mechanisms of P4 are coupled, so the distinctions are not as clear-cut as was first thought [115]. The nuclear PRs are up-regulated by E2 in glial and neural cells, but more in the glial cells, implicating New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection http://dx.doi.org/10.5772/intechopen.74332 287 Figure 1. Progesterone nuclear receptor (pgr) is upregulated by estrogen. Experiments on developing and adult brain of zebrafish, and larvae. Legend: Fold induction of P4 expression after treatment of adult zebrafish with an aromatase inhibitor (A, 10<sup>6</sup> M of ATD) or estradiol (B, 10<sup>7</sup> M of 17β-estradiol), and larvae with E2 (C, 10<sup>8</sup> M of 17β-estradiol). The graphs present the mean value +/ the standard deviation. Asterisk (\) indicates significant differences (p < 0.05, Student's t test). Panel A: the aromatase inhibitor (ATD) leads to a significant decrease of pgr expression in individual brains of adult zebrafish (n = 4). Panel B, the estrogenic treatment leads to a significant increase of pgr expression in pools of 5 brains of adult zebrafish (n = 3). Panel C: the estrogenic treatment leads to a significant increase of pgr expression in pool of 20 larvae (n = 2). crucial progenitor cells, as preferential targets of P4 [116] (Figures 1 and 2). Another tested hypothesis is upon GPR30 that act together with intracellular estrogen receptors to activate cell signaling pathways to promote hippocampal neuron survival after global ischemia [101]. E2 at physiological concentrations intervenes in apoptotic death cascades and ameliorates neuronal death, increasing BCL-2 expression in rat hippocampal neurons [88] (in experimental models of focal and global ischemia), but the proper mechanism is still unclear. Regarding ERs, there are new details about the genes, and mRNA variants of ER-α expressed in different parts of the human brain, and there are specific ER-α mRNA splice variants or isoforms of ER-α in the medial mammillary nucleus (MMN) in AD [102] or in the frontal cortex in AD patients [95], and their relationship to cognitive impairment. There are some therapeutic indications for cognition and memory support, which are partially controversial. The first is regarding the benefits from the upregulation of the expression of ER-α, but not of ER-β, in the hippocampus of aged animals, in order to restore the potential of E2 treatments and rejuvenate E2-induced hippocampal plasticity [78]. The second are from the results of a multinational study [103] sustaining the beneficial effects of long-term treatment with diarylpropionitrile (DPN, 0.05 mg/kg/day, sc.), a selective ER-β agonist, on the hippocampal transcriptome in ovariectomized, middle-aged (13 months) rats. The results reveal the contribution of ER-β-mediated processes to the regulation of transcription, translation, neurogenesis, neuromodulation, and neuroprotection in the hippocampal formation, and the authors concluded that the findings are supporting the notion that selective activation of ER-β may be a viable approach for treating the neural symptoms of E2 deficiency in menopause. There are studies suggesting that DPN—a selective ER-β agonist—mimics many basic effects of E2 in the hippocampus and enhance mice's hippocampus-dependent spatial memory [104, 105]. Recent studies are involving the genetic polymorphism of ERs, especially of ER-β in cognitive impairment and increased risk for AD predominantly in women [106]. It was examined that the role of single nucleotide polymorphisms (SNP) in the ERs genes: rs9340799, rs2234693, rs2228480 (in the ESR1 gene), and rs4986938 (in the ESR2 gene) as a risk factor for amnesic mild cognitive impairment (MCI) and AD. The less represented alleles of SNPs studied are associated with MCI and AD in women APOEε4 allele carriers [107, 108]. Some studies are focusing the association of Eε4 allele of apolipoprotein E gene to obesity, inflammation, and the risk of AD. Although, the pathways underlying this relationship are unclear the sex steroid hormones may be the connection [109, 110]. The epigenetic processes are associated to brain aging. The post-translational histone changes and DNA methylation are modulating the hippocampal memory-enhancing effects of E2 [111, 112]. #### 3.2. Progesterone receptors P4 has neuroprotective effects mediated by various mechanisms P4 or its metabolitesregulated neural responses are mediated by an array of progesterone receptors (PRs) which are broadly expressed throughout the brain, inclusive the hippocampus, and can be detected in every neural cell type [113]. There are known the classic nuclear PR-A (the N-terminally truncated form of PR-B) and PR-B receptors, and splice variants of each, explaining the P4 genomic mechanism of action through specific progesterone response elements (PRE) within the promoter region of target genes to regulate transcription of the genes [114], and the two types of cell surface-associated proteins [membrane PRs (mPRs) and the progesterone membrane receptor component (PGMRC)].These PRs induce classic regulation of gene expression while also transducing signaling cascades that originate at the cell membrane and ultimately activate transcription factors. As for estrogens the genomic and non-genomic mechanisms of P4 are coupled, so the distinctions are not as clear-cut as was first thought [115]. The nuclear PRs are up-regulated by E2 in glial and neural cells, but more in the glial cells, implicating crucial progenitor cells, as preferential targets of P4 [116] (Figures 1 and 2). St. Louis (USA) [99] demonstrated the neuroprotection of the coupling of IGF1 to estrogens and/androgens. It is considered that both steroids are involved in many neuroprotective Another tested hypothesis is upon GPR30 that act together with intracellular estrogen receptors to activate cell signaling pathways to promote hippocampal neuron survival after global ischemia [101]. E2 at physiological concentrations intervenes in apoptotic death cascades and ameliorates neuronal death, increasing BCL-2 expression in rat hippocampal neurons [88] (in experimental models of focal and global ischemia), but the proper mechanism is still unclear. Regarding ERs, there are new details about the genes, and mRNA variants of ER-α expressed in different parts of the human brain, and there are specific ER-α mRNA splice variants or isoforms of ER-α in the medial mammillary nucleus (MMN) in AD [102] or in the frontal cortex There are some therapeutic indications for cognition and memory support, which are partially controversial. The first is regarding the benefits from the upregulation of the expression of ER-α, but not of ER-β, in the hippocampus of aged animals, in order to restore the potential of E2 treatments and rejuvenate E2-induced hippocampal plasticity [78]. The second are from the results of a multinational study [103] sustaining the beneficial effects of long-term treatment with diarylpropionitrile (DPN, 0.05 mg/kg/day, sc.), a selective ER-β agonist, on the hippocampal transcriptome in ovariectomized, middle-aged (13 months) rats. The results reveal the contribution of ER-β-mediated processes to the regulation of transcription, translation, neurogenesis, neuromodulation, and neuroprotection in the hippocampal formation, and the authors concluded that the findings are supporting the notion that selective activation of ER-β may be a viable approach for treating the neural symptoms of E2 deficiency in menopause. There are studies suggesting that DPN—a selective ER-β agonist—mimics many basic effects of E2 in the hippocampus and enhance mice's hippocampus-dependent spatial Recent studies are involving the genetic polymorphism of ERs, especially of ER-β in cognitive impairment and increased risk for AD predominantly in women [106]. It was examined that the role of single nucleotide polymorphisms (SNP) in the ERs genes: rs9340799, rs2234693, rs2228480 (in the ESR1 gene), and rs4986938 (in the ESR2 gene) as a risk factor for amnesic mild cognitive impairment (MCI) and AD. The less represented alleles of SNPs studied are associated with MCI and AD in women APOEε4 allele carriers [107, 108]. Some studies are focusing the association of Eε4 allele of apolipoprotein E gene to obesity, inflammation, and the risk of AD. Although, the pathways underlying this relationship are unclear the sex steroid The epigenetic processes are associated to brain aging. The post-translational histone changes and DNA methylation are modulating the hippocampal memory-enhancing effects of E2 [111, 112]. P4 has neuroprotective effects mediated by various mechanisms P4 or its metabolitesregulated neural responses are mediated by an array of progesterone receptors (PRs) which processes that operate on similar signaling cascades [100]. 286 Sex Hormones in Neurodegenerative Processes and Diseases in AD patients [95], and their relationship to cognitive impairment. memory [104, 105]. hormones may be the connection [109, 110]. 3.2. Progesterone receptors Figure 1. Progesterone nuclear receptor (pgr) is upregulated by estrogen. Experiments on developing and adult brain of zebrafish, and larvae. Legend: Fold induction of P4 expression after treatment of adult zebrafish with an aromatase inhibitor (A, 10<sup>6</sup> M of ATD) or estradiol (B, 10<sup>7</sup> M of 17β-estradiol), and larvae with E2 (C, 10<sup>8</sup> M of 17β-estradiol). The graphs present the mean value +/ the standard deviation. Asterisk (\) indicates significant differences (p < 0.05, Student's t test). Panel A: the aromatase inhibitor (ATD) leads to a significant decrease of pgr expression in individual brains of adult zebrafish (n = 4). Panel B, the estrogenic treatment leads to a significant increase of pgr expression in pools of 5 brains of adult zebrafish (n = 3). Panel C: the estrogenic treatment leads to a significant increase of pgr expression in pool of 20 larvae (n = 2). outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling [122]. Progesterone receptor membrane component-1 (PGRMC-1) and PGRMC-2, with a singletransmembrane domain protein, are mediating the rapid non-genomic effects of E2 and P4, such as the activation of MAPK signaling and intracellular Ca2+ increase [123, 124] mPRβ activates also MAPK cascade, without GPCR signaling, and progesterone-stimulated mPRβ activation did not exhibit the elevation of [Ca2+] [121]. In comparison to the mPRs, the single-transmembrane protein Pgrmc1 (molecular mass 25–28 kDa) and the related Pgrmc2 are a part of a multi-protein complex that binds to P4, other steroids, and to pharmaceutical compounds [123]. Besides the location to membrane surface, Pgrmc1 was reported to have subcellular localization: in endo- New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection http://dx.doi.org/10.5772/intechopen.74332 289 PRs are differentially expressed in neurons, in oligodendrocytes, astrocytes, and reactive microglia, the mPRα expression is observed in oligodendrocytes, astrocytes, and reactive microglia. The increase in mPR expression was proposed to mediate the anti-inflammatory The classical PR and mPRs have overlapping regional expression (e.g., both are expressed in the hippocampus, cortex, hypothalamus, and cerebellum), but their profile of ligand specificity is not identical [126]. The "non-classical/non-genomic" effects of P4 can be initiated rapidly at the cell surface to activate intracellular signaling pathways, through modulation of putative cell surface receptors, ion channels, and cytoplasmic second messenger cascades, the rapid activation of cytoplasmic kinase signaling can result in both transcription-independent and Among the rapid non-genomic signaling pathways activated by P4 are the extracellular signalrelated kinase (ERK) pathways [127], cAMP/protein kinase A (PKA) signaling [128], PKG signaling [129], Ca2+ influx/PKC activation [130], phosphatidylinositol 3-kinases (PI3 K)/Akt pathway [124], and other signal transduction cascades. P4 or its metabolites can act directly and rapidly on neurotransmitter receptors as the GABA-A receptor [131] and Sigma-1/2 recep- The consequences of activation of these signaling pathways are numerous and include influences on neurotrophin release [125], neural progenitor proliferation, regulation of intracellular Ca2+ levels, and regulation of cell viability [57, 127, 131] all of which can contribute to the Androgen neuroprotective effects are mediated both directly by activation of androgen receptors (ARs) pathways, and indirectly by aromatization to estradiol and initiation of protective estrogen signaling mechanisms, but this last action is not totally accepted [133–135]. The Testosterone protects primary human neurons against serum deprivation [134], cultured rat hippocampal neurons against extracellular Aβ toxicity [136], rat neurons against heat shock-mediated knowledge on the effects of testosterone on women cognitive capacities are few. plasmic reticulum, Golgi apparatus, and nuclei [125]. effects of progesterone under conditions of injury [126]. transcription-dependent effects. tors [132] to regulate cellular function. overall health and function of the brain. 3.3. Androgens mechanisms in neuroprotection Figure 2. Experiments on Zebrafish hypothalamicP4 receptors. Legend: Analysis of the P4 staining intensity in the ventral subpallium and in the anterior hypothalamus revealing a stronger intensity (mean value +/ the standard deviation).in the radial cells than neural cells (p < 0.001 Student's t test). Figures 1 and 2 are adapted from [116]. PR-A is exerting a negative control on PR-B-mediated transcription, and the mediated transcription of the ER and glucocorticoid receptors [117], fact that may underlie, at least in part, the mechanism by which progesterone functionally antagonize the effects of estrogen. PRA and PRB can interact as dimers with DNA progesterone responsive element, and with signaling proteins of the Src/Ras/Erk pathway outside the nucleus [118]. The "non-genomic" mechanisms explains the non-reproductive P4 actions, the rapid activation of cytoplasmic kinase signaling that can result in both transcription-independent and transcription-dependent effects. These "nongenomic" actions can be partially explained by membrane transport via nuclear receptor [119]. The mPRs (molecular mass of approximately 40 kDa) had thought to be comprised of three subtypes, mPR α, β, and γ, which belong to the seven-transmembrane domain adiponectin Q receptor (PAQR) family, plus two newly discovered subtypes (mPRδ, and mPRε) [120]. It was shown that cDNAs for the mPRα subtypes of spotted seatrout (st-mPRalpha) and humans (humPRα-) encode progesterone/progestin receptors that display many functional characteristics of G protein-coupled receptors [121], and that mPRβ promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling [122]. Progesterone receptor membrane component-1 (PGRMC-1) and PGRMC-2, with a singletransmembrane domain protein, are mediating the rapid non-genomic effects of E2 and P4, such as the activation of MAPK signaling and intracellular Ca2+ increase [123, 124] mPRβ activates also MAPK cascade, without GPCR signaling, and progesterone-stimulated mPRβ activation did not exhibit the elevation of [Ca2+] [121]. In comparison to the mPRs, the single-transmembrane protein Pgrmc1 (molecular mass 25–28 kDa) and the related Pgrmc2 are a part of a multi-protein complex that binds to P4, other steroids, and to pharmaceutical compounds [123]. Besides the location to membrane surface, Pgrmc1 was reported to have subcellular localization: in endoplasmic reticulum, Golgi apparatus, and nuclei [125]. PRs are differentially expressed in neurons, in oligodendrocytes, astrocytes, and reactive microglia, the mPRα expression is observed in oligodendrocytes, astrocytes, and reactive microglia. The increase in mPR expression was proposed to mediate the anti-inflammatory effects of progesterone under conditions of injury [126]. The classical PR and mPRs have overlapping regional expression (e.g., both are expressed in the hippocampus, cortex, hypothalamus, and cerebellum), but their profile of ligand specificity is not identical [126]. The "non-classical/non-genomic" effects of P4 can be initiated rapidly at the cell surface to activate intracellular signaling pathways, through modulation of putative cell surface receptors, ion channels, and cytoplasmic second messenger cascades, the rapid activation of cytoplasmic kinase signaling can result in both transcription-independent and transcription-dependent effects. Among the rapid non-genomic signaling pathways activated by P4 are the extracellular signalrelated kinase (ERK) pathways [127], cAMP/protein kinase A (PKA) signaling [128], PKG signaling [129], Ca2+ influx/PKC activation [130], phosphatidylinositol 3-kinases (PI3 K)/Akt pathway [124], and other signal transduction cascades. P4 or its metabolites can act directly and rapidly on neurotransmitter receptors as the GABA-A receptor [131] and Sigma-1/2 receptors [132] to regulate cellular function. The consequences of activation of these signaling pathways are numerous and include influences on neurotrophin release [125], neural progenitor proliferation, regulation of intracellular Ca2+ levels, and regulation of cell viability [57, 127, 131] all of which can contribute to the overall health and function of the brain. #### 3.3. Androgens mechanisms in neuroprotection PR-A is exerting a negative control on PR-B-mediated transcription, and the mediated transcription of the ER and glucocorticoid receptors [117], fact that may underlie, at least in part, the mechanism by which progesterone functionally antagonize the effects of estrogen. PRA and PRB can interact as dimers with DNA progesterone responsive element, and with signaling proteins of the Src/Ras/Erk pathway outside the nucleus [118]. The "non-genomic" mechanisms explains the non-reproductive P4 actions, the rapid activation of cytoplasmic kinase signaling that can result in both transcription-independent and transcription-dependent effects. These "nongenomic" actions can be partially explained by membrane transport via nuclear receptor [119]. The mPRs (molecular mass of approximately 40 kDa) had thought to be comprised of three subtypes, mPR α, β, and γ, which belong to the seven-transmembrane domain adiponectin Q receptor (PAQR) family, plus two newly discovered subtypes (mPRδ, and mPRε) [120]. It was shown that cDNAs for the mPRα subtypes of spotted seatrout (st-mPRalpha) and humans (humPRα-) encode progesterone/progestin receptors that display many functional characteristics of G protein-coupled receptors [121], and that mPRβ promotes progesterone-dependent neurite Figure 2. Experiments on Zebrafish hypothalamicP4 receptors. Legend: Analysis of the P4 staining intensity in the ventral subpallium and in the anterior hypothalamus revealing a stronger intensity (mean value +/ the standard deviation).in the radial cells than neural cells (p < 0.001 Student's t test). Figures 1 and 2 are adapted from [116]. 288 Sex Hormones in Neurodegenerative Processes and Diseases Androgen neuroprotective effects are mediated both directly by activation of androgen receptors (ARs) pathways, and indirectly by aromatization to estradiol and initiation of protective estrogen signaling mechanisms, but this last action is not totally accepted [133–135]. The knowledge on the effects of testosterone on women cognitive capacities are few. Testosterone protects primary human neurons against serum deprivation [134], cultured rat hippocampal neurons against extracellular Aβ toxicity [136], rat neurons against heat shock-mediated hyperphosphorylation of tau by modulating glycogen synthase kinase 3 activation [85], cerebellar granule neurons against oxidative stress [133], and rat hippocampal neurons against kainic acidinduced toxicity [135] transiently activate mitogen-activated protein kinase (MAPK) in cultured hippocampal neurons, as evidenced by phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 and by this effect subsequently drives neuroprotection [137]. [4] Brinton RD. Investigative models for determining hormone therapy-induced outcomes in brain: Evidence in support of a healthy cell bias of estrogen action. Annals of the New New Insights for Hormone Therapy in Perimenopausal Women Neuroprotection http://dx.doi.org/10.5772/intechopen.74332 291 [5] Chen S, Nilsen J, Brinton RD. Dose and temporal pattern of estrogen exposure determines neuroprotective outcome in hippocampal neurons: Therapeutic implications. [6] Barha CK, Galea LA. Influence of different estrogens on neuroplasticity and cognition in the hippocampus. Biochimica et Biophysica Acta. 2010;1800(10):1056-1067. DOI: [7] Morrison JH, Brinton RD, Schmidt PJ, Gore AC. Estrogen, menopause, and the aging brain: How basic neuroscience can inform hormone therapy in women. The Journal of [8] Brinton RD. Estrogen regulation of glucose metabolism and mitochondrial function: Therapeutic implications for prevention of Alzheimer's disease. Advanced Drug Deliv- [9] Zhang QG, Han D, Wang R, Dong Y, Yanf F, Vadlamudi KR, Brann WD. C terminus of Hsc70-interacting protein (CHIP)-mediated degradation of hippocampal estrogen receptor-α and the critical period hypothesis of estrogen neuroprotection. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:E617-E624 [10] Henderson VW. Cognition and cognitive aging. Climacteric. 2007;10(Suppl 2):88-91 [11] Scott E, Zhang QG, Brann D, et al. Estrogen neuroprotection and the critical period hypothesis. Frontiers in Neuroendocrinology. 2012;33(1):85-104. DOI: 10.1016/j.yfrne.2011.10.001 [12] Sherwin BB. Estrogen therapy: Is time of initiation critical for neuroprotection? Nature [13] Sherwin BB. The critical period hypothesis: Can it explain discrepancies in the oestrogen- [14] Dye VR, Miller JK, Singer JE, Levine JA Hormone replacement therapy and risk for neurodegenerative diseases. International Journal of Alzheimer's Disease 2012; 18 pgs; [15] Frye CA, Koonce CJ, Walf AA. Progesterone, compared to medroxyprogesterone acetate, to C57BL/6, but not 5α-reductase mutant, mice enhances object recognition and placement memory and is associated with higher BDNF levels in the hippocampus and [16] Sherwin BB, Phillips SJ. Estrogen and cognitive functioning in surgically menopausal [17] Nappi ER, Sinforiani E, Nappi G, et al. Memory functioning at menopause: Impact of age in ovariectomized women. Gynecologic and Obstetric Investigation. 1999;47:29-36 cortex. Neuroscience Letters. 2013;551:53-57. DOI: 10.1016/j.neulet.2013.07.002 women. Annals of the New York Academy of Sciences. 1990;592:474-475 cognition literature? Journal of Neuroendocrinology. 2007;19:77-81 Ed: K. S. Jagannatha Rao. Article ID 258454, doi.org/10.1155/2012/258454 ery Reviews. 2008;60(13–14):1504-1511. DOI: 10.1016/j.addr.2008.06.003 Endocrinology. 2006;147(11):5303-5313. DOI: 10.1210/en.2006-049 York Academy of Sciences. 2005;1052:57-74 10.1016/j.bbagen.2010.01.006 S632–S633 Reviews. Endocrinology. 2000;5:620-627 Neuroscience. 2006;26:10332-10348 #### 4. Perspectives Brain aging and neurodegenerative diseases have a multifactorial nature, metabolic and inflammatory changes from the moment of transition to menopause, blood-brain barrier disruption, and aberrant microglial activation can be modulated or prevented in a moment prior to their onset in the "critical period of opportunity," if the clinicians and the patients are both interested and have a good understanding of very early perimenopausal symptoms. The differences between estrogen types, between progesterone and progestins, between the classes of steroid receptors agonists— NeuroSERMs (novel neuro-selective estrogen receptor modulator) and PhytoSERMs (phytoselective estrogen receptor modulator), and the new molecules tested in high-tech laboratories, will help the clinicians to recommend the best neurotrophic, neuroprotective molecule without any breast or uterine harmful action. #### Author details Manuela Cristina Russu<sup>1</sup> \* and Alexandra Cristina Antonescu<sup>2</sup> \Address all correspondence to: manuela\[email protected] 1 "Dr. I. Cantacuzino" Clinic of Obstetrics and Gynecology, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania 2 Intromed Laboratories, Bucharest, Romania #### References [4] Brinton RD. Investigative models for determining hormone therapy-induced outcomes in brain: Evidence in support of a healthy cell bias of estrogen action. Annals of the New York Academy of Sciences. 2005;1052:57-74 hyperphosphorylation of tau by modulating glycogen synthase kinase 3 activation [85], cerebellar granule neurons against oxidative stress [133], and rat hippocampal neurons against kainic acidinduced toxicity [135] transiently activate mitogen-activated protein kinase (MAPK) in cultured hippocampal neurons, as evidenced by phosphorylation of extracellular signal-regulated kinase Brain aging and neurodegenerative diseases have a multifactorial nature, metabolic and inflammatory changes from the moment of transition to menopause, blood-brain barrier disruption, and aberrant microglial activation can be modulated or prevented in a moment prior to their onset in the "critical period of opportunity," if the clinicians and the patients are both interested and have a good understanding of very early perimenopausal symptoms. The differences between estrogen types, between progesterone and progestins, between the classes of steroid receptors agonists— NeuroSERMs (novel neuro-selective estrogen receptor modulator) and PhytoSERMs (phytoselective estrogen receptor modulator), and the new molecules tested in high-tech laboratories, will help the clinicians to recommend the best neurotrophic, neuroprotective molecule without \ and Alexandra Cristina Antonescu<sup>2</sup> 1 "Dr. I. Cantacuzino" Clinic of Obstetrics and Gynecology, "Carol Davila" University of [1] Rettberg JR, Yao J, Brinton RD. Estrogen: A master regulator of bioenergetic systems in the brain and body. Frontiers in Neuroendocrinology. 2014;35(1):8-30. DOI: 10.1016 [2] Maki PM, Resnick SM. Longitudinal effects of estrogen replacement therapy on PET cerebral blood flow and cognition. Neurobiology of Aging. 2000;21(2):373-383 [3] Kawas C, Resnick S, Morrison A, et al. A prospective study of estrogen replacement therapy and the risk of developing Alzheimer's disease: The Baltimore Longitudinal \Address all correspondence to: manuela\[email protected] Study of Aging. Neurology. 1997;48(6):1517-1521 Medicine and Pharmacy, Bucharest, Romania 2 Intromed Laboratories, Bucharest, Romania (ERK)-1 and ERK-2 and by this effect subsequently drives neuroprotection [137]. 4. Perspectives Author details References Manuela Cristina Russu<sup>1</sup> any breast or uterine harmful action. 290 Sex Hormones in Neurodegenerative Processes and Diseases [18] Vearncombe KJ, Pachana NA. Is cognitive functioning detrimentally affected after early, induced menopause? Menopause. 2009;16(1):188-198. DOI: 10.1097 [31] Yan Y, Cheng L, Chen X, Ai J, et al. Estrogen deficiency is associated with hippocampal morphological remodeling of early postmenopausal mice. 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Distribution of membrane progesterone receptor alpha in the male mouse and rat brain and its regulation after [127] Nilsen J, Brinton RD. Impact of progestins on estrogen-induced neuroprotection: Synergy by progesterone and 19-norprogesterone and antagonism by medroxyprogesterone [128] Petralia SM, Frye CA. In the ventral tegmental area, cyclic AMP mediates the actions of progesterone at dopamine type 1 receptors for lordosis of rats and hamsters. Journal of [129] Peluso JJ. Progesterone as a regulator of granulosa cell viability. The Journal of Steroid Biochemistry and Molecular Biology. 2003;85:167-173. DOI: 10.1016/S0960-0760(03)00192-4 [130] Swiatek-De Lange M, Stampfl A, Hauck SM, Deeg CA, et al. Membrane-initiated effects of progesterone on calcium dependent signaling and activation of VEGF gene expres- [131] Ishihara Y, Kawami T, Ishida A, Yamazaki T. Allopregnanolone-mediated protective effects of progesterone on tributyltin-induced neuronal injury in rat hippocampal slices. The Journal of Steroid Biochemistry and Molecular Biology. 2013;135:1-6. DOI: 10.1016 [132] Xu J, Zeng C, Chu W, Pan F, Rothfuss M, Zhang F, et al. Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site. Nature Communications. [133] Ahlbom E, Prins GS, Ceccatelli S. Testosterone protects cerebellar granule cells from oxidative stress-induced cell death through a receptor mediated mechanism. Brain [134] Hammond J, Le Q, Goodyer C, Gelfand M, Trifiro M, LeBlanc A. Testosterone-mediated neuroprotection through the androgen receptor in human primary neurons. Journal of sion in retinal glial cells. GLIA. 2007;55:1061-1073. DOI: 10.1002/glia.20523 traumatic brain injury. Neuroscience. 2013;231:111-124. DOI: 10.1016 acetate. Endocrinology. 2002;143:205-212. DOI: 10.1210/en.143.1.205 Neuroendocrinology. 2006;18:902-914. DOI: 10.1111/ 5168. DOI: 10.1038/s41598-017-05423-9 300 Sex Hormones in Neurodegenerative Processes and Diseases 10.1210/en.2011-2177 2011;2:380. DOI: 10.1038 Research. 2001;892:255-262 Neurochemistry. 2001;77:1-9 [149] Nilsen J, Brinton RD. Mitochondria as therapeutic targets of estrogen action in the central nervous system. Curr Drug Targets CNS Neurol Disord. 2004;3:297-313 Chapter 13* Provisional chapter Clinical Use of Progesterone and Its Relation to DOI: 10.5772/intechopen.73311 Studies to determine the physiological effects and functions of progesterone started in the twentieth century. Progesterone is a steroid-structured hormone with 21 carbon atoms originating from cholesterol. The corpus luteum, formed after ovulation in ruminants, secretes progesterone, which plays a role in the continuity of the pregnancy. Progestagens can be used for estrus synchronization in cows and heifers. Similarly, they are used for estrus synchronization during the breeding season or outside the breeding season by taking advantage of the negative feedback effect of progesterone in small ruminants. It is applied for the treatment of embryonic deaths due to luteal insufficiency in cows with high milk yield. In anovulatory anestrus, exogenous progesterone applications can be very useful. Progesterone treatment contributes to the resolution of the anestrus by rearranging hypothalamic functions in cattle with follicular cysts. The oxidative stress index in the luteal phase, when progesterone is high in ruminants, is higher than in the follicular phase. In the critical period of pregnancy, a high index of oxidative stress-induced progesterone causes embryonic death. Factors that cause stress in high milk-yielding cows can affect the amount of progesterone synthesis by inhibiting luteal cell function due to excessive free radical production. Keywords: progesterone, ruminants, oxidative stress, estrus synchronization, embryonic Many methods have been developed for controlling reproduction in farm animals. Among these methods, synchronization protocols to increase reproductive efficiency have an important > © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants Oxidative Stress in Ruminants Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.73311 Metin Öğün Metin Öğün Abstract death 1. Introduction Mushap Kuru, Abdulsamed Kükürt, Hasan Oral and Mushap Kuru, Abdulsamed Kükürt, Hasan Oral and #### Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants DOI: 10.5772/intechopen.73311 Mushap Kuru, Abdulsamed Kükürt, Hasan Oral and Metin Öğün Mushap Kuru, Abdulsamed Kükürt, Hasan Oral and Metin Öğün Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.73311 #### Abstract [149] Nilsen J, Brinton RD. Mitochondria as therapeutic targets of estrogen action in the central nervous system. Curr Drug Targets CNS Neurol Disord. 2004;3:297-313 [150] Nilsen J, Irwin RW, Brinton RD. Brain mitochondria as therapeutic target for the prevention of Alzheimer's pathology. Alzheimer's and Dementia. 2006;2[Suppl 3] [151] Simpkins JW, Wang J, Wang X, Perez E, Prokai L, Dykens JA. Mitochondria play a central role in estrogen-induced neuroprotection. Curr Drug Targets CNS Neurol Disord [152] Brewer GJ, Reichensperger JD, Brinton RD. Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons. Neurobiology of Aging. 2006;27:306-317 [153] Zhao L, Wu T-W, Brinton RD. Estrogen receptor subtypes alpha and beta contribute to neuroprotection and increased Bcl-2 expression in primary hippocampal neurons. Brain [154] Rudick CN, Gibbs RB, Woolley CS. A role for the basal forebrain cholinergic system in estrogen-induced disinhibition of hippocampal pyramidal cells. The Journal of Neu- [155] Tinkler GP, Tobin JR, Voytko ML. Effects of two years of estrogen loss or replacement on nucleus basalis cholinergic neurons and cholinergic fibers to the dorsolateral prefrontal and inferior parietal cortex of monkeys. The Journal of Comparative Neurology. 2004; 2005;4:69-83 469:507-521 Research. 2004;1010:22-34 302 Sex Hormones in Neurodegenerative Processes and Diseases roscience. 2003;23:4479-4490 Studies to determine the physiological effects and functions of progesterone started in the twentieth century. Progesterone is a steroid-structured hormone with 21 carbon atoms originating from cholesterol. The corpus luteum, formed after ovulation in ruminants, secretes progesterone, which plays a role in the continuity of the pregnancy. Progestagens can be used for estrus synchronization in cows and heifers. Similarly, they are used for estrus synchronization during the breeding season or outside the breeding season by taking advantage of the negative feedback effect of progesterone in small ruminants. It is applied for the treatment of embryonic deaths due to luteal insufficiency in cows with high milk yield. In anovulatory anestrus, exogenous progesterone applications can be very useful. Progesterone treatment contributes to the resolution of the anestrus by rearranging hypothalamic functions in cattle with follicular cysts. The oxidative stress index in the luteal phase, when progesterone is high in ruminants, is higher than in the follicular phase. In the critical period of pregnancy, a high index of oxidative stress-induced progesterone causes embryonic death. Factors that cause stress in high milk-yielding cows can affect the amount of progesterone synthesis by inhibiting luteal cell function due to excessive free radical production. Keywords: progesterone, ruminants, oxidative stress, estrus synchronization, embryonic death #### 1. Introduction Many methods have been developed for controlling reproduction in farm animals. Among these methods, synchronization protocols to increase reproductive efficiency have an important © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. place. The desired level of pregnancy rates is often not obtained because of the difficulty of following the estrus cycle in each animal on large farms. For this reason, estrus synchronization use in large farms becomes inevitable. Thus, progesterone-assisted estrus synchronizations are implemented intensively in farm animals. In addition, progesterone can also be used for the treatment of reproductive problems such as anestrus, cystic ovarian disease, and luteal insufficiency [1, 2]. stored in plasma membranes. The major source of cholesterol for luteal cells in ruminants is Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants http://dx.doi.org/10.5772/intechopen.73311 305 Cholesterol is primarily a hydrophobic molecule, and this makes it difficult for cholesterol to diffuse freely through hydrophilic environments such as the cytoplasm. In addition, cholesterol has a hydroxyl group at the 3 position that produces a discrete hydrophilic region making it difficult for the "flip-flop" of cholesterol between membrane surfaces within the lipid bilayer of cellular membranes. Therefore, movement of cholesterol in the circulatory system (lipoproteins) or within the cell is dependent upon transport proteins. Addition of another hydroxyl group at the other end of the cholesterol molecule alleviates the need for transport proteins [5]. Progesterone is a steroid hormone primarily secreted by the corpus luteum and placenta. Production of progesterone in luteal cells is dependent more on transport of cholesterol within the cell than to changes in the activity of steroidogenic enzymes. The P450 cholesterol side chain cleavage enzyme (P450scc) is located on the inner mitochondrial membrane and catalyzes the conversion of cholesterol to pregnenolone [6]. This enzyme catalyzes three oxidation steps: hydroxylations at the 20 and 22 positions and then cleavage between these two carbons. Pregnenolone has two hydrophilic residues that increase mobility through cellular membranes. Pregnenolone diffuses from the mitochondria to the smooth endoplasmic reticulum where it is converted to progesterone by the enzyme 3-β hydroxysteroid dehydrogenase (3β-HSD). This final reaction produces a double bond between the 4 and 5 carbon of the molecule and is the basis for the abbreviation of pregnenolone as progesterone (Figures 1 and 2). Progesterone then diffuses from the luteal cells to the bloodstream for transport to target tissues [7, 8]. Progesterone is synthesized from pregnenolone in the corpus luteum, the placenta during pregnancy, and the adrenals as a step in androgen and mineralocorticoid synthesis. Its actions are primarily mediated by an intracellular progesterone receptor, whose numbers increase in The products of hormone synthesis vary with the menstrual cycle; estradiol is the main product during follicular maturation, whereas progesterone is the main product in the luteal phase following ovulation. Progesterone is secreted by ovarian follicular cells prior to ovulation; it is also secreted in larger amounts by the corpus luteum, which forms from follicular granulosa cells following ovulation. The corpus luteum will grow for 10–12 days and then regress if fertilization does not occur; if fertilization does occur, the corpus luteum is maintained for the first 2–3 months of pregnancy. Progesterone plays several important actions in the normal female reproductive cycle. Progesterone prepares the uterus for pregnancy by shifting the endometrium from proliferation to secretion. Withdrawal of progesterone in the absence of pregnancy leads to organized shedding (menstruation) and it helps to mediate sexual response in the brain. After fertilization, progesterone organizes the vasculature of the endometrium to prepare for implantation. It promotes enzymatic digestion of the zona pellucida to allow the oocyte to implant into the uterine wall. In addition, it inhibits contractions of the uterine myometrium (smooth muscle layer) and counteracts the effects of oxytocin on contractility. Progesterone promotes lobuloalveolar growth in the breasts to prepare for lactation, but suppresses premature milk protein synthesis prior to parturition. Some of the effects of progesterone may be related to its ability to antagonize estrogen by decreasing expression of estrogen receptors, e.g. the ability of progesterone to inhibit estrogen-mediated endometrial proliferation. It also has a potent effect as a mineralocorticoid circulating high-density lipoproteins [3, 4]. the presence of estrogen [9]. This section provides information on the structure of progesterone, its role in physiological events in ruminants, its use in clinical practice, and its relation to oxidative stress. #### 2. The structure and biochemical synthesis of progesterone Steroid hormones are lipophilic organic compounds with a low-molecular weight derived from cholesterol (Figure 1). Steroid hormones are synthesized in the mitochondria and smooth endoplasmic reticulum in gonads, such as the ovary and testis, and then released into the bloodstream. The steroid hormones are broadly classified into three categories based on their physiological functions: glucocorticoids, mineralocorticoids, and sex steroids [3]. Cholesterol is an obligate intermediate used for steroid hormone synthesis by the adrenal gland, ovary, testis, and placenta that can be obtained from three principal sources: de novo synthesis of cholesterol from acetate, cholesterol from circulating high-density lipoproteins, and cholesterol Figure 1. Sources of cholesterol for progesterone biosynthesis. stored in plasma membranes. The major source of cholesterol for luteal cells in ruminants is circulating high-density lipoproteins [3, 4]. place. The desired level of pregnancy rates is often not obtained because of the difficulty of following the estrus cycle in each animal on large farms. For this reason, estrus synchronization use in large farms becomes inevitable. Thus, progesterone-assisted estrus synchronizations are implemented intensively in farm animals. In addition, progesterone can also be used for the treatment of reproductive problems such as anestrus, cystic ovarian disease, and luteal insuffi- This section provides information on the structure of progesterone, its role in physiological Steroid hormones are lipophilic organic compounds with a low-molecular weight derived from cholesterol (Figure 1). Steroid hormones are synthesized in the mitochondria and smooth endoplasmic reticulum in gonads, such as the ovary and testis, and then released into the bloodstream. The steroid hormones are broadly classified into three categories based on their physiological functions: glucocorticoids, mineralocorticoids, and sex steroids [3]. Cholesterol is an obligate intermediate used for steroid hormone synthesis by the adrenal gland, ovary, testis, and placenta that can be obtained from three principal sources: de novo synthesis of cholesterol from acetate, cholesterol from circulating high-density lipoproteins, and cholesterol events in ruminants, its use in clinical practice, and its relation to oxidative stress. 2. The structure and biochemical synthesis of progesterone Figure 1. Sources of cholesterol for progesterone biosynthesis. ciency [1, 2]. 304 Sex Hormones in Neurodegenerative Processes and Diseases Cholesterol is primarily a hydrophobic molecule, and this makes it difficult for cholesterol to diffuse freely through hydrophilic environments such as the cytoplasm. In addition, cholesterol has a hydroxyl group at the 3 position that produces a discrete hydrophilic region making it difficult for the "flip-flop" of cholesterol between membrane surfaces within the lipid bilayer of cellular membranes. Therefore, movement of cholesterol in the circulatory system (lipoproteins) or within the cell is dependent upon transport proteins. Addition of another hydroxyl group at the other end of the cholesterol molecule alleviates the need for transport proteins [5]. Progesterone is a steroid hormone primarily secreted by the corpus luteum and placenta. Production of progesterone in luteal cells is dependent more on transport of cholesterol within the cell than to changes in the activity of steroidogenic enzymes. The P450 cholesterol side chain cleavage enzyme (P450scc) is located on the inner mitochondrial membrane and catalyzes the conversion of cholesterol to pregnenolone [6]. This enzyme catalyzes three oxidation steps: hydroxylations at the 20 and 22 positions and then cleavage between these two carbons. Pregnenolone has two hydrophilic residues that increase mobility through cellular membranes. Pregnenolone diffuses from the mitochondria to the smooth endoplasmic reticulum where it is converted to progesterone by the enzyme 3-β hydroxysteroid dehydrogenase (3β-HSD). This final reaction produces a double bond between the 4 and 5 carbon of the molecule and is the basis for the abbreviation of pregnenolone as progesterone (Figures 1 and 2). Progesterone then diffuses from the luteal cells to the bloodstream for transport to target tissues [7, 8]. Progesterone is synthesized from pregnenolone in the corpus luteum, the placenta during pregnancy, and the adrenals as a step in androgen and mineralocorticoid synthesis. Its actions are primarily mediated by an intracellular progesterone receptor, whose numbers increase in the presence of estrogen [9]. The products of hormone synthesis vary with the menstrual cycle; estradiol is the main product during follicular maturation, whereas progesterone is the main product in the luteal phase following ovulation. Progesterone is secreted by ovarian follicular cells prior to ovulation; it is also secreted in larger amounts by the corpus luteum, which forms from follicular granulosa cells following ovulation. The corpus luteum will grow for 10–12 days and then regress if fertilization does not occur; if fertilization does occur, the corpus luteum is maintained for the first 2–3 months of pregnancy. Progesterone plays several important actions in the normal female reproductive cycle. Progesterone prepares the uterus for pregnancy by shifting the endometrium from proliferation to secretion. Withdrawal of progesterone in the absence of pregnancy leads to organized shedding (menstruation) and it helps to mediate sexual response in the brain. After fertilization, progesterone organizes the vasculature of the endometrium to prepare for implantation. It promotes enzymatic digestion of the zona pellucida to allow the oocyte to implant into the uterine wall. In addition, it inhibits contractions of the uterine myometrium (smooth muscle layer) and counteracts the effects of oxytocin on contractility. Progesterone promotes lobuloalveolar growth in the breasts to prepare for lactation, but suppresses premature milk protein synthesis prior to parturition. Some of the effects of progesterone may be related to its ability to antagonize estrogen by decreasing expression of estrogen receptors, e.g. the ability of progesterone to inhibit estrogen-mediated endometrial proliferation. It also has a potent effect as a mineralocorticoid synthesizes >2 ng/mL progesterone. The progesterone level is highest during days 8–10 of the estrous cycle in cows. If pregnancy does not occur, prostaglandin F2 alpha (PGF2α), which is synthesized in the uterus, passes to the ovarian arteries from the uterine vein inducing corpus luteum degeneration. As a result, the blood progesterone level decreases rapidly to less than 0.5 ng/mL [11]. If pregnancy occurs, blood progesterone levels in the cows can be 6–8 ng/mL Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants http://dx.doi.org/10.5772/intechopen.73311 307 In sheep, the corpus luteum following ovulation begins to secrete progesterone after the third day of the estrous cycle. Because the corpus luteum formation is rapid, the blood progesterone level rises rapidly and reaches measurable levels [13]. The progesterone level is the highest (4 ng/mL) between days 9 and 13 of the cycle. If there is no pregnancy, the corpus luteum starts to shrink and the amount of PGF2α in the blood starts to increase on the 12th day of the cycle. On the 14th day of the cycle, the progesterone level is 10 ng/mL but is <0.2 ng/mL on the 16th day of the cycle [14]. Pregnancy in sheep is maintained by the corpus luteum until the 50th day and later by placenta-derived progesterone. Progesterone levels are highest between 60 and 130 days of gestation. As long as pregnancy continues, the concentration of progesterone never The corpus luteum forms after ovulation and progesterone levels begin to increase in goats. Maximum levels of progesterone (6–10 ng/mL) are found in the middle of the cycle. This level starts to decrease on the 15th day of the cycle and descends to basal levels on the 19th Progestagens (ear implants and intravaginal devices [progesterone-releasing intravaginal device (PRID) and controlled internal drug release (CIDR)]) can be administered for 5–20 days for the purpose of estrus synchronization in cows and heifers without uterine infection. Progesterone applications can stimulate puberty in some heifers and initiate normal cyclic activities in anestrus cows. In addition, following the first ovulation after progesterone treatment, normal length After the end of progesterone application, synchronized estrus is observed within 3 days [2, 17]. However, when progesterone is used for a prolonged period, the fertility rates decrease due to the persistence of the follicle. In addition, when melengestrol acetate (MGA) is used for estrus synchronization, subfertile estrus occurs initially because of the persistent follicles and artificial insemination is not recommended [24]. Long-term use negatively affects the intrauterine environment and spermatozoon transport. For this reason, estrus synchronization with According to studies conducted in recent years, the pregnancy rate may be 10–15% higher in short-term progesterone use protocols than long-term protocols. However, other researchers progesterone for 5–9 days is more suitable for increasing fertility [25, 26]. argue that there is no difference between pregnancy rates [2, 26–28]. on day 21 of pregnancy [12, 13]. falls below 1 ng/mL [12]. 4. Clinical use of progesterone in ruminants sexual activities may continue [2, 17–24]. 4.1. Use of progesterone for estrus synchronization in cattle day [15, 16]. Figure 2. Pathway synthesis of progesterone from cholesterol. receptor antagonist that reduces sodium retention when present and increases sodium retention when progesterone is withdrawn [8, 9]. The role of progesterone in males is less clear, but it is believed to play a role in activating sperm in the female reproductive tract. It has also been implicated as a modulator of male sexual response and behavior [7]. #### 3. Progesterone synthesis and secretion during the sexual cycle and pregnancy in ruminants The corpus luteum formed after ovulation in ruminants is a functional structure formed by membrane granulosa in the wall of Graaf follicles. Hypertrophy and luteinization of the theca interna cells play a role in the continuity of the pregnancy, secrete progesterone for a temporary period, and have endocrine activity. In particular, there is a close correlation between corpus luteum development and blood progesterone levels [10, 11]. Progesterone levels in the proestrus and estrus stages of the estrous cycle in cattle are very low. Newly developing corpus luteum cannot produce sufficient progesterone after ovulation (in the period of metaestrus). Therefore, a significant increase in blood progesterone levels cannot be detected. In the diestrus period, the corpus luteum acquires a functional structure and synthesizes >2 ng/mL progesterone. The progesterone level is highest during days 8–10 of the estrous cycle in cows. If pregnancy does not occur, prostaglandin F2 alpha (PGF2α), which is synthesized in the uterus, passes to the ovarian arteries from the uterine vein inducing corpus luteum degeneration. As a result, the blood progesterone level decreases rapidly to less than 0.5 ng/mL [11]. If pregnancy occurs, blood progesterone levels in the cows can be 6–8 ng/mL on day 21 of pregnancy [12, 13]. In sheep, the corpus luteum following ovulation begins to secrete progesterone after the third day of the estrous cycle. Because the corpus luteum formation is rapid, the blood progesterone level rises rapidly and reaches measurable levels [13]. The progesterone level is the highest (4 ng/mL) between days 9 and 13 of the cycle. If there is no pregnancy, the corpus luteum starts to shrink and the amount of PGF2α in the blood starts to increase on the 12th day of the cycle. On the 14th day of the cycle, the progesterone level is 10 ng/mL but is <0.2 ng/mL on the 16th day of the cycle [14]. Pregnancy in sheep is maintained by the corpus luteum until the 50th day and later by placenta-derived progesterone. Progesterone levels are highest between 60 and 130 days of gestation. As long as pregnancy continues, the concentration of progesterone never falls below 1 ng/mL [12]. The corpus luteum forms after ovulation and progesterone levels begin to increase in goats. Maximum levels of progesterone (6–10 ng/mL) are found in the middle of the cycle. This level starts to decrease on the 15th day of the cycle and descends to basal levels on the 19th day [15, 16]. ### 4. Clinical use of progesterone in ruminants receptor antagonist that reduces sodium retention when present and increases sodium retention The role of progesterone in males is less clear, but it is believed to play a role in activating sperm in the female reproductive tract. It has also been implicated as a modulator of male The corpus luteum formed after ovulation in ruminants is a functional structure formed by membrane granulosa in the wall of Graaf follicles. Hypertrophy and luteinization of the theca interna cells play a role in the continuity of the pregnancy, secrete progesterone for a temporary period, and have endocrine activity. In particular, there is a close correlation between Progesterone levels in the proestrus and estrus stages of the estrous cycle in cattle are very low. Newly developing corpus luteum cannot produce sufficient progesterone after ovulation (in the period of metaestrus). Therefore, a significant increase in blood progesterone levels cannot be detected. In the diestrus period, the corpus luteum acquires a functional structure and 3. Progesterone synthesis and secretion during the sexual cycle and corpus luteum development and blood progesterone levels [10, 11]. when progesterone is withdrawn [8, 9]. Figure 2. Pathway synthesis of progesterone from cholesterol. 306 Sex Hormones in Neurodegenerative Processes and Diseases sexual response and behavior [7]. pregnancy in ruminants #### 4.1. Use of progesterone for estrus synchronization in cattle Progestagens (ear implants and intravaginal devices [progesterone-releasing intravaginal device (PRID) and controlled internal drug release (CIDR)]) can be administered for 5–20 days for the purpose of estrus synchronization in cows and heifers without uterine infection. Progesterone applications can stimulate puberty in some heifers and initiate normal cyclic activities in anestrus cows. In addition, following the first ovulation after progesterone treatment, normal length sexual activities may continue [2, 17–24]. After the end of progesterone application, synchronized estrus is observed within 3 days [2, 17]. However, when progesterone is used for a prolonged period, the fertility rates decrease due to the persistence of the follicle. In addition, when melengestrol acetate (MGA) is used for estrus synchronization, subfertile estrus occurs initially because of the persistent follicles and artificial insemination is not recommended [24]. Long-term use negatively affects the intrauterine environment and spermatozoon transport. For this reason, estrus synchronization with progesterone for 5–9 days is more suitable for increasing fertility [25, 26]. According to studies conducted in recent years, the pregnancy rate may be 10–15% higher in short-term progesterone use protocols than long-term protocols. However, other researchers argue that there is no difference between pregnancy rates [2, 26–28]. #### 4.1.1. Estrus synchronization with progestagens + prostaglandins Progesterone administration allows the synchronization of estrus in ruminants in different stages of the cycle. PGF2α may be injected on the last day of progesterone administration (5– 10 days) or 1–2 days before the end of progesterone. After the application of progesterone, estrus occurs and artificial insemination is done [21, 29–31]. Some researchers obtained 54% pregnancy rates in the first estrus in dairy cows (n = 102) given progesterone for 7 days (CIDR) with PGF2α administered on day 6. In addition, 49% pregnancy rates were obtained when a similar protocol was applied to non-cyclic and cyclic heifers [29]. In a similar study, the pregnancy rate was 47% in beef heifers (n = 247) and 59% in dairy heifers (n = 129) [32]. 4.2. Use of progesterone in estrus synchronization in small ruminants body condition score, lactation, age, temperature, light, and breed [56–60]. in sheep and goats [48–55]. In small ruminants, progesterone suppresses GnRH and luteinizing hormone (LH) release by negative feedback [45]. There may be up to 30-fold increases in LH concentration with a decrease in the plasma level of progesterone. In addition, the dominant follicular LH receptors are sensitive and thus ovulation occurs. As a general principle, these effects of progesterone can be used in estrus synchronization protocols [46–48]. Medroxyprogesterone acetate (MAP), fluorogestone acetate (intravaginal sponge), melengestrol acetate, levonorgestrel, and intravaginal progesterone-releasing devices (CIDR and DICO) are used for estrus synchronization Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants http://dx.doi.org/10.5772/intechopen.73311 309 In sheep, progestagens are used effectively in the control and synchronization of estrus. Equine chorionic gonadotropin (eCG) injection is performed in addition to progestagen administration for 12–14 days in order to obtain high estrus rates and ovulation, especially during anestrus. Nevertheless, these types of manipulations may also vary depending on nutrition, In sheep synchronized with MAP during the breeding season, follicle size and LH pulse increase after ram introduction [61, 62]. Intravaginal CIDR or chronolone intravaginal sponge and 500 IU During the breeding season, estrus start and end times were different in sheep treated with MAP or CIDR for 12 days [64]. High estrus rates and similar fertility rates were determined in sheep that applied short-term (6 days) CIDR-G or fluorogestone acetate (FGA, intravaginal sponge) [52]. Short-term (5 day) FGA and eCG administration produce higher estrus rate than long-term FGA and eCG treatments in sheep during the breeding season [50]. Injections of eCG at different doses (300, 400, and 500 IU) to Awassi sheep in estrus synchronization with progesterone similarly affect fertility parameters [65]. The testosterone antibody, β-carotene, and vitamin E administration did not change the estrus and pregnancy rates 7 days before intravaginal 40 mg FGA administration in Tuj ewes during the non-breeding season [66]. Vaginal sponges with progesterone for 11–14 days were applied to Pirlak ewes during the nonbreeding season and 92–100% entered estrus. The pregnancy rate was 37.7–44%. In the study, the fertility parameters did not change for 11 or 14 day vaginal sponge application. Progesterone administration at different day lengths may be effective for the onset of estrus [48]. Short or long-term progesterone treatment for estrus synchronization in goats can be done depending on the breeding season. At the end of progesterone treatment, the protocol is Intravaginal levonorgestrel for 10 days and intramuscular PGF2α administration were used in goats causing high estrus rates (95%) during the breeding season [70]. In a study conducted on Abaza goats during the breeding season, the first estrus pregnancy rate was 73.3% and the pregnancy rate was 93.3% after estrus synchronization using CIDR for 11 days. In addition, the same pregnancy rate was achieved in goats that did not receive any intervention in the study. However, progesterone treatment and estrus synchronization contribute positively to repro- terminated by eCG administration [54, 58, 67–69]. ductive parameters and increase twinning rates [55]. eCG did not affect the LH wave and peak in Tuj sheep outside the breeding season [63]. In some studies, artificial insemination was performed according to PGF2α injection and estrus follow-up (4–6 days) 17–19 days after 14 days of MGA application [25, 28]. In this protocol, the purpose of PGF2α injection is to lyse the corpus luteum that can be formed at the end of 14 days of MGA application [33]. #### 4.1.2. Ovulation synchronization protocols + progesterone Progesterone is administered as an ear implant or an intravaginal device between days 0 and 7 in order for the Ovsynch (GnRH/PGF2α/GnRH) protocol to be successful and the pregnancy rate to increase [27]. In a study performed on heifers (n = 383), a 47% pregnancy rate was obtained [34]. In another study, progesterone administration in Ovsynch did not increase the pregnancy rate in cows with corpus luteum [35]. Others reported that premature estrus was not observed due to progesterone used between 0 and 7 days for ovulation synchronization protocols and full synchronization was achieved [36, 37]. Progesterone administration is also performed in the Cosynch protocol. On day 0, the progesterone device is inserted and gonadotropin-releasing hormone (GnRH) is injected. Progesterone devices are removed during PGF2α injection (day 7). Fixed-time artificial insemination is performed in 48, 56, or 60 hours after GnRH is injected [38–40]. Such protocols may be applied for 12–14 days with progesterone [41, 42]. #### 4.1.3. Five-day Cosynch + progesterone protocols in the heifer Ovulation synchronization methods have a high pregnancy rate, especially in cows, although this rate is lower in heifers [2]. Synchronization protocols have been developed to stimulate ovulation, which do not require heat detection. Progesterone administration occurs for 5 days. During progesterone removal, PGF2α is administered. GnRH is administered 72 hours later and fixed-time artificial insemination is performed [19, 21, 22, 26, 27]. A 10.5% higher pregnancy rate can be obtained with this protocol [43]. The pregnancy rate improves between 45.9 and 54.2% in the 5-day Cosynch + PRID protocol and fixed-time artificial insemination with sexed semen in cyclic heifers [26]. It was determined that vaginitis and mucopurulent discharge were observed after use of intravaginal progesterone devices in heifers. The incidence of vaginitis in the heifers may be around 70% or more [22, 23, 26, 44]. #### 4.2. Use of progesterone in estrus synchronization in small ruminants 4.1.1. Estrus synchronization with progestagens + prostaglandins 308 Sex Hormones in Neurodegenerative Processes and Diseases 14 days of MGA application [33]. 4.1.2. Ovulation synchronization protocols + progesterone protocols and full synchronization was achieved [36, 37]. 4.1.3. Five-day Cosynch + progesterone protocols in the heifer for 12–14 days with progesterone [41, 42]. sexed semen in cyclic heifers [26]. around 70% or more [22, 23, 26, 44]. Progesterone administration allows the synchronization of estrus in ruminants in different stages of the cycle. PGF2α may be injected on the last day of progesterone administration (5– 10 days) or 1–2 days before the end of progesterone. After the application of progesterone, estrus occurs and artificial insemination is done [21, 29–31]. Some researchers obtained 54% pregnancy rates in the first estrus in dairy cows (n = 102) given progesterone for 7 days (CIDR) with PGF2α administered on day 6. In addition, 49% pregnancy rates were obtained when a similar protocol was applied to non-cyclic and cyclic heifers [29]. In a similar study, the pregnancy rate was 47% in beef heifers (n = 247) and 59% in dairy heifers (n = 129) [32]. In some studies, artificial insemination was performed according to PGF2α injection and estrus follow-up (4–6 days) 17–19 days after 14 days of MGA application [25, 28]. In this protocol, the purpose of PGF2α injection is to lyse the corpus luteum that can be formed at the end of Progesterone is administered as an ear implant or an intravaginal device between days 0 and 7 in order for the Ovsynch (GnRH/PGF2α/GnRH) protocol to be successful and the pregnancy rate to increase [27]. In a study performed on heifers (n = 383), a 47% pregnancy rate was obtained [34]. In another study, progesterone administration in Ovsynch did not increase the pregnancy rate in cows with corpus luteum [35]. Others reported that premature estrus was not observed due to progesterone used between 0 and 7 days for ovulation synchronization Progesterone administration is also performed in the Cosynch protocol. On day 0, the progesterone device is inserted and gonadotropin-releasing hormone (GnRH) is injected. Progesterone devices are removed during PGF2α injection (day 7). Fixed-time artificial insemination is performed in 48, 56, or 60 hours after GnRH is injected [38–40]. Such protocols may be applied Ovulation synchronization methods have a high pregnancy rate, especially in cows, although this rate is lower in heifers [2]. Synchronization protocols have been developed to stimulate ovulation, which do not require heat detection. Progesterone administration occurs for 5 days. During progesterone removal, PGF2α is administered. GnRH is administered 72 hours later and fixed-time artificial insemination is performed [19, 21, 22, 26, 27]. A 10.5% higher pregnancy rate can be obtained with this protocol [43]. The pregnancy rate improves between 45.9 and 54.2% in the 5-day Cosynch + PRID protocol and fixed-time artificial insemination with It was determined that vaginitis and mucopurulent discharge were observed after use of intravaginal progesterone devices in heifers. The incidence of vaginitis in the heifers may be In small ruminants, progesterone suppresses GnRH and luteinizing hormone (LH) release by negative feedback [45]. There may be up to 30-fold increases in LH concentration with a decrease in the plasma level of progesterone. In addition, the dominant follicular LH receptors are sensitive and thus ovulation occurs. As a general principle, these effects of progesterone can be used in estrus synchronization protocols [46–48]. Medroxyprogesterone acetate (MAP), fluorogestone acetate (intravaginal sponge), melengestrol acetate, levonorgestrel, and intravaginal progesterone-releasing devices (CIDR and DICO) are used for estrus synchronization in sheep and goats [48–55]. In sheep, progestagens are used effectively in the control and synchronization of estrus. Equine chorionic gonadotropin (eCG) injection is performed in addition to progestagen administration for 12–14 days in order to obtain high estrus rates and ovulation, especially during anestrus. Nevertheless, these types of manipulations may also vary depending on nutrition, body condition score, lactation, age, temperature, light, and breed [56–60]. In sheep synchronized with MAP during the breeding season, follicle size and LH pulse increase after ram introduction [61, 62]. Intravaginal CIDR or chronolone intravaginal sponge and 500 IU eCG did not affect the LH wave and peak in Tuj sheep outside the breeding season [63]. During the breeding season, estrus start and end times were different in sheep treated with MAP or CIDR for 12 days [64]. High estrus rates and similar fertility rates were determined in sheep that applied short-term (6 days) CIDR-G or fluorogestone acetate (FGA, intravaginal sponge) [52]. Short-term (5 day) FGA and eCG administration produce higher estrus rate than long-term FGA and eCG treatments in sheep during the breeding season [50]. Injections of eCG at different doses (300, 400, and 500 IU) to Awassi sheep in estrus synchronization with progesterone similarly affect fertility parameters [65]. The testosterone antibody, β-carotene, and vitamin E administration did not change the estrus and pregnancy rates 7 days before intravaginal 40 mg FGA administration in Tuj ewes during the non-breeding season [66]. Vaginal sponges with progesterone for 11–14 days were applied to Pirlak ewes during the nonbreeding season and 92–100% entered estrus. The pregnancy rate was 37.7–44%. In the study, the fertility parameters did not change for 11 or 14 day vaginal sponge application. Progesterone administration at different day lengths may be effective for the onset of estrus [48]. Short or long-term progesterone treatment for estrus synchronization in goats can be done depending on the breeding season. At the end of progesterone treatment, the protocol is terminated by eCG administration [54, 58, 67–69]. Intravaginal levonorgestrel for 10 days and intramuscular PGF2α administration were used in goats causing high estrus rates (95%) during the breeding season [70]. In a study conducted on Abaza goats during the breeding season, the first estrus pregnancy rate was 73.3% and the pregnancy rate was 93.3% after estrus synchronization using CIDR for 11 days. In addition, the same pregnancy rate was achieved in goats that did not receive any intervention in the study. However, progesterone treatment and estrus synchronization contribute positively to reproductive parameters and increase twinning rates [55]. In sheep and goats, short-term progesterone-impregnated sponge therapy changed the microbial flora of the vagina and formed vaginitis. Staphylococcus is usually detected in these cases of vaginitis [13, 71, 72]. Four different types of anovulatory anestrus may be encountered in the postpartum period. The first (type I) is characterized by follicle development remaining "emergent" (~ 4 mm) and not progressing to the "deviation" (~ 9 mm) phase. This type of anestrus is classically referred to as "inactive ovary." In type II, the "deviation" phase is passed, and after the "growth" phase, the follicle undergoes atresia. In type III, the follicle develops to the preovulatory stage but does not ovulate, thus becoming permanent follicles or follicular/luteal cysts. In type IV, the follicles develop and ovulate and the corpus luteum is formed, but the corpora lutea cannot Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants http://dx.doi.org/10.5772/intechopen.73311 311 In anovulatory anestrus, exogenous progesterone applications can be very useful [84]. The use of intravaginal progesterone devices, such as PRID or CIDR, may induce the restart of cyclic activity in the ovary [13]. Especially at the end of this application, the use of PGF2α and analogues may increase the pregnancy rate [73]. At the end of 10 days of progesterone therapy, injection of eCG, estradiol, or PGF2α is beneficial [83]. In this context, successful results were obtained after 9 days of PRID and intramuscular cloprostenol injection 1 day before PRID removal. Higher pregnancy rates can be achieved by adding progesterone to ovulation syn- Cystic ovarian follicles are non-ovulated preovulatory follicles that maintain long-lasting persistence without any luteal structure on the ovary. With progesterone administration, the number of LH pulses, and thus the LH level, is reduced and maintained at a luteal phase level PRID or CIDR can be administered intravaginally for 7–14 days in cows with cystic ovarian disease [86]. In addition, progesterone is frequently used in the treatment of follicular cysts that cannot be mitigated with GnRH or human chorionic gonadotropin (hCG) injections. Progesterone therapy in cows with follicular cysts restarts hypothalamic functions and con- Combination of progesterone administration for 9 days with GnRH and PGF2α may improve outcomes in cows with cystic ovarian disease. GnRH is injected and a progesterone-releasing device is applied intravaginally for 9 days. On day 7, PGF2α is administered intramuscularly, and 2 days later, the vaginal progesterone-releasing device is removed. Artificial insemination The induction of lactation is a procedure that is applied to infertile heifers and non-lactating cows. The main purpose is to generate profit by initiating milk production. In particular, the combination of progesterone and estradiol in this type of cattle helps to develop the lobule alveolar system. Lactation can be successfully induced in 60% of cows treated with a combina- When 50 mg progesterone and 20 mg 17β-estradiol are injected subcutaneously twice a day for 7 days in repeat breeders or aborted heifers, milk synthesis starts 10–21 days after the treatment [91]. In another study, lactation started 11–21 days after the first 17β-estradiol and regress and become permanent [13, 83]. chronization protocols in acyclic cows [13, 85]. throughout the cycle [73]. 4.5. Use of progesterone in cystic ovarian disease tributes to the resolution of the problem [83, 87]. 4.6. Use of progesterone to induction of lactation tion of estrogen and progesterone for 7 or 10 days [89, 90]. is done when the cows are in heat [88]. #### 4.3. Use of progesterone to prevent embryonic death Progesterone is the most important hormone for the continuity of pregnancy. Progesterone affects oocyte quality by affecting LH wave frequency and persistent follicle formation. Again, progesterone plays a vital role in influencing the endometrium and creating the appropriate environment for the survival of the embryo [73]. The rate of embryonic deaths in cows may range from 7 to 16% in the first week, 6–44% in the second week, 3–33% in the third week, and 19–42% after the fourth week. Embryonic deaths are caused by genetic and environmental factors. Most of the embryonic deaths, especially due to hormone insufficiency, are the result of luteal insufficiency. For this reason, progesterone or its analogues are applied to reduce embryonic losses before or after artificial insemination [74–76]. In ovulation synchronization protocols, such as Ovsynch and Cosynch, progesterone administration for 7 days between the first GnRH and PGF2α injections reduces the embryonic loss rate due to luteal phase deficiency [13]. Gestational losses were 3.6–6.8% after the 5-day Cosynch + progesterone protocol [26]. Progesterone-assisted estrus synchronization protocols can prevent early embryonic losses and increase the pregnancy rate. In many studies, there are reports that progesterone-assisted applications increase the pregnancy rate and decrease the embryonic loss rate. However, some researchers disagree [2, 35, 77, 78]. One of the most important causes of embryonic deaths in high milk-yielding cows is inadequate embryo development prior to implantation due to insufficient progesterone concentrations. Progesterone application performed between 3, 5, and 10 days after artificial insemination caused a statistically significant increase after CIDR administration compared with the control group. The pregnancy rate was 35% (22/63) in the control group and 48% (32/67) in the progesterone-treated group. The effect of exogenous progesterone is important for the development of pregnancy, especially in cows with first and second lactation [79]. Some researchers report an overall increase of 5% in pregnancy rates following progesterone administration. Progesterone administration time is critical to success. Progesterone treatment for 6 days after artificial insemination can increase pregnancy rates (10% more) [80]. Cows in the CIDR groups, which were administered progesterone for 6 or 12 days after the 5th and 7th days following artificial insemination, had higher pregnancy rates than the control group [81]. Intravaginal progesterone administration for 7 days starting 14 days after artificial insemination can reduce both embryonic deaths and fetal losses [77]. Similarly, post-mating treatment of FGA (intravaginal sponge) in sheep has been reported to reduce embryonic mortality [82]. #### 4.4. Use of progesterone in anestrus or anovulatory anestrus Anestrus is a situation in which a sign of heat cannot be detected in beef or dairy herds. Many forms of functional infertility result in anestrus in cows. This leads to serious economic losses for large farms [73]. Four different types of anovulatory anestrus may be encountered in the postpartum period. The first (type I) is characterized by follicle development remaining "emergent" (~ 4 mm) and not progressing to the "deviation" (~ 9 mm) phase. This type of anestrus is classically referred to as "inactive ovary." In type II, the "deviation" phase is passed, and after the "growth" phase, the follicle undergoes atresia. In type III, the follicle develops to the preovulatory stage but does not ovulate, thus becoming permanent follicles or follicular/luteal cysts. In type IV, the follicles develop and ovulate and the corpus luteum is formed, but the corpora lutea cannot regress and become permanent [13, 83]. In anovulatory anestrus, exogenous progesterone applications can be very useful [84]. The use of intravaginal progesterone devices, such as PRID or CIDR, may induce the restart of cyclic activity in the ovary [13]. Especially at the end of this application, the use of PGF2α and analogues may increase the pregnancy rate [73]. At the end of 10 days of progesterone therapy, injection of eCG, estradiol, or PGF2α is beneficial [83]. In this context, successful results were obtained after 9 days of PRID and intramuscular cloprostenol injection 1 day before PRID removal. Higher pregnancy rates can be achieved by adding progesterone to ovulation synchronization protocols in acyclic cows [13, 85]. #### 4.5. Use of progesterone in cystic ovarian disease In sheep and goats, short-term progesterone-impregnated sponge therapy changed the microbial flora of the vagina and formed vaginitis. Staphylococcus is usually detected in these cases Progesterone is the most important hormone for the continuity of pregnancy. Progesterone affects oocyte quality by affecting LH wave frequency and persistent follicle formation. Again, progesterone plays a vital role in influencing the endometrium and creating the appropriate The rate of embryonic deaths in cows may range from 7 to 16% in the first week, 6–44% in the second week, 3–33% in the third week, and 19–42% after the fourth week. Embryonic deaths are caused by genetic and environmental factors. Most of the embryonic deaths, especially due to hormone insufficiency, are the result of luteal insufficiency. For this reason, progesterone or its analogues are applied to reduce embryonic losses before or after artificial insemination [74–76]. In ovulation synchronization protocols, such as Ovsynch and Cosynch, progesterone administration for 7 days between the first GnRH and PGF2α injections reduces the embryonic loss rate due to luteal phase deficiency [13]. Gestational losses were 3.6–6.8% after the 5-day Progesterone-assisted estrus synchronization protocols can prevent early embryonic losses and increase the pregnancy rate. In many studies, there are reports that progesterone-assisted applications increase the pregnancy rate and decrease the embryonic loss rate. However, some One of the most important causes of embryonic deaths in high milk-yielding cows is inadequate embryo development prior to implantation due to insufficient progesterone concentrations. Progesterone application performed between 3, 5, and 10 days after artificial insemination caused a statistically significant increase after CIDR administration compared with the control group. The pregnancy rate was 35% (22/63) in the control group and 48% (32/67) in the progesterone-treated group. The effect of exogenous progesterone is important for the develop- Some researchers report an overall increase of 5% in pregnancy rates following progesterone administration. Progesterone administration time is critical to success. Progesterone treatment Cows in the CIDR groups, which were administered progesterone for 6 or 12 days after the 5th and 7th days following artificial insemination, had higher pregnancy rates than the control group [81]. Intravaginal progesterone administration for 7 days starting 14 days after artificial insemination can reduce both embryonic deaths and fetal losses [77]. Similarly, post-mating treatment of FGA (intravaginal sponge) in sheep has been reported to reduce embryonic mortality [82]. Anestrus is a situation in which a sign of heat cannot be detected in beef or dairy herds. Many forms of functional infertility result in anestrus in cows. This leads to serious economic losses for 6 days after artificial insemination can increase pregnancy rates (10% more) [80]. ment of pregnancy, especially in cows with first and second lactation [79]. 4.4. Use of progesterone in anestrus or anovulatory anestrus of vaginitis [13, 71, 72]. 4.3. Use of progesterone to prevent embryonic death 310 Sex Hormones in Neurodegenerative Processes and Diseases environment for the survival of the embryo [73]. Cosynch + progesterone protocol [26]. researchers disagree [2, 35, 77, 78]. for large farms [73]. Cystic ovarian follicles are non-ovulated preovulatory follicles that maintain long-lasting persistence without any luteal structure on the ovary. With progesterone administration, the number of LH pulses, and thus the LH level, is reduced and maintained at a luteal phase level throughout the cycle [73]. PRID or CIDR can be administered intravaginally for 7–14 days in cows with cystic ovarian disease [86]. In addition, progesterone is frequently used in the treatment of follicular cysts that cannot be mitigated with GnRH or human chorionic gonadotropin (hCG) injections. Progesterone therapy in cows with follicular cysts restarts hypothalamic functions and contributes to the resolution of the problem [83, 87]. Combination of progesterone administration for 9 days with GnRH and PGF2α may improve outcomes in cows with cystic ovarian disease. GnRH is injected and a progesterone-releasing device is applied intravaginally for 9 days. On day 7, PGF2α is administered intramuscularly, and 2 days later, the vaginal progesterone-releasing device is removed. Artificial insemination is done when the cows are in heat [88]. #### 4.6. Use of progesterone to induction of lactation The induction of lactation is a procedure that is applied to infertile heifers and non-lactating cows. The main purpose is to generate profit by initiating milk production. In particular, the combination of progesterone and estradiol in this type of cattle helps to develop the lobule alveolar system. Lactation can be successfully induced in 60% of cows treated with a combination of estrogen and progesterone for 7 or 10 days [89, 90]. When 50 mg progesterone and 20 mg 17β-estradiol are injected subcutaneously twice a day for 7 days in repeat breeders or aborted heifers, milk synthesis starts 10–21 days after the treatment [91]. In another study, lactation started 11–21 days after the first 17β-estradiol and progesterone injection. The highest milk yield was reached 30–35 days after the start of lactation [92]. the reaction of unsaturated fatty acids in the structure of phospholipids, glycolipids, glycerides, and steroids in the membrane by free oxygen radicals to various products such as peroxides, alcohols, aldehydes, hydroxy fatty acids, ethane, and pentane [106]. The major intracellular antioxidants found in organisms include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione S transferase, glucose 6 phosphate dehydrogenase, and paraoxonase enzymes. Vitamin E, ferritin, transferrin, haptoglobin, uric acid, ceruloplasmin, glutathione, albumin, bilirubin, and β-carotene are antioxidant defenses in the Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants http://dx.doi.org/10.5772/intechopen.73311 313 Every month, the oocyte grows and begins to develop in the ovary. However, meiosis-I continues only in the dominant oocyte. This process is inhibited by antioxidants causing an increase in ROS. Thus, antioxidants promote meiosis-II [108]. The ROS produced by the preovulatory follicle is considered an important inducer of ovulation [109]. Thus, ROS do not always cause adverse effects [110]. Recently, ROS have been reported to regulate cell function by controlling the production or activation of substances with biological activity [111]. Oxygen deprivation stimulates follicular angiogenesis, which is important for the growth and development of the follicle in the ovary. While follicular ROS increases apoptosis, glutathione (GSH) and follicle stimulating hormone (FSH) act as a balance in the growing follicle. FSH increases in response to estrogen, which triggers catalase production in the dominant follicle, thereby preventing apoptosis [108]. After ovulation, the corpus luteum synthesizes progesterone. Likewise, ROS, which has a key role in reproduction, is also produced in the corpus luteum [108, 112], and antioxidants play an important role in corpus luteum physiology during the cycle [112–114]. When pregnancy does not occur, the corpus luteum shrinks. During pregnancy, progesterone is continuously synthe- In mammalian species, the main function of the corpus luteum (CL) is the synthesis of progesterone, which is required for the establishment of a uterine environment suitable for the development of the peri-implantation conceptus and the successful progression and maintenance of pregnancy. Progesterone acts on the endometrium to regulate the synthesis of growth factors, cytokines, transport and adhesion proteins, protease inhibitors, hormones, and enzymes, which are primary regulators of conceptus implantation, survival, and development. Thus, compromised CL progesterone production is a potential risk factor for prenatal devel- There should be a rapid decline in the progesterone level for good follicular growth. During the middle luteal phase, superoxide dismutase 1 (SOD1, cofactor Cu or Zn) increases in the CL and decreases during regression [108]. PGF2α is defined as luteolysin because it increases in the CL during regression [115] and inhibits progesterone production by luteal cells. The inhibitory effect of PGF2α on progesterone production by the CL is due in part to increased ROS. In addition, progesterone levels decrease due to the destructive effects of oxidative stressors on luteal cell steroidogenesis [116–118]. ROS can also inhibit progesterone synthesis through inhibition of cytochrome P450, mitochondrial intracellular transport of cholesterol, Although the mechanisms of CL rescue from cell death and maintenance of progesterone production are very complex and vary among mammalian species, substantial evidence extracellular environment [107]. opment and pregnancy outcomes [114]. and degradation of LH receptors [112]. sized [112]. Lactation can be induced in non-pregnant ewes using progesterone and estrogen treatment. Progesterone and 240 mg estradiol benzoate injection once every 3 days for 60 days increased udder size. At the same time, the development of the udder is stimulated daily by injecting 10 mg dexamethasone trimethylacetate or injecting 5 mg estradiol benzoate and 12.5 mg progesterone for 6 days. After this protocol, milk synthesis starts from the udder with physiological size [93]. #### 5. Relationship between progesterone and oxidative stress in ruminants Oxygen is necessary for metabolism in living organism, but oxygen can be damaging to the living organism when it generates reactive oxygen species [94]. Thus, living organism face an oxygen paradox. During vital biochemical reactions in living organisms, intermediate metabolic products called reactive oxygen species (ROS) are generated that cause oxidative damage in many tissues by reducing oxygen. Oxygen is a potentially toxic molecule that is necessary for aerobic organisms to survive. Oxygen species are called "oxidants" or "free radicals" because of the oxidative destruction they provoke. Free radicals occur in all living organisms that metabolize molecular oxygen [95]. Free radicals carry a single number of unshared electrons in their outer orbitals [96]. They are very short-lived reagents, which disrupt the structure of other electrons in the environment of highly energetic electrons. Therefore, free radicals are dangerous to the organism [97]. Free radicals can occur as a by-product in all parts of aerobic cells, during metabolism, or in pathological conditions and they can cause various changes in the cells. As a result, serious cell, tissue, and/or organ damage can occur [98]. Free radicals are highly reactive molecules. Electrons interact with other molecules in the cell generating oxidative damage. They also damage many biological materials such as proteins, lipids, DNA, and nucleotide coenzymes [99]. There are many defensive mechanisms in place to prevent the formation of ROS and damage to the organism. These mechanisms are generally referred to as "antioxidant defense systems" or "antioxidants" [100]. Antioxidants control the metabolism and free radical levels that occur in normal metabolic or pathological conditions and prevent or repair damage that may be caused by these radicals [101–103]. In the organism, the formation rate of free radicals and the rate of their removal are in balance. This condition, called "oxidative balance," prevents the organism from being affected by free radicals. An imbalance between free radical formation and the antioxidant defense mechanism in favor of free radicals is termed "oxidative stress," which in turn leads to tissue damage [104]. Antioxidants are known to have protective effects on lipids, proteins, nucleic acids, and other macromolecules. Antioxidants affect ROS in four ways: scavenger, quencher, restorative, and chain breaker [103]. All biomolecules are exposed to free radicals. However, lipids are most easily affected [105]. The membranes surrounding the cells and organelles contain a large amount of unsaturated fatty acids. The oxygen molecule has a high affinity for lipids in these unsaturated fatty acids in the cell membrane. The binding of oxygen to the double bonds in the unsaturated fatty acids found in tissues is the result of lipid peroxidation. Lipid peroxidation is the reaction of unsaturated fatty acids in the structure of phospholipids, glycolipids, glycerides, and steroids in the membrane by free oxygen radicals to various products such as peroxides, alcohols, aldehydes, hydroxy fatty acids, ethane, and pentane [106]. The major intracellular antioxidants found in organisms include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione S transferase, glucose 6 phosphate dehydrogenase, and paraoxonase enzymes. Vitamin E, ferritin, transferrin, haptoglobin, uric acid, ceruloplasmin, glutathione, albumin, bilirubin, and β-carotene are antioxidant defenses in the extracellular environment [107]. progesterone injection. The highest milk yield was reached 30–35 days after the start of Lactation can be induced in non-pregnant ewes using progesterone and estrogen treatment. Progesterone and 240 mg estradiol benzoate injection once every 3 days for 60 days increased udder size. At the same time, the development of the udder is stimulated daily by injecting 10 mg dexamethasone trimethylacetate or injecting 5 mg estradiol benzoate and 12.5 mg progesterone for 6 days. After this protocol, milk synthesis starts from the udder with physio- 5. Relationship between progesterone and oxidative stress in ruminants the cells. As a result, serious cell, tissue, and/or organ damage can occur [98]. and prevent or repair damage that may be caused by these radicals [101–103]. Oxygen is necessary for metabolism in living organism, but oxygen can be damaging to the living organism when it generates reactive oxygen species [94]. Thus, living organism face an oxygen paradox. During vital biochemical reactions in living organisms, intermediate metabolic products called reactive oxygen species (ROS) are generated that cause oxidative damage in many tissues by reducing oxygen. Oxygen is a potentially toxic molecule that is necessary for aerobic organisms to survive. Oxygen species are called "oxidants" or "free radicals" because of the oxidative destruction they provoke. Free radicals occur in all living organisms that metabolize molecular oxygen [95]. Free radicals carry a single number of unshared electrons in their outer orbitals [96]. They are very short-lived reagents, which disrupt the structure of other electrons in the environment of highly energetic electrons. Therefore, free radicals are dangerous to the organism [97]. Free radicals can occur as a by-product in all parts of aerobic cells, during metabolism, or in pathological conditions and they can cause various changes in Free radicals are highly reactive molecules. Electrons interact with other molecules in the cell generating oxidative damage. They also damage many biological materials such as proteins, lipids, DNA, and nucleotide coenzymes [99]. There are many defensive mechanisms in place to prevent the formation of ROS and damage to the organism. These mechanisms are generally referred to as "antioxidant defense systems" or "antioxidants" [100]. Antioxidants control the metabolism and free radical levels that occur in normal metabolic or pathological conditions In the organism, the formation rate of free radicals and the rate of their removal are in balance. This condition, called "oxidative balance," prevents the organism from being affected by free radicals. An imbalance between free radical formation and the antioxidant defense mechanism in favor of free radicals is termed "oxidative stress," which in turn leads to tissue damage [104]. Antioxidants are known to have protective effects on lipids, proteins, nucleic acids, and other macromolecules. Antioxidants affect ROS in four ways: scavenger, quencher, restorative, and chain breaker [103]. All biomolecules are exposed to free radicals. However, lipids are most easily affected [105]. The membranes surrounding the cells and organelles contain a large amount of unsaturated fatty acids. The oxygen molecule has a high affinity for lipids in these unsaturated fatty acids in the cell membrane. The binding of oxygen to the double bonds in the unsaturated fatty acids found in tissues is the result of lipid peroxidation. Lipid peroxidation is lactation [92]. 312 Sex Hormones in Neurodegenerative Processes and Diseases logical size [93]. Every month, the oocyte grows and begins to develop in the ovary. However, meiosis-I continues only in the dominant oocyte. This process is inhibited by antioxidants causing an increase in ROS. Thus, antioxidants promote meiosis-II [108]. The ROS produced by the preovulatory follicle is considered an important inducer of ovulation [109]. Thus, ROS do not always cause adverse effects [110]. Recently, ROS have been reported to regulate cell function by controlling the production or activation of substances with biological activity [111]. Oxygen deprivation stimulates follicular angiogenesis, which is important for the growth and development of the follicle in the ovary. While follicular ROS increases apoptosis, glutathione (GSH) and follicle stimulating hormone (FSH) act as a balance in the growing follicle. FSH increases in response to estrogen, which triggers catalase production in the dominant follicle, thereby preventing apoptosis [108]. After ovulation, the corpus luteum synthesizes progesterone. Likewise, ROS, which has a key role in reproduction, is also produced in the corpus luteum [108, 112], and antioxidants play an important role in corpus luteum physiology during the cycle [112–114]. When pregnancy does not occur, the corpus luteum shrinks. During pregnancy, progesterone is continuously synthesized [112]. In mammalian species, the main function of the corpus luteum (CL) is the synthesis of progesterone, which is required for the establishment of a uterine environment suitable for the development of the peri-implantation conceptus and the successful progression and maintenance of pregnancy. Progesterone acts on the endometrium to regulate the synthesis of growth factors, cytokines, transport and adhesion proteins, protease inhibitors, hormones, and enzymes, which are primary regulators of conceptus implantation, survival, and development. Thus, compromised CL progesterone production is a potential risk factor for prenatal development and pregnancy outcomes [114]. There should be a rapid decline in the progesterone level for good follicular growth. During the middle luteal phase, superoxide dismutase 1 (SOD1, cofactor Cu or Zn) increases in the CL and decreases during regression [108]. PGF2α is defined as luteolysin because it increases in the CL during regression [115] and inhibits progesterone production by luteal cells. The inhibitory effect of PGF2α on progesterone production by the CL is due in part to increased ROS. In addition, progesterone levels decrease due to the destructive effects of oxidative stressors on luteal cell steroidogenesis [116–118]. ROS can also inhibit progesterone synthesis through inhibition of cytochrome P450, mitochondrial intracellular transport of cholesterol, and degradation of LH receptors [112]. Although the mechanisms of CL rescue from cell death and maintenance of progesterone production are very complex and vary among mammalian species, substantial evidence suggests ROS are key factors in determining the CL lifespan and antioxidants play significant roles in CL physiology during the estrous/menstrual cycle. Luteal ROS production and propagation depend upon several regulating factors, including luteal antioxidants, steroid hormones and cytokines, and their crosstalk. However, it is unknown which of these factors have the greatest contribution to CL function. In addition, the sequence of events leading to the functional and structural luteal regression at the end of the estrous/menstrual cycle is still not clear. The scarce in-vivo reports studying the CL of rats and sheep have shown the importance of antioxidant enzymes in the control of CL function during the peri-implantation period. As a luteal phase defect can impact fertility by preventing implantation and early conceptus development in livestock and humans, this review attempts to address the importance of ROSscavenging antioxidant enzymes in the control of mammalian CL function and integrity [119]. P450scc. Luteal ROS are generated via enzymatic pathways of the mitochondrial cytochrome P450. In the CL, macrophages and luteal cells produce ROS where they can affect progesterone production. Indeed, there is substantial evidence to indicate that ROS regulate steroid hormone biosynthesis in the CL. The induction of ovarian SOD by LH, which in turn could lead to the production of H2O2, suggests that this action is involved in the LH stimulation of progesterone secretion in the CL. Thus, ROS can function beneficially to control the production of progesterone by luteal cells over the course of the reproductive cycle and inhibit progesterone synthesis at the end of the cycle. The O2d radical is reported to be involved in the mechanism Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants http://dx.doi.org/10.5772/intechopen.73311 315 Oxidative stress may affect various physiological functions, such as folliculogenesis and steroidogenesis, in the female reproductive system. High ROS levels may also cause adverse pregnancy outcomes or embryonic/fetal losses [120–122] and are implicated in the etiopathogenesis of cystic ovarian disease [123]. ROS and the oxidative stress index in cows may be higher in the luteal phase than follicular phase, especially when progesterone is high. Again, in the luteal phase, the antioxidant status can be high or low. Imbalances, especially in oxidant and antioxidant capacity, can cause cystic ovarian disease by disturbing physiological events High free radical and low progesterone concentrations were detected in cows identified as repeat breeders. Infertility problems such as repeat breeder are encountered due to a low progesterone level in the critical period of pregnancy and the short life of the CL. All kinds of stress factors cause excessive radical production in high milk-yielding cows. This may be a determining factor for the amount of progesterone synthesized by inhibiting luteal cell function [125, 126]. In another study, the complex arrangement of antioxidant enzymes and compounds in the bovine CL was discussed. In particular, the correlation between antioxidant capacity and progesterone concentration was determined in the luteal phase of the estrous cycle. Findings show that antioxidative mechanisms are activated to cope with oxidative stress, which has a negative effect on steroid hormone synthesis [127]. In support of the previous study, it has been suggested that an antioxidant substance (astaxanthin) promotes progesterone synthesis in bovine luteal cell culture. However, attention has been drawn to the Anestrus is a problem of infertility in which cyclic activity is absent and therefore estrogenprogesterone hormones are not expressed. Non-cyclic Murrah buffaloes were found to have low concentrations of antioxidants such as β-carotene and vitamin E [129]. Oxidative stress biomarkers change in cow milk during the anovulatory and ovulatory estrous cycles. In particular, the SOD levels in cyclic cows are significantly higher than levels in the anovulatory cycle, while the concentrations of lipoperoxides, GSH-Px, and GSH are lower. A low level of lipoperoxides, GSH-Px, and GSH is assumed to be an important event prior to the ovulation response, with high levels of milk SOD concentration in the ovulatory cycle cows [130]. Nitric oxide is synergistic with progesterone and may reduce relaxation by relieving uterine contraction during the paracrine-style secretion phase. In sheep, the regulation of reproductive physiology is related to the effects of oxidative stress [117]. Increased levels of progesterone during pregnancy in sheep and goats as well as increased levels of malondialdehyde (MDA) in fact that the use of antioxidant material at low doses is beneficial [128]. by which LH stimulates progesterone secretion [94, 119]. necessary for ovulation [124]. The production of ATP is derived from the mitochondrial respiratory chain oxidative phosphorylation, which is the main source of oxygen-free radicals and non-radical ROS. The ROS include superoxide anion (dO2d), hydroxyl radical (dOH), nitric oxide (NO), hydrogen peroxide (H2O2), and peroxynitrite (ONOOd). ROS are also produced via enzymatic pathways, including the activity of membrane-bound NADH and NADPH oxidases, the activity of xanthine oxidase, the metabolism of arachidonic acid by lipoxygenases and cyclooxygenases (COX), and the mitochondrial cytochrome P450 [119]. The cause of the ROS concentration increase in the regression phase may be a decrease in the SOD1 concentration. A decrease in SOD1 concentration may be due to an increase in PGF2α or macrophages, or a decrease in blood flow to the ovaries [108]. In the CL, luteal cells and phagocytic leukocytes stimulate the production of a superoxide anion. With decreased blood flow to the ovaries, ROS production increases and causes tissue damage. Concentrations of superoxide dismutase 2 (SOD2, cofactor Mn) in the CL increase to clear the ROS produced in the mitochondria during regression. Along with the complete lysis of the CL, the regressor decreases significantly in the SOD2 cells [110]. The SOD1 enzyme is closely related to progesterone production. SOD2 protects luteal cells from oxidative stress induced inflammation [108]. Aerobic cells are equipped with antioxidant enzymes that control ROS production and prevent their propagation to toxic ROS. The conversion of dO2d to H2O2 by superoxide dismutase (SOD) is the first enzymatic antioxidative pathway. Two different SOD enzymes were identified: copper-zinc-containing SOD (SOD1) is predominantly localized in the cytosol and can be found in mitochondria, and manganese-containing SOD (SOD2) localizes in the mitochondrial matrix. Glutathione peroxidase (GPX) is a group of selenium-containing enzymes that belong to the first antioxidant mechanism preventing the propagation of highly reactive ROS by catalyzing the conversion of H2O2 to H2O and O2. NADH and NADPH are key elements in the control of ROS production and maintenance of the cellular redox state. The mitochondrial NADP+-dependent isocitrate dehydrogenase generates NADPH via oxidative decarboxylation of isocitrate [119]. Like any aerobic cells, those of the CL produce ATP through the respiration of O2 with the consequence of luteal ROS production. The rate-limiting step in steroidogenesis in all steroidogenic organs, including the CL, is the transfer of cholesterol from the outer to the inner mitochondrial membrane where it is converted into pregnenolone by the enzyme cytochrome P450scc. Luteal ROS are generated via enzymatic pathways of the mitochondrial cytochrome P450. In the CL, macrophages and luteal cells produce ROS where they can affect progesterone production. Indeed, there is substantial evidence to indicate that ROS regulate steroid hormone biosynthesis in the CL. The induction of ovarian SOD by LH, which in turn could lead to the production of H2O2, suggests that this action is involved in the LH stimulation of progesterone secretion in the CL. Thus, ROS can function beneficially to control the production of progesterone by luteal cells over the course of the reproductive cycle and inhibit progesterone synthesis at the end of the cycle. The O2d radical is reported to be involved in the mechanism by which LH stimulates progesterone secretion [94, 119]. suggests ROS are key factors in determining the CL lifespan and antioxidants play significant roles in CL physiology during the estrous/menstrual cycle. Luteal ROS production and propagation depend upon several regulating factors, including luteal antioxidants, steroid hormones and cytokines, and their crosstalk. However, it is unknown which of these factors have the greatest contribution to CL function. In addition, the sequence of events leading to the functional and structural luteal regression at the end of the estrous/menstrual cycle is still not clear. The scarce in-vivo reports studying the CL of rats and sheep have shown the importance of antioxidant enzymes in the control of CL function during the peri-implantation period. As a luteal phase defect can impact fertility by preventing implantation and early conceptus development in livestock and humans, this review attempts to address the importance of ROSscavenging antioxidant enzymes in the control of mammalian CL function and integrity [119]. The production of ATP is derived from the mitochondrial respiratory chain oxidative phosphorylation, which is the main source of oxygen-free radicals and non-radical ROS. The ROS include superoxide anion (dO2d), hydroxyl radical (dOH), nitric oxide (NO), hydrogen peroxide (H2O2), and peroxynitrite (ONOOd). ROS are also produced via enzymatic pathways, including the activity of membrane-bound NADH and NADPH oxidases, the activity of xanthine oxidase, the metabolism of arachidonic acid by lipoxygenases and cyclooxygenases The cause of the ROS concentration increase in the regression phase may be a decrease in the SOD1 concentration. A decrease in SOD1 concentration may be due to an increase in PGF2α or macrophages, or a decrease in blood flow to the ovaries [108]. In the CL, luteal cells and phagocytic leukocytes stimulate the production of a superoxide anion. With decreased blood flow to the ovaries, ROS production increases and causes tissue damage. Concentrations of superoxide dismutase 2 (SOD2, cofactor Mn) in the CL increase to clear the ROS produced in the mitochondria during regression. Along with the complete lysis of the CL, the regressor decreases significantly in the SOD2 cells [110]. The SOD1 enzyme is closely related to progesterone production. SOD2 protects luteal cells from oxidative stress induced inflammation [108]. Aerobic cells are equipped with antioxidant enzymes that control ROS production and prevent their propagation to toxic ROS. The conversion of dO2d to H2O2 by superoxide dismutase (SOD) is the first enzymatic antioxidative pathway. Two different SOD enzymes were identified: copper-zinc-containing SOD (SOD1) is predominantly localized in the cytosol and can be found in mitochondria, and manganese-containing SOD (SOD2) localizes in the mitochondrial matrix. Glutathione peroxidase (GPX) is a group of selenium-containing enzymes that belong to the first antioxidant mechanism preventing the propagation of highly reactive ROS by catalyzing the conversion of H2O2 to H2O and O2. NADH and NADPH are key elements in the control of ROS production and maintenance of the cellular redox state. The mitochondrial NADP+-dependent isocitrate dehydrogenase generates NADPH via oxidative decarboxylation Like any aerobic cells, those of the CL produce ATP through the respiration of O2 with the consequence of luteal ROS production. The rate-limiting step in steroidogenesis in all steroidogenic organs, including the CL, is the transfer of cholesterol from the outer to the inner mitochondrial membrane where it is converted into pregnenolone by the enzyme cytochrome (COX), and the mitochondrial cytochrome P450 [119]. 314 Sex Hormones in Neurodegenerative Processes and Diseases of isocitrate [119]. Oxidative stress may affect various physiological functions, such as folliculogenesis and steroidogenesis, in the female reproductive system. High ROS levels may also cause adverse pregnancy outcomes or embryonic/fetal losses [120–122] and are implicated in the etiopathogenesis of cystic ovarian disease [123]. ROS and the oxidative stress index in cows may be higher in the luteal phase than follicular phase, especially when progesterone is high. Again, in the luteal phase, the antioxidant status can be high or low. Imbalances, especially in oxidant and antioxidant capacity, can cause cystic ovarian disease by disturbing physiological events necessary for ovulation [124]. High free radical and low progesterone concentrations were detected in cows identified as repeat breeders. Infertility problems such as repeat breeder are encountered due to a low progesterone level in the critical period of pregnancy and the short life of the CL. All kinds of stress factors cause excessive radical production in high milk-yielding cows. This may be a determining factor for the amount of progesterone synthesized by inhibiting luteal cell function [125, 126]. In another study, the complex arrangement of antioxidant enzymes and compounds in the bovine CL was discussed. In particular, the correlation between antioxidant capacity and progesterone concentration was determined in the luteal phase of the estrous cycle. Findings show that antioxidative mechanisms are activated to cope with oxidative stress, which has a negative effect on steroid hormone synthesis [127]. In support of the previous study, it has been suggested that an antioxidant substance (astaxanthin) promotes progesterone synthesis in bovine luteal cell culture. However, attention has been drawn to the fact that the use of antioxidant material at low doses is beneficial [128]. Anestrus is a problem of infertility in which cyclic activity is absent and therefore estrogenprogesterone hormones are not expressed. Non-cyclic Murrah buffaloes were found to have low concentrations of antioxidants such as β-carotene and vitamin E [129]. Oxidative stress biomarkers change in cow milk during the anovulatory and ovulatory estrous cycles. In particular, the SOD levels in cyclic cows are significantly higher than levels in the anovulatory cycle, while the concentrations of lipoperoxides, GSH-Px, and GSH are lower. A low level of lipoperoxides, GSH-Px, and GSH is assumed to be an important event prior to the ovulation response, with high levels of milk SOD concentration in the ovulatory cycle cows [130]. Nitric oxide is synergistic with progesterone and may reduce relaxation by relieving uterine contraction during the paracrine-style secretion phase. In sheep, the regulation of reproductive physiology is related to the effects of oxidative stress [117]. Increased levels of progesterone during pregnancy in sheep and goats as well as increased levels of malondialdehyde (MDA) in placentomas have been reported [131]. Significant reductions in antioxidant substances may occur in placentomas during early gestation in sheep. These changes in the antioxidant enzymatic defenses of the placenta are thought to be an adaptation to the oxidative stress caused by ROS in early pregnancy [132]. According to the results obtained, pregnancy may be a stressor and it may be beneficial to support progesterone production with antioxidants in order to mitigate oxidative stress effects [131, 132]. The application of antioxidant vitamins in estrus synchronization during the breeding season reduces free radical levels, increases pregnancy performance, and increases the litter size in Tuj sheep [133]. However, β-carotene and vitamin E applications before estrus synchronization did not cause significant changes in plasma MDA levels in sheep during the breeding season [66]. Serum progesterone concentration increases after administration of intravaginal progesterone-releasing devices for estrus synchronization in goats increases oxidants such as eNOS activity, NO, MDA, and total oxidation status total oxidation status decreased [68, 69]. Short-term PRID treatments increase serum progesterone levels but decrease total antioxidant capacity in dairy heifers [23]. [2] Kuru M. Holstein ırkı düvelerde progesteron ile kombine edilen Cosynch protokolünde ovulasyonun uyarılması amacıyla hCG veya GnRH hormonu kullanılmasının gebelik Clinical Use of Progesterone and Its Relation to Oxidative Stress in Ruminants http://dx.doi.org/10.5772/intechopen.73311 317 [3] Salmon AB, Richardson A, Pérez VI. Update on the oxidative stress theory of aging: Does oxidative stress play a role in aging or healthy aging? Free Radical Biology and [4] Brewer GJ: Epigenetic oxidative redox shift (EORS) theory of aging unifies the free radical and insulin signaling theories. Experimental Gerontology. 2010;45:173-179. DOI: [5] Descamps A, Cespuglio R. Influence of aging on the sleep rebound induced by immobilization stress in the rat. Brain Research. 2010;1335:14-23. DOI: 10.1016/j.brainres.2010.03.087 [6] Garrido P, de Blas M, Del Arco A, Segovia G, Mora F. Aging increases basal but not stress-induced levels of corticosterone in the brain of the awakened rat. Neurobiology of [7] Durieux J, Wolff S, Dillin A. The cell-non-autonomous nature of electron transport chain- [8] Woo DK, Shadel GS. Mitochondrial stress signals revised an old aging theory. Cell. [9] Huang N, Pandey AV, Agrawal V, Reardon W, Lapunzina PD, Mowat D, Jabs EW, Van Vliet G, Sack J, Flück CE, Miller WL. Diversity and function of mutations in P450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogene- sis. American Journal of Human Genetics. 2005;76:729-749. DOI: 10.1086/429417 [10] Miyamoto A, Shirasuna K: Luteolysis in the cow: A novel concept of vasoactive mole- [11] Kuru M, Oral H, Kulaksiz R. Mechanism of luteolysis and vasoactive agents in cows. Atatürk Üniversitesi Veteriner Bilimleri Dergisi. 2014;9:141-148. DOI: 10.17094/avbd.01918 [12] Alaçam E, Saban E, Ay SS. The evaluation of various radioimmunoassay progesterone kits for different stage of reproductive period in the cow, sheep and bitch. Ankara Üniversitesi Veteriner Fakültesi Dergisi. 2009;56:37-41. DOI: 10.1501/Vetfak\_0000002183 [13] Öcal H, Doğan H, Saat N, Aydin M. Progesterone, progestins and antiprogestins. Turkiye Klinikleri Journal of Veterinary Sciences – Obstetrics and Gynecology – Special [14] Alaçam E. Koyun ve keçilerde döl verimi. In: Aytuğ CN, Alaçam E, Özkoç Ü, Yalçın BC, Gökçen H, Türker H, editors. Koyun-Keçi Hastalıkları ve Yetiştiriciliği. İstanbul: TÜM VET Hayvancılık Hizmetleri Yayını, Teknografik Matbaası; 1990. pp. 355-366 (Book in Turkish) [15] Kalkan C, Horoz H. Pubertas ve seksüel sikluslar. In: Alaçam E, editor. Evcil Hayvanlarda Doğum ve İnfertilite. Ankara: Medisan, 2015. pp. 23-40 (Book in Turkish) oranları üzerine etkisi [thesis in Turkish]. Kars: Kafkas University; 2015 Medicine. 2010;48:642-655. DOI: 10.1016/j.freeradbiomed.2009.12.015 Aging. 2010;33:375-382. DOI: 10.1016/j.neurobiolaging.2010.02.015 2011;144:11-12. DOI: 10.1016/j.cell.2010.12.023 cules. Animal Reproduction. 2009;6:47-59 Topics. 2015;1:60-86 mediated longevity. Cell. 2011;144:79-91. DOI: 10.1016/j.cell.2010.12.016 10.1016/j.exger.2009.11.007 #### 6. Conclusion Progesterone is synthesized in the luteal phase and is an important hormone required for the continuity of pregnancy in ruminants. It is widely used in cattle for the purpose of estrus synchronization. In addition, this hormone, which has many uses in clinical practice, continues to be explored in ruminants. As time progresses, more detailed facts about the complex effects of progesterone in the organism, its use in clinical practice in ruminants, and the relationship of progesterone to oxidative stress in ruminants will be revealed. #### Author details Mushap Kuru1 \, Abdulsamed Kükürt<sup>2</sup> , Hasan Oral<sup>1</sup> and Metin Öğün2 \Address all correspondence to: [email protected] 1 Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Kafkas University, Kars, Turkey 2 Department of Biochemistry, Faculty of Veterinary Medicine, Kafkas University, Kars, Turkey #### References [1] Helbling IM, Luna JA. Progesterone administration in planned reproduction of cattle. In: Rivera C, editor. Progesterone: Functions, Uses and Research Insights. USA: Nova Science Publishers; 2017. pp. 69-125 [2] Kuru M. Holstein ırkı düvelerde progesteron ile kombine edilen Cosynch protokolünde ovulasyonun uyarılması amacıyla hCG veya GnRH hormonu kullanılmasının gebelik oranları üzerine etkisi [thesis in Turkish]. Kars: Kafkas University; 2015 placentomas have been reported [131]. Significant reductions in antioxidant substances may occur in placentomas during early gestation in sheep. These changes in the antioxidant enzymatic defenses of the placenta are thought to be an adaptation to the oxidative stress caused by ROS in early pregnancy [132]. According to the results obtained, pregnancy may be a stressor and it may be beneficial to support progesterone production with antioxidants in order to mitigate oxidative stress effects [131, 132]. The application of antioxidant vitamins in estrus synchronization during the breeding season reduces free radical levels, increases pregnancy performance, and increases the litter size in Tuj sheep [133]. However, β-carotene and vitamin E applications before estrus synchronization did not cause significant changes in plasma MDA levels in sheep during the breeding season [66]. Serum progesterone concentration increases after administration of intravaginal progesterone-releasing devices for estrus synchronization in goats increases oxidants such as eNOS activity, NO, MDA, and total oxidation status total oxidation status decreased [68, 69]. Short-term PRID treatments increase serum progesterone Progesterone is synthesized in the luteal phase and is an important hormone required for the continuity of pregnancy in ruminants. It is widely used in cattle for the purpose of estrus synchronization. In addition, this hormone, which has many uses in clinical practice, continues to be explored in ruminants. As time progresses, more detailed facts about the complex effects of progesterone in the organism, its use in clinical practice in ruminants, and the relationship of 1 Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Kafkas 2 Department of Biochemistry, Faculty of Veterinary Medicine, Kafkas University, Kars, [1] Helbling IM, Luna JA. Progesterone administration in planned reproduction of cattle. In: Rivera C, editor. Progesterone: Functions, Uses and Research Insights. USA: Nova Sci- , Hasan Oral<sup>1</sup> and Metin Öğün2 levels but decrease total antioxidant capacity in dairy heifers [23]. 316 Sex Hormones in Neurodegenerative Processes and Diseases progesterone to oxidative stress in ruminants will be revealed. \, Abdulsamed Kükürt<sup>2</sup> ence Publishers; 2017. pp. 69-125 \Address all correspondence to: [email protected] 6. Conclusion Author details University, Kars, Turkey Mushap Kuru1 Turkey References [16] Canooğlu E, Sarıbay K. 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DOI: 10.1590/S1516-35982010001300038 [118] Hayashi K, Miyamoto A, Konari A, Ohtani M, Fukui Y. Effect of local interaction of reactive oxygen species with prostaglandin F2α on the release of progesterone in ovine corpora lutea in vivo. Theriogenology. 2003;59:1335-1344. DOI: 10.1016/S0093-691X(02) [119] Sugino N, Karube-Harada A, Taketani T, Sakata A, Nakamura Y. Withdrawal of ovarian steroids stimulates prostaglandin F2α production through nuclear factor-κB activation via oxygen radicals in human endometrial stromal cells: Potential relevance to menstruation. The Journal of Reproduction and Development. 2004;50:215-225. DOI: 10.1262/ [120] Agarwal A, Gupta S, Sharma R. Role of oxidative stress in female reproduction. Reproductive Biology and Endocrinology. 2005;3:1-21. DOI: 10.1186/1477-7827-3-28 [121] Celi P, Merlo M, Da Dalt L, Stefani A, Barbato O, Gabai G. Relationship between late embryonic mortality and the increase in plasma advanced oxidised protein products (AOPP) in dairy cows. Reproduction, Fertility, and Development. 2011;23:527-533. DOI: nal. 2006;77:556-565. DOI: 10.1111/j.1740-0929.2006.00386.x tion. 1990;42:792-800. DOI: 10.1095/biolreprod42.5.792 559-568. DOI: 10.1677/joe.1.06550 326 Sex Hormones in Neurodegenerative Processes and Diseases Press; 2004. pp. 261-265 jrf.0.1200239 01173-1 jrd.50.215 10.1071/RD10268 United States of America. 2011;108:1462-1467. DOI: 10.1073/pnas.1017213108 Chapter 14 Provisional chapter Sex Hormones and Inner Ear Sex Hormones and Inner Ear Additional information is available at the end of the chapter There are increasing evidence of interactions between sex hormones and the structure and function of inner ear, especially in hearing impairment and balance disorders. In this chapter, we will discuss the mechanism of sex hormones on the inner ear, describe both clinical and basic research that has led us to our current understanding, and conclude with future perspectives on avenues of investigation that may lead to innovative treatments on the hearing loss, tinnitus, and dizziness resulted from the changes in estrogen and progesterone levels. The presence of estrogen receptors α and β has earlier been shown in the inner ear of mice. Expression of estrogen receptors (ER) correlates with the protection of auditory function. Estrogen may have certain protective effects on the hearing. Evidence for the treatment of sex hormone-induced symptoms is principally restricted to case reports and retrospective studies. Recognition and understanding of sex hormone-related inner ear problems will allow otologists to notice and manage these patients. Also, basic studies on the mechanism of how sex hormones act on inner ear provide the way to further prevent and treat on hearing impairment and balance disorders. High-quality DOI: 10.5772/intechopen.74157 evidence for their management is limited, with further research required. Keywords: sex hormones, inner ear, hearing, balancing functions, mechanism, treatment Hearing loss, vertigo, dizziness, and tinnitus are the common symptoms in otology clinics. The cochlea and vestibule in the inner ear are filled with endolymph and perilymph, and the homeostasis of the water and blood circulation in the inner ear is essential for maintaining its > © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Additional information is available at the end of the chapter Zi-Yu He and Dong-Dong Ren Zi-Yu He and Dong-Dong Ren http://dx.doi.org/10.5772/intechopen.74157 Abstract methods 1. Introduction #### Chapter 14 Provisional chapter #### Sex Hormones and Inner Ear Sex Hormones and Inner Ear Zi-Yu He and Dong-Dong Ren Zi-Yu He and Dong-Dong Ren Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.74157 #### Abstract There are increasing evidence of interactions between sex hormones and the structure and function of inner ear, especially in hearing impairment and balance disorders. In this chapter, we will discuss the mechanism of sex hormones on the inner ear, describe both clinical and basic research that has led us to our current understanding, and conclude with future perspectives on avenues of investigation that may lead to innovative treatments on the hearing loss, tinnitus, and dizziness resulted from the changes in estrogen and progesterone levels. The presence of estrogen receptors α and β has earlier been shown in the inner ear of mice. Expression of estrogen receptors (ER) correlates with the protection of auditory function. Estrogen may have certain protective effects on the hearing. Evidence for the treatment of sex hormone-induced symptoms is principally restricted to case reports and retrospective studies. Recognition and understanding of sex hormone-related inner ear problems will allow otologists to notice and manage these patients. Also, basic studies on the mechanism of how sex hormones act on inner ear provide the way to further prevent and treat on hearing impairment and balance disorders. High-quality evidence for their management is limited, with further research required. DOI: 10.5772/intechopen.74157 Keywords: sex hormones, inner ear, hearing, balancing functions, mechanism, treatment methods #### 1. Introduction Hearing loss, vertigo, dizziness, and tinnitus are the common symptoms in otology clinics. The cochlea and vestibule in the inner ear are filled with endolymph and perilymph, and the homeostasis of the water and blood circulation in the inner ear is essential for maintaining its © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hearing and equilibrium functions [1]. There are more evidence of interactions between sex hormones and the function of the inner ear, especially in the mechanism of hearing impairment and balance disorders in old women and pregnant women. Is the female sex steroid estrogen the key to preserved hearing in the aging human? Is the hearing loss more profound in elderly males than females? Is the hearing loss easier spontaneously recovered in pregnant women? All these questions remain unanswered. In this chapter, we will discuss the mechanism of sex hormones on the inner ear, describe both clinical and basic research that has led us to our current understanding, and conclude with future perspectives on avenues of investigation that will contribute to stratification strategies on the hearing loss, tinnitus, autophony, and dizziness resulted from the changes in sex hormone levels. may counteract this effect. Estrogen may act as an auditory protectant, which influences the inner Sex Hormones and Inner Ear 331 http://dx.doi.org/10.5772/intechopen.74157 Estrogens are mediated through estrogen receptors (ERs). There are at least three and possibly four distinct estrogen receptors. The most common estrogen receptors are ERα, which is encoded by a gene on chromosome 6, and ERβ, encoded by a gene on chromosome 14. Other ERs include G protein-coupled receptor (GFER, GPR30, and a putative receptor (ER-X), which has been studied mostly in brain [5]. Estrogen receptors α and β containing cells were also found in the inner ear, with a specific distribution pattern, both in auditory pathways and in the water/ion regulating areas. The presence of ERs in the inner ear cell nuclei implies that estrogen may have an influence on the inner ear and auditory functions. Estrogen receptors have been found in the inner ear of rats and mice in different cell types, including inner and outer hair cells, stria vascularis, spiral ligament, Reissner's membrane, and spiral ganglion cells especially type I cells [5]. Moreover, estrogen receptors in the auditory epithelium of vertebrates also occur in fishes [6], cichlid [7], songbird [5], and rodents [8], which suggest a widespread occurrence of steroid-dependent auditory plasticity among the vertebrates. But in adult human inner ear, ERα-containing cells were only found in the spiral ganglion, and ERβ-containing cells in the stria vascularis selectively, which are important for hearing transmission and inner ear homeostasis [8]. It has been shown that there is less expression of ERα in strial marginal cells, outer hair cells, and type II ganglion cells [9]. ERα and ERβ are regulated depending on the stage of maturation, development, and pregnancy, suggesting that estrogen may have an effect on the cochlea during different life time. ERs were not found in the cochlea of the growing fetus, which implied that estrogen does not have an influence on the cochlea during the period of gestation [10]. Estrogen receptor mRNA is expressed in supporting cells The expression of ERα was found in forebrain nuclei, including anterior parvocellular (PPa) and anterior tuberal (AT), which are sites of the integration between auditory and vocal motor system [11], and anterior tuberal is densely innervated by the neuropeptides, arginine vasotocin and isotocin, which modulate vocal motor patterning in midshipman [12–14]. Also, in some gene knockout studies, estradiol plays an important role in the regulation of both vasopressin and oxytocin, especially by ERα in limbic regions [15, 16] and by ERβ in hypotha- The expression of ERs in known target organs is influenced by the amount of estrogen in the serum. Lots of studies have shown a gender difference in hearing function. And some of them suggested that part of this variation was because of the difference in estrogen levels between the two genders. As for the expression pattern of ERs, there is no gender- or age-related difference to be found. However, the fluorescence intensity of ERα was stronger in female mice than in young male ones. To compare with, ERβ showed no significant difference. Also, the expression of ERs decreased with age. In the old mice, the fluorescence intensities of ERs Many clinical and basic studies have proved that estradiol plays an important role in auditory physiology, neuronal plasticity, and the metabolic levels of neurotransmitters [18, 19]. ERα might change cochlea and vestibular sensory transduction, and ERβ may have a neuroprotective effect with similar functions within the saccular epithelium of midshipman fish [6]. were significantly decreased in both male and female [9]. lamic areas [17]. ear at different levels, at the cochlea, vestibular organs, and more proximal levels. #### 2. How the different sex hormones may influence the inner ear The sex hormones, including estrogen, progesterone, and androgen, are known to be implicated in normal auditory function in different proportions [2]. It is the balance of all three hormones in the body that promotes human health and vitality, including inner ear functions. Estrogen is usually thought of a "female" hormone; it may act as an auditory protectant; it is made in the ovaries, adrenal glands, and fat cells; and its levels are higher in those of reproductive age, which is helpful to prevent bone loss and works toward maintaining good cholesterol levels. Estrogens are known to facilitate the loss of intravascular fluid into the extravascular space, producing edema; however, blood vessel permeability, blood circulation, or inflammation has been reported to be related to the inner ear diseases. Progesterone is the sex steroid frequently mentioned for sexual health. In women, it is produced in the ovaries and through ovulation, which performs different benefits in balancing the unwanted effects of estrogen, helping the body use fat for energy, maintaining healthy weight, promoting restful sleep, and protecting against breast and uterine cancer; however, it may have a negative effect on hearing [3]. Androgen is a "male" hormone, the primary and most well-known androgen is testosterone, produced in the testicles and to a lesser degree in the adrenal glands, which helps build muscle tome, increases energy, contributes to a healthy libido, and aids in sperm production [4]. Healthy levels are also important in women; testosterone is produced in the female ovaries and a small amount is made in the adrenal glands, which helps to increase libido, promote musculoskeletal tone and strength, and raise energy levels. When testosterone is too high, however, it can lead to acne, unwanted hair on the face and body, polycystic ovaries with resulting interference of ovulation, and aggression among other concerns. Getting all three sex hormones balanced can be helpful for both men and women, and the results often offer clues on how to prevent unwanted inner ear symptoms in the future. #### 2.1. Estrogen Estrogens influence physiological functions of many organs and systems in both female and male, including the skeletal, cardiovascular, and nervous systems, as well as the male urogenital tracts, mammary glands, and female reproductive organs. Estrogen could lead to neural excitation and thus facilitate auditory transmission, but the possible increase in neurosteroids in the brainstem may counteract this effect. Estrogen may act as an auditory protectant, which influences the inner ear at different levels, at the cochlea, vestibular organs, and more proximal levels. hearing and equilibrium functions [1]. There are more evidence of interactions between sex hormones and the function of the inner ear, especially in the mechanism of hearing impairment and balance disorders in old women and pregnant women. Is the female sex steroid estrogen the key to preserved hearing in the aging human? Is the hearing loss more profound in elderly males than females? Is the hearing loss easier spontaneously recovered in pregnant women? All these questions remain unanswered. In this chapter, we will discuss the mechanism of sex hormones on the inner ear, describe both clinical and basic research that has led us to our current understanding, and conclude with future perspectives on avenues of investigation that will contribute to stratification strategies on the hearing loss, tinnitus, autophony, and dizzi- ness resulted from the changes in sex hormone levels. 330 Sex Hormones in Neurodegenerative Processes and Diseases on how to prevent unwanted inner ear symptoms in the future. 2.1. Estrogen 2. How the different sex hormones may influence the inner ear The sex hormones, including estrogen, progesterone, and androgen, are known to be implicated in normal auditory function in different proportions [2]. It is the balance of all three hormones in the body that promotes human health and vitality, including inner ear functions. Estrogen is usually thought of a "female" hormone; it may act as an auditory protectant; it is made in the ovaries, adrenal glands, and fat cells; and its levels are higher in those of reproductive age, which is helpful to prevent bone loss and works toward maintaining good cholesterol levels. Estrogens are known to facilitate the loss of intravascular fluid into the extravascular space, producing edema; however, blood vessel permeability, blood circulation, or inflammation has been reported to be related to the inner ear diseases. Progesterone is the sex steroid frequently mentioned for sexual health. In women, it is produced in the ovaries and through ovulation, which performs different benefits in balancing the unwanted effects of estrogen, helping the body use fat for energy, maintaining healthy weight, promoting restful sleep, and protecting against breast and uterine cancer; however, it may have a negative effect on hearing [3]. Androgen is a "male" hormone, the primary and most well-known androgen is testosterone, produced in the testicles and to a lesser degree in the adrenal glands, which helps build muscle tome, increases energy, contributes to a healthy libido, and aids in sperm production [4]. Healthy levels are also important in women; testosterone is produced in the female ovaries and a small amount is made in the adrenal glands, which helps to increase libido, promote musculoskeletal tone and strength, and raise energy levels. When testosterone is too high, however, it can lead to acne, unwanted hair on the face and body, polycystic ovaries with resulting interference of ovulation, and aggression among other concerns. Getting all three sex hormones balanced can be helpful for both men and women, and the results often offer clues Estrogens influence physiological functions of many organs and systems in both female and male, including the skeletal, cardiovascular, and nervous systems, as well as the male urogenital tracts, mammary glands, and female reproductive organs. Estrogen could lead to neural excitation and thus facilitate auditory transmission, but the possible increase in neurosteroids in the brainstem Estrogens are mediated through estrogen receptors (ERs). There are at least three and possibly four distinct estrogen receptors. The most common estrogen receptors are ERα, which is encoded by a gene on chromosome 6, and ERβ, encoded by a gene on chromosome 14. Other ERs include G protein-coupled receptor (GFER, GPR30, and a putative receptor (ER-X), which has been studied mostly in brain [5]. Estrogen receptors α and β containing cells were also found in the inner ear, with a specific distribution pattern, both in auditory pathways and in the water/ion regulating areas. The presence of ERs in the inner ear cell nuclei implies that estrogen may have an influence on the inner ear and auditory functions. Estrogen receptors have been found in the inner ear of rats and mice in different cell types, including inner and outer hair cells, stria vascularis, spiral ligament, Reissner's membrane, and spiral ganglion cells especially type I cells [5]. Moreover, estrogen receptors in the auditory epithelium of vertebrates also occur in fishes [6], cichlid [7], songbird [5], and rodents [8], which suggest a widespread occurrence of steroid-dependent auditory plasticity among the vertebrates. But in adult human inner ear, ERα-containing cells were only found in the spiral ganglion, and ERβ-containing cells in the stria vascularis selectively, which are important for hearing transmission and inner ear homeostasis [8]. It has been shown that there is less expression of ERα in strial marginal cells, outer hair cells, and type II ganglion cells [9]. ERα and ERβ are regulated depending on the stage of maturation, development, and pregnancy, suggesting that estrogen may have an effect on the cochlea during different life time. ERs were not found in the cochlea of the growing fetus, which implied that estrogen does not have an influence on the cochlea during the period of gestation [10]. Estrogen receptor mRNA is expressed in supporting cells with similar functions within the saccular epithelium of midshipman fish [6]. The expression of ERα was found in forebrain nuclei, including anterior parvocellular (PPa) and anterior tuberal (AT), which are sites of the integration between auditory and vocal motor system [11], and anterior tuberal is densely innervated by the neuropeptides, arginine vasotocin and isotocin, which modulate vocal motor patterning in midshipman [12–14]. Also, in some gene knockout studies, estradiol plays an important role in the regulation of both vasopressin and oxytocin, especially by ERα in limbic regions [15, 16] and by ERβ in hypothalamic areas [17]. The expression of ERs in known target organs is influenced by the amount of estrogen in the serum. Lots of studies have shown a gender difference in hearing function. And some of them suggested that part of this variation was because of the difference in estrogen levels between the two genders. As for the expression pattern of ERs, there is no gender- or age-related difference to be found. However, the fluorescence intensity of ERα was stronger in female mice than in young male ones. To compare with, ERβ showed no significant difference. Also, the expression of ERs decreased with age. In the old mice, the fluorescence intensities of ERs were significantly decreased in both male and female [9]. Many clinical and basic studies have proved that estradiol plays an important role in auditory physiology, neuronal plasticity, and the metabolic levels of neurotransmitters [18, 19]. ERα might change cochlea and vestibular sensory transduction, and ERβ may have a neuroprotective effect in the inner ear [9]. ERβ protects the auditory system from acoustic trauma in young male and female mice. ERβ in accordance with brain-derived neurotrophic factor (BDNF) promotes neuronal plasticity and protection against trauma in the auditory system [20]. But experimental estrogen-induced hyperprolactinemia leads to hearing loss in the guinea pig. It suggests that otic capsule and hair cell pathology related with estrogen-induced prolonged hyperprolactinemia and conditions such as pregnancy may lead to similar auditory pathology [21]. 17β-estradiol leads to adjustments in the molecular biology of the cochlea and the inferior colliculus of mouse accompanied with behavioral alternations [22]. Estrogen-related receptor gama (ESSR) plays a role in maintenance of hearing in both humans and mice [23]. androgens is testosterone, and by far the highest production of testosterone is in the testes. Testosterone has two main actions: the initiation of spermatogenesis and the development and maintenance of secondary sexual characteristics. In order to achieve the second group of actions, testosterone must be converted to 5α-dihydrotestosterone (DHT). This conversion happens outside the testes, in peripheral tissues. Furthermore, both testosterone and DHT act Sex Hormones and Inner Ear 333 http://dx.doi.org/10.5772/intechopen.74157 In contrast to these well-known effects of estradiol on hearing function, relatively little is known about how androgens might influence hearing or whether androgen receptors (AR) are also expressed in the inner ear of vertebrates. The lack of regenerative ability of adult mammalian cochlea and the irreversible degeneration of cochlear sensory hair cells leads to permanent hearing loss. Whether the androgen receptors (ARs) establish in the inner ear, there are many studies on it, in a transcriptomic analysis of the developing and adult mouse cochlear sensory epithelia, the adult cochlear sensory epithelium overexpressed 2542 transcripts including new transcripts, such as AR, which previously were not reported to be In all major vertebrate animals, androgen receptors have been identified in neural circuits that shape vocalization. Many of those nuclei mentioned above are part of the known vocal and auditory circuit in midshipman. The distribution of androgen receptor mRNA supports that androgens modulate behaviorally defined vocal, auditory, and neuroendocrine circuits in Additionally, testosterone in serum increased neural thresholds in females in a frequency-specific way [29]. And hyperandrogenism may be responsible for the elevation of hearing threshold, particularly in the high frequency, in patients with polycystic ovary syndrome [30–32]. On the contrary, hyperandrogenism did not seem to affect otoacoustic emission levels or the medial In audiology, the usage of biomedical interventions and biotherapeutic methods could play an important role in modulating or preventing some kinds of hearing loss. Planar cell polarity is of high importance as it regulates cochlea extension and coordinates orientation of sensory hair cells in the inner ear. If we could use the effect of sex hormone in the inner ear, the establishment of ectopic hair cell-like cell polarity could be built. Testosterone is related to the neuroprotection and regeneration in central nervous system. We could promote an increase in hair cell-like cell polarity in the LER through proliferation and transdifferentiation by using testosterone-3-(Ocarboxymethyl) oxime bovine serum albumin and Math1 treatment [34]. In the treatment of immune-mediated sensorineural hearing loss, it was confirmed that testosterone has the preventive and therapeutic effects induced by sensitization using bovine inner ear antigens [35]. 3. Physiological variation in sex hormones and effect on inner ear The levels of sex hormones vary in response to endogenous and exogenous stimuli and many vary in a cyclic fashion. The endocrine changes related to reproductive function (ovarian cycle, pregnancy, and menopause) could in turn affect auditory and balance function. Additionally, on the same receptor, the androgen receptor (AR). expressed in the adult cochlea [27]. teleost fish and vertebrates in general [28]. olivocochlear reflex response in adult female subjects [33]. #### 2.2. Progesterone Progesterone is secreted principally by the granulosa lutein cells of the corpus luteum, which are formed from granulosa cells after the luteinizing hormone surge. Progesterone is the main hormone of pregnancy, and in pregnancy, after week 8, the placenta replaces the corpus luteum as the main source of progesterone. Several steroids have similar properties and are together classified as the "progestogens." These include 17α-hydroxyprogesterone and pregnenolone as well as progesterone itself. The two main progesterone receptors are progesterone receptor-A and progesterone receptor-B. And, there are two isoforms of the progesterone receptor encoded by the same gene, but with different start sites for transcription, hence the increased size of progesterone receptor-B compared with progesterone receptor-A. Expression of the progesterone receptor is regulated by estrogens, while progesterone receptors have an important effect, mediated by progesterone receptor-A, in inhibiting the proliferative actions of estrogen. For this reason, progesterone is nearly always given in addition to estrogen therapy, for example, in the oral contraceptive pill and in hormone replacement therapy. The presence of progesterone as a component in hormone replacement therapy leads to poorer hearing in aged women, affecting both the peripheral and central auditory system, and it interferes with the perception of speech in background noise [21]. Nevertheless, there is no direct nuclear effect of progesterone in the inner ear. There is no nuclear progesterone receptor being found in human or rat stria vascularis, organ of Corti or spiral ganglion with immunohistochemistry, or polymerase chain reaction (PCR). But, progesterone receptor-B is being found with Western blot in the cochlea. It probably indicates the staining in the cochlea bone. In this case, the effect of progesterone on hearing is probably not relevant to the action in the inner ear [25]. Progesterone receptors are important for integration of external signals and internal physiological cues in the brain to output an appropriate behavior. In a study using the frog, Physalaemus pustulosus, as a model system, progesterone receptor immunoreactivity was found in key brain regions known to modulate the processing of auditory clues [26]. #### 2.3. Androgen Androgens, which are produced by Leydig cells, like all steroid hormones, are made from cholesterol. A range of androgens is made in the body and, although most of these come from the testes, some are made in the adrenal cortex. The most potent and important of these androgens is testosterone, and by far the highest production of testosterone is in the testes. Testosterone has two main actions: the initiation of spermatogenesis and the development and maintenance of secondary sexual characteristics. In order to achieve the second group of actions, testosterone must be converted to 5α-dihydrotestosterone (DHT). This conversion happens outside the testes, in peripheral tissues. Furthermore, both testosterone and DHT act on the same receptor, the androgen receptor (AR). in the inner ear [9]. ERβ protects the auditory system from acoustic trauma in young male and female mice. ERβ in accordance with brain-derived neurotrophic factor (BDNF) promotes neuronal plasticity and protection against trauma in the auditory system [20]. But experimental estrogen-induced hyperprolactinemia leads to hearing loss in the guinea pig. It suggests that otic capsule and hair cell pathology related with estrogen-induced prolonged hyperprolactinemia and conditions such as pregnancy may lead to similar auditory pathology [21]. 17β-estradiol leads to adjustments in the molecular biology of the cochlea and the inferior colliculus of mouse accompanied with behavioral alternations [22]. Estrogen-related receptor gama (ESSR) plays a Progesterone is secreted principally by the granulosa lutein cells of the corpus luteum, which are formed from granulosa cells after the luteinizing hormone surge. Progesterone is the main hormone of pregnancy, and in pregnancy, after week 8, the placenta replaces the corpus luteum as the main source of progesterone. Several steroids have similar properties and are together classified as the "progestogens." These include 17α-hydroxyprogesterone and preg- The two main progesterone receptors are progesterone receptor-A and progesterone receptor-B. And, there are two isoforms of the progesterone receptor encoded by the same gene, but with different start sites for transcription, hence the increased size of progesterone receptor-B compared with progesterone receptor-A. Expression of the progesterone receptor is regulated by estrogens, while progesterone receptors have an important effect, mediated by progesterone receptor-A, in inhibiting the proliferative actions of estrogen. For this reason, progesterone is nearly always given in addition to estrogen therapy, for example, in the oral contraceptive pill and in hormone replacement therapy. The presence of progesterone as a component in hormone replacement therapy leads to poorer hearing in aged women, affecting both the peripheral and central auditory system, and it interferes with the perception of speech in background noise [21]. Nevertheless, there is no direct nuclear effect of progesterone in the inner ear. There is no nuclear progesterone receptor being found in human or rat stria vascularis, organ of Corti or spiral ganglion with immunohistochemistry, or polymerase chain reaction (PCR). But, progesterone receptor-B is being found with Western blot in the cochlea. It probably indicates the staining in the cochlea bone. In this case, the effect of progesterone on hearing is probably not Progesterone receptors are important for integration of external signals and internal physiological cues in the brain to output an appropriate behavior. In a study using the frog, Physalaemus pustulosus, as a model system, progesterone receptor immunoreactivity was found Androgens, which are produced by Leydig cells, like all steroid hormones, are made from cholesterol. A range of androgens is made in the body and, although most of these come from the testes, some are made in the adrenal cortex. The most potent and important of these in key brain regions known to modulate the processing of auditory clues [26]. role in maintenance of hearing in both humans and mice [23]. 2.2. Progesterone nenolone as well as progesterone itself. 332 Sex Hormones in Neurodegenerative Processes and Diseases relevant to the action in the inner ear [25]. 2.3. Androgen In contrast to these well-known effects of estradiol on hearing function, relatively little is known about how androgens might influence hearing or whether androgen receptors (AR) are also expressed in the inner ear of vertebrates. The lack of regenerative ability of adult mammalian cochlea and the irreversible degeneration of cochlear sensory hair cells leads to permanent hearing loss. Whether the androgen receptors (ARs) establish in the inner ear, there are many studies on it, in a transcriptomic analysis of the developing and adult mouse cochlear sensory epithelia, the adult cochlear sensory epithelium overexpressed 2542 transcripts including new transcripts, such as AR, which previously were not reported to be expressed in the adult cochlea [27]. In all major vertebrate animals, androgen receptors have been identified in neural circuits that shape vocalization. Many of those nuclei mentioned above are part of the known vocal and auditory circuit in midshipman. The distribution of androgen receptor mRNA supports that androgens modulate behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general [28]. Additionally, testosterone in serum increased neural thresholds in females in a frequency-specific way [29]. And hyperandrogenism may be responsible for the elevation of hearing threshold, particularly in the high frequency, in patients with polycystic ovary syndrome [30–32]. On the contrary, hyperandrogenism did not seem to affect otoacoustic emission levels or the medial olivocochlear reflex response in adult female subjects [33]. In audiology, the usage of biomedical interventions and biotherapeutic methods could play an important role in modulating or preventing some kinds of hearing loss. Planar cell polarity is of high importance as it regulates cochlea extension and coordinates orientation of sensory hair cells in the inner ear. If we could use the effect of sex hormone in the inner ear, the establishment of ectopic hair cell-like cell polarity could be built. Testosterone is related to the neuroprotection and regeneration in central nervous system. We could promote an increase in hair cell-like cell polarity in the LER through proliferation and transdifferentiation by using testosterone-3-(Ocarboxymethyl) oxime bovine serum albumin and Math1 treatment [34]. In the treatment of immune-mediated sensorineural hearing loss, it was confirmed that testosterone has the preventive and therapeutic effects induced by sensitization using bovine inner ear antigens [35]. #### 3. Physiological variation in sex hormones and effect on inner ear The levels of sex hormones vary in response to endogenous and exogenous stimuli and many vary in a cyclic fashion. The endocrine changes related to reproductive function (ovarian cycle, pregnancy, and menopause) could in turn affect auditory and balance function. Additionally, there are multiple interactions between the sex hormones involved in these physiological changes and this enhances the possible multidirectional effects on the inner ear. menopausal women with tibolone for 6 months, the improvement was more prominent on the right side in audiometry results at low frequency. It may be explained by differences in distribution of ER in the ear. ERs might be more dense in the right ear, so give better response to estrogen therapy [47]. Many studies have showed that estrogen affects hearing function, especially in the postmenopausal women; a recent study gets a result that there may be hearing lateralization in menopausal women, especially significant improvement on right ear can be explained by lower BMD on that side ear bones in turn better response to estrogen therapy due to this, which may be related to ER concentration and the more dense of type ERα Sex Hormones and Inner Ear 335 http://dx.doi.org/10.5772/intechopen.74157 According to the vestibular function, the level of estradiol and progesterone decreases obviously in postmenopausal women with benign paroxysmal positional vertigo, which can cause Although estrogen has been expected to be benefits on auditory system, both clinician and patients need to take into concern that estrogen may have some unwanted side effects, such as increased risk of uterine cancer. Because in the central nervous system, ERβ is highly expressed in neurons and glial cells. And there is little ERβ in the mature uterus, selective ERβ agonists, Hearing loss appeared in pregnancy is not a commonly reported problem. Some investigators have noticed reversible and physiological sensorineural hearing loss at low frequencies during the period of pregnancy [52, 53]. In some case reports, sudden onset of sensorineural hearing loss during pregnancy has been described [54], and one report concerning a patient who had the hearing loss with each serial pregnancy [55]. But a nationwide populationbased study suggested that sudden sensorineural hearing loss (SSNHL) in pregnancy is rare. SSNHL is defined as sudden, idiopathic, usually unilateral deafness developed at most in 72 hours in previously healthy people [56]. It often happens in the third trimester. And SSNHL in pregnancy does not increase the risks of delivery or subsequent stroke [57]. As for the mechanism, there is a hypothesis implied that SSNHL is connected with the changes in cardiovascular system, hematological system, and/or some other systems because of pregnancy. These changes in pregnancy may evoke disorders of cochlea circulation or cochlea fluid homeostasis resulting in SSNHL [57]. Otosclerosis is one of the most common causes of acquired hearing loss and is widely supposed as being related with pregnancy. Another study revealed that resonance frequency of middle ear was found to be low during the third trimester of the pregnancy. And low resonance frequency informs that the acoustic Tinnitus is another auditory symptom in pregnant patients, with proposed theories of pathogenesis, including hyperdynamic circulation, increase in perilymphatic fluid pressure, and hormonal changes. Clinically, it appears that the hearing loss and tinnitus related to pregnancy can spontaneously recover. As for treatment, it depends on the otorhinolaryngologic doctors to decide whether they should administer steroid drugs for acute hearing loss, as it may recover the inner ear microcirculation disorder, may be a risk factor of BPPV [49]. immittance of the middle ear changes during pregnancy [58]. and/or ERβ in the right ear [48]. then they become available [50, 51]. 3.3. Pregnancy after the delivery [54]. #### 3.1. Ovarian cycle In the ovarian cycle, the levels of estrogen and progesterone in the body have a dynamic regulation. Both clinical and basic studies have proved that the changes of auditory and balance system are attributed to estradiol and progesterone. In other words, the fluctuating hearing levels are evident in females during the ovarian cycle. Across the life span, both women and men undergo transitions in reproductive status related in part to changes in sex hormone levels. There is controversy over how hormonal conditions influence cerebral physiology related to evoked potentials and perceptual speech processing in women during ovarian cycle. Hearing thresholds change upon different sex hormone levels during the menstrual cycle [36]. And hearing conduction, measured by auditory brainstem response, is better in postovulatory phase compared with preovulatory phase of menstrual/ovarian cycle [37]. Also, brainstem auditory evoked potentials change in the mid follicular and the mid luteal phases of the ovarian cycle [38]. Moreover, ovarian cycle effects on postural stability but not optokinetic function, and this needs to be considered when conducting studies of postural stability in women [39]. Studies of dichotic listening in women of the reproductive age also show that there is variation in laterality as a function of menstrual cycle phase. The perceptual speech processing of women is highly plastic and operates at varying states of functional asymmetry across days of the menstrual cycle, which are consistent with other works showing menstrual cycle-related changes in lateralized neurocognitive systems in the language domain [40–42]. #### 3.2. Menopause More organs are found to be influenced by the positive effects of estrogen, and estrogen has been expected to be benefit on auditory system by many investigators. As for the postmenopausal women, many studies suggested that the hearing and balancing problem appeared might be related to their sex hormone levels. Hearing loss in older people usually affects the highest frequencies early on and gradually affects the lower frequencies. Progesterone may have negative effects on the hearing of pre- and postmenopausal women and aging mice. On the contract, estrogen was found in some situation to have a positive influence [25]. The auditory brainstem response thresholds of postmenopausal female are higher than younger men or women [43, 44]. And the lower level of circulating serum estradiol possibly impedes hearing sensitivity in postmenopausal women, which has no relationship with bone mineral densities [45]. Intrinsic estrogen at physiological levels might slow down hearing loss in aging women [46]. At the same time, estrogen therapy may slow down the hearing loss in aging postmenopausal women [47]. Tibolone, a synthetic steroid drug with estrogenic, progestogenic, and weak androgenic actions, is often used in the hormone replacement therapy for menopausal or premenopausal women. And tibolone had no negative effect on hearing function and might decelerate hearing loss in aging postmenopausal women, intrinsic estrogen at physiological levels might slow down hearing loss in aging women [46]. After treatment of healthy menopausal women with tibolone for 6 months, the improvement was more prominent on the right side in audiometry results at low frequency. It may be explained by differences in distribution of ER in the ear. ERs might be more dense in the right ear, so give better response to estrogen therapy [47]. Many studies have showed that estrogen affects hearing function, especially in the postmenopausal women; a recent study gets a result that there may be hearing lateralization in menopausal women, especially significant improvement on right ear can be explained by lower BMD on that side ear bones in turn better response to estrogen therapy due to this, which may be related to ER concentration and the more dense of type ERα and/or ERβ in the right ear [48]. According to the vestibular function, the level of estradiol and progesterone decreases obviously in postmenopausal women with benign paroxysmal positional vertigo, which can cause the inner ear microcirculation disorder, may be a risk factor of BPPV [49]. Although estrogen has been expected to be benefits on auditory system, both clinician and patients need to take into concern that estrogen may have some unwanted side effects, such as increased risk of uterine cancer. Because in the central nervous system, ERβ is highly expressed in neurons and glial cells. And there is little ERβ in the mature uterus, selective ERβ agonists, then they become available [50, 51]. #### 3.3. Pregnancy there are multiple interactions between the sex hormones involved in these physiological In the ovarian cycle, the levels of estrogen and progesterone in the body have a dynamic regulation. Both clinical and basic studies have proved that the changes of auditory and balance system are attributed to estradiol and progesterone. In other words, the fluctuating hearing levels are evident in females during the ovarian cycle. Across the life span, both women and men undergo transitions in reproductive status related in part to changes in sex hormone levels. There is controversy over how hormonal conditions influence cerebral physiology related to evoked potentials and perceptual speech processing in women during ovarian cycle. Hearing thresholds change upon different sex hormone levels during the menstrual cycle [36]. And hearing conduction, measured by auditory brainstem response, is better in postovulatory phase compared with preovulatory phase of menstrual/ovarian cycle [37]. Also, brainstem auditory evoked potentials change in the mid follicular and the mid luteal phases of the ovarian cycle [38]. Moreover, ovarian cycle effects on postural stability but not optokinetic function, and this needs to be considered when conducting studies of postural stability in women [39]. Studies of dichotic listening in women of the reproductive age also show that there is variation in laterality as a function of menstrual cycle phase. The perceptual speech processing of women is highly plastic and operates at varying states of functional asymmetry across days of the menstrual cycle, which are consistent with other works showing menstrual cycle-related changes in lateralized neurocognitive systems in the language domain [40–42]. More organs are found to be influenced by the positive effects of estrogen, and estrogen has been expected to be benefit on auditory system by many investigators. As for the postmenopausal women, many studies suggested that the hearing and balancing problem appeared might be related to their sex hormone levels. Hearing loss in older people usually affects the highest frequencies early on and gradually affects the lower frequencies. Progesterone may have negative effects on the hearing of pre- and postmenopausal women and aging mice. On the contract, estrogen was found in some situation to have a positive influence [25]. The auditory brainstem response thresholds of postmenopausal female are higher than younger men or women [43, 44]. And the lower level of circulating serum estradiol possibly impedes hearing sensitivity in postmenopausal women, which has no relationship with bone mineral Intrinsic estrogen at physiological levels might slow down hearing loss in aging women [46]. At the same time, estrogen therapy may slow down the hearing loss in aging postmenopausal women [47]. Tibolone, a synthetic steroid drug with estrogenic, progestogenic, and weak androgenic actions, is often used in the hormone replacement therapy for menopausal or premenopausal women. And tibolone had no negative effect on hearing function and might decelerate hearing loss in aging postmenopausal women, intrinsic estrogen at physiological levels might slow down hearing loss in aging women [46]. After treatment of healthy changes and this enhances the possible multidirectional effects on the inner ear. 3.1. Ovarian cycle 334 Sex Hormones in Neurodegenerative Processes and Diseases 3.2. Menopause densities [45]. Hearing loss appeared in pregnancy is not a commonly reported problem. Some investigators have noticed reversible and physiological sensorineural hearing loss at low frequencies during the period of pregnancy [52, 53]. In some case reports, sudden onset of sensorineural hearing loss during pregnancy has been described [54], and one report concerning a patient who had the hearing loss with each serial pregnancy [55]. But a nationwide populationbased study suggested that sudden sensorineural hearing loss (SSNHL) in pregnancy is rare. SSNHL is defined as sudden, idiopathic, usually unilateral deafness developed at most in 72 hours in previously healthy people [56]. It often happens in the third trimester. And SSNHL in pregnancy does not increase the risks of delivery or subsequent stroke [57]. As for the mechanism, there is a hypothesis implied that SSNHL is connected with the changes in cardiovascular system, hematological system, and/or some other systems because of pregnancy. These changes in pregnancy may evoke disorders of cochlea circulation or cochlea fluid homeostasis resulting in SSNHL [57]. Otosclerosis is one of the most common causes of acquired hearing loss and is widely supposed as being related with pregnancy. Another study revealed that resonance frequency of middle ear was found to be low during the third trimester of the pregnancy. And low resonance frequency informs that the acoustic immittance of the middle ear changes during pregnancy [58]. Tinnitus is another auditory symptom in pregnant patients, with proposed theories of pathogenesis, including hyperdynamic circulation, increase in perilymphatic fluid pressure, and hormonal changes. Clinically, it appears that the hearing loss and tinnitus related to pregnancy can spontaneously recover. As for treatment, it depends on the otorhinolaryngologic doctors to decide whether they should administer steroid drugs for acute hearing loss, as it may recover after the delivery [54]. Autophony is a classic complaint of patients suffering from a patulous Eustachian tube (PET). The typical patients with PET have lost a drastic amount of weight, resulting in shrinkage of the peritubal mucous membranes. One third of the patients with PET are either pregnant or taking an estrogen replacement therapy [59]. In order to resolve the symptoms postpartum, management should consist of informative reassurance alone [60]. 4. Sex hormones and auditory and vestibular pathology Menière's disease. 4.2. Presbyacusis 4.1. Hearing disorders in Turner syndrome which often develop an early presbyacusis. Considerable anecdotal evidence and limited information from previous studies suggest that auditory and vestibular functions may be influenced by sex hormones resulting in pathological conditions such as hearing disorders in Turner syndrome, Presbyacusis, Otosclerosis, and Sex Hormones and Inner Ear 337 http://dx.doi.org/10.5772/intechopen.74157 Hearing disorders are obvious in mice and women with Turner syndrome (total or partial loss of one X chromosome) [68]. Approximately one-half of women with TS have a 45,X karyotype, about 20% have 45,X/46,XX mosaicism, and the remainder have structural abnormalities of the X chromosome such as X fragments, isochromosomes, or rings. TS is characterized by bilateral streak gonads, short stature, primary amenorrhea, streak ovaries, and no estrogen production, The hearing loss features in patients with Turner Syndrome should be taken into consideration. The common clinical complaints are recurrent otitis media, dysfunction of the Eustachian tube, conductive hearing loss during infancy, and sensorineural hearing loss in the adolescence. The karyotype appears to be important in the hearing loss, with studies demonstrating an increased prevalence in patients with monosomy 45,X or isochromosome 46,i (Xq). It is necessary of morphologic studies of the cochlea to help out in clarifying the etiology of the sensorineural hearing loss [69]. And sensorineural hearing loss is the most common type of hearing loss. It is mostly characterized by a high-frequency loss and/or a mid-frequent dip. It is uncommon for conductive hearing loss in young women with Turner Syndrome. But in a TS cohort, 91% of patients suffered middle ear disease, but the incidence of SNHL was 9%. It is suggested that TS patients should be screened for onset and progression of hearing loss [70]. Consequently, there is a need for hearing rehabilitation in these patients. Questions about hearing must be asked by physicians when treating women with Turner Syndrome to identify those who need hearing rehabilitation, even if they have an audiogram with a normal pure tone average [71]. Both the karyotype and sensorineural dip in hearing could be used to predict the future course of hearing levels for TS patients. And estrogen may have an influence on hearing loss in TS patients [72]. Progressive hearing loss is relatively common in human without a clear molecular basis and medical therapies. A new gene, WBP2, was defined to be involved in the molecular pathway linking hearing impairment to hormonal signaling and provides new therapeutic targets. WBP2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing [73].WBP2 encodes the protein that acts as a transcriptional coactivator for ERα (ESR1) and progesterone receptor. The loss of Wbp2 expression leads to progressive high-frequency hearing loss in mouse, as well as in two deaf children, each carrying two different variants in the WBP2 gene [73]. Presbyacusis or age-related hearing loss is a complex degenerative disease that affects many people worldwide. Gender does play a role in age-related hearing loss. Longitudinal studies of aging have There is an increase in incidence of Bell's palsy (BP) during pregnancy [61]. One of the reasons could be a brain stem synaptic impairment caused by estrogen, presumably because of ischemic changes [62]. And most of them seem to be concentrated in the third trimester. The most likely explanation about the concentration in the third trimester may be the altered susceptibility to herpes simplex viral reactivation during pregnancy. And the prognosis of pregnant patients may be poorer [63]. #### 3.4. Gender differences in auditory function Many authors have shown that a gender differences in auditory function and some of them implied that part of this variation was due to the difference in estrogen levels between females and males. There are well-known sex differences in the auditory brainstem response, with women having shorter latencies than men [64]. Hearing loss is more profound in elderly males than females. Many early studies on otoacoustic emissions revealed the existence of sex and ear differences in human beings. Some also revealed that the sex and ear differences in adults are evident in newborns as well. These differences are in the direction of human females having stronger and more numerous spontaneous otoacoustic emissions and stronger click-evoked otoacoustic emissions than do males; also, human right ears have stronger and more numerous spontaneous otoacoustic emissions and stronger click-evoked otoacoustic emissions than do left ears. There is a so-called prenatal-androgen-exposure explanation. Prenatal androgen exposure apparently can alter auditory evoked potentials. The sex difference in otoacoustic emissions in newborns may be that the prenatal androgen exposure in some way weakens the cochlea amplifiers and thereby weakens spontaneous otoacoustic emissions and click-evoked otoacoustic emissions (and perhaps distortion product otoacoustic emissions less markedly) [65]. Sex differences are limited to frequency ranges, which are related to the processing of natural vocalizations and depend on the type of stimulus. In a research using green tree frog, Hyla cinera, sex did not change audiogram best frequencies, although sex did make a difference in the sensitivities at those frequencies with males more sensitive in the lower frequency range [29]. The auditory system exhibits differences by sex and by sexual orientation, and the implication is that relevant auditory structures are altered during prenatal development, possibly by exposure to androgens [66]. Gender differences also occur in some pathological situations. In the presence of sex hormone receptors in human middle ear cholesteatoma, stronger expression of progesterone receptor was found in samples from male patients, while stronger expression of estrogen receptor was found in samples from female patients. It suggests that female sex hormones may stimulate proliferation of middle ear cholesteatoma keratinocytes [67]. Estrogen levels between females and males in different ages may influence the function of the auditory systems, and the details of the mechanism should be studied in the future. #### 4. Sex hormones and auditory and vestibular pathology Considerable anecdotal evidence and limited information from previous studies suggest that auditory and vestibular functions may be influenced by sex hormones resulting in pathological conditions such as hearing disorders in Turner syndrome, Presbyacusis, Otosclerosis, and Menière's disease. #### 4.1. Hearing disorders in Turner syndrome Autophony is a classic complaint of patients suffering from a patulous Eustachian tube (PET). The typical patients with PET have lost a drastic amount of weight, resulting in shrinkage of the peritubal mucous membranes. One third of the patients with PET are either pregnant or taking an estrogen replacement therapy [59]. In order to resolve the symptoms postpartum, There is an increase in incidence of Bell's palsy (BP) during pregnancy [61]. One of the reasons could be a brain stem synaptic impairment caused by estrogen, presumably because of ischemic changes [62]. And most of them seem to be concentrated in the third trimester. The most likely explanation about the concentration in the third trimester may be the altered susceptibility to herpes simplex viral reactivation during pregnancy. And the prognosis of pregnant Many authors have shown that a gender differences in auditory function and some of them implied that part of this variation was due to the difference in estrogen levels between females and males. There are well-known sex differences in the auditory brainstem response, with women having shorter latencies than men [64]. Hearing loss is more profound in elderly males than females. Many early studies on otoacoustic emissions revealed the existence of sex and ear differences in human beings. Some also revealed that the sex and ear differences in adults are evident in newborns as well. These differences are in the direction of human females having stronger and more numerous spontaneous otoacoustic emissions and stronger click-evoked otoacoustic emissions than do males; also, human right ears have stronger and more numerous spontaneous otoacoustic emissions and stronger click-evoked otoacoustic emissions than do left ears. There is a so-called prenatal-androgen-exposure explanation. Prenatal androgen exposure apparently can alter auditory evoked potentials. The sex difference in otoacoustic emissions in newborns may be that the prenatal androgen exposure in some way weakens the cochlea amplifiers and thereby weakens spontaneous otoacoustic emissions and click-evoked otoacoustic emissions (and perhaps distortion product otoacoustic emissions less markedly) [65]. Sex differences are limited to frequency ranges, which are related to the processing of natural vocalizations and depend on the type of stimulus. In a research using green tree frog, Hyla cinera, sex did not change audiogram best frequencies, although sex did make a difference in the sensitivities at those frequencies with males more sensitive in the lower frequency range [29]. The auditory system exhibits differences by sex and by sexual orientation, and the implication is that relevant auditory structures are altered during prenatal development, Gender differences also occur in some pathological situations. In the presence of sex hormone receptors in human middle ear cholesteatoma, stronger expression of progesterone receptor was found in samples from male patients, while stronger expression of estrogen receptor was found in samples from female patients. It suggests that female sex hormones may stimulate proliferation of middle ear cholesteatoma keratinocytes [67]. Estrogen levels between females and males in different ages may influence the function of the auditory systems, and the details management should consist of informative reassurance alone [60]. patients may be poorer [63]. 3.4. Gender differences in auditory function 336 Sex Hormones in Neurodegenerative Processes and Diseases possibly by exposure to androgens [66]. of the mechanism should be studied in the future. Hearing disorders are obvious in mice and women with Turner syndrome (total or partial loss of one X chromosome) [68]. Approximately one-half of women with TS have a 45,X karyotype, about 20% have 45,X/46,XX mosaicism, and the remainder have structural abnormalities of the X chromosome such as X fragments, isochromosomes, or rings. TS is characterized by bilateral streak gonads, short stature, primary amenorrhea, streak ovaries, and no estrogen production, which often develop an early presbyacusis. The hearing loss features in patients with Turner Syndrome should be taken into consideration. The common clinical complaints are recurrent otitis media, dysfunction of the Eustachian tube, conductive hearing loss during infancy, and sensorineural hearing loss in the adolescence. The karyotype appears to be important in the hearing loss, with studies demonstrating an increased prevalence in patients with monosomy 45,X or isochromosome 46,i (Xq). It is necessary of morphologic studies of the cochlea to help out in clarifying the etiology of the sensorineural hearing loss [69]. And sensorineural hearing loss is the most common type of hearing loss. It is mostly characterized by a high-frequency loss and/or a mid-frequent dip. It is uncommon for conductive hearing loss in young women with Turner Syndrome. But in a TS cohort, 91% of patients suffered middle ear disease, but the incidence of SNHL was 9%. It is suggested that TS patients should be screened for onset and progression of hearing loss [70]. Consequently, there is a need for hearing rehabilitation in these patients. Questions about hearing must be asked by physicians when treating women with Turner Syndrome to identify those who need hearing rehabilitation, even if they have an audiogram with a normal pure tone average [71]. Both the karyotype and sensorineural dip in hearing could be used to predict the future course of hearing levels for TS patients. And estrogen may have an influence on hearing loss in TS patients [72]. Progressive hearing loss is relatively common in human without a clear molecular basis and medical therapies. A new gene, WBP2, was defined to be involved in the molecular pathway linking hearing impairment to hormonal signaling and provides new therapeutic targets. WBP2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing [73].WBP2 encodes the protein that acts as a transcriptional coactivator for ERα (ESR1) and progesterone receptor. The loss of Wbp2 expression leads to progressive high-frequency hearing loss in mouse, as well as in two deaf children, each carrying two different variants in the WBP2 gene [73]. #### 4.2. Presbyacusis Presbyacusis or age-related hearing loss is a complex degenerative disease that affects many people worldwide. Gender does play a role in age-related hearing loss. Longitudinal studies of aging have shown that hearing declines more rapidly in males than females. Elderly people with presbyacusis not only have a loss in sensitivity to sound but also have significant difficulties understanding speech in background noise at supra-threshold, conversational levels. Many researchers have identified sex-specific differences in presbyacusis in humans and animal models [74]. show that women with Menière's disease were identified as having premenstrual phase of their monthly cycle or during pregnancy [82]. Genetic factors could contribute, at least partially to it. Many researchers have identified that in some women with Meniere's disease, attacks of vertigo, low frequency hearing loss, aural fullness and tinnitus are exacerbated in the premenstrual phase, when estrogen levels are low with edema in the endolymphatic spaces due to the loss of intravascular fluid into the extravascular space facilitated by estrogens [83–85]. One possible explanation may be estrogen-induced hyperprolactinemia, which was Sex Hormones and Inner Ear 339 http://dx.doi.org/10.5772/intechopen.74157 Significant associations have been reported between Menière's disease and genetic polymorphisms. Polymorphisms associated with blood vessel permeability, blood circulation, or inflammation have been reported to be related to the inner ear pathology. AQP5 is known as an exocrine-type water channel with the roles in conveying a high degree of membrane water permeability [86]. Mice lacking AQP5 show lower frequency hearing impairment [87]. Some researchers demonstrated identified AQP5 as an ESα target gene in the mouse uterus using Sex hormone-related symptoms of auditory and vestibular systems are common in clinic. Here, we address by recognizing that interactions between sex hormones and sensory systems can be beneficial or detrimental to the peripheral and central auditory and vestibular systems. Knowing how sex steroids can alter hearing ability may give important clues as to how estrogen can preserve hearing in humans. The postmenopausal women have slightly better hearing if administered estrogen replacement therapy, physiological levels of estrogen would seem to have a possible protective effect on hearing function. The association described here shed light to the role of sex hormones and their receptors in the inner ear and behavior and underline the therapeutic potential of specific sex hormone agonists and antagonists. Programs (2016YFC0905200, 2016YFC0905202), and Zhuo-Xue Plan of Fudan University. Department of Otology and Skull Base Surgery, Eye, Ear, Nose and Throat Hospital (EENT This study was supported by grants from the National Natural Science Foundation of China (NSFC 81420108010, 81370022, 81570920, 81771077 and 81000413), Key Project of Chinese National reported to provoke hearing loss and otic capsule dysmorphology in guinea pig [24]. chromatin immunoprecipitation and DNA microarray analyses [88]. 5. Summary Acknowledgements Author details Zi-Yu He and Dong-Dong Ren\* \Address all correspondence to: [email protected] Hospital), Fudan University, Shanghai, China There is growing evidence that interactions between sex hormones and sensory systems are sometimes beneficial, but oftentimes detrimental, such as progesterone with negatively affect hearing in older women, whereas in some cases, estrogen may have positive effects [24]. Data from a large cohort of adults (48–92 years) in the Beaver Dam Epidemiology of Hearing Loss Study show significant age effects in word recognition scores in competing messages for both men and women, but performance is consistently poorer in men than in women at all age groups and hearing loss categories [75]. #### 4.3. Otosclerosis Otosclerosis is a major cause of acquired hearing loss in adult life affecting exclusively the human temporal bone, which is reported to worsen during periods of intense hormonal activity. Many researchers show a possible link between aggravation of otosclerosis and pregnancy is still debated. Thus, sex hormones were believed to be involved in the progression of the disease. Estrogen deficiency is considered to be a cause of osteoporosis in menopause women, and estrogen substitute therapy has shown beneficial effect in those cases [76]. Otosclerosis becomes manifest between the ages of 20–50 years and is usually bilateral. It affects twice as many females as males [77]. A retrospective study on a sample of 479 women with otosclerosis showed that the risk of subjective hearing deterioration with bilateral otosclerosis increased from 33% after one pregnancy to 63% after six pregnancies [78]. Several reports have suggested that oral contraceptives may increase the risk of hearing loss and in particular otosclerosis, although no clear conclusion has been drawn [79]. Estrogen has an inhibitory effect on bone resorption by directly inhibiting osteoclast activity as well as decreasing auto and paracrine production of cytokines such as interleukin (IL) 1 and IL-6 and tumor necrosis factor, TNF [80]. Researchers investigated the effect of 17β-estradiol on bone remodeling via diastrophic dysplasia sulfate transporter (DTDST) in otosclerosis and in a human osteoblast-like cell line, and they have demonstrated that the response to estrogens in terms of DTDST activity might be related to the expressed receptor type. It is possible that exacerbating effects of estrogens in patients with otosclerosis may be mediated by peculiar profiles of estrogen receptor in otosclerotic cells [81]. However, the regulatory mechanisms of Otosclerosis related to the estrogen receptor profile in the otosclerotic cells need to be further analyzed. #### 4.4. Menière's disease Menière's disease is characterized by hearing loss, tinnitus, and vestibular dysfunction. It is thought that endolymph malabsorption is the underlying cause of the swelling of the endolymphatic spaces. Estrogens are known to facilitate the loss of intravascular fluid into the extravascular space, producing edema. Endogenous alterations in concentrations of estrogen and progesterone in the premenstrual syndrome or with the use of exogenous hormones such as oral contraceptives may trigger vertigo in patients with Menière's disease. Many reports show that women with Menière's disease were identified as having premenstrual phase of their monthly cycle or during pregnancy [82]. Genetic factors could contribute, at least partially to it. Many researchers have identified that in some women with Meniere's disease, attacks of vertigo, low frequency hearing loss, aural fullness and tinnitus are exacerbated in the premenstrual phase, when estrogen levels are low with edema in the endolymphatic spaces due to the loss of intravascular fluid into the extravascular space facilitated by estrogens [83–85]. One possible explanation may be estrogen-induced hyperprolactinemia, which was reported to provoke hearing loss and otic capsule dysmorphology in guinea pig [24]. Significant associations have been reported between Menière's disease and genetic polymorphisms. Polymorphisms associated with blood vessel permeability, blood circulation, or inflammation have been reported to be related to the inner ear pathology. AQP5 is known as an exocrine-type water channel with the roles in conveying a high degree of membrane water permeability [86]. Mice lacking AQP5 show lower frequency hearing impairment [87]. Some researchers demonstrated identified AQP5 as an ESα target gene in the mouse uterus using chromatin immunoprecipitation and DNA microarray analyses [88]. #### 5. Summary shown that hearing declines more rapidly in males than females. Elderly people with presbyacusis not only have a loss in sensitivity to sound but also have significant difficulties understanding speech in background noise at supra-threshold, conversational levels. Many researchers have There is growing evidence that interactions between sex hormones and sensory systems are sometimes beneficial, but oftentimes detrimental, such as progesterone with negatively affect hearing in older women, whereas in some cases, estrogen may have positive effects [24]. Data from a large cohort of adults (48–92 years) in the Beaver Dam Epidemiology of Hearing Loss Study show significant age effects in word recognition scores in competing messages for both men and women, but performance is consistently poorer in men than in women at all age Otosclerosis is a major cause of acquired hearing loss in adult life affecting exclusively the human temporal bone, which is reported to worsen during periods of intense hormonal activity. Many researchers show a possible link between aggravation of otosclerosis and pregnancy is still debated. Thus, sex hormones were believed to be involved in the progression of the disease. Estrogen deficiency is considered to be a cause of osteoporosis in menopause women, and estrogen substitute therapy has shown beneficial effect in those cases [76]. Otosclerosis becomes manifest between the ages of 20–50 years and is usually bilateral. It affects twice as many females as males [77]. A retrospective study on a sample of 479 women with otosclerosis showed that the risk of subjective hearing deterioration with bilateral otosclerosis increased from 33% after one pregnancy to 63% after six pregnancies [78]. Several reports have suggested that oral contraceptives may increase the risk of hearing loss and in Estrogen has an inhibitory effect on bone resorption by directly inhibiting osteoclast activity as well as decreasing auto and paracrine production of cytokines such as interleukin (IL) 1 and IL-6 and tumor necrosis factor, TNF [80]. Researchers investigated the effect of 17β-estradiol on bone remodeling via diastrophic dysplasia sulfate transporter (DTDST) in otosclerosis and in a human osteoblast-like cell line, and they have demonstrated that the response to estrogens in terms of DTDST activity might be related to the expressed receptor type. It is possible that exacerbating effects of estrogens in patients with otosclerosis may be mediated by peculiar profiles of estrogen receptor in otosclerotic cells [81]. However, the regulatory mechanisms of Otosclerosis related to Menière's disease is characterized by hearing loss, tinnitus, and vestibular dysfunction. It is thought that endolymph malabsorption is the underlying cause of the swelling of the endolymphatic spaces. Estrogens are known to facilitate the loss of intravascular fluid into the extravascular space, producing edema. Endogenous alterations in concentrations of estrogen and progesterone in the premenstrual syndrome or with the use of exogenous hormones such as oral contraceptives may trigger vertigo in patients with Menière's disease. Many reports particular otosclerosis, although no clear conclusion has been drawn [79]. the estrogen receptor profile in the otosclerotic cells need to be further analyzed. identified sex-specific differences in presbyacusis in humans and animal models [74]. groups and hearing loss categories [75]. 338 Sex Hormones in Neurodegenerative Processes and Diseases 4.3. Otosclerosis 4.4. Menière's disease Sex hormone-related symptoms of auditory and vestibular systems are common in clinic. Here, we address by recognizing that interactions between sex hormones and sensory systems can be beneficial or detrimental to the peripheral and central auditory and vestibular systems. Knowing how sex steroids can alter hearing ability may give important clues as to how estrogen can preserve hearing in humans. The postmenopausal women have slightly better hearing if administered estrogen replacement therapy, physiological levels of estrogen would seem to have a possible protective effect on hearing function. The association described here shed light to the role of sex hormones and their receptors in the inner ear and behavior and underline the therapeutic potential of specific sex hormone agonists and antagonists. #### Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (NSFC 81420108010, 81370022, 81570920, 81771077 and 81000413), Key Project of Chinese National Programs (2016YFC0905200, 2016YFC0905202), and Zhuo-Xue Plan of Fudan University. #### Author details Zi-Yu He and Dong-Dong Ren\ \Address all correspondence to: [email protected] Department of Otology and Skull Base Surgery, Eye, Ear, Nose and Throat Hospital (EENT Hospital), Fudan University, Shanghai, China #### References [1] Nishio N, Teranishi M, Uchida Y, Sugiura S, Ando F, Shimokata H, Sone M, Otake H, Kato K, Yoshida T, Tagaya M, Hibi T, Nakashima T. Polymorphisms in genes encoding aquaporins 4 and 5 and estrogen receptor α in patients with Meniere's desease and sudden sensorineural hearing loss. Life Sciences. Mar 21, 2013;92(10):541-546 [14] Goodson JL, Evans AK, Bass AH. Putative isotocin distributions in sonic fish: Relation to vasotocin and vocal-acoustic circuitry.The Journal of Comparative Neurology. Jul 14, Sex Hormones and Inner Ear 341 http://dx.doi.org/10.5772/intechopen.74157 [15] Choleris E, Ogawa S, Kavaliers M, Gustafsson JA, Korach KS, Muglia LJ, Pfaff DW. Involvement of estrogen receptor alpha, beta and oxytocin in social discrimination: A detailed behavioral analysis with knockout female mice. Genes, Brain, and Behavior. Oct [16] Scordalakes EM, Rissman EF. Aggression and arginine vasopressin immunoreactivity regulation by androgen receptor and estrogen receptor alpha.Genes, Brain, and Behavior. [17] Nomura M, McKenna E, Korach KS, Pfaff DW, Ogawa S. Estrogen receptor-beta regulates transcript levels for oxytocin and arginine vasopressin in the hypothalamic paraventricular nucleus of male mice. Brain Research. Molecular Brain Research. Dec 30, 2002;109(1–2):84-94 [18] Lee JH, Marcus DC. Estrogen acutely inhibits ion transport by isolated stria vascularis. [19] Caruso S, Cianci A, Grasso D, Agnello C, Galvani F, Maiolino L, et al. Auditory brainstem responses in postmenopausal women treated with hormone replacement therapy: A pilot [20] Meltser I, Tahera Y, Simpson E, Hultcrantz M, Charitidi K, Gustafsson JA, Canlon B. Estrogen receptor β protects against acoustic trauma in mice animal study. Journal of [21] Horner KC, Cazals Y, Guieu R, Lenoir M, Sauze N.Experimental estrogen-induced hyperprolactinemia results in bone-related hearing loss in the guinea pig.American Journal of Physiology. Endocrinology and Metabolism. 2007 Nov;293(5):E1224-E1232 [Epub [22] Charitidi K, Meltser I, Canlon B. 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Otolaryngology and Head [64] Jerger J, Hall J. Effects of age and sex on auditory brainstem response. Archives of [65] McFadden D. Masculinization of the mammalian cochlea. Hearing Research. Jun 2009; [66] McFadden D. Sexual orientation and the auditory system. Frontiers in Neuroendocrinol- [67] Raynov AM, Choung YH, Moon SK, Park K. Expression of female sex hormone receptors in human middle-ear cholesteatomas. The Journal of Laryngology and Otology. Dec 2005; [68] Hultcrantz M, Stenberg AE, Fransson A, Canlon B. Characterization of hearing in an X,0 [69] Alves C, Oliveira CS. Hearing loss among patients with Turner's syndrome: Literature review. Brazilian Journal of Otorhinolaryngology. May–Jun 2014;80(3):257-263 nancy. Irish Journal of Medical Science. 2011;180(1):79-84 Ginekologia Polska. 2005 Mar;76(3):225-227 Wochenschrift (1946). 2000;150(22):454-456 and Neck Surgery. Dec 2007;137(6):858-861 'turner mouse'. Hearing Research. 2000;143:182-188 Otolaryngology. 1980;106:387-391 ogy. Apr 2011;32(2):201-213 ogy Research. Apr 20, 2016;6(1):147 344 Sex Hormones in Neurodegenerative Processes and Diseases 2004;329(7465):553-557 1995;113(1):32-35 252(1–2):37-48 119(12):941-945 loss? Acta Oto-Laryngologica Jul 2011;131(7):779-786 [84] Morse GG, House JW. Changes in Meniere's disease responses as a function of the [85] Merchant SN, Adams JC, Nadol JB. Pathophysiology of Meniere's syndrome: Are symptoms caused by endolymphatic hydrops? Otology and Neurotology. 2005;26:74-81 [86] Kawedia JD, Nieman ML, Boivin GP, Melvin JE, Kikuchi K, Hand AR, Lorenz JN, Menon AG. Interaction between transcellular and paracellular water transport pathways through aquaporin 5 and the tight junction complex. Proceedings of the National Academy of [87] Merves M, Krane CM, Dou H, Greinwald JH, Menon AG, Choo D. Expression of aquaporin 1 and 5 in the developing mouse inner ear and audiovestibular assessment of an Aqp5 null mutant. Journal of the Association for Research in Otolaryngology. 2003;4: [88] Kobayashi M, Takahashi E, Miyagawa S, Watanabe H, Iguchi T. Chromatin immunoprecipitation-mediated target identification proved aquaporin 5 is regulated directly by estrogen in the uterus. Genes to Cells. Oct 2006;11(10):1133-1143 menstrual cycle. Nursing Research. 2001;50:286-292 346 Sex Hormones in Neurodegenerative Processes and Diseases 264-275 Sciences of the United States of America. 2007;104:3621-3626 ## Edited by Gorazd Drevenšek* The book provides chapters on sex hormones and their modulation in neurodegenerative processes and pathologies, from basic molecular mechanisms, physiology, gender differences, to neuroprotection and clinical aspects for potential novel pharmacotherapy approaches. The book contains 14 chapters written by authors from various biomedical professions, from basic researchers in biology and physiology to medicine and veterinary medicine, pharmacologists, psychiatrist, etc. Chapters sum up the past and current knowledge on sex hormones, representing original new insights into their role in brain functioning, mental disorders and neurodegenerative diseases. The book is written for a broad range of audience, from biomedical students to highly profiled medical specialists and biomedical researchers, helping them to expand their knowledge on sex hormones in neurodegenerative processes and opening new questions for further investigation. Published in London, UK © 2018 IntechOpen © nnorozoff / iStock Sex Hormones in Neurodegenerative Processes and Diseases Sex Hormones in Neurodegenerative Processes and Diseases Edited by Gorazd Drevenšek	doab	2025-04-07T03:56:57.614279	1-12-2023 17:26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0004847e-ab80-435d-9a35-50f7f1b13b00", "url": "https://mts.intechopen.com/storage/books/5994/authors_book/authors_book.pdf", "author": "", "title": "Sex Hormones in Neurodegenerative Processes and Diseases", "publisher": "IntechOpen", "isbn": "9781789230154", "section_idx": 6 }
0004e4bd-422d-4a23-9686-437c4a3af25b	# Gathering Ecologies Thinking Beyond Interactivity Andrew Goodman ## Gathering Ecologies Thinking Beyond Interactivity ## Immediations Series Editor: SenseLab "Philosophy begins in wonder. And, at the end, when philosophic thought has done its best, the wonder remains" – A.N. Whitehead The aim of the Immediations book series is to prolong the wonder sustaining philosophic thought into transdisciplinary encounters. Its premise is that concepts are for the enacting: they must be experienced. Thought is lived, else it expires. It is most intensely lived at the crossroads of practices, and in the in-between of individuals and their singular endeavors: enlivened in the weave of a relational fabric. Co-composition. "The smile spreads over the face, as the face fits itself onto the smile" – A. N. Whitehead Which practices enter into co-composition will be left an open question, to be answered by the Series authors. Art practice, aesthetic theory, political theory, movement practice, media theory, maker culture, science studies, architecture, philosophy … the range is free. We invite you to roam it. ### Gathering Ecologies ### Thinking Beyond Interactivity Andrew Goodman London 2018 OPEN HUMANITIES PRESS First edition published by Open Humanities Press 2018 Copyright © 2018 Andrew Goodman This is an open access book, licensed under Creative Commons By Attribution Share Alike license. Under this license, authors allow anyone to download, reuse, reprint, modify, distribute, and/or copy their work so long as the authors and source are cited and resulting derivative works are licensed under the same or similar license. No permission is required from the authors or the publisher. Statutory fair use and other rights are in no way affected by the above. Read more about the license at creativecommons.org/licenses/by-sa/4.0 Cover Art, figures, and other media included with this book may be under different copyright restrictions. Cover Illustration © 2018 Leslie Plumb Cover Design by Leslie Plumb Typeset in Open Sans, an open font. Print ISBN 978-1-78542-052-8 PDF ISBN 1-78542-053-5 Freely available online at: http://openhumanitiespress.org/books/titles/gathering-ecologies OPEN HUMANITIES PRESS Open Humanities Press is an international, scholar-led open access publishing collective whose mission is to make leading works of contemporary critical thought freely available worldwide. More at http://openhumanitiespress.org ### Contents For three teachers who encouraged me to write: Max Balchin Karen Ward Edward Colless ### Acknowledgements Thanks must go to the many people who have contributed their knowledge, support and enthusiasm to the making of this book. First and foremost my thanks go to all the members of the Senselab with whom I have had the pleasure of collaborating and learning from, and without whom I could not have thought through these ideas. Amongst many, many Senselab members who deserve thanks, this includes Erin Manning, for her unending enthusiasm and warmth and for sharing art making and writing with me, Nathaniel Stern for various collaborations and his unbounded energy, Alanna Thain for her writing on my work and key conversations, Sam Spurr for artistic collaborations, Anna Munster for her valuable feedback, support and many helpful suggestions, and Andrew Murphie and Lone Bertelsen for their support, suggestions and feedback, and for sympathetic reading of papers in progress. Thanks also to a several people who contributed to the editing of earlier versions of various chapters: Din Heagney for his sterling efforts editing chapters and controlling footnotes, and Trinh Vu and Leonie Cooper for their many constructive suggestions. Thanks to a number of people who have contributed to my own artistic processes during this time through support, collaboration and feedback, including Oliver Cloke, Tony Falla, Samantha Bews, Luhsun Tan, Andrea Ekersley, Kent Wilson, Jude Anderson and Caroline Kennedy-McCracken. Thanks must also go to the anonymous reviewers of the manuscript for their careful reading and efforts to improve the writing style and content of the book. Finally, thanks and love to my daughter Lucy for her humour and patience throughout the long process. Earlier versions of some chapters have appeared in print before. Sections of Chapter One and Chapter Two appeared as "Rethinking Interactivity." ACMC Interactive Conference Proceedings, Victoria, 2012. Edited by Matthew Hitchcock, Australasian Computer Music Association. A version of Chapter Five appears as: "Walking with the World: Towards an Ecological Approach to Performative Art Practice". Walking and the Aesthetics of Modernity: Pedestrian Mobility in Literature and the Arts, edited by François Specq and Klaus Benesch. New York: Palgrave MacMillian, 2016. An earlier version of Chapter Six appears as: "Entertaining the Environment: Towards an Ethics of Art Events." AJE: Australasian Journal of Ecocriticism and Cultural Ecology, vol. 3, 2013/2014. A conference paper version of Chapter Eight appeared as "A Thousand Tiny Interfac(ing)s." Proceedings of the 19th International Symposium on Electronic Art, ISEA2013, Sydney. Edited by K Cleland, L Fisher and R Harley, 2013. ### Introduction: Thinking beyond interactivity Imagine you are out walking in the street. To go for a walk is to create, through the endless flow of interaction, bodily and spatially. With each step – and within each step – perceptual, sensorial and social possibilities are opened up, assemblages of forces gathered, altered and reconnected, complexities multiplied, memories activated. The moment is saturated with affectual relations and intensities (Lorrainne 2005, 73–4). With the fall of the same step, previous possibilities perish, simultaneously propelling the endless opening of fresh possibilities of connection (Manning 2009, 38–9). Try to map all the relations that go to make up one instant, one occasion: within your body, between body and world, mind and body, object and object – all the various 'machinic' combinations producing experience. You will have to consider subatomic, atomic and molecular forces with their general disregard for what we view as discreet bodies. You will want to account for the way the texture and gradient of the terrain shapes movement, rhythm and posture; how sensory perception, vision, hearing and touch and so on begin to ready the body for the next step; how the force of physical habits and body memory shape patterns of movement in the moment. Also present will be all the events of relation that have gone into making each tree, stone, person and sound you are interacting with, affecting your body more or less forcefully. Then there are the mental forces – 'inextricably intertwined' with the physical (Whitehead 1978, 325) – memories, anticipations, evaluations, random associations made and forgotten, affects that will subtly or bluntly alter you, the myriad mental processes that sit behind conscious perception, yet nevertheless shape and reshape your body. Beyond that instant, in the next occasion, the concrescence of all these forces creates anew this simple act of walking the street. It is a constant, complexly enmeshed act of creativity: when we look honestly, all things, as Whitehead says, are vectors of relations (1978, 309). Such an everyday act is saturated with complexity and invention, and is rich with potential. But now imagine you are in a gallery, in some interactive installation. Things happen as you move around – sounds, lights or video. Perhaps triggered by your presence, the work pretty much does its own thing and its actions seem somewhat random, or perhaps it continues to develop as you engage, with a concentration on a demonstration of how your actions affect its workings. Either way, this type of work often lacks the complex, intertwined-ness of body and work, the perceptual nuance, the fluidity, the surprising originality of connection and thickness of experience of a simple walk outside. In general in this book my intention is to focus on a productive move towards exploring positive developments in the field, and so the critique of the poor state of interactive art is painted with broad strokes. I will, however, begin with a brief description of a here-unnamed work I encountered early in the process of writing that contrasts with this imagined walk and illustrates some of the problematic areas that concern me. This particular work formed part of a large exhibition of a broad range of interactive works. Inside the gallery were various pieces that responded to touch, movement or other interactions with the audience, with shifts in sound or video projections and so on. These included a couch that purred as you stroked it, a series of pot plants that made sounds as the audience moved amongst them, and a digital 'mirror' that reflected a greatly aged version of the participant's face. All were at least mildly amusing works, if a little one dimensional in their aesthetics and outcomes, with very direct and limited links between the actions of the viewer and the changes in the works these gestures triggered. The particular work that affected me most negatively was situated in a bare shipping container outside the main gallery. It was set up as a very small rave club that would respond to audience members' dance moves to produce sound. It was silent and empty, other than a large speaker system, when I entered the space as the exhibition opened on a chilly 10-degree morning. The young invigilator then approached and enthusiastically encouraged me to begin dancing in order to trigger sounds. As I hesitated she became more forceful in her pleas for participation. I hovered near the door, unwilling to make a fool of myself in the service of as yet unheard music. Eventually the invigilator gave up in disgust and began to throw herself around the room as the very loud beats began, still pleading with me to join in. Needless to say I beat a hasty retreat, having not only not participated but having been made to feel guilty for my lack of enthusiasm and willingness to sacrifice dignity for the sake of this artist's work. The sense of obligation and potential humiliation of the experience was certainly powerful compared to the mild amusement of the works inside the gallery, however it was mostly a feeling of distaste for the genre that was evoked for me. It was closer to the uncomfortable duty of a work presentation than the more open-ended exploration and play that one might wish from an art-experience, whether as a solitary pleasure to be enjoyed in one's own time, or as a collective investigation with a feeling of relational connection and trust. My concern with this work is not only that the interactive component (movement triggering sound, with the sound's volume and speed relating in some way to the size and speed of the gestures) was somewhat limited in its aesthetic imagination, with little variation in reaction from participant to participant or over time within one interaction (although I think this is a valid criticism of the work). The distributed agency frequently attributed to interactivity is often lacking in these linear, prescribed constructions of relation. At best, as Brian Massumi argues, the interactive experience might seek to expand awareness of the processes of perception and relation (2011, 45), yet too often remains programmatic and replays the same standardised reactions, lacking in subtle and surprising combinations of associations, sensations, affects and prehensions. This is not to suggest that the role of interactive art is to mimic life, but rather that many such works display a paucity of life's rich, heightened experience of connection and potential. My larger critique, however, is an ethical one, deeply concerned with the politics of an enforcement of power relations that instrumentalise bodies and seek control rather than explore the possible expansion of expressive capacities. While there was certainly a series of relations established between the viewer or participant and the artwork in this example, these were highly problematic. The piece demanded 'work' from the participant, prescribing the types of relations and interactions that would be recognised by the technology and requiring a high level of energy from this viewer in order to produce itself (replicating the neoliberal dynamics of society that require the constant donation of immaterial labour). The participant was clearly in the service of the artist, not collaborating in any meaningful way. 'Choice' here became limited to opting in or out, with little possibility of nuanced and singular participation. Like many interactive art experiences, this event did not work to enhance my 'life-world' through any exploration of further potential combinations of bodies and artwork components, but instead replicated the dominant power relationships of society through obligation, control of gestures and the limitation of expression. The limitations of this work made me wonder: what would happen if we were to radically shift our notions of what interactivity is or might be? What would happen to 'interactivity' if we expanded the concept of it greatly and explored its essentially environmental or ecological potential? What would happen if we stopped limiting interactive potentials to human subjects, or to these subjects in conversation with an artwork across the abstracted and artificial divide of the 'interface'? What if we shifted scales – and worked across scales – and thought also the potential for interactive or relational development between, for example, an algorithm and a sensor, a sound vibration and a foot, an affect and a perception and so on? In highlighting this example I don't mean to suggest that interactive art should be 'nice', or that it should promote a bland positivity. Certainly there must be a place to explore tricky, slippery or challenging relations and propositions in art. However, there is a difference between the relational entanglement created by a work such as this example that offers only a heightened precarity to the individual ego in the face of obligation, and one based on a collective 'positive' extension of potential. The relationality that the neoliberal world already offers us is one of shared ecological, social, economic and psychic precarity that certainly creates a connectivity between people, but this is chiefly one of a shared vulnerability not an enjoyment of collective potential. Nor would it be enough for such a work to merely deterritorialise and delocalise or create and capitalise on speculative movement or reconfigurations of these already toxic connections (Guattari 2008, 33). Capitalism already operates successfully in this field of speculative and preemptive control, and, as I argue in Chapter One, the politics of relation in interactive work too often homogenise and constrict experience and orient the participant towards these dominant power structures. Nor can we truly imagine a work that would help us to 'escape' from such networks. Rather, to remain ethical, relational works need to pay attention to and care for what else might be going on: for the differential seeds or 'isolated and repressed singularities' (Guattari 2008, 34) that might suggest transversal movements and other, hidden potentials. Such potentials, rather than re-individualising and controlling yet another aspect of living, might, as I advocate throughout this book, instead be situated in an emphasis on Whiteheadian 'novelty' that is enjoyed at an ecological level and not on a level of individualised or subjective human enjoyment. Terms such as positivity, collective enjoyment, novelty and connection may well raise alarm bells. They will remind some readers of exactly the empty promises of consumerist entertainment already on offer, and the bland, identity-based and resolutely neoliberal iterations of relational aesthetics available for consumption at any major gallery. Here perhaps the concept of affirmation better describes the particular direction I am seeking to head towards in this discussion. Affirmation is speculative, seeking not to confirm the already-prescribed and thought relational possibilities, but to experiment freely and immanently (Manning 2016b, 201). Affirmation seeks to potentialise, thus it moves towards an increase in intensity or differentiation in the event (novelty) rather than homogenisation. To be clear, affirmation is of the event, not pitched at the level of an individual, subjective positive or negative emotional response. The event enjoys its expressions of novelty, which involve both the explorations of new conjunctions and disjunctions. An affirmative interactive practice might seek to expand and explore how components of an event can interact. This does not necessarily imply a concern for any individual component; rather it might seek to affirm ongoing ecological differentiation. Affirmation pitches discussions at a very different level to that of criticism. In the context of this book, it will become apparent to the reader that the works are not 'critiqued' in the negative sense of this term. That is, they are not there to be evaluated against some predetermined criteria of the new face of interactivity that the book might, from the outside, be mistakenly seen to be proposing. Their role is not to have the opinions or judgments of the author bestowed upon them, but, as Brian Massumi has written of this affirmative and 'immanent' style of critique, to perform a 'dynamic evaluation that is lived out in situation' (2010, 338).1 This is 'eventful' and seeks to engage with and acknowledge the singularities of a particular situation rather than resort to generalisations. Rather than leading to a shoring up of established positions, immanent critique might instead 'foster unforeseeable differentiations' (Massumi 2010, 338). In the context of the various discussions of artworks within this book, their inclusion implies neither any attempt to 'assess' or qualify the art, nor a ringing endorsement for all aspects of each work. Nor does it imply any assumption that these works form a 'canon' of important, new or ideal interactive models. Rather, they are there because there are some aspects of them that might productively help both the author and reader to think through the various concepts in their particular intersection with a singular art event, and potentially to lead such thinking into both unexpected and ever more diverse readings of the conceptual material at hand. Such thinking happens in the middle of the majoritarian events and theories, as minor undercurrents or dérives. It suggests a particular attention to what else might be happening: to transversal events that begin to split, fracture or unsettle expected outcomes or thoughts. In line with this, the aspects of the artworks that are examined are often incidental to their main focus. For example, in Chapter Five I discuss aspects of Nathaniel Stern's Compressionism, focusing on the particular and awkward assemblages and rhythms of bodies, spaces and technical objects, rather than on the undoubtedly beautiful photographic outcomes of these performances. Similarly, in discussing Rafael Lozano-Hemmer's Re:Positioning Fear: Relational Architecture 3 in Chapter Eight I focus on an accidental incidence of disruption to the original work that overlaid the existing event with new tonalities and intentions. Affirmation is performative or processual, and thus in this book the interrogation of the concept of relation is performed through the lens of what is broadly termed 'process' philosophy. Process is a creative event of formation of an entity through the 'transformation of the potential into the actual' (Guattari and Rolnik 2005, 311). Whitehead terms the placement of process as primary within thinking a shift from the 'material' to the 'organic'.2 Process philosophy's focus is ontogenetic, concentrating on how events (which here includes objects, relations and forces) come into being, rather than with the states they pass through (Massumi et al 2009, 37). Philosophically, this entails a shift from a hylomorphic view of the world as composed of discrete objects and subjects enduring in relative stability over time and which then interact with each other, to a view of the world as an ongoing, continually unfolding series of events of relation. This is an expanded notion of relation as emerging within an art event, concerned not with its demonstration or metaphoric representations, but with the power of conjunctive and disjunctive relational forces to creatively differentiate. That is, with the capacities of entities to affect and be affected in order to advance events. Thus it replaces ideas of transcendence – where development is focused on the achievement of an ideal, pre-described form – and focuses instead on the drive towards novelty and further differentiation.3 As Whitehead puts it, this is a novelty conditioned by its relationship to past events – 'an urge towards the future based on an appetite in the present' (1978, 21).4 In this approach, all relations need to be considered for their role in forming events, and thus William James' 'radical empiricism' forms an important base here, in asserting that only that which is experienced and all that is experienced must be admitted into its construction of the world (2010, 18). In this expanded model, thoughts and concepts are events in and of themselves, rather than projections or representations, and are as much a part of this enaction as objects. As such a process philosophy approach not only eradicates ideas of preformed or ideal subjects, it also, as Whitehead notes, 'abolishes the detached mind' (1978, 56). Relations that connect experiences, as James states, 'must also be admitted' as real and a place 'found' for them in the system (2010, 18). As Massumi notes, an implication of this system is that most of these relations exist only as potential, and therefore the virtual must also be considered as 'real' (2008, 39–40), with both actualised and potential relations being crucial to an understanding of the ability of relations to develop openly. Thus, expanded empiricism provides, as will be argued in the first chapters of this book, a path to 'thinking beyond' the purely mechanical and overt interactive elements between stable objects, and into a richer and more complex series of formative forces operating within a field; while still grounding thinking in lived experienced and avoiding the traps of transcendence and representation. With this position of the primacy of forces, an expanded and open definition of what constitutes a body is possible. The body referred to here is not limited to the subject, or to a fixed or post-individuated stable entity, but is itself 'a process of intersecting forces (affects) and spatio-temporal variables (connections)' (Braidotti 2002, 21). That is, bodies not only have capacities to interact with external forces and entities, but also are in themselves formed from the ongoing meeting and conversation of forces, and are therefore 'continuous' with the external world (Whitehead 1968, 21), as they also have 'internal resonances' and plays of forces (Simondon 1992, 305). Bodies are creative systems or emergent ecologies themselves, always more than any stable subjectivity, which might be better seen as a partial resolution in ongoing individuation that has always the potential for further movement. Rather than define a body by its representational qualities, the term body is here defined, as Massumi has described it, by 'what capacities it carries from step to step' (c2001, 4): in other words, by its performativity and its abilities to interact within an ecology of which it is an active participant (Grosz 1994, 194). Within this process-orientated view, not only bodies but also other entities – including inanimate objects – can also be defined by their abilities to interact with their environment, and they too can be thought of as complex negotiations of relational forces or events in themselves (Whitehead 1978, 73, 41). If entities all have their own capacities to affect and be affected by other forces and entities (Whitehead 1978, 856, 230), they are therefore always capable of further changes, of influencing and being influenced. This gives an opportunity to consider the interactive potential of not only human bodies, but also the affective capacities of all components of an art event's ecology. This thinking has the potential to greatly expand interactivity within a system, and suggests an obligation to begin to think about how non-human components of a system have capacities to interact with each other. In other words, it implies the necessity to consider a larger ecology at work, rather than focus purely on artwork-participant relations while assuming that other relational forces and objects will remain fixed or are less important to the developing relational meshwork. Here we might, for example, begin to consider the emergent relations (and collective becomings) of and between a speaker vibration, a floor and a diffracted sound wave as examined in Chapter Seven. Or we might pay attention to the relations between movement, shadows, a light sensor and an electrical current, and so on, whilst at the same time thinking about their connections to various bodily assemblages – sense organs, surfaces, forces of movement. This, I argue, has scope to expand notions of interactivity through thinking the potential of much more complexly intermeshed and collectively emergent tensions within an art event, activated through acts of prehension and transduction across its many registers. At the same time, the implications of these ideas potentially move the discussion on interactivity beyond 'new media' artworks.5 As will become apparent both from the choice of works and the aspects of these works discussed, interactive potential should not be limited to works obviously mechanically interactive in their enaction, nor to work necessarily involving 'technologies' in the most obvious sense of the term. This positions the actual artworks discussed and the implications of the discussion within a wider framework and history of relational and participatory artwork.6 Through these discussions on an expanded and heterogeneous relationality I develop a concept of emergent self-organisation in interactivity, which I term a 'gathering' ecology. This broadened concept of interactivity emphasises an event's ability to move towards the generation of its own outcomes out of emerging difference within relations. This self-motivated or ecological gathering is always on the level of the virtual – a gathering of potential – as much as it is an actual entanglement of relations. While in general, process philosophy is always concerned with the becoming of events, this extends becoming in the sense that it concerns not simply the idea of the becoming of an event within a field, or even that the field is co-emergent with the event. Here the very rules and potentials governing these acts of organisation are emerging or gathering as one, although this is a fragmentary, heterogeneous whole. In this sense I argue that there is a shared immanence running through an entire ecology, and autonomy of any component entity is always emergent not only with other entities that parasite it, but with the subjective forces of the ecology with which it nests. In developing this concept I utilise several related or overlapping concepts that argue for the primary role of intensive differentiation in the becoming of events. In Chapter Three this entails a close examination of Whitehead's concept of 'feeling', a complex and abstracted ontology of relation that is for him at the basis of all becoming.7 Here, in order to become, an emergent entity selects 'datum' from other actualised entities and from the virtual plane, intensively valuating and patterning these feelings as 'one complex feeling' (Whitehead 1978, 22) that constitutes its very 'concrescence', or event of becoming. In rethinking interactivity the particular usefulness of this system, which I outline in much more detail in the chapter, is that in this act of feeling an entity has autonomy from what has come before, in that it selects and incorporates only some of the possible information from the actualised world and some of the potentials. It therefor self-generates novelty in the world, but is at the same time always relational. There are many similarities between this Whiteheadian concept and Gilbert Simondon's system of individuation, and in the later chapters of this book this connection is explored through the idea of transduction in relation to micro-perception and sound, interfacing and generative software programming. Transduction, as 'the foundation of individuation' is for Simondon a process whereby an entity (again, in the broadest sense) generates itself through an intensive gathering of incompatible external forces into an intensive communication 'without loss, without reduction, in newly discovered structures' (2009, 12). Feeling and transduction, which cut across forces and forms to generate new intensive and extensive relations, are at the core of the thinking my exploration of the capacity of differential operations within an art event to be activators of co-causal relation within interactivity. In thinking difference, particularly from the pragmatic perspective of the construction and interrogation of interactive artworks, the third key philosophical tool, which is examined in Chapter Four and put to use throughout the book, is Michel Serres' concept of the parasite. This he defines as the essential noise in any system of relations.8 The parasitic disruption to relation that produces new relational connections from within an existing system is proposed as a mechanism for intensively generating change while also drawing elements into more complex interdependence. The parasite, which Serres argues is always present within relations (2007, 79) (and which both Whitehead and Simondon also argue to be constitutive of becoming in the form of the held and productive intensive differentials of an entity), problematises simple connections with its ever-present potential to further differentiate. It transforms stable systems into evolving systems of co-causality.9 Again, all these concepts are affirmative in their focus on the processes of speculative advance towards future novelty. They are speculative in that the outcomes are not prescribed, and positive in that such advance does not erase difference but intensifies it. Thus difference here is not oppositional or negative, but a dynamic creative force for both extensive exchange and intensive development, binding heterogeneous elements into the production of the event (Deleuze 1994, 57). While a discussion that takes process-based ideas of the emergent and intertwined nature of all events is necessarily one about relation, enthusiasm for the 'relational' must be tempered by a closer consideration of the nature of these relations. As I argue in Chapter One, the politics of relation in interactive work too often homogenise and constrict experience and curtail open experimentation. To remain ethical, relational works need to instead concentrate on enabling expressive capacities,10 and to position heterogeneous elements in dynamic or productively noisy relation. Here within the writing I identify and emphasise the imperative to give particular attention to how the various components of an art event begin to gather and intertwine in each other's and a collective creative advance. Amongst this search for an ethical 'equality' of interactive potentiality, we must consider the 'technical equality' that Simondon calls for, which implies 'equal technical participation, even as it assumes difference' (Combes 2013, 92).11 In this sense the 'health' of the whole ecology – in sustaining and extending its expressive capacities – is always a premium consideration in an ethical interaction. It is a question of how ecologies as sets of 'complex dynamics of relations in a given situation' (Bertelsen 2012, 41) begin to form through interactions – not only between participant and work, but between all material, conceptual and affectual components. This interest in the ethics of emergence must also, it seems to me, be extended to include the emergence of thought and concepts, and in this the book adopts a particular methodology that could also be termed transversal. If the task of this discussion is to utilise an affirmative experimentation across conceptual and practical registers to examine the creative role of differentiation within interactive art events, then here this entails a methodology of multiple readings, multiple configurations of concepts, and multiple propositional relational encounters. This is proposed as a potential politics: an ethics addressing immanent construction. This is an ontogenetic approach to the text, practicing a tactical and parasitic method of research that could be described as a 'meta-modeling' or 'study'. Stefano Harney and Fred Moten have proposed the term 'study' as a type of collective learning without end that is resistant to the academic disciplining through policy, reward and identity (2013, 67–8). Study is the experience itself – something already going on – sometimes underneath or inside or in spite of the structuring of knowledge and thinking that it destabalises. Study does not 'call to order' along the lines of an established hierarchy or knowledge (Harney and Moten 2013, 125–6). In this it suggests that we must be careful about not only the content of what is studied, but the methodologies employed, recognising that they are not simply organisational, but can, as Manning states, have a deleterious disciplinary affect on thought in constraining it to the alreadyknown (2016b, 34). Rather, Manning says, study and associated becoming-methodologies such as research-creation might allow us to think beyond the known and to instead experience the act of knowledge becoming out of the unknown (Manning 2016b, 30–1). Just as process philosophy asks us to think objects and subjects as experiences or events, study asks us to consider the larger generation of the conditions of knowledge's emergence. Study, in this sense is not a usual kind of methodology – rather it might be thought of as a becoming-methodology immanent with the problem that we wish to think: a tactical approach. A tactic is open-ended and opportunistic. It reuses elements of a system (as both feeling and the parasite move towards novelty but are formed from a reconfiguration of already present relation), 'without taking over [the system in] its entirety' (de Certeau 1988, xix). The tactic therefore destabilises from within as a minor movement, without necessarily imposing new order. It remains essentially per-formed. A tactic (such as an immanent critique) is always singular, forming in relation to the specific set of conditions within which it arises, and must be reinvented for each new set of events. Various tactics also fold into and complicate one other, so that the range and exact terrain of their productive operation can never be fully defined. In this regard, tactics must be reinvented through practice, avoiding the rigidity of sets of rules or manifestos, being co-composed with events in which they seek to intervene. In Chapter Two, the concept of the tactic is utilised to think the re-invigoration of interactive systems from within, through concepts of molecularisation and drift. In Chapter Four the parasite as a tactic is proposed as molecularising in its production of difference or movement (Guattari and Rolnik 2005, 311) within a dominant form of interactivity. A 'tactical' approach is clearly in line with a process philosophical view of the world, centered on propositions, the gathering of forces and the immanent nature of events, rather than outcomes and closure. A methodology consistent with this impetus within a process-philosophy stream must also address a tactical use of process philosophy. That is, concepts must be reinvented and investigated for and within each singular occasion, not relied on as established truths. A concept itself always individuates with and within a field, and if it has a distinct 'consistency' that 'renders its components inseparable within itself', then this is at best a 'fragmentary whole' (Deleuze and Guattari 1994, 19, 16, emphasis in the original). Here, as Serres states, rather than assuming the possibility of a 'universal method', one should instead seek to compose 'an appropriate method from the very problem one has undertaken to resolve' (Serres and Latour 2011, 91). Within this style of inquiry the invention of knowledge is, as Simondon notes, 'neither inductive or deductive, but transductive', corresponding to a discovery of the dimensions or field of inquiry in conjunction with the specific question (2009, 11, emphasis added).12 Thus a tactical approach avoids the use of models, but rather immanently and speculatively models the problem at hand. For Guattari models are problematic in that they are 'reductions of a diagrammatic space made of intersections and disjunctions' (Parisi 2013, 4). As Manning and Massumi argue, models are 'prescriptive templates' that limit and control the discourse on actual events, which have potential beyond their iterations (2010, 28). In a related discussion, Janell Watson outlines two essentially negative ways that modelling circumvents discourse. Firstly, she criticizes the way models encourage the tendency to analyze actual events only in relation to a perceived 'norm' rather than thinking outside the restrictions of such 'dominant social order[s]' (Watson 2008, 1). Secondly, by prescribing processes, models necessarily curtail possible outcomes – that is, they reduce the freedom of the virtual to a limited set of possible outcomes (Watson 2008, 2). In this book I have a desire to open up space for multiple potential analyses, and Guattari's concept of metamodelling, which bypasses 'the imperative of representation' (Parisi 2013, 4), is thus proposed as a suitable methodology for creating a 'becoming' model of inquiry. Metamodelling, Guattari states, is 'to render palpable lines of formation, starting from no one model in particular, actively taking into account the plurality of models vying for fulfillment' (cited in Manning and Massumi 2010, 25).13 Metamodelling, as Guattari says, places the emphasis on the way ideas interact or have the potential to interact to produce new associations (1995a, 59). To establish a model for the analysis of interactive art risks the exclusion of elements that do not fit, such as aesthetic qualities, an under-discussed area of much interactive art criticism. Fixed models might also imply the creation of a 'check-list' of necessary elements that an artwork must contain to be called interactive; the bracketing into stabilized categories of problems and solutions; and the uncritical promotion of potentially insidious social norms. In contrast, the process of metamodelling abandons attempts at establishing set models, accepting potential in all possible models – providing, Guattari states, they 'abandon all universalizing pretensions' (cited in Watson 2008, 3). Rather than creating a 'didactic program', metamodelling involves a disentangling of oneself from systems of modelling that 'pollute our ways of thinking', creating instead a contingent critical 'bricolage' of possible approaches to be utilised for the particular analysis at hand(Watson 2008, 3). In this sense, metamodelling clearly experiments with a re-energising and reconnecting of existing elements (whether conceptual or physical). Metamodels are resolutely singular – that is, they allow the possibility of constructing a usable model for any given situation by 'taking bits and pieces of other models in an attempt to solve a specific, singular problem' (Watson 2008, 8). This requires embracing increasing complexity and contingency. It demands a preparedness to act contingently and cobble together usable discourses as necessary, and it also requires one to allow this assemblage to perish after the event,14 starting afresh each time. Thus, in relation to interactivity, this methodology enables the taking of any productive path of critique necessary to accommodate new input (and the jumping from path to path), rather than setting up fixed criteria for interactivity and either ignoring contradictory information, or dismissing artworks for not living up to established definitions. I want to suggest, as Manning and Massumi do, that this freedom to adapt and change direction – to critique immanently and speculate affirmatively – be viewed as a positive move, which might 'energize new models of activity…[and] offer a potential to escape or overspill readymade channelings into the dominant system' (2010, 7).15 Metamodelling might be seen as being both speculative and pragmatic, in that it refuses methods or models imposed from without and instead encourages 'a rigor of experimentation' (Manning 2016b, 38). It might also be transversal, seeking to invent new associations and collective potentials. A particular methodology of use here is that of a 'research-creation' framework, which seeks to create resonating lines of inquiry through writing on concepts and artistic experimentation. At its best,16 research-creation might, as Manning writes, exhibit a 'transversality', proposing 'new forms of knowledge, many of which are not intelligible within current understandings of what knowledge might look like', therefore staging 'an encounter for disparate practices, giving them a conduit for collective expression' (2016b, 27). Research-creation is of particular interest here as the writing of this book (perhaps inevitably given my own practice as an artist working within participation and interactivity) has been centrally informed by this history of my continued practical wrestling with the problems of how to turn a general idea of a relational work into an actual work that engages with technologies and bodies in more expansive and ecological ways. At many points these struggles with various artworks threw up possibilities that shifted or troubled my theoretical ideas and which suggested new possibilities for philosophical enquiry. To give one simple example out of many, Chapter Nine, while always informed by reading and writing on the subject of generative algorithms, could not have been conceived of or written in its current propositional form without my deep engagement with the practical task of trying to write a software patch that in some way enacted the concepts proposed. The concepts at this early stage were 'extra-linguistic' (Manning 2016b, 27), tenuously co-emergent with some confluence of code, software, hands, instinct, sounds, maths and the enabling constraints of the sensors and shapes of the artwork as it began to take form. In parallel, and at many other times, reading and writing on the subject opened the beginnings of practical experiments. This is particularly true of the concept of the parasite, and my ongoing attempts within artworks to create parasitic (and therefore conjunctive and disjunctive) relations of connections between various components of such works. In this way while the book is a 'study' of the problem of interactivity, it also seeks to work beyond being something to be studied, and instead to become 'the occasion for study' (Harney and Moten 2013, 109). This a transversal approach that is an ongoing, open and collective activity. This collectivity of study might be more than a collection of subjects, and rather might include collective, diverse activities of 'thinking through' such as parallel making and writing (research-creation), a continued problematisation and multiplicitous approach to the questions (a metamodelling), and the continued intensive movement of these questions (a tactical molecularisation). Here, in 'the crafting of problems greater than their solutions' (Manning 2016b, 10) this study seeks not to conclude to a single point, but rather to build 'machines' to explore the potentials of parasitic actions and feeling, and to push the limits of interactivity, attempting to allow such speculative thinking and immanent connection of ideas on the part of the reader as well as the author. This is a mode of study in which we might find, as we read, that we (collectively) have already been in the middle of. This study, as Harney and Moten argue, is a place where 'the incessant and irreversible intellectuality of these activities is already present', and where the recognition and participation in this multi-leveled approach might allow one 'to access a whole, varied, alternative history of thought' (2013, 110) about interactivity. 1 ### Interactivity and relation: The myth(s) of interactivity 'Interactivity is a very dubious idea.' Woody Vasulka In this chapter I want to chart some of the criticism surrounding the term 'interactivity', and the move towards the concept of 'relational' art. The intention of this critique is to move towards more of a productive engagement with the expanded potential of interactive art rather than to dwell on its past crimes. This problematic history has led many writers and artists to move away from the term interactivity and towards one of relationality to distinguish themselves from the narrow scope of these works. One of the first difficulties we encounter in discussing interactivity might be that the term itself has no readily agreed upon definition. While some authors use it derogatively to condemn programmatic, simple to-and-fro exchanges of an object-orientated communicational model (Massumi 2002, xv)17, others use the same term to imply a much wider range of participatory experiences that might be broadly termed relational. In Towards an Aesthetic of the Interactive, Alan Peacock defines interactivity as 'experiences that include a feedback loop and mutually (self-) modifying sequences and choices within the sequences that form a particular from many possibilities' (2010, 1). Simon Penny also argues for the necessity of feedback loops and demonstrably developmental aspects in design, stating that 'the fundamental requirement of an interactive system is that it correlates in a meaningful way data gathered about its environment (usually a user's behavior) with output'. Without this, he says, there is no perception of interaction (Penny 2011, 80). These definitions, while somewhat limited compared to the more complex and subtle combinations of forces available for consideration within a relational model, do capture a popular idea of interactive art. Here interactivity is conceived of as modification over time of the work itself, and possibly the behavior of the participant, in a way that is perceptible and comprehensible to the participant.18 As Nathaniel Stern points out, these definitions of interactivity tend to concentrate on explanation of the fact that 'a given piece is interactive and how it is interactive, but not on how we interact' (2009, 240)19. That is, Brian Massumi says, there is a concentration on function, rather than quality, that limits the debate (cited in Lozano-Hemmer 2000, 201). This is tied to a focus on the representation of interactivity that fixes relation to preconceived models rather than allowing the immanent production of new ways of experiencing (Murphie 1996, 4–5). Many writers and artists therefore prefer to move from the term interactivity to one of relationality to escape such narrow definitions. In this light Erin Manning proposes that the relational is 'active with the tendencies of interaction, but not limited to them' (2013, 29). Others have attempted a reconditioning of the term, and continue to use interactivity while implying a much wider range of qualitative potentials, believing, as Kelli Fuery states, that a prescriptive view 'must be resisted, and it can be resisted…if we view interactivity as an unstable and uncertain process' (2009, 45).20 Limited and functionally based discussions of interactivity do, however, still contain some pertinent critique, even if in some cases they fail to grasp the potential of a wider reaching and more qualitatively based discussion. Proponents of interactivity have promoted the existence of some qualitative – and indeed moral – judgment of difference between 'interactive' and 'non-interactive' forms. Simone Osthoff argues, for example, that Lygia Clark's work utilises the viewer's own energy, synthesising mind and body to explore the sensorial, and thus replaces the object with the experience. This experience, Osthoff argues, essentially differentiates the interactive experience from the type of engagement that painting and sculpture allow (279–80). Perhaps here there is an implication of an essential moral superiority in interactive artwork, echoed by Victor Stoichita's statement that in Rafael Lozano-Hemmer's work we 'are no longer before the (interactive) work, we are in the work' (Lozano-Hemmer 2007, 129). Similarly, both Pierre Levy's assertion that interactivity 'actualizes the decline of totalization' (2001, 131), and Roy Ascott's claim of 'moving beyond the object' from observed effect to participation, consider participatory art to be somehow in opposition to more 'traditional' forms that might distance one from the process (2003, 237, 328). What then are our expectations of the functioning of interactive art? That it expands the range of art experiences available to the audience, offering levels of 'free choice' and embodied experience seemingly unavailable in more traditional art forms? That it will be participatory on some level unavailable in the supposedly more passive enjoyment of traditional forms; or that it will be experiential rather than representational? The question of (free) choice is, as Alan Peacock argues, one on which the success and failure of interactivity commonly balances, stating that 'decision making of some kind is a necessary condition of the interactive' (2010, 3).21 But are there levels of experience in which there is really open-ended decision-making or 'free will' in generative or interactive art? Can interactivity really offer more choice than, for example, a painting? Can it offer as many options to the viewer, either in the way they assimilate content or in the choices of level of involvement in the work? An exhibition of paintings might offer the viewer relatively free reign in their manner of experiencing the space: the choice to skim over some works, view them in any order, dip in and out of concentration and so on – all fairly banal choices that one would take for granted. Interactive works on the other hand, as Massumi cautions, often dictate prescribed and limited actions in order to achieve results, creating 'a kind of tyranny to interaction' (2008, 1, 3). Such interactive works can then enclose us, as Louise Poissant says, 'into a schema of manipulation rather than propos[ing] a real space for dialogue' (2007, 245)22. In these situations, Mona Sarkis argues, the participant in interactive art remains a passive 'user', assembling the artist's vision without any real free choice (1993, 13). Thus she claims the interactive possibilities of technologies promoted by their producers are often 'adopted in a careless and uncritical manner by…artists and philosophers' (Sarkis 1993 13). It should therefore not be taken for granted that participation in interactive art events necessarily grants freedom from the normative viewer–artwork paradigm. Rather, participation potentially co-opts art practice into the construction of mutable, exploitable bodies (Stern 2012, 26–7). As Manning pointedly states: > To be forced to play is like being forced to touch. Not only does it potentially do violence to the complex relational field in co-composition, it also presupposes an already homogenous arena of engagement (2013a, 129). These contentious elements of interactivity and control might be broadly thought of as three problematic and overlapping 'socialassemblages': productivity, linearity, and histories of control and power embedded in the technologies. Behind these assemblages lies the issue of the naturalisation of a representationalist or essentialist modelling of experience.23 Critiques of these aspects of interactivity link it to bio-politics, consumable entertainment, demonstrability and its didactic applications. Here interactivity, in failing to escape such discourses might become the 'dubious idea' of Woody Vasulka's comment. #### Productivity and exchange The productive structuring of interactive art experiences is situated within the history of the commercialisation of its aesthetics and technologies. While we might commonly think that artists repurpose commercial technologies into more artistic production, Penny argues that there is an historical dialogue between the two that is largely ignored. The 'techno-formalist' concerns (Penny 2009, 4) at the center of 1990s' media art explorations laid much of the groundwork for gaming interfaces, for immersive training systems utilised by the militarily and commercial sectors (such as flight simulators), and for social media platforms on the internet (Penny 2009, 21). These nonart world technical advances – combined with new media works themselves – were, according to Penny, 'informed by the previous thirty years of "art and technology", installation art, performance art and video art' (2009, 11). He proposes that not only do artists recommission technologies of control, but that many of these more prescriptive and troubling applications have arisen, if inadvertently, out of artistic experiments in manipulation.24 The concentration on technical advance, alongside the necessary collaboration with companies and research laboratories invested in the commercial applications of such advances has lead, as Penny points out, to the adoption of a certain philosophical stance that has leant itself to the development of interactive systems based on the dynamics of consumerist exchange.25 These applications promoted certain Platonic ideas about the division of mind and body, naturalising '"objective external real", "sense-data" and "representation"', and the thinking of participants as 'users' or consumers (Penny 2009, 22). Here, Penny argues, the concentration on a distancing vision, produces a 'scopophilic obsession with the eye and vision…[producing] a technology of the phallic gaze, the conquering eye, in which the holistic nature of embodied being [is] elided' (Penny 2009, 22). As such, certain power structures have become a largely unquestioned norm of interactivity: stable systems of objects and bodies exchanging via an interface; users responding to already-formed sets of information; systems that draw attention to their mechanics through reward for behavior; and a focus on representations and exchanges of content within predefined parameters rather than co-emergence (Manning 2009, 63). This might be a focus on 'being – as a generalized ontological equivalent' rather than 'manner of being' (Guattari 1995a, 109). Such a conception of interaction, Stengers argues, implies 'terms that make a difference for one another, but a difference that does not modify their identity' (2011, 514),26 and thus, in this context, an interactivity that fails to challenge the roles of consumer and consumable object that might perhaps begin to be questioned by more open-ended relational works.27 Participatory works sometimes claim to escape this paradigm through a certain freedom from representational content28 – aiming for visceral experience over narrative, contemplation or reflection. While in one sense it is true that a painting's content is constructed by the artist prior to the encounter with a viewer, even in the most didactic, narrative-driven image, there presents the possibility, one could argue an inevitability, for a freedom of association, for the viewer to link elements to memories. For example, a viewer might make personal and cultural associations, such as colours reminiscent of a flag, facial features associated with a friend, lighting effects that trigger memories of a half-forgotten film, muscle memory or a prehension of movement made conscious through an association with a figure's awkward pose. This association is not simply a reliving of old memories, but an actualisation of potential that creates new thought in the event of artwork and viewer, matter and memory. These subtle connections are exactly the kind of 'interaction' that fits with Manovich's argument that the notion of interactivity must become inclusive of notions of psychological processes, and mental as well as physical or temporal connections (2001, 56–7). Many artworks might therefore be read in this psychological sense as loosely 'generative' – not 'mechanically' as in some participatory works,29 but in that the event or experience still emerges from the combination of viewer and work that in its singularity inevitably begins to escape the confines of the artist's control. Interactivity, however, can often struggle to allow such excessive layering and complicating of dialogues. Productive interaction is often lacking the multitude of potential connections and struggles to become excessive, to outstrip function and destabilise orderly systems of exchange.30 The 'tyranny' of interactivity is that it is based not just on required participation, but also on the reduction of such participation to the parameters of linear, programmatic and productive exchanges. The 'connection' promised through interactive participation can often remain at a level of relation that stays safely within systems of information exchange, harnessing participation into the circulation of flows of desire within capitalism (Massumi 1992, 200–1). Here the dynamics of interactivity can be seen to contribute to the construction of exploitable bodies within such a paradigm (Stern 2012, 26–7). There is a danger here that these problematic dynamics might work not only to construct the body as a kind of databank of new information to be fed into the workings of the system, but that as such systems become 'naturalised'. That is, productive and limited exchange becomes the anticipated relationship with a work to which participants then moderate their actions and expectations. As discussed below, this relationship becomes an internalised response to the environment and bodies are problematically performed as data or 'immaterial labour' to be exploited (Foucault 2010, 206–13). It is perhaps then no wonder that interactive technologies form the basis of much entertainment industry spectacle, and interactive systems and displays sit so comfortably in didactic museum displays. Ironically, the very participation that in art is intended to free the viewer from constraints instead operates very effectively to contain, direct and lecture them.31 As Massumi argues, to utilise such technologies in a becoming and emergent fashion, they need to be freed from 'exchange-value', to move beyond 'prodding a participant to gain a response', and take on a more speculative nature that allows an excess to emerge (2008, 9)32. The artist involved in developing interactive systems might be charged here with an obligation to think beyond these co-optable dynamics of relation that so easily lend themselves to dominant power structures, and develop more complex ecologies of relation that begin to resist or at least question productivity and spectacle. #### Linearity: riding the interactive train Interaction can become trivial, as Roy Ascott suggests (2003, 378), in a closed, linear system with finite data – a flicking of an 'on' switch with the viewer's presence, or a prompting of a software program to jump to the next prearranged scene, as in a video game. Preprogrammed events here lack emergent qualities that might help shape the actualized events through the immanent creation of further potential (Manning 2009, 74). The lack of physical or psychological tension created by such experiences is often in hollow contrast to everyday lived experience, as the excess of the virtual is replaced by the probable, while open-endedness is replaced by specific purposes. What space for contemplation does the interactive installation allow? Perhaps the curse of interactive art is that often the viewer must either abandon midway through boredom, endure to a set endpoint, or at least move through in a set direction. That is, the experience remains essentially linear, 'prepackaged and predigested', as de Mèredieu states (2003, 213)33. Levy argues that 'cyber art' systems of interactivity operate against the totalising forms of traditional media, allowing new and greater potentials for coproduction (2001, 115–6, passim). Similarly, Ascott claims that interactivity offers empowerment and greater participation in the workings of the art event (2003, 284). Here the experience can be rather like the participation in riding a train: certainly we are bodily involved in the machinations of travel, but with limited entrance and exit points and heading inexorably in a prescribed direction. It is a kind of roller-coaster experience that contains a certain level of visceral thrill and manipulation without allowing any greater level of co-authorship of the experience (Poissant 2007, 245). The risk is that our movements loose their incipient qualities, and the many and varied levels of potential participation are instead reduced to a role of merely 'performing the software' (Manning 2009, 63). Interactivity here becomes 'Pavlovian', as both Penny and Lozano-Hemmer have noted: a 'trivial' modality based on an action and reward system (Penny 2011, 78; Lozano-Hemmer, Boucher and Harrop 150).34 As de Mèredieu comments, the predetermined nature of such interactive systems 'confines the spectator's actions and reactions to a well mapped art path', and the ubiquity of such forms as 'an art trapped in prefabricated "networks" [running] the risk of being transformed into a kind of global, collective "art in kit form"' (2003, 230–1). While some artists may argue that interactive works have moved beyond this paradigm,35 many would argue that this issue is still pressing today as much work continues to focus on the performance of its mechanisms rather than an investigation of a 'becoming' of such mechanisms (Fuery 2009, 43–4)36. While these issues with escaping linearity arise partially from the use of technologies created for specific, productive purposes, it would be wrong to simply attribute this, as Wood does, to any inherent or inescapable properties of such technologies (2007, 16). Rather, we might see this issue as arising more specifically out of the technologies being primarily harnessed to represent relation within the interactive encounter (in itself a 'productive' use). This tends to promote the demonstration of interaction over experiential emergence (Murphie 1996, 5) and to instrumentalise the user to represent the potentials of the technologies (Penny 2011, 73, 87). This is not to say that the comprehension of relational factors in itself denies a rich involvement in immediate sensorial experience,37 but that the desire to clearly demonstrate to the participant that they are indeed interacting with and causing change or growth in the artwork can prevent the riskier task of enabling the performative exploration of emergent relation. Such relations may or may not reach a level of perceptible representation, and may indeed remain at the level of the virtual. In the sacrifice of the uncertainty of emergent relation for demonstrable connection, what is lost is the 'elasticity' of the larger potentiality of the event. Relations are then often made rigid and linear to ensure the pay-off of a quick and simple explicated exchange for the participant. This focus on demonstration imposes 'self-completing lines through representations that trace existing conditions and attempt to repeat them', as Andrew Murphie argues of representation and virtual reality (1996, 6),38 through its need, even anxiety, to facilitate the perception of an interactive experience. As artist David Rokeby notes, the 'Simplified representations [of interactivity] replace the relationships to which they initially referred. This substitution turns the interesting ambiguities of control and subjectivity in interactive art into serious issues of control, manipulation and deception' (NDb).39 This serves to bring, once again, the modality of interactivity back to the language of gaming, where 'unprescripted' potential is replaced by variations of the possible (Massumi 2002, 9).40 Exploration of 'becoming' in any larger sense, which is essentially non-linear, is replaced by the rehearsal of the already formulated and comprehended (Braidotti 2002, 118). #### Histories and networks of control It is certainly true, as Manning warns, that the sensory technologies at the base of many interactive works have 'problematic pasts, both as displacers of the corporeal body and in assemblages of control' (2007, 118). As I have argued above, Penny presents a potentially even more troubling history where artists must share some of the burden for the ways their technological experiments have been put to use. Mark Dery optimistically advocates that the repurposing of such oppressive technologies within artworks is a potentially political act that displaces the power dynamics by making art with such tools of control (1996, 14). More pessimistically systems in which an artist employs these tools to control the interactions between bodies and artwork could be thought to mirror the political utilisation of surveillance by governments to create systems of control. Therefore in such systems the work could still be said to celebrate the power of the technology.41 Indeed, Penny argues, while these technologies are deployed in novel ways, they retain many of their original functions, including the potential for control inherent in the representation of relation (whether body–technology, body–body, or body–subject) (2003, 268)42. Such representational modes of production can be linked to the reinforcement of the status quo – static systems of discrete subjects incapable of escaping a pre-constructed mode of being. There is no doubt that these technologies have at least the potential to reproduce such power relations, and it is disingenuous for artists to simply assume that art can avoid such pitfalls without a close examination of whether there has been a true shift in the dynamics.43 However any argument that such technologies necessarily have only the capability to produce these power relationships seems flawed. Despite the undoubted links between surveillance and interactivity, this would tend toward a 'technological determinism', as Murphie and Potts argue, framing understanding of technologies as objects capable of independently creating certain relations of power within society, rather than considering them for their functions within certain contexts (2003, 13, 32). Whether inherent or not, surveillance might be thought to 'capture' the body, both in the flattening of the experience of a body to a fixed identity or subjectivity, and the fixing of it within a readable space.44 The reduction of the potential of a body in some virtual reality (VR) immersions to a representation divorced from the complexity of embodied sensory immersion in the world leads, Dery argues, to a 'static body' locked into 'observation mode' (1996 234–5). This is, Penny states, a 'thinning out' experience in an action of 'standardization, reductivism, efficiency [and] instrumentality' (2013, 7–8). Although these critiques are both specifically aimed at virtual reality, while interactive art events utilise the same structures to fix or interpret bodies,45 they will remain subject to the danger of falling into similar power relationships, despite their claims to a greater level of embodied participation than other forms of art. Such representational use of bodies denies their everindividuating nature, and can contribute to disengagement with the corporeal – the separation of images from body that is part of the operation of surveillance.46 These are not inherent properties of the components of the art assemblage, but arise because the components combine to produce similar problematic networkcontrol paradigms. It is through the performance and repetition of these 'specific bodily acts that bodies are reworked and that power takes hold of the body' (Barad 2007, 63). Matteo Pasquinelli's analysis delves further into the problematic and essentially neoliberal aspects of network culture and the digital. He interrogates the 'post-Fordist' move to immaterial labour and charts ways in which, far from escaping capitalist systems of exchange, creative commons and immaterial property always have material property implications that both remain exploitative of labour and absorb innovative and 'revolutionary tendencies' (2008, 87–9, 23–4).47 Pasquinelli argues that far from freeing bodies, the rise of the digital has lead to a new sphere of exploitation as digital machines parasitize the labour of living bodies: a new dynamic model of 'cognitive capitalism' applicable across dimensions or modes (2008, 97).48 What happens, we might wish to ask here, when an interactive work demands the labour of the viewer for its operations? How might the enthusiastic turn in major art events and galleries towards relational aesthetics be at the very least inadvertently creating exploitative relations that, rather than questioning power, work to enhance its operations on highly personal levels, both physically and cognitively. Are there other ways in which a participant might be engaged that do not reinforce existing (or indeed invent new) methods of inequality? Certainly as a beginning point, where any technologies of control (including capitalist exchanges) are concerned, we need to interrogate, interrupt or shift the kinds of power dynamics that are enacted and the networks that are constructed. Within this we need to question the ways that such technologies encourage the replacement of embodied experience with representational models, and the imposition of normative subject–object relations. Here it is not enough to simply claim that the end product differs from the original design aims of those technologies. More detailed and critical examination, particularly of the larger structures and systems within which such technologies are operating, is required. It is important that artists investigate ways to escape the mechanics of the production of exchange, subjectivity and networks of control, in order to allow a rethinking not just of the component parts or productions of these machines, but also of the ways in which these parts form problematic relations. This is an interactivity that moves beyond the performance of a mechanism, as Fuery suggests, becoming itself immanently interactive as a technique for the processes of individuation (2009 43–4).49 As Deleuze notes, it is never enough to trace a line away from something, but rather lines of flight need to continue to be generated for the work to remain performative (Deleuze and Parnet 1987, 29). #### Art as event: a relational model The arguments above begin to suggest some of the problematic ethics of interactivity that are present not just in individual explorations of the genre, but whenever the underlying structuring of the production of the experience is unquestioned. Rather than dwell on these points at length or critique individual works, the purpose here is to propose potential tactics for the thinking beyond those kinds of relations identified in the previous section. Here, more than a critique of specific iterations of the modality, I would suggest that the issues raised are the inevitable outcome of an essentialist system of interactions, which attempts to stratify the reality of co-emergent change (Massumi 2002, 207). Thus it is not enough to simply demand more from the interactive artist and critic: more complexity, more imagination, more inventive solutions, citing that it is a relatively 'young' art form and arguing for its inherent qualities. More imaginative creativity will always have its place, but the issue underlying the limitation of interactive artworks lies primarily, I would argue, in a philosophically limited conception of an object, a subject, and a work of art. It is this thinking that underlies the narrowness of both the invention and critique of interactive art – a narrowness in the selection of evidence as Whitehead might argue, which, in its attempts to reduce the field of discussion to a manageable stability, succeeds only in denying the actual nature of the event.50 What happens to interactivity when rethought through the prism of a process philosophy? As Barad states, a 'dynamic conception of matter is an unsettling of nature's presumed fixity and hence an opening up of the possibilities for change' (2007, 63). If we encourage an ecological approach that emphasises co-emergence and inter-dependence, could we rediscover a fluidity and layered inventiveness and begin to both think and construct interactive art differently? ### From material to organic thinking Massumi argues that interactivity describes a simple back and forth between two elements that remain discrete (in Lozano-Hemmer 2005, 201),51 reflecting a material view of the world in which the viewer is a stable subject and the artwork is a stable object. Seen through process philosophy, however, the scenario is very different as these stable and persistent subjects and objects are replaced by entities that are themselves processes (Whitehead 1978, 41, 309).52 For Whitehead these actualised entities are atomic. That is, they do not change in themselves; rather they exist only in the instance of their becoming, perishing in actualisation to be replaced by new actualisations, an endless advance towards intensity and invention. Viewed in this way, 'objects' are 'cuts' in processes of concrescence of complex events of relation, while 'subjects' (or 'superjects' in Whitehead's preferred term) arise out of experience, rather than interacting with the world in a transcendent manner (Whitehead 1978, 155). This can be related to Simondon's concept of individuation – an ongoing process of development of an entity that always 'contains latent potentials'. Individualisation is here thought of as a 'cut' in this ongoing process (Simondon 1992, 300). Individuation is not, however, a single process of development, but rather 'overlapping phasings happening in non-linear time'. A 'dephasing' or cut occurs when events 'tune toward…a discrete iteration, a remarkable point' that is a 'shift in level from individuation to individual' (Manning 2013a, 17–18). Such concepts can begin to challenge how we think of, make and experience interactive art. They imply the need to view art objects, events and subjects as produced through, and as a result of, the complex play of forces. This does not deny that objects, bodies and subjects exist prior to the art event, but that further potential can be activated through the event relational engagement. Here relationality immerses entities in a field that might be quite distinct from the back and forth conversational model of the interactive paradigm (Manning 2013a, 130). The processual is crucial in this expansion of interactivity, in that it opens the forming relations and the entities they initiate to a multiplicity of becoming that necessarily outstrips any unity of subjectivity.53 It brings into play the ongoing, overlapping individuations – states of constant generation rather than progression to one particular endpoint. These processes of individuation are, as Massumi says, forward looking and rich with potential (cited in Manning 2013a, xi). Manning poses a question about life in general that applies here to participatory art: 'what if, instead of placing self–self interaction at the centre of development, we were to posit relation as the key to experience?' (2013a, 2). As in life in general, the artwork here is the encounter: art as an event of relations. This notion of relationality, as Massumi says, addresses objects and bodies from the point of view of their ability to change and respond – 'a coming together in a fusional event…a telescoping into a potential becoming' (in Lozano-Hemmer 2005, 4). The relational is an immediate 'emergent process', where something new occurs out of the relations (Manning and Massumi 2011, 8; Couze 2010, 139). Thus when Lozano-Hemmer insists that his work is not interactive but 'relational' he means that the focus is not on the fixed or mechanical elements of interaction, but on the potential for establishing relations that always have an immanent, virtual quality to them (Gorschluter 2009, 103).54 This approach allows him 'to think of the computer and technology as potential language with which you can make relationships emerge, as opposed to preconceiving the outcome' (Lozano-Hemmer, Boucher and Harrop 2012, 152). A number of artists have attempted to move beyond the potentially limiting paradigm of interactivity by adopting a relational approach. As noted in the introduction, there is a 'prehistory' to the discussion of a relational model, notably in philosophical writings and texts produced by artists Roy Ascott and Lygia Clark, and scientist-artist Gordon Pask that emerged in the earliest days of discussion on 'interactivity'.55 As Ascott states: 'now that we see that the world is all about process, constant change, we are less surprised to discover that our art is all about process too' (2003, 157). His concept of 'telematic art' (Ascott 2003, 231) proposes a move away from the object to the examination of process – an art that explores an 'interconnectedness' of interweaving fields and forces, able to evolve in an unpredicted, heterogeneous manner – an art that is a state of 'perpetual play' (158–9, 11). Gordon Pask's work, according to Usman Haque, shows an interest in unspecified goals that moves it beyond the realm of much contemporary interactive art (2007, 58)56. Haque argues that Pask's artworks and his elaborate 'conversation model' of interaction demonstrate an interest in an active, shared field and 'mutually constructive' relationships (2007, 55).57 In his own dense and complex writing Pask emphasises the importance of creating work where both technical systems and human participants might cooperatively adjust their relational capacities in an emergent ecology (1961b, 230–4; 1961a, 102).58 Clark's writing provides a more lucid understanding of the scope of a process-based view of the world, and the relational potential of an art practice. Clark writes of her work as non-object based – 'an experience that does not leave a trace' but is an act that 'contains…its own becoming' (cited in Suchan 2008, 6).59 She writes of dissolution of the space between subject and object, a 'vibrating body' affected by worldly forces (cited in Martin, Ruiz and Rolnik 2000, 73, 104), and 'relational objects' designed to instigate affectual connections that might 'launch the spectator into unforeseeable becomings' (Clark cited in Suchan 2008, 12, 10). Here Clark calls for art to evolve beyond 'the simple manipulation and participation of the spectator' and for it to engage in 'the process of bringing the participant's freedom of action to light' (Clark cited in Suchan 2008, 12, 10). A relational approach is explicitly adopted, at least theoretically,60 by a number of more contemporary artists. In the field of architecture Greg Lynn could be cited, particularly his calls for a practice based on theories of complexity that engage with multiplicities to escape both identity and contradiction (1998, 161). One might also cite the more far-reaching explorations of emergent body-space by Arakawa and Gins discussed later in this book,61 and Penny writes as a new media artist about the shift towards an enactive, performative approach to participation (2011, 83). This 'performative ontology' Penny says, expands interactivity towards that of 'machine ecology' (2011, 94–5, 100). Similarly, Nathaniel Stern describes the body and world as 'implicit in one another', a 'per-formed' rather than 'preformed' relationship – a body which is emergent 'through its active relations to other matter-and-matters in progress' (2011, 233; 2012, 34). As such, for Stern the creation of relation is 'continuous; it is embodiment's…always ongoing formation', and he compares this to the 'more finite' possibilities of interactivity that are responsive but restrictive (2012, 8). Likewise, Rokeby argues for a complex interactivity that resonates between participant and artwork (cited in Penny 2011, 84). Participation, Manning states in summing up 'relational' art, differs from the programmatically interactive in its tending towards the virtual and gathering of forces from the field. Manning writes that it is not about 'the plan of the movement or the partitioning of the individual bodies in space. It is the relational force that persists from the collective movement's incipient cueings and alignings, the incipient priming gathered as a force field not of the bodies per se, but of the active intervals their relational movement creates, intervals that in turn propose multiplicities in the moving' (2013b 342). While it is not the purpose of this discussion to provide such a critique, these concepts of the relational in art should be noted as easily distinguishable from the 'relational' as conceived in 'relational aesthetics', which, as Stern remarks, considers and limits itself only to relations between already constituted subjects (2012, 48). The embodied relational approach referred to here, while it still considers the social as a force contributing to the individuations of the body and subject, must also consider a much broader spectrum of relational possibilities. Similarly, an embodied model of relation is in marked contrast to artists such as Stelarc, for instance, who invests in the transcendence of the body through the bio-technological melding. Stelarc, Dery argues, upholds a Cartesian distinction between body and mind, reducing bodies to the position of machinic commodity and making them the ideal subject for power (1996, 232, 164, 154–235).62 #### A relational model(ing) The operational politics of the relational are, on the other hand, improvisational, fluid and emergent, as Manning states (2009, 41),63 a 'becoming' connectivity that moves with and is attentive to the force of the field with which one co-emerges (2013a, 212– 3). The event of the connections and their co-emergence with bodies is co-causal.64 This describes the way relations develop between the body and the work as a 'mutual incipiency' that is a process of change and response (Massumi in Lozano-Hemmer 2005, 201). This may be considered as self-evident information, for if, as process philosophy proposes, all things are events of relation, are not all artworks thus composed, regardless of the artist's intentions? The way many interactive works operate, however, is to attempt to stabilise such unfoldings, erase the connections to the virtual – the future potential for 'immergence'65 – and establish enduring actualised connections and representations of connections. The shift in emphasis to the relational concerns affording an emergent or potential event that may occur or is occurring. A work might still be thought of as existing beforehand, as an object or proposition for an event, but it exists as an event only in a temporal relationship – or rather as a nexus of relationships – with the viewer, enfolded and unfolded through interaction, and each nexus of relations creates a singular event. As a 'proposition' the potential event of art-objects/spaces and bodies can move beyond obstacles that 'delimit the event according to pre-constituted interiorities' to act instead as 'propositions for an ecology of participation' (Manning 2013a, 114, 185). Embodied enaction of an event is always directed towards the 'next' – further potential differentiations – the continuing evolution of the event (Varela, Thompson and Rosch 1992, 205), and therefore open always to the pull of the virtual. Such events create body-artwork assemblages – contingent networks of interconnections with multiple, unplanned, potentially contradictory variables of relation.66 The participant's concentration shifts to the buildup of energy and rhythm between and within body and work; how the event moves beyond a mapping of simple cause and effect and into something that enables its own generative tendencies (Massumi 2008, 13). Such complex multiple actions and potential relations might catalyse a singular experience, moving beyond what can be articulated. Thus what is felt or perceived here in the moment might be intensities of pure sensation, a building of energies expressed through ever reconfiguring combinations of movement, sound, image, posture, and so on – while also including potentially contradictory affectual relations that push and pull at the body. A relational art event might begin to concentrate on enabling the conditions for new connections to arise, a richer palette that might include slippery, hard to define, conjunctive and disjunctive forces: affects, inarticulate sensations, microperceptions, and emotional tonalities. Such fuzzy and inarticulate forces, which can never be fully compressed into productive perception, might move the work further away from any prescribed outcomes, outstripping functionality as an inarticulate remainder affecting the body beyond cognition. This philosophical stance of relationality, O'Sullivan states, points (perhaps optimistically) away from 'consumption' and towards an 'art practice as a process…always producing' (2006, 24). #### Bridge: Into the midst: immersion immersive While in theory it is easy to agree on a general shift to relational modelling, it remains problematic for the practising artist engaged with an interactive or 'relational' art to structure fluidity and maximise open-ended potentiality in more practical terms. This is particularly true when working with interactive technologies designed with other outcomes in mind. An example of some of the practical issues involved in attempting this shift to the relational can be seen in Into the Midst, a five-day, collaborative research-creation workshop and public presentation in the SATosphere – the Society for Art and Technology's interactive and immersive projection dome in Montreal, Canada.67 The project sought to explore alternative potentials of a space constructed with seemingly rigid divisions between the artists' technical and spatial control of events and the viewers' lack of control of the space.68 Key to the usual operation of the dome was that the scale of the space and the configuration of the seating constrained its use to an undoubtedly spectacular, but somewhat passive, viewing space. The design encouraged all viewers to recline while focusing their attention on relating to the surround sound and giant images that wrapped around and cocooned them. The Into the Midst artists hoped to activate more varied experiences within the space, with general tactics including encouraging attention to the edges of the space, the projection of images and sounds that disrupted the smooth illusion of immersion, and creating opportunities for participants to directly relate to one another beyond simply sharing the viewing of the projections.69 Thus the series of interventions that were employed within the space were designed to disrupt the habitual configurations of relation between audience members, artists and audience, and the audience and the spatial dynamics of the dome. In these aims the artists in the project sought not to Figure 1.1 Senselab collaborative project, Into the Midst: Immersion Immersive (performance documentation), Society for Art and Technology, Montreal, 2012. Photo: Hannah Buck simply ignore or diffuse the various technical mechanisms built into the space to provide spectacle, but to reuse them in a more speculative and unconventional manner (see Figure 1.1). However despite the concerted efforts to extend the potential of the dome's mechanisms in its public presentation, the normative paradigm of the dome as a space for relatively passive consumption of immersive imagery continued to overwhelm the efforts of the artists. The event too easily became an extension to, rather than an interruption of, the 'entertainment' space and habits that such places tend to encourage. The lure of the projected imagery continued to centralise the viewers' focus. The design of the space seemed to suggest that it primarily concerned itself with a relationship between a relatively passive subject and events predicated on 'out of body' experiences (such as spectacles of virtual travel reminiscent of nineteenth-century panoramas), rather than with any embodied potential that might be exploited within such a large area. The (deliberately) ephemeral interventions failed to sufficiently disrupt these dynamics to allow new configurations to arise and disturb the stratification. The lack of differentiation in the layout and clear divide between projection space and viewing space were all elements that contributed towards this rigid structuring.70 My own participation in this project crystallised some of the key issues around the difficulties in moving the interactive experience beyond habitual divisions of artwork and subject, and in enabling relations to operate outside the (again habitual) paradigm of the passive consummation of the demonstration of the spectacular. It proved extremely difficult to utilise the technologies built into the space without creating a work that ended up principally demonstrating the undoubtedly impressive capacities of the technology. Potentially disruptive transversal relations that might have interrupted the centralised focus were too easily overwhelmed by the force of attraction of the overhead light show and the 36-speaker surround-sound system, and those viewers who attended the public showing found themselves, for the most part, adopting this passive position within the space, despite the various activities designed to disrupt this action. Even the artists involved found it difficult to not succumb to the lure of the projected spectacle above, despite our shared interest in moving beyond this experience. Technologies of interaction demonstrated in this project that they have the potential to control and limit relation when not carefully constructed to operate otherwise, and that habits of operating within a known paradigm can be hard to shift, even for those with such intent. Here it became evident that the construction of relation in and of itself can still easily conform to dominant and perhaps constrictive paradigms, and that any ethical platform of emergent relation must find new ways to interrupt the habitual means of engagement. Participants' bodies similarly needed to be addressed in individual ways, and encouraged to engage on multiple levels, rather than as a generalised ideal. The kind of dominant relation between fullyformed subject and work that the SAT's dome space assumes as primary also needs to be put into question by relations that allow movement of differing kinds and scales of connection and disconnection to emerge. For me, this project highlighted that, as Penny notes, there is, at times for all of us, a considerable gap between the theory and practice and between broad intention and outcomes (2011, 72).71 While the relational model previously outlined is the one pursued within this research, much of this theory on broader philosophical level only begins, at best, to address the more practical concerns of how to enact such systems within a participatory framework. How to structure a work to allow for multiple, surprising outcomes, and how to create organic movement – the complex flow of prehension, synthesis and perishing, pursued endlessly by further such creation – remains a question. These issues are at the heart of this research, and the next chapter begins to address these more forward-looking and practical concerns in detail: considering the question of 'how to' think beyond interactivity and constructing some of the potential tools that might be required to realise such an aim. Here chaos in itself does not seem to be an answer, and nor is mimicry of the everyday. Rather, it is that particular 'thickness of experience' – the surprise of unusual connection and revelation that the art event can offer – which needs to be retained without losing the kind of underlying complexity and entanglement gained from everyday experiential involvement in the environment. 2 ### Thinking action and event #### Introduction The creation of a chaosmos is what interactive art and art with new technologies should head towards, as only then can outcomes be protected from chaos without turning interaction into a choice of alternative stratified options. Andrew Murphie A reimagining of interactivity along relational lines introduces the possibility of a 'minor' interactivity. This involves a continued activation or problematisation of the major form, in order to avoid a return to any oppressive stasis. Here the concept of the assemblage and the notion of art as an event and as a machine are introduced to enable a closer investigation of something at the heart of this research: the creative power of noise or interference in relation and its role in increasing the selforganising capacities of the interactive event. This rethinking must also involve more practical tools that allow an interrogation of singular instances of relation. It must be remembered that 'relation' in itself is not an answer, since, as I have argued, much interactive work is relationally oppressive in working to fix and contain relational difference and generation along programmatic lines. As Claire Bishop points out in her critique of current trends in socially relational art, relational works are quite capable of enforcing the status quo through blind promotion of social inclusiveness in the works while 'the structural inequalities of society remain uninterrogated' (2009, 241). It is important therefore to think of relational propositions that might allow a certain freedom to reinvent or mobilise existing relation – to produce potential movement. #### Minor interactions As Simon O'Sullivan argues, 'minor' and 'major' are not polar opposites. Rather, the minor can be thought of as a reactivation of the components of a system from within (2006, 71), allowing the system to become something other than its major or established form. The 'becoming-minor', for Gilles Deleuze and Félix Guattari, is therefore a tactic with which to pervert or trouble the structure of an oppressive system in order to explore ways to allow the oppressed qualities of the major to oppose its oppressive qualities (1986, 10)72. The minor, as O'Sullivan says, breaks with the habitual formations, and challenges dominant regimes of the form to allow further movement or open change in the system (2006, 69). In this sense, 'becoming' is always minoritorian, as Erin Manning states (2015, 3), in that it is about the activation, movement or further individuation beyond a stable form. Using the concept of the minor suggests a thinking of the relational potential of interactivity that, rather than being oppositional or reactive to the critiqued dominant paradigm, seeks to explore the further potential of the components of the systems, utilizing the same elements but with a different structural logic. That is, if the major or normative form of interactive artworks tends towards control and signification of subjects and objects, subordinating the wider relational potential, then the becoming-minor of interactivity might be a turn towards the relational that encourages these controlled forces or qualities to flourish. Here the expressive, expansive pull of relations might be utilised to problematise the major structure.73 This might re-energise interactivity's potential, giving rise to an uncertainty within what was fixed in order (Murphie 1997, 68), and allowing new productive capacities to be explored. It is not about the production of a new stabilised 'form' of interactivity but the production of the conditions that enable continued agitation of the elements (an 'expressive machine') (Deleuze and Guattari 1986, 28). Thus the minor here does not designate specific productive outcomes, but rather the 'revolutionary conditions' in which continued exploration might be produced. It is, as Massumi states, directional in that it moves away from stasis, but not 'directed-to' any particular endpoint (1992, 103, 18). Potentially, this disturbs any stabilisation and instead emphasises the productive nature of disorganisation itself. It allows for consideration of the particulars of an event, and the relations and entities co-composed with it, rather than following any established path (Murphie 1997, 72–3). In this, it has specific disruptive implications for fixed or linear interactivity. The move to the relational here is a tactic with which to reactivate and charge (interactive) structures with new potential.74 #### Molecularisation and the assemblage The concept of molecularisation is closely linked to the minor, as an opening up of stratified relation. The 'becoming-molecular' of a system is the decentering of a formally stabilised whole into parts. This both decentres the system and allows new communications or exchange between components (Deleuze and Guattari 1986, 50, 41): a hyper-differentiation that encourages new potentials, intensities and complexities to arise.75 In a 'molar' configuration, as Brian Massumi says, a set of entities are molded to a prescribed set of connections, becoming a 'disciplined' or 'dominated' group of individuals that have a fixed identity imposed upon them. As Massumi notes, the molecular still exists within this molar regime, but it is controlled and free relational movement is contained (1992, 55). A molecular configuration of the same entities allows local activations: transient and improvised connections to take place (and perish).76 Thus, becoming-minor is also always becoming-molecular (Deleuze and Guattari 1987, 272), an increase in movement or intensity within a stratified system. Within the paradigm of interactive art, the 'molar' perspective might be seen, firstly, as the discrete body of the viewer taken as a whole, and the artwork similarly viewed as one idea or fixed assemblage of components. Secondly, it might be the fixed relations between work and viewer that prescribe the types of relations and outcomes possible between them. Thirdly, the molar position might also prescribe the event overall according to a preconceived notion of interactivity. A molecular approach to the same art event would open up the potential of new ways of relating inside these 'wholes', filling the systems with fluctuations, uncertainties and tentativeness that are its opening up to new singular expressions (Guattari and Rolnik 2005, 162).77 Here the site(s) of interaction might become mobile and multiple, delimiting the resultant events of interaction. Pragmatically, any such artwork will be composed of both molar and molecular components or tendencies, and the aim might be to encourage an increase in potential for internal movement and change.78 In this sense, as Deleuze and Guattari state, molecularisation tends towards the creation of 'machinic' assemblages (1986, 37) – collections of entities functioning immanently and pragmatically, rather than being 'subordinate to the laws of resemblance' (Massumi 1992, 192). Assemblages do not create fixed bonds between components; rather the entities are linked through shared collective potentials (Guattari 1995a, 35). An assemblage is 'ad-hoc' in that it is composed of available material, and it is dynamic – as all its relations remain active – a 'volatile mix' of forces, part and materials (Bennett 2010, 24–5). Assemblages maintain the individual qualities of components and the differences between them – rather than repressing these for the sake of the whole – while at the same time collectively and potentially producing or becoming something else. The assemblage is an organisation of relations, though not reducible to this, and is also multiplicitous: it has an internal dynamism that always keeps its relational fields open to potential recombination (Buchanan 120, 129). In this one might say, as Bennett does, that the individual components and the assemblage together exhibit 'agency' (2005, 31–2), and components are 'molecularised' in an assemblage in that they are able to individually modulate their relations while maintaining collective coherence. Importantly for this argument, assemblages are able to operate without resolving or erasing internal tensions. In fact, such internal differences might be seen to drive both creative organisation and production of the assemblage. These tensions saturate the assemblage with intensive potential for derivation from any realised or emergent form, as they relate 'difference to difference' and maintain an adaptive potential: 'a capacity to further differentiate differences' (DeLanda 2005, 23–4). Here relation can be considered to exist not only between stable objects and subjects, but also within and across such idealised forms, initiating and potentialising them. Now the room for continued movement within the seemingly continuous whole begins to become apparent – the infinite gaps and discontinuities that can be activated to drive change within the event. Within an art-event-as-assemblage, such internal modulation provides an open-endedness that enriches, rather than destroys, the now mobile whole. What also becomes apparent is that the privileging of viewer-work relations is no longer necessary. Instead, any discussion of relation can – indeed must – consider all relations as being equally open to change. This includes relations between various body organs, between these organs and technical entities, and between and within technical entities themselves. This decentering of the human in favour of a wider approach to relation is essential in order to more fully consider the forming of the larger ecology of the art event. It acknowledges the dynamic role that all the elements bring to bear on the playing-out of relational forces across the various scales and assemblages in which the interactive event is activated. #### Differential machines Guattari's concept of the machine provides a useful way of conceiving of an artwork or event as a productive assemblage. From this basis the mechanics of self-organisation might be examined. Machines, Guattari tells us, are any system that produces an effect.79 There are, for example, social, logical, biological and linguistic machines, and machines that are combinations of these systems, such as cities (Guattari, 1995b, 9).80 There are also machines that are conglomerates of technical objects and, as Murphie describes, machines that are assemblages of technical objects-plus-bodies such as the 'cardriver' machine that produces travel (1996, 89). The 'machinic' is therefore not the mechanical (a fixed technical system), nor is it specifically linked to the technical (non-organic), but is a productive assemblage, another configuration of the nonunified subject (Braidotti 2002, 254). Its cohesion (such as it is) is achieved through a shared potential (Maturana and Varela 1980, 77). Like assemblages, machines can be broken down into smaller machines, or sets of components held together through some kind of productive relation (Murphie 1997, 265).81 Machines act molecularly in resisting the collapse back into any irreducible whole, or series of wholes, through their continued potential activation of relation. A machinic connection or relation might therefore be one that is pragmatic, flexible and local, always with further potential iteration or expression available to it. This thinking gives us three very useful ideas that help to expand any technologically based concept of the machine in a decidedly non-humanist direction. Firstly, the need to understand the role that the wider ecology in which technical objects are embedded in (or unfold from) has in determining what potential is actualised. Technology, as Andrew Murphie explains, is always only one aspect of a larger notion of the machinic, requiring a larger physical/social field within which to operate (1997, 80).82 Secondly, as Guattari describes, technical machines inherently contain potential beyond their immediate actualization – 'ontogenetic elements' (1995b, 8). Thus they are held together not so much by any physical bond, but by a shared potential, an 'assemblage of possible fields' (1993, 35)83 that develops through the process of 'concretisation' or interdependence. Thirdly, that we must consider machines not through utility or representation, but in terms of their productive capabilities. Guattari's conception of the machinic shifts the discussion of the assemblage from: 'what is it composed from/what is it an aggregate of?' to 'what does it produce?'.84 That is, machines are performative, concerned with 'matters of practices, doings and actions' (Barad 2007, 135). Within a machinic assemblage, Manuel DeLanda explains, components explore their capacities to connect with other component, their abilities to affect and be affected, which is separate (if related) to their 'intrinsic properties'.85 Such machines are necessarily multiplicities, with 'no need whatsoever of unity in order to form a system' (Deleuze 1994, 182), preserving internal differences between components. Their potential lies in a productive 'opening out to heterogeneity and alterity' (Murphie 1996, 92). An interactive art assemblage might be usefully viewed as machinic. This places the focus on how the work as a machinic whole is composed of various smaller machine components – bodies, technical entities and combinations of parts of these entities – that interrupt, modulate or transduce forces they come into contact with or are subjected to (Deleuze 2004, 219). The larger art assemblage or machine is then brought into existence and organised through these productive and provisional relationships between these smaller parts and by their shared modulation of a particular force.86 Each component within an assemblage productively affects and is affected differently by any force, increasing internal difference or molecularity.87 Thus interaction with and transduction of forces is here the process by which such 'an activity sets itself in motion', and at the same time generates 'processes of modification'. These transductions instigate further individuation of the machine while at the same time potentially reconfigure its internal relations (Simondon 1992, 313). In the work A Chorus of Idle Feet, analogue sensors were set up in a public walkway that were capable of transducing the movement of bodies through the space to produce variations in the syncopation of sounds.88 Here, various components might be thought of as forming assemblages, expressing a capacity to connect and produce modulations in forces, and then combining to produce more such machines built on intensive differentiation. Body, movement and light together expressed the capacity to produce shadows in the space – becoming a shadow-machine modulating light – while light sensors modulated the flow of electrons in a light-light sensor-electron machinic assemblage.89 While these were capacities of the two machines, when combined they began to make a machine that transduced the force of movement to the flow of electrical current, as shadows produced changes in electrical resistance in the sensors. This machine, in turn, combined with other components to form another machine that expressed its capacities to connect movement into changes in sound pitch, rhythm, tempo or tone. For example, this machinic assemblage combined with an assemblage that converts electrical resistance to computer code such as MIDI signals that control sounds on a computer (an electrical flow-MIDI code-vibration machine). These machines were productively transducing movement into modulation of light waves, light waves into modulations of electrical current, and flow of electrons into modulated flows of sound waves. All these component machines then nested within a larger assemblage that collectively transduced the force of movement into these sound waves. In the same work, other sensors (such as proximity and movement detection sensors focused on particular areas of the walkway, detecting changes in the position or number of bodies present), linked with the capacities of the movement to produce variations in the spatial distribution of bodies, which linked into larger productive relation with software triggering more sound pulses. This again nested within a larger machine, producing modulations in syncopation of the sounds as they combined. Here all the components provisionally came together as an expressive machine, producing an emergent quality of rhythmic syncopation. This was a collective expression formed through interaction of all parts to create an event that retained the dynamic qualities of modulation of the machinic assemblage. As such it was concerned with the 'viscosities' of the transduction of various forces through the system: the styles and speeds of affectation of components by forces and visa versa. The work operated through an ongoing production of both internal connections and differences in the flow of forces. It was a 'fuzzy aggregate' composed of counterpoints, inequalities and tensions in the processing of forces between the parts (Deleuze and Guattari 1987, 328–9). Here the larger machinic assemblage obtained a level of consistency in production (it continued to express relations between movement and sound Figure 2.1 Andrew Goodman, A Chorus of Idle Feet, 2010. Digital video still. Allans Walk ARI, Bendigo. rhythms) not through the submission of internal difference and organisation, as in a molar system, but precisely because it was internally flexible enough to accommodate intensive modulations. The initial force of movement driving the event was also molecularised, being transduced by various component machines into multiple new and potentially competing forces (see Figure 2.1). What such a machine begins to produce is an event that is an exploration of its collective expressive capacities through the transduction of forces. At the same time, these explorations produce the machine itself. Thus, the two are, to some extent, co-produced, becoming implicit in each other's actualisation and potential: a 'concretisation' or structural unity and interdependence of components of the assemblage (Simondon 1980, 21). Such a shift in an interactive art-machine begins to move it away from the limited capacities of individual components, and from prescribed notions of either outcomes or of particular, pre-thought or fixed relations. This manner of thinking about machinic expressions performs a molecularisation onto the interactive event described. It splits the larger art machine into a series of smaller nested machines. Each has their own internal logic of working and are co-causally relational to other entities that their workings affect and are affected by. This understanding opens up a potential for thinking through both an increased movement of relations within the machine, and movement or transduction of the forces it modulates. Overall this remains a fairly simple example. When considered on their own, most components of this interactive event – such as each individual light sensor – remained relatively predictable in their transduction of forces. However as forces were immanently transduced through multiple nested components over time, thus developing more and more relational entanglement, the assemblage evolved complexity at a higher level and began to generate potential beyond the capacities of these smaller components. My point here is that novelty might be achieved not through designing more technologically complex components, but through 'self-conditioning emergence' (Massumi et al 2009, 40). This requires a rethinking of the philosophical basis of design strategies so that relatively simple components might interact with one another to increase internal differences in the assemblage. ### Structuring action and flow: drift, autopoiesis and concretisation These concepts of the minor, molecularisation, assemblage and machine form something of a basis from which to explore self-organisation in the participatory artwork, in essence being propositional to an event of the production of relation. From this point, in this chapter I address the questions of how an art event generates its own 'satisfaction'90 through consideration of the concepts of drift and concretisation. This is extended in the next two chapters through the question of how the drive towards novelty might be maximised in the event through the key concepts for this book: a more detailed examination Whitehead's concept of 'feeling' as a productive differentiation from what has been, and the noise or parasite within relation. #### Propositional invitations To think of a relational art event in an open-ended fashion, we might think of the practicality of building it out of propositions. These propositions might be multiple, possibly contradictory. If sound 'A' can happen, or sound 'B' can occur, but not both sounds together, the sound that is not actualised still has, as Whitehead says, a creative role to play – both as a 'giveness' that shapes paths of potentiality, and as a continuing link to the virtual. The negated proposition remains a link, both to what might have happened or might in the future happen, and to the unrealised potential of an entity that 'vibrate(s) against the conformal' (1978, 188). An entity, Whitehead states, 'feels as it does feel in order to be the actual entity it is' (1978, 222). The propositions composed within the art event are launching points, 'lures towards feelings' (259). These feelings are the prehensions (220) in which the drive toward 'satisfaction' is the realisation of some potentiality for the entity.91 A feeling here is the potential for affectual connection, that is, an entity's potential capacity to be affected by, and affect other forces, entities or events. Thus, an inanimate entity might be seen as capable of a feeling (affecting and being affected by forces), and of driving towards its own satisfaction, as a sentient being. A sensor, for example, might have the proposition of a tendency to notice movement. This movement may not happen. It is a potentiality, constrained by the given: its position, the mechanics of its construction, and so on. It has 'sensitivity' towards searching for this movement, a potential capacity to form a machinic connection with this force, the incoming sense data that drive its completion. It reaches a point of satisfaction of an occasion when it expresses this capacity for connection, whether it senses movement or not. These are exclusive potentials – and in any occasion, only one potential can be actualised while the other remains virtual. Even simple and linear propositions are, in themselves, never fully conclusive. Any actualisation is only a singular iteration of that proposition's potential, and does not preclude further iterations arising. In this sense, although the outcome is conclusive for the particular event that actualises, the conclusion to a proposition is only approached, never realised. Thus while the individual event of the movement being sensed reaches satisfaction or an end-point, the proposition retains potential for further actualised iterations (DeLanda 2005, 75). In a system with multiple exclusive and inclusive propositions, the outcomes become decidedly more non-linear and the virtual more evident as a factor within the system. The 'other alternatives are there all the time, coexisting with the one that happens to be actualized' (DeLanda 2005, 75, emphasis in the original) and creating a tension or problematisation that pulls the event towards further 'incompossible' actualisations. This increase in intensity is the line of flight from the prescribed event, in that it is a qualitative increase in relational potential within the system. Propositions guide the dynamics of an event, though not in a prescriptive manner, creating tendencies (Bennett 2010, 103) and providing ongoing invitations or lures toward the potentialities of the event they condition. They instigate a 'second phase' of the virtual: that of a 'real' rather than the 'general' potentiality (Whitehead 1978, 65), conditioning the potential by inclusion of the circumstances of the emergent event.92 This is a gathering of, and complex negotiation between, the various individuated propositional potentials of all the machinic components, and it creates a collective propositional potential. Thus while we might think of the artwork as a single entity or event, it is perhaps better viewed as a 'society'93 of entities, divisible into multiple, overlapping and simultaneous events or entities, each seeking and competing for its own satisfaction. During actualisation, the event is always at a point of unfolding, facing multiple potential paths towards various satisfactions. These multiple and fluid assemblages – eyes/brain/image, ears/ noise/speakers/current, software/sensor/movement data and so forth – are each divisible again, each seeking resolution of their feelings. This philosophical stance emphasises that art events are composed from the ground up. It provides an understanding that the concrescence of forces builds towards an endpoint of an actual event, discovered and motivated within the occasion itself by complexities of virtual and actual forces. Seeing art as a propositional event begins to deflect the emphasis away from any final representational form, and to instead emphasise the ongoing role of the internal tension of the negated propositions and emergent differences in enriching the virtual of the event. Within interactive art, this suggests an experience focused on emergent qualities of relations in and of themselves. Here interactivity might begin to distinguish itself from goal-orientated 'gaming' directed towards solving a puzzle, moving through levels or controlling a space, and also from 'didactic' works directed towards a learning outcome, whether based on perception or content. Instead it might move into riskier areas, concerning itself with the setting of conditions that allow events to begin, and accepting the inherent danger that some desired and interesting outcomes and directions may not always eventuate.94 #### Self-organisation How does the art event 'choose' which prehensions it follows through to satisfaction, and which entities will actualise? How can we think of this without falling back into prescriptive models? Having set itself into motion through its propositional structuring, and gained through feeling its own collective movement, the event is no longer beholden to any external intentions or drive – it must sort itself out internally. But it does not strive to be the best event it can – the most efficient, original or surprising. That would again imply some kind of transcendent motivation, a 'neo-Darwinist' thinking that assumes that entities or events are invested in, and capable of, striving for some preconceived ideal form or an outcome of maximum efficiency (Bogue 2003, 69–73).95 Rather, we could say, it 'drifts'. This implies a system, as Francesco Varela says, that 'makes do', seeking the 'viable' rather than the 'optimal' (1992, 205).96 Such a system is 'pragmatic': its motivation is to find a satisfaction, not the satisfaction.97 That is, it makes do with what it has, and cobbles together a solution. As Ronald Bogue states, systems self-organising through drift emphasise change or creativity over 'fitness' (2003, 74–5)98. They experiment with 'assemblage[s] of heterogeneous forms for no other reason than that they are possible' (75). Processes of drift enable systems to be truly interactive, as they are composed through that activity, rather than being representative of determined function or outcome (Varela 207, 209). Here drift is a molecular modelling of an event gathering and accentuating relational intensity within the emergent system, rather than containing such relational play in order to serve a central or molar design aim. In drifting, a system demonstrates an agency that is clearly not attributable to any one (or indeed all) of its component parts that might then direct the unfolding of events. Rather any agency – if agency is viewed simply as the modulating and distributing of forces and relations – can be seen to be a collective expression of the event itself. Julian Yates terms this 'agentive drift', an agency that is a 'dispersed or distributed process in which we participate rather than a property which we are said to own' (2002, 48). Here drift is the dynamics of relations as they gather, a collective individuation with its own emergent and global virtual and actual organization (Bak 1997, 121; Varela, Thompson and Rosch 1992, 65). Drift does not imply that such systems operate through random or chaotic connections, but that they create systems of intensive and local connection. For Murphie, this is a chaosmic interactivity that sits between chaos and stratification (2005b, 42). This might replace a system organised through a single dominant relational pull towards a future 'useful' and externally projected outcome – as much interactive art is designed – where differences becoming suppressed or flattened to serve a larger or dominant purpose. Systems in drift may lack or mitigate external motivation, but they gain a set of competing heterogeneous and intensive motivations. This encourages an immanent expressive exploration of the multiple potentials of relation within the assemblage through the play of subtle and complex dynamic modulation of internal forces. In A Chorus of Idle Feet, changes to a small assemblage within this interactive system could be seen to affect the productive workings of many component assemblages, and the event as a whole. A change in light, for example, would affect the way electrons passed through the assemblage of a particular sensor, while also affecting other assemblages linking the sensor to sound vibrations emitted through speakers.99 These vibrations potentially affected the larger assemblage of the art event by combining and diffracting with other sound waves being emitted, producing local shifts in the expressions of the speaker systems.100 These might then affect both the rhythmic pulls of combinations of sounds, and the affective tonalities of the event.101 Thus the productive expressions of the other component sensor-machines – those that were not directly affected by the changes in light – were potentially still altered through a series of complex implications that were relational, but not entirely predictable. In such a system, localised agitations or changes to flows affected surrounding assemblages and had a run-on effect, potentially spreading through and shifting much of the system's workings. Each component remained primarily responsive to its local connections, with no prescribed aim or outcome dictated by the original movement. A larger movement or circulation of forces in the system was created through emergent difference – contagious and rhizomic – instigating and gathering new combinations and potential combinations of co-dependent relations that the systems needed to negotiate.102 As a system operating through drift this was an open or dissipative system, 'in which momentary deployments of forces produce[d] systemic orderings, local eddies or drifts' (Yates 2002, 50). The system here sacrificed self-preservation as it drove towards creativity through the continued recombining of forces (Whitehead 1978, 103–5).103 Such changes did not necessarily force a collapse in the system,104 as there was a degree of consistency or dynamic equilibrium within the assemblage.105 That is, it was a 'dynamic whole' with an ability to accommodate intensive changes, without necessarily causing destruction to the ability of the machines to communicate productively, even as it caused variations to the productive outcomes of the event.106 Drift has lured into being a system that is productive in a machinic sense, but not at all about a directed, idealised or maximised productivity. As each component assemblage responded to changes in its local systems of forces, there was a flow-on of repercussions that was not always entirely linear or predictable – an excess and freedom of relation that may, as Massumi and Manning state, reorientate exchange. Such systems are therefore principally about self-production, the experience of the components gathering together, an 'emergence of [a] field of relation' (Manning and Massumi 2014, 128). It is also always a 'minor' act that is a reorganization of available entities into new relationships, and more than the inclusion of new factors. A system in drift is involved in a process of increased 'concretisation'. As Simondon thinks the concept, concretisation is a process exclusive to technical (as opposed to 'natural') entities. However here I would argue that it is possible to see it more generally as a process by which a set of entities are brought into increased co-causal relationship with each other. For Simondon concretisation involves a system in which each component 'is part of a system in which a multitude of forces are exercised and in which effects are produced that are independent of the design plan' (1980, 31). In this, concretisation relates directly to a process of drift in what Simondon terms a 'natural object'. That is, both set up circular, coherent systems of distributed agency expressing potentials rather than being driven by external factors (Simondon 1980, 40–1). Such systems attain some level of structural unity, Simondon states, with each element co-determining, becoming implicit in what other elements become. It requires that the component parts develop a 'plurality of function' and negotiate their operations, rather than fulfilling a predesigned or 'ideal' function (Simondon 1980, 20–1).107 It is precisely because of the presence of potential indeterminacy – a flexibility of future relations, rather than a fixed and linear set of actualised relations – that machines are able to develop such self-organizing capacities (Simondon 1980, 13–14).108 While the components in a machine retain their individual potentials, it is the shared potentials that they develop through machinic operations – their shared 'associated milieu' – that forms a base for their collective individuations through drift.109 This is the drawing of elements from a field as a 'system of virtualities, of potentials, of moving forces' (Simondon 1980, 51) into a field of relation. These processes of drift do not just happen within established concrete assemblages. Rather, the drift itself can be seen to draw disparate components into productive relation. Creating dynamic systems of drift must strive to be not simply making connections between component parts through actualised systems of feedback and (flexible) causal chains, but also need to enable conditions for the continued disruption of relations.110 In the example above, the light sensor-machine began to exhibit an ongoing potential to form a relationship with, for example, the sound waves produced by the pressure sensorelectron flow-computer-speaker assemblage that moderated both expressions of vibration. It was not limited in the ways or number of actualisations of the expression of this relationship; nor was it limited to this particular multiplicitous set of light sensor-machine to pressure-sensor machine relations. Entities gathered from a field of potential relation, into an actualised relation with each other, retaining potential for different future individuations.111 It is at this level of potentiality that such a system continues to exhibit its molecular or minor nature. Such a gathered, collective, virtual milieu it is always sensitively balanced on the point of reorganisation – that is, a deterritorialisation and a reterritorialization. Relational art events capable of drift might take many forms, creating many differing events. For interactivity, this does not mean that drift drives towards making events necessarily different. Such systems are indifferent to the quality or quantity of difference they generate. Importantly, they are indifferent to the demonstration of change and relation that haunts so many interactive works – the problematic focus on representation over open exploration. Systems in drift settle where they settle. On some days, the events generated in a work may be markedly variable, on others the work might seem to settle around the same outcomes. The artist must relinquish some control over this, leaving or encouraging it to work itself out: it does what it does, whether disappointing on one occasion and surprising the next. Perhaps this is the most challenging shift in thinking for an artist: creating a place for the participant in an event that is an 'active ecology' without, as Manning says, 'necessarily putting the participant in the role of direct activator of change' (2013a, 130). Thinking in terms of drift requires designing interactive systems that are composed of components capable of retaining flexibility in the order in which they affect other entities, the ways in which they affect entities, and the direction in which such affectual relations operate. What is required is the invention of 'techniques for the proliferation of drifts', rather than the placement of a specific drifting in the event (Manning 2013a, 200). None of this is to promote self-organisation as the be-all and end-all, as it is of course a dominant characteristic of capitalism, subsuming all to an equivalence of exchange (in this sense it is molar while still self-organising).112 Here an ethics of interactivity and self-organisation needs careful consideration. This leads to the key questions of this book: how to propose systems that can continue to express creative potential of differentiation, while maximising their relational interdependence. It is in seeking practical solutions to this issue that the next two chapters examine the capacities of entities to express feeling, and the potential of noise within relation to act as a force of intensive differentiation. ### 3 ### Once more with feeling: Whitehead's concept of feeling and a trans-human ethics Each task of creation is a social effort, employing the whole universe. Alfred North Whitehead Whitehead's world is one of worlds, plural. Andrew Murphie #### Introduction The turn towards the relational as outlined in the previous chapters is still far too general and abstract for the practical task I wish to explore in this book – that of a pragmatic thinking through of how the relational might be utilised across a number of scales of activity in applicable artworks. For Whitehead, as Andrew Murphie notes, 'relation' is a term abstracted from the held 'contrasts' that are constitutive of an entity's becoming (2016, 21).113 Here what we might generally call 'relations' are rather 'the gathering together, maintenance and creation [of] new contrasts – differential intensities' (Murphie 2016, 21). In this view the world is composed of these gatherings of feelings, intensity and differentials (Murphie 2016, 21), and Whitehead's schema investigates such gatherings in detail. In this chapter I wish to outline this system of prehension (positive and negative feeling) – emphasising the role of intensity in Whitehead's ontology. In the next chapter I refine it for my purposes through the use of Serres' concept of the parasite as a particular way of conceiving of these held and productive differentials that Whitehead argues are the constitutive dynamics of relation. These concepts will then be put to use in the following chapters as the basis of a rethinking of interactivity along the lines of differential ecologies. If interactive art has had a tendency to present rigid models with very prescribed and contained notions of relational potential, Whitehead's concept of 'feeling' perhaps offers a way to think beyond this. This might be particularly useful in assisting a move beyond the predictable or prescribed result and into a realm that is more open-ended and process based. It might also assist a move towards an expanded concept of interactivity or participation that thinks both of the relative freedom of the participants in this encounter, while also considering all the various components of the artwork and their freedom of expression. Thus, as Murphie proposes, there is a need to develop a 'syntax of feeling', through which we might 'open up the world to itself, or, more correctly, open up the possibility of participating differentially in the dynamic ecologies of the world' (2016, 12). In this sense such a concern might broadly be thought of as post-human, though perhaps the term 'trans-human' is more suitable,114 since the interest here is the expansion of the human beyond fixed identity, alongside the expansion of the potential of the field and all participant components of an event to co-produce novelty or 'individuate'.115 I will refer to this as an ethics, in that it seeks a right for all components to fully express their capacity to 'feel'. This is a concept I return to later in the chapter and which permeates the more general rethinking of interactivity in the book as a whole. In this chapter the concept of feeling and its trans-human implications are, after a more general discussion of Whitehead's theory, read first through thinking the potential of inanimate entities (rocks) to feel, secondly through the implications of Charles Darwin's study of worms for ideas of the location of a non-human 'intelligence', and lastly through a discussion of the induction drawings of Australian artist Joyce Hinterding. The Whiteheadian concept of 'feeling', as Judith Jones states, repudiates the idea 'that existing objects have determinate, sharp existential boundaries' (1998, 162). This is replaced by a complex system of relation that emphasises the autonomy (or 'subjectivity' in Whitehead's terms) of the becoming of an entity. Whiteheadian autonomy, however, always acknowledges the ways in which such becoming draws on both the actualised environment from which an entity emerges, and the role of the virtual as an equally real, if differently composed and operative, influence on becoming. For Whitehead – as for James with his concept of radical empiricism that admits all experience, including relations, as equally real – it is of utmost importance to develop an ontology with a consistency in philosophical abstraction of reality without resort to exceptions, applicable to all entities and events (Stenner 2008, 99). Feeling, as the basis of his system of becoming, must then be applicable to all entities, as must the acknowledgment that each entity feels in its own particular way.116 And thus, from this world-view, if you and I can 'feel' and make 'choices', so, in their own specific way, can a rock, a bird, an electron, or as Darwin argued, a worm. #### Feeling For Whitehead's organic philosophy, nothing is inert: everything is engaged in processes of becoming, changing, emerging, marching towards novelty. All things, Whitehead states, are capable of feelings (1978, 220), sensitivities that allow them to navigate, to form workable assemblages, and to become with their environment. Such feelings are not necessarily conscious, and in fact the vast majority are not conscious. Thus feelings are as relevant to entities without consciousness and they do not privilege sentience or the living over inanimate entities. Nor is feeling attached to preformed entities (Manning 2013a, 21). Rather, this is feeling as a force gathering towards form, immanent with the occasion, moving with the event. In this sense for Whitehead's 'atomic' philosophy, actuality is only this act of an entity's in-forming or 'concrescence': the gathering of physical and conceptual feelings into one subjective form.117 Once this resolves into the 'satisfaction' of the entity (though such resolution, as discussed below, does not involve the erasure of the differences between the gathered feelings but their productive contrast from the point of view of the entity), it in one sense ceases to be, although it continues to exert influence through its potential to be felt by future entities in their own concrescent processes.118 In this complex theory of 'prehension' Whitehead outlines processes of becoming based on this concept of feeling.119 Two aspects of this theory are of particular important for the purposes of this discussion. Firstly, the concept emphasises the inevitable emergent condition of existence. That is, there is an ongoing, unceasing process of individuation that all actualising events are involved in. Secondly, this emergence involves choice or selection from a larger potential (on a largely if not entirely non-conscious level), and therefore it is always a differentiation. Here process is a creative event of formation of an entity as potential is transformed into actuality (Guattari and Rolnik 2005, 311). Thus feelings are always cuts – choices, points of divergence or nascent novelty – that differentiate both from the potential data drawn from that which is already actualised and from the data drawn from the larger virtual or ongoing planes of potential. These cuts are made intensively, by an entity for its own satisfaction rather than in any way beholden to external interests. To explain this, here I briefly outline aspects of the process of 'concrescence' as Whitehead calls becoming or actualisation. Concrescence is a process by which an emergent entity, occasion or event120 draws a disparate or disjunctive selection of datum from the world into 'the real unity of one actual entity' (Whitehead 1978, 22). Thus in an ecology this entity is a nexus or 'one complex feeling' (Whitehead 1978, 44) at which a number of potentials meet. From the perspective of the field this nexus represents one novel solution to a feeling for the field in its entirety121 that reflects its own perspective on the universe and has some connection, however remote, to all that is actualised.122 Every prehension consists for Whitehead of three factors, the 'subject' prehending, the datum prehended and the 'subjective form' or the way in which that datum is prehended by the subject – that is, the selection or choice that is made and that leads towards novelty (Whitehead 1978, 23). Feeling is a force gathering towards form, immanent with the occasion, moving the event (Manning 2013a, 21). Feelings are not relations between things. Rather the entity is a singular concrescence of feelings: an event of synthesising or patterning of formally disparate relations at one point in the field (Whitehead 1978, 232). The relation, from the point of view of the subject prehending, is a feeling: its own perspective or subjective take on the other entity. As Whitehead states, feelings 'aim at their subject' rather than being 'aimed at' their subject (1978, 222)123: they are generated and owned by the forming entity, not projected by any external agent. For Whitehead, becoming is an act of self-enjoyment, and the entity is in this sense self-realizing, transcending the entities that already exist and adding to the novelty of the universe (1978, 222). It is important, however, not to think of this gathering of various feelings (concrescence) or the end-point resolution of this gathering (satisfaction) as erasing differences between those feelings, either extensively or intensively. In the former case the datum prehended is objectively available for other events of concrescence, within which portions of it will be accommodated and other portions excluded (negatively prehended), creating with this datum new complex feelings that express their difference to previous entities. The potential for further differentiation always exists. In the case of intensive difference, this is a more complex matter that might be overlooked. In her writing Jones attempts to bring this to the fore, positing 'intensity', defined as 'the compression of multiplicity in an individual unity', as the key to understanding Whitehead's ontology (1998, 157).124 Whitehead uses the term 'contrast', which Jones defines as 'the positive relation of two or more discrete elements in the complex feeling involved in concrescence' (1998, 12). That is, differing select datum from the world are made compatible (though only in the instance of a particular concrescence). Here the 'richness' of an entity's becoming depends on its ability to positively involve the maximum amount of datum in this pattern of contrasts and therefor maximise intensity (Jones 1998, 12, 17, 36). Contrast is therefore 'unity in difference', preserving these differences but finding a 'self-consistency' of the many in the one (Jones 1998, 56).125 These 'held' contrasts do not, as Murphie notes, need to be resolved, but exist productively as a differential 'pattern' (2016, 20),126 internal to and constitutive of the subjective form (Jones 1998, 102). This patterning consists of four factors contrasted into a pattern of relevance: 'triviality and vagueness' (the contrast terms for the prehension of a background of a becoming), and 'narrowness and width' (contrasts of which determine the foreground) (Jones 1998, 38). Here relation is this gathering and holding of contrasts, not a simple connectivity. Events come together but remain atomic (a 'disparate multitude' and a subjective self-creation) (Massumi 2011, 20–1). Without this intensity, there can be no relation.127 Intensity or contrast is therefore a problematic structure, a partial solution that is productive (of variation) rather than resolvant. Such intensities continue to be productive after the perishing of an entity in that these patterns are inherited through their ingression into future acts of concrescence (creating 'contrasts of contrasts') (Jones 1998, 40). This intensive organisation is always the entity's self-motivated choice or selection from potential. This point cannot be over emphasised I think, as it provides a way out of a static universe of things in which only select 'special' entities, such as humans, are seen to have choice or agency. This separation of the human from the rest of matter underpins our supposed authority to command nature, and denies the validity of listening to the expressions and forming relations of all events. In this sense Whitehead's philosophy might be seen to be post (or trans-) human, though in reality it is also post object in emphasising relation and process over form. As Murphie points out, Whitehead's system flips traditional Western notions of subjectivity around: no longer is the subject 'somewhat separate from the world', but rather 'it begins to head towards an actual occasion' (2016, 11). Subjectivity is not exactly erased in this thinking, but is seen as emerging from the acts of selection that take place in concrescence.128 Indeed to look for truly individual subjects begins to look like the wrong question.129 Firstly, this is true because, for Whitehead, an entity perishes on completion of its concrescence ('it never really is') (Whitehead, cited Jones 1998, 101). Secondly, this 'character' is trans-entity, in that it is then present in other entities' 'achieved intensive character' (Jones 1998, 100). Subjectivity exists here as 'subjective form' or 'character' in the act of concrescence: the acts of feeling, valuation and patterning of physical datum and virtual forces into an entity's own particular take on the universe. Thus it would seem to more radically complicate notions of agency than philosophical moves simply assigning agency to non-human and inanimate objects.130 'Agency' can be a problematic term, with a tendency to imply the primacy of 'agents' as discrete, independent and stable entities positively exerting force, while somehow remaining internally immune to change.131 This isolates objects, Jones claims, and is Whitehead's key issue with philosophies of substance, which again position relation (and its component parts) as secondary (Jones 1998, 95). Indeed, Jones argues, the search for agency either naively seeks this false separateness, or pessimistically abandons any concept of will or power as per some post-modern approaches. Whitehead, on the other hand, maintains what she terms 'an ethical spirit of hope and adventure' (1978, 176–7), and it is this ethical spirit of the potential for novelty in all events that this discussion of feeling is attempting to channel. In Whitehead's system agency is situated in the event. This does not imply an externality of control, but that agency is intensive, in the making of evaluations as to the relevance of datum for an entity's own concrescence (Jones 1998, 88). That is, the agency is in the ability to create productive contrasts. Thus it is both 'borrowed and new at the same time': drawing on the valuations and ordering of prehensions by previous entities and imposing in some manner 'on all subsequent process' (Jones 1998, 129, 131). Therefore it never truly belongs to objects or entities, even with Whitehead's expanded field of creativity. At best agency is a condition of the emerging ecology itself. Entities indeed do have a very real connection to all of the emerging ecology. As Whitehead states categorically, it takes a whole universe to make an event or entity. This can be seen, firstly, in that as an entity establishes a relationship both to those objects that it directly draws data from, it also draws in a 'second-hand' manner from entities whose data went into the formation of this object. Therefore, in a more and more mediated and remote fashion, the entity forms a relation to all actualised entities and their histories. Thus a prehension is always complex, capable of being divided into other prehensions in indefinite numbers that reach a singular resolution as a pattern of contrasts in any particular entity but never preclude other potential resolutions from arising. Secondly, relation includes not only the complex layering of selected prehensions as discussed above, but also 'negative' prehensions. That is, prehension consists not only of positive relations or feelings – whose selection constitutes the data that forms its concrescence – but also the act of not selecting other data, again a choice or differentiation from what has been. In this, it establishes a richness and complexity that allows it to (negatively) retain relation to all, if only as the 'scars' or 'impressions of what it might have been' (Whitehead 1978, 226–7).132 Lastly, as well as drawing on these actualised objects for data ('physical' prehensions), an in-forming entity also draws conceptually on some of what Whitehead terms 'eternal objects'. These are pure and indefinite qualities such as 'redness', 'hardness' and 'warmth' or, for the 'higher level' entities amongst us, 'lust' and 'despair'.133 These qualities are felt as objects. That is, what is felt is their 'capacity for being a realised determinant of a process' (Whitehead 1978, 239). Through this ingression of eternal essences or qualities (and contrasts between them) an entity conceptually modifies or evaluates its feelings of the actual world, creating another layer of complexity in the concrescence (Jones 1998, 45–6, 59; Whitehead 1978, 240–1).134 Once actualised an entity continues to ingress on proceedings by acting as datum for the concrescent processes of other entities, functioning 'as an object' to be felt in these events (Whitehead 1978, 220). Here it transcends itself and is further enmeshed or integrated into the ecology out of which it has emerged. The seemingly contradictory values of processes of personal satisfaction and objectification (being 'a unity' in its own subjective form, but in being 'divisible' objectification by other concrescences) of course overlap in complex ways and cannot, Jones' argues, be sharply separated on an ontological level, as individuation is never a linear or simple process (1998, 52, 88). Here feelings are never entirely clear and distinct, 'overlapping, subdividing [and] supplementary to each other' (Whitehead 1978, 235), as well as being divisible into other feelings. Conceptual and physical feelings may also 'hybridize' each other through treating other feelings in the event as objects to be felt (Whitehead 1878, 246). In this way they might be thought of as 'nested': as a series of contrasts or intensities that are 'implicated in one another, each in turn both enveloped and enveloping' (Deleuze 1994, 252; Jones 1998 48–50). #### Rocks If we accept Whitehead's challenge and carry this concept to its limit – beyond entities with attributes easy to anthropomorphise, such as animals and plants – we can ask instead: what does a rock in a stream feel? To which forces are its sensitivities tuned: rain, salts, acids, wind, tides, heat? How does the becoming form of the rock instigate new force – shape the wind, give new direction to the current, absorb or dissolve salt solutions? We begin to see the rock-world relation anew: the rock's continued fielding in the world – its continued effect on or transduction of the ecology's forces – and the field's continuous expression through the force of the rock, becomes an ecology of operations. We learn from the 'wisdom of rocks, from which we can derive an ethics involving the notion that, ultimately, we too are fluxes of matter and energy' (DeLanda 1992, 143).135 But the rock does not only feel the flow of the river, its chemical composition and the rain. Through mediation136 it also feels the waterfall further upstream through the waterfall's effects on the flow and mix of sediment stirred up from the river bottom. It feels the mountain at the birth of the river through its various effects on the river over time. It feels the change of season on this mountain top through the melting of the snow, the birth of fish and their thrashing, the splash of a bird hunting in the water and so on, all at various degrees of remove and impact, through the water. And conceptually the rock also feels and selects from qualities of rockiness, hardness, wetness, and heat, exploring some potentiality of these concepts through a selection that inserts them into actual events, valuating to some degree its experience of the actual. And, the rock selects not to feel other events: both the actual, such as the ticking of the clock in your house and your thoughts of lunch, and the conceptual: lust, anger, depression. The valuation and admission of the various positive prehensions is more than a modifying factor; they constitute the rock's becoming – for it to 'not only have but to be a perspective on the world' (Jones 1998, 36, emphasis in the original). The rock has its own singular take on or positive logic of all the feelings that are deemed relevant to its concrescence. Its neighboring rock may also feel the melting of the snow and the movement of the fish upstream, but each evaluates these factors from its own perspective and according to its own appetite for becoming.137 Each rock valuates the positive feelings according to their importance for its concrescence (its triviality, vagueness, narrowness and width), and scales or contrasts these into a pattern of relevance, seeking the required depth of held intensity: the 'inequalit[ies] by which it is measured' (Deleuze 1994, 222). Thus for our rock the movement of a upstream fish may be a trivial feeling, felt through its momentary effect in the very slight variation in current, but a smaller stone at the epicenter of this disturbance feels the full force of the fish's fins and is lifted off the riverbed and flows downstream – not a trivial feeling at all from its perspective. Similarly backgrounded for the rock in question may be the vaguely felt movements of the individual leaves of a tree in the water – 'vague' in their indistinctness from one another (Jones 1998, 35, 38). 'Narrowness' and 'width' constitute the more foregrounded assessments of feelings (though all the terms are interrelated). It is a balancing act between a certain 'narrowness' of scope that is necessary – a value judgment that allows prehensions deemed less relevant to be backgrounded sufficiently (becoming the relatively trivial and vague) – and sufficient 'width' of ingression of those feelings deemed important for satisfactory depth of feeling and complexity of intensity to be achieved, and for sufficient 'thematic unity' for the rock to achieve the status of this particular novel entity in the world. These feelings are contrasted and patterned according to the rock's entertaining of its own 'ideal of itself' – in other words, in relation to its conceptual prehension of relevant eternal objects such as rockiness, solidity and so on (Jones 1998, 38). We might easily accept this conditioning of the rock in relation to its given actual circumstances and its potential expressions during its overt formation, such as rock formed by lava flows molded in relation to landscape, water and climate. But part of the challenge Whitehead demands is that we consider the rock to be continually recomposed through events of concrescence and perishing.138 Here the rock as a whole is a 'society' of smaller and briefly becoming events or entities (Whitehead 2014, Chapter III). Admittedly this is a society without great internal difference and, from a human scale of attention, with a very slow rate of differentiation, but it is a significant step from thinking of an entity as that which goes through a becoming phase to arrive at a 'pure' object status that is outside of this individuation (a downfall in the logic of many philosophies, as Whitehead seeks to demonstrate). Here Whitehead's 'societies' are nexus 'with social order'. That is, they have 'common element[s] of form' that are 'imposed' on all the member entities by their positive prehension of other members of the same society (Whitehead 1978, 34).139 Within this 'atomic' conception of entities the rock retains at least some potential for change and ongoing selection or choice.140 There is always room for differentiation to occur, and there are always prehensive relations of the world that tie the becomingrock to the ecology with which it gathers.141 #### Worms Another humble entity worth consideration, although in this case an animate one, is the earthworm. Charles Darwin devoted many years of study and an entire book to the worm, utilising a rigorously empirical methodology that lead him to an understanding of the very real and complex interactions and forces informing the worms' lives and their impact on the human world.142 However of most interest here are the sections of the study in which Darwin explores the 'intelligence' of the worm, an intelligence evidenced by their creative and non-teleological interactions with their ecology. While worms clearly have limited capacities to sense and interact with their environment, being blind, deaf (though sensitive to vibration), and possessing a limited sense of smell, to Darwin this does not mean that their actions cannot move beyond pure instinct or habit. Through a detailed study of the way that worms utilise leaves, Darwin seeks to show that worms have a capacity for creative selfdeterminacy. The worms appear to be able to adapt very quickly to unfamiliar types of leaves and to invent ways to attach to leaves, to drag them along the ground and to utilise them to plug the entrances and line their burrows. In doing this they develop novel capacities for solving the problems that are incurred with particular leaves – for example, plugging a burrow with pine needles for the first time rather than a broad leaf, or handling a leaf with an unfamiliar petiole (Darwin 1881, 59–60). Here perhaps we could say that the worms are able to successfully assess and pattern their prehensions of the capacities of the leaves in relation to the various conceptual qualities needed carry and to plug their burrows. Darwin concludes firstly that the worms' approach to these new challenges cannot be explained by any logic of inheritance or habit. Secondly, he argues that the worms' abilities to utilise novel materials successfully are not based on methods involving either significant elements of chance or trial and error, which might also eventually result in the worms discovering a suitable method for handling the leaves. Rather, the worms appear able to perceive and creatively engage with difference in thinking through the best way to drag and utilise the leaves (Dawrin 1881, 73–4, 92–3). Any argument that the lowly worm acts always or entirely purely for survival as an ultimate aim seems to be unsustainable when faced with this evidence. Whereas a neo-Darwinist approach might argue that a worm's capacities are all pre-formulated with survival as its entire domain of enquiry (a worm is blind because it has no 'use' for sight, and so on), in fact the worm is involved in a creative enquiry with its environment. Here it pays to remember that in Whitehead's system the leaves have feelings too, as does the borrow, and that the worm's prehension of leaf and burrow opening are, through mediation, also prehensions to some degree of these other entities' subjective take on their worlds. The worm's capacities are always forming-with or immanent to its emergent ecology through acts of feeling-with. These capacities are creative in that they are open-ended to a certain extent, never reaching a state of full formation, desirous of novelty rather than limited to survival or directed towards any one simple perfect worm-form143 (an ur- or uber-worm perfectly able to exploit its environment144). The worm, in other words, has plenty of wriggle room. For Darwin the worms demonstrate 'some degree of intelligence' in their activities (98).145 But the worms are not writing philosophy or sonnets on love, so in this context, what exactly does the intelligence mean? I would suggest that what the worms demonstrate is an ability to tune with their environment on a profound level. In other words, they have the capacity to 'feel' or prehend the potential in worm-leaf-burrow interactions (a resonance or intensification of potentials146), to compose relevant contrasts out of this information (to pattern), and to make creative selections or choices immanent with their actions, tastes and needs (to valuate). Here the usage of the leaves in the burrow is a nexus of possibilities that resolves some potential of the worms, leaves and burrow's collective capacities into an event with its own subjective expression (necessarily composed also of all the negative prehensions of what is not selected or actualised). These collecting feelings making up an ecological intelligence that is immanent with, and belongs more to, the event, as a bringing together of potentials and capacities, rather than belonging to the worms147 – just as the intelligence of juggling belongs in the moment of conversation between balls, hands, gesture, gravity and performativity rather than the identity of the juggler. In this we might also say, in the styles of interior decorating the worms develop in collaboration with the leaves to line their burrows, that they exhibit a (pragmatic) creativity, which again goes beyond simple need or survival. As much of Alphonso Lingis' writing seeks to demonstrate, here there is a joy in living or self-production – in feeling with the world – that is an essential creativity.148 Just as the individuation of the rock becomes more complex and resonate the more we consider its capacities and their expressions-with its ecology, so the worms' choices can never be fully explained in simple terms of need: there is a nonteleological, qualitative desire at work in the event, composed as it is of feelings that prehend and play with potentials. #### Ethics If morality is the relinquishing of individual freedom in favour of alignment with a prescribed concept of good that then constrains as it is applied blanket-like over all (a generalised righteousness), then ethics is perhaps in many ways the opposite, an 'augmentation of the power to live in this world' (Massumi 1992, 108; Deleuze and Guattari 1987, 256–7).149 That is, it deals not with the system-wide or universal but with the specific, local and emergent: with, in other words, a politics. This, as Manning states, is a procedural and emergent politics rather than a politics of control exercised by subjects over life (Manning 2013a, 147). It is therefore 'attentiveness to the conditions of the event expressing itself' (Manning 2013a, 148): a force of becoming that is also always in itself open to new feelings that might affect and overlay its individuation. In this, ethics is affirmative. The negative movement of morality might diminish or flatten relationality, composed as it is of capacities to 'express the high levels of interdependence' of entities. Ethics, on the other hand, emphasises the expression of entities' capacities to prehend and the power of their positive ingression to create novelty (Braidotti 2010, 226). For Whitehead, as Jones states, the system of prehension is a system of ethics in that it is a 'commitment to attentiveness about our world', both in terms of acknowledgment of the role of broad experience, and of the singular intensive character of each event. It does not level out experiences of the world as morality might seek to do, but adds to it, if addition is thought of as the addition of further contrast or difference (1998, 85).150 Here Whitehead's system approaches ethics in that it requires attention to 'the general good' (Whitehead 1978, 15). This attention to other realities and perspectives is the very contrast that is 'internal to the being of the agent, and thus integral and ultimate to any action'. This concept of ethics eliminates both notions of the passivity of the non-sentient or non-biological, and 'the exaggerated sense of ourselves in as some kind of special freedom-nature in the cosmos' (Jones 1998, 85). As Lone Bertelsen has suggested, such ethics might be the beginning of 'ecological responsibility', a 'shared attentiveness and an affective field established across space, bodies and objects' (2012, 39). Perhaps, as Darwin did, there is a need for humans to listen more closely to the non-human and consider more carefully the potential of non-human capacities. If we think these capacities and individuations from the perspective of the field more than from any object, then we may arrive at a transhuman sympathy that recognises not only the capacities of rocks and worms, but also a human potential for greater resonance with the ecology. This might be the very tentative beginnings of a different kind of sympathetic exploration that could also instigate our own adventure into greater expressive freedom: an ethics as relevant to rocks and worms (more specifically of rocking and worm-ing), that is an expanded ethics of differential 'life potentials' (Massumi and McKim 2009, 12). #### Joyce Hinterding's Induction Drawings When it comes to applying these ideas of capacities to feel (powers to select and self satisfy) to art, what is it that we might be looking for? Perhaps it is an art that plays with and off specific difference, concerns itself with flux, with an in-forming. Perhaps it is to seek an artwork that allows space for the emergence of feelings of its component events, which might seek not to impose a human-centered perspective or entertainment but instead might favour an entertaining of an environment by (and for) itself. This might be a paying-attention-to (a listening) and making space and/or time for different scales of interaction, different capacities to ingress into proceedings as nascent eruptions of difference. Fig. 3.1 Joyce Hinterding, Soundwave: Induction Drawings, 2012 Joyce Hinterding's Induction Drawings (see Figure 3.1), I propose, might exhibit just such characteristics and concerns. In these works Hinterding makes continuous graphite drawings on paper, wires them up (sans microphone), via amplification to a speaker system. The graphite forms a continuous conductive loop through which an electrical charge can move, when such a charge occurs its fluctuations are converted into speaker vibrations and thus heard as sound.151 The sounds heard in the work are the result of the phenomenon of electromagnetic induction: sensitivity within the drawing loops to the differentials of fields of magnetic flux that causes a current to be generated in the graphite 'circuit' (induction loop). The natural phenomena of induction occurs when one potentially electro-conductive closed system (such as the graphite drawing) is in close proximity to an electromagnetic field (such as a body, a voltage loop or magnet), and a sympathetic or parasitic resonance occurs that causes a transient current in the first system. It is a resonance – two energies in communication (Deleuze 2002a, 65) – not the same electromagnetic force flowing between the systems. Each is selfdetermining. This resonance occurs only through differentiation: it is only change in the original electromagnetic field that causes a sympathetic voltage to flow in the induction system. Multiple factors are drawn together – selected and felt – by an induction loop (in this case the graphite drawing) in order to create the inductive current. That is, the event of the inductive charge is drawn from a concrescence or patterning of a number of other events or actualisations that act as objects for the insipient induction: the strength of the electromagnetic field, the area that this covers, and its rate of change (and the electromagnetic field is, as an event itself, already a subjective synthesis of all the various emitters of magnetic force in the vicinity that are valuated as relevant – bodies, machines, electronic devices, magnets and so on). This is Faraday's law of induction, where induced electromotive force in any closed circuit is equal to the rate of change of the magnetic flux enclosed by the circuit.152 This law is a differential equation, expressing an immanent contrast (difference differing) between the three key objects from which datum is prehended by the induction event: an event that expresses a particular and subjective patterning of this datum as intensity. The induction is atomic, constantly reinventing itself in relation to the changing conditions. Like the worms, this induction is a kind of ecological intelligence. It is not in any way related to human or worm intelligence and experience, but the induction loop's own ingression into the ecology with which it individuates, an expression of its capacity to prehend relevant differences differing and to put this datum to use to satisfy its own concrescence. Two contrasts are needed to produce this induction: firstly, a rate of change in magnetic field from the position or subjective view of the induction loop<sup>153</sup> (an individuating difference that is qualitative and intensive), and secondly, a contrast in the angles of the two fields that meet (that is, between the electromagnetic field and the induction loop, with the two flows at an angle to each other). A new contrast is always produced in that the resulting induction loop flows in opposition to the original electromagnetic field. Such held contrasts operate problematically (being unresolved) and productively (producing the novelty of induction out of this ongoing prehensive differentiation or intensity). The potential for such contrasts is, according to Whitehead, the precondition of relations (1978, 228–31). That is, this potential operates as a future resonance (the necessary conditions of the field for prehension to occur), or intensity, which is the drawing together or nexus of contrasts or differences brought together as the subjective feelings – in this case, of this particular event of induction.154 But, in order to be true to an ethics of attention to all potential events in their own right, we must also acknowledge that the induction/flow of electrons is not simply a property belonging to the graphite drawing/induction circuit, but is an event in and of itself. It has its own selfish relationship to viewer, drawing, electromagnetic field and so on, and its own patterning of contrasts (as on an atomic level there is a patterning of electrons – a ferromagnetic ordering – in magnetised materials under the influence of electro-magnetic force155). As Isabelle Stengers notes, since Faraday's discoveries, 'the electromagnetic field has exhibited properties irreducible to those of a force "between" two charged and localized bodies' (2011, 101), and the charge here is 'a quantitative character' of the event rather than a property of any electron (Whitehead cited Stengers 2011, 101).156 It is this 'character' that Hinterding lets loose – allowing its voice to be heard, its ingression into events to be expressed. Alongside this event of induction, every component entity of the work has, amongst other feelings, its own subjective feeling of the other components to draw from: the speakers feel and pattern variation in current in the graphite loop, human movements are affected by eruptions of sound, and molecules of graphite feel the negative charge of electrons. Whilst every element draws datum from the others (and draws conceptually from the abstract qualities of flux, vibration, line, volume and so on), it is not possible to say that one exactly follows another in a linear manner. That is, they cannot draw in a simple way from resolved entities as 'objects'. The activities of concrescence are nested within each other. Potentials (individuations) are intertwined and co-dependent, it is an emergence that gathers (contrasts, relations, feelings, concrescence), and a concreteness that continues to become. The induction loop-event is one particular tuning into the world that this artwork highlights. But it also, I think, encourages other sensitivities to be explored, allowing other expressions to be heard and expanded on such as touch, movements and sounds. All of these are events that can be thought of as not only between entities but constituting the very feelings of which these entities are (re)composing. What is foregrounded and made felt, by both the drawing and through mediation by the viewer, is the continued ingression of one event into another. This is communication across (trans) entities – a resonance between certain qualities (orderings of feelings). This might operate between a viewer's electromagnetic field and a potential of the graphite line to carry charge, or between the speed – the style of movement – and the volume and pitch of acoustic vibrations of the speakers. What might be felt in part by the viewer as they interact with the installation is their presence as an object, in the Whiteheadian sense, for the graphite loop. That is, as datum to felt by another, very distinctly non-human but vital entity: desirous and selfish in its exercising of capacities to feel and incorporate some element of the human into its becoming, alive in its self-satisfaction and independence. But what is felt and made apparent in the human is also trans-human – a mobile field of electromagnetism that is itself an event drawing on elements of the human that can be utilised, alongside other electromagnetic forces (the mobile phone in the viewer's pocket, static from a nylon shirt, ambient charges and so on). Thus the work asks not only who or what other than the human might engage or feel with their eventness, but also how other modes of human engagement might occur. While it does not deny the visual pleasure of the drawings or a distanced listening, it engages across the human with an unseen and largely unfelt more-than-human component of us all, the field of our gathering electromagnetic expressions. It requires that we pay attention to forces that can be felt in their effects, but which cannot ever be fully grasped – an oblique attentiveness to differential intensity known only as a continued expression of held contrasts. These induction artworks enable, I would suggest, an act of listening rather than performing, emphasising the singularity of feeling as well as the its collectivity.157 We listen to the drawing system's expressions of a particular capacity (to feel flux), as the graphite listens to our more than human electromagnetic fielding. Unlike in much interactive and new media work where such non-human or more than human components are drowned out by performance and the instrumentation of non-human components that tends to ignore their capacities, perhaps Hinterding, like John Cage amplifying cacti, is listening to the components, allowing the space or time for the resonances between gesture, drawing, movement and flux to arise, giving attention to the various manners in which these acts of feeling by all components to make themselves felt in the event: the graphite's desires, the electrons' future-feelings, the speakers' negative prehensions. Jane Bennett has stated that the 'ethical task' at hand is to 'cultivate the ability to discern non-human vitality', to become affectually open to the larger ecology (2010, 14) (though again I think this is often mistakenly interpreted as a call to acknowledge the agency of objects rather than the field). To me works such as Hinterding's might, in their own humble way, contribute towards an ecological turn and towards thinking the more-thanhuman. The Induction Drawings make us aware of how ecologies emerge across entities, and the importance of non-human scales (the slowness of the rock, the micro-activities of the worm with their macro-resonances, the continued liveliness of a graphite gesture). The works remind us that we must begin to think the ecological not as preservation for human use – indeed not preservation at all, but an ethics of positivity, that is, of a move towards greater involvement, feeling and creativity, as a 'veritable theatre of metamorphoses and permutations. A theatre where nothing is fixed…[leaving] the domain of representation in order to become "experience"…a transcendental empiricism of the multiple, chaos and difference' (Deleuze 1994, 56–7). 4 ### Thinking parasitic action Life degenerates when enclosed within the shackles of mere conformation. A power of incorporating vague and disorderly elements of experience is essential for the advance into novelty. Alfred North Whitehead A turn towards a minor form of interactivity might be seen as a move to an ethical configuration of such events. For Simondon, an ethical approach to relation addresses not its relation but its immanent construction, enabling an opening to further expression and connectivity, and an ability to affect and be affected: to affirm both the singular nature of events and openness of relational potential (Combes 2013, 65). As seen in the discussion of Joyce Hinterding's work in the previous chapter, such a definition of an ethical interactivity might concern not only the ability of relation to remain open in its connective potential, but also the way relation emerges out of a play of affectual forces and subjective feelings collectively taken into consideration. Murphie defines ethics in art as a 'series of practices…which promote expression and machinic connections' (1996, 105). As Murphie argues, the problem for an interactive art event is that a work will always re-stratify after an event of deterritorialisation. Therefore, to retain this ethical potential to explore collective creative expression and defer stasis, the pull of continued potential movement or change is required (Murphie 1996, 105). How then might a continuous and vigorous drive towards reinvention be structured into an event? Rather than just concentrate on the power of the event to establish layers of relation, how can their perishing and replacement also be driven internally? In a minor assemblage, its 'health' lies, as Murphie says, in an ability to conserve creative possibilities (1997, 164–5). In the previous chapter I argued, after Judith Jones' analysis of Whitehead's ontology, that just such a 'preservation' of difference could be seen to operate through the held contrasts (productive differences) that constitute an event's becoming (and indeed constitute and are indivisible from the whole of the event's subjective life). Yet this 'holding' of contrasts should not be conceived as static, it is always in-forming, keeping the actualizing entity at the point of 'supersession by novel actual things' (Whitehead 1978, 45–6). For a system to continue to approach a molecular state, it is not enough to establish relation. It must continue to agitate – even if this molecular agitation exists on a virtual plane as an 'unrealized potential' (Whitehead 1978, 45–6), luring prehension towards further individuation. To become an event that gains the power of continual selfinvention of the everyday experience might require a system that is able to include not just a positive connectivity, but disconnections and failed, disruptive, competing and destructive relations (as Whitehead's system includes both the negative in terms of negative prehensions, and 'competition' in the singular subjective ingressions of an entity into all other entities). What is needed to activate a machine capable of drift is potential machinic difference – a capacity to intensively produce change that then acts on a local level to agitate and destabilise (Deleuze and Guattari1986, 50). To remain intensively relational here, we must look for a disruptive movement that has a causal logic, however complex. Michel Serres proposes that 'noise' in a relation is a necessary condition of its existence, stating that 'if a relationship succeeds, if it is perfect, optimum and immediate; it disappears as a relation' (2007, 79). That is, relations are a condition of difference in a system or assemblage, rather than arising out of harmony or equilibrium. As Serres states, relations are full of 'losses, flights, wear and tear, errors, accidents, opacity' that are their creativity. Without this differential capacity composed of excesses, surpluses, interferences and disruptions, such systems collapse back into a molar configuration (Serres 2007, 92, 127). That is, they becomes at best patterns of stratified or ossified relations, with a loss of the intensity that opens systems to novelty.158 Serres terms these noises within relation 'parasites', and explores the parasite as a potential mechanism to complicate and expand the idea of co-causality (2007, passim). The parasite here has multiple meanings, being both a literal parasite – feeding off the energy (both physical and social) of another – but also more importantly as the noise in the system of relations. Thus in a 'relational' system there is a potential third position – the parasite – (and then a noise within this parasitic relation as a third position of this third, and so on) that creatively interferes from within the assemblage. As the noise or disruption to a force, the parasite is the emergent difference in relation; relation's potential to differentiate from itself. It is a force that pulls towards a more-than, towards a continued individuation or movement of the system that differentiates from the actualised. The parasite, as Yates says, acts against any 'fantasy of control or mastery' (2002, 50). It demonstrates how systems generate their own subjective 'open or dissipative' differentiation through interdependence produced by disruption: 'systematic orderings, local ecologies or drifts' produced by 'momentary deployments of forces' (Yates 2002, 50). The parasite is essentially creative, in that it forces into existence new logics, new combinations, and new orders of exchange (Serres 2007, 35), as a difference that unifies through the production of relation (Deleuze 1994, 56). It disrupts clear communications, but produces something else through its (mis)translation of relations. This third position in the system is itself unstable, Serres argues, as the roles are interchangeable and fluid – each position is potentially noise for the other two. Therefore parasites lie in between any absolute or fixed position, always fuzzy and multiple, contradictory and irresolvable. This destabilises any hierarchy or relational equilibrium, making each position implicit in the relation of the other two (a nesting or quasi-causality) (Serres 2007, 182). This is the 'disorder' or unpredictability of relational systems in drift that is inclusive of the disjunctions and failures that are always initiating new orders (Whitehead 1978, 91). The parasitic proposition is a machine that produces a continued evolution of difference: a difference in relation and then further difference within this difference. As a movement or molecularisation within any system, the parasite is potentially an engine capable of driving drift through its continued problematisation of relation. Parasites turn any linear system of relations into a complex and intertwined set that is never fully resolvable, making 'chains of contingency' (Serres 1995, 71) and then continuing to activate or reactivate these chains (Yates 2002, 51) so that they are more a 'series of frictions' than a linkage – 'tangential, contingent [and] unstable' (Serres 1995, 73). It should be evident that this productive concept of noise is very different to its position within communication theory, as Shannon and Weaver conceptualise it. Within their systemisation, noise is only the 'unfortunate and unwanted additions': distortions, static and errors (Shannon and Weaver 1967, 7–8). For them information exists as in a pure, abstracted form (the medium is not the message), that can ideally be transmitted between a stable source and receiver. Here message, sender and recipient transcend the actual conditions of the event of communication in an artificial separation of semantics and information as signal (Shannon and Weaver 1967, 8). This problematically supposes that information is simply replicated across the divide between two objects, rather than needing to be reproduced (Ingold, 159).159 Any 'freedom' created by the presence of noise is, in Shannon and Weaver's thinking an 'undesirable uncertainty', as uncertainty can only be desirable if it is located in the agency of the sender to make choices (1967, 19). Thus, from a process philosophy viewpoint that seeks a productive problematisation or intensity of relation, their communication theory fails not only to acknowledge the essential role of differentiation in producing novelty, but also seems to deny noise its own status as an event with its own subjective prehension of the sender, signal and receiver – all of which are given an (artificially stable) agential status.160 Serres' parasite, however, is more than a simple disruption to established relation. It is a potential that is immanent to relation in-the-making. This is a potential at the stage of prehensive lure towards connection that always positions relation at the point of splitting and differentiating. In this the parasite is, as Serres states, 'a third [that] exists before a second' (2007, 63). This is a system of differenciation161 – potential difference – as much as actualised differentiation. It is a system of internally organising and foregrounding the lure of instability and difference in creation. The parasite is a self-organising multiplier of relations – it bifurcates any stable exchange as a derivation from equilibrium, with 'abuse-value' rather than exchange-value (Serres 2007, 17).162 This creates new relationships through the eruption of difference that 'recharges the activity of relating from which all experience emerges', as Massumi states, it is not deconstruction but 'continued construction – reconstruction on the fly, not interruption, but recharging and resaturation with potential' (2011, 102).163 This implies creating a propositional structure where relations not only layer, but also have the inbuilt potential to interrupt each other. Even as virtual noise, parasites create open-endedness – potential disruptions that can create a tension acting on any actualised relation to keep it on the verge of change or collapse, multiplying its virtual qualities rhizomically.164 On an interactive design level, the productive implications of the parasite might involve firstly the acknowledgment and encouragement of a wider range of potentially disruptive relations. Secondly, utilising the flexibility in relational positioning that the parasite forces into existence, and, thirdly, the more concrete construction of generative systems – with the inbuilt potential to interrupt and distort each other on multiple scales, and within many differing types of relational forces. The first of these factors involves understanding ways in which sensorial, affective and social relations can creatively alter and disrupt the actual individuated experience in any event, for example: Secondly, the parasitic model embraces fluidity in relation to any art event, enabling numerous interchangeable parasitic diagrams that could be described. For example, if we return to A Chorus of Idle Feet, the artwork example from Chapter Two in which the movements of the viewers comingled with the sensor infrastructure, software and the production of rhythmic pulses of sound, we can see the interchangeability of the three positions within parasitic relations. From one position, the participant is the host; the software draws energy from their body, and the parasite is the rhythmic sounds that disrupt the participant's movements. From another position, the software can be the host, in relation with the sound that draws the energy to mutate from its wave patterns, while the participant is the parasite, interfering with their simple communication through speed and rhythm of the body's movement. The sound might also be considered the host, in communication with ears/brain/ kinesthetic functioning that draw stimulation from the vibrations, with this communication disrupted by the additional difference in rhythm that the software insistently implants in the relation. The exploitation and enhancement of these naturally slippery relations brings to the event an unpredictability of any planned interaction – continual, subtle re-tunings of relations that modulate and invent. Thirdly, the parasite provides a focus in the more overtly concrete design of sensor-machine interactions, factoring ``` Sensor ➝ wire ➝ interface ➝ program ➝ wire ➝ speaker ➝ sound ``` Figure 4.1 A linear chain of relations. in potential perishings or negations as primary creative propositions within intensively active systems. In A Chorus of Idle Feet, the various sensor-machine produced vibrations could be seen to be parasitic in their potential diffractive actions on each other and to involve a drifting. Much of the system still seems linear and predicable in its relational connectivity – with a trigger from a sensor activating a sound via connecting wires, computer interface, sound program, and speaker system (see Figure 4.1). In the design, however, this was complicated through building in multiple competing relations with the potential to act parasitically on each other. The application of a series of parasitic propositions, in even one small part of this chain, altered the nature of relation. For example, in the relations between the sensor output that triggered sounds, a series of competing propositional potentials were designed that complicated any actualisation of a sound. Other sensor events had the potential to turn off the sound sample, and/or swap it for a different sound, and/or modulate its volume so that it might be inaudible or dominant, and so on (see Figure 4.2). Here in the latter example the eventual sound event involved a complex series of prehensions, both positive and negative, and a patterning of this datum. In the linear example however, while there were still prehensions of other events, there was less tension between the potential and actualised relations and ingressions of datum. In such relatively simple ways, the design moved from a linear causation of relation of movement-equals-sound – a realisation of the possible – to multiple complex potential events intermeshed within a nexus of relations. Here the Figure 4.2 Parasitic potential relations. 'noise' of disruption, a continual force moving the process into reconfiguration. These two designs need not be seen in oppositional terms, rather, there is a distinction between a differentiation that leads only to the possible, and a 'hyperdifferentiation' that might 'seethe with fractal future-pasts' of the unactualised potentials (Massumi 1992, 91).166 The nexus of relations here can be seen to operate not just as independently self-satisfying, but also as complexly and fluidly interrelated through disjunctive events of emergence. These are potential noises within relations that construct through disrupting create intensity and the potential for novelty. An enriched connection to the virtual proposed relation as more than just complex vector relations of physical interdependence. Here a technological system utilising simple components began to approach a relational modelling, as each trigger became a factor within a complex series of interrelated events that were concerned with rhythms, intervals and disruptions that built an 'ecology' of interdependent components (Manning 2009, 74). This complex system of relations was then multiplied exponentially for each sound event, and its virtual potentials also added to the equation.167 Triggers that shifted the sound emitted from one speaker to another also disrupted the spatial relations of the sounds. Other triggers proposed competing shifts in the tonal qualities of the sound produced – changes to the equalisation, reverberation, and so on – potentially disrupting the perception of sound by bodies. In this example, the parasitic potentials of the system drew the various machines into implication in each other's individuation through its entangled chains of cause and effect. Not only were these machines all concerned with the production of sounds, they are also involved in the actualisation of each other, as they began to affect the success or failure of each other's productive expressions. Differentiation here was the unifying element – activating the individuation of relation between entities and assemblages that were implicated within each other's actualisation. This was, at the same time, a differenciation that created a shared potential or priming for further disruptions and relational entanglement, and reveals the potential of disruptive noise to open a system. The parasitic embraces Deleuze's concept of a 'difference without negation', it operates as a productive or 'positive' differentiation, rather than an oppositional difference (1994, xx, 205). That is, rather than acting as a negation that 'subordinates difference to itself', it creates problems within a system that are positively productive (Deleuze 1994, 266–7). This means, firstly, that all the differences have a productive or creative role to play in the drive towards novelty of the system. Secondly, it means that those differences not actualised in any one event remain open to further potential influence on the future of the event. The competing forces of the parasitic potential disruptions within the system create a logic by which the system intensively 'works out' what sound will actualise. It is a self-creative unity that in each instance creates a set of competing propositions, which then drift according to local and singular conditions in any one instance, rather than according to any preconceived outcome. Relations within a parasitically activated system have a new intensity. They continue, even after splitting, to contain the tension of potential further such actualisations of disruption. This both molecularises and concretises the system by demanding a reconfiguration of each relational pull in relation to every other actual and potential force. There are always tendencies towards multiple, incompatible future splits, and therefore the relational forces remain in a problematised state that cannot be resolved into stasis. Here parasitic tendencies evolve, not simply in reaction to established relation, but as a force of relation. The virtual and the actual parasite are emergent events in and of themselves. While there is always difference contained within a system, constructing an event that accentuates the parasitic tendencies of relations to creatively disrupt themselves perhaps shifts it further towards a state of hyper-differentiation. This parasitic modelling remains emergent, embracing change and contradiction, constantly at a point of rearranging. Again, it is a way of enabling the conditions for difference to arise within the event, rather than a prescription of actualised differences. This conception of the parasite allows a way of describing a dynamic, emergent and complex series of relations, a methodology that embraces the potential fluidity. The point to such design is, in a sense, to not have a point: to rescue such art-events from purposefulness, to encourage growth, mutation and destruction, to enable an event to generate its own forces of concrescence, and find its own satisfaction. This does not imply an absence of artistic input in any negative sense, but a shift towards propositional, speculative structuring.168 It places emphasis on the intensification of relation through differentiation, a shift that embraces the richness and lure towards future creativity of a dynamic virtual milieu. The task for the artist is to steer interactivity towards the propositional, to invent ways to keep the event and the temporal experience of participation unstable, to keep assemblages fluidly creative. The point of this multiplication of the virtual is twofold. Firstly, it makes the work as the event, the temporal experience of participation, unstable; it keeps the assemblage fluid and emergent – always reconfiguring, inventing new relationships of connection depending on the specifics of involvement. Secondly, this instability begins to apply not just to the actual experience, but to the language that is used to articulate the event – it becomes a kind of meta-modelling of the experience, which combines various potential relations and interferences into a model that describes the event. This combination is an immanent critique, always at a point of change or dissipation; it applies only to a specific viewpoint, and a specific moment, and must always be reinvented. As a model, it remains emergent, embracing change and contradiction, always needing to be rearranged. What this language of the parasite then begins to allow is a way of describing the dynamic, emergent and complex events of relation that embrace their potential fluidity, rather than a concentration on the form and comprehendible movement. The remainder of this book is dedicated to such an open exploration, with a series of different parasitic tactics across a number of registers, all potentially capable of driving interactive events through the intensive production of difference. 5 ### Walking with the world: towards a minor approach to performative art practice One walks down the path to get somewhere, but one enjoys walking, and one leaves one's house just to walk. Alphonso Lingis #### Walking Walking is intrinsically inventive and relational: to space, to the body itself, and to the potential that it both creates and differentiates. Walking moves us beyond a stable configuration of relations between a subject and objects, and towards a more complex experience that begins to escape such boundaries. It is, in the broadest sense, a parasitic tactic for the disruption of social, physical and mental structuring, capable of folding the body into the world – and world into body – a molecularisation that excites and disrupts. This chapter considers the potential of walking as a 'minor' practice. For Michel de Certeau, cities are excessively stratified and homogenising systems that might be troubled through a technique of walking. Walking, de Certeau argues, is a 'soft resistance' that seeks a creative flight through reactivating connections between bodies and their environment. As Ben Highmore articulates, such walking is 'minor' in that it is positioned less as direct opposition to structure, and more as that which 'hinders and dissipates the energy flows of domination' (Highmore 2002, 152). Every walk we set out on, even the most mundane and functional, is inherently an adventure into the unknown, into improvisation and discovery. If we are too jaded or numb to notice, then we have only to invite a small child or dog to accompany us to realise or invent creative and connective possibilities. With a child in tow or towing us, our walk can never be simply a blinkered move from A to B. Instead, it is rich with potential. It splits to become multiple: consisting of many foci, intensities, and heterogeneous singularities (Manning 2009, 7). A particular smell, a pretty tree, a siren, and a cat, a game instantly evoked out of the walk: all layers of an experience that is being continually reinvented in response to stimuli. Our bodies rearrange and respond to the affordances of the rock underfoot, a cold wind, the effort of a hill, the anticipation of a busy road ahead, the pull of the dog's leash. As Erin Manning says, in moving, the body and the space vibrate with potential relationships and affects (Manning 2009, 13). Such a walk is capable of being expansive without necessarily getting lost – a becoming-with the environment. It is for de Certeau a spatial practice that 'slips into the clear text of the planned and readable city' (1988, 93). Stratifying and restricting forces exist not only within cities, but also within bodies that are unified and ordered by habit and subjection, succumbing to stasis and a loss of connectivity and breadth of expression. This Deleuze and Guattari term a body's own capacity for 'micro-fascism' (1987, 215). As movement complicates and disrupts established spatial relations, multiplying and creating new immanent connections to extend the potential of the body in space, it might also allow a becoming-minor of a body. Walking, as Manning argues, is a temporal, re-combinatory operation of becoming that decentres subjectivity and troubles stasis (2009, 23); thus a moving body is always more than a fixed identity (2009, 63–4). Arakawa and Madeline Gins conception of the 'landing sites' (2002, 5–22) – nodes of attention that the moving body produces – further explores minor procedures where bodies and environment fold into one another and disturb boundaries. The intermeshing of body-world potential that Arakawa and Gins articulate is always in-process, a performative exploration within an established system, be it a body or a place. Here movement fundamentally disturbs boundaries. It complicates relations as it multiplies and creates new immanent connections – relation in-the-act. Walking differentiates and intensifies life, folding the body into the world and world into body (an environmental or ecological engagement169), exciting and operating processes of creative disruption. It is, in the broadest sense, a parasitic tactic for the disruption of social, physical and mental structuring, turning a rote exercise into an attentive adventure. In this chapter, Nathaniel Stern's Compressionism performance is examined for its ability to enable exploration of a minor potential of walking. The configuration of technical objects and bodies in Compressionism contributes to a reactivation of the streets as de Certeau proposes, and allows a reconfiguration of intensive bodily relations through the activation of new internal and external sites of attention. I argue that the technical components of Compressionism help to transport the body beyond habit. While this assembling of bodies and technologies helps to constitute an 'augmented awareness' that might be cynically viewed as a postmodern counterpart to some romantic or mythical past of 'pure' non-stratified relation to place, my interest here in the work is rather that it problematises the habitual acts of walking and engaging with the environment. In this, Compressionism demands that the participant's body seek out new intensive and extensive minor relational potential. From this perspective Compressionism can be viewed as a procedure to 'escape or "reenter" habitual patterns of action' in order to reinvigorate our attention to these processes of contraction (Arakawa and Gins 2002, 62), to explore alternative routes, reinvent both processes and outcomes, and to embody a minor practice.170 #### Making the world/performing space For de Certeau, walking through the streets recreates the city as more than a fixed 'geometrical or geographical space of visual, panoptic or theoretical constructions' (1988, 93).171 The immanent movements and 'tactics' of everyday life produce a relational, contingent experience. In 'walking the city', de Certeau examines ways that deterritorialisation of spatial order is enabled through the act of walking, and the positive personal and social implications of these movements (1988, vii). This is positioned as a 'tactic' that destabilises, a fragmentary insinuation into place to reappropriate it 'without taking over in its entirety' (de Certeau 1988, xiv). It is a destabilisation that does not necessarily impose new order, remaining immanent and essentially per-formed rather than a preformed strategy (de Certeau 1988, xix, xx). In walking's immanent recomposition of static place as 'vectors of direction, velocities, time variables…intersections of mobile elements' (de Certeau 1988, 117), it molecularises or reenergises these territorialised 'places' (de Certeau 1988, 117.).172 As Tim Ingold notes, in such a conception of walking and the space itself, both lack any real 'essence' or idealised form, but rather the act of movement explores emergent differential capacities held between bodies and environment (2011, 49, 24). Here de Certeau sets up a clear distinction between an abstracted and disembodied or distanced concept of a space (such as the view of a city from the heights of a skyscraper) (92), and the embodied interactions within such spaces that movement enables. A number of other authors have explored these distinctions, if not always utilising the terms in the same way.173 James J Gibson argues that there is no abstract space or time that is then 'filled in' by lived experience, but rather a 'flow of ecological events' that are heterogeneous and differentiated (1979, 93). Similarly, Ingold describes the emergent space of movement as 'unclassifiable', consisting not of stable and inherent properties, but a dynamic relational field of 'meshwork' that is always 'under construction', reproduced and reinvented (rather than replicated) through movement (2011, 159–60).174 Manuel DeLanda makes a clear distinction between such 'meshworks' and 'state' structuring of space that is useful here as it avoids the naïve concept that a deterritorialisation of space is either sustainable or necessarily positive. Rather, he defines the latter as a centrally organised and rigidly controlled space, and the former as a 'bottom up' approach to the organisation of space that consists of complex, intertwined heterogeneous elements largely self-organising (DeLanda 2011, 257–274 and passim). Indeed, as Ingold notes, any expanded concept of space could be misconstrued to still imply a static order that positions livable places inside of static space. This would, he argues, retain the concept of living as bound to the landscape rather than positing them as existing 'through, around, to and from [places], from and to places elsewhere' (Ingold 2011, 148). Movement for Ingold, after Deleuze and Guattari, is not a connection between points or places, but always runs between: an uncontainable line of flight breaking boundaries (2011, 83). The point here is the need to replace or reinvigorate the imposed structuring with potential for greater novelty and interrelatedness (to molecularise). This is the potential that the tactic of walking proposes in the city space for de Certeau. Michelle Lamant comments that de Certeau's tactics allow pedestrians to 'create for themselves a sphere of autonomous action within the constraints that are imposed on them' (1987, 720). The walker, she argues, reconfigures the impersonal, visible and knowable space of the city streets through minor methods born of creativity rather than passive or active resistance (Lamant 1987, 720), replacing the productive and pre-structured place with an improvisational experience that operates inside the established systems. Of interest here is not the problematic and romantic return to the flâneur, as de Certeau's argument can be read.175 Rather, it is that de Certeau's walker reactivates their relationship to a space by emphasising the reconfiguration of relations out of existing entities, and the continual differential action of movement that keeps these relations at this point of splitting, rejoining and re-layering. Walking invites an intimacy and active engagement with the singularities composing an experience that enriches the homogenising actions of a place. The streets we navigate or describe through remembered movements and sensations might perhaps disrupt any idea of an absolute organisation of space with our shifting experience over time. Instead, as de Certeau says, they become a 'story, jerrybuilt out of elements' that is both 'allusive and fragmentary' (1988, 102),176 layering and splitting the existing structure, filling the streets with 'forests' of 'desires and goals' (1988, xxi) to make the world habitable. An 'in-between' is created that allows a movement, a flow of forces, bodies and affects. In walking, the experience of the city is always an intimate and shifting engagement, as feet selectively prehend the qualities rather essential properties of the street. The street is 'a course cloth of patchwork woven from the comings and goings of its manifold inhabitants' (Ingold 2011, 16), that splits the homogenising actions of the city through a continued gathering of singular ecologies of feelings. It is an immediate engagement with materiality, a creative coming together of surfaces. Mobility here activates the productive potential of life, giving it 'it its seemingly infinite range of specific virtual and actual individuations' (Murphie 2005a, 1). Thus walking becomes a technique of differentiation that extends and complicates, positioned as a creative derivation from that which is already in existence (Deleuze 1994, xx). It is a positive parasitism that is 'molecular' in allowing new communication or composition in the spaces between components (Deleuze and Guattari 1986, 41). #### Differentiating the body While walking can disrupt and reconfigure relations to space, Manning argues that it can also work to differentiate bodies through movement, allowing exploration of new potential intensive connections. Imagine that you are standing stationary in a doorway, about to walk out. Except that 'stillness' undermines itself: you are already always moving in two important ways (Manning 1996, 43–7). Firstly in a literal and physical sense, the body is always in a state of intensive micro-movement. Heart, lungs, eyelids, and eyes are the more obvious aspect of this, even though for the most part they operate below an overtly conscious, willful level. There are also the efforts of the muscles as they continue to exert force in opposition to gravity to keep one upright, and as the body performs constant micro movements and adjustments to keep balanced. The relatively still body, Manning states, is in fact a series of 'micro-postures that move in tandem with the rejigging of micro-movements' (1996, 44), perceptual disruption and differentiation.177 Here, one could argue, the body is always in a process of perceptually differentiating, in that it has its own differential machines – technics – built into the sensory distributions of the body. These operate in the interval – the differential. Again it is this gap between – a qualitative intensity – that is meaningful: the felt experience between the data processed from one ear/eye/nostril/foot and another, a held contrast before a relation.178 Movement here activates the continuous streams of noise that are perceptual differentials, and this 'perception/action continuum' of differentiation is emergent with movement, intrinsically composed of and with such movement (Murphie 2005a, 6).179 Secondly, the continuous gathering and incipient pull of the virtual also undermines stillness. As you are about to begin, Manning proposes, milieus of virtual possibilities are composing themselves, creating tensions, an 'elasticity' that is released as the possibilities resolve into an actual movement. The choices are not infinite, in that not everything is physically possible, but are limitless in that they are being endlessly created, and each choice generates another equally complex series of choices. They resolve in the satisfaction of an actual event (your left foot takes a small step straight ahead), and all the virtual movements perish. This event 'propels the preacceleration of a new occasion' (Manning 1996, 38–9). New sets of virtualities begin composing possibilities for the next step, shaped by many things, such as the limits of body, habits, responses to the space, and so on, and it is movement that both generates and selects from the potential actions. Movement here, Manning says, cuts across the body (2013a, 46), connecting and disrupting the actualised body's relation to its larger potential, which is always also reconstituted by the activity. It is a technique by which a body accomplishes the shifting beyond itself of ongoing individuation. This evolving potential for new connections is a minor 'flight' from stasis, a flight that is not an escape from oneself, but an increase in intensity, or richness of potential (Deleuze and Guattari 1986, 13). #### Landing sites: worlding the body Walking the space of the city is never without constraints: proposing and conditioning movement, the body's projection and diffusion into space. Environments provide conditions – platforms of potential actions – that affect the actions of the walker. As Massumi argues, a park bench, for example, creates anticipation of a certain habitual action (sitting), and in this way works to order the movement in the space. The bench is a 'storage of repose' that creates suggestions of actions. While one could sit on the ground or stand on the bench, Massumi argues that the image of the bench creates anticipation of a certain habitual action (bench-equals-sitting), and in this way works to order the movement in the space (2003b, 5).180 These conditions can enable as much as they constrain, proposing new actions. Massumi relates this both to Gibson's concept of 'affordances' and Arakawa and Gins' concept of 'landing sites'. Affordances are what the environment 'offers' or 'provides or furnishes' an animal (Gibson 1979, 119). Affordances are about productive relationships. What is prehended is not the object as such but the relational or interactive potential between objects and bodies (Gibson 1979, 126). That is, affordances are perceived potential machinic couplings-through-movement. Affordances, Gibson writes, are not neutral or abstract but complementary and specific to an individual animal's tendencies. An affordance 'points two ways, to the environment and the observer' (Gibson 1979, 132). They are a prehension of the potential of the body as much as of the environment (Gibson 1979, 132). Affordances then exist and interact with each other in complex and nested sets or 'niches' that are ecologies in and of themselves, whereby 'the niche implies a kind of animal, and the animal implies a kind of niche' (Gibson 1979, 120).181 Such affordance are propositions, 'lures towards feelings' (Whitehead 1978, 259), constructing potential from which events can draw. For example, a patch of grass might afford many responses from the walker: a place to lie down, the danger of snakes in summer, wetness to be avoided after rain, the smell of the countryside, an opportunity to sit and talk, and so on. These propositions potentially operate on multiple levels – sensorial (softness underfoot/wetness/smells), affectual (inviting tiredness and an urge to rest, fear of hidden danger, joy of free space to play), kinesthetic (sitting, lying, running, walking), and social (conversation, solitary contemplation). The conditions of the space do not necessarily impose a habitual bodily response; rather, they can lure a range of potential actions into being, triggered by common constraints (Gibson 1979, 119). These constraints are immanently performed by the body-incomposition as it walks. The ground, for example, is an 'enabling constraint' of movement intrinsically related to the form and practice of walking (Manning 2009, 70),182 as gravity plays a role in shaping some movements (exertion increasing up a steep hill) as much as it precludes others (leaping walls), wrapping the feet into sensorial relationship with surface textures and resistances of various materials underfoot. This active making of movementbody-space is not limited to, nor even primarily located in, conscious mental activity, as propositions 'are not primarily for belief, but for feeling at the physical level of unconsciousness' (Whitehead 1978, 186). Conscious – mindful183 – and preconscious movements are capable of both the habitual and improvisatory. Certain activities and spaces more forcefully and productively disrupt habits by requiring an active and attentive care that brings to the fore the processes of connection and projection into the world. The urgency of movement and the complex negotiations required to enter or exit a peak hour train, for example, both instinctively causes one to edge into a gap between bodies that affords passage and brings to our consciousness the continual negotiations and collective reconfiguring of space required by moving in the city. We must calculate who will allow passage, who must be edged around, intuiting minute adjustments of tempo and posture to keep a free space ahead. Positional information comes at the body from all directions as we compose a provisional line through the chaos. With every step, the space available and the potential for the next move shift, and both body and path must always be renegotiated, making premeditated paths redundant. It is in such moments of intensely improvised movement that the space and body might together begin to approach a contingent, immanent quality.184 Such an encounter with the city is far from the free and idle wandering of the flâneur; it is a series of conversations between competing forces and potentials affecting both the configuration of the space and the composition of the body (Manning 2009, 15). Arakawa and Gins concept of 'landing sites' provides a useful refining of affordances (2002, 7). This is a process of 'portioning out' space to provisionally deposit sited awareness around the body (Arakawa and Gins 2002, 5). The body, they state, takes cues from the environment to 'assign volume and a host of other particulars to the world' (Arakawa and Gins 2002, 7, 9). These sites are a way that the body contributes to and distributes itself into the world: a 'holding of the world' in attention, an attention with dispersed foci composed of all perception – 'a bit of substance, a segment of atmosphere, an audible anything, a whiff of something, whatever someone notices' (Arakawa and Gins 2002, 81). Landing sites are a process by which differentiation of the field occurs, to different degrees of specification and diffusion.185 This, Arakawa and Gins argue, is a process by which, perceptually and kinesthetically, the world and body are immanently enfolded. In this sense, the body not only differentiates the space through movement, but also distributes itself within the space, contributing its awareness towards things in the world.186 Processes of landing sites productively disrupt the limits of the body, constructing through dispersion a new extended and enriched potential bodying. These projected landing sites fold, nest, diffuse and focus dynamically while the body moves. It is a constant, creative, noisy process of splitting stable relations.187 Returning to the space of the peak-hour train, where spatial relations shift quickly, this process by which the space-bodymovement relations enfold the body and object/world into shared individuations becomes more consciously attended.188 Entering the train carriage with one's own particular style of moving189, we begin to create landing sites. We distribute awareness on both the more physically concrete (arrangements of bodies and objects), and on more vague and diffuse levels, such as the ephemeral (reflections of light on surfaces or affectual tonalities). A change in height or texture underfoot as we enter creates a foot-floor site, a commuter's headphones or conversation sites attention vaguely in one direction, the line of bodies exiting the train deposits attention towards this flow. The vacant seat in front of us concentrates attention not only on the object itself and the seat/body kinesthetic potential (stopping, sitting, a virtual becoming-with of seat/body that makes the seat also part body and body part seat), but also on the kinesthetic possibilities of surrounding floor space (the potential of moving to or beyond the seat). Landing sites thus move through, over, around, and inside other landing sites, each divisible into smaller sites, continually complicating relations as the body moves and redistributes itself in the environment. The point we are pressed against other bodies in the train carriage becomes a shared site of focused attention,190 located within a general awareness of the other passengers. As we move through the space, the sites make such navigation possible, and begin to propose relational and kinesthetic possibilities. The landing site on the exit opposite not only creates another site of attention, but also wraps both body and door in potential future kinesthetic relation (an exit from the train). These landing sites are in-the-making – as Manning says, a 'tending towards relation' (2013a, 12). This again is a process of becoming-minor, a decentering through movements that recombine components of an event (Deleuze and Guattari1986, 50) and create new intensities through prehensive selection. The act of depositing landing sites agitates or molecularises boundaries between body and world – destabilising distinctions through the creation of shared potential collective individuation. In the theories of both affordances and landing sites, vision is construed as haptic and kinesthetic, and this is far from the role de Certeau assigns to vision as inextricably linked to power.191 Both Gibson and Arakawa and Gins suggest that it has other potential operations of an enactive and synesthetic nature, with, as James Gibson states, 'the optic array…not only provid[ing] base information but also the possibilities for action on the basis of that information' (Gibson cited in Mock 2009, 96). 'Ecological' vision, as Gibson describes it, is not about a distancing through the reduction of the world to retinal images, but an ongoing engagement with the world (1979, 61, 244–6, passim). Here vision is a perceptual system, involving 'eye-headbrain-body' rather than passively received messages to be decoded by the brain. Thus for Gibson, vision begins not with the head fixed and the eye exposed to a series of snapshots 'like a camera', but with 'the flowing array of the observer who walks from one vista to another, moves around an object of interest, and can approach it for scrutiny' (1979, 290). Ecological vision is sensed throughout the integrated sensations of muscles and body structure and through the movement within the environment (an 'ambulatory vision') as an embodied, lived experience, not the translation of stimuli (Gibson 1979, 291–2). Manning also elaborates a synesthetic operation of vision that is part of a co-mingling of the various senses that themselves are linked to movement and also kinesthetic (2009, 49). As we move towards some landmark – a tree for example – vision operates not just to recognise the image of a tree, but also proprioceptively to create the feeling of self within the space (Manning 2009, 49).192 This we might think of as a landing site that has been deposited, situating part of the body at the landmark ahead. As we move, we see continual variation in image of the tree – parts come into the field of vision or disappear, become larger or smaller, so that our eyes as they move across the tree might act not as 'a capturing of the world, but a captivating by it' (Manning 2009, 86).193 Furthermore, as Gibson also notes, an essential component of vision is proprioceptive, involving a registering of 'movements of the body as much as does the muscle-joint-skin system and the inner ear system' (1979, 175).194 #### Compressionism Transdisciplinary artist Nathaniel Stern's ongoing Compressionism performances (2005–)195 comprise a customised, scanner-battery pack-laptop assemblage worn or carried by one participant, while she or another person holds and moves the scanner surface across objects to 'perform images into existence' (Stern 2013b) through a kind of shared seeing-moving within an environment. These scans are literally a 'compression' of the temporal act into a two-dimensional image (see Figure 5.2), seeking, as Stern says, to 'accent the relationships between the performance, myself, my subjects and the tools' (Stern 2013a). What does the performance of Compressionism add to the already dynamic becomings of the moving body in space, or, rather, how does it reinvent and re-molecularise these processes, doubling them with new levels of awareness? Compressionism, I want to argue, does not alter being, but the manner of being (Guattari 1995a, 109): it creatively performs the body (and space) in a new way, not to return it to an imagined pre-stratified form, nor to replace previous space-body modulations, but to enfold it with existing relations. The work here challenges habits, provoking participants to intuit new minor ways of being. #### Compressing the city Performing Compressionism was an awkward act. The size and weight of the scanner required that it be held in both hands away from the body, with feet braced to maintain balance. This created a tension running through the body, stretching toward objects to be scanned. Keeping the scanner steady required a clumsy cooperation between both scanner and bodyweight as counterbalance, and also between the holder of the scanner and the person carrying the battery pack and laptop capturing the image (see Figure 5.1). There was a zone of intimacy established, both between the collaborating bodies and between the scanner-body assemblage and the objects being scanned. Scanner, body and space all conjoined through the act of moving. Compressionism here involved a close investigative walking – through back alleys, parks, along surfaces of objects, architecture and bodies. It was an exploration of texture, colour and contrast, held together by the collective movement of the bodies-scanner machine. The intensive, explorative, close-visioning movement in the city enacted through the 'Compressionist' event was remembered through the personal, composed from actions, disjunctions and sensations. One's experience of the event was composed of particular colours, surface textures and variations. The colour of a particular leaf, the textural shifts on a building surface, the passage from tree to wall to doorway, the incidental sounds heard while waiting for the scanner to warm up, the effort of a particular stretching of the body – each of these coloured one's experience of the event. It was a fragmentary mapping of a space – a haptic or closely focused narration of layering intimate, personal actions onto the surface of the city space. The haptic here showed its potential in bringing attention not just to the surface of the object, but also in its engagement with multiple sensations, and with participants' interior/exterior boundaries (Marks, cited Jones 2006, 143). Figure 5.1 Compressionism Documentation, Montreal, 2012. Digital photograph. Photo: Bianca Scliar Participants performing the scans improvised new literal connective passages that opened gaps between systems of Figure 5.2 Nathaniel Stern, Compressionism Scan, Montreal, 2012. Courtesy the artist. Digital Image. place.196 The scanning travelled up walls or through holes, over horizontal, vertical and angled surfaces, backtracking to points of interest – inviting and improvising new affectual connections as much as the equipment's awkwardness precluded usual styles of movement through the space. The space scanned was understood as a series of overlapping and angled surfaces, as the scanner was moved parallel to these surfaces, emphasising their shifts as the body coordinated the changes in angle – a point of 'touch' between body and environment.197 Space was understood not through a stable image or representation, but through practice of engagement: a dynamic expression of the relationships between moving bodies and environment that was felt through the meshing of forces (rhythm, tempo, shifts and variations) (Ingold 2011, 211). Compressionism's movements insinuated into the city the experience of a 'plurality of centres, a superposition of perspectives, a tangle of points of view' (Deleuze 1994, 56). #### Dancing objects As participants slowly moved the scanner over the surface of an object, these actions were translated into a larger movement of the hands and arms – creating an awareness of contours and small deviations that was heightened by the fact that the object itself was always at least partially obscured from view by the scanner. This was a blind, groping approximation of the shape that was performed: a scramble of image memory, a drawing of the shape with the hands, a constant reforming of posture and balance, an attention to the sound of the scanner's processes that resonated with the rhythms of bodies moving. Each object invited potential movements in relation to its form. For the minute or two before the laptop compressed the data into a viewable image, the event existed on its own as an awkward dancing of the object, an approximation of vision performed by a loose assemblage of other senses, drawn together by movement. Compressionism afforded new connections between senses through movement. Vision became situated 'along the tendons and the muscles' (Serres ND, unpaginated), and the event approximated a new eye-organ out of hands/feet/balance.198 What would normally be felt as the small-scale movement of the eyes traversing an object was explicitly performed (danced) as a full body movement, and brought to attention through this shift in registers. The body-scanner assemblage performed sight, inscribing it in space.199 Here the primary link between an ecological perception and movement was made evident to the participants – an engagement in an emergent environment that demanded time spent along a 'path of observation' (Ingold 2011, 46) rather than an assessment from a fixed point. This embracing of the scanning/visioning technics was a minor tactic in that it consisted of 'adding to' and 'perverting' habitual configurations of sense organs to increase the intensity of felt experience (Deleuze and Guattari 1986, 10). The Compressionism event deposited a series of (mobile) landing sites in addition to those that walking the space might normally require. Attention landed on the new and mobile collaborations of sensory input informing the movements (a shared sensory experience residing between and linking body/scanner/ world) that caused unexpected intensified conjunctions, and cooperation between surfaces beyond their usual functioning (Deleuze cited in Grosz 2008, 198). Part of the conscious attention landed on the held scanner, as the mechanics of holding and operating the equipment forced new improvisation of relations and landing sites in the muscles of the hands and arms, in the feet maintaining balance – depositing more defined sites of attentiveness onto the surface, gradient and texture of the ground. Less qualified sites were also deposited in the vague attention given to those carrying the rest of the equipment, and to the space around the object or surface being scanned. The more defined and useful landing sites were in the mobile spaces between object and scanner surfaces, while the unseen object itself remained a more generalised 'imaging' landing site, in Arakawa and Gins' terms, nesting within the particular, while resisting definition. In these ongoing differentiations Compressionism perhaps molecularised and multiplied local connections through a splitting and re-siting of attention (Deleuze and Guattari 1986, 50, 37). Compressionism might be seen to address a heightened awareness of, and engagement with, the processes of the virtual in two ways. Firstly, it literally created new potential that the assemblage's heterogeneous component parts did not hold on their own – such as new capacities for seeing, new postural explorations, and new prehensive potential to trigger actualisations. Secondly, through continued disruption of any developing habit, it promoted a suspension in its own continued unfolding that made the ongoing individuations perceptually felt. Here the assembling of body and scanner equipment provided new levels of potential intensive sensory difference, for example: the rhythms of the scanner head moving that the body attempted to follow, but never quite duplicated; the new decentering weight pulling on bodies that had to be resisted or followed; and new restrictions on the range of movements of the limbs. All these factors created tensions and difficulties. The technological components were not specifically the producers of these new relations, but were a technique to activate the conditions under which bodies began to explore minor 'sideways and decentered movements' (Deleuze and Guattari 1986, 50). #### Conclusion What Compressionism produced as its primary outcome were new expressions of movement – new improvisational collaborations between bodies/scanner/objects /surfaces/space that reconstituted each as enactive and extensively relational, both collective and singular (Manning 2009, 22). The event demanded an augmented or composite awareness, larger than that of the body on its own and prior to the event, reconstructing the body's field of sensitivities, and requiring new cooperation between bodies, equipment and space. In thinking more generally of the potentially generative role of movement in interactive art, what might Compressionism have to offer? Firstly, while many interactive works involve movement as a key factor in participation, in this instance, I would suggest, there is never a sense that it is a functional movement designed simply to link pre-defined capacities of bodies and objects (handto-joystick, gesture-to-sensor). Rather mobile capacities might begin to emerge as 'properties of systems of relations' (Ingold 2011, 49) that continue to differentiate from actual conditions in a lively relation with the virtual. In developing this type of emergent relation, as Massumi comments, a work might attain a dynamic co-causality that is often missing in the triggers of sensor-based works that instrumentalise movement (2011, 45–6). Secondly, unlike many interactive works that employ everyday and unthinking movements, Compressionism foregrounds these habitual gestures through the awkwardness of the constraints of the mechanism, the disruptions to the visual perceptual system, and the delays between gesture and its representation in the final scanned image. While walking, as both de Certeau and Manning argue, might already allow a body to exercise potential to produce minor iterations of streets, bodies and their relations, here such activity is made strange from within at the same time as its relationship to the surfaces of the environment is troubled. Perhaps then the key to this artwork's capacity to activate minor potential lies in its problematisation of any mastery of conditions or movement, creating awkwardness in the negotiations between limbs, bodies and space that made the performers strangers within their own movement capacities.200 Compressionism might then be seen as neither an attempt to return to pre-stratified states, nor as some new prosthetic melding of bodies and technologies to take us beyond the limits of the biological, but as a technique for bodies to disorganise their own forms in order to experiment with new expressions of relations (Massumi 2011, 28). If the 'minor' is concerned not with outcomes but enabling the conditions for new connections to arise (Massumi 2011, 18), then this artwork suggests that the role the technological component of the work plays might be less about creating new relations itself, than with disrupting habit and turning the body's attention to the capacity of movement to gather bodies into emergent and dynamic new ecologies. #### Bridge: Psychopomp In Psychopomp, 201 two performers moved around a darkened space inside two costumes that generated internal light and sounds that played through four speakers arranged around the edges of the space. The costumes worn during the performance were embedded with sensors so that movement, contact and pressure and shifts in posture generated the soundscape and caused coloured LEDs in the costumes to operate (see Figure 5.3). Each individual's actions had the potential to affect the lights in both costumes and to displace sound samples triggered by their movements. The headpieces of the outfits curtailed participants' vision, so that they could only make out bright spots of light, thus they were more reliant than usual on touch and hearing to navigate the space. Their ability to fix stable positions was complicated by the disruptive actions of their movements, which triggered changes in sounds and shifts in the locations of sounds from one speaker to another. In addition, the lights they could see in the costumes altered in response to both body movements and the volumes of sound from various speakers. Navigation further complicated by the weight, volume, and soft texture of the new 'skin' wrapping their bodies, which made tactile sensations vague and somewhat alien. All this created a scenario in which movement was necessary as a means to any level of cognition in body–body and body–space relations, yet movement simultaneously kept these relations highly mobile and caught in a web of co-causality. Here the primary role of movement in understanding both the space of performance and the capacities of bodies was made evident. Stripped of any possibility to overview and quickly grasp the space, each step consisted of a tentative reconstruction as participants tested their new capacities to interact and relate to sounds, lights, surfaces and bodies (both their own, newly made strange and the other participant's body as a potential site of connection). With this reduced vision and unreliable hearing, participants were forced to turn attention to new and mobile collaborations of sensory input that distributed prehension throughout the body and space. This was an imaging that was in the service of, and serviced by, a synaesthetic coalescence of sensations – touch, hearing, balance, temperature of another body (the body as a perceptual system) (Gibson 1979, 61) – that were cobbled together as a workable alliance. Peripheral sensations were brought to attention by bodies attempting to make connections: perceptions composed of the intensities held between contrasting surfaces.202 This cooperation between surfaces beyond their usual functioning caused unexpected and intensified conjunctions to arise – an arm pressed against the weight of a back, a foot cautiously feeling out the terrain underfoot, the slight vibrational ripple and noise of costumes brushing lightly past each other, all became central to any comprehension of spatialisation and the boundaries of the performers' own bodies. As bodies reached out, groping in darkness for certainty, they battled with the problematics of their new clumsy relation to the field. With such compromised and unstable sensory input, affordances became more transient and slippery. Landing sites could be tentatively projected here – onto the new augmented surfaces of the body, the spots of light perceived on the other performer's costume, a particular sound emanating from a speaker, a shared site between foot and floor, and so on – distributing attention onto the surface of the body, the collaborator, and into the space. But these alliances quickly dissipated as the conditions continued to shift. In this way, senses cautiously turned out to these edges in an unresolvable searching for a stable point of location, an attention to these new shared but fuzzy spaces between body, costume and world: an attunement to the collective event in its unfolding. Such tentativeness might be a suspension in the gathering of the event, an emergence of form, or, perhaps even less definite, an emergence of the conditions for form to begin to arise. Perhaps it was the inability to filter or prioritise sense information – to order and stabilise the field of experience – rather than a lack of information, which caught participants in a looping state of 'always just beginning' to make sense of world. The flooding with sensation of something not yet comprehensible is described by Manning as the 'activation in the here-now of the notyet' (2013a 179) – a tuning towards and slowing down of the process of 'parsing the object from the field' (2013a, 277).203 This disruption to the usual processes of perception separated causal comprehension from the richness of undifferentiated sensual immersion. It was a stretching of perception that provoked, as Manning has written of such experiences, an encounter with the shaping of the 'more than' of the event (2013a, 179), of the crystal point at which the actual and its larger potential begins to split, and the pull or lure of the virtual can be felt. This tentativeness might approach what Arakawa and Gins have termed a 'biotopological thinking', encouraging an attention to the field, as much as to the body proper (2006, 60). Such Figure 5.3 Andrew Goodman, Psychopomp Costume documentation. 2012. Digital photograph. thinking they describe as a 'self-diagraming', a coordinating of one's world that portions spatial relations both approximately (as it was always evolving), and at the same time rigorously (as it was intensely relational across multiple scales of engagement) (Arakawa and Gins 2006, 73–4). Psychopomp accentuated a felt quality of 'not knowing' – not quite knowing what delineated one's boundaries anymore, where either oneself or the other performer were positioned in the space, where a sound emanated from, how movement translated into sound events. This might be viewed not as a 'lack' as such, but, as Stengers notes, a 'characterization of a mode of working' (Stengers 2011, 286) that foregrounded the multiplicitous nature of the point of actual/virtual at which bodies moved. The 'not knowing' was a parasite within the knowable – the alreadyformed relation, the stable object of representation – disrupting and advancing through differentials with which movement problematised and molecularised the body. Not knowing was here commissioned as a tactic of production, positioning bodies at the 'edge of virtuality' (Manning 2009, 35) that movement then stretched out. In this it was perhaps a system 'advanc[ing] through problems and not through victories, through failures and rectifications rather than by surpassing' (Serres and Latour 2011, 188); a system charged with new indeterminacy. It required a new in-process attention that drew the creative and ecological processes of 'worlding' and bodying that are always occurring, bringing the gathering of relation to a perceptible level. ### 6 ### Entertaining the environment #### Introduction In the late eighteenth century, the Abbé Nollet created entertainment by passing electric current from a Leyden jar (an early battery prototype) through a line of 300 Carthusian monks holding hands, causing them to simultaneously jump in the air (Elsenaar and Scha 2002, 19). This was one of a series of early experiments exploring a fascination with this newly discovered force in the world, capable of passing through and rearranging subjects and objects. Such works demonstrated a shift in positioning the human and the environment: an enthusiasm for exploration of a distinctly non-human agency active in a lively world of forces, and an entrancement with the capability of such forces to traverse and reorganise human body potential into a decidedly 'post-human collective body/assemblage' (Goodman and Manning 2012, 2). Erin Manning has proposed 'entraining' and 'entertaining' the environment (Goodman and Manning 2012, 6)204 as a way of thinking through Alfred North Whitehead's perceptual categories of 'causal efficacy' and 'presentational immediacy' (Whitehead 1978, 310–21; 2014, passim). 'Entrainment' concerns the 'immanently relational intertwining of perception with action' (Goodman and Manning 2012, 6), and as causal efficacy can be thought of as a 'lure' towards prehension – 'call(ing) forth new immanent associations and new assemblages' (Manning 2013a, 23). 'Entertainment', on the other hand, is indifferent to these causal relationships (Whitehead 1978, 324).205 Entertainment concentrates on 'the direct perception of the fielding of experience such that it brings its qualitative resonances to the fore' (Goodman and Manning 2012. 1). It centres on the felt quality of the experience of the activities of the field organising itself, rather than on the resulting objects or subjects. 'Entertainment' is resolutely concerned with the immediate activities of the field or environment and the collective individuations of an event that might arise. Perception, Whitehead states, 'is the catching of a universal quality in a particular substance' (1978, 158). Here perception moves beyond mere feeling, subjectively 'rooting' the 'blind' and 'vague' feeling in the 'immediacy of the present occasion' (Whitehead 1978, 163). For Whitehead, as Jones states, it is the bringing together of the perceptual components of causal efficacy and presentational immediacy as the display for the subject of 'an extended environment contemporary to the percipient', that allows for comprehension through symbolic reference of 'the way in which causally connected organisms in temporalised relationship…are apprehended in genuine community in the present moment' (1998, 151, emphasis in the original).206 That is, the two aspects of perception 'intersect' to provide sufficient understanding of 'a contemporary world of extended actual things', which, Whitehead argues contra to Hume, presentational immediacy cannot provide on its own (Whitehead 2014, unpaginated, II: 5, II: 1).207 In this 'intersection' of the two factors intensity is achieved through subjective patterning, in that there is a contrast felt between the perception of the moment and comprehension of what has come before it, and that there is a contrast between what is in the moment and prehension of that which has potential to come to be (Whitehead 2014, unpaginated, II: 4). In thinking through Whitehead's dissection of acts of perception into these two categories there are three points that I would argue are worth noting. Firstly, that the two components are essential to perception and are never fully separable. Nor, despite the term presentational 'immediacy', does one necessarily precede the other in a clear linear sense (Whitehead 2014, unpaginated, I: 8). Secondly, perception in this sense does not necessarily mean conscious perception, though it certainly is a factor for, as Whitehead terms them, the 'higher grade' organisms. These organisms have greater access to presentational immediacy and therefore have a greater capacity to condition the causal information prehended from the environment (Whitehead 2014, unpaginated, I: 8).208 Thirdly, we must remember that for Whitehead perception is never the passive imaging of an established environment by a subject, but an act of self-production through the prehension of different components of actual and virtual datum and their constitution into the organism's concrescence (Whitehead 2014, unpaginated, I: 6). Isabelle Stengers explains causal efficacy as a construction of chains of cause and effect, often based on prior knowledge or habitual response to sense data (2011, 401). This is a succinct description, in line with Whitehead's own initial description of causal efficacy as a subjective perception of the relation between the organism perceiving and relevant concurrent and precedent events.209 But it also something of a simplification of the greater potential of causal efficacy, which more expansively also places events within a temporal and spatial relational patterning. This is not concerned with notions of time as 'pure succession', but the 'concrete' relational time marking the passages from events to events (the objectification of events that 'establish the conditions' to which subsequent events conform) (Whitehead 2014, unpaginated, II: 1). Similarly, causal efficacy is not a relationship to abstract space, but, in defining actual geometrical relationships to the environment, causal efficacy explicates another aspect of subject–event relations, and importantly is also a perception of the intertwining of the body with the environment, as we 'see with our eyes, we taste with our palates, we touch with our hands' (Whitehead 1978, 170). Through these aspects, causal efficacy provides not only a grounding of the body in the space and time of the past, but 'a sense of the implications…of the present on the future' (Whitehead 2014, unpaginated, II: 4). For Whitehead causal efficacy belongs 'to the fundamental constitution of an occasion', and is therefore available in some form 'even to organisms of the lowest grade', whilst presentational immediacy is a 'more sophisticated activity' and available only to 'organisms of a relatively high grade' (1978, 172)210. Presentational immediacy is 'our immediate perception of the contemporary external world', and the knowledge it provides is, Whitehead says, 'vivid, precise and barren' (2014, I: 12). That is, whereas much if not all of the information gleaned from causal efficacy is vague and trivial, in the qualitative force of the immediate sensation we find both the precision and directness needed for a deeper perception. On its own however (if such a thing is truly possible), presentational immediacy remains 'barren' or disconnected from the full realisation of the occasion because these qualitative factors are not linked with intrinsic characteristics of that which is prehended until combined with causal efficacy (Whitehead 2014, unpaginated, I: 12).211 As Whitehead notes, it is hard to imagine, at least for a human organism, a situation in which one might experience presentational immediacy on its own. Firstly this is because 'the present fact is luminously the outcome from its predecessors, one quarter of a second ago', at the very least in terms of having laid the conditions for events, however surprising, to arise from (Whitehead 2014, unpaginated, II: 4). Secondly, the very act of sensing provides some spatial information, as there is a spatial relationship established between, for example, a sound and the ears that hear this sound (Whitehead 2014, unpaginated, I: 12, II: 6). Even in a moment of blind anger, he reasons, 'it is the man we hate' – a 'causal and efficacious' object, and 'not a collection of sense data' (Whitehead 2014, unpaginated, II: 4), and thus not blind to the way actual things are at all. However, perhaps one might occasionally experience a moment in which immediate and unqualified sensation briefly overwhelms the causal,212 and it is one such experience in an artwork that this chapter examines. Art events, like all other events of perception, necessarily contain causal efficacy and presentational immediacy to various degrees. However, as Massumi has articulated, interactive artworks have tended to overshadow direct experience in their insistence on demonstrating and fixing relational connections, foregrounding 'causal efficacy, instrumentality, [and] affordance' at the expense of their 'own artistic dimension' (2008, 7–8)213. This, Massumi says, is 'why you so often hear the comment from participants that [interactivity] feels like a video game' (2008, 8). Massumi argues that this reduces and contains relation in problematic and prescriptive ways as representational (2008, 8–10). Here it again becomes evident that a call to a 'relational' turn in interactive art is not enough on its own, without a careful consideration of the types and qualities of relation, and particularly an intention to encourage open-ended relational pulls towards the future rather than reinforcing existing conditions. The question of how to foreground the felt qualities and intensities of an interaction over causal comprehension is therefore a pertinent one for interactivity – the kind that wishes to step beyond the representation of existing relation toward an experience of its felt emergence. While an emergent awareness of the processes by which causal efficacy folds into presentational immediacy does provide, as Whitehead states, a sense of the 'withness of the body (as) an ever present' (1978, 312), here, as a means to immerse within the immediacy of sensation of the event, I propose disruptions to the qualifications and validations of sensation that causal efficacy provides214. This parasitic disruption is examined through Lygia Clark's propositional artwork Caminhando, where the lack of causal comprehension within the work disrupts habitual perceptive processes and instead works to activate a felt resonance with environmental fields. This is produced through processes of transduction, bringing a new engagement with other entities in the environment and felt implication in a larger shared potential. This chapter attempts to 'think with' Manning's concept of entertaining the environment in order to unpack the experience of Caminhando, concentrating on its potential for the opening of the body to a wider transductive field of play, and for the production of a phasing. This phasing might be a moment of slippage, a crack through which to escape the limitations of subjectivity. The question of how to think beyond the human subject is, as Simon O'Sullivan states, not as simple as a turning away from the human. Rather, it is a becoming-minor that is 'a kind of stretching or twisting, a rupturing and stammering, a releasing of forces from within and the contact of forces that are without' (2006, 64). I relate Caminhando to a concept of an ecological ethics in that the work addresses not the representation of relation but its immanent construction. I argue that the work is ethical in that it enables an opening to further expression and connectivity. That is, it encourages an increased ability to recognise and respond to the force of other components of the event (to affect and be affected).215 #### From agency to transduction As discussed in Chapter Three, the term 'agency' is problematic for a relational approach that seeks to resist collapsing back into the philosophies of substance of which Whitehead is justly critical. Once subjectivity is seen as only emergent in the act of concrescence, another, more ecologically compatible or event-based term is needed that is capable of acknowledging ongoing individuation and emphasising the ongoing and positive ingression of forces into new individuations. In Vibrant Matter, Jane Bennett addresses these issues by thinking such acts of forces as a 'distributed agency', a 'swarm of vitality at play' (2010, 31–2). We might also think it as a process of transduction by which we can understand individuation that 'operates beneath all forms [and] is inseparable from a pure ground that it brings to the surface' (Deleuze 1994, 152). It is an ongoing and, in itself, multiple process that underlies individualisation. Individuation is the 'more than of becoming' (Massumi in Manning 2013a, xi) – becomings being dephasings of ongoing field-entity relations, singular expressions or differentiations of larger ecologies of forces. Transduction then is the process by which such 'an activity sets itself in motion' at the same time as it generates 'processes of modification' (Simondon 1992, 313; Simondon 2009, 11). For Simondon, it is a way of understanding and expressing the ongoing relation of a gathering of pre-individualised forces to an individualised entity that then exists as a 'partial and relative resolution' to these internal tensions (Simondon 1992, 300), while still allowing potential for further change. Transduction describes the integration of formerly disparate things within a concrete system, the evolution of a shared associated milieu. It is how the becoming of an entity generates further unfoldings: becoming a force for further change, though not as a linear progression, but a series of overlapping, always transforming forces of differing viscosities, driving ongoing individuation. Whitehead's theory of prehension similarly describes such a process as a system of concrescence and continuity: an entity, having achieved actualisation, becomes an 'object' for other entities, potentially influencing these entities' unfolding concrescence (Whitehead 1978, 235). Thus an entity draws prehensively on every other actualised entity and the further potentials of the system, by whatever degree of separation, becoming a dynamic point in a complex ecology of relations. Here such feeling can be seen as transduction, a continual moving of force from subjective gathering to objective datum, and in such a complex and intertwined system, the transduction that triggers prehension must be seen as a vast nexus of complex forces, rather than a simple cause and effect paradigm. #### Caminhando Make yourself a trailing: you take the band of paper wrapped around a book, you cut it open, you twist it, and you glue it back together so as to produce a Mobius strip. Then you take a pair of scissors, stick one point into the surface and cut continuously along the length of the strip…When you have gone the circuit of the strip, its up to you whether to cut to the left or to the right of the cut you've already made. This idea of choice is capital. The special meaning of this experience is in the act of doing. Lygia Clark Following Caminhando's instructions creates a body-tool-object machine producing movement or an expression of connectivity rather than representation.216 The work is per-formed rather than pre-formed, opening potential for a process of collective individuation to occur – a new event of assembling between its component parts – a drawing together through the force of shared movements between hands, eyes, scissors and paper (see Figure 6.1). As Clark says, 'at the outset, the Trailing is only a potentiality' (Bois and Clark 1994, 99); the paper and the cutting are, in themselves, nothing substantial. In the end, the result seems inconsequential and leaves little trace (Clark in Suchan 2008, 6). The art exists as a moment of resonate intensity, of prehended phasing, its beauty lying in the delicate capacity to activate and foreground transduction. Process philosophy clearly views transduction as a ubiquitous event, enabling the 'drive towards novelty' in the universe that Whitehead describes. What then differentiates Caminhando from the everyday? It reveals the process of the translation of forces moving through the hands, scissors and paper, but it does not make the process 'conscious' in any articulate manner. It makes the effects of transduction felt by slowing down the process of phasing, provoking a suspension in the flow, and making evident the potentiality of the event. With the opportunity for re-construction and invention, it brings attentiveness to the environment, not as 'other', but as a collective gathering of a potential dynamic ecology. At the point where you have cut an entire loop of paper and are back to the beginning, the scissors are no longer next to the original incision, they are somehow on the other side. Sight contradicts expectation, hand/scissors contradict paper: the habitual perceptual schema is problematised and cohesion falls apart. The causal efficacy gleaned from the skin/hand sense datum leads one to expect that the cuts in the paper will match up, but this is contradicted by the presentational immediacy. It is an 'error in symbolic reference', exploited here to promote 'imaginative freedom' (Whitehead 2014, I: 10). The link between these two components of perceptual processes is felt through their failure to smoothly orchestrate. Any stable sense of fixed space instantly dissolves, briefly becoming purely relative to the movement. It is a sudden plunge into the depths of presentational immediacy – an immediacy of sensuous perception that does not yet have the 'solidarity' that its qualification by causally efficious information will provide (and thus on its own it resists division 'into delusions and notdelusions') (Whitehead 2014, I: 12). Figure 6.1 Lygia Clark, Caminhando,1964. Photo: Beto Feliciano. Courtesy of "The World of Lygia Clark" Cultural Association. #### Tentativeness This jolt shifts one out of habitual inattention and preformed assumptions, forcing a new concentration on what is going on in the moment. A similar sensation of disorientation might be experienced in the everyday when there is an unexpected loss or distortion of sense perception – such as a sudden change of auditory conditions like the disorientating effects of echoes in a tunnel, or the tactile strangeness of one's mouth after dental anesthesia. Such occurrences make the familiar world uncanny, and force improvisations with new combinations of sense information. For the sighted person, for example, sudden darkness might trouble any sense of stability of objects and their relations and boundaries, and force a temporary fluidity and experimentation as the body cobbles together some kind of workable new 'organ' to make sense of the available data. In such a space, to those habitually reliant on sight to make quick spatial decisions, the whole body surface becomes a groping hand. Skin feels the edge of an object – as a resistant force – to gain information about the object, but never really know it as a whole. An edge could as well belong to a table, as a bookcase or doorway. Nerves respond only to the immediacy of the hard flatness, reinventing the object and body in relation at the next, cautious groping forward. As Whitehead says, sense relations here become 'vague', losing spatial definition yet retaining and even amplifying the emotional tonality of the event (1978, 176). Causal efficacy becomes less distinct here, while the immediate sensory information – and its felt lack – is drawn to the fore. This process of re-gathering and reconfiguration that follows such a shift is the focus of Caminhando. Faced with a sudden loss of causal logic and a confusion of sensory data, completing the delicate task at hand requires a response to the unfoldings of the event in the present – and, indeed, to care more for what is being felt in the moment. The work demands a slowing down, a care towards the developing relationships between hands, paper and scissors, and how their potentials begin to merge and interact: sympathy with their own particular capacities.217 We are asked to pay careful attention to what is being felt: to be immersed in the feeling of a re-gathering of forces. In navigating such conditions, 'tentativeness' naturally arises, as Arakawa and Gins might say (2002, 45), as both cause and affect of a body rearranging. Such tentativeness might be thought as a feeling-out of the future potential of the event, an immersion in its goings-on. It requires that we gather what sense information we can, and backtrack from assumptions. This slowing down the shift from shaping to content allows a felt awareness of the pull of forces towards recomposition to arise (Manning 2013a, 189), feeling out the ongoing transductions of the ecology. Caminhando problematises any sense of subjective control over the event; it begins to evoke tentativeness into a simple habitual cutting action. For an art event seeking to re-energise relations to the evolving ecology, we might ask how this kind of tentativeness evokes or makes evident the momentum of future potential and its relation to the field. Caminhando enacts Manning's proposition by unlinking the processes of entertainment and entrainment (however briefly or incompletely) in order to become submerged in the flow of individuation, of the gathering and transduction of forces from the field.218 It perhaps asks us to develop a sensitivity to proceedings outside of habitual, so that the 'delicate and fragile value-realities' – those first tentative prehensions of the gathering of the ecology – do not 'die under our feet' as we march toward the already known (Jones 1998, 195). If there is an ethical need to think-with and feel-with the individuations of the non or more-than human ecology (Bennett 2010, 14), then art might have a potential role to play in engaging us in this increased attention and sensitivity towards emergent relation. #### Paper, scissors, hands As discussed in Chapter Three, for Whitehead the feelings of all entities are shared with the environment in the ingression of datum to form the entity and in the entity's gifting of itself as datum for other acts of concrescence (1978, 220). These feelings allow entities to become with their environment, if the environment itself is taken to be an event or series of enmeshed events. In Caminhando, affects pass through and initiate assemblages, new forms, and instigate new forces. The arrangement of fibers in the paper form tendencies (tearing in one direction, resisting in another way), that shapes the displacement from the hand-scissors' force. The kinesthetic tendencies of the scissoring action collect and direct the expressed pressure of muscle energies; the rhythm of vibrations of the cutting of paper is transduced by the ear and skin. Caminhando engages with not only the extension of what is perceptible to the participant, but also the dynamic negotiation between what is felt by all components of the event, and the feelings not immediately perceptible but essential to the forming of the event (Goodman and Manning 2012, 1). The event requires attention to how scissors, fingers and paper feel, to the sensitivities that form their worlds. It questions how their combined individuation – their folding into one another, their eventful assembling – creates, mixes and shapes their shared responsibility for events and further potential. In itself, this is a potential extension of interconnectedness with the larger ecology of the event. The forces instigating the unfolding individuation flowing through the entities – the event of cutting and their intertwined affectual relations, their ability to feel – form the assemblage. These flows distribute the agency, not within objects per se, but in the event itself, contradicting the animate/inanimate divide. The 'environment' here is not some stage for a theatre of operations, but the field of forces resonating with entities. Here we might say that rather than things having feelings or sensitivities to an environment, entities have types of forces that can pass through them, that can transduce them, activating phasings, and that an increase in affectual sensitivity is therefore an increase in involvement with a larger ecology. #### Multiplicity The Caminhando assemblage is more than a binary machine. It is more than a multiple; the event is a multiplicity with its own logic, a concrete system of objects and field that exists in its entirety or not at all (Deleuze and Parnet 1987, 2). This multiplicity lies in the gaps between molar opposites – between hand/scissors, body/ paper, subject/artwork – and in the transduction, the movement of forces through simultaneous individuations that pull apart the molar, making sieves of its boundaries and, in the excess of ongoing further differentiation, its shared potentiality. Such transduction integrates disparate realities into a system of relation (Simondon1992, 315). This is a relation not only of the actual, but also the virtual. Multiplicities are irreducible: the sound of the ocean, wind, fog, and flocking birds. The earth's multiplicities are 'nebulous set(s)...whose exact definition escapes us, and who's local movements are beyond observation' (Serres 1995, 103), that we are thrust into or born out of (already always re-phasing): always from the middle of things (Deleuze and Parnet 1987, 23).219 In the middle are the lines of flight (lines of 'growth and movement'), as immanent and symbiotic connections between that which is in the midst of comingto-be and the larger potential (Ingold 2011, 71, 83).220 Leaderless birds, for example, can collectively navigate so gracefully because their shared individuation brings into being not only the individual, but also an associated milieu, a collective pool of potentiality (Mitchell 2012, 73). Subjects themselves are not communicating, but rather are 'regimes of individuation that meet' (Debaise 2012, 7). Caminhando places us in the middle of the tension of events tending towards further becoming, as always in-process, a reaching towards the next. Paper, scissors, skin each become dynamic points in a system, singular expressions implicated in the modulation of a shared multiplicity. This is the agencement of the assemblage (which is also always the assembl-ing221), more than its component parts, where cause and effect are lost in concrete inter-determined, co-causal transindividuation (Manning 2013a, 24–6). The becoming-scissors of the hand, the becoming-paper of the scissors, or the becoming-cutting of all the components, are combined in their shared potential – indeterminacy that is the richness of the event. To begin to feel part of such a gathering of future potential of forces might be a lure tending towards, or giving attentive care to, the qualities of how and what emerges, towards a shared responsibility in an ecology. The power of the forming multiplicity here is that it takes us beyond the stalemate of the dichotomous, denouncing 'simultaneously the One and the many, the limitation of the One by the many and the opposition of the many to the One' (Deleuze 1994, 203). Caminhando draws attention to our shared individuation with the ecology of the event, and that our individualisation is an expression in and of this individuation that neither halts nor contradicts the latter process, but is a partial solution to an ongoing field of negotiations. Here it is made evident that we cannot have the individual without environment, that the two are points on a path of symbiotic enaction, individuation driven by transduction that is the becoming of the whole system, both the actual and the virtual with which it resonates. Assemblages in Caminhando create a shared ecology in the largest sense – a shared milieu or potential alongside a connected actuality – a system with 'internal coherence' (Simondon 1980, 40), because the enaction of the assemblage is co-causal with its field of potential: field and individual are a multiplicity. #### Tactics Clark says that, through participation, Caminhando causes the figure of the participant to 'deterritorialize itself' (cited in Martin, Ruiz and Rolnik 2000, 76). Deleuze and Guattari state that everything can have a microbrain (1994, 213), a topological system of forces for a nervous system. While Arakawa and Gins say we are organisms that 'choose to person'; such individuations are routines of expected behaviors (2002, 1–5). Implicit in Caminhando's instructions are challenges: choose something else; embrace your multiplicity, your connections with the world, the forces that exceed your body, invent procedures, tactics to free yourself, learn to 'swim' in the tentativeness that is the 'more than' of bodying (Arakawa and Gins 2002, 84) that move us beyond stable subjectivity. Arakawa and Gins' work shows how bodying makes 'landing sites', mobile points of connection penetrating the world, dispersing the body and intertwining with environment. Caminhando is such a technique for reaching into the world, transducing the body into emergent assemblages, to spark new individuations. It is a procedure that gives rise to new microbrains: in the hands-scissors, in the ears-eyes-paper, and so on. The art event here is a machine that might open up a gap in the subject. It is in this gap that moments of 'felt phasing' begin to create a flight path: an option to embrace multiplicity, to accent individuation over fixed identity. Caminhando begins to question the containment of the subject; it begins to activate an awareness of a dynamic relation both the actual environment and to the virtual, 'the combination of mutating fluxes, on their productions of speed' (Deleuze and Parnet 1987, 88). #### Conclusion: Towards a new politics This relationship to an environment is not something separate with which to engage, but is enactive: formed through collective individuations always occurring from and in the middle of other processes. This is not to say that the everyday does not contain subtle but strange occurrences when the body schema becomes momentarily confused. These are moments where causal efficacy and presentational immediacy fail to align and the body has to scramble to reassemble itself, allowing a brief glimpse into the processes of exchange and emergence in individuations (the confusion of tying a tie while looking in a mirror, where right becomes left, for example). But it is in Caminhando's ability, despite the banality of the actions, to detach the event from the habitual inattention to transduction, and instead create a 'semblance', that such processes are drawn to the fore. Semblance, as Massumi uses the term, is the virtual's felt ingression into the event (2011, 15–16), its felt presence allowing a diagramming to take place, a thinking-feeling of the 'dynamic form' of relation and its connection to ongoing potentiality (Massumi 2011, 15). All this, I suggest, is a step towards a new politics of art that attempts to engage in the creation of lines of flight, with the composing of techniques for inventing (new) potentials for existence (Massumi 2011, 14). It is political in that it 'connects up different aspects of life' – new lines of causality and experience (O'Sullivan 2006, 74).222 Here Caminhando's politics are those of the 'micro-political', as Lone Bertelsen defines it, working at the level of bodily habits (2012, 43), in which the event focuses attention on the continued felt emergence from which neither body nor field can be detached. This is an ethical art in Deleuze's definition, a practice of pursuing expression and connection, rather than representation (Murphie 1996, 105). It is an ecological approach that activates attentiveness to life and the field, to the conditions of the event expressing itself (Manning 2013a, 147–8), an ontogenetic 'technicity'223 for living. This is an ecology-in-themaking: body-becoming-environment, environment-becomingbody. It is ecologically sensitive in assisting the formation of a trans-subjective attentiveness to an affective field across the becoming of space, time, bodies and objects (Bertelsen 2012, 39). Art events here, as Guattari states, can create an 'ecology of the virtual' capable of engendering 'conditions for the creation and development of unprecedented formations of subjectivity' (1995a, 91). Throughout this book I have suggested that interactive art could at times do with less emphasis on the efficacy of relation between participant and artwork, and particularly on the conscious comprehension of these relations. Instead it might focus on further exploration of the potential of an immersion in sensation that stretches perceptual processes and makes felt the viewer's own emergent role in an environment's individuations.224 I am proposing that the agencies driving this are best understood as the flow of forces and their transduction. These forces pass through and trigger the individuations of entities, gathering such individuations into an intensive ecology that drives invention. This is an experience of a trans-human and lively world in the widest possible sense. This is a move beyond the subjectivity and agency of the human participant. In this move an investigation into what might trigger the environment's own capacity to generate forces of becoming would seem to me to be paramount, and I begin to approach this through Manning's concept of the 'minor gesture' in the following section. #### Bridge: Pnuema and the minor gesture In the installation Pnuema, 225 things happened as participants moved around – lights came on and faded or their rhythmic pulses quickened, and stormy sounds erupted and swirled around the space. Things happened too when the participant stood perfectly still or left the space, as elements of the work responded to other components' actions (such as feedback from light variations affecting the tonal qualities of sounds, or the movement of the hanging sculptural objects triggering the sensors), and complex 'behind the scenes' algorithmic processes continued to activate changes calculated from both current and previous sensor input. Here, rather than linear connections between movement and light or sound, complex combinations of triggers determined what changes were generated, so that the effects of a particular action continued to reverberate through the work over some time. For example, the composition and development of the layers of singing sounds that occurred when the space had no participants present was shaped by the system's 'memory' of bodily actions that had occurred earlier, and any stormy sound sample required particular sequences of triggers within certain time limits in order to be played. Thus while the generative aspects of the work related to bodily movement, the participant was not able to discern a direct link between their gestures and what was generated. In this sense, the event began to have an (automated or algorithmic) life of its own, entering into relation not only with human bodies, but also into a series of temporal conversations between various elements of the work. Participants were able to feel some qualitative connection between their actions and how events evolved. For example, striding quickly around the space would over time increase the speed at which the aural storm developed and the lights took on more complex and resonate patterns of movement. But as the work played out connections and disruptions, it also resisted the demonstration of interaction. The complexity of the relation between an event – a movement or a change in light, the effects on other components of the work, the built in time-lag between a sensor event and its repercussions, the variations in the effects of a particular movement – meant that while the art event itself could, in its own way, 'feel' the relational implications, such quantitative understanding was denied to human participants.226 What filled this space that was formerly central to the relational or interactive event? Perhaps the immediacy of sensation began to assert itself? Perhaps it was something subtler that resolutely refused to address the human, and instead addressed the formation of the work from the field at an imperceptible and undemonstrative level? In this, the effects began to edge into vague perception – a fuzzy awareness of the incipient gathering of an event that was the event's ability to feel and respond to itself, to prehend potential individuation. Manning has defined such relational pulls that 'lead the field of experience' and 'open [it] to its differential' as 'minor gestures' (Manning 2016a, 48).227 A minor gesture is not exactly contained in any entity (algorithm, sensor or person), or event (movement, calculation, sound, light or relation), though, in order to individuate, these draw on the potential such gestures open.228 A minor gesture 'introduces a kind of continuous variability into the work's progress, a variability that is durational', as Manning states, where what is felt is variability in itself, a sense of an opening to (parasitic) potential (2016a, 49). This is a 'tuning' of the event to its future that is felt qualitatively, as an aliveness of an event forming. In Pnuema, this might be felt through the immediate and sensual connection with the expanded relational value of the lights and sounds as they form new complexities of connections. That is, it might be felt as a variation in connective or transductive potential sitting alongside any material or actualised variation. The minor gestures at the heart of Pnuema's self-tuning made both the actualised and potential relations mobile, always in flux – though not comprehensively demonstrated to the participant. Rather, such causal efficacy addressed, and was sensed by or resonated across, the ecology as a whole. This was an intensive exploration of the 'environment's own capacity to make felt the complex ecologies at work' (Manning 2016a, 54, emphasis in the original) – an ecological sensitivity not fully located in any one body, but as a plane with which the event itself engaged. Participants were addressed here, but not only on a subject-toobject level. They might have tuned to the shifts in the affective tonality, alongside other components that also tuned and aligned in their own ways with such field effects. There were forces or wills at work that were not only dispersed but that resisted residing in objects and remained instead gestures incipient with the event. This allowed components to begin to address each other directly rather than only via human mediation. Did participants feel these gestures? Perhaps as an excess of relation beyond understanding, as a displacement of will, a loss of agency when compared to a normative interactive experience, as a sense of something lurking just beyond comprehension but nevertheless broadly affective: as an immediate but indistinct sense of variation and of a gathering of a more-thanhuman ecology. ### 7 ### The noise in the noise: micro-perception as affective disruption to listening and the body Sounds…dematerialize the substance of things they resounded and extend their own patterns…they drift off things and link up with one another. Alphonso Lingis #### Introduction: vibrational symbiosis The pitcher plant and the wasp have come to an arrangement: when the wasp enters the plant's flower and buzzes at a specific pitch the stamen release their pollen in an emphatic burst of rhythmic (vibrational) sympathy. No other pitch will do, the flower is indifferent to all other notes. It waits; it listens, attentively, for the wasp's particular calling card. And yet…this is a plant – it has no ears, no brain. How is it able to listen, with what does it hear, how does it pay attention? And, one must ponder, how is it that it knows what it hears when it has no brain to perceive with? Perhaps, just as the brittle star has no eyes and yet is all eyes,229 the pitcher is all ears – its entire surface attuned to the potential of a frequency, sensitive to the particular oscillations of the one vibrational speed for which it has an appetite. The dance of the pitcher plant and wasp hints at the microperceptive potential enriching heard sounds. The transversal agency of sound as vibrational force courses through ecologies at pre-subjective, pre-content and pre-contextual levels, enveloping all in resonance that is the 'combat of energies' confronting each other (Deleuze 2002a, 65–8).230 This is the vibrational diffraction of enmeshed relational difference. At this affective level, interactions – immanent relations – with sounds are not limited to the ear and the brain. They stretch across the entire surfaces of bodies attuned to the sensations of their particular ecologies: a 'listening' independent of cognitive capacities and body boundaries. This strange pitcher-wasp symbiotic relation seems to indicate that sound contains, or is contained within, sonic excess (Goodman 2010, 9): a silent, contagious life as force and as potential force, enveloping all in the ecology of the unheard. This chapter considers some of the disruptive potentials of sound – that is, micro-perceptive sound's potential as a parasitic activator of change. It considers ways in which affective force produces ecologies through vibrational diffraction. #### Micro-perception The term 'micro-perception' refers not just to perceptions that are literally too small to be recognised, though the physical presence of the unheard begins to indicate some of the potential of micro-perception in relation to sound. Rather, as Brian Massumi asserts, it refers to a 'perception of a qualitatively different kind' (Massumi and McKim 2009, 4). It is pitched at the level of affect: 'hitting' the body, not with perceivable content but as a noise or interruption. Micro-perception can be perceived only as this interruption and transition, thus it is a 'purely affective re-beginning of the world' (Massumi and McKim 2009, 5). Affect is a primary creative force,231 as Jonas Fritsch argues, that unifies an event as it is also its extension or excess. Affect is, he argues, 'pre-personal, pre-individual and non-conscious but real in so far as it offers potential for action' (Fritsch 2009, 5). As such, it questions easy distinctions between event, subject and field (ibid. 6). It is a transitive force that connects and remains in excess of its effects, thus retaining further capacity to affect as it moves cross-temporally towards the future (Bertelsen and Murphie 2010, 140, 145). Micro-perceptive sound then might be seen to offer potential as a transductive force, disrupting boundaries as it drives creativity through a resonance that connects through intersecting and knotted together 'diverse realities' (Mackenzie 2002, 13). Understanding the act of hearing as one of transduction potentially alters our whole conception of the act. A single sound pulse is micro-perceptible. It is a singular shock to a surface that on its own cannot be understood as sound. It can be perceived only when in contrast: in relation to the interval, rhythm or difference between pulses. That is, it is not so much the single high or low point of a sound wave that is comprehensible, but the variations in pressure over time or the differences between a high and low point of a wave (the amplitude), and the distance between waves (the frequency, or number of waves in a given time). These then require firstly an internal or intensive differential logic (in the subjective comparison between the components of the larger sound event).232 Secondly perception requires an external differential between the ambient pressure of the medium through which the wave travels and the pressure of the wave itself. In both cases if there is no contrast there will be no perceived sound, although clearly a single force or pressure will still be felt by the body as a micro-perception (though, as I will argue further on in the chapter, this can be expanded on in several ways). As indicated briefly in the previous chapter, a direct connection can be made between Whitehead's conception of the role of prehension and held contrasts in the concrescence or individuation of an entity, and Simondon's concept of transduction. Simondon argues that transduction is more than the operation of forces on objects and the subsequent transformation of these forces through those operations, in that it is forces transformed into other forces through structuring (Combes 2013, 14–15; Mackenzie 2002, 50). We can therefore say that forces as held resonance constitute becoming (or individuation or concrescence):233 affectivity that is 'the relational layer constituting the centre of individuality' (Combes 2013, 31). These 'resonances' are the internal contrast of different relations that, like the held contrasts of prehensions for Whitehead, are constitutional of the event of concrescence (Simondon cited Combes 2013, 18–19).234 As in Whitehead's system whereby there is a subjective ingression of selected datum from the world into the concrescence of an event, the resonate contrasts are internal but also forge a link to the actualised world. These contrasts also resonate with the preindividualised potential of the event (which is also in a sense exterior to the individual at any one point in time) (Combes 2013, 31; Simondon 2009, 5, 7–9). Thinking of this link between transduction, affect and resonance emphasises that it is never a resolution of affective energies that occurs in becoming, but as Deleuze says, there is an ongoing 'confrontation', frisson or interaction between energies (2002a, 65–8). A collective or trans-individual and concrete example of this is discussed in this chapter in relation to the phenomenon of diffraction, in which complex resonances between sound waves constitute new and collective sound events. In thinking of resonance as a key to the production of an event, one might argue that we do not even 'hear' the sound per se. Rather, the sound waves activate a sympathetic resonance in the mechanisms of the ear, which in turn are transduced into impulses in the nerves and then to neural firings in the brain. This suggests that while in the act of hearing sound in the environment is prehendable by a body, the actual 'hearingevent' is self-contained and self-actualised as a subjective event that is intensively driven and satisfied. Deleuze's example of this thinking is that of a needle piercing the thigh: the pain felt is not the needle, but the actions of the nerve endings in the flesh (1993, 96). In this, as in Whitehead's theory, an entity is responsible for its own 'satisfaction' or concrescence, even as it draws prehensively on its relation to other entities (1978, 126, 153–6, 236–8). In this sense, sound does not pass into the body as such, but perception might be said to occur through a productive sympathetic tension or held resonance between the two systems.235 In terms of an act of hearing or listening, this means that the hearing event is separate from, though influenced by, the sound event, and is never simply a passive ingression of data. Thus there is always the potential for creative divergence. #### Interlude: Artaud's scream Artaud's radio play To be done with the judgment of God pierces the air and vibrates the listener with wild screams and glossolalia (that are parasites to language, 'ruptures' and 'stoppages of flow') (Serres, 2007, 189). These can never be confused for nor contained within representational meanings. But more than this: the words themselves that are sung, shouted, agonised and whispered are so charged with affective power as to 'illuminate the entire nervous system' (Artaud cited Weiss 1992, 275). Here objects (bodies and meanings of words) become again force. The play's broadcast is an act of transduction as its transmission disperses the actors' bodies through the airwaves as disruptive vibrations, enacting Artaud's philosophy that man 'is not only dispersed within his body, he is also dispersed in the outside of things' (Artaud cited Weiss 1992, 253). Artaud incites this ability of sound to transverse the body, turn it inside out, to make organs of its surfaces, to empty its interior of meaning: Artaudthe-alchemist236 cruelly uses 'radio magnetism as a countershock to achieve…the destruction of bodily hierarchies through vibrations' (Lucaciu 2010, 72). It is a cruelty, as Deleuze says, made not of horrible things, but rather 'the action of forces upon the body' (2002, 45). Artaud's scream is made a 'physical substance in space' (Barber 1999, 106), a disruptive vibrational force that encapsulates his 'vast project of physical transformation' (Barber 1999, 93). Broadcast, it seeks to invade the sanctity of the listener's home and body. But the scream is transmitted not just in the literal scream that punctuates the radio play, but also saturates every sound of the event, as micro-perceptible affect coursing through and tearing open bodies it encounters.237 It proposes to fold out the listener's body, makes their whole surface an organ that is invited to resonate in sympathy with the a-perceivable force of the sounds. The problem Artaud addresses through his particular use of language/vocalisation is one of how to extend the tension of contrast of the micro-perceptive without providing resolution.238 He develops a technics to suspend the body within the processes of multiple 'tendential unfoldings', as Massumi phrases it (Massumi and McKim 2009, 11), while also making felt the potential for 'different capacities for existence' (Massumi and McKim 2009, 12) outside of the major and the molar. It is an 'exploratory dancing of the extremities of the body' (Barber 1999, 103) an adventure into excess, a plunging into the multiplicity, where body, home, language as ground are contaminated and shattered. #### Body as ear While micro-perception is a pre-bodily force of the world, it must also be recognised that it is always implicated in the bodily, in that it acts on and through a body.239 It affects bodies through the creation of a felt difference, both prior to and after the micro-perceptive event: an affective attunement (Bertelsen and Murphie 2010, 5, 6). Affects can be known as such only through their effects on bodies (Bertelsen and Murphie 2010, 4), and such bodies – be they speakers, walls or animals – all have an 'appetite': that is, a potential to affect and be affected240. Each, in its own way, performs a particular way of 'knowing' the world – a specific engagement with certain vibrational frequencies (Barad 2007, 379). The human ear could be thought to engage with vibrations roughly between 20Hz and 20 KHz (Roads 2001, 7), but the human body is, in fact, receptive to a much wider spectrum. Outside of this audible frequency range lies 'unsound': the infrasonic and ultrasonic (Goodman 2010, 17).241 To this list of the imperceptible, we might add, as Curtis Roads does, the subsonic (sounds too soft to be perceptibly heard), and ultra-loud sounds (those that are 'felt by the exposed tissues of the body as a powerful pressure wave' more than they are recognised or processed through the ears) (Roads 2001, 7). Such vibrations might be said to act synaesthetically on bodies – they affect the body at a base level of vibrational force that disrupts and stimulates multiple sensory capacities. This is the pain of high volume shock waves forcibly vibrating flesh, the infrasonic beat of a sub-woofer that reaches you through the soles of the feet, the prickling sensation on the skin of high frequencies, and the physiological effects of these frequencies in stimulating neural activity (Goodman 2010, 184). To this we might add the emotional effects of such unsound: the anxiety or edginess that might be evoked by either the very high or loud, the coercive effects of deep beats, the lure of the just-too-quiet to be heard. As affects, these unsounds come to be known to us through their formative effects on our emergent bodies. #### Space-Shifter Entering the environment of Sonia Leber and David Chesworth's Space-Shifter <sup>242</sup> the viewer is bombarded by strange voices – part language, part guttural exclamation – that saturate and resonate every surface, as much unsound as sound in their violent a-rhythmic shaking of the entire space (see Figures 7.1 and 7.2). Floor, walls, air, speakers, sheets of metal, and bodies are invaded, vibrated, penetrated, turned outward, and made into surface. Metal buzzes with secondary resonances, feet become ears as they oscillate with the floor. Waves of vibrations bounce of windows, walls and flesh, taking on new and singular speeds through their interactions with the differing viscosities of surfaces. The speakers, room, floor, metal, and bodies all (re)perform or express these vibrations in their own way, transducing according to their own affordances. Thus, a speculative vibration launched into the space by the speakers proposes to these various surfaces a multiplicity of responses, combining their various and singular capacities to resonate into a machine that produces vibrational difference. The event of vibrational penetration of the space makes these new and contingent surface assemblages: machines that attract and modulate sound and unsound.243 It rearticulates all bodies/entities into 'shifters',244 new combinatory propositions glued together by the force of vibration. Its 'choral' sounds245 are 'launched like missiles' to 'act directly on the space'246 and entities. #### Parasitic diffraction: the vibrational as differential force In his essay entitled Four Objections to Sound Art Tim Ingold (2011, 136–9) sets out to argue that we need to make use of sound in artworks in ways that do not simply replicate the representational models of much visual art in presenting a soundscape to be 'played back', made 'aural' as painting can make a landscape 'visible' (2011, 136). This, he states, denies the fact that 'the ears, like the eyes, are organs of observation… just as we use our eyes to watch and look, so we use our ears to listen as we go forth in the world' (Ingold 2011, 137). Rather than compare sound to vision, Ingold says, we should associate it with light, emphasising the experiential nature of sound that involves a 'commingling with the world' that is both an 'embodiment' and 'emplacement' (2011, 137, 138)247. Here, I think, Ingold argues effectively for the need to think beyond the naïve idea that merely making an immersive sound recording is enough to create meaningful interaction or avoid the traps of representation. He instead suggests a more provocative approach that might emphasise the potential of vibration to move bodies beyond themselves and instigate to new and collective processes of individuation. What then happens then when we think of Space-Shifter not as 'sound art', but as a series of vibratory propositions encouraging trans-body resonances – focussing on the productive disruptive potential that such micro-sound initiates, rather than its aesthetic or representational qualities? How can we think of such vibratory events for their ethical potential as disruptive relational forces that breach thresholds, folding and splitting entities? To begin this, we need to first understand something of vibrational diffraction, and its role in producing difference through parasitic disruption. To include micro-perception in any discussion on sound is to acknowledge a more expansive definition of sound as vibrational force. Here it is a 'variation in pressure over time' (Evans in Massumi 2002, 171) encompassing all the elements of a sound that will be contracted into a perception – tone, pitch, rhythm, volume (composed from waves that differentiate in frequency, amplitude, phase and shape) (Evans in Massumi 2002, 171) – and the unsound, the microperceptible remainder. The physics of sound, Roads argues, clearly demonstrate that the basis of all these components of sounds is events of vibrational difference,248 where rhythms of contrast disrupt any continuum: questions of speed and interval of oscillation. Sound itself is then an expression of this modulating difference (Evans in Massumi 2002, 171).249 But a vibration's actualisation must also always act parasitically on other waves in the space through the physics of diffraction. Diffraction 'has to do with the way waves combine when they overlap and the apparent bending and spreading of waves that occurs when waves encounter an obstruction' (Barad 2007, 74).250 As waves, sound then 'intra-acts' in this manner,251 with individual wave patterns engaging in disruption and interference with one another. These entangle in complex ecologies, always immanently expressing their differences in producing novelty in the torsion between these forces. In Space-Shifter, for example, a sound wave generated by the speakers hits and reflects off a surface, returning as a repetition but at a different speed. These reflections diffract with the incoming wave, producing new modulations that then also interfere and combine with both incoming and reflected waves, producing further modulations, and so on. What consistency of relation there is here is the consistency of events surpassing themselves (Combes 2013, 41), as each wave is implicated in the individuation of all the others. Such noisily productive enfoldings, disruptions, complications and interferences are parasitic actions. It is the noise in relation that is its creative force – a third and mobile position252 that multiplies vibrational difference, blurring distinctions between cause and effect (Serres 2007, 57) as a resonance of a resonance. Due to diffraction, we can say that a vibration in Space-Shifter always also produces parasitic vibrational forces intrinsic to its event. Space-Shifter proposes to construct vibration-surface assemblages that form parasitic machines operating on multiple fronts: producing intensive difference within wave events through diffraction that multiplies and drives towards novelty. The work here employs micro-perception tactically in several different ways, revealing the experience of Space-Shifter primarily as an event that explores the parasitic potential of sound and unsound. The heard and unheard components of the sounds affectively engage the body with vibration in ways that create new contingent bodies from components of the body-artwork assemblage (machines within machines). Over and above the sound that is perceived by the ear itself, there is also the vibrational excess of sensation experienced by the 'skin-asear drum' that envelops the body (Serres 2008, 119).253 This creates a shared vibrational zone of feedback loops between skin and world, an intra-active ecology of diffractions. Surfaces are implicated in each other's becoming(s): speaker surfaces affecting and affected by the vibrational capacities of the metal plates, floorboard oscillations meeting and conversing with vibrations of shoes, skin and walls bifurcating each other's projected vibrations in the shared space in-between, bodies remade as speakers, receivers, reflectors – together resonating surfaces. Space-Shifter proposes space, floor, feet and metal as the ears, as they act as conductive surfaces, transducing vibration. Sound waves differentially connect surfaces to make vibrational ecologies that nest within ecologies. This is a doubling of the surface into a field-body machine, an in-between that is alive with productive potential – a 'sound envelope' that is as much a sieve as a container, a 'sensate surface' of connection (Anzieu 1989, 62–9).254 The force of this sensorial meeting of surfaces – pressure/resistance meeting pressure/resistance – is a vibrational interaction with another that leads us out of ourselves (Lingis 1998, 135). It is a worlding that the sympathetic resonances enact: our surfaces taut drum skins.255 This is a collective perception, as performed by the body as a sensate organ in sympathy with the forces of the world (re)generating.256 Figure 7.1 Sonia Leber & David Chesworth, Space-Shifter (detail view), 2009 Steel with 2G pack enamel paint, 14 channel audio, audio transducers, speakers. Courtesy the artists. This project was supported by the Australian Government Through the Australia Council for the Arts. Here Space-Shifter makes explicit the vibrational forces surrounding and interpenetrating the body. The diffractional resonances with, and resistances to, the power of the external vibrational rhythms are folded into the body's own rhythms and speeds to create a third shared potential – a parasitic body disrupting prescribed boundaries. This is an ecological resonance, a collective and generative contrasting or transductive event. Both audible and inaudible elements of a sound set up diffractive patterns with each other (Roads 2001, 33), a resonance that Goodman terms the 'hypersonic effect' (2010, 184). This parasitic noise operates on the audible range, modulating unheard vibrations, producing what we perceive as tonal colour or timbre (the layering of tones, overtones, intra- and ultrasonic frequencies that give qualitative breadth and openness Figure 7.2 Sonia Leber & David Chesworth, Space-Shifter (detail view), 2009 Steel with 2G pack enamel paint, 14 channel audio, audio transducers, speakers. Courtesy the artists. This project was supported by the Australian Government Through the Australia Council for the Arts. to perceived sounds). In addition, these diffractions produce a rhythmic multiplication or syncopation, with surfaces acting as attractors in the system of modulation of beats.257 Here Space-Shifter becomes an affective 'rhythm machine', where connections between entities are assembled via sympathetic rhythms (Goodman 2010, 111), organising or patterning relations between pulsating bodies. Rhythm plays out the problem of the disjunction of differing vibrational speeds, as a gathering of these differences on a plane.258 As such, the parasitic actions of wave diffraction more than multiply the vibrations to be experienced through diffraction. They are micro-perceptive machines that produce a multiplicity, virtuality, to the sound event, a system of potential disruptive production of 'new rhythms, resonances, textures and syntheses' (Goodman 2010, 191) that is immanently produced with the audible. Micro-perceptive sounds are parasites on cognition, on the hegemony of perceptive reduction of sensation of vibration,259 and on the easy distinction between listener and the listened-to (receiver and received) as all become entangled in ongoing transductions. The insistent force of vibration in its not-fullyformed or cognisable state requires of a body that it compose organs to cut or actualise perception from a virtual plane of vibration. It also keeps vibration on the edge of the virtual, still at its most open to different combinatory possibilities, suspended in the not-quite decided. This is the parasite as creator of 'fuzzy' relation (Serres 2007, 57). Here sounds in Space-Shifter lose their beginnings and ends through refolding and held dispersion. There is unease in the encounter with these heightened disturbances, an edginess that the lure of the unheard performs, a heightened sense of the presence of an excess that cannot be contained within the audible, that refuses contraction but insistently is felt on the body. This invades enjoyment or contemplation of the work as one is thrust into the middle of its machinations (Deleuze 1993, 93). In this way Space-Shifter acts parasitically on one's emotion state – a metaphorical diffraction – disrupting the contraction of sound to signification, acting heterogeneously on established language-sound hierarchies. On all these levels, Space-Shifter is insistently not just 'sound' to be contemplated and comprehended, but affective force in the event. It is a 'performance of the world in its ongoing articulation' (Deleuze 1993, 93), a way of 'knowing', a 'specific engagement of the world' (Barad 2007, 379) across a vibrational plane. #### Refrain: Parasitic unsounds In the installation Momo260 (see Figures 7.3 and 7.4), unheard but affectually forceful vibrations were layered to produce a sound ecology that might impact on bodies beyond the perceptual processes afforded by the ears. Within a sound design – one that already recombined sounds through cutting, layering, echoing and volume shifts in response to fluctuations of light and movement in the space – each sound sample was itself a layered combination of perceptible and micro-perceptible sounds. Samples consisted of both a dominant sound (a word, phrase, or other vocalisation), and approximately four to eight 'unsounds' (see Figure 7.5). These sounds were manipulated to sit below a humanly perceptible threshold by virtue of their high or low frequency range, and/or because their volume sat below an audible level, and consisted of both altered versions of the dominant sound, and other found sounds chosen for their particular affectual qualities.261 Here another layer of held difference or contrast was added to each sound event through these new and competing samples that began to move the dominant sound beyond itself, recreating the sample as a machine capable of intensively differentiating. While the viewer could not audibly comprehend these additional layers, they did create affects on bodies in ways somewhat more difficult to articulate. These affects could be felt by listening to the difference between the main sample on its own and the layered composite sound. When combined, what was heard gained an unsettling quality that heightened the already abrasive qualities of the vocalisation. A sense of uneasiness was added that could be described as a shift and increase in richness or intensity of the affectual tonality – a prehension of the unsaid/ unheard. In addition, certain frequencies in the background sounds produced subtle physical affects on the body (such as a slight prickly feeling on the skin or a tension in certain muscles) that added to the emotional response, and to the feeling of a 'more-than' qualitatively combining with the perceived sound. These layers of the experience might be thought of as disrupting through creative multiplication, a 'checking' of the process of clear perception that allowed micro-perceptions to 'invade' consciousness (Deleuze 1993, 93). This felt presence of an unheard excess within the sounds perceived might be proposed as the beginnings of a bodily awareness of a larger vibrational ecology at work. In this way, the design sought to experiment with heightening sensitivities to both the excess of sound in the sonic environment, and to the sensitive capacities of parts of the body that interact with vibration. Rather than focusing on communication via the ears, the design experimented with the disruptive qualities of vibrations to encourage listening in a larger bodily sense. The utilisation of micro-perceptible sound also began to work towards a more complex ecology of interactions. These were concerned not just with ear-to-speaker connections, but also with multiple sound wave-to-sound wave and surface-to-surface connections and combinations. A non-human level of dynamic interaction played out within the work, as vibrations of both sounds and unsounds interfered with each other – as they always do – but were multiplied and complicated by the greatly increased percentage of unsounds present. These played out, on an environmental plane, the combinatory, diffractory and essentially molecular nature of vibration. Should we still term these as sounds? Certainly they acted on bodies, making connections between surfaces, but perhaps they began to disturb the boundaries between sound and other forces, between one kind of sensation and another, between the capacities of the ear and the potential of the surface of a body to be coopted into an expanded listening machine. These microperceptible vibrations remained, to some extent at least, at a level of affect, of trans-objective and trans-subjective force. These active forces played out their differential equations below a perceivable level. What was perceived were the effects of this battle of 'wills' (Deleuze 2002b, 61), but the vibrational here extended to a more-than-human plane, beginning to position the work as being concerned with a larger play of force within Figure 7.3 Andrew Goodman, Momo, (detail view). 2011. Paradise Hills Gallery, Melbourne. the environment. Micro-perception here operated at the level of the minor gesture, emerging 'from the field itself', as Manning says, concerned with an expressive variation not held within an Figure 7.4 Andrew Goodman, Momo, (installation view). 2011. Paradise Hills Gallery, Melbourne. object as a perceived sound, but within the environment's own capacity to prehend and interact with its intensive differential (2016a, 48, 54). #### Multiplicity: the aliveness of the virtual How can we perceive sound – whether through the ears or body as a whole –and construct a useable set of vibrations from the multiplying 'noise' of diffracting micro-perceptions? Clear perceptions, as Deleuze argues, are actualised out of the potential of the micro-perceptions that form their virtual – the multiplicity from which they concresce. Each perception is a singular configuration of 'compossible minute perceptions' that yields perception as a cut in the multiplicity of such potential combinations (a 'zone of clear expression') (Deleuze 1993, Figure 7.5 Sound layering in a sample from Momo. In this example, the dominant sound is highlighted, while other manipulated copies of this sample, and samples from other sources, sit below a perceivable threshold. 90).262 These enmeshed micro-relations form an affective entanglement, without themselves being distinctly expressed. It is the act of perception, productive resonance with vibration, which cuts into this virtual plane and actualises a particular contrast of the relations between micro-perceptions. That is, the perception expresses a distinct diffractive combination of microperceptions in a particular way that yields a focus, but retains also some relation to all the micro-perceptions of the multiplicity. Each perception then is a singular and subjective expression of its relationship to the entire field in its intensive and selective patterning or contrasting of these micro-perceptions (Deleuze 1993, 90).263 As always, perception is a result of the differentials of differential equations, that is, what is perceived is the modulation of difference over time (Evans in Massumi 2002, 177). This dynamic (unheard) virtual of the perceived sound actively disrupts its stable status as 'object' with determinate or idealised status.264 Instead it becomes the product of differential relations of affects expressed in conscious perception. There is always a multiplicity that is alive in its ever-diffracting evolution in each heard or felt sound – a future-feeling drawing the sound towards further perceptive concrescence. Shape-Shifter draws these unheard relations into a clearer zone of expression, just as it positions what would habitually be clear into a zone of indeterminacy. This makes evident the dynamic complexity of vibrational forces present, and makes felt something of their relation to the perceived sound as it invites us to suspend ourselves in this individuating process. One is thrust into – or emerges tentatively out of – a seething ecology of sensations: the body reconstructed as synesthetic machine, drawing vibratory sensation from it's various surfaces-as-organs to construct a perception.265 In this respect, the work might be seen to be 'ethical' in sympathy with Simondon's proposition of ethics as the 'sense of individuation' (Simondon cited Combes 2013, 64) that links or makes felt the preindividual component of an event and affirms the relational nature of this event (Combes 2013, 65). Here, Shape-Shifter's ethical relationality encourages an awareness of a 'vitality' of nonhuman composition, and the ability 'to become perceptually open to it' (Bennett 2010, 14). But the 'non-human' here must be thought of, not as an exclusion of the human dimension, but as the affectual forces that course through and are felt by both the human and other entities, making evident the transindividual elements of any concrescence (Mackenzie 2002, 117). The transindividual, Mackenzie says, cannot be conceived of as being interior or exterior to the individual, but as a 'continuing folding and unfolding limit between inside and out' (2002, 137). This positions the individual as 'one provisional [outcome] of a collective individuation in process' (Mackenzie 2002, 207). This transductive stance, which Mackenzie takes from Simondon, again places an emphasis on the forces and minor gestures within the field that motivate the further collective gathering or individuation, and of which the vibrational ecology of Shape-Shifter provides an exemplary case. Shape-Shifter achieves this ethical sympathy with the ecology, I would argue, through its ability to make problematic the experience and concrescence of a remarkable or clear perception (Deleuze 1993, 91). 'Perception' of sound is revealed as contingent and in-process, a process of differentials differentiating, which is 'an expression of the in-between' (Murphie 1997, 326). Sounds that have denied representation on a more superficial level – by emphasising part words and vocal expression over easy signification – work to draw the participant into implication in the processes of diffraction and production, as a series of interactive surfaces that assemble as differential machines.266 Micro-perception is configured as a problem, which finds an expression in perception (though not as a 'solution' as such, more a 'working through'). When engaging with Shape-Shifter we try to comprehend, to make the vibrations coalesce into readable 'sounds'. But the magnitude of the differentials, the speeds at which they move, and the unbalanced relationship between the heard and 'unheard', disrupts this contraction. The richness of the work's affectual force leaves us disorientated, perceptually unresolved, still searching for a defined body, space and sound. This process of disruption of vibrational wave by vibrational wave is not only foregrounded but stretched or preserved. It is the vibrational 'aliveness' of the event that the body of the participant comes to feel itself explicitly implicated in. Thus as feelings – as prehensive resonance with other entities (Whitehead 1978, 220) – the affectual qualities of microperceptible vibrations become evident, and new sensitivities to the vibrational ecology in which we are immersed are proposed and can be experimented with.267 #### Conclusion Shape-Shifter emphasises the temporal through the mediation of rhythms of the vibrations disrupting and combining (Braidotti 2002, 154). The work addresses listening as an act of combining and disrupting relational vibrational processes and of sensing the inherent further parasitic potential, rather than as the perception of individual 'completed' or explicated sounds. In this it approaches what Braidotti has proposed as a 'nomadic music', concerned with a becoming-interval and a dynamic relation to the field, to the inaudible and imperceptible (Braidotti 2002 155). 268 A 'nomadic' music suggests shifting ideas of sound design from completed or wholly realised sounds to mobile assemblages of micro-sounds as micro-perceptions. This might enable a shift from a representational model to one of production. That is, a shift towards a focus on enabling conditions for the production of perception of sounds out of the field of micro-perceptions, with their inherent and parasitic diffractive resonances. Shape-Shifter approaches the limit of what can be heard or understood as sound, and in addressing this limit of the perceptible, it proposes new organisations of surfaces (assemblages) with which to perceive. Here sound in an art event is potent, not for its ability to extend meaning and communication beyond the capabilities of the eye, as it is so often how it is utilised, but rather to problematise such notions of communication-between. Thus it is harnessed at the level of affect to open potential for new bodily individuations, and it is the space of the body that Shape-Shifter vibrates as much as the air or floor. It sets these bodies resonating to awaken new appetites, new sympathetic resonances and dissonances, as they tune into the multiplicity of the vibrational ecology within which they become. 8 ### A thousand tiny interfacings: fertile acts of resistance #### Introduction These spaces between are more complicated than one might think…less a juncture under control than an adventure to be had. Michel Serres Brian Massumi has argued that the interface is an unsustainable concept within a process-centered world. In its usual understanding, the interface is positioned as a 'privileged site of mediation' within a system, Massumi states (1995, 7). This is evident in the various definitions of the interface as 'a bridge and a channel' (Hansen 2011, 68), a distinct 'point of contact' (Grau 2003, 198), or as 'devices that link humans to machines' (Poissant 2007, 236).269 Such ideas of the interface as a prime site of creativity, interaction and communication deny what in process philosophy might be seen as the relationally enmeshed nature of all entities. Massumi's philosophical stance emphasises the 'primacy of processes of becoming over the states of being through which they pass' (Massumi, De Boever and Rolfe 2009, 38), that is, that any entities interfacing with each other are themselves composed of relations. As such, discrete interfaces are problematic in that they might be seen to imply a world inhabited by ideal, internally stable objects, between which interactions occur. The interface's role, in such modes of thinking, is to rejoin entities that are by implication discrete, where the complexity of continued unfolding and relation to the dynamic virtual or potential is greatly diminished. There is much to be critical of in the privileging of the interface. As Massumi notes, it can promote a naïve excitement in undifferentiated flows of information, an unquestioning, utopian promotion of interface 'for interfaces sake' (1995, 1), that fits in perfectly with capitalist models of circulation and surplus value (Massumi 1995, 9). To this, one might add the cybernetic conflation of the biological and technical, of which Simondon is so dismissive,270 and which Massumi describes as the 'industry philosophy' (1999, 33). The extension of the 'prosthetic function' of the interface, is utilised as a method of controlling, 'a relay point in the dissemination of human ordering activity into space…transform[ing it] into a realm of expansion onto which the human projects itself', with real difference erased as the body 'disappears behind a techno-logical shield' (Massumi 1995, 3). This subjectification of the technical object, which Anna Munster has pointedly termed 'interfaciality', is a codification as face to face, rather than body to machine relation (2006, 122–4). Interfaces here contain potentially problematic elements of power and control in their stratification and limitation of relationships, denying, as Matthew Fuller says, a user's engagement with the internal operations of computer software (2003, 142). In this, he argues, they potentially not only codify relations and subjectify technical elements (treating technical assemblages as a stable and ordered 'whole' with fixed interrelations between these elements), but also work to model human subjectivities in relation to the particular and pre-coded interactions that an interface demands of them (Fuller 2003, 113–4). Here the disciplining operations of interfaces operate not only to refuse certain levels or types of engagement, but also to enforce or require other types of homogenous interaction, such as the ubiquitous 'swipe' of the smartphone or tablet that encodes and limits bodily gestures. Such disciplining of relations, may indeed, as Fuller notes, exhibit tendencies to break down as 'control folds in upon control, mess[ing] with its too-easy seriality', creating 'opportunity for something else to emerge' (2003, 113). However, the primary sticking point for discussion of the interface within process philosophy remains: that the very concept of a distinct interface relies on hylomorphic thinking that see it as a privileged site of interaction within an otherwise inert representational system of 'scientific materialism' that seeks to explain 'all change in terms of changes in "external" relations between beings that do not change in themselves' (Stengers 2011, 128).271 These clear cut boundaries between things become hard to sustain with closer inspection: electrons migrate, charges pass, affects flow, bacteria course freely through us – the separation and discretion of objects and forces becomes more and more relative and intertwined. Within a process-based conception of the world that recognises the primacy of forces and relation over form, all is interface; everything is dynamic communication, incipiently co-forming. So here we have our paradox: maintaining clear and distinct interfaces between things requires us to ignore the actual flow and enmeshed quality of lived experience, while acknowledging the primacy of the relational means everywhere we look are a thousand tiny interfaces. Neither proposition is of much use for either thinking or constructing dynamic, immanent art events. In this chapter, I want to show some ways in which we might think through the process of interfacing as a creative force within an art event without succumbing to the type of static, representational models of which Massumi is justifiably critical. To do this I will examine a particular incidence of interfacing that occurred in Raphael Lozano-Hemmer's work, Re:Positioning Fear: Relational Architecture 3, in order to consider ways in which unplanned interfacings between a public and the technical assemblages of the work helped to develop a greater level of both self-organisation and openness in the event, potentially operating across a social, artistic and technical level. An interesting shift in the forces generating in the work occurred – moving from those preconceived by the artist to new, shared and emergent individuations developed through an interfacing of a public bringing their own intentions and tonalities to the event. But, while these events are of particular interest here, I do not wish to overstate the uniqueness of the case. As Lozano-Hemmer has said, the events were certainly significant in his rethinking of the ways in which he staged further Relational Architecture iterations, however this does not necessarily imply that the occurrences were extraordinary for such large-scale interventions, which are by their nature always composed of multiple and often contradictory intentions and forces, and can potentially head in numerous directions, both predictable and surprising.272 Rather, this example provides an opportunity to consider the creative potential of interfacing, and its ability to complicate and re-energise the event. In putting the interface to productive use as a differential tactic within an art process, I propose that it might provide a logic of self-regulation, one capable of internally driving the creation of intensities of resonance or disturbance through connection. #### Interfacing If we begin by thinking temporally rather than spatially, it is possible to consider these interfaces as moments rather than points of action or relation. This suggests that the interface might now be thought of more as a process of interfacing, 273 as an unfolding or contingent process within a larger nexus of relation, as an in-action moment of intensity of disruption, contrast and invention rather than a privileged or static position within an art event. As noted in Chapter Two, a machinic conception of both bodies and technical objects allows us to think of them as assemblages that are productively relational, rather than fixed – always capable of further expression of some potential. These organic and non-organic machinic assemblages are mobile, in that they can contain other machinic combinations nesting within them, and can also co-operate with other assemblages to form larger (though resolutely non-unified) machines (Braidotti 2002, 254). I will briefly consider the idea of an art event as a machine producing transductions of forces, before attempting to unpack the creative role of interfacings in Re:Positioning Fear by suggesting that interfacing might productively parasitise. #### Transduction It is common to think of interfaces as translators of code, points of information exchange, from digital to analogue or vice versa, or as a 'point of contact where humans and machines meet in order for exchange to take place' (Grau 2003, 198). However to assert the primacy of the flow of forces, rather than the secondary exchanges of text, I have begun to argue that transduction is a better way to fully think the event of interfacing. Transduction positions interfacing as the integration, through the flow of forces of differing viscosities, of formerly disparate things within a becoming-concrete system.274 As Thomas LaMarre notes, a move towards concretisation implies an increase in the complex inter-determination of the individuations of the entities that comprise a larger ecology. It also implies a greater openness or indeterminacy in the 'charging' (with potential) of the relationship between the potentials of an entity and its field (the internal and external milieu) as the 'associated milieu' of the event that 'runs through or across inside and outside as a transductive potential' (LaMarre in Combes 2013, 93, emphasis in the original). This 'charging' with potential is, as I will argue is the case with the events that disturbed and ultimately transformed Re:Positioning Fear, a problematisation within a field that acts as the catalyst for new individuations to arise. These are partial or ongoing solutions that individuate not only relations between the entity and field, but also new sets of rules or planes on which individuations might operate (Vollrath 46). That is, such problematisation enables the 'discovery of the dimensions according to which [the] problematic can be defined' (Simondon 2009, 11, 12). Transductions individuate or evolve these dimensions, Simondon states, over time (2009, 12), bringing the concept in line with interfacing as an unfolding of an ongoing productive relationship rather than a pre-structured or instantaneous ordering of relations. In transduction then, as argued in Chapter Seven, we have a way of thinking how components relate that, rather than resolving or fixing relation, emphasises the ongoing productive and speculative internal and external tension of resonances between potentials brought into conjunctive and nonlinear relation (Mackenzie 2008, unpaginated). Transduction, for Simondon, generates the dimensions in which components can communicate 'without loss, without reduction' (Simondon 2009, 12): a 'solution' that conserves rather than limits or reduces information and potential as in the traditional notion of the interface. Thus transduction might expand interfacing from a limited notion of fixed actualised relations mediated through an interface, to one that emphasises dynamic and ongoing interactive potential on a virtual plane. An art-event might be a transductive machine: regulating and producing affectual flows, a 'machinic of expression rather than a signifying apparatus' (Murphie 1996, 104), a producer of movement (Munster 2006, 15) or difference. Again, transduction must be thought of as occurring not only on a concrete physical level, but also – as will become particularly pertinent in the art examined in this chapter – at social and psychological levels (Simondon 2009, 11). In conjunction with this, the individuation of the individual is also trans-individual in that it is inherently intertwined in larger, collective individuations (Simondon 2009, 9). If the transduction that occurs through interfacing produces difference, then this positions interfacing as a prime creative force-form.275 Seeing interfacing as a machinic action implies a shift in designing art events in order to emphasise their machinic potential, their productive capacity or capability to produce difference. It is this operation of the interface as a differential machine that is addressed below through an unpacking of Re:Positioning Fear, in light of three related actions of creative differentiation: parasitic noise, folding and the resonance of the incompossible, and concretisation. ### Re:Positioning Fear Re:Positioning Fear consisted of an orchestrated shadow dance composed of a projected conversation thrown onto the architecture of the city. This text was made visible within the shadows participants cast on the surface, creating silhouettes of differing sizes depending on their distance from the light sources (see Figures 8.1 and 8.2). Here the bodies of the participants performed disruptive interfacings within a machine composed otherwise of technical objects and public architectural components. This melding of technical objects with the unpredictable input of a public presents one possibility of providing the technical elements with an expanded potentiality. Its 'relation with elements outside itself' provides a level of indeterminacy (Munster 2006, 14). The body, as Combes states, is always in an ideal position to make connections with the technical, to 'become with', to play the role of 'transducer between machines' as it has an 'always active virtual' (2013, 60).276 #### Parasitic noise Part of the appeal of this work is undoubtedly the inbuilt complexity with which it enables or creates potential to engage various components of the city in a new and playful manner. This, as Andreas Broeckman writes of the work, was a dynamic 'social interfacing', as Re:Positioning Fear constructed a 'fragmented and heterogeneous system of engaging different publics in a variety of specific ways' (Broeckmann 2004, 381). Thus personal imagery was re-inscribed on architecture burdened with often-oppressive histories, public spaces re-commissioned into dialogues with the performative, bodies unproductively intertwined with technologies of surveillance and control, and so on. However a much more interesting and radical disruption also occurred in the particular unfolding of this work, which was already primed for playful intervention and evolution. It was in this catalysing moment, through parasitic action, that a new and more complex machine was produced. Alongside the positioning of their shadows on the façade to activate the hidden text, participants began to synthesise a different work out of the components by engaging specifically in play between their projected silhouettes. They utilised the potential to radically alter the size of their shadows by moving closer and further away from the projected light source to engage creatively with one another. For example, a wheelchair bound participant created a giant image of himself and 'ran down' everyone else (Lozano-Hemmer 2005, 6), while other participants played with shadow puppetry of smaller bodies, and the making of multi-limbed combinatory beings.277 Figure 8.1 Rafael Lozano-Hemmer, Re:positioning Fear, Relational Architeture 3, 1997. Landeszeughaus, Architecture and Media Bienale, Graz, Austria. Photo: Joerg Mohr. Figure 8.2 Rafael Lozano-Hemmer, Re:positioning Fear, Relational Architecture 3, 1997. Landeszeughaus, Architecture and Media Bienale, Graz, Austria. Photo: Joerg Mohr. This free shadow play was, I would suggest, a kind of parasitic noise, feeding off the energy already flowing through the work to create new paths, and to creatively bifurcate relations.278 It continued to qualitatively express something of the original relation (moving shadows revealing text on the building's surface), while at the same time producing a new (minor) relation through the same initial forms. The contemplative and reflective rhythm of movement in the large-scale text was overlaid with the noise of a quick and teasing play of shadows, creating a tension, a clash of intentions and tonalities: gaps and miscommunications. These parasitic actions existed on multiple levels and at different scales. They operated throughout the transductions of formforce taking place, wherever interfacing occurred, producing excess. For example, as bodies overtly disrupted light to create new imagery, there was also a more subtle disruption of intention. Artist's intentions (or perceived potential of the work) interfaced with the participants' disparate motivations, to create a third, more mobile position. This composed indeterminacy within prescribed events of relation. This is not intended as a metaphor – within process thinking intentions, urges, feelings, desires are not phantasms, but forces and lures towards forces in and of the world (as James states, process thinking must not 'exclude from [its construction] any element that is directly experienced') (2010, 18).279 Such conceptual forces are, I am suggesting, as capable of interfacing as anything more materially substantive – of immanently joining and modulating together to produce new movement, to drive differentiation/ bifurcation. Parasitic machinics here produced not a linear evolution of the work, but rather enabled processes of transversal connectivity and entanglement (O'Sullivan 2006, 17). The parasitic action of interfacing was an agent of difference in that it continued to transduce relation. It kept the event always on the point of splitting and moving into multiple new forms, suspending it in unfolding differentiation, disrupting any simple or sustained connectivity. As such this contingency operated both on the level of actualised relations (how a given participant's presence and gestures interfaced within the larger event), but also on a level of the development of potential dimensions of operation. This affected how the very rules that constituted the larger event began to evolve and complicate, beginning to creatively disrupt some of the more potentially problematic interfacing that might occur in the work to fix participants in the gaze of the technology's eye. While disruptions to intention are not unusual within works such as this, designed to accommodate interference, what is notable is the degree to which such interference overtook the original structures. #### Folds – the vibration of the incompossible If parasitic action was, in a sense, a continually performed splitting of relation, the interfacing that occurred in Re:Positioning Fear might also be thought as producing difference through connecting, through incitation or a 'dynamics of infection' (Stengers 2011, 160). That is, through a folding of technological objects and bodies in interfacing, something new was produced (art). As Andrew Murphie writes, this is a doubling that technologies can perform (1996, 89), in this case the body becoming-with the lights, the façade becoming-with shadows, portraits becoming-with movement and so on. Rather than collapsing difference to produce a new homogenous history or façade, this folding multiplied difference to produce new singularities that were performed alongside, throughout and in the gaps of the previously existing iterations.280 Thus, for example, in folding shadows that had a single purpose now performed (at least) two operations. But this was not simply a doubling of function, as folding overlaid and intertwined the two actions: to complement, overlap, interrupt, and fragment each other, creating multiple shifting moments of differentiation out of what was initially a fairly simple folding. Interfacing here was a performative act by which the machine continued to re-fold its internal systems, and fold elements outside itself (various bodies, intentions, movements, tonalities, and so on) into its workings. This created, as Deleuze writes of such actions, a 'forced movement' or 'internal resonance' within the system (1994, 118). As discussed in the previous chapter, resonance acts like 'contrast' in Whitehead's system of concrescence to conserve 'tensions in the form of a structure' (Simondon 2009, 6)281 – differences that are transduced through being 'topologically and temporally restructured across an interface' (Mackenzie 2002, 25; Sauvagnargues 2102, 66–7). This ongoing and continually productive resonant intensity-withoutresolution was a 'machine' producing ongoing and new potential. The new shadow play on the façade overlaid the original projections; as the artist's intentions and the new tonalities participants brought to the event continued to question one another; and as one participant's shadow intentionally and/ or accidentally overlapped in new combinations with other pedestrians' movements, gestures and poses. Such enfolding and resultant resonance might suggest that the event of Re:Positioning Fear had moved on from a relatively stable state of equilibrium where potentials of the event were actualised and 'no more force exist[ed]' (Simondon 2009, 6, 8), and where each component was kept at a regular spacing or relationship to one another that did not significantly develop over time. Instead it had developed into what Simondon terms a 'metastable' system, 'supersaturated' with potential that was always individuating (2009, 6). This potential that was always immediately available 'without distance and without delay' (Simondon 1995b, 225), recharging itself through the re-enfolding of components. This might be a diagrammatic system that was a 'place only of mutation', whereby forces were 'in a perpetual state of evolution', and were 'inseparable from the variations in their relations' (Deleuze 1988, 71). Here folding implicated machinic components in each other's becoming through an ongoing and inventive process of variation and re-articulation – repetitions that produced difference.282 In this entanglement or nesting the event became 'polyphasic', in Simondon's terms, a condition whereby there is a 'persistence of the primitive and original phase in the second phase, and this persistence implies a tendency towards a third phase' (cited in Combes 2013, 46).283 This third phase is, as Combes explains, the genesis of collective individuation. It is by drawing on the 'preindividual shares [of nature]' of potential remaining post-individualization that 'individuals can give birth to a new reality': a collective individuation that 'reunites these shares of nature charged with potential' (2013, 47–8). Such collectivity 'is not a result of relation [between individuals]…it is relation that expresses individuation of the collective' as 'its own operation of individuation' (Combes 2013, 47). Thus the new shadow play in Re:Positioning Fear gave rise to a new collective event with its own set of transindividual relations between participants, façade, lights and projections. This drew on the un-actualised potentials remaining within these components, folding these virtual remainders together; addressing collectively those problems that were not resolvable on an individual level (Grosz 2012, 50). Here we can see the event itself taking on a new level of self-generative power – new intensity – through a resonance that drew on but surpassed the potential of participants, artist, technological objects, architecture and so on.284 Thus the event's creative power might be in both the creation of actualised and potential foldings that the interfacing opened up, and in a bifurcating of future enfoldings that resonated within the event. I want to suggest here that this more radical folding occurring in the interruption of Re:Positioning Fear might also be seen as a fold of the outside. The 'outside' or 'incompossible' is force in non-relation (Deleuze 1988, 72; Deleuze 1993, 60) – itself a disruptive gap in the relational field – that 'eats into the interval and forces or dismembers the internal' (Deleuze 1988, 72). This can produce a reorganisation that is a 'trans-formation…to the composing forces, [which] enter in to a relation with the other forces which have come from the outside' (Deleuze 1988, 73).285 The participants' shadow-body play was an outside of the event, which was folded into emergent relation, at the level of force as well as form. This folding began to transform the affects of the event, since affect is what is experienced in the transduction of force (Deleuze 1988, 60). The new affective tonality that was folded into the event coursed through, transducing, infecting all the systems and delimiting the event.286 This was a force of qualitative change, of affective tonality. Interfacing here might be viewed as a 'vitality affect' on a force, 'elicited by changes in motivational states, appetites, and tensions' (Stern cited Manning 2013a, 5),287 producing a felt moment of creative differing. What is it that can be conceived of as truly outside of the event? Not the participants themselves (it cannot be any 'composed form', Deleuze argues), but these emergent and composing affectual forces outside of any form (Deleuze 1993, 73, 72). This again is more than the individuation of an event within an established field – the remarkable point within an already constituted ecology. It is the force of individuation that makes both the event and its paired environment appear (Simondon 2009, 4–5, 14 n.2): an impersonal individuating force that precedes relation, preceding but acting to gather an ecology. The new dimensions of the shadow-play event in Re:Positioning Fear did not exist within the initial registers of the systems (technological, psychological, social). The plane connecting one participant to another, to the lights, to the artist, composed itself as components folded, gathering force for a collective individuation and the evolution of a shared associated milieu.288 This was interfacing as not only unimagined prior to the event, but 'unreasoned' and unthinkable in its entirety, in that it's potential might be 'impossibly enveloped in a…still undefined experience, compoundly unpreviewed' (Massumi 2002, 97): known and knowable only in its performative individuation, in its relativity to the genesis of its collective invention.289 #### Concretisation and the diagrammatic I suggest that it was through these particular interfacings that the machine of Re:Positioning Fear underwent a process of concretisation: shifting systems from a limited, linear or closed functioning, towards self-regulation and sustenance, and consequently, towards a 'solidarity of openness', an increase in self-generative capacities (LaMarre in Combes 92–3).290 This might be a critical point in the folding in of the outside at which new system-level rules begin to operate.291 Interfacing here might be seen to have incited a phase or register-shift through transduction, implicating the external. That is, these radical interfacings acted to create completely new milieus.292 More than modulating transduction, a new machine was produced from the field when the system passed a 'threshold of [qualitative] intensity' (DeLanda 2005, 18–19), forcing new flows, with their attendant individuations, to begin. The interfacing of the incompossible, here 'vibrating against the conformal' (Whitehead 1978, 188), instigated a leap or jump of registers, whereby a point of 'absolute origin' (LaMarre in Combes 2013, 86) of a new event (and a new type of event) was produced.293 As I have hinted at in the previous section, one might term such an ongoing and provisional and essentially co-producing and becoming-concrete type of event 'diagrammatic': a system of 'ongoingly organized and redistributing gatherings' of its own making (Arakawa and Gins 2006, 56). Here what Re:Positioning Fear becomes as it moves from the linear to the concrete is a super-charged, dynamic ecology. This is not the replacement of order with chaos, but a different mode of operation that perhaps 'leaps over chaos', a 'catastrophe' (for the original artwork) that is also a 'germ of (new) order or rhythm' (Deleuze 2002a, 84, 83).294 A diagrammatic modelling (for it is never a 'model' in any fixed sense but an ongoing process of re-inquiry into its own status or dimensions – a metamodelling) moves towards an immanence of production and connection, across (and in conversation with) both actualised and potential planes, and can only be understood on a global or ecological level. Simondon argues that Euclidean notions of relation between fixed entities are inadequate to think the dynamics of over-lapping, intertwined and ongoing individuations, and describes this new and precarious mode of operation as topological (1995b, 223). Topological relationality creates a diagrammatic connectivity passing at once between such fixed points while at the same time expressing the whole event, not as a reunified object, but as the system of rhythms of contrasts or tensions or forces (Deleuze 2002a, 85–6). In the new 'charged grounding' (LaMarre in Combes 2013, 93) between internal spacing and external contrast a larger machine ecology began to gather or self-modulate on another scale295. Not only the event, but also the field itself had changed. Re:Positioning Fear had changed its nature, not only by actualising a previously un-actualised potential, but also by rewriting the very field of potential available to it. This meant that the work gained a greater capacity to generate its own emergent difference – a parasitic operation – and in this the parasitic actions on relation lead to a state of greater self-regulation and sustenance.296 #### Conclusion The shifts that occur in Re:Positioning Fear as a result of interfacing were both materially (ontologically) slight and processually (ontogenetically) significant. What the participants brought to the event that instigated such a shift was, in a sense, no more than a new intention, or perhaps even less distinctively, a new tonality that infected the work to produce something new. This is not to suggest necessarily that what it shifted to was in itself significant, but that the way that interfacings performed such a shift was of philosophical and artistic interest, in that it provides a potential tactic towards the thinking of more open-ended systems of interactivity, suggesting a potential machinic, 'minor' art event, concerned less with signification than a collective becoming (O'Sullivan 2006, 69–71). This performative interfacing perhaps acted as a 'lure' towards feeling or transduction, as a pull towards the future (Manning 2013a, 57), a pre-relational tendency towards affectual relation or as a productive diagrammatic tension. Re:Positioning Fear was concerned not with utility in technology, but with, as LaMarre articulates, Simondon's plea for relations with machines that might instigate sustained inventive engagement (LaMarre in Combes 2013, 97). The objects, such as they were, in Re:Positioning Fear – lights, buildings, shadows – can then be seen to move towards what Manning has termed the 'objectile': propositions for engagement 'emphasiz[ing] the temporal and qualitative' (2013a, 148, 149). The event, one might say, answered Stern's call for interactive art to move away from privileging signs and images at the interface, and the demonstration or fetishisation of the technology in the work. Instead the event engaged, as Stern proposes, with the invention of styles or qualities or emergence, with the implicit and the potential – to construct new ways of relating through interfacing (2012, 10). #### Refrain: Fuzzy interfacing In Momo interfacing occurred between bodies and the sculptural forms (see Figure 8.2) through a series of light sensors embedded in the main form, and movement sensors positioned throughout the space.297 Shadows cast by bodies on the central sculpture increased the volume of various audio tracks, providing a fluid mix of sounds.298 This operation of interfacing was qualitative in its nature as multiple light sensors spread over the surface of the sculpture registered subtle variations in the intensity of shadows falling across its form. These variations were dependent on such factors as the distance of bodies from the sculpture, the density of materials blocking light (a thin fabric versus a limb, for example), the exact angle of a particular light sensor in the folds of fabric, or the collective volume of the shadows of bodies momentarily overlapping, alongside subtle potential changes in the overall light in the room. Such qualitatively based sensor interfacings were perhaps a step towards a more fluid connection of components that began to move away from a focus on delineating and capturing or interpreting individual bodily actions and towards a fuzzy collective expression of the movement of the event itself. By this I do not mean to imply here any set division between analogue and digital sensors. Rather, there might be some distinction between motion capture systems such as those utilised in Wii or Xbox to translate body part movements onto a Cartesian grid (and that seek to address not only the participant's body to the exclusion of any other environmental changes, but also to focus rigidly on a relation between the intentional actions of the subject and the software), and the fuzziness of a qualitative sensor registering the variation in the collective sum of a particular force over time. Beyond this hardware-based interfacing of the sensors, Momo also proposed more ephemeral interfacings. These speculated on the resonance of the meeting of affectual tonalities between the participant and aspects of the work: the infective tonal qualities of the vocal qualities and the garish colour palette, for example. Such partially unintentional interfaces began to capture difference on an environmental level. These were not only well outside subjective consciousness, but also outside a larger sense of a single body. This fluidity created inexact, unstable connections: a disruption of clear relation through a vagueness that might be 'due to an excess of identification', not a lack of connection, as Whitehead states (1978, 111–12). Here the contrasts between groups of actual objects that the sensors sought to hold a relation to were indistinct and appeared as 'one extensive whole' (though this whole was divisible), and the feelings prehended were therefore sensed as 'chaotic factors' (Whitehead 1978, 111–12). Given the somewhat abrasive and confrontational nature of the sounds emanating from the sculpture, and its increased vocal 'agitation' in reaction to the proximity of the participant, these sounds then might be seen to have begun to feed back into the styles of movement of bodies within the space. In this way, the event perhaps began to take on its own collective energy, a folding in the meeting of affectual tonalities of the event and the participant – a resonating of different moods and intensities – a collective shifting and gathering. This interfacing gathered, to some extent at least, qualitative gestures within the event, rather than enforcing privileged conversations. That is, the event became sensitive to collective sums of reactions, directions, styles and speeds. These might be seen as transversal connections, as ongoing acts of the transduction of flows of forces across bodies and objects that co-implicated them in a collective, performative emergence leading towards concretisation – a shared potential or transindividuality (Simondon 2009, 9). In this, it began to gather a collective field for the event to draw on, beyond the combined individual potential of the component parts. More than simply being entities communicating across an interface, bodies, sounds, colours and lights became fluid (topological) genetic components intensively driving an event of collective expression. Such concretisation, through a shared responsibility for the emergent event, neither subsumed the will of the work to that of the participant, nor vice versa. Though participants affected the modulation and flows of sound, as the installation contained the potential to coax certain styles of behaviour from bodies, the expressions of both added further variation and intensive movement. Connective possibilities generated and intensified, rather than collapsed, difference. For example (on the most concrete level), a gesture of one arm created subtle variations in shadows across a number of light sensors and simultaneously triggered the switching of audio samples through sensed movement. Meanwhile the counter-movements of the other arm might temporally combine with areas of shadow and send contradictory sample-swapping messages to the computer system (again an increase of resonance or held contrast in and driving the individuations of event). Interfacing here potentially directed intentional and accidental movements into multiple and overlapping chains of causality – creating multiple relations between a body part and the work – and also provided mechanisms for variation through instability of its relations. Again, it is important to note that the event was not concerned with representing these interfacings to the participant, or with enforcing any one particular set of relations, style of movement or feeling of connection, but with affording a variety of potential connections. In this instance interactive interfacing at least began to move towards the consideration of the infective potential of a series of resonating or contrasting styles and tonalities. It began to consider interfacing as an intensive (and therefore parasitic) action within an event – a folding back of the event into itself to gather collective forces – with inexact edges and eddies at which difference might pool. Figure 8.3 Andrew Goodman, Momo, (detail), 2011. Paradise Hills Gallery, Melbourne. 9 ### Sacrificial RAM: locating feeling and the virtual in software One of the questions ahead of us now is this: what are the conditions of digitization and binarization? Can we produce technologies of other kinds? Is technology inherently a simplification and reduction of the real... What might a technology of process, of intuition rather than things and practices look like? Elizabeth Grosz How is it possible to think through from a normative freeze-frame of representational to a more machinic or rhizomic approach to technology? Andrew Murphie #### Introduction: towards a technical ontogenesis In 1996, Rafael Lozano-Hemmer published a short article entitled 'Perverting Technological Correctness' in which he suggests a number of potential 'misuses' of technology to trouble the aura of 'technological correctness' surrounding the promotion of digital technologies within art practice (1996, 5). While the suggestions themselves are lighthearted (they include wearing a hollowed-out computer on one's head), they reveal a commonly held suspicion about the mechanical role of the computer in art, and the dangers of 'perfect replication' through the use of the digital (Lozano-Hemmer 1996, 6). How to make a computer program in itself behave in anything remotely approaching a 'relational' mode rather than simply working around such issues is important, and inherently political (Fuller 2003, 29–30; Dery 1996, 14). That is, in order to question and pursue the further molecularisation of an interactive artwork across all its registers, the speculative and non-totalising potential of algorithms must be addressed. In this we might seek to liberate digital programming from representation and productive 'purposefulness' that are its legacy in disciplinary structures. Within an expanded empiricist framework, all relations demand to be seen as real forces that must be accounted for within an ecology. Yet the actual nature of algorithmic events, as Luciana Parisi argues (2013, 10–11), is often denied adequate explanation within the schema of relations. In order to remain true to a process philosophy view of the world the becoming potential of an algorithm must be explored, alongside that of the potential of all the other components of an interactive artwork. Here we might seek a way of thinking the primacy of technical process or techno-genesis within computers. Whitehead seeks to develop a process-based philosophy 'applicable to any kind of actual occasion' (Stenner 2008, 99). For this to be consistent, as Whitehead aims, I would argue that it is necessary that it be as applicable to the workings of a set of code as to any other occasion. Thus rethinking software interactions demands the finding of a becoming-minoritarian potential of computational processes – an ability to disrupt structuring and destabilise any 'whole' that is based on the transcendent replicability of software process. Again, as Parisi advocates, it is necessary to question the whole philosophical basis of thinking about code in order to find a new and specific way of tackling the problem at hand (2013, 3–5). Therefore, contrary to notions of code as a mechanical process incapable of further potentiality, or as immaterial representations that are transcendent of empirical dynamics,299 an algorithm must be shown to be 'machinic'. That is, it must be capable of acting as an assemblage primed and therefore capable of shaping it's becoming as a real event, in and of itself, rather than a mechanical assemblage that produces a repeatable result. If we want to truly concern ourselves with the 'ethics of relation', that is, an attention to the event in its emergence that does not deny the potency of any of the composing forces (Manning 2013a, 213, 171), then we need to consider seriously how to afford the performativity of algorithms. This involves thinking through how the potential written into code can become temporal events of actualisation, and addressing an algorithm's ongoing potential for engagement with both actualised entities and 'eternal objects' – the infinite potential variety within these entities (Parisi 2013, 63). In this chapter, I will attempt to think the machinic potential of an algorithm (a 'step by step procedure for calculations') (Parisi 2013, 259)300 and a software patch (a set of sequences of algorithmic processes created within a software program). This discussion, unlike most of the preceding main chapters that have other artists' work as their primary discussion points, moves for practical reasons directly to focus on a software patch developed for Orgasmatron – one of the works made in conjunction with this book.301 After a brief description of the relevant aspects of the work, I discuss the software patch in relation to some relevant common aspects of generative software design in order to discuss both these concepts' relevance to the artwork, and how the software design attempts to move beyond these paradigms. In thinking beyond these concepts, I then discuss the work in relation to the more promising potential of algorithmic prehension in order to argue for an algorithm's acceptance as an entity in its own right, and then examine how the design utilises systems of parametrically linked multiple attractors to modulate data in non-linear ways. ### Orgasmatron In Orgasmatron, 302 data from pairs of sensors303 embedded in the structure of the work was fed into the computer to be utilised by the software patch, created in the Isadora program.304 Through a series of algorithmic processes this data drove ongoing variations in light, sound, sound spatialisation and vibration. The processes by which incoming data was modulated are briefly described here (see Figure 9.1).305 Firstly, 'Differential' actors: here the data from a pair of sensors was processed in an algorithm utilising a differential equation to calculate the rate of their difference differing over time. For example, two pressure sensors (embedded in opposite sides of the floor of the work) measured the shifts in pressure as a body moved across the surface. As an equation this can be expressed by (xi – x)/(yi – y), where 'x' and 'y' are the initial readings of the two pressure, and 'xi ' and 'yi ' are the pressure sensor readings taken 0.1 of a second later. This provided a series of numbers that reflect the rate of change of pressure on one side of the structure relative to the rate of change of pressure on the other side. The result of this equation was then constrained within a range of 0–100. Secondly, 'Watching' actors: Here a set of algorithmic actors watched the numbers outputted from these equations, looking for a particular range of numbers with which they interacted, and then counted the incidence of such numbers within the constraining parameters. For example, one such algorithm might look for numbers between 0.001 and 1.0, or between 10 and 20, and so on. In this sense, these algorithms acted as a 'gate', allowing the flow-on of certain data through to the rest of the system, while ignoring other data. That is, the watching actor had the capacity to be positively affected by, or interact with, certain data and had a relation of non-relation with other data, as it actively ignored data outside certain ranges, dividing data into two groups, creating a 'positive' relation with data accepted, and a 'negative' relation to rejected data. As will become important to the argument that develops below, each evolving set of differentials was 'watched' by (or was capable of interacting with) more than one of these 'watching' actors, each with gates of different parameters, so that the affectual potential of the flow of data was split in ways that might also overlap. Thirdly, 'Triggering' actors: Once the watching actor had counted to a set number of positive interactions, this triggered the sending of data to the next series of algorithmic actors for further modulation. This next set of actors also watched for numbers within certain parameters with which they could interact, while similarly rejecting other data. These actors counted a certain number of interactions, and then sent the data flow to further algorithms that triggered a range of video projections and sound events.306 Fourthly, within both the watching and triggering algorithmic actors, the ranges of data looked for, and the numbers of such incidents counted, were designed with variable parameters. While each of these parameters had an initial set range or number, they were linked to both its own and each other's outputs, so that they changed over time. That is, the range of numbers being accepted at each 'gate' increased or decreased in response to the amount of stimuli received by the set of actors, while the threshold number of such events being counted before triggering the flow-on of data also changed in response to the activities of the system. In this way, the ability of an algorithm to be affected developed complexly in relation to its neighboring algorithms.307 Fifthly, amongst the triggering set of actors described above were actors whose outcomes triggered the activation of additional watching and triggering actors, thus potentially utilising and splitting the data flow-on in further directions. This will be discussed later in the chapter in terms of a 'bifurcation' of the system that created a new set of relations inclusive of previous relational factors within the system. #### Generative software design Any discussion of the programmatic nature of computer operations and codes, within any artwork that is attempting a generative or open-ended approach, must acknowledge some of the strategies that have been previously employed and their (at least) partial success in creating larger systems that have open-ended characteristics. In most cases, however, these strategies do not adequately address the non-linear potential of algorithmic process itself. While it is not within the scope of this chapter to provide a detailed account of the various approaches that have been taken, I want to here very briefly discuss three areas that retain relevance to the larger system utilised in the Orgasmatron project.308 These (related) approaches, at their simplest, concern: firstly, attempts to 'diffuse' the linear nature of computer processes through their integration into larger and principally analogue based systems; secondly, the use of complex feedback systems interacting with software processes to create biologically imitative self-generative systems (secondorder cybernetics); and thirdly, attempts to make code itself behave in a generative or evolutionary manner through the use of parametric feedback. #### Diffusion In practice, many software-generative works are in fact assemblages of software, sensors, participants' bodies, and other aesthetic elements such as larger environments of sound, light or sculpture. Many such works rely on the integration of these components with software to 'diffuse' the digital technologies. The supposedly prescriptive digital data is 'diffused' within an analogue field, as qualitative flows of data stimulate movement in the software through the transduction of analogue signals into the digital, acting parasitically on each other. The analogue qualitative flow is disrupted by its digitisation and translation into binary code, while the excess of the analogue disrupts the digital. As such, it is easy to argue that in the larger context of its place within assemblages that include other elements, and within the larger social field within which it must also be seen to operate, an algorithm or code begins to become extensively indeterminate.309 In these tactics the injections of data might be said to be relational rather than purely chaotic. But while they may have clear creative potential in opening systems to novelty, in isolation (that is, when they are proposed as the only generative tactics rather than perhaps operating as one element on a particular scale in conjunction with other generative propositions ), these approaches still rely on working around algorithmic prescriptiveness and ignore Parisi's more radical proposition that an algorithm itself might be thought of as intensively indeterminate. Potentially implied in this approach is the problematic acceptance of an always-clear analogue/digital divide. As Anthony Wilden argues, distinctions can be made between the continuous qualities of analogue variation, and the discontinuous scales of digital differentiation that then operate through different kinds of differentiation (1980, 158). However, he also argues that discrete definitions of the two are problematic, and more concerned with the ways in which entities relate than any innate qualities. Many processes in the world involve both analogue and digital on differing scales within the one event of communication (1980, 188–9).310 In addition, when viewed as events of relation, the digital is always saturated with the rhythms of the analogue in the form of gaps, interruptions, processing time, and signaletic noises (Wilden 1980, 158).311 Thus, as Wilden acknowledges, the translation from analogue to digital can result in loss of ambiguity and meaning (1980, 163), implying that the digital Figure 9.1 Simplified graphic representation of Actors in Orgasmatron patch. is just a poor replica of the 'real' analogue experience. I would argue that within an expanded empiricism the digital might also be thought of as a different but potentially creative and real mode in its own right.312 #### Second-order cybernetics A second-order cybernetics approach involves assemblages of positive feedback chains between components in order to develop complex systems of relation from the 'bottom up' (Parisi 2013, 261). Such systems, as Francisco Varela examines in his discussion of drift, create connections of 'viable coupling' with no regard for an end point (Varela, Thompson and Rosch 1992, 205).313 Here, feedback emphasises the processual – the ways in which elements are drawn into relation and the fact that all these components play an active role in this emergent organisation (Murphie and Potts 2003, 192). In the Orgasmatron system, the enmeshing of data from the larger artwork assemblage created relations between the workings of the computer and the other component parts through systems of feedback. Feedback loops were established across the various inputs and outputs, for example, as shifts in pressure triggered sound events, causing vibrations to be sensed, which then triggered light events, causing light variations to be sensed, that then cause vibrations to be sent, and so on.314 In addition, the bodies of participants' became implicated in these systems, adding their own rhythms and disruptions to the speakers' vibrations, varying the pressure applied on parts of the floor, and creating variations in light through shadows cast (as the system also worked to disrupt the bodies' rhythms). Bodies were then drawn into relation with other components of the larger assemblage, and the system was primed to afford the gradual development of such relational complexity, as various components became further implicated in each other's expressions. Without particular concern for any endpoint, the system was always in a state of reconfiguring its feedback loops. This transduction of forces within feedback systems emphasised movement or circulation over established relations and, in this, might be seen to be heading towards becoming-molecular configurations. Parisi, however, critiques such self-organising, second-order cybernetic models as relying on the actions of biological elements directly animating algorithmic objects to build a responsive environment (Parisi 2013, 33).315 Again, these models might tend to imply that the environment exists only outside of algorithms, rather than seeing these objects themselves as being composed of environments of relations, and thus 'discard the possibility that change could concern the formal logic of computation' (Parisi 2013 36, 11,13). Parisi argues that such systems still treat computation as a passive, non-aesthetic component and potentially infer that aesthetics can only be found within sensation and not within algorithmic processes (Parisi 2013, xv). Thus feedback systems, Parisi argues, contain computational potential by demanding that its primarily relation is to an external environment that it responds to (2013, 155). If such systems also allow only positive and enduring connections between the components then this limitation in the rules governing their relations may well, as Parisi argues, prime them towards the organisation of a stability of connection and a molar thinking, rather than a continued emergence and ongoing potential for relational movement to be expressed (2013, 35). However I am not entirely convinced by this aspect of Parisi's critique of autopoiesis and its reliance on feedback to maintain a whole, which I read as a narrow definition of the possible range of potential becoming-autopoietic systems, and possible qualifications are discussed below. #### Parametric feedback Such systems might, for a start, be extended by a 'parametric' approach within the software patch itself. Where generative software seeks to create complex forms through sets of simple rules and variations of forms, a parametric approach, as argued by Portanova, shifts the emphasis towards the programming of relations between these rules or algorithmic processes, affording another scale on which feedback operates and co-implication develops (Portanova 2013, 87). This concept of 'parametricism' might be in line with the 'ecological' approach that Jon McCormack, amongst others, has advocated. It involves the creation of a field of what McCormack terms 'conditions and resources' (that might be better termed as a series of environmental propositions) where heterogeneously distributed 'mortal organisms' draw both from the field (in some cases including the presence of viewer's bodies) and their relations to each other, and have some ability to develop their interactive parameters (McCormack 2012, 51).316 These systems, as McCormack argues, draw components into interdependent relations through feedback on a component-to-component level, and are self-organising and dynamic in their modulations (McCormack 2012, 45), developing system-level relationality as a by-product of these interactions (McCormack 2012, 48). As described above, the Orgasmatron software patch linked some of the parameters of the operations of its 'watching' and 'triggering' algorithms to each other, so that they changed over time in relation to the amount of stimulation various parts of the system received. This in itself was a parasitic disruption to established relations, as it replaced stable capacities to be affected with the vagueness and fuzzy logic of contingent and evolving parameters of potential relation. As it gathered parameters into co-implication in each other's modulation, it also created a rolling or gathering of excitation of the system. Here stimulation lead to increased potential to be stimulated – leading the system towards a 'far-from-equilibrium' state, rather than a stasis of connectivity that cancelled further potential movement. Such far-from-equilibrium systems, Manuel DeLanda states, maintain intensive differences, 'mesh[ing] difference' rather than cancelling it, and thus the potential for change remains active within the system (2005, 74–5). Accentuating this non-equilibrium state, he argues, puts systems in a condition of heightened potential, what he terms a 'zone of intensity' of operation that moves away from linearity (DeLanda 2005, 76).317 Here, parametric systems might begin to escape the purely positive feedback of second-order cybernetics in utilising feedback within algorithmic relations. Instead they begin to draw together and further complexify the computational conditions in which such relational play might occur – encouraging an intensive movement in the shifting of relations between the component algorithms.318 In Orgasmatron, local algorithmic excitations infected the parameters of neighboring algorithms, creating a molecular movement, and it is only through these complex and speculative chains that effects on the system as a whole emerged. In emphasising models of interference and parts over wholes, parametricism (as an extension of feedback systems) can, as Parisi argues, begin to escape pre-emptive control, and the smoothing or flattening of novelty that is problematic in topological systems. Parametricism thus interferes with the smooth 'capitalization of change, futurity and potentiality' (Parisi 2013, 92–3). In her critique of the problematic smoothness of such topological models of self-organising systems, which she considers as a form of 'post-cybernetic control' (Parisi 2013, xvii),319 Parisi contrasts them to 'mereotopological' systems, which she suggests, consist of this consideration of the whole as divisible space, and a concern with its interior parts and the relations between the two (Parisi 2013, 123–4). Mereotopology, as Portanova writes, emphasises 'not only wholes and parts, but the boundaries and interiors of wholes [and] the relations of contact and connectedness between wholes and parts' (2013, 79, 76–80). Potentially still implicit in this parametric approach is the idea that the digital can only be made to behave more openendedly by making it operate in a pseudo-biological manner. This refusal to acknowledge the potential indeterminacy at the very heart of coding processes reflects, Goodman and Parisi write, the 'anthropocentrism of interactivity, which pervades recent conceptions of digital architecture' (2009, 1).320 Technical machines, as Pickering argues, have their own singular ways of relating (1995, 186–7) and algorithmic processes have their own specific modes of thought (Parisi 2013, 186).321 Their processes should not be erroneously conflated with representations of the biological world, as happens in cognitive approaches.322 Instead of constructing algorithms as 'tools for thinking' in order to enhance abilities to plan and control – a 'mechanics of possibilities' (Parisi 2013, 169) – Parisi advocates for a 'soft(ware) thought…producing computational space-time' (2013, 169). This 'software thought', which she describes as the architecture of a new, specifically digital, mode of thought (Parisi 2013, 169), can be clearly linked to the need to rethink interactivity and its use of computer technology, moving it away from systems of control and manipulation that curtail potential, and towards more openended and collectively creative expressions. The Orgasmatron assemblage clearly utilised combinations of analogue and digital processes and feedback systems – including parametric feedback – to varying degrees in order to create multiple systems of relation, and as such it is open to the criticisms of such approaches mentioned above. However, as I will attempt to show in the following discussions, employing these tactics did not necessarily occur at the expense of ignoring the creative potential within algorithmic processes, but as a supplement to it – allowing a range of complementary tactics within various component parts of the overall system and across relations between these parts. In the following section, I explore how it might be possible to move beyond these limited conceptions of the operations of code through a utilisation of Whitehead's concept of prehension. In the third section of the chapter I then use this concept of algorithmic prehension, and its implication of the existence of an algorithmic potential, to lay the groundwork for the exploration of generative systems that utilise parasitic disruptions to drive creativity. This is explored through the concepts of attractors and bifurcations – emphasising the parasitic potential within generative computer processes that might move towards machine ecologies. #### Algorithmic feelings: a digital mode of thought In order to establish that algorithms are more than 'simulators of material dynamics' (Parisi 2013, 1), it is necessary to demonstrate how they are actualised entities in their own right, with accompanying obligations and powers within a schema of the play of forces. To do this, Parisi draws on Whitehead's system of prehensions, as it is, she argues, an entity's prehensive capabilities that define 'what an entity is and how it relates to others' (2013, xii). A system of prehensive feeling describes 'how any actuality…grasps, includes and excludes, and transforms data' (Parisi 2013, xii).323 As discussed in earlier chapters, Whitehead argues that in prehending an entity creates a system of relation or 'extensive connection' – including both conjunctive and disjunctive connection – that connects it to all other actual entities (1978, 41). At the same time, this is reciprocated, as any entity also acts as an 'object' to be prehended by all other entities.324 Thus, an actualised entity must influence the individuation of entities it forms relationships with, as they must influence it, 'however trivial or faint' this influence is (Whitehead 1978). It should be noted that there is no essential distinction in this ontology between conceptual and material entities, living and non-living, or between what constitutes a subject (that prehends) and an object (that is prehended) (Shaviro 2009, 23). Whitehead is adamant, however, that despite drawing on the datum of objects, each new entity is 'freed from those entities' histories', having its own subjective feeling that is different to the previous entity's feeling on which it draws, and that translates (transduces), rather than simply duplicating the original force (1978, 238, 236).325 In this there is a creative but atomic advance that builds on what exists, but which is also always capable of movement and further complexity. It is also always a singular point of complex negotiations between all the entities whose forces influence it.326 In this system, Parisi says, Whitehead manages to conceive an understanding of relations as being 'both more than effects and less than the projections of a perceiving subject' (Parisi 2013, 59). Here prehensions form the 'indissolvable atomic architecture of any occasion' that is therefore both actual while never complete or static (Parisi 2013, 60). In this sense, no entity (including an algorithmic entity) can be said to be purely predetermined, but selects the manner and degree to which it is influenced by other events – it gathers singular and particular relations to the world that define its existence.327 Actual entities are therefore always individual, actualised realisations of potentialities, but never fully stable or 'whole', and process here is conceived of not as a self-modulating whole, but as a system of parts that are nevertheless all related and capable of affecting each other (Parisi 2013, 61). Process is therefore self-organising but molecular, as each component has its own subjective power to relate and ingress into other entities without regard to any overall design or configuration. Algorithms, Parisi argues, are necessarily engaged in prehension, or selection of numbers with which to interact from a larger field or potential that contains incomputable numbers – the actual and discrete passages between and combinations of the zeros and ones that make up binary code (Parisi 2013, 64–5) and that can be endlessly arranged and extended (Portanova 2013, 127). Parisi draws on mathematician Gregory Chaitlin's theories that classify these incomputable objects as 'Omega' (Ω) (Chaitlin 2011, 126; Parisi 2013, 17–18).328 Chaitlin's theorem draws on earlier work by Gödel and Turing that shows that, contra to common sense, most real numbers can never be entirely or definitively calculated or known. That is, they remain problematic: incomplete and uncomfortably 'ugly',329 and resistant to axiomatisation.330 Omega is a real number between zero and one,331 yet it cannot be calculated through any other, smaller processes or patterns and is therefore 'algorithmically and logically irreducible' information (Chaitlin 2011, 137).332 Thus Omega shows that calculation of particular sequences of ones and zeroes cannot fully precede the event of that code's coming into being itself – the entity and its process of actualisation are reciprocal, belonging 'to the register of creation itself' (Stengers 2010, 42). Omega proves that there remains an excess – an incalculable and problematic uncertainty lurking behind binary code (Portanova 2013, 126). The potential in Omega lies in that it represents not simply 'an empty repetition of the same' ones and zeros (a 'self-varying deformation' or topology), but rather the potential novel and 'infinite addition of one more possibility' (an infinite number of ones and/or zeros to the sequence) (Portanova 2013, 127). As these infinite, real infinitesimals (endlessly divisible fractions between zero and one) and sequences (endless combinatory possibilities of zeros and ones) cannot be compressed into any one algorithmic operation, Parisi says, they are best thought of as an 'incomputable virtuality'. They operate as a multiplicity or 'eternal object' – that is 'patternless and random, objective and undetermined' and that cannot be contained into any smaller set of rules (Parisi 2013, 126, 65).333 These are 'indeterminate conditions within which algorithmic objects are able to exist', they are unsynthesisable quantities that disrupt and open algorithms to a greater potential (Parisi 2013, 204). Here there is a 'strain' between limitless (both virtual and incalculable) and limited (specific algorithmic functions) (Portanova 2013, 57). Any algorithm speculatively contracts potential and determines positive and negative relations with numbers it both can and cannot contain – 'demarcating an immanent, actual space of disjunctions and conjunctions' (Parisi 2013, 240–1).334 In the Orgasmatron software patch, not only were these incomputable and disruptive transitions inherently present within each algorithmic process, each 'watching' algorithm selected, evaluated and produced data for use by other such entities, thus becoming a 'performing extensive actuality' (Parisi 2013, ix). The 'watching' actor made a selection of some data to interact with – a positive prehension – while rejecting interaction with data outside set parameters. This selection established a positive prehensive relation with some real numbers, and a negative prehensive relation to both other real numbers and incomputable numbers: it drew positively on some of the potential, but never all of it. This was an act of selecting that was an unseen but nevertheless a real moment of transition and therefore indeterminacy between actualised determined occasions. In exercising its capacities to prehend and utilise data – in order to realise potential and resolve its satisfaction as that particular temporal and spatial algorithmic process – the 'watching' actor established itself as a singular vector of actualised relations. Given that the parameters of numbers the 'watching' actor prehended were themselves modulating, in this it performed a certain choice or capacity to connect or feel that was not purely prescribed or linear (that is, a simple positive connectivity). We must remember that this was an 'automated prehension' (Parisi 2013, xii), with its own particular algorithmic type of prehension, rather than a simulation of other entities' ways of feeling. Moreover, in that each 'differential' algorithm in the system was watched by multiple algorithms with differing parameters, at any particular moment in the process, a number calculated could be 'felt' and prehensively utilised to drive the various 'watching' algorithms' processes in multiple different ways. This established extensive, but speculative, immanent connections between not only a 'differential' algorithm and each watcher, but between the watchers themselves, in that a number positively or negatively prehended by one was also either positively or negatively prehended by all the others. In this, algorithmic prehensions allowed new complexity in the form of emergent contrasts to enter into the system, which then increased in differential intensity. These causal chains were 'ordinally' specific – they had a specific order in which their operations were linked – but left open other dimensions such as time and actual processes. Ordinal numbers ('firstness', 'secondness', and so on) specify an order but not an actual number. That is, they specify one rule governing a set of numbers, but leave all other parameters open to change, as numbers can be any quantity as long as they follow in order. Ordinal distances, DeLanda states, connect entities, creating a relation between, whereas metric distances separate events (2005, 126). Ordinal numbers are 'anexact yet rigorous', having a single determined spatial quality that allows them to function – 'this' is next to or after 'that' – while never strictly metric in leaving other spatio-temporal parameters open. This leaves as many factors as possible open to further individuation, retaining enough practical specificity to allow their structuring into a software patch (DeLanda 2005, 68, 81–2). The 'gate' function of the watcher algorithms was ordinal, specifying a position ('bigger than', 'smaller than' or 'between' numbers), while leaving the specification of these numbers open to change. Furthermore, the ordinal links ran not only from differential-to parallel watchingto-triggering algorithms, but also in multiple lines across from watching-to-watching-to-watching as they sequentially influenced each other's parameters. This constructed chains of causation that no longer prescribed to simple linear chains of events. A set of algorithm processes can be argued to have operated here within Whitehead's system of prehensive connection. Each actively determined its own actualisation by selectively drawing on data from multiple algorithms acting as objects for it, and was an object who's felt datum affected the ways other algorithms actualise. This, one might suggest, demonstrated a logic of infection that governed algorithmic operations with an open potentiality, rather than a fixed law that remained transcendent of the play of temporal forces – a process of temporal selection that makes immanent extensive connections (Whitehead 1978, 294; Portanova 2013, 10–11). When we consider an algorithm as an actualised machinic process, and not simply an abstract set of instructions, it is possible to argue that it is a temporal processing of data, no matter how infinitesimal that timespan is (Miyazaki 2012, 1).335 As Shintaro Miyazaki argues, algorithms and assemblages of algorithms must all have their own singular passages or rhythms of operation that are analogue noises within the digital process, delineating a rhythmic actuality from a field of potential (2012, 10).336 When this temporal quality of processing is taken into account, numbers produced by algorithmic process are always singular spatiotemporal actualities, infected with a parasitic analogue: with the micro-rhythms of transition that express a temporal ordering of processing, gathering a new relationship between the actions.337 Intensive rhythmic differences began to arise in the Orgasmatron patch as multiple 'watcher' algorithms waited for, and then actualised, the processing of data selected from a 'differential' algorithm. That is, each began at its own starting time – the moment it prehended a usable number range – and then took its own specific time to process. Thus what was a single flow of data was split (parasitically) into multiple nested cyclical timespans.338 These potential syncopations creating new rhythmic patterns of operation were evolving algorithmic refrains (patternings of temporal contrasts).339 As a system utilising parallel processing, and given that the 'watchers' could also affect changes in each other's operation, these relative processing times were critical to how the system developed as a whole, as well as to how its parts processed data flows.340 The syncopation in relations between algorithmic cycles could open new potential and actual configurations of relation to invent new modulations of data. Potential rhythms of operation are one multiplicity of qualities and quantities on which an actualised algorithmic process draws upon, along with potential ordinal sequences, potential parameters, and potential sets of numbers. In line with Whitehead's system of eternal objects, it is possible to argue that an algorithm draws on the potential of various numbers and mathematical functions as concepts, expressing some – whilst never exhausting all – of their potential. Here an algorithm 'nests' 'infinite parts of infinities' (concepts of numbers and functions341) within itself (Parisi 2013, 63), but these eternal objects – as 'the pure potentials of the universe' (Whitehead 1978, 149)342 – are never fully able to be contained or compressed within any one algorithm. These incomputable quantities are a non-linear 'second order' of relation, as the algorithmic entity expresses a relation to various (but not all) potentialities.343 In Orgasmatron, each actualisation of a differential algorithm produced a specific and temporal mathematical process.344 Here actual ordinal sequences arose out of cuts in larger potentials (this watcher algorithm next accepted the data, rather than that watcher), potential ranges of numbers were expressed and prehended, and so on. The excess of ongoing relational potential to the virtual was never exhausted by any particular actualisation. An algorithm then had a 'dipolar' relationship, drawing prehensively on both relations to the actual, determined world, and conceptually prehending 'the indeterminateness of the eternal world' (Whitehead 1978, 45).345 This potential was irreducible data – inexpressible in its entirety – that again moved algorithms beyond being merely 'systemization[s] of the possible' (Massumi 2002, 137), and demonstrated that they were always infected with an indeterminacy of the incomputable (Massumi 2002, 62). Each enaction of code was a singular and limited nexus of both physical prehensions and prehensions expressing a particular relation to larger potentials, and a material and conceptual realisation of some of its potential to interact with other material and conceptual actualities – the electrical and mechanical components of the computer and data flows, and the mathematical concepts. Here it is possible to argue that each algorithmic event was engaged in feeling in the Whiteheadian sense, a spacing or patterning of sets of external and internal differential relations or contrasts that constituted its very becoming as an actualised event. This applied not only within each smaller algorithmic event (a 'watcher' or 'differential' or 'triggering' algorithm), but in the relations between these events that became further entangled and intensified through their effects on each other's becoming (a collective individuation, producing both new relations between events and a shared field out of which such individuations emerged) (Grosz 2012, 42–3). This was then a speculative logic of algorithmic process, acknowledging a vagueness in its operations,346 that positions the processing of data as an open expression of the concrescence of algorithmic entities, not because the code itself necessarily altered, but because there was a level of indeterminacy in the potentials and processes that governed its operations (Parisi 2013, 144). This could never be fully positively accounted for in any iteration of the algorithm. Exploitation of prehensive potential in algorithmic processing of data enabled not a smooth modulation, subsuming all to a continuous whole (of design function) (Parisi 2013, 167), but a series of cuts that interrupted, contradicted and problematised. These cuts molecularised relations by creating further intensification or differentiation within the data-algorithm machine. In the materiality of actualisation, with its disruptions and rhythms, and in its continued non-linear relations to the further potentials, algorithms exercised particular capacities (ways of prehending), and became charged with indeterminacy. Here, algorithmic prehension was a parasitic action within the computer's operations, in that it broke with clear and absolute transference of data between algorithms, inserting difference into these relations. #### Systems modulating through disruption In order to further articulate the intensive noise within algorithmic processes, in this next section I discuss the concept of multiple attractor systems. I want to explore how accentuated intensive disruption can drive an open-ended futurity through systems of attractors. In this, I want to move further into the concept of speculative transitions between software processing events to continue to think through the software patch developed for the Orgasmatron project. #### Attractors To begin this thinking through of attractors, I want to consider software patches as non-metric (that is, 'projective, differential, topological') (DeLanda 2011b, 18) 'state spaces'– consisting of a system of 'attractors' that act on and organise the potential flow of force within the system.347 States are 'meta-stable', in that they are capable of self-organisation through their interaction with forces to accommodate change. They also have a 'tipping point', at which they 'bifurcate' and move to a new, related state with a new set of organising parameters or potentialities.348 Here it is important to remind ourselves that these state spaces are themselves only momentary cuts in ongoing processes of individuation of a system. That is, the system is involved in ongoing exploration and genesis of its potential, rather than any state representing a final or fixed organisation of forces and relations (DeLanda 2011b, 13).349 States organise through intensive differentiation, and the 'attractors' condition or influence the system and its modulations by influencing the longterm tendencies of differential trajectories.350 States then are the outcomes of differential processes, with attractors implicated in the genesis of the system (DeLanda 2011b, 15), in that they condition or lure the potential of forces as potential becomings, or pulls towards change (Massumi and McKim 2009, 9).351 An attractor is a tendency towards a terminus of a trajectory, and, while real, is never reached or fully actualised (DeLanda 2005, 29).352 The lure of attractors explains, without resorting to concepts of essences but instead through process, why different inputs can have a tendency to result in similar trajectories. Attractors propose a particular way of thinking through the dynamics of the modulation or differential negotiations of forces in a system in a non-prescriptive manner. They suggest, rather than prescribe, outcomes and relations. They are also impersonal or non-subjective tendencies that belong to the field and are therefore directly implicated in how events begin to gather within ecologies. Systems with multiple attractors 'break the link between necessity and determinism, giving a system a "choice" between different destinies' (DeLanda 2005, 35).353 That is, since multiple attractors might lure towards different becomings, the actualised differences or modulations in the system have complex causes that remain relational but cannot be reduced to linear causality or replication. Attractors themselves are not fixed. They might themselves be viewed as becoming-states, with their own set of attractors that condition their genesis. While some attractors are steady (that is, a constant lure), others can be cyclical or chaotic. Thus, states can move periodically between relatively stable and far-from-equilibrium conditions. An attractor itself might also develop or modulate the way it pulls within an event. Multiple attractors here create open, problematic and never more than partially resolved states composed of the contrasts (intensity) of contradictory potentials354 and, as such, are of use within thinking through of open-ended algorithmic processes. Each state might then be seen as a machine, modulating flows according to the play of the intensive dynamics of its competing attractors on forces. They are also potentially capable of moving from one particular self-organising solution into another related state that is therefore not fixed. DeLanda warns, however, that in order to actively engage with the virtual – and therefore exhibit non-linear behavior – a system of attractors also needs to maintain a far-from-equilibrium state. That is, a state in which intensive difference, as a continuous flow of energy, or data 'traverses the system…acting as a constraint maintaining intensive differences alive' (DeLanda 2005, 75).355 Such nonequilibrium causing flow 'reveals the potentialities hidden in the non-linearities, potentialities that remain dormant at or near equilibrium' (DeLanda 2005, 75). In other words, such systems depend not only on the pull of multiple attractors to move beyond the predictable, but on the high degree of intensity that makes the system sensitive to switching between the various lures of the attractors (DeLanda 2005, 76).356 A dynamic system, as DeLanda suggests, also needs high degrees of connectivity, which, as with parametric systems, allows the potential for various component parts to mutually influence each other's relationship to attractors (DeLanda 2005, 65). When the Orgasmatron system was 'activated' by the incoming differentials provided by a participant's body,357 it moved from a state of high stability to one in which the increased flows of data from sensors became intensified (more differentialised), shifting rapidly between ranges of numbers. This data was processed by a differential algorithm, and was then subject to the 'pull' of multiple watcher algorithms. That is, the data had a potential to become through interaction with a watcher that drew it towards that watcher's particular modulation of the flow. Here the watcher algorithms were the collective potential futures of the data, multiplicities towards with which it could engage and actualise its transduction. The tension of the potential for the data to be drawn instead towards relationship with one of the other watcher algorithms, or to be split and interact with two or more simultaneously was always inherent. These watchers were constant attractors for a flow, and the data could oscillate between the potential pulls of them because it was a set of unstable or changing numbers.358 As with the prehensive capacity of the algorithms, the lure of attractors was here automated,359 yet it retained its dynamic potential through the unresolvable tensions of multiple attractors. While these watcher actors were stable attractors operating throughout the Orgasmatron's processes, the 'triggering' algorithms could be considered to be cyclical attractors. That is, they counted interactions before triggering a further event; luring interactions with data flows until a limit point was reached. Then the cycle of attraction effectively reset and began again, creating multiple and overlapping rhythms of operation within the system. Alongside this, the cross-links between the parameters of watching attractors' inputs and other watchers' outputs meant that a gate parameter of an algorithm, acting as an attractor for a flow of data, was itself attracted towards realising a potential in its continued development.360 A more complex system arose here that moved towards concretisation, as potential was intertwined and co-produced. That is, it was a system of causality that was irresolvable into a linear chain, as attractors effectively nested inside each other by co-producing each other's parameters: 'A' nested in 'B' while 'B' was also nested within 'A' simultaneously. This was a parasitic mode of operation, with each attractor held together by the dynamic and potentially disruptive pull of the forces of other attractors on it. The relation between an attractor and the system or field within which it nested was 'charged', as attractor and field became implicit in each other's production.361 #### Limits and bifurcations State systems can move further away from self-preservation by incorporating the ability to undergo phase transitions or bifurcations. Phase transitions 'are events which take place at a critical value of some parameter…switching a physical system from one state to another' (DeLanda 2005, 18). That is, these bifurcations shift a system from one particular set of attractors to another set, though this may include the attractors of the previous system as well as new attractors. As such phase transitions are another potentiality with which a system might engage. They are instigators of, and meaningful to, the emergence of new relations within systems, rather than necessarily changes to individual component parts (Prigogine and Stengers 1996, 45). Besides occurring within a system as a whole, a bifurcation might occur within an attractor, causing an evolution to its affectual capacities.362 Thus systems might potentially bifurcate in multiple directions at once, without dissolving the assemblage. In the Orgasmatron software patch, some of the triggering algorithms had the potential to trigger the activation of new sets of attractors that operated in addition to those already active. These algorithms were triggered into action when a certain limit of intensity of a particular activity was reached363. As there were multiple triggering algorithms counting and multiple new attractors waiting to be activated, this had multiple potential outcomes. These limits were relative thresholds of the system, 'above which [it] cease[ed] to be itself but [got] a new lease on life in a different mode' (Massumi 1992, 36). Thus, the bifurcatory potential created limits that became creative factors, drawing new potential from the field.364 These transitions of both the whole state and parts that made up a state were always a partial expression of its many potentials, both of the system as a whole, and the parts that exceeded this actualised state. Once again, increased excitement of the system primed it for change, through a system of potential shifts and disruptions to chains of causality as new relational factors arose in the system365. This was not a smooth modulation of the system, but an ongoing potential of sudden shifts, interruptions to established tendencies, and renegotiation of relational pulls. As algorithmic prehension demonstrates a way that such process engaged selectively and creatively with that which preceded it, concepts of attractors and bifurcations here indicate a creative and open engagement with a futurity. #### Towards an ecology of patching Within her concept of the 'minor gesture', Manning poses the question of how technology might be able to 'activate a field event without making the field about the technology itself' (2016a, 18). The challenges implied in a process-driven approach to software design might be seen here as twofold. Firstly, this might involve taking Whitehead's expanded empiricism seriously, and seeking to explicate how all components of a work, including any computer operations, can be viewed as entities or events emergent within a relational system, capable of exercising some of their potential to affect and be affected. Secondly, as Manning suggests, there is always a need to consider the ethics of not only what emerges, but also how it emerges. In the computer processes, this must then be concerned firstly with how the computer's operations affect the gathering of the larger artworkecology – its minor potential to move beyond predictive control and representation. Secondly attention must be given to how these operations are able to move towards an intensively minor state; a concretisation of a 'machine ecology' (Penny 2011, 100) that preserves potential as it draws algorithmic processes into collective individuation. The tactics explored here begin to suggest ways in which a software patch might remain intensively problematic: always irresolvable as a whole, while also immanently offering partial solutions. For Simondon, the ontogenetic power of a system – its capacity for emergent novelty – depends on this ability to generate problems that force the actualisation of partial solutions as new sets of relations that establish a milieu (Vollrath 2013, 46). Thus the role of intensity is crucial here in gathering an emergent ecology within the software patch while still keeping the system open to the disruptive pull of multiple eternal entities that it can then draw from. The Orgasmatron system attempted to provide this intensity through the multiple tipping points that were always cycling: through the constant unresolvable pull of the stable attractors; through the entanglement of parameters with algorithmic actualisations; and, through the strain of the cut of negative and positive prehensions. In this, it was an assemblage of 'non-linear combinatorics' – various selforganising structural operations negotiating to produce novel structures (DeLanda 2011a, 16, 277 n.5), producing algorithmic processes through a differential or parasitic approach, or 'new ways of folding the world into itself' (O'Sullivan 2006,143). Clearly, an ethics of computer process needs to do more than just consider ways to make complex relational webs that move beyond linear causality, and allow new software modes of thinking to arise. It also needs to avoid the trap of creating topological or autopoietic systems that, in their ability to anticipate and influence future modulations, enhance rather than curtail the predictive and controlling potential of the digital.366 Here, again, the intensively parasitic has a role to play. The proposed tactics are a gathering, but also a splitting of data or force – a continuity of becoming, rather than a smooth modulation that can be predicted and controlled. They involve a concretisation of the assemblage of the various algorithmic and analogue data, but not necessarily a preservation of the assemblage over other relational potential. Instead, such systems have component parts and processes that remain larger than any actualised whole. Through their relation to eternal objects, and through the dynamics of bifurcation, these systems are always on the verge of exceeding their limits, and become the gathering, generative collective force that catalyses new ecologies of relation. Once again, these tactics are, to a certain extent, about enabling drift: forgoing control over outcomes, and instead concentration on the setting of conditions for events to emerge from. If it is an automated emergence, then this is because it is an algorithmic mode of thought that needs to be given its own space, style and rhythms. Algorithms are events in themselves, co-emergent with and co-causal ecologies of relation that begin to gather. Their actualisations are digital becomings that begin to draw the collective expression beyond not only the biological, but also outside of the analogue. Perhaps here, a software patch can approach a diagrammatic meta-modelling, 'strategically return[ing] its process to the quasi-chaotic field of its own emergence, in order to regenerate itself as it generates new figures, forms and contrasts, for itself and others' (Massumi 2011, 103). #### Coda: Towards a gathering ecology In Orgasmatron, participants entered an intimate environment designed for one or two, where their presence within the space contributed – through disruptions and additions – to the generation of rhythmic pulses of coloured light, sounds that surrounded them, and vibrations that coursed through the base of the structure (see Figure 9.2). Participants lay in the Orgasmatron, relinquishing, to some extent, the possibility of feeling in control, and accepting this new posture that emphasised the pull of gravity and what at first might have felt like 'passivity' within the event. Movement shifted in register, being restricted to small, seemingly inconsequential gestures – eye movements, breath expanding the torso, a fractional turning of the head, reflex reaction to vibration under their body, a hand raised, subtle shifts in weight: small adjustments and micro-movements in sympathy with the rhythms of sound, light and vibration affecting the participant. This was a rearrangement and testing of the potential of the body that perhaps began to challenge habitual ways of moving through an interactive work, as the spatial configuration and the shift in postural schema constricted movement, bringing to attention the way forces challenged the body's freedom of action. Lying in the Orgasmatron, connection to the ecology of operations in process was slowed down. There was nothing productive to 'do': no obvious action that would activate events, with a clear or immediate pay-off or resultant change in the work. Here participants were given the time to tune in to the events building around them, allowing such minor forms of bodies to be noticed and evolve. This was less a space to command, and more one to listen with one's body, to seek new connections and open out to an awareness of the gathering rhythm of events in which participants were becoming implicated. This required a new sensitivity to the prehensive pull of the event that was activated at the surface of bodies. Textures, the pressure of the base of the structure, and the vibrations building and coursing through the base of the Orgasmatron brought attention to the skin and the activated shared space in between, beginning to combine body, equipment and space. This was a listening with the whole surface: the body an expanded listening machine (an ear). The experience conflated senses, as it was perhaps also a new reflexive listening to or doubling of experience, a reflexive consciousness of this disruption of habits. Micro-perceptive vibrations addressed various sensory organs; pulses of light, sound and the participants' own bodily rhythms combined and syncopated in this surface-to-surface interfacing. Thus the body itself was reconfigured in a minor form as a 'sensor' – transducing different vibrational forces from the event – testing and opening up its affectual capacities to new intensities. The Orgasmatron itself was a combined 'sensor', its components tuned, not only towards the presence of the participant's body entering, and their micro-movements that reflected slight shifts in attention, but also always tuning towards the multiple expressions of its own machinations. While the Orgasmatron was sensitive to a participant's weight, vibrations, gestures, sounds and shadows that were a source of disruption to the systems, it also had sensors capable of interacting with its own expressions of light, sound and vibration. Here, in a complex series of feedback circuits, some sensors fed data from changing pulses of light into the development of sound events, others collected vibrational permutations that then affected lighting, while others sensed pressure changes in the floor of the pod that caused further expressions of vibrations, sound and/or lighting. This was a constantly shifting web of parasitic actions – a molecularisation of components: as pressure differentials disrupted light; light differentials disrupted sound (cutting, layering spatialising); and sound differentials altered vibration. The actions of bodies within this environment provided further parasitic disruptions to these emerging causalities: further variations in pressure, light or vibration as the Orgasmatron listened to and fed on (in its own way) its own constant permutations and exploratory combinations. These sensory capacities (capacities to feel) of the machine (bodies and technical components) folded into one another, to begin the collective individuation of the event: a mixing and shaping of a shared potential and responsibility. Such a turn towards a collective listening and expressing might be a tending towards a 'self-tuning': the will of the event to emerge and to carry forward. This questioned the position of the participants as the focus of the gathering of forces, as the work perhaps began to trouble distinctions between the subject of the event and the field from which it drew its energies. Rather, participants shared responsibility for this gathering, adding their own attention, care and potential to the attention and sensitivities that the Orgasmatron was itself able to generate. The concern here was less with being, and more concerned with a communication or engagement across a vibrational plane: a collective feeling for the gathering that was distributed throughout the components of the event. The system disrupted the representation and comprehension of causal chains – how a particular rhythm, sound or pulse of light was connected to previous actions or events – as both participants and work were immersed in the ongoing collection of sensations (relationality in its own right). The engagement with affectual forces – both the collectively engagement of the event, and individual engagement by various components with different appetites or capacities – split, folded and remixed causality. The dynamic, complex and qualitative interfacings and parasitic actions cultivated a suspension in the gathering of relation – creating a pull towards further relational iteration. In this, the parasite forced an opening to further expression, connectivity, and an ability to affect and be affected. This was a turning towards immanent construction of relation taking precedence over its stratification (that is, an opening of sense experiences – of both the participant and other components). The 'working out' of these relational disruptions moved the system towards a concretisation. The components of the event were no longer as dependent on 'outside' intervention to facilitate communication between them – whether the participant's body providing this interfacing or the work of a computer that stood outside of the mobile parameters of the work itself. Instead, the components were able to utilise their transductive sensitivities to create their own local relational interactions and to produce affects 'that [were] independent of the design plan' (Simondon 1980, 31). But it was a concretising, in that this was never resolved to a fixed state of intertwined sub-systems, fully subsumed to the functioning of the whole (Simondon 1980, 30), but any move towards resolution continued to be challenged by the disruptions that forced a re-gathering. There was always some further potential for agitation, for the continued parasitic disruption allowing new connections to be performed. This was an agitation that was not reliant on a human participant for its energy, but was able to activate itself, to generate the minor gestures from within the event. This further potential was the tension that drove the transduction of the system, its provisional resolution of multiple potentials, and the ongoing working out of the problem of disruption and reconnection.367 This was the conversation between the various interferences of one force on another that formed a collective individuation located in the event as it gathered. Orgasmatron proposed a field of potential sensitivities and potential disruptions from which provisional connections and disconnections might begin to form a relational web. Here, I term the act of the Orgasmatron tuning into this potential – to begin to become an event – a 'gathering ecology'. A gathering ecology implies a particular attention to the event's own ability Figure 9.2 Andrew Goodman, Orgasmatron (detail), Blindside, Melbourne, 2013. to prehend the potential of the field and gather or implicate components' individual and shared capacities for connection and disruption into a collective event, and to give attention to the 'minor gestures' that are the event's own intensive drivers of individuation. This focus on a gathering ecology shifts interaction further, from the fixed or linear sets of relations between technical objects and bodies, towards what might be thought of as an ethics of relation, in that it places a focus not just on the flexibility and complexity of relations, but squarely on the opening of conditions for the event's emergence. A potential politics of interactive art might be an ethics that addresses not the representation of relation, but its immanent construction, enabling an opening to further expression, connectivity and an ability to affect and be affected: to affirm both the singular nature of events and openness of relational potential (Simondon cited Combes 2013, 65).368 It might seek to encourage 'the suspension of normal co-ordinates of sensory experience' (Rancière 2009, 25), that is, an opening of sense experience towards the new – the preservation of difference (Murphie 1997, 163–5) in a gathering ecology. ### Conclusion Now more than ever, nature cannot be separated from culture; in order to comprehend the interactions between ecosystems…we must learn to think transversally. Felix Guattari #### Parasitic friends and enemies The parasite disrupts and creates; it 'makes life and kills' (Serres 2007, 168). It is the instigator of the new, it is 'an expansion; it runs and grows' (Serres 2007, 253). It causes disruption to gather and multiply. It bifurcates all, driving systems towards the novelty of new connection as it makes new systems. It is the best friend of complex emergent relation. The parasite 'invades and occupies' (Serres 2007, 253); it troubles orders, disrupts connections. It is a noise that 'destroys and horrifies' (127), pulls things apart, confuses and obscures (Serres 2007,12), lays waste to plans. It is the worst enemy of the clear and simple relation. Parasitic procedures trouble totalities, creatively disrupting clear communications, orders, hierarchies and dichotomies. Parasites can be thought not only as a third factor in relation, shifting the already established, but also as a difference that might be original, thrusting us always in to the middle of things going on. In the interactive art event, parasites fragment the simple causal relationship of a participant's intentional action and comprehendible change in the work. They coax into existence minor interactive potentials that are situated within the major, problematising interactivity's boundaries, questioning both its definition and its mechanisms. The parasite is a noise that, though disruptive, is far from being chaotic. Rather it is intensely and complexly relational, implicating elements of systems into each other's ongoing individuation. In this, it is potentialising – saturating the actualised with an inbuilt ability to continue to grow, modulate and add to itself. The parasite is the friend of noise and the noise within friendships, but it is never friendless and never outside of relation. #### Parasitic feelings To feel, for Whitehead, is to be involved in processes of the becoming of novelty, and it is also to be involved in processes of ingression and entanglement. In feeling, an emergent entity grasps datum from the world as a selfish and parasitic activity (reaching into and feeding off not only the actualised world, but also ingressing into the virtual plane of the eternal objects). At the same time, and in the same action of feeling, the world 'steals in' (Jones 1998, 3)369 or ingresses into the entity as its very core of becoming. This enfolding or nesting is problematising: entities parasite for their own selfish means, but are in turn subject to the parasitic ingressions of others. Feeling produces a resonance that is both intensive (valuated and patterned contrasts) and extensive (a differing from what already is and a selection from the larger potential). It utilises the tensions involved not to homogenise and consolidate, but as an adventure that explores potential in novel combinations that intensify difference. In this feelings are always transductive – reaching forward, moving beyond the realised, turning many things into one new thing, again to be folded into another eruption of novelty. The parasitic actions of feeling on the 'world-as-it-is' to individuate the 'world-as-it-will-be' suggest an interactivity of occasions and potentials that is never going to be contained in simplistic viewer-to-artwork conversations, but that is an emergent and parasitic ecology of ingressions, transgressions, interruptions and additions. #### Parasitic ecologies The gathering of an ecology is a machinic act, that is, the immanent production of a symbiotic entanglement,370 no more a bottom-up phenomenon than it is top-down. It is a systemwide productivity (though not totalising), passing through scales and dimensions, a becoming that is a qualitative increase in intensity (Braidotti 2002, 147). It is a gathering (not resolving) of difference, and therefore a gathering of an ecology that is autoparasitic. The generation of its minor gestures is 'in the wind', as the affectual force of the event, an evolving capacity to prehend or tune to the potential of the field (to gather on the level of the virtual). These gestures that produce and drive the ecology are the will of the event. As a gathering ecology is an 'immediatory'371 process, it might not be known through any predetermined configuration, but only through the field's continued exercising of self-productive expressions. A gathering ecology retains this capacity to gather in its connection to the virtual. It is never 'gathered' as a final act that exhausts potential or resolves beyond the provisional differentiation of forces and relations, but is always a question of process (Stengers 2010, 33). In this sense there is never 'an' ecology, but only the transductive process of becoming-ecology or gathering. 'Ecologies' here do not submit to exterior truths but produce and are produced through continued experimentation. Thus the ecological could be described as inherently minor, as it is concerned with 'the production of new, immanent modes of existence, and not the recognition of a more powerful interest before which divergent particular interests would have to bow down' (Stengers 2010, 35). Neither is this a question of the identities of components, which cannot be reduced to, nor deduced by the role each plays in the ecology (Stengers 2010, 34–5). For interactive art this might suggest a move away from the reinforcement of identity of the experimenter and components (what are they, how have they related?). Instead interactivity might be better served to invent ways to embrace the ecological with its 'disparate causalities' and 'unintentional creations of meaning' (Stengers 2010, 34) (how could it become?). This then might move towards an interactive system that creates relation without having the nature of those relations fully prescribed, or the manner in which they might individuate. Nor need the design prescribe the scale or extent of those interactions, or the outcomes for either components parts (participants, affects, sensations, technical objects), or the system as an evolving whole. Thus it is concerned with an 'ecology of the virtual' that can 'engender conditions for the creation and development of unprecedented formations of subjectivity that have never been seen and never been felt' (Guattari 1995a, 91). For this to occur a work needs to be rich with minor gestures as lures towards novelty that 'seed' the potential for further gathering of ecological force. Here we might seek an art that operates as a field through systemlevel dynamics, parasitically inserting difference into such relations, and through this held intensity allows for relations to arise and gather. #### Parasitic politics One might argue that our contemporary world already offers unparalleled connectivity through the globalised economy and the collapse of time and distance through the so-called 'virtual' world of the internet (and of course the effects of this on art have been momentous and neither entirely negative or positive). It is also possible to argue that there has been a proliferation of difference and experimentation – one that can be seen in the post-millennium art world with its furious multiplication of styles, diverging trends and voices. Perhaps too there has already been an unprecedented blurring of human and non-human life and the technical, with increasing incursion of algorithms into the political, social and personal sphere, to name just one example.372 However a closer examination of these conditions of contemporary living shows little to be enthusiastic about. As Braidotti has so eloquently pointed out, the connectivity offered within neoliberalism is principally one of a shared precarity (2014, 40). That is, it is an ecological and political vulnerability that we share in across borders with other social groups, with other animals on whose lives global capitalism encroaches on and into for its own purposes, and with the greater ecology that is also commodified and exploited. This vulnerability is evident in terms of the increasing precarity of a global environment under ecological crisis that affects and connects us all in a decidedly negative manner.373 If advanced capitalism produces difference, it is only for the sake of further commodification. If it encourages experimentation, it is to harness this potential within a subjectivity based on passivity and individualism (Braidotti 2014, 58, 61). So too, if capitalism blurs the human and non-human it is to further advance networks of control in order to exploit and profit from them (Braidotti 2014, 63). Capitalism, for all its relational flexibility, has no ethics, no care for or interest in the potential of the events that emerge beyond their vulnerability to be exploited. It is a 'steamroller' enforcing 'capitalistic subjectivity – the subjectivity of one-dimensionality, generalised equivalence, segregation, and deafness to true alterity' (Guattari 1995a, 91). The connectivity offered in this world tends towards a greater surveillance and biopolitical control of life that now grips the social/political, the personal or psychic and the ecological planes to an unprecedented extent. Ultimately such processes of connectivity collapse difference as they subjugate: they are operations of power that are restrictive and repetitious rather than productive of any larger sense of novelty. Why then would we want an interactive art experience that merely mimics this precarity, one that at once instrumentalises our bodies as it reinforces subjectivity and our separation from the field and its further collective potential? I say all this not to make any great claims for the artworks discussed in this book, but to suggest that they all exhibit, in various and modest ways, the beginnings of different ways of operating. They point towards transversal and immanent practices or technics of becoming-with ecologies of relations that embrace complexity, disruption and novelty. It will be evident that none of these artworks directly address the ecological and political crises we face. These are not 'issue' based works seeking to explore the negative aspects of our situation. This is not to argue that such messages do not have a place in our thinking, but that art, and perhaps interactive or relational artworks in particular, might have a different and more affirmative and forward-reaching or speculative role to play. Affirmation, as Manning articulates, is a very particular type of positivity that is propositional (2016b, 196). That is, it enables invention rather than compliance, and in this it opens up events to further evolution rather than collapsing difference in relational consensus (Manning 2016b, 196). An affirmative relational artwork might seek to move away from a capitalistic model of self-organisation to 'imagine a form of self-organization that is not exploitative' but a 'genuine novelty' (Shaviro 2009, 128 n.16). This is an affirmative ethics that extends care towards the quality of expression of a system. It seeks to nourish the potential for creative movement or exploration within an event, with attention to and care for the conditions of emergence as well as what emerges. This is not the artwork of grand gestures and political sloganeering, but of attention to what else is going on beyond, beside and throughout events of relation. It requires that we develop capacities to listen and give care to the resolutely non-human and the more-than human: to the diffractions and conversations of sound waves with spaces, to the algorithmic appetites, to the rhythms of collaborations between bodies and spaces, bodies and bodies, bodies and technical objects, and within bodies themselves. In this it might seek to experiment with technics that move us beyond shared precarity (and the capitalisation of emergence) and begin to bring new and affirmative potential ecologies into being. Technicity, as Manning states, is 'the associated milieu of technique'. That is, it addresses the generation or gathering of a field of potentiality as much or more than the individual technique (Manning 2013a, 34). Technicity therefore speaks to the gathering ecology in the realm of the political, a realm that permeates relational or interactive art, which always expresses a politics (whether productive or repressive). Affirmative technics addresses the construction of an ethics of immanence: an ethics of experimentation with the construction of the contrasts and differential intensities in relations that enlarges collective potential rather than reverting to individualism or negativity.374 This must be an emergent ethics that operates transindividually, at a collective and ecological level. In this positivity one might seek to move beyond a criticism of the state of interactivity and to seek to generate new concepts of what might constitute an expanded notion of interactivity: that is, to begin to generate new potentials and futures for the genre itself. Such ethics must remain firmly grounded in the 'how' of collective enunciation (Manning 2013a, 35), in the questions of the individuation of the field or ecology, in order not to simply replicate the neoliberal repressive responsibility of the individual towards the state or the status quo (Braidotti 2014, 116). That is, energy and attention needs to be invested in the pre-relational gathering of ecologies – into the nurturing and tentative feeling-out of the minor gestures that emerge from a complex ecology's own feeling of potential and that tend towards a feeling of events in the beginnings of formation (Manning 2013, 6–7). These gestures – as a series of differential events within a field that catalyse a collective tuning towards the field's potential concrescence into a dynamic and enmeshed ecology – are the seeds that ensure that ecologies adventure into the unknown as they individuate. That is, they are performative and belong to the ecology's gathering. The 'how' of this gathering must of course always be open in itself. There can be no definitive answer to this but only a series of practices that continue to explore the problem – that meta-model or 'stay with the trouble' to quote Haraway (2016, passim). In this each well-thought and constructed interactive event can contribute (in its own tentative way and within its own area of concern) to the collective thinkingthrough of the problem of not only how to live in this world, but how to begin minoritarian transformations of it. Politics, or even ethics, may still seem a heavy burden for such simple relational works. But it is, I would argue, a politics of dissention, of reconfiguration and extension, of etching out further space or potential no matter how slight. I have contended that the programmatic tendencies of interactive artworks contain difference and universalise experience – a politics in itself, albeit an oppressive one. Aesthetic acts that extend and prolong contrasts can be seen instead as ethical politics, making felt novel relational connections and new collective capacities (Massumi and McKim 2009, 12). The capacity for these aesthetic acts to produce 'mutant percepts and affects' gives them an important role in the liberation from the merely possible: the cannibalistic moves of capitalism to reiterate and mutate existing relations and subjectivities (Guattari 1995a, 91, 131). Aesthetic acts are transversal, operating through 'affective contamination'. They have the potential not only to operate as the 'nuclei of differentiation', but also to operate 'between the different domains in order to accentuate their heterogeneity' (Guattari 1995a, 93–4). The 'beauty' of the aesthetic act is in its ability to sustain contrasts, to extend differentials. Such aesthetic politics need not be conceived of as necessarily earnest. Rather, they may be better situated in play and the disruptive power of such unproductive action that proposes starting rather than endpoints of relations. Here, again, my argument seeks to arrive not at any solution, but rather to build conceptual machines with which to allow a working through both of the potential of parasitic actions, and a questioning of the limits of interactivity. This book has intentionally examined works that cover a broad range of relational experiences, and that move somewhat away from easy classification as 'interactive', while still involving many elements of such systems. Near these edges or limits, the question must always arise: 'But is this still interactive?' This, I would suggest, is in itself productive, capable of always provoking some uncertainty as to what does or does not constitute an interactive work. It is an interactivity that by its existence challenges interactivity from within, injects tentativeness into its identity. It is a questioning that is productively disruptive to the very concept of interactivity: a parasite. As such, the thinking as a whole might perhaps be positioned as both parasitic and a minor practice; a gathering of an ecology, a rethinking of interactivity that seeds further potential disruptions, always attempting to take it beyond the re-emerging majoritarian forms. ### Notes artworks. On painting and relation, for example, see Irwin (2011); Manning (2009, 55–63); and Massumi (2011, 127–30). and propositional nature of the theoretical discussions is, I would argue, both a philosophical choice in line with process philosophy, and a practical technique for dealing with the necessarily open-ended nature of propositional art events – bringing to attention that 'theory-making itself, [is] a messy, fleshy practice' (Loveless 2012, 95). of the participant's movements. The term 'mechanical' is not here used to infer negative connotations to such processes. sound, vibrations, infra-red waves, pressure, heat, and so on, can be mapped and plotted alongside the visible. aligned with the major, one's flows contained within its antiproductive maneuvering' (Murphie 1996, 17). the forces of relations (2010, 42, n.2). As Francisco Varela argues, such events of relation between the world and bodies are always events of mutual creation – neither wholly internal nor external (Varela, Thompson and Rosch 1992, 198–205). In a similar vein, Karen Barad uses 'intra-action' to describe a system where cause and effect emerges as the differential materialisation of bodies. Barad writes that 'intra-actions are non-arbitrary, nondeterministic causal enactments through which matter-in-process-of-becoming is iteratively enfolded into its ongoing differential materialization' (2007, 176, 169). This Barad terms a system of 'agential realism' (2007, 132–88 and passim). Lone Bertelsen uses the term 'trans-subjective' after Ettinger, to move beyond interactivity and describe responsibility as a shared concern between all emergent aspects of an event (2012, 31–71). the workshop. (This description is adapted from the blurb on the project on the Senselab site, available at: senselab.ca/wp2/events/ into-the-midst/). interaction and interactivity and the complexities of designing interaction as artistic process' (2011, 72). modulation and interaction and the ongoing tensions between the modulating affectual capacities of parts on the force. bring (physical capabilities, tastes, moods), plus the worldly circumstances surrounding the art event (culture, politics, geography, art histories, weather), which all co-create the event's virtual milieu. Seven of this book for an extended discussion of diffraction as a generative differential force within a system. is not linked to a singular form of experience, nor is it linked to a singular mode of organized being that performs a variety of functions' (1998, 134). in regards to a lack of care for any particular individuation, see Adams (2000, 38–42); and Ingold (2011, 77). 'cognitive' capitalism exploiting immaterial labour relations, multiplying modes of exploitation and reaching across many dimensions of production (Pasquinelli 2008, 97, 91–104, passim). See also Massumi on exchange-value (1992, 199–201). causality dispersed, notions of an artist as 'agent' are replaced by a co-causal conversation between competing forces. Within such simple tactics, we begin to understand that sounds within the system become free floating events, inhabiting a virtual soundscape: sounds as societies, vibrating internally and externally with the tensions of relation – they begin to hum with difference and potential. Here it can be seen that in a system that in many ways was a relatively simple construction (utilising twenty or so basic sensors and a dozen sounds), it is possible to design the potential for a move towards greater complexity. The artist's role might be less to design the complex relations that might occur, but more to focus on setting the preconditions for these developments. And, while such a design shift certainly increases component events' implication in each other's various actualisations, this is not in any way presented as a definitive example of the scope of the parasite. Such tinkering represents both small, seemingly inconsequential moderations, and at the same time, a paradigm shift: the death of the (software) author to be replaced by the propositional event. engagement between bodies and environment (de Certeau 1988, 117–18; Ingold 2011, 145–55; Gibson 1979, 35, 93). that the environment exists independently and prior to engagement with inhabitants in some stable form (2011, 78), it seems clear from Gibson's own writing on the subject that his intention is ecological. continual attention to negotiate (Arakawa and Gins 1997). The 'elastic point' at which the body 'culls from the movement's potential its becoming-form' is extended through such propositional spaces that demand a clear and ongoing shifting beyond habit (Manning 2009, 35). and perspectival painting for another way of thinking through the bodily implications of the system (2011, 127–30). noise, available light, traffic, and so on, all became factors folded into the event by the act of walking the performance beyond the gallery, disrupting or mutating the event itself through chance encounters, emotional tonalities, sounds heard, time spent on detours. when triggered through a complex chain of relations. At certain triggers, sounds from the performance were also recorded by the computer system and then looped into increasingly complex layers and replayed into the space, and the system was configured to emphasise the potential for disturbance to any sound event. perception and choice is only available to select organisms, Manuel DeLanda argues that even the earliest bacteria developed internal (that is, subjective) models of their relationship to the environment through combining a primitive sensory system with a motor-driven understanding of their relationship to space – which could be viewed as a germinal version of presentational immediacy and causal efficacy, although he makes no direct mention of Whitehead's two categories of perception (2011, 80). – such as an artist might have – 'if we are to refrain from acting upon it' (2014, unpaginated, I: 3). 'becoming of the being in general [that] produces both the individual and its environment' (Simondon 2009, 14, note 2). types of performances it produced, and the potential from which it was drawn. The introduction of a whole new outside tactic of production, alongside the introduction or infection of the event with new intentions and tonalities of play, then delimited the Re:Positioning Fear event. The tactic initiated new performances and fields of potential to compose with, even as it continued to drive towards its previously instigated concrescence. systems shared no potential. Where does this potential, and the actuality of unfolding connectedness arise from? Simondon's answer, as Massumi explains it, is that it is brought from the future, from a point post-concretisation. Interfacing here is the catalyst that instigates both the actual assemblage and simultaneously creates a new potential, a new milieu created immanently with the assemblage on which it has somehow already drawn, a circularity possible only within a conception of time as non-linear (Massumi, DeBoever and Rolfe 2009, 39–40). in Chorus of Idle Feet in Chapter Two. The computer system also watched for the quantity of light variation within a set timespan that, once a tipping point was reached, could then trigger further shifts in the potential range of volume (so that louder volumes were made possible). See Chapter Nine for some discussion of limits and bifurcation within software patches. changing colour and speed as the Orgasmatron became more excited; speakers surrounding the bodies whispered and spoke; and tiny speakers and a subsonic speaker sent ripples of vibrations through the base on which participants were lying. Sensors embedded in the base captured data from the weight and movement of bodies, light sensors captured shifts in brightness caused by both the projections and shadows from bodies, and vibration sensors captured the vibrations at various points in the base of both sounds and bodies. See www.andrewgoodman.com.au/ orgasmatron-spaces-to-make-love-in/. processes was in itself not a simple linear process, but also engaged with parasitic tactics. As with the example discussed in Chapter Two, these triggers interfered with and disrupted each other, replacing, for example, one sound event with another, or altering its tone, volume, and so on. As with the examples discussed in Chapter Five, within the actualised sound and light events, there were further potential processes of parasitic disruption, such as the 'unsounds' embedded in the sound samples that altered perceived sound events through diffraction, and the extended moments of transition between video projections where colours and rhythms diffractively combine. disregards the benefits or costs to the system of its creativity (2012, 364). McCormack argues that fitness-driven evolutionary art is a contradictory term, being anything but evolutionary in nature (2013, 5). It does, however, fit neatly into goal-orientated, neo-Darwinist theories of transcendence – a working or evolving towards an ideal form, as discussed in Chapter Two in relation to drift. The secondary tactic – employed both within fitness-based systems and on its own – has been to use injections of chaos or external randomness to generate change. Such systems, whereby an unrelated set of parameters are used as raw data converted to some artistic output through computational processes (such as weather data converted to shifts in colours on a screen, for example), are, as McCormack and others argue, a poor 'proxy' for intensive complexity (McCormack et al 2014, 8). While fitnessbased systems concentrate on positive, directed connectivity at the expense of exploratory room to move, random data creates systems concerned with the superficial appearance of complexity rather than its actualization. See also Per Bak's discussion of the misunderstandings of the operation of fitness within much scientific discussion (1997, 142). 309. As Anna Munster states: 'the technical element is always in a relation with elements outside itself, its form is therefore indeterminate and virtual' (2006, 14). Munster argues that bodies are 'the chaos and interruption with which the machine cannot dispense' (2006, 185). See also Murphie and Potts (2003, 31–2). It could be argued that simply through the processes of flows of data translating from software platform to software platform within a computer this data undergoes a transduction, shifting from one coded flow to another, with accompanied and somewhat unpredictable losses through the noise of translation (Newman 2012, 135–7). For example, in the movement of data through the series of patches utilised in the Orgasmatron system, numerical data is transduced from voltage flows (positive numbers between 0 and 5 volts), to midi in the first patch (positive integers between 0 and 127), then in the second patch to numbers between -100 and +100, then back to MIDI in the third patch. The social aspects of code provide another register in which any determinate nature of algorithms might also be disturbed. As Adrian Mackenzie charts in his discussion of Java, the software operates more as an unstable 'collection of resources with multiple potential machinic productions' than as a fixed object (2006, 95). Here Java, with its constant upgrades, user initiated fixes and modifications, independently operating layers of code and ability to work within other coding languages across platforms, operates as an indeterminate 'virtual' that is differentially actualised in each specific operational event (Mackenzie 2006, 96–102). components and electrical charges, is perhaps one of the most interesting experiments in ecological 'programming', concerned with how a field of potential is able to organise its own gathering into an assemblage capable of expressing relation (see Pask 1960; Cariani 1997). This is the type of evolutionary art that Galanter advocates, one capable of creating new sensing machines (and therefore evolving its own parameters) as well as operating machinically (2010, 6). technologies' inputs and outputs while ignoring their internal working structures (1992, 410). temporality, and overlapping potentialities or temporal multiplicities. See DeLanda (2005, 107–8). on a flow of data, either accommodating some of their potential (but in its own way, making it a new process), or differing from it. ### Bibliography Barthes, Roland. Empire of Signs. London: Jonathan Cape, 1982. –––––––. The Posthuman. Cambridge: Polity Press, 2014. –––––––. A Thousand Years of Non-Linear History. New York: Zone Books, 2011a. –––––––. Philosophy and Simulation: The Emergence of Synthetic Reason. New York: Continuum, 2011b. Deleuze, Giles. Bergonism. Translated by Hugh Tomlinson and Barbara Habberjam. Brooklyn: Zone Books, 1988a. –––––––. Mediaarthistories. Cambridge: MIT Press, 2007. –––––––. "The Architectural Relevance of Gordon Pask." Architectural Design, vol. 77, no. 4, 2007: 54–61. Haraway, Donna. Modest\_Witness@Second\_Milleniu.Femalemanc\_ Meets\_Oncomousetm: Feminism and Technoscience. London: Routledge, 1997. –––––––. Staying with the Trouble: Making Kin in the Chthulucene. Durham: Duke University Press, 2016. www.youtube.com/watch?v=AQRHon2eiKc. Accessed October 12, 2014. –––––––. "Interface and Active Space: Human Machine Design." Sixth International Symposium on Electronic Art. Montreal, Canada, 1995. www.brianmassumi.com/textes/ INTERFACE%20AND%20ACTIVE%20SPACE.pdf. Accessed August 12, 2011. –––––––. "Experimenting with Refrains: Subjectivity and the Challenge of Escaping Modern Dualism." Subjectivity, vol. 22, 2008: 38–59. –––––––. Cosmopolitics I. Translated by Robert Bononno. Minneapolis: University of Minesota Press, 2010 Varela, Francisco J. "The Specious Present: A Neurophenomenology of Time Consciousness." Naturalizing Phenomenology: Issues in Contemporary Phenomenology and Cognitive Science. Edited by FJ Varela, J Petitot, JM Roy, B Pachoud. Stanford: Stanford University Press, 1997: 266–314. Varela, Francisco J, Evan Thompson and Eleanor Rosch. The Embodied Mind. Cambridge, MA: MIT Press, 1992. Wilden, Anthony. Systems and Structures: Essays in Communication and Exchange. New York: Tavistock Publications, 1980. Wood, Aylish. Digital Encounters. New York: Routledge, 2007. ### Index Aesthetics 13, 34, 206, 262 note 135. and politics 231, 275 note 213, 276 note 222. Affect 12, 18-20, 47, 50, 61-63, 66, 74, 90-91, 98. Affect theory 91, 157, 278 note 231. Affectual tonality 104, 154, 169, 190, 193, 267 note 165. Affirmation 16-17, 237. Affordance 111, 118-122, 132, 139, 162, 269 note 181. Agency 14, 59, 69-72, 81-82, 96, 102, 135, 141, 147, 152, 156, 256 note 96, 261 notes 129-131, 276 note 222. Algorithm 15, 153, 199-225, 236, 238, 287 notes 300-301, 288 note 305, 290 note 309, 293 notes 321-322, 294 note 329, 294 note 335, 295 note 340, 295 note 344, 298 note 366, 299 note 372. Generative algorithm 28, 103, 203-210, 246 note 33, 277 note 225, 289 note 308, 293 note 321. Analogue 203, 215, and digital 181, 194, 203-5, 209, 215, 225, 255 note 88, 273 note 201, 281 note 259, 291 note 310, 291 note 312. Arakawa and Madeline Gins 47, 112-113, 120-122, 128, 133-134, 145, 149- 150, 191, 250 note 61, 270-271 notes 185-188, 280 note 254. Ascott, Roy 32, 37, 38, 46, 291 note 313. Associated milieu 72, 141, 148, 181, 190, 238, 284 note 285. Artaud 159-160, 279 note 236, 281 note 260. Attractor 167, 210, 218-224, 279 note 243, 281 note 257, 288 note 304, 292 note 319, 297 note 350-351, 297 notes 353-354, 297 note 356, 298 note 359, 298 note 361. Autopoiesis 65-66, 207, 254 note 79, 257 note 103, 258 note 111. Bak, Per 70, 256 note 95, 264 note 144, 285 note 291, 289-290 note 308. Cariani, Peter 292 note 316. Casual efficacy 135-140, 143, 145, 150-151, 154, 246 note 37, 274 note 207, 275 notes 208, 209, 211& 213, 276 note 214, 277 note 226, 295 note 341. see also presentational immediacy Causal chain 73, 100, 137, 196, 205, 214-215, 222, 223, 228, 273 note 201, 298 note 365. Chaitlin, Gregory 212, 294 note 328, 294 notes 330-332. see also Omega Communication theory 101-102. Contrast 75, 78, 80-84, 85-86, 90, 93-96, 99, 116, 124, 136, 157-158, 160, 164, 169, 173, 188, 192, 195, 196, 214, 216, 217, 220, 225, 133, 238, 239-240, 258 note 108, 260 note 125, 262 note 135, 269 note 178, 278 note 232, 278 note 234. Conversation theory 47. Creativity 12, 69-71, 82, 89, 97, 100, 108, 115, 157, 177, 210, 253 note 77, 265 note 147, 279 note 240, 289 note 308. Cybernetics 250 note 62, 258 note 111, 283 note 270. see also second-order cybernetics Cyborg 250 note 62. - and diagram 188, 192, 286 note 294, - and difference 84, 85, 99, 101, 107, 116, 156, 158, 188, 254 note 86, 260 note 124, 266 note 154, 283 note 275, 297 note 351. - and Guattari 25, 56, 57-59, 63, 90, 111, 116, 127, 129, 149, 273 note 200, and the virtual 150, 169, 172-173. Drift 65-66, 69-74, 99, 101, 107, 205, 225, 256 note 96, 257 note 103, 258 note 110, 258 note 111. Ecology 20-21, 49, 74, 79, 82-83, 87-88, 97, 106, 114, 122, 134, 147-149, 151-152, 154, 156, 164-166, 174-176, 191, 207, 223-224, 235, 236-237, 274 note 205, 282 note 265. and intelligence 89, 93, 265 note 147. and responsibility 23, 91, 140, 146, 237-239, 256 note 94. see also gathering ecology, meshwork - and morality 90, 265 note 150, 299 note 368. - and relation 74, 84, 97, 140, 200, 230, 237-238. - see also politics Extensive 22, 23, 79-80, 129, 195, 204, 213-215, 233263 note 140, 282 note 263. see also intensive Far from equilibrium 208, 220, 285 note 291, 298 note 361. Faraday's law 93, 265 note 152, 266 note 156. see also induction Feeling 21-22, 66, 69, 75-97, 118-119, 136, 142, 146-147, 174, 175, 193, 195, 210-211, 217, 228, 233-234, 239, 242 note 9, 255 note 91, 256 note 93, 260 note 119, 260 note 123, 263 note 139, 265 note 146, 269 note 178, 276 note 220, 284 note 279. see also conceptual feeling, complex feeling, negative feeling, prehension, physical feeling Field, the 21, 25, 45, 48, 49, 61, 72-73, 76, 79, 84, 91, 92-96, 114, 120, 132- 133, 136, 141, 146, 147-149, 153-154, 165, 173, 176, 182, 189, 191-192, 212, 217, 219, 222-223, 230, 234, 238-239, 260 note 121, 270 note 187, 274 note 203, 282 note 267, 297 note 350. Fitness criteria 69, 246 note 33, 289 note 308. Foucault, Michel 37, 271 note 191. Fuller, Matthew 187, 179, 199, 283 note 269, 294 note 329. Goodman, Steve 156, 161, 166-167, 209, 280 not 253, 292 note 320. and machinic 58, 60-63, and the three ecologies 232, 235, 274 note 205. Harney and Moten 24, 29. Hapticality 122, 124, 272 note 192. Hinterding, Joyce 91-97. Hylomorphism 18, 179, 260 note 120, 266 note 159. Interface 15, 34, 35, 177-183, 186-188, 193, 194-195, 248 note 45, 278 note 234, 283 note 269. Incompossible 67, 187-191, 284 note 286, 292 note 319. Individuation 19, 45, 62, 73, 78, 89, 99, 100, 107, 115, 117, 141-143, 146-148, 149-152, 164, 174, 181-183, 190-191, 211, 219, 230, 249 note 53, 257 note 101, 258 note 113, 276 note 215, 277 note 228, 285 note 289. see also collective individuation Ingold, Tim 102, 113-115, 126, 127, 148, 259 note 115, 264 note 142, 268 note 169, 268 note 171, 269 note 174. and affordance 269 note 181. and sound 162-163. Induction 92-97, 242 note 12, 265 note 153. see also Faraday's law Intensity 16, 44, 67, 69, 75-76, 80, 85-86, 93-94, 102, 108, 116-117, 127, 136, 143, 188-189, 191, 208, 220, 223, 224, 260 note 124, 260 note 127, 263 note139, 283 note 275. Intensive 21-23, 59, 62, 70-71, 80-82, 93, 107, 112, 116, 152, 157, 164, 173, 196, 208, 218-220, 224, 233, 258 note 111, 262 note 135, 277 note 227. see also extensive Interactivity 20-21, 27, 30-32, 44, 53-54, 55, 56-58, 62, 96, 103, 129- 130, 139, 151, 209, 232-233, 244 note 20, 244, 245 notes 25 & 26, 253 note 71. and control 14, 33-37, 40-41, 49, 178-179, 209, note 22, 248 note 46. and didacticism 37, 39-40, 139. and ethics 73-74, 98-99, 230-231, 235. and generative 63-65, 68, 69, 109, 193. and instrumentality 33-34, 38. and politics 35, 230-231, 236-240. see also minor gesture Manovich, Lev 36, 244 note 22. Maturana, Humberto R 60, 254 note 79, 257 note 103, 298 note 361. Massumi, Brian 14, 16-19, 26-27, 31, 33, 36-37, 44-46, 57-58, 65, 71-72, 90-91, 103, 106, 118, 12-130, 139, 141, 150-151, 156, 160, 177-179, 190- 191, 217, 223, 225, 239, 241 note 3, 242 note 7, 243 note 14, 246 note 30, 248 note 46, 257 note 106, 267 note 164, 272 note 192, 272 note 193, 276 note 219, 285 note 293. Meshwork 105, 114, 126, 208, 257 note 105, 269 note 174. Mereotopology 209. Metamodelling 26-29, 109, 192, 225, 239, 243 notes 13, 14 & 15. Metastability 188, 218, 285 note 292. Microsound 163, 176. see also molar, minor flight Minor flight 117. Minor gesture 152-154, 171-172, 175, 223, 229-230, 234, 235, 239, 277 note 228. Miyazaki, Shintaro 215, 287 note 300, 295 note 339. Molar 57-58, 64, 69, 74, 100, 147, 206, 253 note 76, 254 note 86. Molecular 57-65, 69, 99, 108, 110, 113, 116, 122, 128, 170, 206, 208, 211- 212, 227, 253 note 76, 253 note 77, 254 note 86, 254 note 87. Monad 260 note 121. Morris, Meaghan 253 note 73. Nomadic music 176. - and algorithms 200, 216, 218, 220, 224. - and relation 285 note 293, 297 note 353. - see also linearity Omega 212-213. Pattern, patterning 21, 79-81, 83, 85-87, 89, 93, 94, 105, 136-137, 153, 167, 173, 213, 216-217, 260 note 126263 note 139, 263 note 140. Penny, Simon 31, 34-35, 38-41, 47-48, 54, 224, 245 note 25, 247 note 42, 248 note 49, 250 note 60, 252 note 71, 291 note 312. Perception 14, 39, 50, 120, 127, 132-133, 135-154, 156-157, 159, 165, 168, 172-175, 176. Prehension 66, 78-80, 82-83, 85-96, 99, 105, 118, 135, 137, 141, 158, 169, 210-214, 217, 218, 223, 255 note 91, 256 note 93, 260 note 119, 262 note 135, 293 note 323, 293 note 326, 298 note 359. Physical feeling 78, 81, 84, 106, 217. Physics 163-164, and Newtonian physics 244 note 17. and non-linear physics 261 note 130, 266 notes 155 & 156, 296 note 347. and quantum physics 278 note 231. Pickering, Andrew 209, 256 note 96, 261 note 130. Politics 14, 24, 49, 90, 150-151, 230-231, 235-240, 276 note 222. see also ethics Portanovo, Stamatia 207, 209, 212-213, 215, 287 note 301, 293 note 321, 296 note 345. Posthuman 76, 135, 259 note 114. see also transhuman Potential 12, 15-16, 18, 19, 20-22, 36, 39, 40, 45-46, 49-50, 56-61, 64- 68, 72-74, 78-79, 81-82, 85-87, 89, 94-95, 100-109, 111-112, 116 -122, 127-129, 133, 136, 142-143, 145, 147-149, 153-154, 156, 157, 163, 172, 181-183, 188-189, 192, 195, 199, 206-207, 212-213, 215-218, 219-221, 234, 238-239, 242 note 11, 254 note 87, 257 note 101, 257 note 103, 265note 149, 267 note 164, 270 note 187, 275 note 212, 276 note 223, 285 note 288, 285 note 293, 294 note 338, 295 note 344, 297 notes 353 & 354. Precarity 15, 136-238, 248 note 48. Presentational immediacy 135-139, 143, 150, 274 note 207, 275 note 208, 275 note 211, 275 note 213, 276 note 214. see also casual efficacy Prigogine, Ilya 222, 241 note 2, 266 note 156. Problematisation 22, 29, 55, 57, 67, 80, 94, 101, 102, 112, 130, 134, 143, 175-176, 182, 212, 220, 233. Productivity 34-37, 39, 50, 57, 71, 115, 199, 234, 246 note 35. Proposition 25, 49, 66-69, 101, 103-105, 107-109, 140, 162-163, 237, 257 note 104, 267 note 168, 282 267. and spatial propositions 118, 193, 207, 270 note 188. Proprioception 122-123. Radical empiricism 18-19, 77, 205, 223, 241 note 2, 241 note 3. Rancière, Jacques 231, 275 note 213, 276 note 222, 299 note 368. Refrain 216. and algorithm 215-218, 295 note 339. - and art 18, 20, 43-49, 66-67, 73-74, 108, 126-127, 139, 146, 167, 173- 174, 186-187, 207. - and codification 14, 23, 36-43, 55-56, 178, 179, 192. - and interactivity 30-31, 35, 49-54, 56-58, 65, 205-206, 235, 237, 241 note 6, 246 note 37, 249 note 54. - and politics, ethics 15, 23-24, 33, 90, 98, 174, 200, 224-225, 230- 231, 237-240. - see also relational aesthetics Second-order cybernetics 205-207, 208, 293 note 321. Self-organisation 21, 60, 69-74, 180, 219, 237. see also autopoiesis - and individuation 45, 174, 189-190, 195, 258 note 113, 277 note 228, 285 note 289, 297 note 354. - and resonance 158-159, 188, 284 note 285. - and transduction 22-23, 26, 62, 141, 148, 174-175, 182-183, 229, 242 note 12, 278 note 234. and technical objects 23, 193. Style 63, 89, 95, 121, 193, 195-196, 254 note 85, 271 note 189. Study 24, 29. Subject 15-16, 18-19, 40-41, 43, 44-45, 48, 54, 59, 60, 79, 81, 90, 110-111, 136, 140-141, 147-148, 150, 151-152, 157, 178, 194, 211, 236-237, 240, 244 note 21, 246 note 30, 248 note 46, 248 note 49, 276 note 222. Symbiosis 155-156. Symbolic reference 136, 143. System 19, 22, 23, 36, 37, 40-41, 56-58, 64, 67, 69-74, 92-93, 99-101, 102, 106-108, 122, 123, 132, 134, 141, 147-149, 167, 181, 188, 205-209, 218- 220, 222-225, 229, 254 note 86, 257 note 105, 258 note 108, 285 note 291, 292 note 319296 note 347, 296 notes 348 & 349, 297 note 353. System-level 191-192, 207-208, 234-235, 257 note 102, 258 note 113. - Of control 35, 53, 184, 187, 209, 247 note 43, 247 note 44, 271 note 191, 298 note 366. and fetishisation 40, 193, 241 note 5. Transhuman 91, 259 note 114. see also post-human Trans-individual 158, 174, 183, 189, 195, 238, 284 note 284. Unsound 161-164, 169-170. Varela, Francesco J. 49-50, 60, 69-70, 205, 250 note 64, 257 note 103, 267 note 165, 270 note 183, 298 note 361. see also autopoiesis, drift Vibration 15, 66, 70, 73, 95, 104-105, 111, 146, 155-156, 159-176, 187, 191, 227-228, 280 note 249, 280, 253, 280 note 255, 282 note 265. Vitality 96, 141, 174, 190. Walking 11-12, 110-113, 254 note 85, 268 note 169, 269 note 175, 270 note 182, 272 note 192. and the city 113-130, 268 notes 171 & 172, 271 note 191, 272 note 196. - and ontology 21, 75, 77, 80, 199, 211, 242 note 7, 259 note 116, 275 note 210. - see also concrescence, datum, entity, entrainment, event, feeling, prehension, proposition, satisfaction, societies, symbolic reference Art Criticism & Theory / Art Digital What might an interactive artwork look like that enabled greater expressive potential for all of the components of the event? How can we radically shift our idea of interactivity towards an ecological conception of the term, emphasising the generation of complex relation over the stability of objects and subjects? Gathering Ecologies explores this ethical and political shift in thinking, examining the creative potential of differential relations through key concepts from the philosophies of A.N. Whitehead, Gilbert Simondon and Michel Serres. Utilising detailed examinations of work by artists such as Lygia Clark, Rafael Lozano-Hemmer, Nathaniel Stern and Joyce Hinterding, the book discusses the creative potential of movement, perception and sensation, interfacing, sound and generative algorithmic design to tune an event towards the conditions of its own ecological emergence. Andrew Goodman is a visual artist and writer with an interest in participation, science fiction and process philosophy. Cover Illustration and Design by Leslie Plumb OPEN HUMANITIES PRESS	doab	2025-04-07T03:56:57.700281		{ "license": "Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/", "book_id": "0004e4bd-422d-4a23-9686-437c4a3af25b", "url": "https://library.oapen.org/bitstream/20.500.12657/30501/1/646116.pdf\|\|https://library.oapen.org/bitstream/20.500.12657/30501/1/646116.pdf\|\|https://library.oapen.org/bitstream/20.500.12657/30501/1/646116.pdf\|\|https://library.oapen.org/bitstream/20.500.12657/30501/1/646116.pdf\|\|https://library.oapen.org/bitstream/20.500.12657/30501/1/646116.pdf", "author": "Goodman, Andrew", "title": "Gathering Ecologies", "publisher": "Open Humanities Press", "isbn": "", "section_idx": 0 }
00064f14-4c6c-4123-bed8-b5ae765fbfc8.0	Edited by Salih Mohammed Salih The field of material analysis has seen explosive growth during the past decades. Almost all the textbooks on materials analysis have a section devoted to the Fourier transform theory. For this reason, the book focuses on the material analysis based on Fourier transform theory. The book chapters are related to FTIR and the other methods used for analyzing different types of materials. It is hoped that this book will provide the background, reference and incentive to encourage further research and results in this area as well as provide tools for practical applications. It provides an applicationsoriented approach to materials analysis written primarily for physicist, Chemists, Agriculturalists, Electrical Engineers, Mechanical Engineers, Signal Processing Engineers, and the Academic Researchers and for the Graduate Students who will also find it useful as a reference for their research activities. ISBN 978-953-51-0594-7 Fourier Transform - Materials Analysis Photo by JaysonPhotography / iStock ## Fourier Transform Materials Analysis FOURIER TRANSFORM – MATERIALS ANALYSIS	doab	2025-04-07T03:56:57.755534	1-12-2023 15:59	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "00064f14-4c6c-4123-bed8-b5ae765fbfc8", "url": "https://mts.intechopen.com/storage/books/2270/authors_book/authors_book.pdf", "author": "", "title": "Fourier Transform", "publisher": "IntechOpen", "isbn": "9789535105947", "section_idx": 0 }
00064f14-4c6c-4123-bed8-b5ae765fbfc8.1	FOURIER TRANSFORM – MATERIALS ANALYSIS http://dx.doi.org/10.5772/2659 Edited by Salih Mohammed Salih #### Contributors Erica Mejia, Marco Marquez, Juan Ospina, Alvaro Morales, Xiongwu Wu, Bernard Brooks, Mizi Fan, Hassan Safouhi, Fengxian Xin, Tianjian Lu, Hassen Aroui, Johannes Orphal, Fridolin Kwabia, Teuku Edisah Putra, Shahrum Abdullah, Mohd. Zaki Nuawi, Sei Ueda, Chanel Fortier, Carlos Borges, Paulo J. Amorim Madeira, Bingzheng Li, Nicolae Aldea, Florica Matei #### © The Editor(s) and the Author(s) 2012 The moral rights of the and the author(s) have been asserted. All rights to the book as a whole are reserved by INTECH. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECH's written permission. Enquiries concerning the use of the book should be directed to INTECH rights and permissions department ([email protected]). Violations are liable to prosecution under the governing Copyright Law. Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be foundat http://www.intechopen.com/copyright-policy.html. #### Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. First published in Croatia, 2012 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia Legal deposit, Croatia: National and University Library in Zagreb Additional hard and PDF copies can be obtained from [email protected] Fourier Transform - Materials Analysis Edited by Salih Mohammed Salih p. cm. ISBN 978-953-51-0594-7 eBook (PDF) ISBN 978-953-51-4293-5	doab	2025-04-07T03:56:57.755832	1-12-2023 15:59	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "00064f14-4c6c-4123-bed8-b5ae765fbfc8", "url": "https://mts.intechopen.com/storage/books/2270/authors_book/authors_book.pdf", "author": "", "title": "Fourier Transform", "publisher": "IntechOpen", "isbn": "9789535105947", "section_idx": 1 }
00064f14-4c6c-4123-bed8-b5ae765fbfc8.2	We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists 4,200+ Open access books available 116,000+ International authors and editors 125M+ Downloads 151 Countries delivered to Our authors are among the Top 1% most cited scientists 12.2% Contributors from top 500 universities Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) ## Interested in publishing with us? Contact [email protected] Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com	doab	2025-04-07T03:56:57.756039	1-12-2023 15:59	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "00064f14-4c6c-4123-bed8-b5ae765fbfc8", "url": "https://mts.intechopen.com/storage/books/2270/authors_book/authors_book.pdf", "author": "", "title": "Fourier Transform", "publisher": "IntechOpen", "isbn": "9789535105947", "section_idx": 2 }
00064f14-4c6c-4123-bed8-b5ae765fbfc8.3	Meet the editor Dr Salih Mohammed Salih (Member IEEE) was born in Babylon-1970. He received the B.Sc. degree from the Electrical Department, University of Baghdad (1999- Iraq), M.Sc. and Ph.D. degrees in Communication Engineering from the University of Technology-Iraq in 2003 and 2008 respectively. Part of his Ph.D. was completed at the National Technical University of Athens (Scholar- ship: 2006-2007). Since 2005, he has been working at the University of Anbar-Iraq, where he is a lecturer in Electrical Engineering Department. His research interests include MC-CDMA, OFDM, Wireless Communication, Signal Processing, Optical Communication, Radar, Computer Networks, Security, and Renewable Energy Resources. He published more than 30 papers in different international Journals and Conferences. Contents Preface IX Chapter 1 Fourier Series and Fourier Transform with Florica Matei and Nicolae Aldea Chapter 2 High Resolution Mass Spectrometry Chapter 3 Fourier Transform Infrared Chapter 5 Fourier Transform Spectroscopy Chanel Fortier Hassan Safouhi Chapter 7 Molecular Simulation with Bingzheng Li Applications in Nanomaterials Structure 1 Using FTICR and Orbitrap Instruments 25 the Measurement of Spectral Line Profiles 69 Hassen Aroui, Johannes Orphal and Fridolin Kwabia Tchana NMR Integrals over Exponential Type Functions 121 Surface Chemistry of Activated Carbons – A Review 165 E. R. Mejía, J. D. Ospina, M. A. Márquez and A. L. Morales Spectroscopy for Natural Fibres 45 Mizi Fan, Dasong Dai and Biao Huang Chapter 4 Fourier Transform Infrared Spectroscopy for of Cotton and Cotton Trash 103 Chapter 6 Fourier Transformation Method for Computing Discrete Fast Fourier Transform 137 Xiongwu Wu and Bernard R. Brooks Chapter 8 Charaterization of Pore Structure and Chapter 9 Bioleaching of Galena (PbS) 191 Paulo J. Amorim Madeira, Pedro A. Alves and Carlos M. Borges ## Contents ## Preface XI E. R. Mejía, J. D. Ospina, M. A. Márquez and A. L. Morales Preface This book focuses on the Fourier transform applications in the analysis of some types of materials. The field of Fourier transform has seen explosive growth during the past decades, as phenomenal advances both in research and application have been made. During the preparation of this book, we found that almost all the textbooks on materials analysis have a section devoted to the Fourier transform theory. Most of those describe some formulas and algorithms, but one can easily be lost in seemingly incomprehensible mathematics. The basic idea behind all those horrible looking formulas is rather simple, even fascinating: it is possible to form any function . as a summation of a series of sine and cosine terms of increasing frequency. In other words, any space or time varying data can be transformed into a different domain called the frequency space. A fellow called Joseph Fourier first came up with the idea in the 19th century, and it was proven to be useful in various applications. As far as we can tell, Gauss was the first to propose the techniques that we now call the fast Fourier transform (FFT) for calculating the coefficients in a trigonometric expansion of an asteroid's orbit in 1805. However, it was the seminal paper by Cooley and Tukey in 1965 that caught the attention of the science and engineering community and, in a way, founded the discipline of digital signal processing (DSP). One of the main focuses of this book is on getting material characterization of nanomaterials through Fourier transform infrared spectroscopy (FTIR), and this fact can be taken from FTIR which gives reflection coefficient versus wave number. The Fourier transform spectroscopy is a measurement technique whereby spectra are collected based on measurements of the coherence of a radiative source, using time-domain or space-domain measurements of the electromagnetic radiation or other type of radiation. It can be applied to a variety of types of spectroscopy including optical spectroscopy, infrared spectroscopy (FTIR, FT-NIRS), nuclear magnetic resonance (NMR) and magnetic resonance spectroscopic imaging (MRSI), mass spectrometry and electron spin resonance spectroscopy. There are several methods for measuring the temporal coherence of the light, including the continuous wave Michelson or Fourier transform spectrometer and the pulsed Fourier transform spectrograph (which is more sensitive and has a much shorter sampling time than conventional spectroscopic techniques, but is only applicable in a laboratory environment). The term Fourier transform spectroscopy reflects the fact that in all these techniques, a Fourier transform is required to turn the raw data into the actual spectrum, and in many of the cases in optics involving interferometers, is based on the Chapter 12 Fourier Transform Sound Radiation 239 F. X. Xin and T. J. Lu ## Preface VI Contents Chapter 10 Application of Hankel Transform for Chapter 11 Eliminating the Undamaging Fatigue Cycles Chapter 12 Fourier Transform Sound Radiation 239 F. X. Xin and T. J. Lu S. Abdullah, T. E. Putra and M. Z. Nuawi Sei Ueda Solving a Fracture Problem of a Cracked Piezoelectric Strip Under Thermal Loading 207 Using the Frequency Spectrum Filtering Techniques 223 This book focuses on the Fourier transform applications in the analysis of some types of materials. The field of Fourier transform has seen explosive growth during the past decades, as phenomenal advances both in research and application have been made. During the preparation of this book, we found that almost all the textbooks on materials analysis have a section devoted to the Fourier transform theory. Most of those describe some formulas and algorithms, but one can easily be lost in seemingly incomprehensible mathematics. The basic idea behind all those horrible looking formulas is rather simple, even fascinating: it is possible to form any function . as a summation of a series of sine and cosine terms of increasing frequency. In other words, any space or time varying data can be transformed into a different domain called the frequency space. A fellow called Joseph Fourier first came up with the idea in the 19th century, and it was proven to be useful in various applications. As far as we can tell, Gauss was the first to propose the techniques that we now call the fast Fourier transform (FFT) for calculating the coefficients in a trigonometric expansion of an asteroid's orbit in 1805. However, it was the seminal paper by Cooley and Tukey in 1965 that caught the attention of the science and engineering community and, in a way, founded the discipline of digital signal processing (DSP). One of the main focuses of this book is on getting material characterization of nanomaterials through Fourier transform infrared spectroscopy (FTIR), and this fact can be taken from FTIR which gives reflection coefficient versus wave number. The Fourier transform spectroscopy is a measurement technique whereby spectra are collected based on measurements of the coherence of a radiative source, using time-domain or space-domain measurements of the electromagnetic radiation or other type of radiation. It can be applied to a variety of types of spectroscopy including optical spectroscopy, infrared spectroscopy (FTIR, FT-NIRS), nuclear magnetic resonance (NMR) and magnetic resonance spectroscopic imaging (MRSI), mass spectrometry and electron spin resonance spectroscopy. There are several methods for measuring the temporal coherence of the light, including the continuous wave Michelson or Fourier transform spectrometer and the pulsed Fourier transform spectrograph (which is more sensitive and has a much shorter sampling time than conventional spectroscopic techniques, but is only applicable in a laboratory environment). The term Fourier transform spectroscopy reflects the fact that in all these techniques, a Fourier transform is required to turn the raw data into the actual spectrum, and in many of the cases in optics involving interferometers, is based on the #### XII Preface Wiener–Khinchin theorem. In this book, New theoretical results are appearing and new applications open new areas for research. It is hoped that this book will provide the background, references and incentive to encourage further research and results in this area as well as provide tools for practical applications. One of the attractive features of this book is the inclusion of extensive simple, but practical, examples that expose the reader to real-life materials analysis problems, which has been made possible by the use of computers in solving practical design problems. The whole book contains twelve chapters. The chapters deal with nanomaterials structure, mass spectrometry, infrared spectroscopy for natural fibers, infrared spectroscopy to the measurements of spectral line profile, spectroscopy of cotton and cotton trash, computing NMR integrals over exponential type functions, molecular simulation, charaterization of pore structure and surface chemistry of activated carbons, bioleaching of galena, the cracked piezoelectric strip under thermal loading, eliminating the undamaging fatigue cycles, and the Fourier transform sound radiation. Finally, we would like to thank all the authors who have participated in this book for their valuable contribution. Also we would like to thank all the reviewers for their valuable notes. While there is no doubt that this book may have omitted some significant findings in the Fourier transform field, we hope the information included will be useful for Physics, Chemists, Agriculturalists, Electrical Engineers, Mechanical Engineers and the Signal Processing Engineers, in addition to the Academic Researchers working in these fields. > Salih Mohammed Salih College of Engineering Univercity of Anbar Iraq 1 Cluj-Napoca, Romania Fourier Series and Fourier Transform with Applications in Nanomaterials Structure 1University of Agricultural Science and Veterinary Medicine, Cluj-Napoca, 2National Institute for Research and Development of Isotopic and Molecular Technologies, One of the most important problems in the physics and chemistry of the nanostructured materials consists in the local and the global structure determination by X-ray diffraction and X-ray absorption spectroscopy methods. This contribution is dedicated to the applications of the Fourier series and Fourier transform as important tools in the determination of the nanomaterials structure. The structure investigation of the nanostructured materials require the understanding of the mathematical concepts regarding the Fourier series and Fourier transform presented here without theirs proofs. The Fourier series is the traditional tool dedicated to the composition of the periodical signals and its decomposition in discreet harmonics as well as for the solving of the differential equations. Whereas the Fourier transform is more appropriate tool in the study of the non periodical signals and for the solving of the first kind integral equations. From physical point of view the Fourier series are used to describe the model of the global structure of nanostructured materials that consist in: average crystallite size, microstrain of the lattice and distribution functions of the crystallites and microstrain versus size. Whereas the model for the local structure of the nanomaterials involves the direct and inverse Fourier transform. The information obtained consist in the number of atoms from each coordination shell and their radial One of the most often model studied in physics is the one of oscillatory movement of a material point. The oscillation of the electrical charge into an electrical field, the vibration of a tuning fork that generated sound waves or the electronic vibration into atoms that generate light waves are studied in the same mode (Richard et al., 2005). The motion equations related to the above phenomena have similar form; therefore the phenomena treated are analogous. From mathematical point of view these are modeled by the ordinary differential equations, most of them with constant coefficients. Due to the particular form of the equation any linear combination of the solution it is also a solution and the mathematical substantiation is given by the superposition principle. It consists in, if u1, u2, …, uk are 1. Introduction distances. 2. Fourier series and theirs applications Florica Matei1 and Nicolae Aldea2 Florica Matei1 and Nicolae Aldea2 1University of Agricultural Science and Veterinary Medicine, Cluj-Napoca, 2National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca, Romania ## 1. Introduction X Preface Wiener–Khinchin theorem. In this book, New theoretical results are appearing and new applications open new areas for research. It is hoped that this book will provide the background, references and incentive to encourage further research and results in this area as well as provide tools for practical applications. One of the attractive features of this book is the inclusion of extensive simple, but practical, examples that expose the reader to real-life materials analysis problems, which has been made The whole book contains twelve chapters. The chapters deal with nanomaterials structure, mass spectrometry, infrared spectroscopy for natural fibers, infrared spectroscopy to the measurements of spectral line profile, spectroscopy of cotton and cotton trash, computing NMR integrals over exponential type functions, molecular simulation, charaterization of pore structure and surface chemistry of activated carbons, bioleaching of galena, the cracked piezoelectric strip under thermal loading, eliminating the undamaging fatigue cycles, and the Fourier transform sound radiation. Finally, we would like to thank all the authors who have participated in this book for their valuable contribution. Also we would like to thank all the reviewers for their valuable notes. While there is no doubt that this book may have omitted some significant findings in the Fourier transform field, we hope the information included will be useful for Physics, Chemists, Agriculturalists, Electrical Engineers, Mechanical Engineers and the Signal Processing Engineers, in addition to the Academic > Salih Mohammed Salih College of Engineering Univercity of Anbar > > Iraq possible by the use of computers in solving practical design problems. Researchers working in these fields. One of the most important problems in the physics and chemistry of the nanostructured materials consists in the local and the global structure determination by X-ray diffraction and X-ray absorption spectroscopy methods. This contribution is dedicated to the applications of the Fourier series and Fourier transform as important tools in the determination of the nanomaterials structure. The structure investigation of the nanostructured materials require the understanding of the mathematical concepts regarding the Fourier series and Fourier transform presented here without theirs proofs. The Fourier series is the traditional tool dedicated to the composition of the periodical signals and its decomposition in discreet harmonics as well as for the solving of the differential equations. Whereas the Fourier transform is more appropriate tool in the study of the non periodical signals and for the solving of the first kind integral equations. From physical point of view the Fourier series are used to describe the model of the global structure of nanostructured materials that consist in: average crystallite size, microstrain of the lattice and distribution functions of the crystallites and microstrain versus size. Whereas the model for the local structure of the nanomaterials involves the direct and inverse Fourier transform. The information obtained consist in the number of atoms from each coordination shell and their radial distances. ## 2. Fourier series and theirs applications One of the most often model studied in physics is the one of oscillatory movement of a material point. The oscillation of the electrical charge into an electrical field, the vibration of a tuning fork that generated sound waves or the electronic vibration into atoms that generate light waves are studied in the same mode (Richard et al., 2005). The motion equations related to the above phenomena have similar form; therefore the phenomena treated are analogous. From mathematical point of view these are modeled by the ordinary differential equations, most of them with constant coefficients. Due to the particular form of the equation any linear combination of the solution it is also a solution and the mathematical substantiation is given by the superposition principle. It consists in, if u1, u2, …, uk are ( ) cos(2 ) sin(2 ) <sup>2</sup> n n /2 /2 <sup>2</sup> ( ) T <sup>2</sup> ( )cos(2 ) <sup>2</sup> ( )sin(2 ) c x t nf t dt <sup>T</sup> ii. If xt T xt ( /2) ( ) + =− then Fourier series of x has only amplitudes with odd index (Tang, ( ) cos(2 ) sin(2 ) n n iii. In practice the argument of the function x can be a scalar as time, frequency, length, angle, and so on, thus (4) is defined on period [0, T], spectral interval [0,f], spatial ( ) ( ) cos(2 ) sin(2 ) <sup>2</sup> The right term of the above equality represents the energy density of the signal x. Thus the Parseval equality shows that the whole density energy is contained in the squares of the all amplitudes of harmonic terms defined on the interval [ / 2, /2] −T T . The Parseval equality holds for any function whose square is integrable. The next problem is to analyze the convergence of the series. Because the aim of this chapter are the application of the Fourier series it will be only mentioned the basic principles of the Fourier analysis. If the interval [ /2, /2] −T T can be decomposed in a finite number of intervals on that the function f is <sup>a</sup> x t dt x t b nf tc nf t dt x t b nf t c nf t π [ ] 21 0 <sup>0</sup> b x t nf t dt <sup>T</sup> nf t and integration nf t and integration 0 0 π π T a x t dt T − π T /2 /2 /2 T /2 Some observations about physical signal modeled by Fourier series are given below. i. Value a / 2 represents the mean value for the physical signal on[−T T /2, /2] . T − T − π <sup>a</sup> x t b nf t c nf t π 1 = n i. by integration of the previous relation between [−T T /2, /2] n n 0 = + λ ( ) 2 2 ∞ iv. Using the Fourier coefficients the Parseval's equality is given by 1 ⎡ ⎤ =+ + ⎢ ⎥ ⎣ ⎦ 2 2 n n = ∞ ∑ n T a b c n /2 /2 1 2 ∫ ∫ ∑ T T n − − = ∞ The Fourier coefficients are obtained in the following way: ii. by multiplication of (4) with 0 cos(2 ) iii. by multiplication of (4) with 0 sin(2 ) 2007) all the other terms will vanish: interval [0, ] L , wave length [0, /2 /2 2 T T ∞ [ ] 0 0 π = + ∑ <sup>+</sup> (4) <sup>=</sup> ∫ (5) <sup>=</sup> ∫ (6) <sup>=</sup> ∫ (7) π ] or the whole trigonometric circle, respectively. 0 0 π (9) [ ] n n π ⎧ ⎫ ⎪ ⎪ <sup>=</sup> ⎨ ⎬ ++= ⎪ ⎪ ⎩ ⎭ <sup>=</sup> ∑ <sup>+</sup> (8) solutions for the homogenous linear equations L u[] 0 = , then the linear combination (or the superposition) is a solution of [ ] 0 L u = for any choice of the constants. The previous statement shows that the general solution of a linear equation is a superposition of its linearly independent particularly solution that compose a base in the finite dimension space of the solution. The superposition is true for any algebraic equations as well as any homogenous linear ordinary differential equations. #### 2.1 Physical concept and mathematical background The analysis of the linear harmonic oscillatory motion for a material point of mass m round about equilibrium position due to an elastically force F=-Kx it is given by the harmonic equation that is a differential equations which appear very frequently in the analysis of physical phenomena (Tang, 2007) $$m\frac{d^2\mathbf{x}}{dt^2} + \mathbf{K}\mathbf{x} = \mathbf{0} \tag{1}$$ with the solution 0 0 xt A ft ( ) cos(2 ) = π −ϕ where A, K, f0, <sup>K</sup> m ω = , 0 ω = 2πf and φ0 represent the motion's amplitude, elastic constant, fundamental frequency, angular speed and phase shift, respectively. Generalizing let consider the physical signal given by $$\mathbf{x}(t) = \underbrace{a\_1 \sin(2\pi f\_0 t)}\_{f\_0 \text{ line}} + \underbrace{a\_2 \sin(4\pi f\_0 t)}\_{2f\_0 \text{ line}} + \underbrace{a\_3 \sin(6\pi f\_0 t)}\_{3f\_0 \text{ line}} + \dots + \underbrace{a\_n \sin(2\pi n f\_0 t)}\_{nf\_0 \text{ line}}\tag{2}$$ that is periodic but non harmonic process, the physical signal being a synthesis of n spectral lines with the frequencies f0, 2f0, 3f0, …, nf0 and the amplitudes a1, a2, … , an, respectively. The practical problem that had lead to Fourier series was to solve the heat equation which is a parabolic partial differential equation. Before the Fourier contribution no solution for the general form of the heat equation was known. The Fourier idea was to consider the solution as a linear combination of sine or cosine waves in according with the superposition principle. The solution space for of the partial differential equation are infinite dimensional spaces thus there are needed an infinite number of independent solutions. Therefore is not possible to find all independent particular solutions of a linear partial differential equations. The key found by Joseph Fourier in his article "Théorie analitique de la chaleur", published in 1811, was to form a series with the basic solutions. The ortonormality is the key concept of the Fourier analysis. The general representation of the Fourier series with coefficients a, b<sup>n</sup> and cn is given by: $$\mathbf{x}(t) = \frac{a}{2} + \sum\_{n=1}^{\infty} \left[ b\_n \cos(nt) + c\_n \sin(nt) \right] \tag{3}$$ The Fourier series are used in the study of periodical movements, acoustics, electrodynamics, optics, thermodynamics and especially in physical spectroscopy as well as in fingerprints recognition and many other technical domains. It was proved (Walker, 1996) that any physical signal of the period T=1/f0 can be represented as an harmonic function with the frequencies f0, 2f0, 3f0, … $$\exp(t) = \frac{a}{2} + \sum\_{n=1}^{a} \left[ b\_n \cos(2\pi n f\_0 t) + c\_n \sin(2\pi n f\_0 t) \right] \tag{4}$$ The Fourier coefficients are obtained in the following way: 2 Fourier Transform – Materials Analysis solutions for the homogenous linear equations L u[] 0 = , then the linear combination (or the superposition) is a solution of [ ] 0 L u = for any choice of the constants. The previous statement shows that the general solution of a linear equation is a superposition of its linearly independent particularly solution that compose a base in the finite dimension space of the solution. The superposition is true for any algebraic equations as well as any The analysis of the linear harmonic oscillatory motion for a material point of mass m round about equilibrium position due to an elastically force F=-Kx it is given by the harmonic equation that is a differential equations which appear very frequently in the analysis of the motion's amplitude, elastic constant, fundamental frequency, angular speed and phase 0 00 0 1 02 03 0 0 2 3 ( ) sin(2 ) sin(4 ) sin(6 ) ... sin(2 ) <sup>n</sup> f line f line f line nf line x t a f t a f t a f t a nf t = + + ++ ππ that is periodic but non harmonic process, the physical signal being a synthesis of n spectral lines with the frequencies f0, 2f0, 3f0, …, nf0 and the amplitudes a1, a2, … , an, respectively. The practical problem that had lead to Fourier series was to solve the heat equation which is a parabolic partial differential equation. Before the Fourier contribution no solution for the general form of the heat equation was known. The Fourier idea was to consider the solution as a linear combination of sine or cosine waves in according with the superposition principle. The solution space for of the partial differential equation are infinite dimensional spaces thus there are needed an infinite number of independent solutions. Therefore is not possible to find all independent particular solutions of a linear partial differential equations. The key found by Joseph Fourier in his article "Théorie analitique de la chaleur", published in 1811, was to form a series with the basic solutions. The ortonormality is the key concept of the Fourier analysis. The general representation of the Fourier series with coefficients a, b<sup>n</sup> where A, K, f0, <sup>K</sup> [ ] =+ + ∑ (3) ω + = (1) π f and φ0 represent (2) m = , 0 ω = 2π 2 <sup>2</sup> <sup>0</sup> d x m Kx dt homogenous linear ordinary differential equations. physical phenomena (Tang, 2007) and cn is given by: with the frequencies f0, 2f0, 3f0, … with the solution 0 0 xt A ft ( ) cos(2 ) = 2.1 Physical concept and mathematical background π −ϕ π shift, respectively. Generalizing let consider the physical signal given by 1 = n ∞ ( ) cos( ) sin( ) <sup>2</sup> n n The Fourier series are used in the study of periodical movements, acoustics, electrodynamics, optics, thermodynamics and especially in physical spectroscopy as well as in fingerprints recognition and many other technical domains. It was proved (Walker, 1996) that any physical signal of the period T=1/f0 can be represented as an harmonic function <sup>a</sup> x t b nt c nt i. by integration of the previous relation between [−T T /2, /2] $$a = \frac{2}{T} \int\_{-T/2}^{T/2} x(t)dt\tag{5}$$ ii. by multiplication of (4) with 0 cos(2 ) πnf t and integration $$b\_n = \frac{2}{T} \int\_{-T/2}^{T/2} x(t) \cos(2\pi n f\_0 t) dt \tag{6}$$ iii. by multiplication of (4) with 0 sin(2 ) πnf t and integration $$c\_n = \frac{2}{T} \int\_{-T/2}^{T/2} x(t) \sin(2\pi n f\_0 t) dt \tag{7}$$ Some observations about physical signal modeled by Fourier series are given below. $$\mathbf{x}(t) = \sum\_{n=0}^{n} \left[ b\_{2n+1} \cos(2\pi n f\_0 t) + c\_n \sin(2\pi n f\_0 t) \right] \tag{8}$$ $$\begin{aligned} \left[ \int\_{-T/2}^{T/2} x^2(t) \, dt = \int\_{-T/2}^{T/2} x(t) \left\{ \frac{a}{2} + \sum\_{n=1}^{\infty} \left[ b\_n \cos(2\pi n f\_0 t) + c\_n \sin(2\pi n f\_0 t) \right] \right\} dt = \\ = \frac{T}{2} \left[ \frac{a^2}{2} + \sum\_{n=1}^{\infty} \left( b\_{\frac{n}{n}}^2 + c\_{\frac{n}{n}}^2 \right) \right] \end{aligned} \tag{9}$$ The right term of the above equality represents the energy density of the signal x. Thus the Parseval equality shows that the whole density energy is contained in the squares of the all amplitudes of harmonic terms defined on the interval [ / 2, /2] −T T . The Parseval equality holds for any function whose square is integrable. The next problem is to analyze the convergence of the series. Because the aim of this chapter are the application of the Fourier series it will be only mentioned the basic principles of the Fourier analysis. If the interval [ /2, /2] −T T can be decomposed in a finite number of intervals on that the function f is 2 2 ( ) cos sin ; <sup>2</sup> iii. The Fourier coefficients associated to polynomial P are obtained by the least square kt kt P t [ ] B B M B ∫ ∫ ∑ ∫ A A K A B B M B A A K A ∫ ∫ ∑ ∫ cos cos sin cos ( )cos <sup>2</sup> γ m kk kk m m C dt C C C dt Y t C dt m kk kk m m C dt C C C dt Y t C dt [ ] sin cos sin sin ( )sin <sup>2</sup> γ trigonometric polynomial of degree M and m =1, …, M. The system (16) has 2M+1 equations and due to the orthogonality properties of the functions cosCk and sinCk the solution of kt Y t dt π iv. Previous considerations are often used in the process of global approximation of the discreet physical signals. Let consider the sequence of experimental values ( ) 1, , k k k N y P <sup>=</sup> , > 2 2 2 2 ( 1) cos 11 1 ⎡ ⎤ ⎛ ⎞ <sup>−</sup> ≈≈ + ⎢ ⎥ ⎜ ⎟ − − −− ⎣ ⎦ ⎝ ⎠ jA j k P P jA j k P j <sup>M</sup> <sup>2</sup> 2 2 ( 1) sin , 1, ; 1 1 π N NB A N <sup>=</sup> − − <sup>∫</sup> and 2 2 ( )sin ; k t BA N = ( ) /( 1) − − thus ( 1) kt Ak t = + − π γ π i i + + == ∑ (19) = ⇔ ∑ <sup>−</sup> <sup>=</sup> and represents the same condition Pt Yt Cky Cky Y i N k k π BA BA γ π − , Y is the approximated function by the B kt Y t dt <sup>=</sup> − − ∫ (17) π Δ and then (18) A BA BA (16) ⎛ ⎞ ⎛⎞ ⎛⎞ <sup>=</sup> + + ⎜ ⎟ ⎜⎟ ⎜⎟ ⎝ ⎠ ⎝⎠ ⎝⎠ − − <sup>∑</sup> (15) 1 [ ] dt C C dt Y t dt γ <sup>⎪</sup> ⎧ ⎫ ⎪ ⎪ ⎨ ⎨ +− = <sup>⎬</sup> <sup>⎪</sup> ⎪ ⎪ ⎩ ⎭ <sup>⎪</sup> ⎧ ⎫ <sup>⎪</sup> ⎪ ⎪ <sup>⎪</sup> +− = ⎨ ⎬ ⎩ ⎪ ⎪ ⎩ ⎭ π B A the approximate values of Fourier coefficients are given by β 1 1 = β k α The system (16) is equivalent with ( )<sup>2</sup> M = ∑ N j k k 1 1 = = π ⎡ ⎤ ⎛ ⎞ <sup>−</sup> ≈ += ⎢ ⎥ ⎜ ⎟ − −− ⎣ ⎦ ⎝ ⎠ v. If the trigonometric polynomial pass through all the experimental points, that is ( ) 1 i relation 2M+1=N where N represents the number of experimental points; vi. The degree of approximation is given by the residual index defined by = N γ () () cos sin , 1.. <sup>2</sup> k ik i i () 0 i i find in the least squares method for the discreet case. The coefficients are given by (17) relations. In this case the degree of the trigonometric polynomial, M, has to satisfy the Pt Y N N k j k ∑ ∑ k k N B A N BA BA Δ cos sin ( ) <sup>2</sup> B B M B k kk k A A K A ∫∫ ∫ ∑ <sup>⎧</sup> ⎧ ⎫ ⎪ ⎪ ⎪+ − = ⎨ ⎬ <sup>⎪</sup> ⎪ ⎪ ⎩ ⎭ <sup>⎪</sup> β β = β k α 1 = β 1 = method and the linear system is α α α system (16) is α 1 = where , C Ckt <sup>k</sup> = C Cmt <sup>m</sup> = , C BA = 2 /( ) <sup>=</sup> <sup>−</sup> <sup>∫</sup> 2 2 ( )cos β with discretization step defined by k <sup>2</sup> () , B α γ A Y t dt B A M continuous and monotonic, then the function f has a Fourier series representation. The next consideration is connected with the <sup>2</sup>L AB [,] space defined below $$L^2[A,B] = \left\{ \mathbf{x} : [A,B] \to R \left\| \int\_A^B \left\| \mathbf{x}(t) \right\|^2 dt < \infty \right\} \tag{10}$$ The complete system for the <sup>2</sup>L AB [,] is given by $$ \sqrt{\frac{2}{B-A}}, \sqrt{\frac{2}{B-A}} \cos \frac{2\pi t}{B-A}, \sqrt{\frac{2}{B-A}} \sin \frac{2\pi t}{B-A}, \dots, \tag{11} $$ $$ \sqrt{\frac{2}{B-A}} \cos \frac{2\pi kt}{B-A}, \sqrt{\frac{2}{B-A}} \sin \frac{2\pi kt}{B-A}, \dots $$ then any function from <sup>2</sup>L AB [,] can be written as a linear combination of its complete system and the Fourier coefficients are $$a = \sqrt{\frac{2}{B - A}} \int\_{A}^{B} \mathbf{x}(t) dt,$$ $$\beta\_k = \sqrt{\frac{2}{B - A}} \int\_{A}^{B} \mathbf{x}(t) \cos \frac{2\pi kt}{B - A} dt, \quad \gamma\_k = \sqrt{\frac{2}{B - A}} \int\_{A}^{B} \mathbf{x}(t) \sin \frac{2\pi kt}{B - A} dt. \tag{12}$$ #### 2.2 Trigonometric polynomials and Fourier coefficients determination One of the useful mathematical tools in order to apply the Fourier series in data analysis is the trigonometric polynomial. From physical point of view the trigonometric polynomials are used to characterize the periodic signals. The general form of the real trigonometric polynomial of degree M is given by: $$P(t) = \frac{\alpha}{2} + \sum\_{k=1}^{M} \left( \beta\_k \cos\left(\frac{2\pi kt}{T}\right) + \gamma\_k \sin\left(\frac{2\pi kt}{T}\right) \right) \tag{13}$$ where α, β <sup>k</sup> , <sup>k</sup> γ , T being real constants. Let denote by S the square deviation of the function x from trigonometric polynomial P defined on interval of length T $$S(\alpha, \beta\_1, \dots, \beta\_{M'}, \gamma\_1, \dots, \gamma\_M) = \int\_{-T/2}^{T/2} \left(\mathbf{x}(t) - P(t)\right)^2 dt \tag{14}$$ Let enumerate some properties of trigonometric polynomials that will be used in this chapter (Bachmann et al., 2002). continuous and monotonic, then the function f has a Fourier series representation. The next B A x A B R x t dt t t kt kt 22 2 2 2 , cos , sin , , BA BA BA BA − −− − 2 2 2 2 cos , sin , then any function from <sup>2</sup>L AB [,] can be written as a linear combination of its complete <sup>2</sup> () , BA BA BA BA γ One of the useful mathematical tools in order to apply the Fourier series in data analysis is the trigonometric polynomial. From physical point of view the trigonometric polynomials are used to characterize the periodic signals. The general form of the real trigonometric B a x t dt B A π 2.2 Trigonometric polynomials and Fourier coefficients determination 1 function x from trigonometric polynomial P defined on interval of length T = 1 1 β γ coefficient equal with the Fourier coefficients of function x; β k M 2 αβ trigonometric polynomial associated is given by α <sup>=</sup> <sup>−</sup> A B B ∫ A A 2 2 ( ) cos sin kt kt P t ( , , , , , , ) () () Let enumerate some properties of trigonometric polynomials that will be used in this i. Let x be a function in [ ] <sup>2</sup>LT T − /2, /2 , then from all polynomials of degree M the minimum of the square deviation is obtained for the trigonometric polynomial with the ii. If the physical signal is defined on an arbitrary interval [A B, ] with period B-A then the γ M M S x k k π T T , T being real constants. Let denote by S the square deviation of the /2 T /2 T − γ π ⎛ ⎞ ⎛⎞ ⎛⎞ = + ⎜ ⎟ <sup>+</sup> ⎜⎟ ⎜⎟ ⎝ ⎠ ⎝⎠ ⎝⎠ <sup>∑</sup> (13) ( ) = − � � ∫ (14) t P t dt 2 ∫ ∫ <sup>=</sup> <sup>=</sup> −− −− 2 2 2 2 ( )cos , ( )sin . kt kt x t dt x t dt π BA BA BA BA BA − − −− − π <sup>⎧</sup> <sup>⎫</sup> <sup>⎪</sup> <sup>⎪</sup> <sup>⎨</sup> → < ∞⎬ ⎪⎩ ⎭⎪ <sup>∫</sup> (10) π � … π (11) (12) π <sup>2</sup>L AB [,]= <sup>2</sup> :[ , ] ( ) consideration is connected with the <sup>2</sup>L AB [,] space defined below k k The complete system for the <sup>2</sup>L AB [,] is given by system and the Fourier coefficients are β polynomial of degree M is given by: chapter (Bachmann et al., 2002). where α, β <sup>k</sup> , <sup>k</sup> γ $$P(t) = \frac{\alpha}{2} + \sum\_{k=1}^{M} \left( \beta\_k \cos\left(\frac{2\pi kt}{B-A}\right) + \gamma\_k \sin\left(\frac{2\pi kt}{B-A}\right) \right);\tag{15}$$ iii. The Fourier coefficients associated to polynomial P are obtained by the least square method and the linear system is $$ \begin{aligned} \left\langle \frac{\alpha}{2} \right\rangle^{B} dt + \sum\_{K=1}^{M} \left\langle \left[ \beta\_{k} \cos \mathbf{C}\_{k} - \boldsymbol{\gamma}\_{k} \sin \mathbf{C}\_{k} \right] dt \right\rangle &= \bigcap\_{A}^{B} Y(t) dt \\ \left\langle \frac{\alpha}{2} \right\rangle \cos \mathbf{C}\_{m} dt + \sum\_{K=1}^{M} \left\langle \left[ \beta\_{k} \cos \mathbf{C}\_{k} - \boldsymbol{\gamma}\_{k} \sin \mathbf{C}\_{k} \right] \cos \mathbf{C}\_{m} dt \right\rangle &= \bigcap\_{A}^{B} Y(t) \cos \mathbf{C}\_{m} dt \\ \left\langle \frac{\alpha}{2} \right\rangle \sum\_{A}^{B} \sin \mathbf{C}\_{m} dt + \sum\_{K=1}^{M} \left\langle \left[ \beta\_{k} \cos \mathbf{C}\_{k} - \boldsymbol{\gamma}\_{k} \sin \mathbf{C}\_{k} \right] \sin \mathbf{C}\_{m} dt \right\rangle &= \prod\_{A}^{B} Y(t) \sin \mathbf{C}\_{m} dt \end{aligned} \tag{16} $$ where , C Ckt <sup>k</sup> = C Cmt <sup>m</sup> = , C BA = 2 /( ) π − , Y is the approximated function by the trigonometric polynomial of degree M and m =1, …, M. The system (16) has 2M+1 equations and due to the orthogonality properties of the functions cosCk and sinCk the solution of system (16) is $$\alpha = \frac{2}{B-A} \int\_{A}^{B} Y(t)dt, \ \beta\_k = \frac{2}{B-A} \int\_{A}^{B} Y(t) \cos \frac{2\pi kt}{B-A} dt \ \text{and} \ \gamma\_k = \frac{2}{B-A} \int\_{A}^{B} Y(t) \sin \frac{2\pi kt}{B-A} dt; \tag{17}$$ iv. Previous considerations are often used in the process of global approximation of the discreet physical signals. Let consider the sequence of experimental values ( ) 1, , k k k N y P <sup>=</sup> , with discretization step defined by Δt BA N = ( ) /( 1) − − thus ( 1) kt Ak t = + − Δ and then the approximate values of Fourier coefficients are given by $$\begin{split} \alpha & \approx \frac{2}{N-1} \sum\_{k=1}^{N} P\_k \quad \beta\_j \approx \frac{2}{N-1} \sum\_{k=1}^{N} \left[ P\_k \cos \left( \frac{2\pi jA}{B-A} + \frac{2\pi j(k-1)}{N-1} \right) \right] \\ \gamma\_j & \approx \frac{2}{N-1} \sum\_{k=1}^{N} \left[ P\_k \sin \left( \frac{2\pi jA}{B-A} + \frac{2\pi j(k-1)}{N-1} \right) \right] \quad \quad j = \overline{1, M}; \end{split} \tag{18}$$ v. If the trigonometric polynomial pass through all the experimental points, that is $$P(t\_i) = Y(t\_i) \quad \Leftrightarrow$$ $$\frac{a}{2} + \sum\_{k=1}^{M} \left(\beta\_k \cos \text{Cky}\_i + \gamma\_k \sin \text{Cky}\_i\right) = Y\_{i\prime} \quad i = \overline{1...N} \tag{19}$$ The system (16) is equivalent with ( )<sup>2</sup> 1 () 0 N i i i Pt Y = ∑ <sup>−</sup> <sup>=</sup> and represents the same condition find in the least squares method for the discreet case. The coefficients are given by (17) relations. In this case the degree of the trigonometric polynomial, M, has to satisfy the relation 2M+1=N where N represents the number of experimental points; vi. The degree of approximation is given by the residual index defined by average crystallite size, microstrains of the lattice, total probability of the defaults and the 10 20 30 40 50 60 70 80 90 100 harmonic index The broadening of X-ray line profiles can be determined from the first derivative of the experimental spectrum analysis. The first derivative of the nickel foil spectrum in Fig. 3 is 26 28 30 32 34 36 38 40 42 44 46 48 Theta [degree] 2.4 Application of the trigonometric polynomial in the integration of partial differential One of the most important roles of the trigonometric polynomial is that played in the solving of the partial differential equations. The first example of this procedure is Fig. 3. The first derivative for the computed signal obtained through Fourier synthesis distribution functions of the crystallites and the microstrains determination. 0 The first derivative 10000 20000 30000 0 Fig. 2. The square magnitudes of the Fourier coefficients 50 100 150 200 Square magnitudes of Fourier coefficients given. equations 250 300 350 400 $$R = 100 \sum\_{i=1}^{N} \frac{\left\| \mathcal{Y}\_i^{\text{exp}} - P\_i \right\|}{\mathcal{Y}\_i^{\text{exp}}} \tag{20}$$ where exp <sup>i</sup> y represents the sequence of experimental values; vii. The first derivative of the physical signal approximated by the trigonometric polynomial is given by $$\frac{dP(t)}{dt} \approx -\frac{2\pi}{B-A} \sum\_{i=1}^{M} \left( k\rho\_k \sin\frac{2\pi kt}{B-A} - k\gamma\_k \cos\frac{2\pi kt}{B-A} \right) \tag{21}$$ Previous relation can be useful only when the physical signal is less affected by the noise; viii. Other application of the trigonometric polynomials is the determination of the integral intensity, I, of the physical signal $$I \approx \frac{a}{2}(B - A). \tag{22}$$ #### 2.3 Application of the Fourier series in X-ray diffraction The spectrum for X-ray diffraction for the nickel foil is represented in Fig.1. It has been registered with the Huber goniometer that used an incident fascicle with synchrotron radiation with the wavelength 1.8276 Å. The experimental data was recorded with constant step <sup>0</sup> Δθ= 0.033 and its number of pairs is n=766. Fig. 1. Experimental spectrum of the nickel foil The Miller indexes of the X-ray line profiles measurements are (111), (200), respectively (220). Data analysis of the experimental spectrum was realized by our package program (Aldea & Indrea, 1990). The Fig. 2 shows the square magnitude of Fourier coefficients versus the harmonic indexes. They are used in the Warren – Averbach model (Warren, 1990) for the 100 R 1 i = π 2.3 Application of the Fourier series in X-ray diffraction = 0.033 and its number of pairs is n=766. 0 Fig. 1. Experimental spectrum of the nickel foil 1000 2000 3000 Relative intensity 4000 5000 6000 M <sup>i</sup> y represents the sequence of experimental values; where exp is given by step <sup>0</sup> Δθ intensity, I, of the physical signal exp <sup>N</sup> <sup>i</sup> <sup>i</sup> y P k k γ ⎛ ⎞ ≈ − <sup>−</sup> ⎜ ⎟ − −− ⎝ ⎠ <sup>∑</sup> (21) π ( ). <sup>−</sup> <sup>=</sup> <sup>∑</sup> (20) π ≈ − (22) Experimental Fourier synthesis exp <sup>1</sup> i i vii. The first derivative of the physical signal approximated by the trigonometric polynomial () 2 2 2 sin cos Previous relation can be useful only when the physical signal is less affected by the noise; viii. Other application of the trigonometric polynomials is the determination of the integral > 2 I BA α The spectrum for X-ray diffraction for the nickel foil is represented in Fig.1. It has been registered with the Huber goniometer that used an incident fascicle with synchrotron radiation with the wavelength 1.8276 Å. The experimental data was recorded with constant 25 27 29 31 33 35 37 39 41 43 45 47 49 The Miller indexes of the X-ray line profiles measurements are (111), (200), respectively (220). Data analysis of the experimental spectrum was realized by our package program (Aldea & Indrea, 1990). The Fig. 2 shows the square magnitude of Fourier coefficients versus the harmonic indexes. They are used in the Warren – Averbach model (Warren, 1990) for the Theta [degree] β dP t kt kt k k dt B A B A B A <sup>=</sup> y average crystallite size, microstrains of the lattice, total probability of the defaults and the distribution functions of the crystallites and the microstrains determination. Fig. 2. The square magnitudes of the Fourier coefficients The broadening of X-ray line profiles can be determined from the first derivative of the experimental spectrum analysis. The first derivative of the nickel foil spectrum in Fig. 3 is given. Fig. 3. The first derivative for the computed signal obtained through Fourier synthesis #### 2.4 Application of the trigonometric polynomial in the integration of partial differential equations One of the most important roles of the trigonometric polynomial is that played in the solving of the partial differential equations. The first example of this procedure is (0, ) ( , ) 0 (boundary condition) The function y represents the position of each oscillating point versus Ox axis. The function f describes the initial string position and g describes the initial speed of the string. The constant c2 represents the ratio between straining force and the linear density of the string; the force has the same direction as the movement of the string element. The solution for the problem (30) is obtained using the same technique as in the case of the heat equation and it (30) π 0 <sup>2</sup> (0) ( )sin L n x a B <sup>f</sup> x dx L L = = ∫ and π <sup>=</sup> ⎡ ⎤ <sup>+</sup> ∑⎣ ⎦ (31) n n ( ,0) ( ) (initial conditions) ( ) π time and space of a particle with m mass inside the potential V and it is given by Ψ i ⎪ ⎪⎩ Ψ Ψ Ψ ( , ) cos / sinc( / ) sin n n n x y x t a nc t L b t nct L <sup>L</sup> Schrödinger equation is the other example presented that describes the quantum behavior in 2 2 ∂ ∂ =− + ∂ ∂ Ψ i V x t m x π equation becomes the free particle equation and this case will be analyzed forward. Ψ <sup>⎪</sup> <sup>=</sup> <sup>⎪</sup> <sup>2</sup> ( ) <sup>2</sup> 2 2 <sup>2</sup> 2 (0, ) ( , ) 0 ( ,0) ( ) ( ,) 1 Ψ t m x t Lt x fx Ψ From physical point of view Ψ represents a probability density generator and <sup>2</sup> \* 2 2 describes the existence probability of a particle of mass m at position x and time t, thus 0 Ψ Analogous with the previous two examples the solution of the problem (33) is given by L ⎨ = = <sup>⎧</sup> ∂ ∂ <sup>⎪</sup> = − <sup>∂</sup> <sup>∂</sup> <sup>⎪</sup> � � Ψ � � (32) . If the potential energy is vanished the previous <sup>=</sup> x t dx <sup>=</sup> ∫ (34) (33) Ψ ΨΨ= 2 2 2 2 2 ⎧∂ ∂ <sup>⎪</sup> <sup>=</sup> ⎪ ∂ ∂ ⎪⎪ = = <sup>⎨</sup> ⎪ = ⎪∂⎪ <sup>=</sup> ⎪⎩ <sup>∂</sup> 1 = n where "sinc" represents the normalized sinc function, π= = ∫ for all 0,1,2, <sup>n</sup> <sup>=</sup> … where � is Planck constant divided by 2 ∞ has the form ' n n 0 <sup>2</sup> (0) ( )sin L n x b B g x dx L L y y <sup>c</sup> t x y t yLt yx f x ( ,0) ( ) <sup>y</sup> x gt <sup>t</sup> applied to the heat equation in one dimensional space in the conditions of the following problem: $$\begin{cases} \frac{\partial \boldsymbol{u}}{\partial t} = \boldsymbol{a}^2 \frac{\partial^2 \boldsymbol{u}}{\partial \mathbf{x}^2} \\ \boldsymbol{u}(0, t) = \boldsymbol{u}(L, t) = \boldsymbol{0} \text{ (boundary condition)} \\ \boldsymbol{u}(\mathbf{x}, t) = \mathbf{g}(\mathbf{x}) \text{ (initial condition)} \\ \end{cases} \tag{23}$$ where a2 is the diffusion coefficient, it represents the heat conductibility of the material expressed in cm2/s. The solution of (23) is obtained using the separation of variables and the Fourier series technique. The solution has the form $$\mu(t,\mathbf{x}) = \sum\_{n=1}^{\infty} B\_n(t) \sin \frac{n \pi \chi}{L} \tag{24}$$ where Bn are determined using the Fourier series technique. $$B\_n(t) = \frac{2}{L} \Big\| \int\_0^L \mu(\mathbf{x}, t) \sin \frac{n \pi \chi}{L} d\mathbf{x} \tag{25}$$ Replacing (24) in the heat equation is obtained $$B\_n^\dagger(t) + \left(\frac{n\pi a}{L}\right)^2 B\_n(t) = 0, \quad n = 0, 1, 2, \cdots \tag{26}$$ From the initial condition is obtained $$b\_n = B\_n(0) = \frac{2}{L} \Big\| \int\_0^L \mu(\mathbf{x}, 0) \sin \frac{n \pi \chi}{L} d\chi \quad n = 0, 1, 2, \cdots \tag{27}$$ The relation (27) shows that the right member represents the n Fourier coefficient for the function that gives initial temperature g. By solving the ordinary differential equation (26) is obtained $$B\_n(t) = b\_n \exp\left(\left(-n\pi \, a \,/\, L\right)^2 t\right) \quad n = 0, 1, 2, \cdots \tag{28}$$ Replacing (28) in (24), the general solution of the problem (23) for the heat equation is $$u(t, \mathbf{x}) = \sum\_{n=1}^{\infty} b\_n \exp\left(-\left(\frac{n\pi a}{L}\right)^2 t\right) \sin\frac{n\pi \mathbf{x}}{L} \tag{29}$$ where the gaussian part plays the role of damping factor. Let consider, as the second example, a vibrating string of length L fixed on both ended in the absence of any external force, 0 ≤ x L ≤ and t>0, its motion is describes by the wave equation applied to the heat equation in one dimensional space in the conditions of the following (0, ) ( , ) 0 (boundary condition) where a2 is the diffusion coefficient, it represents the heat conductibility of the material expressed in cm2/s. The solution of (23) is obtained using the separation of variables and the ∞ 1 ( , ) ( )sin <sup>n</sup> n n x utx B t n x B t u x t dx L L ( ) ( ) 0, 0,1, 2, n n <sup>2</sup> (0) ( ,0)sin 0,1, 2, The relation (27) shows that the right member represents the n Fourier coefficient for the function that gives initial temperature g. By solving the ordinary differential equation (26) is > (( ) ) <sup>2</sup> ( ) exp / 0,1, 2, Bt b na L t n n n =− = π Replacing (28) in (24), the general solution of the problem (23) for the heat equation is ( , ) exp <sup>n</sup> sin na nx utx b <sup>t</sup> Let consider, as the second example, a vibrating string of length L fixed on both ended in the absence of any external force, 0 ≤ x L ≤ and t>0, its motion is describes by the wave equation ⎛ ⎞ ⎛ ⎞ = −⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ ⎝ ⎠ π 2 π ∞ L L π ∑ (29) + == ⎜ ⎟ = 0 <sup>2</sup> ( ) ( , )sin L 2 n a Bt Bt n L ⎛ ⎞ π n x b B u x dx n L L 0 1 = n where the gaussian part plays the role of damping factor. L L π π <sup>=</sup> ∑ (24) <sup>=</sup> ∫ (25) ⎝ ⎠ " (26) " (28) = = <sup>=</sup> ∫ " (27) (23) ( , ) ( ) (intial condition) 2 2 2 u u a t x u t uLt uxt gx ⎨ = = ⎧∂ ∂ <sup>⎪</sup> <sup>=</sup> <sup>∂</sup> <sup>∂</sup> <sup>⎪</sup> <sup>⎪</sup> <sup>=</sup> <sup>⎪</sup> where Bn are determined using the Fourier series technique. ' n n n ⎪ ⎪⎩ Fourier series technique. The solution has the form Replacing (24) in the heat equation is obtained From the initial condition is obtained obtained problem: $$\begin{cases} \frac{\partial^2 y}{\partial t^2} = c^2 \frac{\partial^2 y}{\partial x^2} \\ y(0, t) = y(L, t) = 0 \quad \text{(boundary condition)} \\ y(\mathbf{x}, 0) = f(\mathbf{x}) \quad \text{(initial conditions)} \\ \frac{\partial y}{\partial t}(\mathbf{x}, 0) = \mathbf{g}(t) \end{cases} \tag{30}$$ The function y represents the position of each oscillating point versus Ox axis. The function f describes the initial string position and g describes the initial speed of the string. The constant c2 represents the ratio between straining force and the linear density of the string; the force has the same direction as the movement of the string element. The solution for the problem (30) is obtained using the same technique as in the case of the heat equation and it has the form $$y(\mathbf{x},t) = \sum\_{n=1}^{\alpha} \left[ a\_n \cos \left( nc\pi t \,/\, L \right) + b\_n t \sin \left( nc\pi t \,/\, L \right) \right] \sin \frac{n\pi \, \chi}{L} \tag{31}$$ where "sinc" represents the normalized sinc function, 0 <sup>2</sup> (0) ( )sin L n n n x a B <sup>f</sup> x dx L L π= = ∫ and $$b\_n = B\_n^\dagger(0) = \frac{2}{L} \Big\| \int\_0^L g(\alpha) \sin \frac{n \pi \alpha}{L} d\alpha \quad \text{for all } n = 0, 1, 2, \dots$$ Schrödinger equation is the other example presented that describes the quantum behavior in time and space of a particle with m mass inside the potential V and it is given by $$i\hbar\frac{\partial\Psi}{\partial t} = -\frac{\hbar^2}{2m}\frac{\partial^2\Psi}{\partial x^2} + V(\mathbf{x})\Psi \tag{32}$$ where � is Planck constant divided by 2π . If the potential energy is vanished the previous equation becomes the free particle equation and this case will be analyzed forward. $$\begin{cases} i\hbar \frac{\partial \Psi}{\partial t} = -\frac{\hbar^2}{2m} \frac{\partial^2 \Psi}{\partial x^2} \\ \Psi(0, t) = \Psi(L, t) = 0 \\ \Psi(\mathbf{x}, 0) = f(\mathbf{x}) \\ \mathbf{x} \end{cases} \tag{33}$$ From physical point of view Ψ represents a probability density generator and <sup>2</sup> \* Ψ ΨΨ = describes the existence probability of a particle of mass m at position x and time t, thus $$\left\\|\Psi'\right\\|^2 = \int\limits\_{0}^{L} \left\|\Psi'(\mathbf{x}, t)\right\|^2 d\mathbf{x} = 1\tag{34}$$ Analogous with the previous two examples the solution of the problem (33) is given by h t H f i f t df ( ) ( )exp 2( ) The argument of the exponential function from relation (40) is dimensionless. From physical point of view this is very important to emphasize it. For instance, if the argument represents time [s] or distance [m] thus the argument of Fourier transform has dimension [1/s] and [1/m], therefore the product of dimensions for the arguments of Fourier transform is From physical point of view the difference between the Fourier series and the Fourier transform is illustrated considering two signals one periodic, g, and the other non periodic, > 0, if 0 ( ) exp( / 2)exp(2 ), if ( 1) T , 1,2, ; 0 t > > 0, if 0 ( ) exp( / 2)exp(2 ), if 0, 0 The Fourier coefficients An and theirs magnitudes associated with periodical signal (42) are n 0 0.5 1 1.5 2 2.5 3 3.5 4 t [ms] Fig. 5 represents the spectral distribution of g signal that it is defined by the square Fig. 4. shows the graphic for the real part of the periodical g signal for a=10, γ=5 ms-1 and <sup>⎧</sup> <sup>&</sup>lt; <sup>=</sup> <sup>⎨</sup> <sup>−</sup> ≥ > <sup>⎩</sup> γπ and if t n - t nT n 0 <sup>⎧</sup> <sup>&</sup>lt; <sup>=</sup> <sup>⎨</sup> <sup>−</sup> ≤≤ = > <sup>⎩</sup> " (42) t if t t 2 2 0 π <sup>f</sup> A a γ 22 2 0 f n 4 (1 ) <sup>4</sup> − + exp 1 <sup>2</sup> ⎛ ⎞ ⎛ ⎞ ⎜ ⎟ ⎜ ⎟ − − ⎝ ⎠ ⎝ ⎠ <sup>=</sup> γ h. The non periodical signal, h, is often find in NMR spectroscopy. 0 π a t 0 − − γ exp 1 <sup>2</sup> γ ⎛ ⎞ ⎜ ⎟ <sup>−</sup> <sup>−</sup> 2 (1 ) <sup>2</sup> if n 0 ⎝ ⎠ <sup>=</sup> <sup>f</sup> A a π Fig. 4. The periodical physical signal g magnitude of the Fourier coefficients. dimensionless. given by relation f0=2 KHz. g t a t n Real[g(t)] γ h t <sup>∞</sup> π −∞ <sup>=</sup> ∫ (41) γ 2 2 γ (43) (44) $$\Psi^{\nu}(\mathbf{x},t) = \sum\_{n=1}^{\infty} \exp\left[-\frac{i\hbar}{2m} \left(\frac{n\pi}{L}\right)^{2}t\right] c\_{n} \sin\frac{n\pi\pi}{L}.\tag{35}$$ where (0) C c n n = , n =±± 0, 1, 2," and $$\mathbf{C}\_{n}(t) = \frac{2}{L} \Big\| \!\!\Psi(\mathbf{x}, t) \sin \frac{n \mathbf{x} \pi}{L} \mathbf{dx} \tag{36}$$ ### 3. Generalization of the Fourier series for the function of infinite period In the physical and chemical signals analyzing it is often find a non periodical signals defined on the whole real axis. There are many examples in the physics spectroscopy where the signals damp in time due in principal by the absorption process thus there can not be modeled by the periodical functions. The nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR) as well as X-ray absorption spectroscopy (XAS) dedicated to K or L near and extended edges are based on non periodical signals analysis. #### 3.1 Mathematical background of the discreet and inverse Fourier transform Let consider the complex form of the Fourier series for a signal h defined on the interval [−T T /2, /2] it will be introduced the Fourier transform of h based on the concept of infinite period (T → ∞ ) $$h(t) = \frac{1}{T} \sum\_{n=-\alpha}^{\alpha} \underbrace{\left\{ \int\_{-T/2}^{T/2} h(s) \exp\left(-2\pi i \frac{n}{T} s\right) ds \right\}}\_{\text{Fourier coefficients}} \exp\left(2\pi i \frac{n}{T} t\right) \tag{37}$$ where the fundamental frequencies f0 is expressed by 0f = 1 /T . If it be denoted by f nT <sup>n</sup> = / and 1 Δff f T = −= n n <sup>+</sup> 1 / then the relation (37) becomes $$h(t) = \sum\_{n=-\infty}^{\infty} \left( \int\_{-T/2}^{T/2} h(s) \exp\left(-2\pi i f\_n s\right) ds \right) \exp\left(2\pi i f\_n t\right) \Delta f \tag{38}$$ If Δf → 0 then period T goes to infinity and $$h(t) = \int\_{-\alpha}^{\alpha} \underbrace{\left(\int\_{-\alpha}^{\alpha} h(s) \exp\left(-2\pi ifs\right) ds\right)}\_{H(f)} \exp\left(2\pi ift\right) df\tag{39}$$ The function h will be found in the scientific literature named as the Fourier integral (Brigham, 1988) and the expression $$H(f) = \int\_{-\infty}^{\infty} h(s) \exp(-2\pi ifs) \, ds \tag{40}$$ represents the Fourier transform of the function h. From the relation (40) is it possible to obtain the function h by inverse Fourier transform given by 10 Fourier Transform – Materials Analysis ( , ) exp sin . 2 <sup>n</sup> <sup>i</sup> n nx x t t c 2 ( ) sin dx L In the physical and chemical signals analyzing it is often find a non periodical signals defined on the whole real axis. There are many examples in the physics spectroscopy where the signals damp in time due in principal by the absorption process thus there can not be modeled by the periodical functions. The nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR) as well as X-ray absorption spectroscopy (XAS) dedicated to K or L near and extended edges are based on non periodical signals analysis. Let consider the complex form of the Fourier series for a signal h defined on the interval [−T T /2, /2] it will be introduced the Fourier transform of h based on the concept of Fourier coefficients where the fundamental frequencies f0 is expressed by 0f = 1 /T . If it be denoted by n n <sup>T</sup> <sup>n</sup> h t h s i f s ds i f t f π { ( ) } ( ) /2 { ( ) } ( ) π<sup>f</sup> s ds <sup>∞</sup> π The function h will be found in the scientific literature named as the Fourier integral H( ) ( )exp 2 f hs i ( ) represents the Fourier transform of the function h. From the relation (40) is it possible to ( ) ( ) ( )exp 2 exp 2 ht hs i H f n n h t h s i s ds i t T TT π ⎧ ⎫ ⎛ ⎞ ⎛⎞ <sup>=</sup> ⎨ ⎬ −⎜ ⎟ ⎜⎟ ⎩ ⎭ ⎝ ⎠ ⎝⎠ <sup>∑</sup> <sup>∫</sup> <sup>1</sup> ( ) ( )exp 2 exp 2 <sup>T</sup> ff f T = −= n n <sup>+</sup> 1 / then the relation (37) becomes /2 ( ) ( )exp 2 exp 2 <sup>T</sup> ⎡ ⎤ ⎛ ⎞ = −⎢ ⎥ ⎜ ⎟ ⎢ ⎥ ⎝ ⎠ <sup>⎣</sup> <sup>⎦</sup> 0 3. Generalization of the Fourier series for the function of infinite period 3.1 Mathematical background of the discreet and inverse Fourier transform /2 /2 <sup>T</sup> <sup>n</sup> <sup>−</sup> =−∞ ∞ ∞ f → 0 then period T goes to infinity and (Brigham, 1988) and the expression <sup>−</sup> =−∞ <sup>∞</sup> <sup>∞</sup> −∞ −∞ <sup>=</sup> <sup>−</sup> ∫ ∫ obtain the function h by inverse Fourier transform given by nx C t <sup>Ψ</sup>(x,t) L L 1 = n n ∞ Ψ where (0) C c n n = , n =±± 0, 1, 2," and infinite period (T → ∞ ) f nT <sup>n</sup> = / and 1 Δ If Δ 2 π mL L π π <sup>=</sup> ∫ (36) π π πf s ds i ft df −∞ <sup>=</sup> <sup>−</sup> ∫ (40) <sup>=</sup> ∑ <sup>−</sup> ∫ (38) Δ (37) (39) ∑ <sup>=</sup> (35) $$h(t) = \int\_{-\infty}^{\infty} H(f) \exp\{2\pi i f t\} \, df \tag{41}$$ The argument of the exponential function from relation (40) is dimensionless. From physical point of view this is very important to emphasize it. For instance, if the argument represents time [s] or distance [m] thus the argument of Fourier transform has dimension [1/s] and [1/m], therefore the product of dimensions for the arguments of Fourier transform is dimensionless. From physical point of view the difference between the Fourier series and the Fourier transform is illustrated considering two signals one periodic, g, and the other non periodic, h. The non periodical signal, h, is often find in NMR spectroscopy. $$\mathbf{g}(t) = \begin{cases} 0, & \text{if } t < 0 \\ a \exp(-\gamma t / 2) \exp(2\pi i f\_0 t), & \text{if } \quad (n \text{-1}) \text{ T} \le t \le nT, \quad n = 1, 2, \dots; \gamma > 0 \end{cases} \tag{42}$$ $$h(t) = \begin{cases} 0, & \text{if } \ t < 0 \\ a \exp(-\gamma t / 2) \exp(2\pi i f\_0 t), & \text{if } \ t \ge 0, \ \gamma > 0 \end{cases} \tag{43}$$ The Fourier coefficients An and theirs magnitudes associated with periodical signal (42) are given by relation $$A\_n = a \frac{\exp\left(-\frac{\mathcal{Y}}{2f\_0}\right) - 1}{2\pi i f\_0 (1 - n) - \frac{\mathcal{Y}}{2}} \text{ and } \left\| A\_n \right\|^2 = a^2 \frac{\left(\exp\left(-\frac{\mathcal{Y}}{2f\_0}\right) - 1\right)^2}{4\pi^2 \left.f\_0^2 (1 - n)^2 + \frac{\mathcal{Y}^2}{4}} \tag{44}$$ Fig. 4. shows the graphic for the real part of the periodical g signal for a=10, γ=5 ms-1 and f0=2 KHz. Fig. 4. The periodical physical signal g Fig. 5 represents the spectral distribution of g signal that it is defined by the square magnitude of the Fourier coefficients. 0 1 2 3 4 5 6 7 8 f [kHz] From physical reason it is easier to analyze the resonant answer of a physical signal than free damping oscillations. In NMR spectroscoply this signal is known as Free Impulse Decay (FID). Studying the resonant answer it is possible to obtain the relaxation time parameter of the free oscillations. The relaxation time derives from the width of the spectrum obtained from the Fourier transform of the FID. Therefore it is easier to determine the relaxation time Above it was shown that the Fourier series of periodical signal is represented as a sum of periodical functions with discreet frequencies f0, 2f0, 3f0, … as shown in Fig. 5. The amplitudes of the signals associated to each frequency are given by the spectral distribution named Fourier analysis. The difference between Fourier series and Fourier transform is that the latter has the frequencies as argument which continuously varies. Whereas Fourier transform of the signal h allows spectral decomposition of it with frequencies defined on the In practice the function h represents a physical signal resulted from an experiment. The experimental signals can not be acquisitioned on the entire real axis thus the working /2, /2 1 ( − ⎤ ) ⎣ ⎦ (Mandal & Asif, 2007). Instead of the function H there Δ . Between the relaxation time and FWHM there is the relation Δ γ t. From physical reasons, the f represents the discretization step of is named the Fig. 7. The square magnitude of Fourier transform of the signal h τ 3.2 The Fourier transform for discreet signals Δ interval is ⎡−Nt N t Δ are a set of pairs (n fH signal can be discretizated on N samples with a constant step Δ , <sup>n</sup> ) , n N = 0, 1 − where data. Let consider the following relation between discretization steps Some times in signals analysis theory, the inverse of the damping parameter from Fourier transform of h signal instead of fit technique applied to FID. relaxation time denoted by 1 2 π<sup>=</sup> . 1/2 whole real axis. τ Δf Square of \|H(f)\| Fig. 5. The spectral distribution of the signal g The real component of the function (43) is represented in Fig. 6 and the square magnitude of the Fourier transform is given by relation (45) and it is represented in Fig. 7. Fig. 6. The real part of non periodical signal h The maximum value for the spectral distribution take place when 0 f = f , thus 2 2 2 max Hf a () 4 = γ . The full width at half maxim (FWHM) is denoted by 1 2 Δf and 1 2 Δ = f γ 2π 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 harmonic index The real component of the function (43) is represented in Fig. 6 and the square magnitude of )( <sup>2</sup> π 0 2 <sup>2</sup> <sup>2</sup> 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 t [ms] The maximum value for the spectral distribution take place when 0 f = f , thus . The full width at half maxim (FWHM) is denoted by 1 2 Δf and <sup>4</sup> )(4 +− ff 2 γ <sup>a</sup> fH (45) the Fourier transform is given by relation (45) and it is represented in Fig. 7. = 0 2 2 2 γ max Hf a () 4 = 1 2 Δ = f γ 2π Fig. 6. The real part of non periodical signal h 0 2 Real[h(t)] 4 6 8 10 Fig. 5. The spectral distribution of the signal g 2 4 6 Spectral distribution 8 10 12 Fig. 7. The square magnitude of Fourier transform of the signal h Some times in signals analysis theory, the inverse of the damping parameter γ is named the relaxation time denoted by τ . Between the relaxation time and FWHM there is the relation 1/2 1 2 τ Δ f <sup>=</sup> . From physical reason it is easier to analyze the resonant answer of a physical signal than free damping oscillations. In NMR spectroscoply this signal is known as Free Impulse Decay (FID). Studying the resonant answer it is possible to obtain the relaxation time parameter of the free oscillations. The relaxation time derives from the width of the spectrum obtained from the Fourier transform of the FID. Therefore it is easier to determine the relaxation time from Fourier transform of h signal instead of fit technique applied to FID. Above it was shown that the Fourier series of periodical signal is represented as a sum of periodical functions with discreet frequencies f0, 2f0, 3f0, … as shown in Fig. 5. The amplitudes of the signals associated to each frequency are given by the spectral distribution named Fourier analysis. The difference between Fourier series and Fourier transform is that the latter has the frequencies as argument which continuously varies. Whereas Fourier transform of the signal h allows spectral decomposition of it with frequencies defined on the whole real axis. #### 3.2 The Fourier transform for discreet signals π In practice the function h represents a physical signal resulted from an experiment. The signal can be discretizated on N samples with a constant step Δt. From physical reasons, the experimental signals can not be acquisitioned on the entire real axis thus the working interval is ⎡−Nt N t Δ Δ /2, /2 1 ( − ⎤ ) ⎣ ⎦ (Mandal & Asif, 2007). Instead of the function H there are a set of pairs (n fH Δ , <sup>n</sup> ) , n N = 0, 1 − where Δ f represents the discretization step of data. Let consider the following relation between discretization steps 2 2 2 2 20 0 [ ] <sup>1</sup> cos(2 ) cos(2 ) cos(2 ) <sup>2</sup> θ ππ θ θθ θ [ ] − − th t f f tS f tS nn n n 2 2 2 2 1 ππ ∑ (52) θ > θ > > γπΔ 1 sin 2 2sin cos(2 ), ( ) 2 2 2 3 3 2 0 0 π ∫ (53) βπΔ <sup>1</sup> sin(2 ) sin(2 ) sin(2 ) <sup>2</sup> γ θ αθ 21 21 21 2 3 ⎛ ⎞ <sup>+</sup> ⎛ ⎞ = − =− ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ ⎝ ⎠ = = + − C h f t h f t h f t 1 cos sin 2 sin cos () 2 , () 4 2 2 2 2 20 0 [ ] By taking into account the relation (46) used in the FFT method it is not possible to compute the Fourier transform for all value of the frequency. This disadvantage can lead to poor resolution of the Fourier transform H. Meanwhile the Filon algorithm is more time consuming but its application offers a more reliable resolution. A detailed analysis of these algorithms applied in the extended X-ray absorption fine structure (EXAFS) spectroscopy 3.4 Application of the Fourier transform in X-ray absorption spectroscopy and X-ray The study of XAS can yield electronic and structural information about the local environment around a specific atomic constituent in the amorphous materials (Kolobov et Additional, this method provides information about the location and chemical state of any catalytic atom on any support (Miller et al., 2006) as well as the nanoparticle of transition metal oxides (Chen et al., 2002; Turcu et al., 2004)). X-ray absorption near edge structure (XANES) is sensitive to local geometries and electronic structure of atoms that constitute the nanoparticles. The changes of the coordination geometry and the oxidation state upon decreasing the crystallite size and the interaction with molecules absorbed on nanoparticles The EXAFS is a specific element of the scattering technique in which a core electron ejected by an X-ray photon probes the local environment of the absorbing atom. The ejected S h f t h f t h f t ≈ − π sin(2 ). π nj jn n =−+ θ nj jn n =−+ π θ 2 θ θ − (54) 0 j = j n π 0 j can be found in the paper (Aldea & Pintea, 2009). surface can be extracted from XANES spectrum. = ∑ j n = = n ∑ 0 = β θ 2 n t 0 t n ∑ 0 2 and the imaginary component of the Fourier transform is given by ( )sin(2 ) (2 ) cos(2 ) h t t f dt t f t h t f π Δα π Δ Δ 2 1 21 21 −− − nj j S h ft θ π nj j −− − θ C h ft where where diffraction al., 2005), $$Af = \frac{1}{N\Delta t} \tag{46}$$ the Fourier transform associated to the set ( ) /2, /2 1 ( ) kN N hk t Δ =− − is contained in the H vector with the components $$H(n\Delta f) = \Delta t \sum\_{k=-N/2}^{N/2-1} h(k\Delta t) \exp\left(-\frac{2\pi ikn}{N}\right) \quad n = \overline{0, N-1}.\tag{47}$$ It is more convenient, for the computation, if all the indexes are positive, for this it is assumed that qkN = + for k<0. Relation (47) becomes $$H(n\Delta f) = \Delta t \sum\_{k=0}^{N-1} h(k\Delta t) \exp\left(-\frac{2\pi ikn}{N}\right). \tag{48}$$ Using the same consideration, the inverse Fourier transform has the form $$\ln(n\Delta f) = \Delta f \sum\_{k=0}^{N-1} \underbrace{\left[\text{Re}\,H(k\Delta f) + i\,\text{Im}\,H(k\Delta f)\right]}\_{H(k\Delta f)} \exp\left(\frac{2\pi ikn}{N}\right) \tag{49}$$ If the physical signal is recorded on negative arguments then for the numerical computation of the Fourier transform for the components ( ,) <sup>k</sup> k th Δ , kN N = − − / 2... / 2 1 must be arrange in the following order $$(0, 1, 2, \cdots, N \;/ \; 2 - 1) \to (h\_0, h\_1, h\_2, \cdots, h\_{N \;/ \; 2 - 1})$$ $$\left(\frac{N}{2}, \frac{N}{2} + 1, \frac{N}{2} + 2, \cdots, N - 1\right) \to (h\_{-N/2}, h\_{-N/2 + 1}, h\_{-N/2 + 2 \;/ \; 2 + 2 \;/ \; -}, h\_{-1}).\tag{50}$$ #### 3.3 The main algorithms for the Fourier transform. The Filon quadrature and the Cooley-Tukey method Most of the times the physical-chemical signals can not be expressed analytical, thus it is impossible to use the relation (40). Therefore in computation is used the discreet form of Fourier transform given by the relation (48). Generally speaking the physical signals are recorded around thousands of points; additionally the Fourier transform of the signal is important to compute on the same numbers. By using relation (48) the computation time is too long. This problem was solved by Cooley Tukey algorithm (Brigham, 1988) named in the literature as a Fast Fourier Transform (FFT) method. In the case when the physical signal is registered from pairs in a range of hundreds up to few thousand values can be successfully used the Filon algorithm (Abramowitz & Stegun, 1972). The method assumes that the physical signal is defined on the interval [t t 0 2 , <sup>n</sup> ] with step Δt then the real component of the Fourier transform is approximated by $$\begin{aligned} \int\_{t\_0}^{t\_{2n}} h(t) \cos(2\pi t \, f) dt &\approx \mathcal{A}t \, a(2\pi f \, \mathcal{A}t) \mathbb{I}\_{2n} \sin(2\pi t\_{2n} f) - \\\\ &\quad \cdot \mathcal{A}t \Big[ \mathbb{I}\_0 \sin(2\pi t\_0 f) - \beta(2\pi f \, \mathcal{A}t) \mathbb{C}\_{2n} - \gamma(2\pi f \, \mathcal{A}t) \mathbb{C}\_{2n-1} \Big] \end{aligned} \tag{51}$$ where 14 Fourier Transform – Materials Analysis <sup>1</sup> <sup>f</sup> N t Δ <sup>2</sup> ( ) ( )exp , 0, 1. It is more convenient, for the computation, if all the indexes are positive, for this it is <sup>2</sup> ( ) ( )exp . [ ] ⎛ ⎞ <sup>=</sup> <sup>+</sup> ⎜ ⎟ ⎝ ⎠ ∑ ikn hn f f Hk f i Hk f <sup>N</sup> Δ If the physical signal is recorded on negative arguments then for the numerical computation Δ N hhh h , 1, 2, , 1 ( , , , , ). 22 2 Most of the times the physical-chemical signals can not be expressed analytical, thus it is impossible to use the relation (40). Therefore in computation is used the discreet form of Fourier transform given by the relation (48). Generally speaking the physical signals are recorded around thousands of points; additionally the Fourier transform of the signal is important to compute on the same numbers. By using relation (48) the computation time is too long. This problem was solved by Cooley Tukey algorithm (Brigham, 1988) named in the literature as a Fast Fourier Transform (FFT) method. In the case when the physical signal is registered from pairs in a range of hundreds up to few thousand values can be successfully used the Filon algorithm (Abramowitz & Stegun, 1972). The method assumes that the NN N N hh h h " " ⎛ ⎞ <sup>=</sup> −⎜ ⎟ Δ ikn Hn f t hk t <sup>N</sup> ⎛ ⎞ <sup>=</sup> − =− ⎜ ⎟ N π > π Δ 0 1 2 /2 1 NN N /2 /2 1 /2 2 1 Δ n n − γπΔ [ ] − − t h t f f tC f tC 0 0 2 2 1 2 2 βπΔ n n π ∫ (51) t then the real component of " " (50) − − +− + − N − ikn Hn f t hk t n N 1 − N k 0 = <sup>0</sup> ( ) <sup>2</sup> ( ) Re ( ) Im ( ) exp (0,1,2, , / 2 1) ( , , , , ) − → 3.3 The main algorithms for the Fourier transform. The Filon quadrature and the Δ <sup>k</sup> Hk f Δ the Fourier transform associated to the set ( ) /2, /2 1 ( ) kN N hk t /2 1 − N ΔΔ assumed that qkN = + for k<0. Relation (47) becomes ΔΔ of the Fourier transform for the components ( ,) <sup>k</sup> k th ⎛ ⎞ ⎜ ⎟ + + −→ ⎝ ⎠ physical signal is defined on the interval [t t 0 2 , <sup>n</sup> ] with step ( )cos(2 ) (2 ) sin(2 ) h t t f dt t f t h t f Δα π Δ Δ ≈ − π the Fourier transform is approximated by π k N =− /2 ΔΔ Using the same consideration, the inverse Fourier transform has the form 1 − = N with the components in the following order Cooley-Tukey method 2 n t 0 t Δ Δ <sup>=</sup> (46) ⎝ ⎠ ∑ (47) ⎝ ⎠ ∑ (48) π , kN N = − − / 2... / 2 1 must be arrange (49) =− − is contained in the H vector $$\begin{aligned} \mathbf{C}\_{2n} &= \sum\_{j=0}^{n} h\_{2j} \cos(2\pi f \, t\_{2j}) - \frac{1}{2} [h\_{2n} \cos(2\pi f \, t\_{2n}) + h\_0 \cos(2\pi f \, t\_0)] \\ \mathbf{C}\_{2n-1} &= \sum\_{j=0}^{n} h\_{2j-1} \cos(2\pi f \, t\_{2j-1}), \quad a(\theta) = \frac{1}{\theta} + \frac{\sin 2\theta}{2\theta^2} - \frac{2 \sin^2 \theta}{2\theta^3} \\ \boldsymbol{\beta}(\theta) &= 2 \left( \frac{1 + \cos^2 \theta}{\theta^2} - \frac{\sin 2\theta}{\theta^3} \right), \quad \boldsymbol{\gamma}(\theta) = 4 \left( \frac{\sin \theta}{\theta^3} - \frac{\cos \theta}{\theta^2} \right) \end{aligned} \tag{52}$$ and the imaginary component of the Fourier transform is given by $$\begin{aligned} \int\_{t\_0}^{t\_{2n}} h(t) \sin(2\pi t \, f) dt &\approx 4\pi a (2\pi \, f \, \mathcal{A} t) h\_0 \cos(2\pi t\_0 f) - \\\\ - 4\pi \Big[ h\_{2n} \cos(2\pi t\_{2n} f) - \beta (2\pi \, f \, \mathcal{A} t) S\_{2n} - \gamma (2\pi \, f \, \mathcal{A} t) S\_{2n-1} \Big] \end{aligned} \tag{53}$$ where $$\begin{aligned} S\_{2n} &= \sum\_{j=0}^{n} h\_{2j} \sin(2\pi f \, t\_{2j}) - \frac{1}{2} [h\_{2n} \sin(2\pi f \, t\_{2n}) + h\_0 \sin(2\pi f \, t\_0)] \\ S\_{2n-1} &= \sum\_{j=0}^{n} h\_{2j-1} \sin(2\pi f \, t\_{2j-1}). \end{aligned} \tag{54}$$ By taking into account the relation (46) used in the FFT method it is not possible to compute the Fourier transform for all value of the frequency. This disadvantage can lead to poor resolution of the Fourier transform H. Meanwhile the Filon algorithm is more time consuming but its application offers a more reliable resolution. A detailed analysis of these algorithms applied in the extended X-ray absorption fine structure (EXAFS) spectroscopy can be found in the paper (Aldea & Pintea, 2009). #### 3.4 Application of the Fourier transform in X-ray absorption spectroscopy and X-ray diffraction The study of XAS can yield electronic and structural information about the local environment around a specific atomic constituent in the amorphous materials (Kolobov et al., 2005), Additional, this method provides information about the location and chemical state of any catalytic atom on any support (Miller et al., 2006) as well as the nanoparticle of transition metal oxides (Chen et al., 2002; Turcu et al., 2004)). X-ray absorption near edge structure (XANES) is sensitive to local geometries and electronic structure of atoms that constitute the nanoparticles. The changes of the coordination geometry and the oxidation state upon decreasing the crystallite size and the interaction with molecules absorbed on nanoparticles surface can be extracted from XANES spectrum. The EXAFS is a specific element of the scattering technique in which a core electron ejected by an X-ray photon probes the local environment of the absorbing atom. The ejected ( ) ( ) ( )exp( 2 ) . <sup>n</sup> The EXAFS signal is weighted by kn (n=1, 2, 3) to get the distribution function of atom distances. Different apodization windows WF(k) are available as Kaiser, Hanning or Gauss filters. An inverse Fourier transform of the RSF can be obtained for any coordination shell, > 2 1 <sup>1</sup> ( ) ( ) ( )exp(2 ) . <sup>j</sup> j R ( ) ( )sin 2 ( ) j j jj where the index j refers to the jth coordination shell. The structural parameters for the first 1 01 2 ( ) <sup>s</sup> The coefficients c0, c1, c2, c3, a-1, a0, a1 and a2 are derived from the EXAFS spectrum of a compound whose structure is accurately known. The values Ns and rs for each coordination shell for the standard sample are known. The trial values of the eight coefficients can be calculated by algebraic consideration and then they are varied until the fit between the X-ray diffraction pattern of a crystal can be described in terms of scattering intensity as discussed the X-ray diffraction for the mosaic structure model in which the atoms are arranged in blocks, each block itself being an ideal crystal, but with adjacent blocks not accurately fitted together. The experimental XRLP, h, represents the convolution between h s g s s f s ds () ( ) ( ) \* \\ +∞ χ δ 1 2 k WF k r ikr dr Φ r k k WF k ikr dk <sup>∞</sup> function (RSF) can be derived. The single shell may be isolated by Fourier transform, χ <sup>j</sup> <sup>n</sup> <sup>R</sup> j(k) function is given by: k coordination shell are determined by fitting the theoretical function δ δ function of scattering direction defined by the scattering angle 2 χ −∞ <sup>=</sup> <sup>−</sup> ∫ (58) <sup>=</sup> ∫ (59) k A k kr k <sup>=</sup> <sup>⎡</sup> <sup>+</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> , (60) j(k) are conveniently parameterized (Aldea et al., 2007). k a k a ak ak <sup>−</sup> = ++ + <sup>−</sup> (62) <sup>3</sup> 2 c 0 12 F (k,r, <sup>s</sup> <sup>π</sup> )=c exp(c ) /k ⎡ ⎤ k ck <sup>+</sup> ⎣ ⎦ (61) , where λ is the wavelength of the incident radiation. It is χ j(k) derived from relation (59). In the empirical θ −∞ = − ∫ (63) HL GL FL ( ) = ( ) ( ) , (64) , or by the scattering j(k) given by the (k), the radial structure generally proceeds by the use of the Fourier transform. From Φ χ χ relation (60) with the experimental signal Eight coefficients are introduced for each shell: observed and calculated EXAFS is optimized. θ λ the true sample f and the instrumental function g The equation (63) is equivalent with the following relation EXAFS calculation, F(k,r,π) and 3.4.2 XRD analysis parameter s = (2sin ) / χ The theoretical equation for photoelectron backscattered by the neighboring atoms around the absorbing atom interferes constructively with the outgoing electron wave, depending on the energy of the photoelectron. The energy of the photoelectron is equal to the difference between the X-ray energy photon and a threshold energy associated with the ejection of the electron. X-ray diffraction (XRD) line broadening investigations of nanostructured materials have been limited to find the average crystallite size from the integral breadth or the FWHM of the diffraction profile. In the case of nanostructured materials due to the difficulty of performing satisfactory intensity measurements on the higher order reflections, it is impossible to obtain two orders of (hkl) profile. Consequently, it is not possible to apply the classical method of Warren and Averbach (Warren, 1990). On the other hand we developed a rigorous analysis of the X-ray line profile (XRLP) in terms of Fourier transform where zero strains assumption is not required. The apparatus employed in a measurement generally affects the obtained data and a considerable amount of work has been done to make resolution corrections. In the case of XRLP, the convolution of true data function by the instrumental function produced by a well-annealed sample is described by Fredholm integral equation of the first kind (Aldea et al., 2005; Aldea & Turcu, 2009). A rigorous way for solving this equation is Stokes method based on Fourier transform technique. The local and global structure of nanosized nickel crystallites were determined from EXAFS and XRD analysis. #### 3.4.1 EXAFS analysis The interference between the outgoing and the backscattered electron waves has the effect of modulating the X-ray absorption coefficient. The EXAFS function χ(k) is defined in terms of the atomic absorption coefficient by $$\chi(k) = \frac{\mu(k) - \mu\_0(k)}{\mu\_0(k)} \tag{55}$$ where k is the electron wave vector, μ(k) refers to the absorption by an atom from the material of interest and μ0(k) refers to the atom in the free state. The theories of the EXAFS based on the single scattering approximation of the ejected photoelectron by atoms in the immediate vicinity of the absorbing atom gives an expression for χ(k) $$\mathcal{L}\mathcal{X}(k) = \sum\_{j} A\_{j}(k) \sin\left[2kr\_{j} + \delta\_{j}(k)\right] \tag{56}$$ where the summation extends over j coordination shell, rj is the radial distance from the jth shell and δj(k) is the total phase shift function. The amplitude function Aj(k) is given by $$A\_{\dot{j}}(k) = \left(\frac{N\_{\dot{j}}}{k r\_{\dot{j}}^2}\right) F(k, r\_{\dot{j}}, \pi) \exp\left(-2\left(r\_{\dot{j}} \ne \lambda\_{\dot{j}}(k) - k^2 \sigma\_{\dot{j}}^2\right)\right) \tag{57}$$ In this expression Nj is the number of atoms in the jth shell, σ<sup>j</sup> is the root mean squares deviation of distance about rj, F(k, rj, π) is the backscattering amplitude and λj(k) is the mean free path function for the inelastic scattering. The backscattering factor and the phase shift depend on the kind of atom responsible for scattering and its coordination shell (Aldea et al., 2007). The analysis of EXAFS data for obtaining structural information [Nj, rj, σj, λj(k)] generally proceeds by the use of the Fourier transform. From χ(k), the radial structure function (RSF) can be derived. The single shell may be isolated by Fourier transform, $$\mathcal{O}(r) = \int\_{-\infty}^{\infty} k'' \, \mathcal{X}(k) \mathcal{W} \mathcal{F}(k) \exp(-2ikr) dk \,. \tag{58}$$ The EXAFS signal is weighted by kn (n=1, 2, 3) to get the distribution function of atom distances. Different apodization windows WF(k) are available as Kaiser, Hanning or Gauss filters. An inverse Fourier transform of the RSF can be obtained for any coordination shell, $$\mathcal{L}\_j \mathcal{X}\_j(k) = \frac{1}{k^{\text{tr}}} \text{V} \mathbf{V} \mathbf{F}(k) \int\_{R\_{1j}}^{R\_{2j}} \Phi(r) \exp(2ikr) dr. \tag{59}$$ The theoretical equation for χj(k) function is given by: $$\mathcal{X}\_{j}(k) = A\_{j}(k)\sin\left[2kr\_{j} + \delta\_{j}(k)\right] \tag{60}$$ where the index j refers to the jth coordination shell. The structural parameters for the first coordination shell are determined by fitting the theoretical function χj(k) given by the relation (60) with the experimental signal χj(k) derived from relation (59). In the empirical EXAFS calculation, F(k,r,π) and δj(k) are conveniently parameterized (Aldea et al., 2007). Eight coefficients are introduced for each shell: $$F\_s(\mathbf{k}, r, \pi \mid \mathbf{\bar{r}}\_0 \left[ \exp(\mathbf{c}\_1 k + c\_2 k^2) \right] / \mathbf{k}^{c\_3} \tag{61}$$ $$\mathcal{S}\_s(k) = a\_{-1}k^{-1} + a\_0 + a\_1k + a\_2k^2\tag{62}$$ The coefficients c0, c1, c2, c3, a-1, a0, a1 and a2 are derived from the EXAFS spectrum of a compound whose structure is accurately known. The values Ns and rs for each coordination shell for the standard sample are known. The trial values of the eight coefficients can be calculated by algebraic consideration and then they are varied until the fit between the observed and calculated EXAFS is optimized. #### 3.4.2 XRD analysis 16 Fourier Transform – Materials Analysis photoelectron backscattered by the neighboring atoms around the absorbing atom interferes constructively with the outgoing electron wave, depending on the energy of the photoelectron. The energy of the photoelectron is equal to the difference between the X-ray X-ray diffraction (XRD) line broadening investigations of nanostructured materials have been limited to find the average crystallite size from the integral breadth or the FWHM of the diffraction profile. In the case of nanostructured materials due to the difficulty of performing satisfactory intensity measurements on the higher order reflections, it is impossible to obtain two orders of (hkl) profile. Consequently, it is not possible to apply the classical method of Warren and Averbach (Warren, 1990). On the other hand we developed a rigorous analysis of the X-ray line profile (XRLP) in terms of Fourier transform where zero strains assumption is not required. The apparatus employed in a measurement generally affects the obtained data and a considerable amount of work has been done to make resolution corrections. In the case of XRLP, the convolution of true data function by the instrumental function produced by a well-annealed sample is described by Fredholm integral equation of the first kind (Aldea et al., 2005; Aldea & Turcu, 2009). A rigorous way for solving this equation is Stokes method based on Fourier transform technique. The local and global structure of nanosized nickel crystallites were determined from EXAFS and XRD analysis. The interference between the outgoing and the backscattered electron waves has the effect of μ 0 0 () () ( ) ( ) k k <sup>k</sup> k 0(k) refers to the atom in the free state. The theories of the EXAFS ( ) ( ) 2 2 λσ σ k A k kr k <sup>=</sup> <sup>⎡</sup> <sup>+</sup> <sup>⎤</sup> ∑ <sup>⎣</sup> <sup>⎦</sup> (56) δ μ μ where k is the electron wave vector, μ(k) refers to the absorption by an atom from the based on the single scattering approximation of the ejected photoelectron by atoms in the ( ) ( )sin 2 ( ) j jj where the summation extends over j coordination shell, rj is the radial distance from the jth <sup>2</sup> ( ) ( , , )exp 2 / ( ) <sup>j</sup> j j jj j π al., 2007). The analysis of EXAFS data for obtaining structural information [Nj, rj, A k Fkr r k k = −− ⎜ ⎟ free path function for the inelastic scattering. The backscattering factor and the phase shift depend on the kind of atom responsible for scattering and its coordination shell (Aldea et j(k) is the total phase shift function. The amplitude function Aj(k) is given by χ <sup>−</sup> <sup>=</sup> (55) (k) is defined in terms of (57) j(k) is the mean σj, λj(k)] <sup>j</sup> is the root mean squares λ modulating the X-ray absorption coefficient. The EXAFS function χ immediate vicinity of the absorbing atom gives an expression for χ(k) χ j N ⎛ ⎞ kr In this expression Nj is the number of atoms in the jth shell, ⎜ ⎟ ⎝ ⎠ j deviation of distance about rj, F(k, rj, π) is the backscattering amplitude and energy photon and a threshold energy associated with the ejection of the electron. 3.4.1 EXAFS analysis material of interest and shell and δ the atomic absorption coefficient by μ X-ray diffraction pattern of a crystal can be described in terms of scattering intensity as function of scattering direction defined by the scattering angle 2θ, or by the scattering parameter s = (2sin ) / θ λ , where λ is the wavelength of the incident radiation. It is discussed the X-ray diffraction for the mosaic structure model in which the atoms are arranged in blocks, each block itself being an ideal crystal, but with adjacent blocks not accurately fitted together. The experimental XRLP, h, represents the convolution between the true sample f and the instrumental function g $$h(s) = \int\_{-\infty}^{+\infty} g\left(s - s^\\right) f\left(s^\\right) ds^\* \tag{63}$$ The equation (63) is equivalent with the following relation $$H(L) = G(L)F(L) \,. \tag{64}$$ In according with relation (55) EXAFS signals modulated by Hanning and Gauss filters were performed in the range 2 Å -14 Å and they are shown in Fig. 9. In order to obtain the atomic distances distribution it was computed the RSF, using the relation (58) and the Filon algorithm. Ni sample EXAFS signal chi\wk^3 Hanning filter\chi\wk^3 Gauss filter\chi\wk^3 2 4 6 8 10 12 14 wave vector [1/A] The mean Ni-Ni distances of the first coordination shell for standard sample at room temperature are closed to values of R1=2.49Å. Based on relation (46) between Δk* and Δr steps, the computation of the RSF using the FFT of the EXAFS signal gives a non reliable resolution. To avoid this disadvantage it used the Filon algorithm for Fourier transform procedure. Based on this procedure the Fourier transform of k3χ(k)WF(k), performed in the range 0.51 Å and 2.79 Å, are shown in Fig. 10 for the standard Ni foil investigation. In order to minimize the spurious errors in the RSF it was considered Gauss filter as the window FT of EXAFS signal - Filon algorithm <sup>X</sup> FT[k^n\WF\chi] Band limit r1 = 0.38 A r2 = 2.74 A k^n; n = 3 0 1 2 3 4 5 6 X X Fig. 10. The Fourier transform of the EXAFS spectrum for the nickel foil Distance [A] function. Fig. 9. EXAFS signal for the nickel crystallites EXAFS signal where F(L), H(L) and G(L) are the Fourier transforms of the true sample, experimental XRLP and instrumental function, respectively. The variable L is the perpendicular distance to the (hkl) reflection planes. The generalized Fermi function (GFF) (Aldea et al., 2000) is a simple function with a minimal number of parameters, suitable for the XRLP global approximation based on minimization methods and it is defined by relation: $$h(s) = \frac{A}{e^{a(s-c)} + e^{-b(s-c)}} \, \, \, \, \tag{65}$$ where A, a, b, c are unknown parameters. The values A, c describe the amplitude, the position of the XRLP and a, b control its shape. In the case when X-ray line profiles h and g are approximated by GFF distribution then the solution of Fedholm integral equation of the first kind represents the true sample function and it is given by $$f(s) = \frac{2A\_h \rho\_\mathcal{g}}{\pi A\_\mathcal{g}} \frac{\cos \frac{\pi \rho\_h}{2} \cosh \rho\_h s}{\cos \frac{\pi \rho\_h}{\rho\_\mathcal{g}} + \cos 2\rho\_h s} \tag{66}$$ where the arguments of trigonometric and hyperbolic functions depend on the shape parameters of the h, g signals, respectively. They are expressed by ( )/2 h hh ρ = + a b and ( ) /2. g gg ρ= + a b #### 3.4.3 EXAFS results The extraction of the EXAFS signal is based on the threshold energy of the nickel K edge determination followed by background removal of pre-edge and after-edge base line fitting with different possible modeling functions where μ0(k) and μ(k) evaluation are presented in Fig. 8. Fig. 8. The absorption coefficient of the nickel K edge In according with relation (55) EXAFS signals modulated by Hanning and Gauss filters were performed in the range 2 Å -14 Å and they are shown in Fig. 9. In order to obtain the atomic distances distribution it was computed the RSF, using the relation (58) and the Filon algorithm. Fig. 9. EXAFS signal for the nickel crystallites 18 Fourier Transform – Materials Analysis where F(L), H(L) and G(L) are the Fourier transforms of the true sample, experimental XRLP and instrumental function, respectively. The variable L is the perpendicular distance to the (hkl) reflection planes. The generalized Fermi function (GFF) (Aldea et al., 2000) is a simple function with a minimal number of parameters, suitable for the XRLP global approximation > () () ( ) as c bs c <sup>A</sup> h s e e <sup>−</sup> − − <sup>=</sup> <sup>+</sup> where A, a, b, c are unknown parameters. The values A, c describe the amplitude, the position of the XRLP and a, b control its shape. In the case when X-ray line profiles h and g are approximated by GFF distribution then the solution of Fedholm integral equation of the 2 2 ρ A f s <sup>A</sup> π hg g g h where the arguments of trigonometric and hyperbolic functions depend on the shape parameters of the h, g signals, respectively. They are expressed by ( )/2 h hh The extraction of the EXAFS signal is based on the threshold energy of the nickel K edge determination followed by background removal of pre-edge and after-edge base line fitting with different possible modeling functions where μ0(k) and μ(k) evaluation are presented in Ni sample 8200 8300 8400 8500 8600 8700 8800 8900 9000 9100 9200 X-ray energy [eV] cos cosh πρ > ρ πρ ρ h h ρ s h ρ s cos cos2 + g , (65) (66) = + a b and ρ X Background correction Back(x)=v0+v1\x v0 = 2.99098 v1 = -0.00032 "Ni.abs" Background Post edge based on minimization methods and it is defined by relation: first kind represents the true sample function and it is given by ( ) ( ) /2. g gg* 3.4.3 EXAFS results 0 X X Fig. 8. The absorption coefficient of the nickel K edge X 0.5 1 1.5 Absorption coefficient 2 2.5 = + a b ρ Fig. 8. = The mean Ni-Ni distances of the first coordination shell for standard sample at room temperature are closed to values of R1=2.49Å. Based on relation (46) between Δk and Δr steps, the computation of the RSF using the FFT of the EXAFS signal gives a non reliable resolution. To avoid this disadvantage it used the Filon algorithm for Fourier transform procedure. Based on this procedure the Fourier transform of k3χ(k)WF(k), performed in the range 0.51 Å and 2.79 Å, are shown in Fig. 10 for the standard Ni foil investigation. In order to minimize the spurious errors in the RSF it was considered Gauss filter as the window function. Fig. 10. The Fourier transform of the EXAFS spectrum for the nickel foil and the nickel foil as instrumental broadening effect are shown in Fig. 12. The next steps consist in the background correction of the XRLP by the polynomial procedures and the determination of the best parameters of GFF distributions by nonlinear least squares fit. In order to determine the average crystallite size, the lattice microstrain and the probability of defects were computed the true XRLP by the Fourier transform technique and it is > Experimental Instrumental True sample signal 25.5 26 26.5 27 27.5 28 diffraction angle [theta] s-s0 In this contribution it has presented the mathematical background of Fourier series and illustrated in Fig. 13, the curve is centred on its mass centre s0. 0 0 Fourier transform used in nanomaterials structure field. Fig. 13. The true sample signal (f) 4. Conclusions 0.5 1 1.5 Relative intensity 2 2.5 Fig. 12. The experimental XRLP (h) and the instrumental signals (g) 500 1000 1500 Relative intensity 2000 2500 Each peak from \|Φ(r)\|is shifted from the true distance due to the phase shift function that is included in the EXAFS signal. We proceed by taking the inverse Fourier transform given by relation (59) of the first neighboring peak, and then extracting the amplitude function Aj(k) and the phase shift function δ(k) in according with the relations (61) and (62). By Lavenberg-Marquard fit applied to the relation (60) and from the experimental contribution for each coordination shell, are evaluated the interatomic distances, the number of neighbors Fig. 11. Experimental and calculated EXAFS signals of the first coordination shell of the nickel foil and the edge position. Fig. 11 shows the calculated and the experimental EXAFS functions 1 χ( ) k of the first shell for the investigated sample. #### 3.4.4 XRD results Practically speaking, however, it is not easy to obtain accurate values of the crystallite size and the microstrain without extreme care in the experimental measurements and analysis of XRD data. The Fourier analysis of XRLP validity depends strongly on the magnitude and the nature of the errors propagated in the data analysis. In paper (Aldea et al., 2000) are treated three systematic errors: the uncorrected constant background, the truncation and the effect of the sampling for the observed profile at a finite number of points that appear in discrete Fourier analysis. In order to minimize propagation of these systematic errors, a global approximation of the XRLP is adopted instead of the discrete calculations. Therefore, the analysis of the diffraction line broadening in X-ray powder pattern was analytically calculated using the GFF facilities. The reason of this choice, as described above, was simplicity and the mathematical elegance of the analytical Fourier transform magnitude and the integral width of the true XRLP. The robustness of the GFF approximation for the XRLP arises from possibility of using the analytical form of the Fourier transform instead of the numerical FFT. The validity of the microstructural parameters are closely related to accuracy of the Fourier transform magnitude of the true XRLP. The experimental relative intensities with respect to θ values and the nickel foil as instrumental broadening effect are shown in Fig. 12. The next steps consist in the background correction of the XRLP by the polynomial procedures and the determination of the best parameters of GFF distributions by nonlinear least squares fit. In order to determine the average crystallite size, the lattice microstrain and the probability of defects were computed the true XRLP by the Fourier transform technique and it is illustrated in Fig. 13, the curve is centred on its mass centre s0. Fig. 12. The experimental XRLP (h) and the instrumental signals (g) Fig. 13. The true sample signal (f) #### 4. Conclusions 20 Fourier Transform – Materials Analysis Each peak from \|Φ(r)\|is shifted from the true distance due to the phase shift function that is included in the EXAFS signal. We proceed by taking the inverse Fourier transform given by relation (59) of the first neighboring peak, and then extracting the amplitude function Aj(k) By Lavenberg-Marquard fit applied to the relation (60) and from the experimental contribution for each coordination shell, are evaluated the interatomic distances, the number of neighbors The first shell - experimental and calculated of Ni sample EXAFS single domain k1 =3.92 1/A k2 =14.24 1/A EXAFS - experimental EXAFS - calculated Backscatering amplitude Back\_amp\N/r^2 > a\_1 =-5.06688 a0 =-4.11355 a1 =-1.38168 a2 =0.04043 c0 =-0.00016 c1 =-2.94194 c2 =0.06417 c3 =-11.74119 4 6 8 10 12 14 wave vector [1/A] Fig. 11. Experimental and calculated EXAFS signals of the first coordination shell of the and the edge position. Fig. 11 shows the calculated and the experimental EXAFS functions Practically speaking, however, it is not easy to obtain accurate values of the crystallite size and the microstrain without extreme care in the experimental measurements and analysis of XRD data. The Fourier analysis of XRLP validity depends strongly on the magnitude and the nature of the errors propagated in the data analysis. In paper (Aldea et al., 2000) are treated three systematic errors: the uncorrected constant background, the truncation and the effect of the sampling for the observed profile at a finite number of points that appear in discrete Fourier analysis. In order to minimize propagation of these systematic errors, a global approximation of the XRLP is adopted instead of the discrete calculations. Therefore, the analysis of the diffraction line broadening in X-ray powder pattern was analytically The reason of this choice, as described above, was simplicity and the mathematical elegance of the analytical Fourier transform magnitude and the integral width of the true XRLP. The robustness of the GFF approximation for the XRLP arises from possibility of using the analytical form of the Fourier transform instead of the numerical FFT. The validity of the microstructural parameters are closely related to accuracy of the Fourier transform magnitude of the true XRLP. The experimental relative intensities with respect to θ* values and the phase shift function δ(k) in according with the relations (61) and (62). ( ) k of the first shell for the investigated sample. EXAFS single shell nickel foil 3.4.4 XRD results calculated using the GFF facilities. 1 χ > In this contribution it has presented the mathematical background of Fourier series and Fourier transform used in nanomaterials structure field. For the next properties are assuming the function h is sufficiently smooth so that can be ( ) (2 ) ( ) dh <sup>h</sup> where Hh represents the Fourier Transform of the function h, and for the n times derivative <sup>n</sup> ( ) (2 ) ( ) ( ) 2 () <sup>h</sup> ( ) (2 ) () <sup>n</sup> dH f iH <sup>f</sup> df = − π h n n t h dH f i H <sup>f</sup> df = − π Abramowitz, M. & Stegun, I. (1972). Handbook of Mathematical Functions, Dover Publication, Aldea, N. & Indrea, E. (1990). XRLINE, a program to evaluate the crystallite size of Communications, Vol.60, No.1, (August 1990), pp. 155-163, ISSN: 0010-4655 Aldea, N.; Gluhoi, A.; Marginean, P.; Cosma, C. & Yaning X. (2000). Extended X-ray Aldea, N.; Barz, B.; Silipas, T. D.; Aldea, F. & Wu, Z. (2005). Mathematical study of metal Aldea, N.; Marginean, P.; Rednic, V.; Pintea, S.; Barz, B.; Gluhoi, A.; Nieuwenhuys, B. E.; supported metal-catalysts by single X-ray profile fourier-analysis. Computer Physics absorption fine structure and X-ray diffraction studies on supported nickel catalysts. Spectrochimica Acta B, Vol.55, No.7, (July 2000), pp. 997-1008, ISSN: 0584- nanoparticle size determination by single X-ray line profile analysis. Journal of Optoelectronics and Advanced Materials, Vol.7, No.6, (December 2005), pp. 3093-3100, Xie Y.; Aldea, F. & Neumann, M. (2007). Crystalline and electronic structure of gold nanoclusters determined by EXAFS, XRD and XPS methods. Journal of Optoelectronics and Advanced Materials, Vol.9, No.5, (May 2007), pp. 1555-1560, ISSN π d h h n th H f if H f = π acceptable the differentiation and the integration. ix. The derivative of the Fourier transform is given by ISBN 0-486-61272-4, NY, USA and for the n times derivative of the Fourier transform n viii. The Fourier transform of the derivative of the function h is given by dt n dt H f = () () () Hf H f H f = o e + (73) (74) (76) (77) i f H f (75) thus 6. References 8547 ISSN: 1454-4164 1454-4164 The conclusions that can be drawn from this contribution are: The experimental data used in analyses consists in measurements that have done to Beijing Synchrotron Radiation Facilities from High Institute of Physics. ### 5. Acknowledgement The authors are grateful to Beijing Synchrotron Radiation Facilities staff for beam time and for their technical assistance in XAS and XRD measurements. This work was partially supported by UEFISCDI, projects number 32-119/2008 and 22-098/2008. ## Appendix In this appendix are given the main analytical properties of the Fourier transform. $$h(-f) = \int\_{-\infty}^{\infty} H(t) \exp\left(-2\pi ift\right) dt\tag{67}$$ iii. Scaling. If h has the Fourier transform H then $$\int\_{-\infty}^{\infty} h(kt) \exp(-2\pi ift) dt = \frac{1}{\|k\|} H(f \;/\; k), \quad k \in \mathbb{R}^\* \tag{68}$$ iv. Shifting. If the Fourier transform of h is H and h is translated with t0 then $$\int\_{-\infty}^{\infty} h(t - t\_0) \exp(-2\pi ift)dt = \exp(-2\pi ift\_0)H(f) \tag{69}$$ v. If the signal he is even function and the Fourier transform of he is He then $$H\_e(f) = \int\_{-\infty}^{\infty} h\_e(t) \cos(2\pi ft) dt\tag{70}$$ vi. If the signal ho is odd function and the Fourier transform of ho is Ho then $$H\_o(f) = -i \int\_{-\infty}^{\infty} h\_o(t) \sin(2\pi ft) dt\tag{71}$$ vii. Any real function defined on a symmetrical interval has the following decomposition $$h(t) = h\_o(t) + h\_e(t) \, . \tag{72}$$ thus 22 Fourier Transform – Materials Analysis i. The physical periodical signals are successfully modeled using the trigonometric polynomial such us global approximation of the XRLP and the spectral distribution ii. The most important tools applied in EXAFS is based on the direct and inverse Fourier iii. The examples presented are based on the original contributions published in the The experimental data used in analyses consists in measurements that have done to Beijing The authors are grateful to Beijing Synchrotron Radiation Facilities staff for beam time and for their technical assistance in XAS and XRD measurements. This work was partially i. Linearity. If the signals x and y have the Fourier transform X and Y then the Fourier h f H t i f t dt ( ) ( )exp 2 ( ) <sup>∞</sup> <sup>1</sup> \* h kt ift dt H f k k R ( )exp( 2 ) ( / ), <sup>k</sup> 0 0 ht t i ( )exp( 2 ) exp( 2 ) ( ) ft dt if t H <sup>f</sup> <sup>∞</sup> ( ) ( )cos(2 ) H f h t ft dt e e ( ) ( )sin(2 ) Ho o f i ht vii. Any real function defined on a symmetrical interval has the following decomposition <sup>∞</sup> π −∞ −= − ∫ (67) −∞ −= ∈ ∫ (68) π π πft dt <sup>∞</sup> −∞ − − =− ∫ (69) −∞ <sup>=</sup> ∫ (70) −∞ = − ∫ (71) () () () o e ht h t h t = + , (72) The conclusions that can be drawn from this contribution are: Synchrotron Radiation Facilities from High Institute of Physics. supported by UEFISCDI, projects number 32-119/2008 and 22-098/2008. ii. Symmetry. If the Fourier transform of the function h is H, then <sup>∞</sup> In this appendix are given the main analytical properties of the Fourier transform. π π iv. Shifting. If the Fourier transform of h is H and h is translated with t0 then v. If the signal he is even function and the Fourier transform of he is He then vi. If the signal ho is odd function and the Fourier transform of ho is Ho then determination based on the Fourier analysis; transform methods; scientific literature. 5. Acknowledgement Appendix transform of α x y + β is α X Y + β. iii. Scaling. If h has the Fourier transform H then $$H(f) = H\_o(f) + H\_e(f) \tag{73}$$ For the next properties are assuming the function h is sufficiently smooth so that can be acceptable the differentiation and the integration. viii. The Fourier transform of the derivative of the function h is given by $$H\_{\frac{dh}{dt}}(f) = (2\pi \,\text{i}\,\,f)H\_h(f) \tag{74}$$ where Hh represents the Fourier Transform of the function h, and for the n times derivative $$H\_{\underbrace{d^n h}}(f) = \begin{pmatrix} \mathbb{Z}\pi \text{ i } f \end{pmatrix}^n H\_h(f) \tag{75}$$ ix. The derivative of the Fourier transform is given by $$\frac{dH\_h(f)}{df} = -2\pi \,\text{i}\, H\_{bh}(f) \tag{76}$$ and for the n times derivative of the Fourier transform $$\frac{d^n H\_h(f)}{df^n} = (-2\pi i)^n H\_{t^n h}(f) \tag{77}$$ #### 6. References 2 Portugal High Resolution Mass Spectrometry Faculdade de Ciências da Universidade de Lisboa Using FTICR and Orbitrap Instruments Paulo J. Amorim Madeira, Pedro A. Alves and Carlos M. Borges Centro de Química e Bioquímica, Departamento de Química e Bioquímica, From the 1950s to the present, mass spectrometry has evolved tremendously. The pioneering mass spectrometrist had a home-built naked instrument, typically a magnetic sector instrument with electron ionisation. Nowadays, highly automated commercial systems, able to produce thousands of spectra per day, are now concealed in a "black box", a nicely designed and beautifully coloured unit resembling more an espresso machine or a Mass spectrometry (MS) is probably the most versatile and comprehensive analytical technique currently available in the chemists and biochemists' arsenal. Mass spectrometry precisely measures the molecular masses of individual compounds by converting them into charged ions and analysing them in what is called a mass analyser. This is the simplest, but somewhat reductionist, definition of mass spectrometry. The days of the simple determination of the m/z ratio of an organic compound are over, today mass spectrometry can be used to determine molecular structures, to study reaction dynamics and ion chemistry, provides thermochemical and physical properties such as ionisation and appearance energies, reaction enthalpies, proton and ion affinities, gas-phase acidities, and Mass spectrometry is so versatile that even several areas of physics, pharmaceutical sciences, archaeology, forensic and environmental sciences, just to state a few, have The history of mass spectrometry starts in 1898 with the work of Wien, who demonstrated that canal rays could be deflected by passing them through superimposed parallel electric and magnetic fields. Nevertheless, its birth can be credited to Sir J. J. Thomson, Cavendish Laboratory of the University of Cambridge, through his work on the analysis of negatively and positively charged cathode rays with a parabola mass spectrograph, the great grandfather of the modern mass spectrometers. (Thomson 1897; Thomson 1907) In the next two decades, the developments of mass spectrometry continued by renowned physicists like Aston, (Aston 1919) Dempster, (Dempster 1918) Bainbridge, (Bainbridge 1932; Bainbridge benefited from the advances in this instrumental technique. and Jordan 1936) and Nier. (Nier 1940; Johnson and Nier 1953) 1. Introduction so on. tumble dryer than a mass spectrometer. ## High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments Paulo J. Amorim Madeira, Pedro A. Alves and Carlos M. Borges Centro de Química e Bioquímica, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa Portugal ## 1. Introduction 24 Fourier Transform – Materials Analysis Aldea, N.; Turcu, R.; Nan, A.; Craciunescu, I; Pana, O; Yaning, X.; Wu, Z.; Bica, D; Vekas, L. Aldea, N.; Pintea, S.; Rednic, V.; Matei, F. & Xie Y. (2009). Comparative study of EXAFS Bachmann, G.; Narici, L. & Beckenstein, E. (2002). Fourier and Wavelet Analysis (2nd edition), Brigham, E. (1988). The Fast Fourier Transform and Its Applications, Prentice Hall, ISBN Chen, L.X.; Liu, T.; Thurnauer, M.C.; Csencsits, R. & Rajh, T. (2002). Fe2O3 nanoparticle Kolobov, A. V.; Fons, P.; Tominaga, J.; Frenkel, A. I.; Ankudinov, A. L. & Uruga, T. (2005). Miller, J.T.; Kropf, A.J.; Zha, Y.; Regalbuto, J.R.; Delannoy, L.; Louis, C.; Bus, E. & Bokhoven, Richard, P.; Leighton, R.B. & Sands M. (2005). The Feynman Lectures on Physics, Vol. 1: Mainly Tang, K-T. (2007). Mathematical Methods for Engineers and Scientists 3, Fourier Analysis, Partial Turcu, R.; Peter, I.; Pana, O.; Giurgiu, L; Aldea, N.; Barz, B.; Grecu, M.N. & Coldea, A. Molecular Crystals and Liquid Crystals, Vol. 417, pp. 719-727, ISSN 1058-725X Walker, J. (1996). Fast Fourier Transforms, (2nd edition), CRC Press, ISBN 978-0849371639, Warren, B. E. (1990). X-Ray Diffraction, Dover Publications, ISBN 0486663175, NY, USA Vol.11, No.12, (December 2009), pp. 2167 – 2171, ISSN: 1454-4164 Springer, ISBN 978-0-387-98899-3, New York, USA No.34, (August 2002), pp. 8539-8546, ISSN 1520-6106. University Press, ISBN 9780521854559, London , UK. (June 2006), pp. 222-234, ISSN: 00219517 3540446958, Berlin-Heidelberg, Germany Boston, USA Boca Raton, USA pp. 1429-1439, ISSN: 1388-0764. 0133075052, New Jersey, USA & Matei, F. (2009). Investigation of nanostructured Fe3O4 polypyrrole core-shell composites by X-ray absorbtion spectroscopy and X-ray diffraction using synchrotron radiation. Journal of Nanoparticle Research, Vol.11, No.6, (August 2009), spectra for close-shell systems. Journal of Optoelectronics and Advanced Materials, structures investigated by X-ray absorption near-edge structure, surface modifications, and model calculations. Journal of Physical Chemistry B, Vol.106, Local Structure of Ge-Sb-Te and its modification Upon the Phase Transition. Journal of Ovonic Research, Vol.1, No.1, (February 2005), pp. 21 – 24, ISSN 1584 - 9953 Mandal, M & Asif, A. (2007). Continuous and Discrete Time Signals and Systems, Cambridge J.A. (2006). The effect of gold particle size on Au{single bond}Au bond length and reactivity toward oxygen in supported catalysts. Journal of Catalysis, Vol.240, No.2, Mechanics, Radiation, and Heat, (2nd edition), Addison Wesley, ISBN 978-0805390469, Differential Equations and Variational Methods (vol. 3), Springer-Verlag, ISBN 978- (2004). Structural and magnetic properties of polypyrrole nanocomposites. From the 1950s to the present, mass spectrometry has evolved tremendously. The pioneering mass spectrometrist had a home-built naked instrument, typically a magnetic sector instrument with electron ionisation. Nowadays, highly automated commercial systems, able to produce thousands of spectra per day, are now concealed in a "black box", a nicely designed and beautifully coloured unit resembling more an espresso machine or a tumble dryer than a mass spectrometer. Mass spectrometry (MS) is probably the most versatile and comprehensive analytical technique currently available in the chemists and biochemists' arsenal. Mass spectrometry precisely measures the molecular masses of individual compounds by converting them into charged ions and analysing them in what is called a mass analyser. This is the simplest, but somewhat reductionist, definition of mass spectrometry. The days of the simple determination of the m/z ratio of an organic compound are over, today mass spectrometry can be used to determine molecular structures, to study reaction dynamics and ion chemistry, provides thermochemical and physical properties such as ionisation and appearance energies, reaction enthalpies, proton and ion affinities, gas-phase acidities, and so on. Mass spectrometry is so versatile that even several areas of physics, pharmaceutical sciences, archaeology, forensic and environmental sciences, just to state a few, have benefited from the advances in this instrumental technique. The history of mass spectrometry starts in 1898 with the work of Wien, who demonstrated that canal rays could be deflected by passing them through superimposed parallel electric and magnetic fields. Nevertheless, its birth can be credited to Sir J. J. Thomson, Cavendish Laboratory of the University of Cambridge, through his work on the analysis of negatively and positively charged cathode rays with a parabola mass spectrograph, the great grandfather of the modern mass spectrometers. (Thomson 1897; Thomson 1907) In the next two decades, the developments of mass spectrometry continued by renowned physicists like Aston, (Aston 1919) Dempster, (Dempster 1918) Bainbridge, (Bainbridge 1932; Bainbridge and Jordan 1936) and Nier. (Nier 1940; Johnson and Nier 1953) High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments 27 Ion Source Mass Analyser Detector High Vacuum Fig. 1. Diagram of the major components common to all typical modern mass spectrometers Samples are introduced in the mass spectrometer and transferred into the gas phase through the inlet system that could be at atmospheric pressure or under vacuum. In the ion source, the gas-phase analytes are ionised and transferred into the mass analyser where they are separated according to their mass-to-charge ratios (m/z). Ion detection can be accomplished by electron multiplier systems that enable m/z and abundance to be measured and displayed by means of an electric signal perceived by the data system, which also controls the equipment. All mass spectrometers are equipped with a vacuum system in order to maintain the low pressure (high vacuum) required for operation. This high vacuum is necessary to allow ions to reach the detector without undergoing collisions with other gaseous molecules. In fact, collisions would produce a deviation of the trajectory and the ion would lose its charge against the walls of the instrument. On the other hand, a relatively high pressure environment could facilitate the occurrence of ion-molecule reactions that would increase the complexity of the spectrum. In some experiments the pressure in the source region or in a part of the mass spectrometer is intentionally increased to study ionmolecule reactions or to perform collision-induced dissociations. The high vacuum is maintained using mechanical pumps in conjunction with turbomolecular, diffusion or cryogenic pumps. The mechanical pumps allow a vacuum of about 10-3 torr to be obtained. Once this vacuum is achieved the operation of the remainder of the vacuum system allows a In the following sections we will briefly describe two types of mass analysers that employ Fourier transforms to determine m/z ratios. We will describe the Fourier Transform Ion Cyclotron Resonance mass spectrometer (FTICR MS) and the Orbitrap in sections 2.1 and 2.2, respectively. The basic aspects of each mass analyser will be dealt with; nevertheless, the interested reader is encouraged to seek more information in the literature. For example, in the case of FTICR mass spectrometry several reviews (Marshall, Hendrickson et al. 1998; Zhang, Rempel et al. 2005) and books are available. (Marshall and Verdun 1990; Gross 2004; Dass 2007; Hoffmann and Stroobant 2007) For the Orbitrap, the operation principles are well described in the papers published by Makarov, its inventor, (Makarov 2000; Hu, Noll et al. 2005; Makarov, Denisov et al. 2006) as well as by other authors, (Perry, Cooks et al. 2008) and in the more recent editions of some mass spectrometry textbooks. (Dass 2007; Hoffmann Inlet System Atmosphere/Vacuum vacuum as high as 10-10 torr to be reached. and Stroobant 2007) 2. Fourier transforms in mass spectrometry Data System In the 1940s, chemists recognised the great potential of mass spectrometry as an analytical tool, and applied it to monitor petroleum refinement processes. The first commercial mass spectrometer became available in 1943 through the Consolidated Engineering Corporation. The principles of time-of-flight (TOF) and ion cyclotron resonance (ICR) were introduced in 1946 and 1949, respectively. (Sommer, Thomas et al. 1951; Wolff and Stephens 1953) Applications to organic chemistry started to appear in the 1950s and exploded during the 1960s and 1970s. Double-focusing high-resolution mass spectrometers, which became available in the early 1950s, paved the way for accurate mass measurements. The quadrupole mass analyser and the ion trap were described by Wolfgang Paul and coworkers in 1953. (Paul 1990) The development of gas chromatography/mass spectrometry (GC/MS) in the 1960s marked the beginning of the analysis of seemingly complex mixtures by mass spectrometry. (Ryhage 2002; Watson and Biemann 2002) The 1960s also witnessed the development of tandem mass spectrometry and collision-induced decomposition, (Jennings 1968) being a breakthrough in structural and quantitative analysis, as well as in the development of soft ionisation techniques such as chemical ionisation. (Munson and Field 1966) By the 1960s, mass spectrometry had become a standard analytical tool in the analysis of organic compounds. Its application to the biosciences, however, was lacking due to the inexistence of suitable methods to ionise fragile and non-volatile compounds of biological origin. During the 1980s the range of applications in the field of the biosciences increased "exponentially" with the development of softer ionisation methods. These included fast atom bombardment (FAB) in 1981, (Barber, Bordoli et al. 1981) electrospray ionisation (ESI) in 1984-1988, (Fenn, Mann et al. 1989) and matrix-assisted laser desorption/ionisation (MALDI) in 1988. (Karas and Hillenkamp 2002) With the development of the last two methods, ESI and MALDI, the upper mass range was extended beyond 100 kDa and had an enormous impact on the use of mass spectrometry in biology and life sciences. This impact was recognised in 2002 when John Fenn (for his work on ESI) and Koichi Tanaka (for demonstrating that high molecular mass proteins could be ionised using laser desorption) were awarded with the Nobel Prize in Chemistry. Concurrent with the development of ionisation methods, several innovations in mass analyser technology, such as the introduction of high-field and superfast magnets, as well as the improvements in the TOF and Fourier transform ion cyclotron resonance (FTICR) enhanced the sensitivity and the upper mass range. The new millennium brought us two new types of ion traps, the orbitrap which was invented by Makarov (Makarov 2000) and the linear quadrupole ion trap (LIT) which was developed by Hager. (Hager 2002) The coupling of high-performance liquid chromatography (HPLC) with mass spectrometry was first demonstrated in the 1970s (Dass 2007); nevertheless, it was with the development and commercialisation of atmospheric pressure ionisation sources (ESI, APCI) that for the first time the combination of liquid chromatography and mass spectrometry entered the realm of routine analysis. (Voyksner 1997; Covey, Huang et al. 2002; Whitehouse, Dreyer et al. 2002; Rodrigues, Taylor et al. 2007) Generally, a mass spectrometer is composed of five components (Fig. 1): inlet system, ion source, mass analyser, ion detector, and data system. In the 1940s, chemists recognised the great potential of mass spectrometry as an analytical tool, and applied it to monitor petroleum refinement processes. The first commercial mass spectrometer became available in 1943 through the Consolidated Engineering Corporation. The principles of time-of-flight (TOF) and ion cyclotron resonance (ICR) were introduced in 1946 and 1949, respectively. (Sommer, Thomas et al. 1951; Wolff and Applications to organic chemistry started to appear in the 1950s and exploded during the 1960s and 1970s. Double-focusing high-resolution mass spectrometers, which became available in the early 1950s, paved the way for accurate mass measurements. The quadrupole mass analyser and the ion trap were described by Wolfgang Paul and coworkers in 1953. (Paul 1990) The development of gas chromatography/mass spectrometry (GC/MS) in the 1960s marked the beginning of the analysis of seemingly complex mixtures by mass spectrometry. (Ryhage 2002; Watson and Biemann 2002) The 1960s also witnessed the development of tandem mass spectrometry and collision-induced decomposition, (Jennings 1968) being a breakthrough in structural and quantitative analysis, as well as in the development of soft ionisation techniques such as chemical ionisation. (Munson and By the 1960s, mass spectrometry had become a standard analytical tool in the analysis of organic compounds. Its application to the biosciences, however, was lacking due to the inexistence of suitable methods to ionise fragile and non-volatile compounds of biological origin. During the 1980s the range of applications in the field of the biosciences increased "exponentially" with the development of softer ionisation methods. These included fast atom bombardment (FAB) in 1981, (Barber, Bordoli et al. 1981) electrospray ionisation (ESI) in 1984-1988, (Fenn, Mann et al. 1989) and matrix-assisted laser desorption/ionisation (MALDI) in 1988. (Karas and Hillenkamp 2002) With the development of the last two methods, ESI and MALDI, the upper mass range was extended beyond 100 kDa and had an enormous impact on the use of mass spectrometry in biology and life sciences. This impact was recognised in 2002 when John Fenn (for his work on ESI) and Koichi Tanaka (for demonstrating that high molecular mass proteins could be ionised using laser desorption) Concurrent with the development of ionisation methods, several innovations in mass analyser technology, such as the introduction of high-field and superfast magnets, as well as the improvements in the TOF and Fourier transform ion cyclotron resonance (FTICR) enhanced the sensitivity and the upper mass range. The new millennium brought us two new types of ion traps, the orbitrap which was invented by Makarov (Makarov 2000) and The coupling of high-performance liquid chromatography (HPLC) with mass spectrometry was first demonstrated in the 1970s (Dass 2007); nevertheless, it was with the development and commercialisation of atmospheric pressure ionisation sources (ESI, APCI) that for the first time the combination of liquid chromatography and mass spectrometry entered the realm of routine analysis. (Voyksner 1997; Covey, Huang et al. 2002; Whitehouse, Dreyer et Generally, a mass spectrometer is composed of five components (Fig. 1): inlet system, ion the linear quadrupole ion trap (LIT) which was developed by Hager. (Hager 2002) Stephens 1953) Field 1966) were awarded with the Nobel Prize in Chemistry. al. 2002; Rodrigues, Taylor et al. 2007) source, mass analyser, ion detector, and data system. Fig. 1. Diagram of the major components common to all typical modern mass spectrometers Samples are introduced in the mass spectrometer and transferred into the gas phase through the inlet system that could be at atmospheric pressure or under vacuum. In the ion source, the gas-phase analytes are ionised and transferred into the mass analyser where they are separated according to their mass-to-charge ratios (m/z). Ion detection can be accomplished by electron multiplier systems that enable m/z and abundance to be measured and displayed by means of an electric signal perceived by the data system, which also controls the equipment. All mass spectrometers are equipped with a vacuum system in order to maintain the low pressure (high vacuum) required for operation. This high vacuum is necessary to allow ions to reach the detector without undergoing collisions with other gaseous molecules. In fact, collisions would produce a deviation of the trajectory and the ion would lose its charge against the walls of the instrument. On the other hand, a relatively high pressure environment could facilitate the occurrence of ion-molecule reactions that would increase the complexity of the spectrum. In some experiments the pressure in the source region or in a part of the mass spectrometer is intentionally increased to study ionmolecule reactions or to perform collision-induced dissociations. The high vacuum is maintained using mechanical pumps in conjunction with turbomolecular, diffusion or cryogenic pumps. The mechanical pumps allow a vacuum of about 10-3 torr to be obtained. Once this vacuum is achieved the operation of the remainder of the vacuum system allows a vacuum as high as 10-10 torr to be reached. #### 2. Fourier transforms in mass spectrometry In the following sections we will briefly describe two types of mass analysers that employ Fourier transforms to determine m/z ratios. We will describe the Fourier Transform Ion Cyclotron Resonance mass spectrometer (FTICR MS) and the Orbitrap in sections 2.1 and 2.2, respectively. The basic aspects of each mass analyser will be dealt with; nevertheless, the interested reader is encouraged to seek more information in the literature. For example, in the case of FTICR mass spectrometry several reviews (Marshall, Hendrickson et al. 1998; Zhang, Rempel et al. 2005) and books are available. (Marshall and Verdun 1990; Gross 2004; Dass 2007; Hoffmann and Stroobant 2007) For the Orbitrap, the operation principles are well described in the papers published by Makarov, its inventor, (Makarov 2000; Hu, Noll et al. 2005; Makarov, Denisov et al. 2006) as well as by other authors, (Perry, Cooks et al. 2008) and in the more recent editions of some mass spectrometry textbooks. (Dass 2007; Hoffmann and Stroobant 2007) High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments 29 Inside the FTICR cell an ion has three natural motions: the cyclotron, the trapping and the magnetron motions. The nature of each motion will be briefly explained in the following Many fundamental aspects of FTICR can be understood from very simple idealised models: • Ion cyclotron frequency, radius, velocity and energy, as a function of ion mass, ion charge and magnetic field strength, follow directly from the motion of an ion in a • Ion cyclotron motion may be rendered coherent (and thus observable) by the application of a spatially uniform RF electric field (excitation) at the same frequency as the ion cyclotron frequency. The ICR signal results from induction of an oscillating "image" charge on two conductive infinitely extended opposed parallel electrodes. A frequency-domain spectrum is obtained by Fourier transformation of the digitised ICR • Confinement of ions by application of a three-dimensional axial quadrupolar DC electric field shifts the ion cyclotron frequency, whereas excitation and detection remain • Collisions broaden the ICR signal in a simple way, and actually make it possible to cool • Although FTICR-MS has been coupled to virtually every type of ion source, most ion sources work best outside the magnet. Thus, several methods have been developed to An ion moving in the presence of a uniform electric and magnetic fields, E and B, is subjected to a Lorentz force given by equation 1, where m, q and v are the mass, charge and Let us now consider only the presence of the magnetic field, B. If the ion maintains constant speed (i.e. no collisions), then the magnetic field bends the ion path into a circle of radius r, > + v > > qvB0 Fig. 2. Ion cyclotron motion for a positive or negative ion due to the presence of a magnetic B0 (1) guide the externally generated ions into the ion trap inside a high-field magnet. • The above features may be combined in various experimental "event sequences" to essentially linear, but with a reduced proportionality constant. perform tandem-in-time mass spectrometry (MS/MS or MSn). and compress an ion packet for improved detection. paragraphs. signal. Cyclotron motion the cyclotron motion (Fig. 2). field (B) perpendicular to the plane of the paper velocity. spatially uniform static magnetic field. ## 2.1 Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) The theory of cyclotron resonance was developed in the 1930s by Lawrence (1951 Nobel Prize in Physics). Lawrence built the first cyclotron accelerator to study the fundamental properties of the atom. Subsequently, Penning devised the first trap for charged particles by using a combination of static electric and magnetic fields to confine electrons. (Vartanian, Anderson et al. 1995) In the 1950s the principle of ion cyclotron resonance was first incorporated into a mass spectrometer, called the omegatron, by Sommer and co-workers, who successfully applied the concept of cyclotron resonance to determine the charge-tomass ratio of the proton. (Sommer, Thomas et al. 1951) Major improvements in ICR awaited McIver's introduction of the trapped ion cell. Unlike the conventional drift cell, the trapped ion cell allowed for ion formation, manipulation and detection to occur within the same volume in space. The trapped ion cell differed from previous ICR cell designs by the inclusion of "trapping" electrodes. By applying small voltages to these electrodes, McIver was able to trap ions for 1-2 ms (approximately 100 times that of the drift cell). These advantages led to a much greater dynamic range, sensitivity and mass resolution. More importantly, the extended trapping capability of the McIver cell was a prerequisite for the FTICR detection technique invented by Comisarow and Marshall later that decade. In the second half of the 1970s, Comisarow and Marshall adapted Fourier transform methods to ICR spectrometry and built the first FTICR-MS instrument. (Comisarow and Marshall 1974; Marshall, Comisarow et al. 1979) Since then, FTICR-MS has matured into a state-of-the-art high-resolution mass spectrometry instrument for the analysis of a wide variety of compounds (biological or not). All FTICR-MS systems have in common five main components: a magnet (nowadays usually a superconducting magnet); analyser cell (placed in the strong magnetic field created by the magnet); ultra-high vacuum system, and ion source (Fig. 2); and a sophisticated data system (many of the components in the data system are similar to those used in NMR). Fig. 2. Schematic representation of an FTICR mass spectrometer. Note that not all components are present in this scheme, for example, the ion optics is not presented, nor the rotary vacuum pumps that are needed for the proper functioning of the turbomolecular pumps In this section, we shall not discuss the magnet, vacuum and data systems, and focus on the ICR cell, which is the heart of the FTICR-MS instrument. It is here that the ions are stored, mass analysed and detected. Inside the FTICR cell an ion has three natural motions: the cyclotron, the trapping and the magnetron motions. The nature of each motion will be briefly explained in the following paragraphs. Many fundamental aspects of FTICR can be understood from very simple idealised models: #### Cyclotron motion 28 Fourier Transform – Materials Analysis The theory of cyclotron resonance was developed in the 1930s by Lawrence (1951 Nobel Prize in Physics). Lawrence built the first cyclotron accelerator to study the fundamental properties of the atom. Subsequently, Penning devised the first trap for charged particles by using a combination of static electric and magnetic fields to confine electrons. (Vartanian, Anderson et al. 1995) In the 1950s the principle of ion cyclotron resonance was first incorporated into a mass spectrometer, called the omegatron, by Sommer and co-workers, who successfully applied the concept of cyclotron resonance to determine the charge-tomass ratio of the proton. (Sommer, Thomas et al. 1951) Major improvements in ICR awaited McIver's introduction of the trapped ion cell. Unlike the conventional drift cell, the trapped ion cell allowed for ion formation, manipulation and detection to occur within the same volume in space. The trapped ion cell differed from previous ICR cell designs by the inclusion of "trapping" electrodes. By applying small voltages to these electrodes, McIver was able to trap ions for 1-2 ms (approximately 100 times that of the drift cell). These advantages led to a much greater dynamic range, sensitivity and mass resolution. More importantly, the extended trapping capability of the McIver cell was a prerequisite for the FTICR detection technique invented by Comisarow and Marshall later that decade. In the second half of the 1970s, Comisarow and Marshall adapted Fourier transform methods to ICR spectrometry and built the first FTICR-MS instrument. (Comisarow and Marshall 1974; Marshall, Comisarow et al. 1979) Since then, FTICR-MS has matured into a state-of-the-art high-resolution mass spectrometry instrument for the analysis of a wide variety of All FTICR-MS systems have in common five main components: a magnet (nowadays usually a superconducting magnet); analyser cell (placed in the strong magnetic field created by the magnet); ultra-high vacuum system, and ion source (Fig. 2); and a sophisticated data system (many of the components in the data system are similar to those used in NMR). Fig. 2. Schematic representation of an FTICR mass spectrometer. Note that not all components are present in this scheme, for example, the ion optics is not presented, nor the rotary vacuum pumps that are needed for the proper functioning of the turbomolecular pumps In this section, we shall not discuss the magnet, vacuum and data systems, and focus on the ICR cell, which is the heart of the FTICR-MS instrument. It is here that the ions are stored, ICR cell Superconducting magnet Turbomolecular pumps Collision cell 2.1 Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) compounds (biological or not). Ion source mass analysed and detected. An ion moving in the presence of a uniform electric and magnetic fields, E and B, is subjected to a Lorentz force given by equation 1, where m, q and v are the mass, charge and velocity. $$F = ma = m\frac{dv}{dt} = qE + q\nu B\tag{1}$$ Let us now consider only the presence of the magnetic field, B. If the ion maintains constant speed (i.e. no collisions), then the magnetic field bends the ion path into a circle of radius r, the cyclotron motion (Fig. 2). Fig. 2. Ion cyclotron motion for a positive or negative ion due to the presence of a magnetic field (B) perpendicular to the plane of the paper High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments 31 Finally, considering that the average kinetic energy of an ion with velocity vxy in equilibrium with its surroundings at a temperature T (in K) is given by equation 6 (where k is the Boltzmann's constant) and that the ion cyclotron orbital radius, r, is given by equation 7 Substituting vxy in equation 7 we obtain an expression that relates the ion cyclotron orbital Considering T=298 K and various magnetic field strengths, we can construct a graphical representation of r as a function of m/z (Fig. 4). This representation allows us to conclude that even large ions are confined by the magnetic field to a small orbital radius. For example, a "modest" superconducting magnet of 3 T confines an ion with m/z 2000 to an orbit with a 0 2000 4000 6000 8000 10000 m/z It is also possible to relate the kinetic energy of a trapped ion with its orbital radius by rearranging equation 7 (equation 9). A graphical representation of the ion's kinetic energy as a function of the orbital radius, depicted in Fig. 5 for a singly charged ion at m/z 400, reveals Fig. 4. ICR orbital radius, r, vs. m/z ratio at 298K and 3 T, 7 T, 9.4 T, 15 T and 21 T that ions can be heated to high kinetic energies even in a relatively small container. (derived from equation 3). radius smaller than 0.5 mm. r (mm) 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 radius as a function of m/z ratio (equation 8). (5) (6) (7) (8) 3 T 7 T 9.4 T 15 T 21 T The angular acceleration and angular velocity, ω, are defined by equations 2 and 3. $$\frac{dv}{dt} = \frac{v\_{\infty}}{r} \tag{2}$$ $$ \omega = \frac{v\_{xy}}{r} \tag{3} $$ Substituting these terms in equation 1 we obtain1 $$ \omega\_c = \frac{qB}{m} \tag{4} $$ Equation 4 is the celebrated "cyclotron" equation that relates the cyclotron frequency, ωc, with the mass and charge of the ion. It is clear that for a given m/z all ions have the same ICR frequency independent of their initial velocity; hence, energy focussing is not required to precisely determine the m/z ratio of an ion. Several useful conclusions arise from the cyclotron equation (eq. 4). Considering an m/z range of 10 to 100 000, the frequencies lie between a few kHz and a few MHz (Fig. 3), which is a very convenient range for commercially available electronics. Fig. 3. ICR orbital frequency (for ions in the m/z range between 10 and 100 000) as a function of m/z at 1.2 T, 3 T, 7 T, 9.4 T, 15 T (highest magnetic field strength commercially available) and 21 T (highest magnetic field strength magnet under development at the National High Magnetic Field Laboratory in the USA) The first derivative of the cyclotron equation with respect to m gives equation 5, which allows us to conclude that frequency resolution and mass resolution are essentially the same (apart from the minus sign). <sup>1</sup> All of the equations will be presented in S.I. units. To convert, for example, m/q to m/z the reader should take into account that q = z×e, where z is in multiples of elementary charge (e = 1.602×10-19 C) and m is in atomic mass units (1u = 1.660×10-27 kg). Equation 4 is the celebrated "cyclotron" equation that relates the cyclotron frequency, ωc, with the mass and charge of the ion. It is clear that for a given m/z all ions have the same ICR frequency independent of their initial velocity; hence, energy focussing is not required to Several useful conclusions arise from the cyclotron equation (eq. 4). Considering an m/z range of 10 to 100 000, the frequencies lie between a few kHz and a few MHz (Fig. 3), which 9.4 T 7 T 10 100 1000 10000 100000 m/z Fig. 3. ICR orbital frequency (for ions in the m/z range between 10 and 100 000) as a function of m/z at 1.2 T, 3 T, 7 T, 9.4 T, 15 T (highest magnetic field strength commercially available) and 21 T (highest magnetic field strength magnet under development at the National High The first derivative of the cyclotron equation with respect to m gives equation 5, which allows us to conclude that frequency resolution and mass resolution are essentially the same 1 All of the equations will be presented in S.I. units. To convert, for example, m/q to m/z the reader should take into account that q = z×e, where z is in multiples of elementary charge (e = 1.602×10-19 C) (2) (3) (4) The angular acceleration and angular velocity, ω, are defined by equations 2 and 3. Substituting these terms in equation 1 we obtain1 precisely determine the m/z ratio of an ion. 1.E+02 Magnetic Field Laboratory in the USA) and m is in atomic mass units (1u = 1.660×10-27 kg). (apart from the minus sign). 1.E+03 1.E+04 1.E+05 νc (Hz) 1.E+06 1.E+07 1.E+08 1.E+09 is a very convenient range for commercially available electronics. 3 T 1.2 T 21 T 15 T $$\frac{d\boldsymbol{\alpha}\_c}{d\boldsymbol{m}} = -\frac{qB}{m^2} = -\frac{\boldsymbol{\alpha}\_c}{m} \Leftrightarrow \frac{\boldsymbol{\omega}\_c}{d\boldsymbol{\omega}\_c} = -\frac{m}{dm} \tag{5}$$ Finally, considering that the average kinetic energy of an ion with velocity vxy in equilibrium with its surroundings at a temperature T (in K) is given by equation 6 (where k is the Boltzmann's constant) and that the ion cyclotron orbital radius, r, is given by equation 7 (derived from equation 3). $$ \frac{1}{2}m\langle v\_{xy}\|^2\rangle \cong kT \Leftrightarrow v\_{xy} = \sqrt{2kT/m} \tag{6} $$ $$ \sigma = \frac{m\nu\_{\chi\chi}}{qB\_0} \tag{7} $$ Substituting vxy in equation 7 we obtain an expression that relates the ion cyclotron orbital radius as a function of m/z ratio (equation 8). $$r = \frac{1}{qB\_0} \sqrt{2mkT} \tag{8}$$ Considering T=298 K and various magnetic field strengths, we can construct a graphical representation of r as a function of m/z (Fig. 4). This representation allows us to conclude that even large ions are confined by the magnetic field to a small orbital radius. For example, a "modest" superconducting magnet of 3 T confines an ion with m/z 2000 to an orbit with a radius smaller than 0.5 mm. Fig. 4. ICR orbital radius, r, vs. m/z ratio at 298K and 3 T, 7 T, 9.4 T, 15 T and 21 T It is also possible to relate the kinetic energy of a trapped ion with its orbital radius by rearranging equation 7 (equation 9). A graphical representation of the ion's kinetic energy as a function of the orbital radius, depicted in Fig. 5 for a singly charged ion at m/z 400, reveals that ions can be heated to high kinetic energies even in a relatively small container. High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments 33 frequency, the so-called trapping motion (or trapping oscillation). The trapping frequency, In general, the trapping frequency is much smaller than the ICR frequency so it is not The combination of the magnetic field and the radial component of the electric field created by the trapping potential induce a third motion: the magnetron rotation. The magnetron frequency (equation 16), ωm, is independent of both the mass and charge of the ion. Nevertheless, it is proportional to the trapping voltage (VT) and the cell geometry factor (α) and inversely proportional to the cell edge length (a) and the magnetic field strength (B). The three natural ion motions (cyclotron rotation, magnetron rotation and trapping oscillation) are depicted in Fig. 6. As mentioned earlier, the magnetron and trapping frequencies are usually much smaller than the cyclotron frequency and generally are not detected. Nevertheless, several reviews on the subject of ion trajectories inside the ICR cell and their influence on the cyclotron frequency were published over the years, e.g. the one by Ion cyclotron motion does not by itself generate an observable electric signal. When the ion packets enter the ICR cell, their ion cyclotron orbits are centred on the z-axis (i.e. they are too small to be detected) and must be made spatially coherent by moving them away from the centre of the cell. For that purpose excitation is needed and this is achieved by applying a spatially uniform electric field oscillating at or near the cyclotron frequency of ions of a • To accelerate ions coherently to a larger (and thus detectable) orbital radius – Fig. 7 a); (13) (15) (14) (16) ωT, and the k' constant are defined by equations 13 and 14, respectively. Substituting k' in equation 13 we obtain detected. Magnetron motion (Vartanian, Anderson et al. 1995) Cyclotron + Trapping + Magnetron Motions Vartanian et al. (Vartanian, Anderson et al. 1995) Excitation is used in three ways in FTICR-MS: Excitation and detection of an ICR signal particular m/z range. Fig. 5. Ion kinetic energy as a function of the ICR orbital radius (singly charged ion at m/z 400) #### Trapping motion The static magnetic field effectively confines ions in the xy plane; nevertheless, ions are still free to escape along the z-axis. To prevent this, a small electrostatic potential (equation 10), usually ≈ 1 V, is applied to the trapping electrodes (positioned at z = ± d/2 from the center of the ion trap). (Vartanian, Anderson et al. 1995) $$V(\mathbf{z}) \cong \frac{V\_t}{2} + \frac{k'\mathbf{z}^2}{2} \tag{10}$$ where k' is a constant. The trapping electric field can be obtained by the negative gradient with respect to z of the electrostatic potential (equation 11). $$E(\mathbf{z}) = -\frac{dV(\mathbf{z})}{d\mathbf{z}} = -k'\mathbf{z} \tag{11}$$ This electric field subjects the trapped ion to a force, F (z), given by equation 12 $$F(\mathbf{z}) = -\frac{d^2 \mathbf{z}}{dz^2} = -qk'\mathbf{z} \tag{12}$$ Equation 12 resembles the harmonic oscillator equation; hence, ions trapped in a quadratic z-potential must oscillate back and forth along the z-direction at a natural trapping frequency, the so-called trapping motion (or trapping oscillation). The trapping frequency, ωT, and the k' constant are defined by equations 13 and 14, respectively. $$ \omega\_T = \sqrt{\frac{k'q}{m}}\tag{13} $$ $$k' = \frac{4V\_T}{d^2} \tag{14}$$ Substituting k' in equation 13 we obtain $$ \omega\_T = \frac{2}{d} \sqrt{\frac{qV\_T}{m}} \tag{15} $$ In general, the trapping frequency is much smaller than the ICR frequency so it is not detected. #### Magnetron motion 32 Fourier Transform – Materials Analysis 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 r (cm) Fig. 5. Ion kinetic energy as a function of the ICR orbital radius (singly charged ion at m/z The static magnetic field effectively confines ions in the xy plane; nevertheless, ions are still free to escape along the z-axis. To prevent this, a small electrostatic potential (equation 10), usually ≈ 1 V, is applied to the trapping electrodes (positioned at z = ± d/2 from the center of The trapping electric field can be obtained by the negative gradient with respect to z of the Equation 12 resembles the harmonic oscillator equation; hence, ions trapped in a quadratic z-potential must oscillate back and forth along the z-direction at a natural trapping This electric field subjects the trapped ion to a force, F (z), given by equation 12 0.0 the ion trap). (Vartanian, Anderson et al. 1995) 400) Trapping motion where k' is a constant. electrostatic potential (equation 11). 1.0 2.0 3.0 4.0 Ek (keV) 5.0 6.0 7.0 8.0 9.0 (9) 21 T 15 T 9.4 T 7 T 3 T (10) (11) (12) The combination of the magnetic field and the radial component of the electric field created by the trapping potential induce a third motion: the magnetron rotation. The magnetron frequency (equation 16), ωm, is independent of both the mass and charge of the ion. Nevertheless, it is proportional to the trapping voltage (VT) and the cell geometry factor (α) and inversely proportional to the cell edge length (a) and the magnetic field strength (B). (Vartanian, Anderson et al. 1995) $$ \mu\_m = \frac{2\alpha V\_T}{a^2 B} \tag{16} $$ Cyclotron + Trapping + Magnetron Motions The three natural ion motions (cyclotron rotation, magnetron rotation and trapping oscillation) are depicted in Fig. 6. As mentioned earlier, the magnetron and trapping frequencies are usually much smaller than the cyclotron frequency and generally are not detected. Nevertheless, several reviews on the subject of ion trajectories inside the ICR cell and their influence on the cyclotron frequency were published over the years, e.g. the one by Vartanian et al. (Vartanian, Anderson et al. 1995) #### Excitation and detection of an ICR signal Ion cyclotron motion does not by itself generate an observable electric signal. When the ion packets enter the ICR cell, their ion cyclotron orbits are centred on the z-axis (i.e. they are too small to be detected) and must be made spatially coherent by moving them away from the centre of the cell. For that purpose excitation is needed and this is achieved by applying a spatially uniform electric field oscillating at or near the cyclotron frequency of ions of a particular m/z range. Excitation is used in three ways in FTICR-MS: • To accelerate ions coherently to a larger (and thus detectable) orbital radius – Fig. 7 a); High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments 35 With the excitation the ion speeds up and its radius increases linearly with time and the rate The integration of equation 20 from t=0 to t=texcite yields the total energy absorbed during the Substituting the cyclotron equation (equation 4) in equation 21 we obtain an expression that An interesting conclusion arises from equation 22 in that the orbital radius of the excited ions is independent of the m/z ratio, which means that ions of different m/z ratios can be The detection of the ions occurs as the ion packets pass two detector plates. As the ion packets have past these plates, charge moves within the detection circuit to counteract the proximity of the ions. The potential change (voltage) between the detection plates can be measured as a function of time and it is from here that the raw data is obtained. It should be noted that the ions repeatedly pass the detector plates for the duration of the acquisition, as non-destructive detection is employed. The magnitude of the signal is proportional to the total charge and to the proximity of the ions to the detection plates (orbital radius), and is independent of magnetic field strength. The raw data will represent the detections of all the ions at the same time, with their different cyclotron frequencies. It is therefore necessary to extract data concerning the different ion packets. This is done through the usage of a mathematical procedure known as Fourier transform (FT) where frequency information is obtained from time-domain data. Fig. 8 illustrates the process of obtaining a mass spectrum from the time-domain data through Fourier transform, conversion to m/z and calibration. Unlike other mass spectrometers (e.g. sector instruments, time-of-flight, quadrupole) where mass analysis and ion detection are spatially separated events, in FTICR all analytical steps are made on the same spatial place but separated in time. Fig. 9 shows a typical sequence of events for a tandem mass spectrometry experiment performed in a FTICR mass spectrometer. Before ion introduction, the ICR cell is emptied with a quench pulse. After the ions have been introduced into the cell a significant amount of time is required for the ion selection, dissociation, excitation, detection, time-domain data storage and Fourier excitation period and assuming a complete conversion into kinetic energy we obtain relates the radius with the excitation electric field and the excitation time. excited to the same ICR orbital radius. of power absorption is given by equation 20. (Marshall, Hendrickson et al. 1998) (18) (19) (20) (22) (21) Fig. 6. Schematic representation of the three natural motions of an ion confined in an ICR cell and the resulting ion trajectory shape Fig. 7. Ion cyclotron excitation uses: a) acceleration of ions to larger orbital radius for detection; b) acceleration of ions to induce ion dissociation or ion-molecule reactions; c) removal of unwanted ions from the ICR cell. (E – Excitation electrode, D – Detection electrode) In this chapter we will focus on the detection; the other two ways of using excitation (to increase the ion's kinetic energy and to remove undesired ions from the instrument) will not be addressed in this chapter. Nevertheless, more information can be obtained in a review by Marshall and co-workers. (Marshall, Hendrickson et al. 1998) Spatial coherence is created by applying an oscillating resonant, ω = ωc, phase-coherent electric field excitation E (t) (equation 17). $$E(t) = E\_0 \cos\alpha t \,\mathrm{j}\tag{17}$$ This linearly-polarised electric field can be decomposed into two counter-rotating components, E (t)=EL (t)+ER (t) (equations 18 and 19). • To increase ion kinetic energy above the threshold for ion dissociation and/or ion- • To accelerate ions to a cyclotron radius larger than the radius of the ion trap, so that Fig. 6. Schematic representation of the three natural motions of an ion confined in an ICR Fig. 7. Ion cyclotron excitation uses: a) acceleration of ions to larger orbital radius for detection; b) acceleration of ions to induce ion dissociation or ion-molecule reactions; c) removal of unwanted ions from the ICR cell. (E – Excitation electrode, D – Detection electrode) In this chapter we will focus on the detection; the other two ways of using excitation (to increase the ion's kinetic energy and to remove undesired ions from the instrument) will not be addressed in this chapter. Nevertheless, more information can be obtained in a review by Spatial coherence is created by applying an oscillating resonant, ω = ωc, phase-coherent This linearly-polarised electric field can be decomposed into two counter-rotating (17) (a) (b) (c) Marshall and co-workers. (Marshall, Hendrickson et al. 1998) components, E (t)=EL (t)+ER (t) (equations 18 and 19). electric field excitation E (t) (equation 17). molecule reaction – Fig. 7 b); cell and the resulting ion trajectory shape ions are removed from the instrument – Fig. 7 c). $$E\_L(t) = \frac{E\_0}{2}\cos\omega t \, f + \frac{E\_0}{2}\sin\omega t \, l \tag{18}$$ $$E\_R(t) = \frac{E\_0}{2}\cos\omega t \, f - \frac{E\_0}{2}\sin\omega t \, t \tag{19}$$ With the excitation the ion speeds up and its radius increases linearly with time and the rate of power absorption is given by equation 20. (Marshall, Hendrickson et al. 1998) $$A(t\_{excite}) = \frac{E\_0^{\,^2} q^2 t\_{excite}}{4m} \tag{20}$$ The integration of equation 20 from t=0 to t=texcite yields the total energy absorbed during the excitation period and assuming a complete conversion into kinetic energy we obtain $$\frac{m\omega\_c^2 r^2}{2} = \int\_0^{t\_{\text{excite}}} A(t) \, dt = \frac{E\_0^{\,^2} q^2 (t\_{\text{excite}})^2}{8m} \tag{21}$$ Substituting the cyclotron equation (equation 4) in equation 21 we obtain an expression that relates the radius with the excitation electric field and the excitation time. $$r = \frac{E\_0 t\_{\text{excite}}}{2B\_0} \tag{22}$$ An interesting conclusion arises from equation 22 in that the orbital radius of the excited ions is independent of the m/z ratio, which means that ions of different m/z ratios can be excited to the same ICR orbital radius. The detection of the ions occurs as the ion packets pass two detector plates. As the ion packets have past these plates, charge moves within the detection circuit to counteract the proximity of the ions. The potential change (voltage) between the detection plates can be measured as a function of time and it is from here that the raw data is obtained. It should be noted that the ions repeatedly pass the detector plates for the duration of the acquisition, as non-destructive detection is employed. The magnitude of the signal is proportional to the total charge and to the proximity of the ions to the detection plates (orbital radius), and is independent of magnetic field strength. The raw data will represent the detections of all the ions at the same time, with their different cyclotron frequencies. It is therefore necessary to extract data concerning the different ion packets. This is done through the usage of a mathematical procedure known as Fourier transform (FT) where frequency information is obtained from time-domain data. Fig. 8 illustrates the process of obtaining a mass spectrum from the time-domain data through Fourier transform, conversion to m/z and calibration. Unlike other mass spectrometers (e.g. sector instruments, time-of-flight, quadrupole) where mass analysis and ion detection are spatially separated events, in FTICR all analytical steps are made on the same spatial place but separated in time. Fig. 9 shows a typical sequence of events for a tandem mass spectrometry experiment performed in a FTICR mass spectrometer. Before ion introduction, the ICR cell is emptied with a quench pulse. After the ions have been introduced into the cell a significant amount of time is required for the ion selection, dissociation, excitation, detection, time-domain data storage and Fourier High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments 37 acquisition of time-domain image current transients, with subsequent fast Fourier Fig. 10. Orbitrap mass analyser. Ions are captured in a quadrologarithmic electrostatic field. An outer electrode enclosing a central spindle electrode consists of two halves separated by a dielectric material. The image current of ions moving as concentric rings along the central electrode is picked up by the outer electrode sections. Image kindly supplied by Thermo Features of the Orbitrap at its present stage of development include high mass resolution (up to 150 000), large space charge capacity, high mass accuracy (2–5 ppm), a mass/charge range of at least 6000, and dynamic range greater than 103. The current commercially available Orbitrap systems are equipped with several features that increase the range of applications. Fig. 11 depicts a hybrid ion trap-Orbitrap mass spectrometer equipped with an Electron Transfer Dissociation (ETD) module. ETD is the ion trap equivalent to Electron Capture Dissociation (a very popular fragmentation technique used in FTICR mass spectrometers) Fig. 11. Schematic representation of a hybrid ion trap-Orbitrap mass spectrometer. The main parts of a commercially available instrument are annotated on the diagram. The blue box highlights an optional ETD module which further extends the versatility of the system. Image kindly supplied by Thermo Fisher Scientific Ceramic Ring Inner Electrode transforms (FFT) being used to obtain the mass spectra. (Hu, Noll et al. 2005) Outer Electrode Fisher Scientific Fig. 8. Illustration of the processing of raw data. A Fourier transform is performed on timedomain data to convert it to the frequency-domain, and the resulting spectrum is then calibrated to m/z values transformation events before the next experiment (i.e. sequence) is started. The time involved in the events that follow ion introduction, greatly depends on the instrument used and on the type of experiment (for example, the acquisition of a normal full scan mass spectrum will take less time than other mass spectra since the ion selection and dissociation steps will not be performed). (Heeren, Kleinnijenhuis et al. 2004) Fig. 9. Example for a tandem mass spectrometry sequence performed in a FTICR mass spectrometer. The sequence shows the order of the different time-separated analytical steps #### 2.2 Orbitrap mass spectrometry The new millennium introduced a "new" mass analyser in the field of mass spectrometry that had not been stirred up since the first principles of FT-ICR. The Orbitrap (Fig. 10) is an ion trap that operates based only on an electrostatic field, by radially trapping ions about a central spindle electrode. An outer barrel-like electrode is coaxial with the inner spindle-like electrode and m/z values are measured from the frequency of harmonic ion oscillations along the axis of the electric field. This axial frequency is independent of the energy and spatial distribution of the ions. Ion frequencies are measured non-destructively by Time-domain spectrum Frequency-domain spectrum Mass spectrum Fig. 8. Illustration of the processing of raw data. A Fourier transform is performed on timedomain data to convert it to the frequency-domain, and the resulting spectrum is then transformation events before the next experiment (i.e. sequence) is started. The time involved in the events that follow ion introduction, greatly depends on the instrument used and on the type of experiment (for example, the acquisition of a normal full scan mass spectrum will take less time than other mass spectra since the ion selection and dissociation steps will not be performed). (Heeren, Kleinnijenhuis et al. 2004) Ion selection Ion dissociation Fig. 9. Example for a tandem mass spectrometry sequence performed in a FTICR mass spectrometer. The sequence shows the order of the different time-separated analytical steps The new millennium introduced a "new" mass analyser in the field of mass spectrometry that had not been stirred up since the first principles of FT-ICR. The Orbitrap (Fig. 10) is an ion trap that operates based only on an electrostatic field, by radially trapping ions about a central spindle electrode. An outer barrel-like electrode is coaxial with the inner spindle-like electrode and m/z values are measured from the frequency of harmonic ion oscillations along the axis of the electric field. This axial frequency is independent of the energy and spatial distribution of the ions. Ion frequencies are measured non-destructively by Ion excitation Ion detection Data storage Fourier transform Time (s) calibrated to m/z values Ion quench (empty cell) 2.2 Orbitrap mass spectrometry Ion introduction Fourier transform Conversion to m/z and calibration acquisition of time-domain image current transients, with subsequent fast Fourier transforms (FFT) being used to obtain the mass spectra. (Hu, Noll et al. 2005) Fig. 10. Orbitrap mass analyser. Ions are captured in a quadrologarithmic electrostatic field. An outer electrode enclosing a central spindle electrode consists of two halves separated by a dielectric material. The image current of ions moving as concentric rings along the central electrode is picked up by the outer electrode sections. Image kindly supplied by Thermo Fisher Scientific Features of the Orbitrap at its present stage of development include high mass resolution (up to 150 000), large space charge capacity, high mass accuracy (2–5 ppm), a mass/charge range of at least 6000, and dynamic range greater than 103. The current commercially available Orbitrap systems are equipped with several features that increase the range of applications. Fig. 11 depicts a hybrid ion trap-Orbitrap mass spectrometer equipped with an Electron Transfer Dissociation (ETD) module. ETD is the ion trap equivalent to Electron Capture Dissociation (a very popular fragmentation technique used in FTICR mass spectrometers) Fig. 11. Schematic representation of a hybrid ion trap-Orbitrap mass spectrometer. The main parts of a commercially available instrument are annotated on the diagram. The blue box highlights an optional ETD module which further extends the versatility of the system. Image kindly supplied by Thermo Fisher Scientific High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments 39 where r and z are cylindrical coordinates (z= 0 being the plane of the symmetry of the field), C is a constant, k is field curvature (or more recently known as axial restoring force determined by the exact shape of the electrodes and applied potential), and Rm is the characteristic radius. For a more detailed description, please refer to literature. (Gillig, This field, shown in equation (22), is the sum of a quadrupole field of the ion trap (which confines ions axially) and a logarithmic field of a cylindrical capacitor (provides orbital ion trapping); therefore, it is also called a quadro-logarithmic field. It is clear from this expression that there are no cross-terms in r and z, thus motion in z is independent of r, The trap consists of an outer barrel-like electrode and a central spindle-like electrode along the axis (see Fig. 10). The geometrical shape of these axially symmetrical electrodes can be where index 1 denotes the central electrode (spindle like), index 2 denotes the outer electrode (with flanges enclosing the trapping volume), z = 0 is the plane of symmetry, and Stable ion trajectories involve both orbiting motion around (rotational) the central electrode (r, φ-motion, where φ is the angular coordinate) and simultaneous harmonic oscillations in For the Rotational Motion, the r, φ–motion, although not used for mass analysis, is still important because ions must be trapped in the radial plane. Equilibrium is reached between For the Axial Motion, the movement back and forth around the central electrode, along z, is (approximately) described by a simple harmonic oscillator. The angular frequency of axial oscillations in rad s-1 (ω) is defined by equation 25 in terms of the charge (q) and mass (m) of the ion, k the field constant, being essential for establishing the fundamental relation for Use of the axial angular frequency as opposed to rotational or radial frequency is critical because only this frequency is completely independent of energy and spatial spread of ions (see below). A mass analyser employing such electrostatic axially harmonic orbital trapping mass analysis. (Makarov 2000; Hu, Noll et al. 2005; Makarov, Denisov et al. 2006) Geometry of the Orbitrap (axially symmetric electrodes and equations of motion) Bluhm et al. 1996; Makarov 2000; Makarov, Denisov et al. 2006) R1,2 are the maximum radii of the corresponding electrodes. the z-direction (coherent axial oscillations). The Ion Trajectories (Equations of motion) is referred hereinafter as the Orbitrap. attraction and centrifugal forces that act on the ions. φ-motion (where φ is the angular coordinate). deduced from eq. 22: (25) (23) (24) that enables, for example, the probing of post-translational modifications of proteins. (Jung, Pasini et al. 2010) ### 2.2.1 Principles of operation of the Orbitrap mass analyser (overview) The principles of operation of the Orbitrap can be summarised as follows: In a very simple way, in this mass analyser, ions are injected tangentially into an electric field between specially shaped electrodes and trapped because their electrostatic attraction to the inner electrode is balanced by centrifugal forces. Thus, ions cycle around the central electrode in rings - rotational motion (elliptical orbiting). In addition, the ions also move back and forth along the axis of the central electrode - axial oscillation. Therefore, ions of a specific mass-to-charge ratio move in rings which oscillate along the central spindle-like electrode. The frequency of these harmonic oscillations is independent of the ion velocity and is inversely proportional to the square root of the mass-to-charge ratio (m/z). By sensing the ion oscillation in a manner similar to that used in FT-ICR (ion image current detection and FFT), the trap can be used as a mass analyser. There are three types of ion trapping linear, segmented and orbital trapping. The latter is the focus of our review. #### Orbital trapping (History) Orbital trapping was first implemented by Kingdon in 1923. (Kingdon 1923) In its classical shape, the Kingdon trap contains a wire stretched along the axis of an outer cylinder with flanges enclosing the trapping volume. When a voltage is applied between the wire and the cylinder, the strong field attracts ions to the wire. Only ions that have sufficient tangential velocity miss the wire and survive. Motion along the wire is restrained by the field curvature caused by the flanges of the outer cylinder. A more elaborate electrode shape has been developed by Knight ("ideal Kingdon trap"). (Knight 1981) #### The modified Kingdon trap Orbital trapping is known to be used in experiments on the spectroscopy of ions. In these applications, ions have been formed within the trap or injected tangentially prior to switching on the field of the trap. (Knight 1981; Lewis 1982; Lisheng and Church 1991; Sekioka, Terasawa et al. 1991) Makarov (Makarov 2000) proposed the concept of orbital trapping with a fresh review for application to mass analysis. A "new" type of mass analyser was described which employs orbital trapping in an electrostatic field. For didactic purposes the orbitrap can be considered as an enhanced 'Knight-style' ideal Kingdon trap, but with specially shaped electrodes. When a DC voltage is applied between the two axially symmetric electrodes it results in the electrostatic field with potential distribution described in equation 23. that enables, for example, the probing of post-translational modifications of proteins. (Jung, • It exclusively uses an electrostatic field to trap the ions (no RF or magnetic fields). • Moving ions are trapped around the central electrode with only electrostatic attraction • Control of frequencies of oscillation (axial in particular) by shape/geometry of In a very simple way, in this mass analyser, ions are injected tangentially into an electric field between specially shaped electrodes and trapped because their electrostatic attraction to the inner electrode is balanced by centrifugal forces. Thus, ions cycle around the central electrode in rings - rotational motion (elliptical orbiting). In addition, the ions also move back and forth along the axis of the central electrode - axial oscillation. Therefore, ions of a specific mass-to-charge ratio move in rings which oscillate along the central spindle-like electrode. The frequency of these harmonic oscillations is independent of the ion velocity and is inversely proportional to the square root of the mass-to-charge ratio (m/z). By sensing the ion oscillation in a manner similar to that used in FT-ICR (ion image current detection and FFT), the trap can be used as a mass analyser. There are three types of ion trapping Orbital trapping was first implemented by Kingdon in 1923. (Kingdon 1923) In its classical shape, the Kingdon trap contains a wire stretched along the axis of an outer cylinder with flanges enclosing the trapping volume. When a voltage is applied between the wire and the cylinder, the strong field attracts ions to the wire. Only ions that have sufficient tangential velocity miss the wire and survive. Motion along the wire is restrained by the field curvature caused by the flanges of the outer cylinder. A more elaborate electrode shape has Orbital trapping is known to be used in experiments on the spectroscopy of ions. In these applications, ions have been formed within the trap or injected tangentially prior to switching on the field of the trap. (Knight 1981; Lewis 1982; Lisheng and Church 1991; Makarov (Makarov 2000) proposed the concept of orbital trapping with a fresh review for application to mass analysis. A "new" type of mass analyser was described which employs For didactic purposes the orbitrap can be considered as an enhanced 'Knight-style' ideal Kingdon trap, but with specially shaped electrodes. When a DC voltage is applied between the two axially symmetric electrodes it results in the electrostatic field with potential being compensated by centrifugal force (from initial tangential velocity). linear, segmented and orbital trapping. The latter is the focus of our review. been developed by Knight ("ideal Kingdon trap"). (Knight 1981) 2.2.1 Principles of operation of the Orbitrap mass analyser (overview) The principles of operation of the Orbitrap can be summarised as follows: • The outer electrode confines ions axially. Pasini et al. 2010) electrodes. Orbital trapping (History) The modified Kingdon trap Sekioka, Terasawa et al. 1991) orbital trapping in an electrostatic field. distribution described in equation 23. $$U(r,z) = \frac{k}{2} \left(z^2 - \frac{r^2}{2}\right) + \frac{k}{2} (R\_m)^2 \ln\left[\frac{r}{R\_m}\right] + \mathcal{C} \tag{23}$$ where r and z are cylindrical coordinates (z= 0 being the plane of the symmetry of the field), C is a constant, k is field curvature (or more recently known as axial restoring force determined by the exact shape of the electrodes and applied potential), and Rm is the characteristic radius. For a more detailed description, please refer to literature. (Gillig, Bluhm et al. 1996; Makarov 2000; Makarov, Denisov et al. 2006) This field, shown in equation (22), is the sum of a quadrupole field of the ion trap (which confines ions axially) and a logarithmic field of a cylindrical capacitor (provides orbital ion trapping); therefore, it is also called a quadro-logarithmic field. It is clear from this expression that there are no cross-terms in r and z, thus motion in z is independent of r, φ-motion (where φ is the angular coordinate). #### Geometry of the Orbitrap (axially symmetric electrodes and equations of motion) The trap consists of an outer barrel-like electrode and a central spindle-like electrode along the axis (see Fig. 10). The geometrical shape of these axially symmetrical electrodes can be deduced from eq. 22: $$z\_{1,2}\left(r\right) = \sqrt{\frac{r^2}{2} - \frac{\left(R\_{1,2}\right)^2}{2}} + (R\_m)^2 \ln\left[\frac{R\_{1,2}}{r}\right] \tag{24}$$ where index 1 denotes the central electrode (spindle like), index 2 denotes the outer electrode (with flanges enclosing the trapping volume), z = 0 is the plane of symmetry, and R1,2 are the maximum radii of the corresponding electrodes. Stable ion trajectories involve both orbiting motion around (rotational) the central electrode (r, φ-motion, where φ is the angular coordinate) and simultaneous harmonic oscillations in the z-direction (coherent axial oscillations). #### The Ion Trajectories (Equations of motion) For the Rotational Motion, the r, φ–motion, although not used for mass analysis, is still important because ions must be trapped in the radial plane. Equilibrium is reached between attraction and centrifugal forces that act on the ions. For the Axial Motion, the movement back and forth around the central electrode, along z, is (approximately) described by a simple harmonic oscillator. The angular frequency of axial oscillations in rad s-1 (ω) is defined by equation 25 in terms of the charge (q) and mass (m) of the ion, k the field constant, being essential for establishing the fundamental relation for mass analysis. (Makarov 2000; Hu, Noll et al. 2005; Makarov, Denisov et al. 2006) $$ \omega = \sqrt{(q/m)k} \tag{25} $$ Use of the axial angular frequency as opposed to rotational or radial frequency is critical because only this frequency is completely independent of energy and spatial spread of ions (see below). A mass analyser employing such electrostatic axially harmonic orbital trapping is referred hereinafter as the Orbitrap. High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments 41 [Ub+7H]7+ [Ub+8H]8+ [Ub+9H]9+ [Ub+10H]10+ 952,7 [Ub+9H]9+ 1071,5 [Ub+8H]8+ 1224,6 [Ub+10H] b) 10+ [Ub+7H]7+ 857,5 [Ub+10H]10+ 779,7 [Ub+11H]11+ [Ub+11H]11+ a) 600 800 1000 1200 1400 1600 1800 m/z 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z [Ub+10H]10+ ion. (High resolution system: 7 T Apex Ultra FTICR-MS from Bruker Daltonics; Low resolution system LCQ-Duo ion trap mass spectrometer from Thermo Fisher Scientific Fig. 12. High resolution (a) and low resolution (b) electrospray ionisation mass spectra of Ubiquitin (Ub). The insets show an expansion of the peak at m/z 857.5 attributed to 753,1 817,5 934,3 1714,1 956,7 1089,7 1258,3 861,1 991,1 591,1 1307,5 1468,0 678,7 1563,1 1618,5 1795,1 1931,7 [Ub+6H]6+ 857,5 1428,3 [Ub+6H]6+ Δ(m/z)=0.10 [Ub+5H]5+ [Ub+5H]5+ 856 857 858 859 860 861 862 863 m/z 855,7 858,8 859,2 861,1 861,6 855,5 859,9 860,3 860,8 861,9 862,3 856,1 856,7 <sup>1</sup> 862,9 856.5 857.0 857.5 858.0 858.5 m/ Resolving power ≈ 30 000 [Ub+10H]10+ This axial frequency may be determined using image current detection and fast FT algorithms. (Dienes, Pastor et al. 1996) The detection of an ion image current due to motion along the Orbitrap axis is only possible as long as the ion packets retain their spatial coherence in the axial direction (i.e., phase coherence and small spatial extent). The outer electrode is split in half at z=0 allowing the ion image current due to axial motion to be collected. The current is differentially amplified from each half of the outer electrode and then undergoes analog-to-digital conversion before processing and collection by customised control and acquisition software. Transient ion image current in the outer electrodes (split at z=0) is acquired as a time-domain transient and transformed to the spectra in the mass spectrometer by applying fast Fourier transform (FFT). The image current is amplified and processed exactly in the same way as in FT ICR; (see previous section) therefore, similar sensitivity and signal-to-noise ratios are expected. The resulting frequency spectrum however differs due to a much slower decrease of ion frequency with mass according to equation 25 when compared with equation 4. As a conclusion, potential advantages of the Orbitrap include: ## 3. Applications The mass analysers described in this chapter are capable of performing high resolution and high accuracy mass measurements on a routine basis. It is worth mentioning that before such mass analysers were developed, accurate mass measurements were performed only on sector instruments with a great deal of work and time behind each analysis within a restricted mass range (basically the masses in the close vicinity of the measured mass). Nowadays, high resolution can be attained in a broad mass range and the spectra are acquired in a couple of minutes (usually the time spent to acquire an HR mass spectrum is well below 1 minute). To demonstrate how useful high resolution is, electrospray ionisation (ESI) mass spectra will be shown that were acquired on two different instruments: an Apex Ultra FTICR mass spectrometer equipped with a 7 T superconducting magnet (Bruker Daltonics, Bremen, Germany) and a LCQ-Duo ion trap mass spectrometer (Thermo Fisher Scientific, Bremen Germany). The high- and low-resolution ESI mass spectra of ubiquitin are depicted in Fig. 12. Even though both systems provide information about charge state distribution, it is perfectly clear that the isotopic envelope is completely resolved when using an FTICR system (see insets in Fig. 12). This axial frequency may be determined using image current detection and fast FT algorithms. (Dienes, Pastor et al. 1996) The detection of an ion image current due to motion along the Orbitrap axis is only possible as long as the ion packets retain their spatial coherence in the axial direction (i.e., phase coherence and small spatial extent). The outer electrode is split in half at z=0 allowing the ion image current due to axial motion to be collected. The current is differentially amplified from each half of the outer electrode and then undergoes analog-to-digital conversion before processing and collection by customised control and acquisition software. Transient ion image current in the outer electrodes (split at z=0) is acquired as a time-domain transient and transformed to the spectra in the mass spectrometer by applying fast Fourier transform (FFT). The image current is amplified and processed exactly in the same way as in FT ICR; (see previous section) therefore, similar sensitivity and signal-to-noise ratios are expected. The resulting frequency spectrum however differs due to a much slower decrease of ion frequency with mass according to • high mass resolution (up to 100 000-200 000) since the quadro-logarithmic field • increased space charge capacity at higher masses due to independence of trapping potential on mass-to-charge ratio and larger trapping volumes in contrast to FT ICR and • high mass accuracy (2-5 ppm internal and external calibration, respectively), dynamic range over which accurate masses can be determined, and upper mass limit; • maintenance free analyser (when compared to the FTICR mass analyser where cryogenic liquids or expensive cooling apparatus are needed to maintain the The mass analysers described in this chapter are capable of performing high resolution and high accuracy mass measurements on a routine basis. It is worth mentioning that before such mass analysers were developed, accurate mass measurements were performed only on sector instruments with a great deal of work and time behind each analysis within a restricted mass range (basically the masses in the close vicinity of the measured mass). Nowadays, high resolution can be attained in a broad mass range and the spectra are acquired in a couple of minutes (usually the time spent to acquire an HR mass spectrum is To demonstrate how useful high resolution is, electrospray ionisation (ESI) mass spectra will be shown that were acquired on two different instruments: an Apex Ultra FTICR mass spectrometer equipped with a 7 T superconducting magnet (Bruker Daltonics, Bremen, Germany) and a LCQ-Duo ion trap mass spectrometer (Thermo Fisher Scientific, Bremen Germany). The high- and low-resolution ESI mass spectra of ubiquitin are depicted in Fig. 12. Even though both systems provide information about charge state distribution, it is perfectly clear that the isotopic envelope is completely resolved when using an FTICR equation 25 when compared with equation 4. • high sensitivity and stability; and superconducting magnet). ion trap; 3. Applications well below 1 minute). system (see insets in Fig. 12). As a conclusion, potential advantages of the Orbitrap include: (equation 22) may be defined with very high accuracy; Fig. 12. High resolution (a) and low resolution (b) electrospray ionisation mass spectra of Ubiquitin (Ub). The insets show an expansion of the peak at m/z 857.5 attributed to [Ub+10H]10+ ion. (High resolution system: 7 T Apex Ultra FTICR-MS from Bruker Daltonics; Low resolution system LCQ-Duo ion trap mass spectrometer from Thermo Fisher Scientific High Resolution Mass Spectrometry Using FTICR and Orbitrap Instruments 43 Hager, J. W. (2002). A new linear ion trap mass spectrometer. Rapid Communications in Mass Heeren, R. M. A., A. J. Kleinnijenhuis, et al. (2004). A mini-review of mass spectrometry using Hoffmann, E. d. and V. Stroobant (2007). Mass spectrometry: Principles and Applications. Hu, Q., R. J. Noll, et al. (2005). The Orbitrap: a new mass spectrometer. Journal of Mass Jennings, K. R. (1968). Collision-induced decompositions of aromatic molecular ions. International Johnson, E. G. and A. O. Nier (1953). Angular aberrations in sector shaped electromagnetic lenses Jung, H. R., D. Pasini, et al. (2010). Quantitative mass spectrometry of Histones H3.2 and H3.3 in Knight, R. D. (1981). Storage of ions from laser-produced plasmas. Applied Physics Letters 38 (4): Lewis, R. R. (1982). Motion of ions in the Kingdon trap. Journal of Applied Physics 53 (6): 3975- Lisheng, Y. and D. A. Church (1991). Confinement of injected beam ions in a Kingdon trap. Makarov, A. (2000). Electrostatic axially harmonic orbital trapping: a high-performance technique Makarov, A., E. Denisov, et al. (2006). Performance evaluation of a hybrid linear ion trap/Orbitrap Marshall, A. G., M. B. Comisarow, et al. (1979). Relaxation and spectral line shape in Fourier transform ion resonance spectroscopy. Journal of Chemical Physics 71 (11): 4434-4444. Marshall, A. G., C. L. Hendrickson, et al. (1998). Fourier transform ion cyclotron resonance mass Marshall, A. G. and F. R. Verdun (1990). Fourier tansforms in NMR, optical, and mass Munson, M. S. B. and F. H. Field (1966). Chemical ionization mass spectrometry. I. General Introduction. Journal of the American Chemical Society 88 (12): 2621-2630. Nier, A. O. (1940). A mass Spectrometer for routine isotope abundance measurements. Review of Paul, W. (1990). Electromagnetic traps for charged and neutral particles. Reviews of Modern Perry, R. H., R. G. Cooks, et al. (2008). 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Analytical Chemistry 72 (6): 1156-1162. mass spectrometer. Analytical Chemistry 78 (7): 2113-2120. spectrometry: A primer. Mass Spectrometry Reviews 17 (1): 1-35. and applications. Mass Spectrometry Reviews 27 (6): 661-699. high-performance FTICR-MS methods. Analytical and Bioanalytical Chemistry 378 (4): Gross, J. H. (2004). Mass Spectrometry: A Textbook. Berlin, Springer-Verlag. Spectrometry 16 (6): 512-526. Chichester, John Wiley & Sons. Spectrometry 40 (4): 430-443. 1048-1058. 221-223. 3980. There are basically two ways to determine the charge state of a given ion. One involves the analysis of the isotope envelope, implying that it can only be used in high-resolution mass spectrometry, while the other relates each peak with the subsequent one and can be used in high- and low-resolution mass spectrometry. The mass difference between two isotopic peaks is related to the charge state by z=1/Δ (m/z), hence, for the ubiquitin ion at m/z 857.47 ([Ub+10H]10+ ion of Fig. 12a) the charge state will be given by z= (1/0.10) = 10. It is worth mentioning that isotopic peaks are useful in the interpretation as long as there is no interference from other ions. Considering that two adjacent peaks in the mass spectrum, for example, m/z 857.5 and m/z 957.7 of Fig. 12b, are due to the same entity (i.e. they reflect different charge states of ubiquitin), one can assume that m = 857.5z' and m = 957.7z (where z' = z+1), i.e. 857.5 (z+1) = 957.7z, hence z = 9. As such the ion at m/z 957.7 corresponds to a 9+ ion while the ion at m/z 857.5 corresponds to a 10+ ion. ### 4. Acknowledgments The authors acknowledge the funding provided by Fundação para a Ciência e a Tecnologia (PEst-OE/QUI/UI0612/2011 and REDE/1501/REM/2005). Pedro A. Alves acknowledges Fundação para a Ciência e a Tecnologia for the PhD grant (SFRH/BD/49069/2008). Thermo Fisher Scientific and Dr. Michaela Scigelova are gratefully acknowledged for supplying the Orbitrap images. #### 5. References There are basically two ways to determine the charge state of a given ion. One involves the analysis of the isotope envelope, implying that it can only be used in high-resolution mass spectrometry, while the other relates each peak with the subsequent one and can be used in The mass difference between two isotopic peaks is related to the charge state by z=1/Δ (m/z), hence, for the ubiquitin ion at m/z 857.47 ([Ub+10H]10+ ion of Fig. 12a) the charge state will be given by z= (1/0.10) = 10. It is worth mentioning that isotopic peaks are useful in the Considering that two adjacent peaks in the mass spectrum, for example, m/z 857.5 and m/z 957.7 of Fig. 12b, are due to the same entity (i.e. they reflect different charge states of ubiquitin), one can assume that m = 857.5z' and m = 957.7z (where z' = z+1), i.e. 857.5 (z+1) = 957.7z, hence z = 9. As such the ion at m/z 957.7 corresponds to a 9+ ion while the ion at m/z The authors acknowledge the funding provided by Fundação para a Ciência e a Tecnologia (PEst-OE/QUI/UI0612/2011 and REDE/1501/REM/2005). Pedro A. Alves acknowledges Fundação para a Ciência e a Tecnologia for the PhD grant (SFRH/BD/49069/2008). Thermo Fisher Scientific and Dr. Michaela Scigelova are gratefully acknowledged for supplying the Aston, F. W. (1919). LXXIV. A positive ray spectrograph. Philosophical Magazine Series 6 38 Bainbridge, K. T. and E. B. Jordan (1936). Mass spectrum analysis 1. The mass spectrograph. 2. 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China Fourier Transform Infrared Mizi Fan1,2, Dasong Dai1,2 and Biao Huang2 Spectroscopy for Natural Fibres 1Department of Civil Engineering, Brunel University, London, UB8 3PH, 2School of Material and Engineering, Fujian Agricultural and Forestry University, Infrared spectroscopy is nowadays one of the most important analytical techniques available to scientists. One of the greatest advantages of the infrared spectroscopy is that virtually any sample in any state may be analyzed. For example, liquids, solutions, pastes, powders, films, fibres, gases and surfaces can all be examined with a judicious choice of sampling technique. The review by Annette, Sudhakar, Ursula and Andrea [1-2] also demonstrates the applicability of dispersion infrared spectroscopy for natural fibres studies. Fourier transform infrared spectroscopy (FTIR) has facilitated many different IR sampling techniques, including attenuated total reflection and diffuses reflectance infrared Fourier transform (DRIFT) spectroscopy. It has dramatically improved the quality of infrared spectra and minimized the time required to obtain data. The increased speed and higher ratio of signal-to-noise of FTIR relative to dispersion infrared has lead to a substantially greater number of applications of infrared in natural fibres research. In addition, the constant advancing of computer and computing science has made infrared spectroscopy techniques striding further: The availability of a dedicated computer, which is required for the FTIR instrumentation, has allowed the digitized spectra to be treated by sophisticated data processing techniques and increased the utility of the infrared spectra for qualitative and quantitative purposes. With interferometric techniques, the infrared spectroscopy is being launched into a new era and interest in this technique is at an all time high. compositions of native natural fibres and the modified natural fibres. Cellulose, which acts as the reinforcing material in the cell wall, is the main constitute in natural fibres. The cellulose molecules are laid down in microfibrils in which there is extensive hydrogen bonding between cellulose chains, producing a strong crystalline structure. Much work has been published on the characterization of the hydrogen bonds in cellulose by using various techniques, among which FTIR has proved to be one of the most useful methods [3-6]. Furthermore, FTIR can provide researchers with further information on the super-molecular structure. FTIR can also be used to determine the chemical This chapter of the book describes the application of FTIR in the hydrogen bonds analysis, determination of structures and chemical compositions, and the morphology characterization 1. Introduction for natural fibres. ## Fourier Transform Infrared Spectroscopy for Natural Fibres Mizi Fan1,2, Dasong Dai1,2 and Biao Huang2 1Department of Civil Engineering, Brunel University, London, UB8 3PH, 2School of Material and Engineering, Fujian Agricultural and Forestry University, 1UK 2P. R. China ## 1. Introduction 44 Fourier Transform – Materials Analysis Rodrigues, J. A., A. M. Taylor, et al. (2007). Mass Spectrometry of Carbohydrates: Newer Aspects, Ryhage, R. (2002). Use of a mass spectrometer as a detector and analyzer for effluent emerging from Sekioka, T., M. Terasawa, et al. (1991). Ion storage in Kingdon trap. Radiation Effects and Sommer, H., H. A. Thomas, et al. (1951). The measurement of e/M by cyclotron resonance. Thomson, J. J. (1907). On rays of positive electricity. Philosophical Magazine Series 6 13 (77): Vartanian, V. H., J. S. Anderson, et al. (1995). Advances in trapped ion cells for Fourier transform ion cyclotron resonance mass spectrometry. Mass Spectrometry Reviews 14 (1): 1-19. Voyksner, R. D. (1997). Combining Liquid Chromatography with Electrospray Mass Spectrometry, Watson, J. T. and K. Biemann (2002). Direct recording of high resolution mass spectra of gas Whitehouse, C. M., R. N. Dreyer, et al. (2002). Electrospray interface for liquid chromatographs Wolff, M. M. and W. E. Stephens (1953). A pulsed mass spectrometer with time dispersion. Zhang, L.-K., D. Rempel, et al. (2005). Accurate mass measurements by Fourier transform mass In Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Thomson, J. J. (1897). Cathode rays. Philosophical Magazine Series 5 44 (269): 293-316. Applications. R. B. Cole. New York, John Wiley & Sons: 323-342. chromatographic effluents. Analytical Chemistry 37 (7): 844-851. and mass spectrometers. Analytical Chemistry 57 (3): 675-679. spectrometry. Mass Spectrometry Reviews 24 (2): 286-309. Review of Scientific Instruments 24 (8): 616-617. Academic Press. Volume 61: 59-141. Defects in Solids 117 (1-3): 253-259. Physical Review 82 (5): 697-702. 764. 561-575. In Advances in Carbohydrate Chemistry and Biochemistry. D. Horton. London, high temperature gas liquid chromatography columns. Analytical Chemistry 36 (4): 759- Infrared spectroscopy is nowadays one of the most important analytical techniques available to scientists. One of the greatest advantages of the infrared spectroscopy is that virtually any sample in any state may be analyzed. For example, liquids, solutions, pastes, powders, films, fibres, gases and surfaces can all be examined with a judicious choice of sampling technique. The review by Annette, Sudhakar, Ursula and Andrea [1-2] also demonstrates the applicability of dispersion infrared spectroscopy for natural fibres studies. Fourier transform infrared spectroscopy (FTIR) has facilitated many different IR sampling techniques, including attenuated total reflection and diffuses reflectance infrared Fourier transform (DRIFT) spectroscopy. It has dramatically improved the quality of infrared spectra and minimized the time required to obtain data. The increased speed and higher ratio of signal-to-noise of FTIR relative to dispersion infrared has lead to a substantially greater number of applications of infrared in natural fibres research. In addition, the constant advancing of computer and computing science has made infrared spectroscopy techniques striding further: The availability of a dedicated computer, which is required for the FTIR instrumentation, has allowed the digitized spectra to be treated by sophisticated data processing techniques and increased the utility of the infrared spectra for qualitative and quantitative purposes. With interferometric techniques, the infrared spectroscopy is being launched into a new era and interest in this technique is at an all time high. Cellulose, which acts as the reinforcing material in the cell wall, is the main constitute in natural fibres. The cellulose molecules are laid down in microfibrils in which there is extensive hydrogen bonding between cellulose chains, producing a strong crystalline structure. Much work has been published on the characterization of the hydrogen bonds in cellulose by using various techniques, among which FTIR has proved to be one of the most useful methods [3-6]. Furthermore, FTIR can provide researchers with further information on the super-molecular structure. FTIR can also be used to determine the chemical compositions of native natural fibres and the modified natural fibres. This chapter of the book describes the application of FTIR in the hydrogen bonds analysis, determination of structures and chemical compositions, and the morphology characterization for natural fibres. Fourier Transform Infrared Spectroscopy for Natural Fibres 47 Fig. 2. Synchronous 2D plot-cross-correlation of in- and out-of-phase spectra [3] Fig. 3. Asynchronous 2D plot-cross-correlation of in- and out-of-phase spectra [3] FTIR is very useful for examining the variation of hydrogen-bonds due to various defects [32]. The Nano-cellulose and Bio-composite Research Centre at Brunel University has investigated dislocations in natural fibres (hemp fibres) by using hydrogen-bonding characteristics under FTIR procedure. The test pieces were made from dislocation cluster (region) with the size of a single dislocation from a few microns to 100μm. The test pieces were then processed and examined by using FTIR measurement by using a Perkin-Elmer spectrometer and the standard KBr pellet technique. The recorded FTIR spectra (e.g. 3660– 3000cm-1) were deconvolved using Peak Fit V.4.12 software (Figure 4) and the peak positions of the major IR bands can be summarized and compared (Table 1). It can be found that the absorbance of hemp fibres without dislocations in the X–H (O–H and C–H) stretching region is much higher than that with dislocations. The peak positions of the four bands for hemp fibres with and without dislocations are 3450cm-1, 3346cm-1, 3262cm-1 and 3161cm-1 for the hemp without dislocations, and 3451cm-1, 3350cm-1, 3264cm-1 and 3167cm-1 for the dislocation regions. These bands are related to the valence vibration of hydrogen ## 2. Hydrogen bonds analysis of natural celluloses by using FTIR A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. Cellulose occurs in the form of long, slender chains, polymer of 1-4 linked β-D-glucose (Figure 1). Hydroxyl groups in C2, C3 and C6 contribute to the formation of various kinds of inter- and intra-molecular hydrogen bonds. The formation of inter- and intra- molecular hydrogen bonds in the cellulose not only has a strong influence on the physical properties of cellulose, including solubility [7-8], hydroxyl reactivity [9-10] and crystallinity [11-12], but also plays an important role in the mechanical properties of the cellulose [13]. Calculated by Tashiro and Kobayashi [14] showed that hydrogen bonds contribute about 20% the strain energy to the cellulose. It is apparent that the investigation of hydrogen bonds on cellulosic fibres and other materials gives rise to great benefits for the research on all other aspects of natural fibres and related materials. Fig. 1. Chemical structure of cellulose X-ray diffraction has been a powerful tool [15-19] to investigate hydrogen bonds visualization, lengths and angles. FTIR is even a more advanced tool to study hydrogen bonds in cellulose. IR was firstly used to investigate hydrogen bonds in cellulose in the 1950s [e.g. 20-22] and then the whole area of OH stretching wave-number in IR spectra for cellulose I and cellulose II [23-24]. The OH stretching region always covers 3-4 sub-peaks and these sub-peaks cannot be determined in the original data set. Some mathematical methods (e.g. deconvolution [25-27] and second-derivative [28-30]) were used to identify the exact peak for hydrogen bonds. Hinterstoisser and Salmén [3, 31] recently used DMA-FTIR to investigate OH stretching vibration regions between 3700 and 3000 cm−1 in the cellulose. In their experiments, cellulose sheets were stretched sinusoidally at low strains while being irradiated with polarized infrared light. For the obtained dynamic IR signals (the in-phase and the out-of-phase responses of the sample), the dynamic IR cross-correlation can be defined. The responses of the OH-groups to an external perturbation can be recorded as in-phase and out-of phase spectra. The cross correlation of these spectra gave the 2D synchronous (Figure 2) and asynchronous (Figure 3) plots, clearly showing the separated bands in the OH-vibration range and the relation of the OH-groups among them. It is apparent that most of the researchers have focused on the establishment of cellulose structure by investigating hydrogen bonds with FTIR. These (the structure of nature fibres) will be discussed in the next section. Few reports have described the correlation of hydrogen bonds with other characteristics of cellulose by using FTIR technologies. A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. Cellulose occurs in the form of long, slender chains, polymer of 1-4 linked β-D-glucose (Figure 1). Hydroxyl groups in C2, C3 and C6 contribute to the formation of various kinds of inter- and intra-molecular hydrogen bonds. The formation of inter- and intra- molecular hydrogen bonds in the cellulose not only has a strong influence on the physical properties of cellulose, including solubility [7-8], hydroxyl reactivity [9-10] and crystallinity [11-12], but also plays an important role in the mechanical properties of the cellulose [13]. Calculated by Tashiro and Kobayashi [14] showed that hydrogen bonds contribute about 20% the strain energy to the cellulose. It is apparent that the investigation of hydrogen bonds on cellulosic fibres and other materials gives rise to great benefits for the X-ray diffraction has been a powerful tool [15-19] to investigate hydrogen bonds visualization, lengths and angles. FTIR is even a more advanced tool to study hydrogen bonds in cellulose. IR was firstly used to investigate hydrogen bonds in cellulose in the 1950s [e.g. 20-22] and then the whole area of OH stretching wave-number in IR spectra for cellulose I and cellulose II [23-24]. The OH stretching region always covers 3-4 sub-peaks and these sub-peaks cannot be determined in the original data set. Some mathematical methods (e.g. deconvolution [25-27] and second-derivative [28-30]) were used to identify the exact peak for hydrogen bonds. Hinterstoisser and Salmén [3, 31] recently used DMA-FTIR to investigate OH stretching vibration regions between 3700 and 3000 cm−1 in the cellulose. In their experiments, cellulose sheets were stretched sinusoidally at low strains while being irradiated with polarized infrared light. For the obtained dynamic IR signals (the in-phase and the out-of-phase responses of the sample), the dynamic IR cross-correlation can be defined. The responses of the OH-groups to an external perturbation can be recorded as in-phase and out-of phase spectra. The cross correlation of these spectra gave the 2D synchronous (Figure 2) and asynchronous (Figure 3) plots, clearly showing the separated bands in the OH-vibration range and the relation of the OH-groups among them. It is apparent that most of the researchers have focused on the establishment of cellulose structure by investigating hydrogen bonds with FTIR. These (the structure of nature fibres) will be discussed in the next section. Few reports have described the correlation of hydrogen bonds with other characteristics of cellulose by using FTIR technologies. 2. Hydrogen bonds analysis of natural celluloses by using FTIR research on all other aspects of natural fibres and related materials. Fig. 1. Chemical structure of cellulose Fig. 2. Synchronous 2D plot-cross-correlation of in- and out-of-phase spectra [3] Fig. 3. Asynchronous 2D plot-cross-correlation of in- and out-of-phase spectra [3] FTIR is very useful for examining the variation of hydrogen-bonds due to various defects [32]. The Nano-cellulose and Bio-composite Research Centre at Brunel University has investigated dislocations in natural fibres (hemp fibres) by using hydrogen-bonding characteristics under FTIR procedure. The test pieces were made from dislocation cluster (region) with the size of a single dislocation from a few microns to 100μm. The test pieces were then processed and examined by using FTIR measurement by using a Perkin-Elmer spectrometer and the standard KBr pellet technique. The recorded FTIR spectra (e.g. 3660– 3000cm-1) were deconvolved using Peak Fit V.4.12 software (Figure 4) and the peak positions of the major IR bands can be summarized and compared (Table 1). It can be found that the absorbance of hemp fibres without dislocations in the X–H (O–H and C–H) stretching region is much higher than that with dislocations. The peak positions of the four bands for hemp fibres with and without dislocations are 3450cm-1, 3346cm-1, 3262cm-1 and 3161cm-1 for the hemp without dislocations, and 3451cm-1, 3350cm-1, 3264cm-1 and 3167cm-1 for the dislocation regions. These bands are related to the valence vibration of hydrogen Fourier Transform Infrared Spectroscopy for Natural Fibres 49 Absorbance 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 3600 3500 3400 3300 3200 3100 3000 Wavenumber (cm-1) <sup>4</sup> <sup>3</sup> <sup>2</sup> 1 3600 3500 3400 3300 3200 3100 3000 3. Structure of natural fibres determined by using FTIR (a) (b) Fig. 4. Deconvolved FTIR spectra of the υOH region of hemp without dislocation (a) and dislocation regions (b). (Solid curves=calculated data; dotted curves=experimental data) [32] It can be seen that the wave-numbers of peak position of dislocations are higher than those of hemp fibre without dislocation. This indicates that the degree of hydrogen bonding in dislocation regions is lesser than that in without dislocation regions. Furthermore, the absorbance of these bands in the dislocation regions is much lower than that in the regions without dislocations: for dislocation regions being about 79.3% for band 1, 64.4% for band 2, 64.9% for band 3 and 75.7% for band 4 those without dislocations respectively. These mean that the number of hydrogen bonds in dislocations is lower than without dislocation regions The structure of cellulose has a remarkable and complex influence on the course of chemical reactions of the polymer (cellulosic materials). Generally, the structure of cellulose consists of three structural levels: namely (i) the molecular level of the single macromolecule; (ii) the supramolecular level of packing and mutual ordering of the macromolecules; (iii) the morphological level concerning the architecture of already rather complex structural entities, as well as the corresponding pore system [33]. This section only focuses on the molecular level and supramolecular level, and the morphological level will be discussed in Molecular orientation is one of the most important parameters, affecting the physical properties of macromolecular systems. It is often introduced in natural macromolecules by the mechanical deformation incurred during their processing. By using FTIR equipped with a microscopic accessory, Kataoka and Kondo [34] determined the molecular orientation of cellulose during the formation of wood cell wall by virtue of the C-O-C stretching mode parallel to molecular chains [23] (Figure 5). It was found that the molecular orientation of cellulose in the primary cell wall coincided with the direction of enlarging cellular growth. It is therefore that the cellulose in the (nascent) primary cell wall might be oriented during Wavenumber (cm-1) <sup>4</sup> <sup>3</sup> <sup>2</sup> 1 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 according to Beer–Lambert law. the final section of this chapter. Absorbance bonded OH groups [26]: i.e. band 1 to the intra-molecular hydrogen bond of O(2)H---O, band 2 to the intra-molecular hydrogen bond of O(3)H---O, band 3 to the intermolecular hydrogen bond of O(6)H---O and band 4 to the O---H stretching respectively. Table 1. Bonds wavenumber related to regions without and with dislocations bonded OH groups [26]: i.e. band 1 to the intra-molecular hydrogen bond of O(2)H---O, band 2 to the intra-molecular hydrogen bond of O(3)H---O, band 3 to the intermolecular > 3327 3332 5 OH stretching 2883 2882 -1 C–H symmetrical 1724 1724 0 C=O stretching 1623 1624 1 OH bending of 1506 disappear - C=C aromatic 1368, 1363 1367,1363 -1/0 In-the-plane CH 1325 1325 0 S ring stretching 1314 1313 -1 CH2 rocking vibration 1259 1261 1 G ring stretching 1232 1231 -1 COH bending at C6 1152 1156 4 C-O-C asymmetrical 662 663 1 C-OH out-of-plane Δν(cm-1) Bonds stretching vibration bending at C6 absorbed water symmetrical stretching HCH and OCH inplane bending vibration C-C plus C-O plus C=O stretch; G condensed > G etherfied C-O-C symmetric stretching, OH plane deformation C-C, C-OH, C-H ring and side group vibrations C-C, C-OH, C-H ring and side group vibrations C-C, C-OH, C-H ring and side group vibrations COC,CCO and CCH deformation and stretching bending stretching hydrogen bond of O(6)H---O and band 4 to the O---H stretching respectively. Peak wavenumber (with dislocation) 1423 1423 0 1245 1244 -1 1204 1199 -5 1046 1043 -3 1020 1018 -2 994 996 2 895 894 -1 Table 1. Bonds wavenumber related to regions without and with dislocations (cm-1) Peak wavenumber (without dislocation) (cm-1) Fig. 4. Deconvolved FTIR spectra of the υOH region of hemp without dislocation (a) and dislocation regions (b). (Solid curves=calculated data; dotted curves=experimental data) [32] It can be seen that the wave-numbers of peak position of dislocations are higher than those of hemp fibre without dislocation. This indicates that the degree of hydrogen bonding in dislocation regions is lesser than that in without dislocation regions. Furthermore, the absorbance of these bands in the dislocation regions is much lower than that in the regions without dislocations: for dislocation regions being about 79.3% for band 1, 64.4% for band 2, 64.9% for band 3 and 75.7% for band 4 those without dislocations respectively. These mean that the number of hydrogen bonds in dislocations is lower than without dislocation regions according to Beer–Lambert law. ## 3. Structure of natural fibres determined by using FTIR The structure of cellulose has a remarkable and complex influence on the course of chemical reactions of the polymer (cellulosic materials). Generally, the structure of cellulose consists of three structural levels: namely (i) the molecular level of the single macromolecule; (ii) the supramolecular level of packing and mutual ordering of the macromolecules; (iii) the morphological level concerning the architecture of already rather complex structural entities, as well as the corresponding pore system [33]. This section only focuses on the molecular level and supramolecular level, and the morphological level will be discussed in the final section of this chapter. Molecular orientation is one of the most important parameters, affecting the physical properties of macromolecular systems. It is often introduced in natural macromolecules by the mechanical deformation incurred during their processing. By using FTIR equipped with a microscopic accessory, Kataoka and Kondo [34] determined the molecular orientation of cellulose during the formation of wood cell wall by virtue of the C-O-C stretching mode parallel to molecular chains [23] (Figure 5). It was found that the molecular orientation of cellulose in the primary cell wall coincided with the direction of enlarging cellular growth. It is therefore that the cellulose in the (nascent) primary cell wall might be oriented during Fourier Transform Infrared Spectroscopy for Natural Fibres 51 Fig. 6. Absorption spectra of the C–O–C vibration peak with increasing stress levels [35] Polarized FTIR accompanied with a vapor-phase deuteration has been used to characterize orientation of the main chains and hence to study the molecular orientation of Nematic Ordered Cellulose (NOC) [36]. A ratio (R) of the absorbance of the band due to the particular molecular moiety for radiation polarized perpendicular to to parallel to the stretching direction was introduced to evaluate the orientation behaviour of the main chains and OH groups. Computation of the FTIR spectra (e.g. Figure 7) shows that R values for the main chain are 0.32, and OH group 0.81 for Intramolecular and 0.91 for intermolecular H.B. Fig. 7. The bridge C–O–C (a) and OH (b) stretching band for the NOC film before deuteration [for (b) (A, B)=before, (C, D)=after, (//)=electric vector parallel to and (⊥)=perpendicular to It is apparent that: (1) the R value for the β-glucan main chains of cellulose molecules is not necessarily in agreement with that for the side chains of OH groups; (2) the uniaxial drawing process to prepare the NOC film gave rise to the oriented main chains toward the stretching direction; (3) the nonoriented OH groups in the noncrystalline regions which (a) (b) the stretching direction] crystallization and subsequent formation of microfibrils due to the drawing stress/effect exerted during cellular enlargement. This force, distributed along molecular chains, can cause β-glucose chains in the nascent cellulose to crystallize in the Iα phase with a higher crystallinity, making the molecules orientated in the enlarging direction. Fig. 5. Changes in FTIR spectra with a rotation of IR polarizer to the tracheid cell axis due to the C-O-C stretching mode: the primary (P) and the mature (P + S1 + S2 + S3) [34] In order to better understand wood and wood fibres for their potential utilization in advanced materials, some researchers have employed FTIR in conjunction with mechanical loading to study the molecular responses to the stress/load, such as for spruce wood and cellulose paper materials [35] (Figure 6), illustrating the shift of the absorption peak at 1,160 cm-1, C–O–C vibration when the materials successively loaded from 0 up to 24 MPa at 0% RH. The decrease of the shift of absorption peak as the stress increased can be observed (6 wavenumbers in Figure 6). This decrease in wavenumber signifies an increase in the length of the covalent bonds involved in the vibration absorption, i.e. a decrease in the force constant of the bond. This demonstrates that FTIR-spectroscopy may be used to monitor molecular straining of cellulosic material under load and the molecular deformation is linearly related to the macroscopic load of the material. Using FTIR technologies, it was found that spectral deformations occurred in cellulose related groups, but no molecular deformation detected for the lignin or hemicelluloses of wood constituents. The molecular straining of the cellulose molecule resulted in greater macroscopic force under moist conditions compared to dry conditions, but an equal macroscopic strain under both conditions. This may be interpreted that moisture accessible regions are arranged tending in parallel with the cellulose load bearing entities, suggesting that the cellulose disordered regions may not exist as large regions across the cellulose aggregate structure, rather that are spread out. In addition, the moisture absorbing area of the cellulose structure is probably related to the surface areas of the cellulose. crystallization and subsequent formation of microfibrils due to the drawing stress/effect exerted during cellular enlargement. This force, distributed along molecular chains, can cause β-glucose chains in the nascent cellulose to crystallize in the Iα phase with a higher > 90º to tracheid axis > > 0º 10º 20º 30º 40º 50º 60º 70º 80º 90º 0º 1180 1160 1140 Wavenumbers (cm )-1 Fig. 5. Changes in FTIR spectra with a rotation of IR polarizer to the tracheid cell axis due to In order to better understand wood and wood fibres for their potential utilization in advanced materials, some researchers have employed FTIR in conjunction with mechanical loading to study the molecular responses to the stress/load, such as for spruce wood and cellulose paper materials [35] (Figure 6), illustrating the shift of the absorption peak at 1,160 cm-1, C–O–C vibration when the materials successively loaded from 0 up to 24 MPa at 0% RH. The decrease of the shift of absorption peak as the stress increased can be observed (6 wavenumbers in Figure 6). This decrease in wavenumber signifies an increase in the length of the covalent bonds involved in the vibration absorption, i.e. a decrease in the force constant of the bond. This demonstrates that FTIR-spectroscopy may be used to monitor molecular straining of cellulosic material under load and the molecular deformation is linearly related to the macroscopic load of the material. Using FTIR technologies, it was found that spectral deformations occurred in cellulose related groups, but no molecular deformation detected for the lignin or hemicelluloses of wood constituents. The molecular straining of the cellulose molecule resulted in greater macroscopic force under moist conditions compared to dry conditions, but an equal macroscopic strain under both conditions. This may be interpreted that moisture accessible regions are arranged tending in parallel with the cellulose load bearing entities, suggesting that the cellulose disordered regions may not exist as large regions across the cellulose aggregate structure, rather that are spread out. In addition, the moisture absorbing area of the cellulose structure is the C-O-C stretching mode: the primary (P) and the mature (P + S1 + S2 + S3) [34] crystallinity, making the molecules orientated in the enlarging direction. P P+S +S +S 1 2 3 Absorbance probably related to the surface areas of the cellulose. Fig. 6. Absorption spectra of the C–O–C vibration peak with increasing stress levels [35] Polarized FTIR accompanied with a vapor-phase deuteration has been used to characterize orientation of the main chains and hence to study the molecular orientation of Nematic Ordered Cellulose (NOC) [36]. A ratio (R) of the absorbance of the band due to the particular molecular moiety for radiation polarized perpendicular to to parallel to the stretching direction was introduced to evaluate the orientation behaviour of the main chains and OH groups. Computation of the FTIR spectra (e.g. Figure 7) shows that R values for the main chain are 0.32, and OH group 0.81 for Intramolecular and 0.91 for intermolecular H.B. Fig. 7. The bridge C–O–C (a) and OH (b) stretching band for the NOC film before deuteration [for (b) (A, B)=before, (C, D)=after, (//)=electric vector parallel to and (⊥)=perpendicular to the stretching direction] It is apparent that: (1) the R value for the β-glucan main chains of cellulose molecules is not necessarily in agreement with that for the side chains of OH groups; (2) the uniaxial drawing process to prepare the NOC film gave rise to the oriented main chains toward the stretching direction; (3) the nonoriented OH groups in the noncrystalline regions which Fourier Transform Infrared Spectroscopy for Natural Fibres 53 bonds in cellulose II contains three intramolecular bonds: O(2)H---O(6) bonding, O(3)H--- O(5) bonding and O(2)H---O(2) bonding, and two intermolecular bonding: O(6)H---O(2) and O(6)H---O(3) (Figure 9b). The IR assignments for OH regions in cellulose I and II are O O O O O5 O1 O O Bonds 3315 OH Intra H-bond 3374 OH Intra H-bond 3486 OH Intra H-bond O O O O3, O5, O O O2, O O <sup>O</sup> <sup>O</sup> O O O O O O O O O O O O O O O O <sup>O</sup> <sup>O</sup> O3 O O6 O O O O2 O O O <sup>O</sup> <sup>O</sup> O O O O O O O O O4 <sup>O</sup> <sup>O</sup> O O O O Peak wavenumber (cellulose II) (cm-1) 3340-3375 O(3)H---O(5) 3405-3460 O(2)H---O(6) Fig. 9. Hydrogen-bonding network: (a) parallel to the bc plane (cellulose I ); (b) in the centre 3175 OH stretching 3230-3310 O(6)H---O(3) 3308 OH Inter H-bond 3309 OH Inter H-bond O O O O O O O O O <sup>O</sup> <sup>O</sup> (a) (b) summarized in Table 2. O O O O6 O2 O O O <sup>O</sup> <sup>O</sup> chains (Cellulose II) Peak wavenumber (cellulose I) (cm-1) O O O O O O O O O4 <sup>O</sup> <sup>O</sup> O O1 O O O O O <sup>O</sup> <sup>O</sup> Table 2. Correlation of bonds and celluloses (structure) [43], [44] O O O O6 O3, O O O O O5 O3 occupy more than 80% of the drawn film samples could be the key for discouragement of the crystallization. Supramolecular level investigated with FTIR mainly focuses on the crystal structure, which includes: 1) hydrogen bonding, 2) crystallinity measurement and 3) cellulose Iα and I<sup>β</sup> determination. Kondo, Togawa and Brown [37] proposed a concept to describe how various states of molecular association can be categorized in cellulose. Figure 8 demonstrates the schematic representation of their concept. Fig. 8. Concept of glucan chain association for cellulose According to two-phase model theory [38], there exist two regions in cellulose chain, namely amorphous and crystalline regions. Crystalline region in cellulose is an idealistic assembly of cellulose molecules in the biological system. There exist four different crystalline forms in cellulose. Researchers have developed various techniques to characterize the crystalline structure of cellulose, e.g. XRD, FTIR, Raman spectroscopy, and 13C CP/MAS NMR. Among them FTIR is a more advanced tool for investigating the structure of cellulose. As mentioned above, since 1950s, some important work had been carried out by researchers and there are a number of literatures reporting on the IR/FTIR data of natural fibres [39]. The hydrogen bonds in cellulose mainly distribute in crystal domains and amorphous domains. It is possible to establish relation between the OH-bands and the cellulose structure. In 1913, Nishikawa and Ono [40] firstly revealed the crystalline nature of cellulose with X-ray diffraction. Cellulose has four polymorphic crystalline structures from cellulose I to cellulose IV. However, cellulose I and cellulose II have been most extensively studied. The other crystalline structures are still in question and yet to be studied further. According to Gardner-Blackwell model [41], hydrogen bonds for cellulose I include two intramolecular bonding, namely, O(2)H---O(6) bonding and O(3)H---O(5) bonding and one intermolecular bonding, O(6)H---O(3) (Figure 9a). Based on the Kolpak-Blackwell model [42], hydrogen occupy more than 80% of the drawn film samples could be the key for discouragement of Supramolecular level investigated with FTIR mainly focuses on the crystal structure, which includes: 1) hydrogen bonding, 2) crystallinity measurement and 3) cellulose Iα and I<sup>β</sup> determination. Kondo, Togawa and Brown [37] proposed a concept to describe how various states of molecular association can be categorized in cellulose. Figure 8 demonstrates the > crystalline • Crystalline lattice • Molecular packing (Energy minimization) Non-crystalline For example NOC, Liquid crystals Ordered domain Non-ordered domain (Amorphous; no preferred orientation) For example Random gels, amorphous solids According to two-phase model theory [38], there exist two regions in cellulose chain, namely amorphous and crystalline regions. Crystalline region in cellulose is an idealistic assembly of cellulose molecules in the biological system. There exist four different crystalline forms in cellulose. Researchers have developed various techniques to characterize the crystalline structure of cellulose, e.g. XRD, FTIR, Raman spectroscopy, and 13C CP/MAS NMR. Among them FTIR is a more advanced tool for investigating the structure of cellulose. As mentioned above, since 1950s, some important work had been carried out by researchers and there are The hydrogen bonds in cellulose mainly distribute in crystal domains and amorphous domains. It is possible to establish relation between the OH-bands and the cellulose structure. In 1913, Nishikawa and Ono [40] firstly revealed the crystalline nature of cellulose with X-ray diffraction. Cellulose has four polymorphic crystalline structures from cellulose I to cellulose IV. However, cellulose I and cellulose II have been most extensively studied. The other crystalline structures are still in question and yet to be studied further. According to Gardner-Blackwell model [41], hydrogen bonds for cellulose I include two intramolecular bonding, namely, O(2)H---O(6) bonding and O(3)H---O(5) bonding and one intermolecular bonding, O(6)H---O(3) (Figure 9a). Based on the Kolpak-Blackwell model [42], hydrogen a number of literatures reporting on the IR/FTIR data of natural fibres [39]. For example Cellulose I-IV the crystallization. schematic representation of their concept. Fig. 8. Concept of glucan chain association for cellulose bonds in cellulose II contains three intramolecular bonds: O(2)H---O(6) bonding, O(3)H--- O(5) bonding and O(2)H---O(2) bonding, and two intermolecular bonding: O(6)H---O(2) and O(6)H---O(3) (Figure 9b). The IR assignments for OH regions in cellulose I and II are summarized in Table 2. Fig. 9. Hydrogen-bonding network: (a) parallel to the bc plane (cellulose I ); (b) in the centre chains (Cellulose II) Table 2. Correlation of bonds and celluloses (structure) [43], [44] Fourier Transform Infrared Spectroscopy for Natural Fibres 55 Compositional variation and physical organization at the microscopic level determine the ability to perform a desired function for most materials. Lignocellulosic fibres from different lignocellulosic materials appear quite different, but the chemical composition is fairly similar although with different magnitudes of constituents. The major compositions of lignocellulosic fibres are cellulose, hemicellulose and lignin (see Figure 10), while the minor constituents include minerals, pectin, waxes and water-soluble components. The application of infrared spectroscopy in lignocellulosic fibres has a long history: The infrared spectroscopy was used to investigate the hydroxyl groups of cellulose in the 1930's [53] and significant efforts were made in the 1950's to assign the different absorption maxima in the IR spectrum of cellulose [54-59]; The absorption maxima in the IR spectra of lignin were investigated from 1940's [60-61] through 1950's [62-64]; The characteristic absorption 4. Chemical composition of natural fibres by using FTIR maxima of hemicellulose were studied during the 50's [65-66]. Fig. 10. IR spectra of cellulose, hemicellulose and lignin of natural fibres [60] takes place on the surface of cellulose fibres. FTIR has been commonly used to characterize natural fibres with various treatments, e.g. grafting [67-68], coupling [69-71], mercerization [72-74]. With the aid of FTIR, researchers are able to obtain much more in-depth information of natural fibres after various modifications. FTIR is also an efficacy technique for the surface and interface characterizations of lignocellulosic fibres [75]. This allows further interpretation of the nature of adhesion between lignocellulosic with other substances. For example, Felix and Gatenholm [76] modified the lignocellulosic fibers with polypropylene–maleic anhydride copolymer. The spectrum of untreated fibres from the spectrum of treated fibres showed two peaks: one located at 1739 cm-1 and one at 1746 cm-1, and the FTIR analysis indicated that the reaction between fibres and copolymer can be divided into two main steps: the copolymer is firstly converted into the more reactive anhydride form and then esterification Hatakeyama and his coworkers firstly studied the hydrogen bond in the amorphous regions of cellulose. These studies focus on investigating the effect of temperature on the formation of interchain hydrogen bonds [45], and the effect of hydrogen bonds on the temperature dependence of the dynamic modulus and the mechanical loss tangent [46]. In 1996, Kondo and Sawatari systematically examined the formation of hydrogen bonds in amorphous cellulose. The substituted amorphous cellulose derivatives, 6-O-, 2,3-di-O-, and tri-O-substituted methylcellulose, were used to model the components of amorphous cellulose. An artificial spectrum for amorphous cellulose was then quantitatively constructed by using compound IR spectra in order to investigate hydrogen bond formation in cellulose. The typical absorption wavenumber for the real and artificial spectra were summarized in Table 3. Table 3. Absorption wavenumber between the real and synthesized IR spectra of amorphous cellulose [43, 47] The traditional two-phase cellulose model describes cellulose chains as containing both crystalline (ordered) and amorphous (less ordered) regions. A parameter termed the crystallinity index (CI) has been used to describe the relative amount of crystalline material in cellulose. The CI of celluloses has been measured using several different techniques including XRD, solid-state 13C NMR, infrared (IR) spectroscopy and Raman spectroscopy. The determination of CI using FTIR spectroscopy is the simplest method. It should be noted that this method gives only relative values, because the spectrum always contains contributions from both crystalline and amorphous regions. In 1958, O'Connor [49] proposed Lateral Order Index (LOI, A1420/A893) to calculate the CI for cellulose. Later, Nelson and O'Connor [49-50] introduced Total Crystallinity Index (TCI, A1375/A2900) to evaluate the CI of cellulose. The absorbance ratio A1420/A893 was defined as an empirical CI. The absorbance at 1420 and 894 cm-1 are sensitive to the amount of crystalline versus amorphous structure in the cellulose, that is, broadening of these bands reflects more disordered structure. As for TCI, various reports seem not to show a coherent result [51-52]. Hatakeyama and his coworkers firstly studied the hydrogen bond in the amorphous regions of cellulose. These studies focus on investigating the effect of temperature on the formation of interchain hydrogen bonds [45], and the effect of hydrogen bonds on the temperature dependence of the dynamic modulus and the mechanical loss tangent [46]. In 1996, Kondo and Sawatari systematically examined the formation of hydrogen bonds in amorphous cellulose. The substituted amorphous cellulose derivatives, 6-O-, 2,3-di-O-, and tri-O-substituted methylcellulose, were used to model the components of amorphous cellulose. An artificial spectrum for amorphous cellulose was then quantitatively constructed by using compound IR spectra in order to investigate hydrogen bond formation in cellulose. The typical absorption wavenumber for the real and artificial 669 671 W OH out-of-phase bending 899 892 M Nonsymmetric out-phase 1070 1075 S Skeletal vibrations C-O 1108 1108 S Nonsymmetric in-phase ring 1159 1154 S Nonsymmtric bridge C-O-C 1420 1425 W CH2 symmetric bending Table 3. Absorption wavenumber between the real and synthesized IR spectra of amorphous The traditional two-phase cellulose model describes cellulose chains as containing both crystalline (ordered) and amorphous (less ordered) regions. A parameter termed the crystallinity index (CI) has been used to describe the relative amount of crystalline material in cellulose. The CI of celluloses has been measured using several different techniques including XRD, solid-state 13C NMR, infrared (IR) spectroscopy and Raman spectroscopy. The determination of CI using FTIR spectroscopy is the simplest method. It should be noted that this method gives only relative values, because the spectrum always contains contributions from both crystalline and amorphous regions. In 1958, O'Connor [49] proposed Lateral Order Index (LOI, A1420/A893) to calculate the CI for cellulose. Later, Nelson and O'Connor [49-50] introduced Total Crystallinity Index (TCI, A1375/A2900) to evaluate the CI of cellulose. The absorbance ratio A1420/A893 was defined as an empirical CI. The absorbance at 1420 and 894 cm-1 are sensitive to the amount of crystalline versus amorphous structure in the cellulose, that is, broadening of these bands reflects more disordered structure. As for TCI, various reports seem not to show a coherent result Absorbance Bond stretching ring spectra were summarized in Table 3. Peak wavenumber (artificial) (cm-1) 1040 1040 S C-O 2892 2903 M CH 3420 3457 S OH 1374 1375 M CH bending Peak wavenumber (real) (cm-1) cellulose [43, 47] [51-52]. ## 4. Chemical composition of natural fibres by using FTIR Compositional variation and physical organization at the microscopic level determine the ability to perform a desired function for most materials. Lignocellulosic fibres from different lignocellulosic materials appear quite different, but the chemical composition is fairly similar although with different magnitudes of constituents. The major compositions of lignocellulosic fibres are cellulose, hemicellulose and lignin (see Figure 10), while the minor constituents include minerals, pectin, waxes and water-soluble components. The application of infrared spectroscopy in lignocellulosic fibres has a long history: The infrared spectroscopy was used to investigate the hydroxyl groups of cellulose in the 1930's [53] and significant efforts were made in the 1950's to assign the different absorption maxima in the IR spectrum of cellulose [54-59]; The absorption maxima in the IR spectra of lignin were investigated from 1940's [60-61] through 1950's [62-64]; The characteristic absorption maxima of hemicellulose were studied during the 50's [65-66]. Fig. 10. IR spectra of cellulose, hemicellulose and lignin of natural fibres [60] FTIR has been commonly used to characterize natural fibres with various treatments, e.g. grafting [67-68], coupling [69-71], mercerization [72-74]. With the aid of FTIR, researchers are able to obtain much more in-depth information of natural fibres after various modifications. FTIR is also an efficacy technique for the surface and interface characterizations of lignocellulosic fibres [75]. This allows further interpretation of the nature of adhesion between lignocellulosic with other substances. For example, Felix and Gatenholm [76] modified the lignocellulosic fibers with polypropylene–maleic anhydride copolymer. The spectrum of untreated fibres from the spectrum of treated fibres showed two peaks: one located at 1739 cm-1 and one at 1746 cm-1, and the FTIR analysis indicated that the reaction between fibres and copolymer can be divided into two main steps: the copolymer is firstly converted into the more reactive anhydride form and then esterification takes place on the surface of cellulose fibres. Fourier Transform Infrared Spectroscopy for Natural Fibres 57 The S ring (CH2 rocking at C6 in cellulose) and G ring stretching (C–C plus C–O plus C O stretch and COH bending at C6 in cellulose) could normally be observed in bands at 1325, 1314, 1259, 1245 and 1232cm-1 respectively for the hemp fibres without dislocation. Due to the overlapping of bands, only two peaks can be seen in Figure 11b. Lignin is composed of three basic units, namely p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) [78]. Guaiacyl (G) and syringyl (S) are the main units of lignin, but the ratio of S/G varies from one to another plant. It was reported recently by del Río et al. [79] that S/G values calculated upon FTIR were in agreement with those calculated upon Py-GC/MS at the bands of 1271cm-1 and 1327cm-1 respectively. However, the study on hemp fibre showed that the bands at 1271cm-1 and 1327cm-1, assigned as G-ring stretching and S ring stretching respectively, were shifted to lower wavenumbers: for the hemp fibres without dislocations (Figure 12a), the G ring and S ring stretching appear at the bands of 1259cm-1 and 1325cm-1 and for the (a) (b) Fig. 13. FTIR spectra of various types of composites dislocation regions at 1261cm-1 and 1325cm-1 (Figure 12b). FTIR has recently been found most promising to examine the change of the chemical compositions of natural fibres (hemp fibres) due to inherent defects. An example of the results is given in Figure 11. A scrutiny of the IR spectra from 1370cm-1 to 1330cm-1 shows that the band at 1368cm-1 and 1363cm-1 almost disappears in dislocation regions (Figure 11a). These two bands, assigned as the in-plane CH bending, may be from hemicelluloses or cellulose, the near disappearance of these may be due to the removal of the hemicelluloses in dislocation regions. Hemicelluloses can form a linkage between cellulose and lignin, and lignin-carbohydrate complex with lignin by ether bonds [77]. The removal of hemicelluloses in dislocation regions may cause the decrease of transfer of shear stress under tensile loading and loss of lignin as well. Fig. 11. FTIR spectra of hemp fibres from 1370 cm-1 to 1330 cm-1(a) and from 1330 cm-1 to 1215 cm-1 (b) with and without dislocation [32] Fig. 12. Deconvolved FTIR spectra without dislocation (a) and dislocation regions (b). (Solid curves=calculated data; dotted curves=experimental data) [32] FTIR has recently been found most promising to examine the change of the chemical compositions of natural fibres (hemp fibres) due to inherent defects. An example of the results is given in Figure 11. A scrutiny of the IR spectra from 1370cm-1 to 1330cm-1 shows that the band at 1368cm-1 and 1363cm-1 almost disappears in dislocation regions (Figure 11a). These two bands, assigned as the in-plane CH bending, may be from hemicelluloses or cellulose, the near disappearance of these may be due to the removal of the hemicelluloses in dislocation regions. Hemicelluloses can form a linkage between cellulose and lignin, and lignin-carbohydrate complex with lignin by ether bonds [77]. The removal of hemicelluloses in dislocation regions may cause the decrease of transfer of shear stress under tensile > Hemp with dislocation (a) (b) 5 (a) (b) 4 Fig. 11. FTIR spectra of hemp fibres from 1370 cm-1 to 1330 cm-1(a) and from 1330 cm-1 to Fig. 12. Deconvolved FTIR spectra without dislocation (a) and dislocation regions (b). (Solid 0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 1 Absorbance 1317 1317 1320 1300 1280 1260 1240 1220 1340 1320 1300 1280 1260 1240 1220 Wavenumber (cm-1 ) 3 1247 1234 5 4 Wavenumber (cm-1) Hemp with dislocation 2 Hemp without dislocation loading and loss of lignin as well. 1363 2 1 1363 1367 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 Absrobance 1368 1370 1360 1350 1340 1330 Hemp without dislocation Wavenumber (cm-1) 1215 cm-1 (b) with and without dislocation [32] 1340 1320 1300 1280 1260 1240 1220 Wavenumber (cm-1 ) curves=calculated data; dotted curves=experimental data) [32] 3 The S ring (CH2 rocking at C6 in cellulose) and G ring stretching (C–C plus C–O plus C O stretch and COH bending at C6 in cellulose) could normally be observed in bands at 1325, 1314, 1259, 1245 and 1232cm-1 respectively for the hemp fibres without dislocation. Due to the overlapping of bands, only two peaks can be seen in Figure 11b. Lignin is composed of three basic units, namely p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) [78]. Guaiacyl (G) and syringyl (S) are the main units of lignin, but the ratio of S/G varies from one to another plant. It was reported recently by del Río et al. [79] that S/G values calculated upon FTIR were in agreement with those calculated upon Py-GC/MS at the bands of 1271cm-1 and 1327cm-1 respectively. However, the study on hemp fibre showed that the bands at 1271cm-1 and 1327cm-1, assigned as G-ring stretching and S ring stretching respectively, were shifted to lower wavenumbers: for the hemp fibres without dislocations (Figure 12a), the G ring and S ring stretching appear at the bands of 1259cm-1 and 1325cm-1 and for the dislocation regions at 1261cm-1 and 1325cm-1 (Figure 12b). Fig. 13. FTIR spectra of various types of composites Fourier Transform Infrared Spectroscopy for Natural Fibres 59 found to be a remarkable tool for biological and materials analysis. It can be used extensively to investigate the chemical composition of stem [83-85] and cell wall structure FTIR imaging in conjunction with pyrolysis molecular beam mass spectrometry (py- MBMS) can work as a rapid analysis tool to evaluate difference in the chemical composition, for example, from the bark to the pith of wood stern (Figure 15) [85], and the data can statistically be processed to establish the correlation of the change in chemical features and Fig. 15. (a) Visible image of the bark, cambium, and xylem of the control aspen stem. The area in the box was selected for FT-IR spectral analysis. (b) Spectral image of a portion of the outer bark [o], inner bark [I], cambium [c], xylem [x] showing the relative concentration of Fig. 16. PLS model predicting the distance from the bark to pith based on changes in the chemical composition. (Filled circles=calibration and open circles =test set) [85] [86] of natural fibres, and natural fibre composites [87]. the distance across the xylem (Figure 16). phenolic in these anatomical features [85] The different molar contents of G-lignin and S-lignin of the hemp with and without dislocations gave rise to the ratio of S/G 0.9 for the former and 1.1 for the latter fibres. The lignin network in the parts without dislocations would be more rigid than that in dislocation regions. The lower absorbance in dislocations means that the lignin was removed from dislocation regions, and such the cellulose content in dislocations would be higher than that without dislocations. FTIR can further be used to investigate the interfacial properties of natural fibre composites [80]. For example, Figures 13a and b exhibits the spectra for different types of composites containing 40% aspen fibres. The highest absorbance value corresponds to the untreated composites and the lowest value to that of composites modified with maleated polypropylene. The FTIR examination on the interface of wood fibre-reinforced polypropylene composites has also confirmed the efficacy of the technique [81]. The spectra are able to illustrate that the coupling agent was located around the wood fibers rather than randomly distributed in the polypropylene matrix, and the compatabilizer was attached to the wood fibers either by ester or hydrogen bonds. ## 5. Morphologies of natural fibres by using FTIR FTIR spectroscopic imaging is the complete synthesis of FTIR spectroscopy with sample visualization and greatly extends the capabilities of conventional FTIR spectroscopy. Figure 14 illustrates a general configuration of an FTIR imaging micro- spectrometer. Spectral data can be represented as a picture, showing chemical information simultaneously from thousands of pixels. The main advantages of FTIR imaging are noninvasiveness, fast data collection and the ability to create visually appealing display. FTIR imaging not only provides new scientific capabilities, but it is also a compact and informative way to present results. It can collect more than 10,000 spectra in a few minutes. FTIR imaging has been Fig. 14. Schematic of a typical FTIR imaging spectrometer [82] The different molar contents of G-lignin and S-lignin of the hemp with and without dislocations gave rise to the ratio of S/G 0.9 for the former and 1.1 for the latter fibres. The lignin network in the parts without dislocations would be more rigid than that in dislocation regions. The lower absorbance in dislocations means that the lignin was removed from dislocation regions, and such the cellulose content in dislocations would be higher than that FTIR can further be used to investigate the interfacial properties of natural fibre composites [80]. For example, Figures 13a and b exhibits the spectra for different types of composites containing 40% aspen fibres. The highest absorbance value corresponds to the untreated composites and the lowest value to that of composites modified with maleated polypropylene. The FTIR examination on the interface of wood fibre-reinforced polypropylene composites has also confirmed the efficacy of the technique [81]. The spectra are able to illustrate that the coupling agent was located around the wood fibers rather than randomly distributed in the polypropylene matrix, and the compatabilizer was attached to FTIR spectroscopic imaging is the complete synthesis of FTIR spectroscopy with sample visualization and greatly extends the capabilities of conventional FTIR spectroscopy. Figure 14 illustrates a general configuration of an FTIR imaging micro- spectrometer. Spectral data can be represented as a picture, showing chemical information simultaneously from thousands of pixels. The main advantages of FTIR imaging are noninvasiveness, fast data collection and the ability to create visually appealing display. FTIR imaging not only provides new scientific capabilities, but it is also a compact and informative way to present results. It can collect more than 10,000 spectra in a few minutes. FTIR imaging has been without dislocations. the wood fibers either by ester or hydrogen bonds. 5. Morphologies of natural fibres by using FTIR Fig. 14. Schematic of a typical FTIR imaging spectrometer [82] found to be a remarkable tool for biological and materials analysis. It can be used extensively to investigate the chemical composition of stem [83-85] and cell wall structure [86] of natural fibres, and natural fibre composites [87]. FTIR imaging in conjunction with pyrolysis molecular beam mass spectrometry (py- MBMS) can work as a rapid analysis tool to evaluate difference in the chemical composition, for example, from the bark to the pith of wood stern (Figure 15) [85], and the data can statistically be processed to establish the correlation of the change in chemical features and the distance across the xylem (Figure 16). Fig. 15. (a) Visible image of the bark, cambium, and xylem of the control aspen stem. The area in the box was selected for FT-IR spectral analysis. (b) Spectral image of a portion of the outer bark [o], inner bark [I], cambium [c], xylem [x] showing the relative concentration of phenolic in these anatomical features [85] Fig. 16. PLS model predicting the distance from the bark to pith based on changes in the chemical composition. (Filled circles=calibration and open circles =test set) [85] Fourier Transform Infrared Spectroscopy for Natural Fibres 61 2) hemicelluloses are arranged in parallel with the cellulose microfibrils and accordingly more or less in parallel with the longitudinal axis (the S2 layer of the cell wall) of fibres, 3) only a little degree of orientation can be observed for lignin and 4) the variation in the molecular orientation along the fibres seems to be uniform in the pore-free regions. These results gave rise to a conclusion that all of three main components within fibres may have a clear anisotropic behaviour under mechanical stress, that is, their properties will be different FTIR can be used to examine the structure of natural fibre based composites, such as, examining the surface distribution of polyacrylamide (PAM) or the in-plane distribution of Fig. 18. Average orientation spectra of the two W fibres (W1 and W2) and the two H fibres (H1 and H2): cellulose 1160 cm-1, 1316 cm-1, 1370 cm-1 and 1424 cm-1, glucomannan 810 cm-1, xylan FTIR offers scientists an excellent range of solutions for understanding natural fibres and their related modification technologies and products, such as chemical compositions, microstructures, fibre architectures, characterisation of interface, and properties of both 1734 cm-1, 1460 cm-1 and 1240 cm-1 and lignin 1508 cm-1 [86] 6. Conclusions natural fibres and related composites. in the longitudinal direction (along the fibre axis) and the transverse direction. cellulose within a paper sheet [87]. Fig. 17. Total IR absorbance full-spectral images of the two W fibres (W1 and W2) and the two H fibres (H1 and H2), showing the 25 pixel positions for each fibre used for evaluating the average orientation spectra as well as the three pixel positions for each fibre selected for evaluating the orientation of the different wood polymers in the fibres [86] FTIR spectroscopy imaging has also been used to examine the orientation of the main wood compositions in transverse and longitudinal directions of wood fibres. For example, the examination by using FTIR on spruce fibres (Figures 17 and 18) [86] is able to illustrate that 1) glucomannan and xylan show a predominant orientation in the S2 layer of cell wall, Fig. 17. Total IR absorbance full-spectral images of the two W fibres (W1 and W2) and the two H fibres (H1 and H2), showing the 25 pixel positions for each fibre used for evaluating the average orientation spectra as well as the three pixel positions for each fibre selected for FTIR spectroscopy imaging has also been used to examine the orientation of the main wood compositions in transverse and longitudinal directions of wood fibres. For example, the examination by using FTIR on spruce fibres (Figures 17 and 18) [86] is able to illustrate that 1) glucomannan and xylan show a predominant orientation in the S2 layer of cell wall, evaluating the orientation of the different wood polymers in the fibres [86] 2) hemicelluloses are arranged in parallel with the cellulose microfibrils and accordingly more or less in parallel with the longitudinal axis (the S2 layer of the cell wall) of fibres, 3) only a little degree of orientation can be observed for lignin and 4) the variation in the molecular orientation along the fibres seems to be uniform in the pore-free regions. These results gave rise to a conclusion that all of three main components within fibres may have a clear anisotropic behaviour under mechanical stress, that is, their properties will be different in the longitudinal direction (along the fibre axis) and the transverse direction. FTIR can be used to examine the structure of natural fibre based composites, such as, examining the surface distribution of polyacrylamide (PAM) or the in-plane distribution of cellulose within a paper sheet [87]. Fig. 18. Average orientation spectra of the two W fibres (W1 and W2) and the two H fibres (H1 and H2): cellulose 1160 cm-1, 1316 cm-1, 1370 cm-1 and 1424 cm-1, glucomannan 810 cm-1, xylan 1734 cm-1, 1460 cm-1 and 1240 cm-1 and lignin 1508 cm-1 [86] ### 6. Conclusions FTIR offers scientists an excellent range of solutions for understanding natural fibres and their related modification technologies and products, such as chemical compositions, microstructures, fibre architectures, characterisation of interface, and properties of both natural fibres and related composites. Fourier Transform Infrared Spectroscopy for Natural Fibres 63 [9] Kondo, T. (1997a). The relationship between intramolecular hydrogen bonds and certain [10] Kondo, T. (1997b). The relationship between intramolecular hydrogen bonds and [11] Itagaki, H., Tokai, M. & Kondo, T. (1997). Physical gelation process for cellulose whose [12] Kondo, T., Sawatari, C., Manley, R. S. J. & Gray, D. G. (1994). Characterization of [13] Kondo, T. & Sawatari, C. (1994). Intermolecular hydrogen bonding in cellulose/ [14] O'Sullivan, A. (1997). Cellulose: the structure slowly unravels. Cellulose, Vol.4, No.3, [15] Tashiro, K. & Kobayashi, M. (1991). Theoretical evaluation of three-dimensional elastic [16] Gardner, K. H. & Blackwell, J. (1974). The hydrogen bonding in native cellulose. [17] Stipanovic, A. J. & Sarko, A. (1976). Packing Analysis of Carbohydrates and [18] Woodcock, C. & Sarko, A. (1980). Packing Analysis of Carbohydrates and Polysaccharides. [19] Langan, P., Nishiyama, Y. & Chanzy, H. (1999). A Revised Structure and Hydrogen- [21] Marrinan, H. J. & Mann, J. (1954). A study by infra-red spectroscopy of hydrogen [22] Marrinan, H. J. & Mann, J. (1956). Infrared spectra of the crystalline modifications of cellulose. Journal of Polymer Science, Vol.21, No.98, pp. 301-311, ISSN 1542-6238. Macromolecules, Vol.9, No.5, pp. 851-857, ISSN 0024-9297. Vol.38, No.16, pp. 4201-4205, ISSN 0032-3861. Vol.32, No.8, pp. 1516-1526, ISSN 0032-3861. Vol.13, No.5, pp. 1183-1187, ISSN 0024-9297. 0488. 1099-0488. 210-215, ISSN 0024-9297. 4423-4428, ISSN 0032-3861. pp. 173-207, ISSN 0969-0239. 14300-14306, ISSN 0002-7863. 1934-998X. ISSN 0304-4165. physical properties of regioselectively substituted cellulose derivatives. Journal of Polymer Science Part B: Polymer Physics, Vol.35, No.4, pp. 717-723, ISSN 1099- certain physical properties of regioselectively substituted cellulose derivatives. Journal of Polymer Science Part B: Polymer Physics, Vol.35, No.4, pp. 717-723, ISSN hydroxyl groups are regioselectively substituted by fluorescent groups. Polymer, hydrogen bonding in cellulose-synthetic polymer blend systems with regioselectively substituted methylcellulose. Macromolecules, Vol.27, No.1, pp. poly(ethylene oxide) blends: thermodynamic examination using 2,3-di-O- and 6-O-methylcelluloses as cellulose model compounds. Polymer, Vol.35, No.20, pp. constants of native and regenerated celluloses: role of hydrogen bonds. Polymer, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.343, No.1, pp. 232-237, Polysaccharides. 6. Molecular and Crystal Structure of Regenerated Cellulose II. 11. Molecular and Crystal Structure of Native Ramie Cellulose. Macromolecules, Bonding System in Cellulose II from a Neutron Fiber Diffraction Analysis. Journal of the American Chemical Society, Vol.121, No.43, pp. 9940-9946, ISSN 0002-7863. [20] Nishiyama, Y., Sugiyama, J., Chanzy, H. & Langan, P. (2003). Crystal Structure and Hydrogen Bonding System in Cellulose Iα from Synchrotron X-ray and Neutron Fiber Diffraction. Journal of the American Chemical Society, Vol.125, No.47, pp. bonding in cellulose. Journal of Applied Chemistry, Vol.4, No.4, pp. 204-211, ISSN FTIR is a powerful technique to examine the formation of inter- and intra- molecular hydrogen bonds in cellulose. The detailed database allows the establishment of strong correlation between the nature of hydrogen bonds and physical (e.g. solubility, hydroxyl reactivity, crystallinity) and mechanical properties of cellulose. The capability of accurate examination of hydrogen bonds has lead to an ever increasing uses of FTIR for investigating the defects (e.g. dislocation of hemp fibre) or deterioration (e.g. perturbation) of natural fibres and change of materials after modification. The structure of cellulose has a profound influence on the course of chemical reactions of cellulose materials and the resulted properties. The molecular orientation and crystallization and formation of microfibrils not only vary from one plant to another, but could also change due to various environmental or other physical effects. FTIR is able to examine the nature of molecular chains, crystallinity and their correlations with various bonds. In commons with other materials, the chemical composition at microscopic level determines the ability to perform various functions for the usefulness of natural fibres. FTIR has been mostly successful in accurate analysis of both major (cellulose, hemicellulose and lignin) and minor (mineral, pectin, waxes) constituents of natural fibres. Change in chemical compositions, interface and hence properties of natural fibres and composites could also be effectively identified by using FTIR. FTIR is the most interesting and versatile of all analytical techniques and are well placed to become the technology of the century. ### 7. References FTIR is a powerful technique to examine the formation of inter- and intra- molecular hydrogen bonds in cellulose. The detailed database allows the establishment of strong correlation between the nature of hydrogen bonds and physical (e.g. solubility, hydroxyl reactivity, crystallinity) and mechanical properties of cellulose. The capability of accurate examination of hydrogen bonds has lead to an ever increasing uses of FTIR for investigating the defects (e.g. dislocation of hemp fibre) or deterioration (e.g. perturbation) of natural The structure of cellulose has a profound influence on the course of chemical reactions of cellulose materials and the resulted properties. The molecular orientation and crystallization and formation of microfibrils not only vary from one plant to another, but could also change due to various environmental or other physical effects. FTIR is able to examine the nature of In commons with other materials, the chemical composition at microscopic level determines the ability to perform various functions for the usefulness of natural fibres. FTIR has been mostly successful in accurate analysis of both major (cellulose, hemicellulose and lignin) and minor (mineral, pectin, waxes) constituents of natural fibres. Change in chemical compositions, interface and hence properties of natural fibres and composites could also be FTIR is the most interesting and versatile of all analytical techniques and are well placed to [1] Urban, M. W. (1993). Fourier Transform Infrared and Fourier Transform Raman [3] Hinterstoisser, B. & Salmén, L. (2000). Application of dynamic 2D FTIR to cellulose. Vibrational Spectroscopy, Vol.22, No.1-2, pp. 111-118, ISSN 0924-2031. [4] Kokot, S., Czarnik-Matusewicz, B. & Ozaki, Y. (2002). Two-dimensional correlation [7] Kondo, T. (1994). Hydrogen bonds in regioselectively substituted cellulose derivatives. [8] Kondo, T. (1997). The assignment of IR absorption bands due to free hydroxyl groups in cellulose. Cellulose, Vol.4, No.4, pp. 281-292, ISSN 0969-0239. 0841225257, the University of Virginia, American Chemical Society. [2] Annette, N., Sudhakar, P., Ursula, K. & Andrea, P. Fourier Transform Infrared Spectroscopy of Polymers, In: Urban, M. W. & Craver, C. D. (Ed.),3-40, ISBN Microscopy in Wood Analysis. In: Ursula Kües(Ed.) Wood production, wood technology, and biotechnological impacts. Universitätsverlag Göttingen, 2007. 179- spectroscopy and principal component analysis studies of temperature-dependent IR spectra of cotton–cellulose. Journal, Vol.67, No.6, pp. 456-469, ISSN 1097-0282. [5] Guo, Y. & Wu, P. (2008). Investigation of the hydrogen-bond structure of cellulose diacetate by two-dimensional infrared correlation spectroscopy. Biopolymers, Journal of Polymer Science Part B: Polymer Physics, Vol.32, No.7, pp. 1229-1236, molecular chains, crystallinity and their correlations with various bonds. fibres and change of materials after modification. effectively identified by using FTIR. become the technology of the century. ISBN 978-3-940344-11-3. Vol.74, No.3, pp. 509-513, ISSN 0144-8617. [6] http://en.wikipedia.org/wiki/Hydrogen\_bond ISSN 1099-0488. 7. References Fourier Transform Infrared Spectroscopy for Natural Fibres 65 [40] Salmén, L., Åkerholm, M. (2005). Two-Dimensional Fourier Transform Infrared [41] Nishikawa, S. & Ono, S. (1913). Transmission of X-rays through fibrous, lamellar and [42] Sugiyama, J., Vuong, R. & Chanzy, H. (1991). Electron diffraction study on the two [43] Kolpak, F. J. & Blackwell, J. (1976). Determination of the Structure of Cellulose II. [44] Kondo, T. (2005). Hydrogen Bonds in Cellulose and Cellulose Derivatives. 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Modification of cellulosic fibres with functionalised silanes: development of surface properties. International Journal of Adhesion and Adhesives, Vol.24, No.1, and natural fiber composites. Composites Part A: Applied Science and properties of hemp, sisal, jute and kapok for composite reinforcement. Die Angewandte Makromolekulare Chemie, Vol.272, No.1, pp. 108-116, ISSN 1522-9505 and kapok fibers by alkalization. Journal of Applied Polymer Science, Vol.84, modified wood fibers using FTIR spectroscopy for biocomposites. Journal of characterization of interfaces and fiber surfaces in lignocellulosic fiber-reinforced composites. Composite Interfaces, Vol.12, No.1/2, pp. 95-124, ISSN 0927-6440 [77] Felix, J. M. & Gatenholm, P. (1991). The nature of adhesion in composites of modified cellulose fibers and polypropylene. Journal of Applied Polymer Science, Vol.42, prepared from cellulose in NaOH/thiourea aqueous solution. 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Russian Chemical Bulletin, Vol.7, No.2, pp. 241-243, ISSN 1066-5285. [59] Klein, E. & Snowden, J. (1958). Replacing Hydroxyl Groups in Cotton Cellulose. Industrial & Engineering Chemistry, Vol.50, No.1, pp. 80-82, ISSN 0019-7866. [60] Liang, C. Y. & Marchessault, R. H. (1959). Infrared spectra of crystalline polysaccharides. Science, Vol.39, No.135, pp. 269-278, ISSN 1542-6238. Standards, Vol.45, No.(2, pp. 109-113. ISSN 0002-7863. 690-693, ISSN 0002-7863. 2409-2412, ISSN 0002-7863. Vol.44, No.12, pp. 4074-4081, ISSN 0014-3057 4042 ISSN 1612-8869 infrared transmission measurements. Journal of Research of the National Bureau of Absence of Free Aldehyde Groups in Periodate-oxidized Cellulose1. Journal of the Investigation of some oxidation reactions of cellulose by infrared spectroscopy. II. Native celluloses in the region from 640 to 1700 cm−1. Journal of Polymer Lignin. Journal of the American Chemical Society, Vol.71, No.4, pp. 1297-1299, Isolation and Characterization of Bagasse Native Lignin. Journal of the American on Hardwood Lignin. Journal of the American Chemical Society, Vol.73, No.2, pp. Compounds. I. Characteristic Carbonyl and Hydroxyl Frequencies of Some Flavanones, Flavones, Chalcones and Acetophenones1. Journal of the American Hemicellulose1,2. Journal of the American Chemical Society, Vol.81, No.10, pp. saccharum). Canadian Journal of Chemistry, Vol.37, No.5, pp. 893-898, ISSN 0008- Polymerisation of Functional Acrylic Monomers onto Cotton Fibres Activated by Continuous Ar Plasma. Plasma Processes and Polymers, Vol.3, No.1, pp. 48-57, polycaprolactone by heterogeneous click-chemistry. European Polymer Journal, [61] Jones, E. J. (1948). The infrared spectrum of spruce native lignin. Journal of the American Chemical Society, Vol.70, No.5, pp. 1984-1985, ISSN 0002-7863. [62] Buchanan, M. A., Brauns, F. E. & Leaf, R. L. (1949). Native Lignin. II. Native Aspen [63] Stevens, G. D. & Nord, F. F. (1951). Investigations on Lignin and Lignification. VIII.1 [64] Kudzin, S. F. & Nord, F. F. (1951). Investigations on Lignin and Lignification. IV. Studies [65] Hergert, H. L. & Kurth, E. F. (1953). The Infrared Spectra of Lignin and Related [66] Srivastava, H. C. & Adams, G. A. (1959). Uronic Acid Components of Jute Fiber [67] Timell, T. E. (1959). The constitution of a hemicellulose ffrom sugar maple (acer [68] Castelvetro, V., Fatarella, E., Corsi, L., Giaiacopi, S. & Ciardelli, G. (2006). Graft [69] Krouit, M., Bras, J. & Belgacem, M. N. (2008). Cellulose surface grafting with Chemical Society, Vol.73, No.10, pp. 4622-4625, ISSN 0002-7863. Chemical Society, Vol.75, No.7, pp. 1622-1625, ISSN 0002-7863. 4 Fourier Transform Infrared Spectroscopy for Our knowledge of the process of global change in Earth's atmosphere is primarily based on experimental observations made in situ or by remote-sensing. The data obtained reveal the state of the atmosphere and provide input data for validation of theoretical models of the atmosphere. In this context, high resolution molecular spectroscopy plays a key role as it is at the heart of optical remote sensing measurements. In fact, infrared spectroscopy is a powerful tool to identify and quantify atmospheric trace species, through the use of characteristic spectral signatures of the different molecular species and their associated vibration-rotation bands in the mid- or near-infrared. Different methods, based on quantitative spectroscopy, permit tropospheric or stratospheric measurements: in situ long path absorption, atmospheric absorption/emission by Fourier transform spectroscopy with high spectral resolution instruments on the ground, airborne, balloon-borne or satelliteborne measurements (Camy-Peyret et al., 2001). In all cases, the quality of the analysis and interpretation of atmospheric spectra requires reference spectroscopic information (positions, intensities, broadenings, profiles…) measured in the laboratory. Such information exists and is compiled in databases such as HITRAN, GEISA and ATMOS, Fourier Transform Infrared (FTIR) can be utilized to measure some components of an unknown mixture and is currently applied to the analysis of solids, liquids, and gases. The term FTIR refers to the manner in which the data is collected and converted from an Fourier transform spectrometers have progressively replaced dispersive instruments for most applications due to their superior speed and sensitivity. They have greatly extended 1. Introduction available to the international scientific community. interference pattern to a spectrum. the Measurement of Spectral Line Profiles Hassen Aroui1, Johannes Orphal2 and Fridolin Kwabia Tchana3 Université de Tunis, Ecole Supérieure des Sciences et Techniques de Tunis, 1Laboratoire de Dynamique Moléculaire et Matériaux Photoniques, 3Laboratoire Interuniversitaire des Systèmes Atmosphériques (LISA), UMR CNRS 7583 Universités Paris Est Créteil et Paris- Diderot, 2Institute for Meteorology and Climate Research (IMK), Karlsruhe Institute of Technology (KIT), Institut Pierre Simon Laplace, 1Tunisia 2Germany 3France stems. Journal of the Science of Food and Agriculture, Vol.82, No.7, pp. 685-696, ISSN 1097-0010 ## Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles Hassen Aroui1, Johannes Orphal2 and Fridolin Kwabia Tchana3 1Laboratoire de Dynamique Moléculaire et Matériaux Photoniques, Université de Tunis, Ecole Supérieure des Sciences et Techniques de Tunis, 2Institute for Meteorology and Climate Research (IMK), Karlsruhe Institute of Technology (KIT), 3Laboratoire Interuniversitaire des Systèmes Atmosphériques (LISA), UMR CNRS 7583 Universités Paris Est Créteil et Paris- Diderot, Institut Pierre Simon Laplace, 1Tunisia 2Germany 3France ## 1. Introduction 68 Fourier Transform – Materials Analysis [85] Himmelsbach, D.S.; Khalili, S. & Akin, D.E. (1998) FT-IR microspectroscopic imaging of [86] Labbé, N., Rials, T. G., Kelley, S. S., Cheng, Z.-M., Kim, J.-Y. & Li, Y. (2005). FT-IR [87] Stevanic, J. S. & Salmén, L. (2009). Orientation of the wood polymers in the cell wall of spruce wood fibres. Holzforschung, Vol.63, No.5, pp. 497-503, ISSN 0018-3830 [88] Sakaemura, T., Mihara, I. & Yamauchi, T. (2009). Microscopic Attenuated Total ISSN 1097-0010 ISSN OO37-9875 7719 pp.99-108, ISSN 0270-7306 stems. Journal of the Science of Food and Agriculture, Vol.82, No.7, pp. 685-696, flax (Linum usitatissimum L.) stems. Molecular and Cellular Biology, Vol. 44, No.1, imaging and pyrolysis-molecular beam mass spectrometry: new tools to investigate wood tissues. Wood Science and Technology, Vol.39, No.1, pp. 61-76, ISSN 0043- Reflection/Fourier Transform Infrared Imaging of Paper Containing a Polyacrylamide Dry Strength Resin. Sen'i Gakkaishi, Vol.65, No.9, pp. 252-255, > Our knowledge of the process of global change in Earth's atmosphere is primarily based on experimental observations made in situ or by remote-sensing. The data obtained reveal the state of the atmosphere and provide input data for validation of theoretical models of the atmosphere. In this context, high resolution molecular spectroscopy plays a key role as it is at the heart of optical remote sensing measurements. In fact, infrared spectroscopy is a powerful tool to identify and quantify atmospheric trace species, through the use of characteristic spectral signatures of the different molecular species and their associated vibration-rotation bands in the mid- or near-infrared. Different methods, based on quantitative spectroscopy, permit tropospheric or stratospheric measurements: in situ long path absorption, atmospheric absorption/emission by Fourier transform spectroscopy with high spectral resolution instruments on the ground, airborne, balloon-borne or satelliteborne measurements (Camy-Peyret et al., 2001). In all cases, the quality of the analysis and interpretation of atmospheric spectra requires reference spectroscopic information (positions, intensities, broadenings, profiles…) measured in the laboratory. Such information exists and is compiled in databases such as HITRAN, GEISA and ATMOS, available to the international scientific community. > Fourier Transform Infrared (FTIR) can be utilized to measure some components of an unknown mixture and is currently applied to the analysis of solids, liquids, and gases. The term FTIR refers to the manner in which the data is collected and converted from an interference pattern to a spectrum. > Fourier transform spectrometers have progressively replaced dispersive instruments for most applications due to their superior speed and sensitivity. They have greatly extended Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 71 Excluding the Introduction and the Conclusion, this chapter is divided into three sections. The FTIR spectroscopy technique is described in detail in section 2. In particular, the model used to describe the instrument line shape function of the Fourier transform spectrometer is presented. Measurement of line profiles using Fourier transform spectroscopy is presented in section 3. In this section, the measured broadening and shift coefficients, as well as line intensities and line-mixing parameters are presented and discussed. The rotational dependence of the pressure broadening coefficients is fitted using empirical polynomial Infrared spectroscopy is the absorption measurement of different IR frequencies by a sample located in the path of an IR beam. Using various sampling accessories, the spectrometers can The basic principle for the evaluation in IR spectroscopy is the Beer-Lambert law which was defined in 1852. This law relates the absorption of light to the properties of the material through which the light is traveling. Absorption spectroscopy consists of the application of this law which illustrates that when traversing a measurement cell, the light intensity where α(σ) (in cm-1) is the absorption coefficient of the material, σ is the wavenumber (in cm-1), A is the absorption path length (in cm). I0(σ) and I(σ) are the intensity of a collimated 0 Eq. (1) can be expressed as a function of the absorption cross section σe(σ) (in cm-2 N is the particle density (in molecule cm-3). At standard conditions of pressure (P = 1 atm) and the temperature (T = 273.15 K), N is equal to the Loschmidt number1 (Auwera, 2004): nL = 2.6867661 (47) x 1019 molecule cm-3 atm-1. Computed from the value of the molar gas constant R available in February 2003 on the website of the light beam (in Wm-2) respectively before and after absorption by the sample. The transmission spectrum T(σ) is the ratio of I(σ) by I0(σ): Physics Laboratory of the National Institute of Science and Technology I=I (0 σ)exp -{ α(σ)A} , (1) I(σ) T(σ) = I (σ) . (2) I=I (0 e ν)exp -{ σ (σ)NA} . (3) equations to provide empirical interpolation and extrapolation models. accept a wide range of sample types such as gases, liquids, and solids. 2. Fourier transform spectroscopy 2.1 Quantitative absorption spectroscopy decreases exponentially: molecule-1). 1 (http://physics.nist.gov/). the capabilities of infrared spectroscopy and have been applied to many fields that are very difficult or nearly impossible to study by dispersive instruments. Instead of analyzing each spectral component sequentially, as in a dispersive IR spectrometer, all frequencies are examined simultaneously in FTIR spectroscopy. By interpreting the infrared absorption spectrum, information about the structure and the nature of the chemical bonds in a molecule can be determined. For most common materials, the spectrum of an unknown mixture can be identified by comparison to a library of known compounds. The FTIR technique is a powerful tool for identification of chemical species and their structures, for interpretation of the atmospheric spectra by producing an infrared absorption spectrum that is like a molecular fingerprint. This technique can be used to identify chemicals from spills, paints, polymers, coatings, drugs, and contaminants. It can measure more than 120 gaseous pollutants in the ambient air (U.S. Environmental Protection Agency, 2011), such as carbon monoxide, sulfur dioxide, ozone, and many others. This technique can also quantify toxic organic pollutants, such as toluene, benzene, methanol etc. This quantification is based on the fact that every gas has its own specific spectrum. The FTIR sensor monitors the entire infrared spectrum and reads the different fingerprints of the gases present in the atmosphere. FTIR spectroscopy is also extensively used in the study of absorption line profiles to determine spectroscopic parameters related to line broadening. This chapter describes laboratory measurements of line parameters for atmospheric trace species, using Fourier transform spectroscopy in the infrared spectral range. An illustration is given with the example of atmospheric molecules such as NH3, OCS and CH3Br, for which we have determined line intensities, self- and foreign-gas broadening coefficients, pressure-induced line shifts, as well as line-mixing coefficients for these molecules for more than 300 rovibrational lines located in the spectral range 1000-3000 cm-1, at room and low temperatures. A non-linear least-squares multi-spectrum fitting procedure, including Doppler, and linemixing effects, has been used to retrieve line parameters from more than twenty experimental spectra, recorded at different pressures of the molecules. The accuracies of the results are also estimated and discussed as a function of the measured parameter. The results, obtained for several branches of the molecules considered here, will be presented and analyzed as a function of gas pressure, temperature, rotational quantum numbers and vibrational bands. From the intensity measurements, we have determined effective transition dipole moments, vibrational band strengths as well as Herman–Wallis parameters for some bands taking into account Coriolis and l-type interactions. For the NH3 molecule, the results concerning line-mixing demonstrate a large amount of coupling between the symmetric and asymmetric components of inversion doublets. The pressure shift coefficients and line-mixing parameters are both positive and negative. Some groups of lines illustrate a correlation between line-mixing and line shift phenomena, demonstrated by a nonlinear pressure dependence of the line position. Excluding the Introduction and the Conclusion, this chapter is divided into three sections. The FTIR spectroscopy technique is described in detail in section 2. In particular, the model used to describe the instrument line shape function of the Fourier transform spectrometer is presented. Measurement of line profiles using Fourier transform spectroscopy is presented in section 3. In this section, the measured broadening and shift coefficients, as well as line intensities and line-mixing parameters are presented and discussed. The rotational dependence of the pressure broadening coefficients is fitted using empirical polynomial equations to provide empirical interpolation and extrapolation models. ## 2. Fourier transform spectroscopy 70 Fourier Transform – Materials Analysis the capabilities of infrared spectroscopy and have been applied to many fields that are very difficult or nearly impossible to study by dispersive instruments. Instead of analyzing each spectral component sequentially, as in a dispersive IR spectrometer, all frequencies are By interpreting the infrared absorption spectrum, information about the structure and the nature of the chemical bonds in a molecule can be determined. For most common materials, the spectrum of an unknown mixture can be identified by comparison to a library of known The FTIR technique is a powerful tool for identification of chemical species and their structures, for interpretation of the atmospheric spectra by producing an infrared absorption spectrum that is like a molecular fingerprint. This technique can be used to identify chemicals from spills, paints, polymers, coatings, drugs, and contaminants. It can measure more than 120 gaseous pollutants in the ambient air (U.S. Environmental Protection Agency, 2011), such as carbon monoxide, sulfur dioxide, ozone, and many others. This technique can also quantify toxic organic pollutants, such as toluene, benzene, methanol etc. This quantification is based on the fact that every gas has its own specific spectrum. The FTIR sensor monitors the entire infrared spectrum and reads the different fingerprints of the FTIR spectroscopy is also extensively used in the study of absorption line profiles to This chapter describes laboratory measurements of line parameters for atmospheric trace species, using Fourier transform spectroscopy in the infrared spectral range. An illustration is given with the example of atmospheric molecules such as NH3, OCS and CH3Br, for which we have determined line intensities, self- and foreign-gas broadening coefficients, pressure-induced line shifts, as well as line-mixing coefficients for these molecules for more than 300 rovibrational lines located in the spectral range 1000-3000 cm-1, at room and low A non-linear least-squares multi-spectrum fitting procedure, including Doppler, and linemixing effects, has been used to retrieve line parameters from more than twenty experimental spectra, recorded at different pressures of the molecules. The accuracies of the The results, obtained for several branches of the molecules considered here, will be presented and analyzed as a function of gas pressure, temperature, rotational quantum From the intensity measurements, we have determined effective transition dipole moments, vibrational band strengths as well as Herman–Wallis parameters for some bands taking into For the NH3 molecule, the results concerning line-mixing demonstrate a large amount of coupling between the symmetric and asymmetric components of inversion doublets. The pressure shift coefficients and line-mixing parameters are both positive and negative. Some groups of lines illustrate a correlation between line-mixing and line shift phenomena, results are also estimated and discussed as a function of the measured parameter. demonstrated by a nonlinear pressure dependence of the line position. determine spectroscopic parameters related to line broadening. examined simultaneously in FTIR spectroscopy. compounds. temperatures. gases present in the atmosphere. numbers and vibrational bands. account Coriolis and l-type interactions. #### 2.1 Quantitative absorption spectroscopy Infrared spectroscopy is the absorption measurement of different IR frequencies by a sample located in the path of an IR beam. Using various sampling accessories, the spectrometers can accept a wide range of sample types such as gases, liquids, and solids. The basic principle for the evaluation in IR spectroscopy is the Beer-Lambert law which was defined in 1852. This law relates the absorption of light to the properties of the material through which the light is traveling. Absorption spectroscopy consists of the application of this law which illustrates that when traversing a measurement cell, the light intensity decreases exponentially: $$\mathbf{I} = \mathbf{I}\_0(\mathbf{o}) \exp\left\{-\mathbf{a}(\mathbf{o})\boldsymbol{\ell}\right\},\tag{1}$$ where α(σ) (in cm-1) is the absorption coefficient of the material, σ is the wavenumber (in cm-1), A is the absorption path length (in cm). I0(σ) and I(σ) are the intensity of a collimated light beam (in Wm-2) respectively before and after absorption by the sample. The transmission spectrum T(σ) is the ratio of I(σ) by I0(σ): $$\mathbf{T(o)} = \frac{\mathbf{I(o)}}{\mathbf{I\_0(o)}}.\tag{2}$$ Eq. (1) can be expressed as a function of the absorption cross section σe(σ) (in cm-2 molecule-1). $$\mathbf{I} = \mathbf{I}\_0(\mathbf{v}) \exp\left\{-\mathbf{o}\_e(\mathbf{o})\mathbf{N}\ell\right\}.\tag{3}$$ N is the particle density (in molecule cm-3). At standard conditions of pressure (P = 1 atm) and the temperature (T = 273.15 K), N is equal to the Loschmidt number1 (Auwera, 2004): nL = 2.6867661 (47) x 1019 molecule cm-3 atm-1. <sup>1</sup> Computed from the value of the molar gas constant R available in February 2003 on the website of the Physics Laboratory of the National Institute of Science and Technology (http://physics.nist.gov/). Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 73 spectrometer generates the quantity I'(δ) which is the cosine Fourier transform of the incident light beam of spectral irradiance B(σ) over a range of optical path differences δ defined by the positions of the moving mirror with respect to the fixed mirror (Bell, 1972): I'(δ) = B(σ) 1+ cos(2πσδ) d<sup>σ</sup> <sup>∞</sup> I(δ) = B(σ)cos(2πσδ)d<sup>σ</sup> <sup>∞</sup> The real quantity I(δ) is called the interferogram. An example of such interferogram is shown in Fig. 2 for an infrared source, recorded from –δmax to +δmax, where δmax is the The spectral distribution (spectrum) can be recorded by computation of the Fourier B(σ) = I(δ)exp -i2πσδ <sup>d</sup><sup>δ</sup> +∞ This equation requires that the interferogram is known from δmax=-∞ up to δmax=∞. Since B(σ) = 2 E(δ)cos(2πσδ)d<sup>δ</sup> - 2i O(δ)sin(2πσδ)d<sup>δ</sup> ∞ ∞ This equation shows that the observed spectrum B(σ) is real and even, if the interferogram is an even function. Only the side of the interferogram corresponding to δ ≥ 0 is recorded; the When the spectral analysis takes into account experimental parameters such as the truncation of the interferogram, the finite size of the source, phase errors, optical misalignments, etc., an instrumental line shape function, FApp(σ), is introduced, and the The symbol ⊗ means the convolution of B(σ) with FApp(σ). In the following, we discuss the { } 1 1 I(δ) = I(δ)+ I(-δ) + I(δ) - I(-δ) = E(δ)+ O(δ) 2 2 ⎡ ⎤⎡ ⎤ ⎣ ⎦⎣ ⎦ . (7) ∫ ∫ . (8) B(σ App ) = B(σ)F ( ⊗ σ) (9) ∫ . (6) ⎡ ⎤ ∫ ⎣ ⎦ . (4) ∫ . (5) 0 0 I(δ) can be split into the sum of even and odd functions (Brassewell, 1965): ∞ 0 0 spectrum is then computed using the cosine Fourier transformation only. In FTIR spectroscopy, only the modulated part is retained: maximum optical path difference achieved. transform of I(δ): Eq. (6) can be written as: 2.3.2 Instrumental line shape function recorded spectrum can be written as: effect of FApp(σ) on the recorded spectra. ## 2.2 Fourier transform spectroscopy ## 2.2.1 Introduction Since the invention of the first spectrophotometers in the beginning of 20th century, a rapid technological progress has occurred. The first-generation spectrometers were all dispersive with a prism or grating as dispersive elements. In the mid 1960s infrared spectroscopy has seen the advent of the Fourier transform spectrometer. These second-generation infrared spectrometers have significant advantages compared to dispersive spectrometers. ## 2.2.2 General description The basic experimental set up of the Fourier transform spectrometer is constituted by the Michelson interferometer. As shown in Fig. 1, light from a source (S) is collimated and then divided at a beam splitter (L) into two beams of equal amplitude. These beams are reflected back on themselves by two separate mirrors, one fixed (M2) and the other movable (M1). Each single beam strikes the beam splitter again, where they are recombined and directed to the detector (D). The two components in the recombined beam interfere with each other and form a spot whose intensity depends upon the different paths traversed by the two beams before recombination. As one mirror moves, the path length of one beam changes and the spot on the detector becomes brighter and dimmer successively, in synchronization with the mirror position. It should be noted that the same types of radiation sources can be used for both dispersive and Fourier transform spectrometers. Fig. 1. Michelson interferometer showing three compartments: the radiation source (S), the detector (D), and the interferometer with its two mirrors M1 and M2 and its beam splitter L ## 2.3 Mathematic formulation ### 2.3.1 The interferogram expression Fourier transform spectroscopy principles are extensively described in the literature (Bell, 1972; Davis et al., 2001; Griffiths & Haseth, 1986). Basically, a Fourier transform spectrometer generates the quantity I'(δ) which is the cosine Fourier transform of the incident light beam of spectral irradiance B(σ) over a range of optical path differences δ defined by the positions of the moving mirror with respect to the fixed mirror (Bell, 1972): 0 I'(δ) = B(σ) 1+ cos(2πσδ) d<sup>σ</sup> <sup>∞</sup> ⎡ ⎤ ∫ ⎣ ⎦ . (4) In FTIR spectroscopy, only the modulated part is retained: $$\mathbf{I}(\boldsymbol{\delta}) = \bigcap\_{\alpha} \mathbf{B}(\alpha) \cos(2\alpha \boldsymbol{\delta}) \mathrm{d}\alpha \,. \tag{5}$$ The real quantity I(δ) is called the interferogram. An example of such interferogram is shown in Fig. 2 for an infrared source, recorded from –δmax to +δmax, where δmax is the maximum optical path difference achieved. The spectral distribution (spectrum) can be recorded by computation of the Fourier transform of I(δ): $$\tilde{\mathbf{B}}(\mathbf{o}) = \bigcap\_{\ast \approx}^{+\infty} \mathbf{I}(\delta) \exp\left\{-i2\pi \mathbf{n} \mathbf{o} \mathbf{\delta}\right\} \mathbf{d} \mathbf{\delta} \tag{6}$$ This equation requires that the interferogram is known from δmax=-∞ up to δmax=∞. Since I(δ) can be split into the sum of even and odd functions (Brassewell, 1965): $$\mathbf{I}(\boldsymbol{\Theta}) = \frac{1}{2} \left[ \mathbf{I}(\boldsymbol{\Theta}) + \mathbf{I}(-\boldsymbol{\Theta}) \right] + \frac{1}{2} \left[ \mathbf{I}(\boldsymbol{\Theta}) \cdot \mathbf{I}(-\boldsymbol{\Theta}) \right] = \mathbf{E}(\boldsymbol{\Theta}) + \mathbf{O}(\boldsymbol{\Theta}) \,. \tag{7}$$ Eq. (6) can be written as: 72 Fourier Transform – Materials Analysis Since the invention of the first spectrophotometers in the beginning of 20th century, a rapid technological progress has occurred. The first-generation spectrometers were all dispersive with a prism or grating as dispersive elements. In the mid 1960s infrared spectroscopy has seen the advent of the Fourier transform spectrometer. These second-generation infrared The basic experimental set up of the Fourier transform spectrometer is constituted by the Michelson interferometer. As shown in Fig. 1, light from a source (S) is collimated and then divided at a beam splitter (L) into two beams of equal amplitude. These beams are reflected back on themselves by two separate mirrors, one fixed (M2) and the other movable (M1). Each single beam strikes the beam splitter again, where they are recombined and directed to the detector (D). The two components in the recombined beam interfere with each other and form a spot whose intensity depends upon the different paths traversed by the two beams before recombination. As one mirror moves, the path length of one beam changes and the spot on the detector becomes brighter and dimmer successively, in synchronization with the It should be noted that the same types of radiation sources can be used for both dispersive M1 e D Fig. 1. Michelson interferometer showing three compartments: the radiation source (S), the detector (D), and the interferometer with its two mirrors M1 and M2 and its beam splitter L Fourier transform spectroscopy principles are extensively described in the literature (Bell, 1972; Davis et al., 2001; Griffiths & Haseth, 1986). Basically, a Fourier transform L l M2 spectrometers have significant advantages compared to dispersive spectrometers. 2.2 Fourier transform spectroscopy 2.2.1 Introduction 2.2.2 General description mirror position. and Fourier transform spectrometers. S 2.3 Mathematic formulation 2.3.1 The interferogram expression l $$\tilde{\mathbf{B}}(\mathbf{o}) = \mathbf{2} \int\_0^\mathbf{\hat{E}} \mathbf{\hat{E}}(\mathbf{\hat{\delta}}) \cos(2\mathbf{n}\mathbf{o}\mathbf{\hat{\delta}}) \mathbf{d}\mathbf{\hat{\delta}} - 2\mathbf{i} \int\_0^\mathbf{\hat{O}} \mathbf{\hat{O}}(\mathbf{\hat{\delta}}) \sin(2\mathbf{n}\mathbf{o}\mathbf{\hat{\delta}}) \mathbf{d}\mathbf{\hat{\delta}}\,\tag{8}$$ This equation shows that the observed spectrum B(σ) is real and even, if the interferogram is an even function. Only the side of the interferogram corresponding to δ ≥ 0 is recorded; the spectrum is then computed using the cosine Fourier transformation only. #### 2.3.2 Instrumental line shape function When the spectral analysis takes into account experimental parameters such as the truncation of the interferogram, the finite size of the source, phase errors, optical misalignments, etc., an instrumental line shape function, FApp(σ), is introduced, and the recorded spectrum can be written as: $$\tilde{\mathbf{B}}(\mathbf{o}) = \mathbf{B}(\mathbf{o}) \otimes \mathbf{F}\_{\text{App}}(\mathbf{o}) \tag{9}$$ The symbol ⊗ means the convolution of B(σ) with FApp(σ). In the following, we discuss the effect of FApp(σ) on the recorded spectra. Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 75 where sinc(x)=sin(x)/x. The full width at half maximum (FWHM) of this function defines the theoretical maximum spectral resolution Rth that can be achieved with a Fourier The sinc(x) function exhibits large oscillations: the amplitude of the first lobe is equal to 22% of the maximum amplitude of the function. These oscillations can be rather annoying for the spectral analysis since they lead to distortion of spectral lines. They can be reduced or eliminated through multiplication of the interferogram I(δ) with a so-called apodization function which decrease with increasing optical path difference, thus reducing the A typical apodization function is a triangle function whose Fourier transform is the sinc2(2πδmaxσ) function. However, because Rth is inversely proportional to δmax, apodization has the disadvantage of decreasing the spectral resolution. Then the apodized maximum spectral resolution achievable by a Fourier transform spectrometer is equal to the full width max This definition of the resolution is used for Bruker FTIR spectrometers. For the Bomem For line intensity measurements, the major distortion of spectra may be due to the nonlinearities of the detector (MCT detectors are nonlinear). To remove these nonlinearities, some studies have been performed (Abrams et al., 1994; Auwera, 2004; Guelachvili, 1986). Abram et al. showed that the recorded interferogram can be described as power series of the true interferogram (Abrams et al., 1994). Later Auwera developed an algorithm based on This phenomenon is manifested when the detector is illuminated by a relatively intense source with large spectral coverage. This nonlinearity affects the interferograms near the zero optical path difference where the signal is the largest. Such behaviour affects Note that, using the sensitivity curve of the detector, one should be sure to remain permanently in the interval of linearity of detection by reducing the most possible the explored spectral region and by limiting the intensity of the source to avoid the effects of saturation of the detector. Since the true physical measured quantity is the interferogram, for broad band measurement in absorption spectroscopy, these effects will affect more strongly the center burst of the interferogramm, reducing artificially its amplitude where the signal is largest. Thus, one of the experimental signs signaling the presence of gross nonlinearity effects is the appearance, after Fourier Transform, of an artefactual signal below the detector or beam-splitter cut-off (see for instance the curve below of Figure 2, where no th max 1.2067 R = <sup>2</sup><sup>δ</sup> . (11) 1.79 R = <sup>2</sup>δ (12) th transform spectrometer (Bell, 1972): truncation effect of the interferogram at δmax. at half maximum of the sinc2(x) function. Abram's work to avoid nonlinearities (Auwera, 2004). instruments, R= 0.5/δmax is used. consequently the entire spectrum. signal should be present below 500 cm-1). 2.3.4 Detector non-linearity #### 2.3.3 Truncation of the interferogram Mathematically, the truncation of the interferogram recorded up to a finite maximum optical path difference δmax yields: F ( App σ max max )=2δ sinc(2πσδ ), (10) Fig. 2. Example of an interferogram and its spectrum calculated using a Fourier Transform algorithm where sinc(x)=sin(x)/x. The full width at half maximum (FWHM) of this function defines the theoretical maximum spectral resolution Rth that can be achieved with a Fourier transform spectrometer (Bell, 1972): $$\mathcal{R}\_{\text{th}} = \frac{1.2067}{2\mathcal{S}\_{\text{max}}}.\tag{11}$$ The sinc(x) function exhibits large oscillations: the amplitude of the first lobe is equal to 22% of the maximum amplitude of the function. These oscillations can be rather annoying for the spectral analysis since they lead to distortion of spectral lines. They can be reduced or eliminated through multiplication of the interferogram I(δ) with a so-called apodization function which decrease with increasing optical path difference, thus reducing the truncation effect of the interferogram at δmax. A typical apodization function is a triangle function whose Fourier transform is the sinc2(2πδmaxσ) function. However, because Rth is inversely proportional to δmax, apodization has the disadvantage of decreasing the spectral resolution. Then the apodized maximum spectral resolution achievable by a Fourier transform spectrometer is equal to the full width at half maximum of the sinc2(x) function. $$\mathcal{R}\_{\text{th}} = \frac{1.79}{2\mathcal{S}\_{\text{max}}} \tag{12}$$ This definition of the resolution is used for Bruker FTIR spectrometers. For the Bomem instruments, R= 0.5/δmax is used. #### 2.3.4 Detector non-linearity 74 Fourier Transform – Materials Analysis Mathematically, the truncation of the interferogram recorded up to a finite maximum F ( App σ max max )=2δ sinc(2πσδ ), (10) FT H O lines <sup>2</sup> δ 500 1000 1500 2000 2500 3000 Wavenumber σ in cm-1 Wavenumber in cm σ -1 500 1000 1500 2000 2500 3000 Fig. 2. Example of an interferogram and its spectrum calculated using a Fourier Transform 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500 Optical path difference Optical path difference δ H2 O lines 2.3.3 Truncation of the interferogram 0.0 0.0 algorithm 0.1 0.1 B(σ) σ B( ) 0.2 0.2 0.3 0.3 0.0 0.0 I(d) δ I( ) 0.2 0.2 0.4 0.4 0.6 0.6 optical path difference δmax yields: For line intensity measurements, the major distortion of spectra may be due to the nonlinearities of the detector (MCT detectors are nonlinear). To remove these nonlinearities, some studies have been performed (Abrams et al., 1994; Auwera, 2004; Guelachvili, 1986). Abram et al. showed that the recorded interferogram can be described as power series of the true interferogram (Abrams et al., 1994). Later Auwera developed an algorithm based on Abram's work to avoid nonlinearities (Auwera, 2004). This phenomenon is manifested when the detector is illuminated by a relatively intense source with large spectral coverage. This nonlinearity affects the interferograms near the zero optical path difference where the signal is the largest. Such behaviour affects consequently the entire spectrum. Note that, using the sensitivity curve of the detector, one should be sure to remain permanently in the interval of linearity of detection by reducing the most possible the explored spectral region and by limiting the intensity of the source to avoid the effects of saturation of the detector. Since the true physical measured quantity is the interferogram, for broad band measurement in absorption spectroscopy, these effects will affect more strongly the center burst of the interferogramm, reducing artificially its amplitude where the signal is largest. Thus, one of the experimental signs signaling the presence of gross nonlinearity effects is the appearance, after Fourier Transform, of an artefactual signal below the detector or beam-splitter cut-off (see for instance the curve below of Figure 2, where no signal should be present below 500 cm-1). Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 77 Generally, the original interferogram size should always be at least doubled by zero filling, i.e. zero filling factor (ZFF) of two is chosen. Zero-filling is an interpolation that does not affect the instrument line-shape, and in most cases, is therefore superior to polynomial or In Fourier transform spectroscopy interferograms are apodized prior to the transformation and the calculated spectrum is phase corrected. If the zero point for the Fourier transformation is displaced, a phase error occurs because the apodization remains fixed while the interferogram is shifted by the phase correction. To avoid distortions of the spectrum resulting from phase errors, double-sided interferograms are needed. In practice, single-sided interferograms are recorded double-sided on a small range of optical path difference, allowing determination of the phase error. In such a case, the phase error is determined at low resolution, typically about 1 cm-1 or less. This is not a problem because it Compared with the dispersive spectrometer, the FTIR instrument has several advantages: • Better speed and sensitivity: A complete spectrum is obtained during a single scan of the moving mirror, while the detector observes all frequencies simultaneously. This is • The so-called "Jaquinot advantage" consists of an increase of the optical throughput. A circular optical aperture is used in FTIR systems. The beam area of a Fourier transform instrument is usually about 100 times larger than the small slit width of a dispersive spectrometer. Thus, more radiation energy is made available. This constitutes a major • The so-called "Connes advantage" consists of the use of a helium neon laser as the internal reference. This source radiation provides an automatic and stable calibration for all wavelengths. This eliminates, to some extent, the need for external calibration • FTIR spectrometers are usually equipped with a powerful, computerized data system. It can perform a wide variety of data processing tasks such as Fourier transformation, interactive spectral subtraction, baseline correction, smoothing, integration, spectral line fits and library searching. Development of the fast Fourier transform algorithm (Cooley 3. Measurement of line profiles using Fourier transform infrared spectroscopy Analysis of line profiles are useful for atmosphere monitoring and studying the evolution of its composition including chemical trace species related to the reduction in stratospheric ozone, starting from the analysis of observations obtained by infrared spectroscopy. The retrieved spectroscopic parameters are indispensable for the interpretation of high-resolution infrared spectra of gaseous species, and are of increasing importance for qualitative and and Tukey, 1965) has facilitated these tasks and reduced the computation time. spline interpolation methods that are applied in the spectral domain. 2.3.8 Phase errors 2.4 FTIR advantages sources. 3.1 Introduction varies only slowly with wavenumber. called the "Felgett advantage". advantage for many samples that are energy-limited. #### 2.3.5 Extended size of the source The collimated beam inside the Michelson interferometer is produced using an extended source at the focus of a lens. This beam passes through an iris occulting part of the image of the source. Because of the finite size of this iris, the collimated beam is slightly divergent in the interferometer. When this effect is taken into account, the interferogram <sup>0</sup> I(δ) = Bcos(2πσ δ) of a monochromatic source at wavenumber σ0 becomes $$\mathbf{I(\delta) = B} \frac{\mathbf{Ind}^2}{4\mathbf{F}^2} \text{sinc}\left(\frac{\text{nd}^2 \mathbf{o}\_0 \boldsymbol{\delta}}{8\mathbf{F}^2}\right) \cos\left(2\text{mo}\_0 \boldsymbol{\delta} \left[\mathbf{1} \cdot \frac{\text{d}^2}{16\mathbf{F}^2}\right]\right). \tag{13}$$ In this expression, d is the diameter of the iris and F the focal length of the collimating optic. This equation shows that the beam divergence has two effects: account for by using δ' in all calculations. #### 2.3.6 Sampled interferogram and spectrum First the interferogram is sampled at regular intervals as the moving mirror traverses the optical path difference δ. To avoid the loss of the signal information, for a spectrum with a Δσ extend, the sampling step Δσ must satisfy the Nyquist condition: $$ \Delta\delta \prec \frac{1}{2\Delta\sigma}.\tag{14} $$ Thus the continuous interferogram is multiplied by a sampling comb, modelled theoretically by a comb of delta functions. Sampling the interferogram has obvious effects such as replacing the integrals of Eqs. (6) and (8) by sums over the samples. The number of sample points in the spectrum is equal to 2δ Δσ max + 1 . Practically in the spectrum the wavenumber interval between two points is equal to max <sup>1</sup> δσ <sup>=</sup> <sup>2</sup><sup>δ</sup> . Thus the spectral resolution is chosen such that there are at least two sample points per line width. #### 2.3.7 Zero filling Zero filling is a data processing technique where zero points are added to the end of the interferogram before the digital Fourier transformation. It is the process of interpolating extra data points into a spectrum so that the spectral lines have a smoother shape with a better digital resolution, using the same Fourier coefficients. FTIR software automatically provides zero-filling by extending the length of an interferogram with a zero straight line. However there is no new information added to the spectrum. Generally, the original interferogram size should always be at least doubled by zero filling, i.e. zero filling factor (ZFF) of two is chosen. Zero-filling is an interpolation that does not affect the instrument line-shape, and in most cases, is therefore superior to polynomial or spline interpolation methods that are applied in the spectral domain. ## 2.3.8 Phase errors 76 Fourier Transform – Materials Analysis The collimated beam inside the Michelson interferometer is produced using an extended source at the focus of a lens. This beam passes through an iris occulting part of the image of the source. Because of the finite size of this iris, the collimated beam is slightly divergent in the interferometer. When this effect is taken into account, the interferogram 2 2 2 2 2 0 2 ⎛ ⎞ ⎛ ⎞ <sup>⎡</sup> <sup>⎤</sup> ⎜ ⎟ ⎜ ⎟ <sup>⎢</sup> <sup>⎥</sup> ⎝ ⎠ ⎝ ⎠ <sup>⎣</sup> <sup>⎦</sup> <sup>d</sup> <sup>δ</sup>' = <sup>δ</sup> 1 - 2 2 16F ⎛ ⎞ ⎜ ⎟ ⎝ ⎠ . (13) . The first effect is easy to <sup>2</sup>Δσ . (14) 4F 8F 16F In this expression, d is the diameter of the iris and F the focal length of the collimating optic. First the interferogram is sampled at regular intervals as the moving mirror traverses the optical path difference δ. To avoid the loss of the signal information, for a spectrum with a Thus the continuous interferogram is multiplied by a sampling comb, modelled theoretically by a comb of delta functions. Sampling the interferogram has obvious effects such as replacing the integrals of Eqs. (6) and (8) by sums over the samples. The number of Practically in the spectrum the wavenumber interval between two points is equal to <sup>1</sup> δσ <sup>=</sup> <sup>2</sup><sup>δ</sup> . Thus the spectral resolution is chosen such that there are at least two sample Zero filling is a data processing technique where zero points are added to the end of the interferogram before the digital Fourier transformation. It is the process of interpolating extra data points into a spectrum so that the spectral lines have a smoother shape with a better digital resolution, using the same Fourier coefficients. FTIR software automatically provides zero-filling by extending the length of an interferogram with a zero straight line. 0 <sup>π</sup>d d <sup>π</sup><sup>d</sup> σ δ I(δ) = B sinc cos 2πσ δ 1 - <sup>0</sup> I(δ) = Bcos(2πσ δ) of a monochromatic source at wavenumber σ0 becomes This equation shows that the beam divergence has two effects: Δσ extend, the sampling step Δσ must satisfy the Nyquist condition: 2.3.6 Sampled interferogram and spectrum <sup>1</sup> Δδ <sup>&</sup>lt; sample points in the spectrum is equal to 2δ Δσ max + 1 . However there is no new information added to the spectrum. max 2.3.7 Zero filling points per line width. account for by using δ' in all calculations. 2.3.5 Extended size of the source In Fourier transform spectroscopy interferograms are apodized prior to the transformation and the calculated spectrum is phase corrected. If the zero point for the Fourier transformation is displaced, a phase error occurs because the apodization remains fixed while the interferogram is shifted by the phase correction. To avoid distortions of the spectrum resulting from phase errors, double-sided interferograms are needed. In practice, single-sided interferograms are recorded double-sided on a small range of optical path difference, allowing determination of the phase error. In such a case, the phase error is determined at low resolution, typically about 1 cm-1 or less. This is not a problem because it varies only slowly with wavenumber. ## 2.4 FTIR advantages Compared with the dispersive spectrometer, the FTIR instrument has several advantages: ## 3. Measurement of line profiles using Fourier transform infrared spectroscopy ## 3.1 Introduction Analysis of line profiles are useful for atmosphere monitoring and studying the evolution of its composition including chemical trace species related to the reduction in stratospheric ozone, starting from the analysis of observations obtained by infrared spectroscopy. The retrieved spectroscopic parameters are indispensable for the interpretation of high-resolution infrared spectra of gaseous species, and are of increasing importance for qualitative and Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 79 Carbonyl sulfide (OCS) is one of the principal and most long-lived reservoirs of sulfur in the Earth's troposphere (Watts, 2000) and has been detected in different astrophysical objects (the atmosphere of Venus (Bezard et al., 1990), the Orion molecular clouds (Evans et al., 1991), the comets Hyakutake and Hale-Bopp (Dello Russo et al., 1998; Woodney et al., 1997), The results presented and discussed in the following are obtained using spectra recorded with the high resolution Bruker IFS125HR Fourier transform spectrometer (an upgraded version of the previous version, IFS120HR) located at the LISA facility, in Créteil. A general view of the laboratory is presented in Fig. 3. It shows the Fourier transform spectrometer Bruker IFS125HR, two multiple-reflections (white-type) Pyrex absorption cells and a gas handling system. The Fourier transform spectrometer is characterized by a maximum optical path difference (MOPD) of up to 473.68 cm (maximum resolution = 0.0019 cm-1). The instrument has all the equipment and accessories required to operate from the far Fig. 3. View of the laboratory showing the Fourier transform spectrometer Bruker IFS125HR, two multiple-reflections Pyrex absorption cells and a gas handling system including a turbomolecular pump. In the back is an optical table with a difference frequency generation (DFG) laser system developed at LPPM in Orsay and LISA in Creteil and the starburst galaxies (Martin et al., 2005; Mauersberger et al., 1995)). infrared (20 cm-1, 500 microns) to the ultraviolet region (45000 cm-1). 3.2 Experimental set-up quantitative applications of spectroscopy. Also the pressure effects of gases on spectral lines are of significant interest in modeling and interpretation of radiative transfer calculations in climate models. In this context, inversion of atmospheric spectra to determine column densities of trace atmospheric species requires accurate knowledge of spectroscopic parameters. The needed parameters are, among others, line positions, line intensities, lower state transition energies and line widths as a function of temperature and quantum numbers. However, spectroscopic information contained in the databases such as HITRAN09 (Rothman et al., 2009), GEISA (Jacquinet-Husson et al., 2005), JPL (Pickett et al., 1998) and the more specialized MASTER (Perrin et al., 2005), are often still partly incomplete, e.g. for some species or transitions, experimental or theoretical line pressure shift and line-mixing parameters are missing. For this reason, Fourier transform spectroscopy is extensively used to generate these parameters. Compared to diode laser spectrometers, FT spectrometers have the advantage of recording spectra over a large frequency range. In this context, we present in the following Fourier transform measurements of NH3, CH3Br and OCS line profile parameters (line intensity, line width, line-mixing and line shift). When the gas pressure increases, inelastic collisions transfer populations from one radiative state to another. At low pressure, the lines are isolated; this effect gives a line broadening and shift proportional to the pressure. At high pressure the lines are overlapped, interference effects occur between adjacent lines. The resulting profile can no longer be described by the superposition of Lorentzian profiles. In this case, in addition to the previous parameters, one must determine the line-mixing coefficients. A large amount of laboratory data using various spectroscopic techniques of increasing precision has been assembled over the last two decades. Among the various molecular species, ammonia (NH3) is probably one of the most investigated species. This molecule has always played an important spectroscopic role because of its inversion spectrum and its large molecular dipole, which are important properties for theoretical models. Moreover, ammonia is present in many planetary atmospheres (Ho & Townes, 1983; Kunde et al., 1982). It is also commonly used as an interstellar thermometer and is considered as an industrial and biological pollutant (Brassington, 1988). The methyl halides (i.e. CH3Cl, CH3Br, and CH3I) have been the subject of very detailed studies of the Earth's atmosphere. They are atmospheric components which take part in atmospheric photochemical reactions. Methyl bromide (CH3Br) is a halogen chemical compound used in agriculture as a plant fungicide, and automobile using leaded petrol (Thomas et al., 1977). This molecule significantly contributes to ozone depletion since CH3Br is dissociated by UV radiation, producing Br radicals that catalyze the destruction of ozone (McElroy et al., 1986). These bromide atoms are far more destructive of ozone than the chlorine atoms coming from the chlorofluorocarbons compounds (CFC) (Kurylo & Rodriguez, 1998). For this reason, since 2005 the use of CH3Br has been prevented under the Montreal protocol. Despite the important role of CH3Br in the atmosphere, no spectroscopic data on CH3Br is available in the main databases (Jacquinet-Husson et al., 2005; Perrin et al., 2005; Pickett et al., 1998; Rothman et al., 2009). Carbonyl sulfide (OCS) is one of the principal and most long-lived reservoirs of sulfur in the Earth's troposphere (Watts, 2000) and has been detected in different astrophysical objects (the atmosphere of Venus (Bezard et al., 1990), the Orion molecular clouds (Evans et al., 1991), the comets Hyakutake and Hale-Bopp (Dello Russo et al., 1998; Woodney et al., 1997), and the starburst galaxies (Martin et al., 2005; Mauersberger et al., 1995)). ## 3.2 Experimental set-up 78 Fourier Transform – Materials Analysis quantitative applications of spectroscopy. Also the pressure effects of gases on spectral lines are of significant interest in modeling and interpretation of radiative transfer calculations in climate models. In this context, inversion of atmospheric spectra to determine column densities of trace atmospheric species requires accurate knowledge of spectroscopic parameters. The needed parameters are, among others, line positions, line intensities, lower state transition energies and line widths as a function of temperature and quantum However, spectroscopic information contained in the databases such as HITRAN09 (Rothman et al., 2009), GEISA (Jacquinet-Husson et al., 2005), JPL (Pickett et al., 1998) and the more specialized MASTER (Perrin et al., 2005), are often still partly incomplete, e.g. for some species or transitions, experimental or theoretical line pressure shift and line-mixing parameters are missing. For this reason, Fourier transform spectroscopy is extensively used to generate these parameters. Compared to diode laser spectrometers, FT spectrometers In this context, we present in the following Fourier transform measurements of NH3, CH3Br and OCS line profile parameters (line intensity, line width, line-mixing and line shift). When the gas pressure increases, inelastic collisions transfer populations from one radiative state to another. At low pressure, the lines are isolated; this effect gives a line broadening and shift proportional to the pressure. At high pressure the lines are overlapped, interference effects occur between adjacent lines. The resulting profile can no longer be described by the superposition of Lorentzian profiles. In this case, in addition to the A large amount of laboratory data using various spectroscopic techniques of increasing precision has been assembled over the last two decades. Among the various molecular species, ammonia (NH3) is probably one of the most investigated species. This molecule has always played an important spectroscopic role because of its inversion spectrum and its large molecular dipole, which are important properties for theoretical models. Moreover, ammonia is present in many planetary atmospheres (Ho & Townes, 1983; Kunde et al., 1982). It is also commonly used as an interstellar thermometer and is considered as an The methyl halides (i.e. CH3Cl, CH3Br, and CH3I) have been the subject of very detailed studies of the Earth's atmosphere. They are atmospheric components which take part in atmospheric photochemical reactions. Methyl bromide (CH3Br) is a halogen chemical compound used in agriculture as a plant fungicide, and automobile using leaded petrol This molecule significantly contributes to ozone depletion since CH3Br is dissociated by UV radiation, producing Br radicals that catalyze the destruction of ozone (McElroy et al., 1986). These bromide atoms are far more destructive of ozone than the chlorine atoms coming from the chlorofluorocarbons compounds (CFC) (Kurylo & Rodriguez, 1998). For this reason, since 2005 the use of CH3Br has been prevented under the Montreal protocol. Despite the important role of CH3Br in the atmosphere, no spectroscopic data on CH3Br is available in the main databases (Jacquinet-Husson et al., 2005; Perrin et al., 2005; Pickett et have the advantage of recording spectra over a large frequency range. previous parameters, one must determine the line-mixing coefficients. industrial and biological pollutant (Brassington, 1988). (Thomas et al., 1977). al., 1998; Rothman et al., 2009). numbers. The results presented and discussed in the following are obtained using spectra recorded with the high resolution Bruker IFS125HR Fourier transform spectrometer (an upgraded version of the previous version, IFS120HR) located at the LISA facility, in Créteil. A general view of the laboratory is presented in Fig. 3. It shows the Fourier transform spectrometer Bruker IFS125HR, two multiple-reflections (white-type) Pyrex absorption cells and a gas handling system. The Fourier transform spectrometer is characterized by a maximum optical path difference (MOPD) of up to 473.68 cm (maximum resolution = 0.0019 cm-1). The instrument has all the equipment and accessories required to operate from the far infrared (20 cm-1, 500 microns) to the ultraviolet region (45000 cm-1). Fig. 3. View of the laboratory showing the Fourier transform spectrometer Bruker IFS125HR, two multiple-reflections Pyrex absorption cells and a gas handling system including a turbomolecular pump. In the back is an optical table with a difference frequency generation (DFG) laser system developed at LPPM in Orsay and LISA in Creteil Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 81 The light is then collimated into a 7 cm diameter beam, sent into the Michelson interferometer. Various beam splitters, made of Mylar, KBr/Ge, CaF2/Si and Quartz/TiO2 are available, and used for different spectral regions. Exiting from the interferometer, the light is focused through an optical filter and then through the secondary iris. The beam then goes through the sample. Finally, the beam reaches the detectors compartment. We use four detectors, a Si bolometer operating at 4.2 K, a HgCdTe (MCT) detector, and an InSb detector, both cooled For the studies of the OCS molecule, this spectrometer was equipped with a KBr/Ge beamsplitter, Globar source (Silicon Carbide, SiC), an InSb detector cooled at 77 K, and For NH3 and CH3Br spectra in the region 1000-1800 cm-1, we used also a liquid nitrogencooled HgCdTe detector. These spectra were recorded with an aperture diameter of 1.15 mm, 40 kHz scanner frequency, and a maximum optical path difference MOPD = 225 cm for OCS and NH3, and 450 cm for CH3Br. According to the Bruker definition this corresponds to A White-type multipass absorption cell, made of Pyrex glass and equipped with CsBr windows, was used to record OCS spectra broadened by O2 and N2. The cell is in thermic equilibrium with the air conditioned room with a temperature stabilized at 295 K. For NH3 self-broadening experiments the gas was contained in a stainless steel gas cell equipped with CaF2 windows with a path-length of 2.5 cm. For NH3-H2 collisions, the gas mixture was contained in a Pyrex cell, with a path length of 15 cm, equipped with ZnSe windows. For CH3Br spectra, a multipass cell, of 1m base length equipped with KCl windows, was used For self-broadening studies, the cell was filled with an increasing pressure of the active gas. For foreign gas broadenings, the cell was first filled with the active gas, then, after thermalization, the absorption cell was filled with an increasing pressure of perturber gas. The following procedure was used for measurements: First a background spectrum was collected while the cell was being continuously evacuated. Next, the cell was filled with the considered gas at an increasing pressure. For OCS or NH3 perturbed by N2 or H2, the pressure of these gases was added in stages leading to a series of at least 6 pressures. Table 1 summarizes the experimental conditions of the spectra recorded for OCS-N2 and OCS-O2 collisions. The number of spectra for each gas is chosen in order to have sufficient information on all line parameters when analyzing a transition using the multi-spectrum fitting procedure. The sample pressure in the cell was measured using a calibrated MKS Baratron capacitance manometers (2, 10, 100, 1000 Torr full scale) each of them characterized by its stated uncertainty according to the manufacturer. For all these manometers the uncertainty is less than 1 %. The spectra were recorded at a stabilized room temperature of 295 K with an uncertainty of ± 1 K. All spectra were ratioed against the empty cell, singlechannel background spectrum which was taken in order to ensure the best possible signalto-noise in the ratioed spectra. The spectra were the result of the co-addition of a large number of scans (interferograms). For CH3Br spectra, the number of scans was 200. Every scan has been individually transformed to spectrum using the Fourier transform procedure included in the Bruker software OPUS package (Wartewig, 2003), selecting a Mertz phase optical and electronic filters covering the spectral region 1850 - 2150 cm-1. a resolution of 0.004 cm-1 for OCS and NH3 spectra and 0.002 cm-1 for CH3Br. to 77 K, and a Si photodiode at room temperature. for a total absorption path of 415 cm. error correction (Griffiths & Haseth, 1986; Mertz, 1965). The two multiple reflections cells are of the so-called White-type. They consist of a Pyrex tube containing the mirrors separated by 20 and 80 cm, respectively, thus providing base lengths of 0.849 and 3.249 m (this value takes into account the distance between the surface of the field mirror and the windows of the cell (2 × 2.45 cm)). The largest path length that one can achieve is 7.249 m with the short cell and 32.049 m with the long one. The temperature of the sample in these cells is well defined by an air-conditioning system regulating the temperature of the room, with an uncertainty of ± 1 K. The gas handling system is made of stainless steel tubing and can be evacuated to less than 10-6 mbar by a Pfeiffer Turbomolecular Pumps. Commercial gas lecture bottles with pure samples or buffer gases like N2, He, Ne, Ar etc. can be connected to the system to allow transfer of the gases to be studied into the absorption cells. Fig. 4 presents the optical design of the Fourier transform spectrometer Bruker IFS 125HR. This instrument is a fast-scanning, asymmetrical Michelson interferometer, characterized by a resolving power better than one million. Four different parts can be distinguished: the source compartment (bottom right), the interferometer itself (above the sources), the sample position (where short path absorption cells are placed) and detector compartment. The whole optical path is maintained under vacuum (pressure below 0.06 mbar). This instrument provides three different sources, a high-pressure Hg lamp for the far-infrared, a globar (Silicon Carbide, SiC) heated to about 1100 K for the mid-infrared, and a tungsten lamp for the near-infrared and visible regions. An external light beam can also enter the instrument through the input port. The light is focused onto an iris, whose size ranges from 0.5 to 12 mm (aperture changer). Fig. 4. Optical scheme of the Bruker IFS125HR Fourier transform spectrometer The two multiple reflections cells are of the so-called White-type. They consist of a Pyrex tube containing the mirrors separated by 20 and 80 cm, respectively, thus providing base lengths of 0.849 and 3.249 m (this value takes into account the distance between the surface of the field mirror and the windows of the cell (2 × 2.45 cm)). The largest path length that one can achieve is 7.249 m with the short cell and 32.049 m with the long one. The temperature of the sample in these cells is well defined by an air-conditioning system The gas handling system is made of stainless steel tubing and can be evacuated to less than 10-6 mbar by a Pfeiffer Turbomolecular Pumps. Commercial gas lecture bottles with pure samples or buffer gases like N2, He, Ne, Ar etc. can be connected to the system to allow Fig. 4 presents the optical design of the Fourier transform spectrometer Bruker IFS 125HR. This instrument is a fast-scanning, asymmetrical Michelson interferometer, characterized by a resolving power better than one million. Four different parts can be distinguished: the source compartment (bottom right), the interferometer itself (above the sources), the sample position (where short path absorption cells are placed) and detector compartment. The whole optical path is maintained under vacuum (pressure below 0.06 mbar). This instrument provides three different sources, a high-pressure Hg lamp for the far-infrared, a globar (Silicon Carbide, SiC) heated to about 1100 K for the mid-infrared, and a tungsten lamp for the near-infrared and visible regions. An external light beam can also enter the instrument through the input port. The light is focused onto an iris, whose size ranges from Fig. 4. Optical scheme of the Bruker IFS125HR Fourier transform spectrometer regulating the temperature of the room, with an uncertainty of ± 1 K. transfer of the gases to be studied into the absorption cells. 0.5 to 12 mm (aperture changer). The light is then collimated into a 7 cm diameter beam, sent into the Michelson interferometer. Various beam splitters, made of Mylar, KBr/Ge, CaF2/Si and Quartz/TiO2 are available, and used for different spectral regions. Exiting from the interferometer, the light is focused through an optical filter and then through the secondary iris. The beam then goes through the sample. Finally, the beam reaches the detectors compartment. We use four detectors, a Si bolometer operating at 4.2 K, a HgCdTe (MCT) detector, and an InSb detector, both cooled to 77 K, and a Si photodiode at room temperature. For the studies of the OCS molecule, this spectrometer was equipped with a KBr/Ge beamsplitter, Globar source (Silicon Carbide, SiC), an InSb detector cooled at 77 K, and optical and electronic filters covering the spectral region 1850 - 2150 cm-1. For NH3 and CH3Br spectra in the region 1000-1800 cm-1, we used also a liquid nitrogencooled HgCdTe detector. These spectra were recorded with an aperture diameter of 1.15 mm, 40 kHz scanner frequency, and a maximum optical path difference MOPD = 225 cm for OCS and NH3, and 450 cm for CH3Br. According to the Bruker definition this corresponds to a resolution of 0.004 cm-1 for OCS and NH3 spectra and 0.002 cm-1 for CH3Br. A White-type multipass absorption cell, made of Pyrex glass and equipped with CsBr windows, was used to record OCS spectra broadened by O2 and N2. The cell is in thermic equilibrium with the air conditioned room with a temperature stabilized at 295 K. For NH3 self-broadening experiments the gas was contained in a stainless steel gas cell equipped with CaF2 windows with a path-length of 2.5 cm. For NH3-H2 collisions, the gas mixture was contained in a Pyrex cell, with a path length of 15 cm, equipped with ZnSe windows. For CH3Br spectra, a multipass cell, of 1m base length equipped with KCl windows, was used for a total absorption path of 415 cm. For self-broadening studies, the cell was filled with an increasing pressure of the active gas. For foreign gas broadenings, the cell was first filled with the active gas, then, after thermalization, the absorption cell was filled with an increasing pressure of perturber gas. The following procedure was used for measurements: First a background spectrum was collected while the cell was being continuously evacuated. Next, the cell was filled with the considered gas at an increasing pressure. For OCS or NH3 perturbed by N2 or H2, the pressure of these gases was added in stages leading to a series of at least 6 pressures. Table 1 summarizes the experimental conditions of the spectra recorded for OCS-N2 and OCS-O2 collisions. The number of spectra for each gas is chosen in order to have sufficient information on all line parameters when analyzing a transition using the multi-spectrum fitting procedure. The sample pressure in the cell was measured using a calibrated MKS Baratron capacitance manometers (2, 10, 100, 1000 Torr full scale) each of them characterized by its stated uncertainty according to the manufacturer. For all these manometers the uncertainty is less than 1 %. The spectra were recorded at a stabilized room temperature of 295 K with an uncertainty of ± 1 K. All spectra were ratioed against the empty cell, singlechannel background spectrum which was taken in order to ensure the best possible signalto-noise in the ratioed spectra. The spectra were the result of the co-addition of a large number of scans (interferograms). For CH3Br spectra, the number of scans was 200. Every scan has been individually transformed to spectrum using the Fourier transform procedure included in the Bruker software OPUS package (Wartewig, 2003), selecting a Mertz phase error correction (Griffiths & Haseth, 1986; Mertz, 1965). Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 83 The OCS spectra were calibrated with residual CO2 and H2O lines observed in the spectra. For OCS and NH3 spectra, the analyses have been done taking into account interference effects. Thus within the impact theory of the spectral line shape and for moderately a overlapping line at low pressure considered for these molecules, the collisional absorption <sup>P</sup> PY (<sup>σ</sup> - <sup>σ</sup> )+P<sup>γ</sup> <sup>α</sup>(σ)= S vfJfKf, Sk its intensity, and Yk its line mixing parameter related to the off diagonal element of the relaxation matrix. Spectra were analyzed by means of nonlinear least squares fitting procedures, using the τ (σ)= F (σ - σ') × exp - × α (σ' - σ'') × α(σ'')×dσ'' × dσ' gaussian shape and A is the cell length. Eq. (16) is a convolution of the collisional absorption Otherwise the collisional parameters for a given temperature have been deduced by means of nonlinear least square multipressure fitting in which all spectra at various pressures are successively adjusted using Eq. (16). For a line k, the parameters deduced from the fits are Because of the spectral density for the bands of the molecules considered here and the strong values of the self-broadening coefficient, a line by line study could not have been possible with the relatively high pressure, so the line parameters were derived using the multi-spectrum fitting method applied to the measured shapes of the lines, including the An example of a multi-spectrum fit in the case of PaP(8,7) and the two components of the PasP(8,8) doublet all pertaining to the PP branch of the ν4 band is given in Fig. 6 with the corresponding pressures. Plots (a) and (b) show that the overlapping increases with NH3 pressure. They also demonstrate clearly the influence of line-mixing in the overlapped contours as illustrated by large discrepancies obtained when this process is disregarded in <sup>k</sup>, Sk and PYk. These spectroscopic parameters have been iteratively varied by a fitting program to obtain the best agreement between the experimental and calculated absorption Dop is the Doppler profile, FApp is the Fourier transform instrument function with a ) could be written as (Gentry & Larrabee Strow, 1997; Lévy et al., 1992; Pine, k 2 2 lines k k k σ ⎡ ⎤ ⎢ ⎥ ⎣ ⎦ ∫ ∫ <sup>A</sup> , (16) <sup>π</sup> (<sup>σ</sup> - <sup>σ</sup> ) + (P<sup>γ</sup> ) ∑ , (15) ) for the transmission and the collisional absorption k its wavenumber, γ <sup>k</sup> its halfwidth For CH3Br the calibration was done using the low pressure ν2 band spectrum of NH3. 3.3 Fitting procedure α(σ where k represents the line viJiKi following theoretical expression C coefficient in Eq. (15): where α Pγk, σ coefficients. Fig. 6(a). 1997; Rosenkranz, 1975; Thibault et al., 1992): NH3 kk k → τC(σ + + ∞ ∞ coefficient with the instrument function and the Doppler profile. interference effects caused by the line overlaps. ∞ ∞ App Dop coefficient a The values given in parentheses correspond to the estimated errors. Table 1. Experimental data Fig. 5. Transmittance spectrum of OCS around 2116 cm-1 showing some lines of the R branch of 4ν2 band broadened by N2. The pressures are 0.086 Torr of OCS, and 7.74 and 60.00 Torr of N2 The OCS spectra were calibrated with residual CO2 and H2O lines observed in the spectra. For CH3Br the calibration was done using the low pressure ν2 band spectrum of NH3. #### 3.3 Fitting procedure 82 Fourier Transform – Materials Analysis number OCS pressure (Torr)a N2 pressure (Torr)a OCS pressure (Torr)a O2 pressure (Torr)a 2112 2114 2116 2118 2120 σ(cm-1 ) Fig. 5. Transmittance spectrum of OCS around 2116 cm-1 showing some lines of the R branch of 4ν2 band broadened by N2. The pressures are 0.086 Torr of OCS, and 7.74 and 60.00 Torr of N2 60.00 Torr 7.74 Torr Optical path (m) 3.249 Resolution (cm-1) 0.004 Maximum optical path difference (cm) 225 Collimator focal length (mm) 418 Aperture diameter (mm) 1.15 Useful spectral domain (cm-1) 1850-2150 a The values given in parentheses correspond to the estimated errors. Table 1. Experimental data 0,1 0,2 0,3 0,4 0,5 0,6 Transmitance 0,7 0,8 0,9 1,0 1,1 OCS-N2 OCS-O2 Spectrum 1 0.0859 (1) 7.74 (2) 0.0754 (1) 5.80 (1) 2 0.0859 (1) 15.05 (4) 0.0754 (1) 13.98 (3) 3 0.0859 (1) 25.53 (6) 0.0754 (1) 22.09 (6) 4 0.0859 (1) 34.89 (9) 0.0754 (1) 32.41 (8) 5 0.0859 (1) 46.90 (1) 0.0754 (1) 51.80 (1) 6 0.0859 (1) 60.00 (2) 0.0754 (1) 83.20 (2) For OCS and NH3 spectra, the analyses have been done taking into account interference effects. Thus within the impact theory of the spectral line shape and for moderately a overlapping line at low pressure considered for these molecules, the collisional absorption coefficient α(σ) could be written as (Gentry & Larrabee Strow, 1997; Lévy et al., 1992; Pine, 1997; Rosenkranz, 1975; Thibault et al., 1992): $$\mathbf{a(o)} = \frac{\mathbf{P\_{NH\_3}}}{\mathbf{n}} \sum\_{\text{lines k}} \mathbf{S\_k} \frac{\mathbf{P} \mathbf{Y\_k(o \cdot o\_k)} + \mathbf{P} \mathbf{y\_k}}{\left(\mathbf{o} \cdot \sigma\_k\right)^2 + \left(\mathbf{P} \mathbf{y\_k}\right)^2},\tag{15}$$ where k represents the line viJiKi → vfJfKf, Sk its intensity, σk its wavenumber, γ<sup>k</sup> its halfwidth and Yk its line mixing parameter related to the off diagonal element of the relaxation matrix. Spectra were analyzed by means of nonlinear least squares fitting procedures, using the following theoretical expression τC(σ) for the transmission and the collisional absorption coefficient in Eq. (15): $$\mathbf{r}^{\scriptscriptstyle{\mathsf{C}}}(\mathsf{o}) = \bigcap\_{\scriptstyle \preccurlyeq \atop \rightsquigarrow} \mathbf{F}\_{\scriptstyle \operatorname{App}}(\mathsf{o} \cdot \mathsf{o}^{\scriptscriptstyle \mathsf{I}}) \times \exp\left[ \cdot \ell \times \bigcup\_{\scriptstyle \preccurlyeq}^{\scriptscriptstyle \mathsf{t} \rightarrow \mathsf{o}} \mathbf{a}\_{\textit{Doop}}(\mathsf{o}^{\scriptscriptstyle \mathsf{I}} \cdot \mathsf{o}^{\scriptscriptstyle \mathsf{I}}) \times \mathbf{a}(\mathsf{o}^{\scriptscriptstyle \mathsf{I}}) \times \mathbf{d}\mathsf{o}^{\scriptscriptstyle \mathsf{n}} \right] \times \mathbf{d}\mathsf{o}^{\scriptscriptstyle \mathsf{I}},\tag{16}$$ where αDop is the Doppler profile, FApp is the Fourier transform instrument function with a gaussian shape and A is the cell length. Eq. (16) is a convolution of the collisional absorption coefficient with the instrument function and the Doppler profile. Otherwise the collisional parameters for a given temperature have been deduced by means of nonlinear least square multipressure fitting in which all spectra at various pressures are successively adjusted using Eq. (16). For a line k, the parameters deduced from the fits are Pγk, σ<sup>k</sup>, Sk and PYk. These spectroscopic parameters have been iteratively varied by a fitting program to obtain the best agreement between the experimental and calculated absorption coefficients. Because of the spectral density for the bands of the molecules considered here and the strong values of the self-broadening coefficient, a line by line study could not have been possible with the relatively high pressure, so the line parameters were derived using the multi-spectrum fitting method applied to the measured shapes of the lines, including the interference effects caused by the line overlaps. An example of a multi-spectrum fit in the case of PaP(8,7) and the two components of the PasP(8,8) doublet all pertaining to the PP branch of the ν4 band is given in Fig. 6 with the corresponding pressures. Plots (a) and (b) show that the overlapping increases with NH3 pressure. They also demonstrate clearly the influence of line-mixing in the overlapped contours as illustrated by large discrepancies obtained when this process is disregarded in Fig. 6(a). Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 85 1288,43 1288,44 1288,45 1288,46 1288,47 1288,48 σ(cm-1) Fig. 7. Results of spectrum fits for the QP79(26,E,5) line in the v2 band of CH3Br self-broadened at 296 K. (symbol) and (—) are, respectively, the calculated and measured values. Measured The line intensities presented here are for NH3 in ν2 and ν4 band spectra. As shown in Fig. 8 for the PsP(2,1), PsP(3,1), PsP(4,1), PsP(6,1) and PsP(7,1) lines of the ν4 band, the intensity parameter S (cm-2) derived from the fits is proportional to the real NH3 pressure P'= P × 0.985 (0.985 is the fractional abundance of NH3 for the gas sample). The PsP(5,1) line which has practically the same intensity value as the PsP(3,1) line is not plotted. The line intensities S0 (cm-2 atm-1) are deduced from the slopes of the straight lines obtained from a linear leastsquares procedure and the length A = 2.5 cm of the absorption cell. The results are listed in Refs. (Aroui et al., 2003; Hadded et al., 2001) for symmetric and asymmetric transitions for For the two bands, the intensities of transitions with a nuclear spin statistical weight of the lower level gs = 2 for K = 3n (n = 1, 2,...) are larger than those with gs = 1. S0 increases with K for a given J when dividing by 2 the intensities of the lines with gs = 2. However, for the ν<sup>2</sup> band when K is close or equal to J, line intensities exhibit a slight decrease. On the other hand, for a fixed value of K, S0 decreases significantly as J increases. Otherwise the intensities of the asymmetric transitions are slightly larger than those of symmetric transitions. In the PP branch of the ν4 band, line intensities also increase with K for a given J, but, as shown in Fig. 8, the evolution with J presents a maximum for J = 4 for the PP(J,K=1) minus calculated deviations are shown in the lower part of the graph 1.008 Torr 2.490 Torr 3.626 Torr 6.811 Torr Line:Mesured Symbol:Calculated 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 1,1 3.4 Line intensities 3.4.1 Line intensity results the ν2 and ν4 bands of NH3. lines and decrease monotonically for K ≥ 2. Transmittance Q P79(26,E,5) Fig. 6. Graphic demonstration of line-mixing obtained for pure NH3 spectra for PaP(8,7) line and PasP(8,8) inversion doublet of the ν4 band at various pressures. (⎯) and (•) are the measured and calculated values, respectively. Measured minus calculated deviations are shown in the lower part of graphs. Plots (a) and (b) have been obtained without (Y=0) and with (Y≠0) the line-mixing, respectively. These plots illustrate the shift of PasP(8,8) components line center towards each other Fig. 7 shows the results of multi-pressure spectrum fits for the QP79(26,E,5) line in the v2 band of CH3Br, self-broadened at 296 K at different pressures. In this figure symbols and (—) are, respectively, the calculated and measured values. This line profile was calculated using a Voigt function. Indeed, no characteristic signature due to the presence of collisional narrowing or line-mixing has been observed in any residual. Fig. 7. Results of spectrum fits for the QP79(26,E,5) line in the v2 band of CH3Br self-broadened at 296 K. (symbol) and (—) are, respectively, the calculated and measured values. Measured minus calculated deviations are shown in the lower part of the graph #### 3.4 Line intensities 84 Fourier Transform – Materials Analysis P P(8,8)s <sup>P</sup> P(8,8)a σ / cm-1 1500,4 1500,6 1500,8 1501,0 1501,2 P P(8,8)s σ / cm-1 Fig. 6. Graphic demonstration of line-mixing obtained for pure NH3 spectra for PaP(8,7) line and PasP(8,8) inversion doublet of the ν4 band at various pressures. (⎯) and (•) are the measured and calculated values, respectively. Measured minus calculated deviations are shown in the lower part of graphs. Plots (a) and (b) have been obtained without (Y=0) and Fig. 7 shows the results of multi-pressure spectrum fits for the QP79(26,E,5) line in the v2 band of CH3Br, self-broadened at 296 K at different pressures. In this figure symbols and (—) are, respectively, the calculated and measured values. This line profile was calculated using a Voigt function. Indeed, no characteristic signature due to the presence of collisional with (Y≠0) the line-mixing, respectively. These plots illustrate the shift of PasP(8,8) P P(8,7)a P P(8,7)a 1500,4 1500,6 1500,8 1501,0 1501,2 <sup>P</sup> P(8,8)a 15 mbar 43 mbar 61 mbar 81 mbar 98 mbar 15 mbar 43 mbar 61 mbar 81 mbar 98 mbar 0,0 (b) components line center towards each other narrowing or line-mixing has been observed in any residual. 0,2 0,4 0,6 Transmittance 0,8 1,0 0,0 0,2 (a) 0,4 0,6 Transmittance 0,8 1,0 #### 3.4.1 Line intensity results The line intensities presented here are for NH3 in ν2 and ν4 band spectra. As shown in Fig. 8 for the PsP(2,1), PsP(3,1), PsP(4,1), PsP(6,1) and PsP(7,1) lines of the ν4 band, the intensity parameter S (cm-2) derived from the fits is proportional to the real NH3 pressure P'= P × 0.985 (0.985 is the fractional abundance of NH3 for the gas sample). The PsP(5,1) line which has practically the same intensity value as the PsP(3,1) line is not plotted. The line intensities S0 (cm-2 atm-1) are deduced from the slopes of the straight lines obtained from a linear leastsquares procedure and the length A = 2.5 cm of the absorption cell. The results are listed in Refs. (Aroui et al., 2003; Hadded et al., 2001) for symmetric and asymmetric transitions for the ν2 and ν4 bands of NH3. For the two bands, the intensities of transitions with a nuclear spin statistical weight of the lower level gs = 2 for K = 3n (n = 1, 2,...) are larger than those with gs = 1. S0 increases with K for a given J when dividing by 2 the intensities of the lines with gs = 2. However, for the ν<sup>2</sup> band when K is close or equal to J, line intensities exhibit a slight decrease. On the other hand, for a fixed value of K, S0 decreases significantly as J increases. Otherwise the intensities of the asymmetric transitions are slightly larger than those of symmetric transitions. In the PP branch of the ν4 band, line intensities also increase with K for a given J, but, as shown in Fig. 8, the evolution with J presents a maximum for J = 4 for the PP(J,K=1) lines and decrease monotonically for K ≥ 2. Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 87 For the interpretation of these dependencies, the zero-order theory, where the correction for vibration-rotation interactions is not included, cannot describe the transition dipole moment ( ) { } J K JJ(Q) <sup>2</sup> JJ(PR) 2 KK 2 JK F = 1+A m +A m +A J(J +1) - m +A m +A K +A m m HW <sup>J</sup> <sup>K</sup> <sup>J</sup> <sup>J</sup> J K ⎡ ⎤ ⎣ ⎦ , (19) function describing the dependence of Reff on J and K. For the ν2 parallel band, for example, where the A's are correction parameters. For the R-transitions of this band, we have mJ = J+1, The correction parameters AJ, AJJ(R) and AKK as well as the vibrational transition moment v R0 , were determined, using linear least-squares fits, from the experimental values of effective transition moments. The resulting correction parameters and vibrational transition moments for the ν2 band are listed in Table 2 along with the standard deviation of the fits (Aroui et al., 2003). In this table, the AJ and AJJ(R) parameters cannot be determined for ν2s, since, for this partial band, the fits were performed only for 7 transitions pertaining to the J = 10 manifold, whereas for asymmetric ν2a partial band the fits were performed for 62 For the ν4 band, the correction function, the vibrational transition moments and the correction parameters are reported previously (Aroui et al., 1998). For this band significant differences between the correction parameters obtained for the symmetric and asymmetric transitions, so one can determine the vibrational band strengths, <sup>V</sup> S0S and <sup>V</sup> S0a respectively, for the symmetric and asymmetric partial bands ν4s and ν4a of the ν4 band. The values are Parameter s←a transitions a<sup>←</sup>s transitions v R0 (D) 0.24604 ± 0.00209 0.23868 ± 0.00154 AKK (7.66 ± 0.62)×10-4 (5.03 ± 0.37)×10-4 γ Fig. 9 for the PsP(3,1) line of NH3 in the ν4 band at the temperatures 235 and 296 K. The gases versus the pressure P of hydrogen is shown in symmetric and asymmetric transitions of the ν2 band of NH3 (Aroui et al., 2003) Table 2. Vibrational transition moments and Herman-Wallis correction parameters for the AJ (-3.88 ± 1.47)×10-3 ⎯ AJJ(R) (-1.57 ± 0.72)×10-4 ⎯ is the vibrational transition moment, and FHW is the Herman-Wallis correction <sup>2</sup> J 2 JJ(R) KK <sup>2</sup> F = 1+A (J + 1) + A (J + 1) + A K HW <sup>⎡</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> . (20) <sup>2</sup> 2 v R (J,K) = R F eff 0 HW , (18) 2 variations. By taking in to account the effects of these interactions, one can write using the second-order development of Watson (Watson, 1992) we may write, mK = 0, ( )<sup>2</sup> J(J + 1) = J + 1 and 2 2 K =K , thus we can write FHW in the form: where R<sup>0</sup> ν transitions. reported in previous work (Aroui et al., 1998). 3.5 Pressure broadenings 3.5.1 Pressure broadening coefficients For NH3-H2 collisions, a typical plot of P Fig. 8. Pressure dependence of the intensity parameter S for the PsP(2,1), PsP(3,1), PsP(4,1), PsP(6,1) and PsP(7,1) lines in the ν4 band of NH3. The slopes of the best fit line represent the line intensity As expected the line intensities in the ν2 band (Aroui et al., 2003) are much larger than those reported previously (Aroui et al., 1998; Cottaz et al., 2001; Hadded et al., 2001) for the ν<sup>4</sup> band and also several times larger than the intensities in the ν1 and ν3 bands (Markov, 1993). #### 3.4.2 Vibrational transition moments and correction function For NH3 molecule, the effective transition dipole moments Reff(J,K) in the ν2 band are derived from the line intensities S0 such as (Aliev et al., 1987): $$\mathrm{R}\_{\mathrm{eff}}^{2}\mathrm{(J.K)} = \frac{\mathrm{S}\_{0}\mathrm{TQ} \,\,\exp\left(\mathrm{E(J.K)}/\mathrm{kT}\right)}{3054.8 \,\,\mathrm{g} \,\,\mathrm{o} \,\,\left[1\,\mathrm{-exp}\left(\mathrm{ho}\,\mathrm{/}\mathrm{kT}\right)\right] \,\,\mathrm{H}\_{\mathrm{JK}}\,\mathrm{'} \tag{17}$$ where HJK is the Höln-London factor (Aroui et al., 2003). σ is the wave number of the line under study. E(J,K) is the rotational energies of the lower level of the transitions. Q = QrQv is the total partition function, with the rotational partition function Qr = 563.56 at T = 295 K and the vibrational partition function Qv = 1.011. The effective transition dipole moments Reff, determined from the observed line intensities using Eq. (17) show significant rotational dependencies (Aroui et al., 1998, 2003) which are mainly caused by vibration-rotation interactions (l-type interactions in the ν4 band and Coriolis interaction between ν4 and 2ν2 bands): they decrease with J for a fixed value of K and increase with K for a given J. s P P(2,1) s P P(3,1) s P P(4,1) s P P(6,1) s P P(7,1) 0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 P'(atm) As expected the line intensities in the ν2 band (Aroui et al., 2003) are much larger than those reported previously (Aroui et al., 1998; Cottaz et al., 2001; Hadded et al., 2001) for the ν<sup>4</sup> band and also several times larger than the intensities in the ν1 and ν3 bands (Markov, 1993). For NH3 molecule, the effective transition dipole moments Reff(J,K) in the ν2 band are where HJK is the Höln-London factor (Aroui et al., 2003). σ is the wave number of the line under study. E(J,K) is the rotational energies of the lower level of the transitions. Q = QrQv is the total partition function, with the rotational partition function Qr = 563.56 at T = 295 K The effective transition dipole moments Reff, determined from the observed line intensities using Eq. (17) show significant rotational dependencies (Aroui et al., 1998, 2003) which are mainly caused by vibration-rotation interactions (l-type interactions in the ν4 band and Coriolis interaction between ν4 and 2ν2 bands): they decrease with J for a fixed value of K S TQ exp E(J,K)/kT R (J,K) = 3054.8 g <sup>σ</sup> ⎡ ⎤ ( ) ( ) ⎣ ⎦ 1 - exp h<sup>σ</sup> /kT H , (17) i JK 3.4.2 Vibrational transition moments and correction function derived from the line intensities S0 such as (Aliev et al., 1987): 2 0 eff and the vibrational partition function Qv = 1.011. and increase with K for a given J. Fig. 8. Pressure dependence of the intensity parameter S for the PsP(2,1), PsP(3,1), PsP(4,1), PsP(6,1) and PsP(7,1) lines in the ν4 band of NH3. The slopes of the best fit line represent the 0,000 line intensity 0,008 0,016 0,024 S(cm-2 ) 0,032 0,040 0,048 For the interpretation of these dependencies, the zero-order theory, where the correction for vibration-rotation interactions is not included, cannot describe the transition dipole moment variations. By taking in to account the effects of these interactions, one can write $$\mathbf{R}\_{\rm eff}^2(\mathbf{J}, \mathbf{K}) = \left(\mathbf{R}\_0^\times\right)^2 \mathbf{F}\_{\rm HW} \,\prime \,\tag{18}$$ where R<sup>0</sup> ν is the vibrational transition moment, and FHW is the Herman-Wallis correction function describing the dependence of Reff on J and K. For the ν2 parallel band, for example, using the second-order development of Watson (Watson, 1992) we may write, $$\mathbf{F\_{HW}} = \left(\mathbf{1} + \mathbf{A^{I}m\_{\parallel}} + \mathbf{A^{K}m\_{\mathbf{K}}} + \mathbf{A^{I(Q)}} \left[\overline{\mathbf{J(J+1)}} \cdot \mathbf{m\_{J}^{2}}\right] + \mathbf{A^{I(PR)}m\_{\parallel}^{2}} + \mathbf{A^{KK}\overline{K^{2}}} + \mathbf{A^{IK}m\_{\parallel}m\_{\mathbf{K}}}\right)^{2} . \tag{19}$$ where the A's are correction parameters. For the R-transitions of this band, we have mJ = J+1, mK = 0, ( )<sup>2</sup> J(J + 1) = J + 1 and 2 2 K =K , thus we can write FHW in the form: $$\mathbf{F\_{HW}} = \begin{bmatrix} \mathbf{1} + \mathbf{A^{J}(J+1)} \ + & \mathbf{A^{J(R)}(J+1)}^2 \ + & \mathbf{A^{KK}K^2}^2 \end{bmatrix}^2 \cdot \tag{20}$$ The correction parameters AJ , AJJ(R) and AKK as well as the vibrational transition moment v R0 , were determined, using linear least-squares fits, from the experimental values of effective transition moments. The resulting correction parameters and vibrational transition moments for the ν2 band are listed in Table 2 along with the standard deviation of the fits (Aroui et al., 2003). In this table, the AJ and AJJ(R) parameters cannot be determined for ν2s, since, for this partial band, the fits were performed only for 7 transitions pertaining to the J = 10 manifold, whereas for asymmetric ν2a partial band the fits were performed for 62 transitions. For the ν4 band, the correction function, the vibrational transition moments and the correction parameters are reported previously (Aroui et al., 1998). For this band significant differences between the correction parameters obtained for the symmetric and asymmetric transitions, so one can determine the vibrational band strengths, <sup>V</sup> S0S and <sup>V</sup> S0a respectively, for the symmetric and asymmetric partial bands ν4s and ν4a of the ν4 band. The values are reported in previous work (Aroui et al., 1998). Table 2. Vibrational transition moments and Herman-Wallis correction parameters for the symmetric and asymmetric transitions of the ν2 band of NH3 (Aroui et al., 2003) #### 3.5 Pressure broadenings #### 3.5.1 Pressure broadening coefficients For NH3-H2 collisions, a typical plot of Pγ versus the pressure P of hydrogen is shown in Fig. 9 for the PsP(3,1) line of NH3 in the ν4 band at the temperatures 235 and 296 K. The gases Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 89 m Contrary to the other symmetric top molecules, the broadening coefficients of NH3 increase with K for a given J (Markov et al., 1993; Pine & Markov, 2004; Aroui et al., 2003, 2009). This behavior is more pronounced for the small J values. This increase could be related to the rotational energy separation between NH3 levels which decrease with K. Note that the J = K lines have almost a constant value of broadening coefficient. These J and K dependencies have previously been observed in other bands (Hadded et al., 2001; Markov et al., 1993). For CH3Br molecule, previous works (Gomez et al., 2010, Jacquemart et al., 2007) report the observed and predicted self broadening coefficients of the PP, RQ and RR branches of the ν<sup>6</sup> band of CH3Br. Fig. 11 shows that for a give K, the broadening coefficients of the ν2 band increase with J in the interval J ≤ Jmax = 20 to nearly γ = 0.45 cm-1 atm-1 at J = Jmax. Then these coefficients decrease at higher J values. The same trend is observed for the ν6 band for K = 6 (Jacquemart et al., 2007). This pattern may be easily understood by considering the resonance condition that exists between the energy gaps of the two partners during collision. For the self broadening corresponding to the maximum contribution of the electrostatic interactions, Jmax is given by the resonance condition (Giraud et al., 1971; > 1 1 max 2p 2 2 <sup>B</sup> J» J <sup>B</sup> A J2P is the most populated level of the perturbing molecule at the temperature considered (for CH3Br at room temperature J2p = 18). B1 and B2 are the rotational constants of the two <sup>A</sup> (22) Fig. 10. Quantum number \|m\|dependence of the measured N2, and O2 broadening coefficients for P and R branches of the 4ν2 band of OCS. Uncertainties become larger for 0,03 0,04 0,05 0,06 0,07 0,08 0,09 0,10 0,11 0,12 0,13 0,14 0,15 Pourcin et al., 1981): γ 0(cm-1atm-1 ) OCS-N2 OCS-O2 high values of J (above 30) because of weaker line intensities were contained in a 15 cm path-length Pyrex cell. One can observe that straight line fits go through the measured points very well. The pressure broadening coefficients γ for each temperature are determined as the slopes of the best-fit lines. This figure illustrates an increase of line width with H2 pressure but a decrease of broadening coefficient as function of temperature (Nouri, 2004). This dependence is usually well represented by the simple power law $$\dot{\lambda} = \text{ } \text{y} (\text{T}\_0) \left( \frac{\text{T}\_0}{\text{T}} \right)^n \text{ } \text{ } \tag{21}$$ where γ(T0) is the broadening coefficient at the reference temperature T0 = 296 K. From the measured values of γ at the considered temperatures, one can determine the values of exponent n as the slope of the graphs of lnγ(T) versus lnT. The straight lines obtained for all transitions validate Eq. (21) within the indicated range of T. The derived values of n are given in Ref. (Nouri, 2004) for the PP, RP, PR, RQ, RR, and PQ branches in the ν4 band and for R branch in the 2ν2 band of NH3. Fig. 9. Pressure dependence of the broadening line width Pγ for the PsP(3,1) line of the ν<sup>4</sup> band perturbed by H2 ; (o) T=235 K ; (•) T=296 K. The slope of the best fit line represents the broadening coefficient As shown previously, distinctive dependence on the rotational quantum number m can be observed for the molecules considered here (Aroui et al., 2003, 2009; Bouanich et al., 1986; Cottaz et al., 2001; Domenech et al., 2000; Hadded et al., 2001; Koshelev et al., 2006, 2009; Markov et al., 1993; Mouchet et al., 1985). For OCS-N2 and OCS-O2 systems, Fig. 10 is a plot of the measured broadening coefficients versus \|m\| (m = −J for P(J ) lines and m = J +1 for R(J) lines) for the P and R branches of the 4ν2 band (Galalou et al., 2011). It shows the decrease of these coefficients with increasing \|m\|. were contained in a 15 cm path-length Pyrex cell. One can observe that straight line fits go temperature are determined as the slopes of the best-fit lines. This figure illustrates an increase of line width with H2 pressure but a decrease of broadening coefficient as function of temperature (Nouri, 2004). This dependence is usually well represented by the simple > 0 <sup>T</sup> = (T ) <sup>T</sup> transitions validate Eq. (21) within the indicated range of T. The derived values of n are given in Ref. (Nouri, 2004) for the PP, RP, PR, RQ, RR, and PQ branches in the ν4 band and for 0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 P(atm) band perturbed by H2 ; (o) T=235 K ; (•) T=296 K. The slope of the best fit line represents the As shown previously, distinctive dependence on the rotational quantum number m can be observed for the molecules considered here (Aroui et al., 2003, 2009; Bouanich et al., 1986; Cottaz et al., 2001; Domenech et al., 2000; Hadded et al., 2001; Koshelev et al., 2006, 2009; Markov et al., 1993; Mouchet et al., 1985). For OCS-N2 and OCS-O2 systems, Fig. 10 is a plot of the measured broadening coefficients versus \|m\| (m = −J for P(J ) lines and m = J +1 for R(J) lines) for the P and R branches of the 4ν2 band (Galalou et al., 2011). It shows the γ for the PsP(3,1) line of the ν<sup>4</sup> Fig. 9. Pressure dependence of the broadening line width P decrease of these coefficients with increasing \|m\|. γ ⎛ ⎞ ⎜ ⎟ ⎝ ⎠ n 0 at the considered temperatures, one can determine the values of (T0) is the broadening coefficient at the reference temperature T0 = 296 K. From the γ , (21) (T) versus lnT. The straight lines obtained for all for each through the measured points very well. The pressure broadening coefficients γ γ power law where γ 0,000 broadening coefficient 0,002 0,004 0,006 0,008 Pγ(cm-1 ) 0,010 0,012 0,014 0,016 measured values of γ exponent n as the slope of the graphs of ln R branch in the 2ν2 band of NH3. T=296 K T=235 K Fig. 10. Quantum number \|m\|dependence of the measured N2, and O2 broadening coefficients for P and R branches of the 4ν2 band of OCS. Uncertainties become larger for high values of J (above 30) because of weaker line intensities Contrary to the other symmetric top molecules, the broadening coefficients of NH3 increase with K for a given J (Markov et al., 1993; Pine & Markov, 2004; Aroui et al., 2003, 2009). This behavior is more pronounced for the small J values. This increase could be related to the rotational energy separation between NH3 levels which decrease with K. Note that the J = K lines have almost a constant value of broadening coefficient. These J and K dependencies have previously been observed in other bands (Hadded et al., 2001; Markov et al., 1993). For CH3Br molecule, previous works (Gomez et al., 2010, Jacquemart et al., 2007) report the observed and predicted self broadening coefficients of the PP, RQ and RR branches of the ν<sup>6</sup> band of CH3Br. Fig. 11 shows that for a give K, the broadening coefficients of the ν2 band increase with J in the interval J ≤ Jmax = 20 to nearly γ = 0.45 cm-1 atm-1 at J = Jmax. Then these coefficients decrease at higher J values. The same trend is observed for the ν6 band for K = 6 (Jacquemart et al., 2007). This pattern may be easily understood by considering the resonance condition that exists between the energy gaps of the two partners during collision. For the self broadening corresponding to the maximum contribution of the electrostatic interactions, Jmax is given by the resonance condition (Giraud et al., 1971; Pourcin et al., 1981): $$\mathbf{J}\_{\text{max}} \,\mathrm{\mathcal{P}} \frac{\mathbf{B}\_1}{\mathbf{B}\_2} \frac{\ell\_1}{\ell\_2} \mathbf{J}\_{2\mathbf{p}} \tag{22}$$ J2P is the most populated level of the perturbing molecule at the temperature considered (for CH3Br at room temperature J2p = 18). B1 and B2 are the rotational constants of the two Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 91 Some least squares regressions fail to fit the observed values for particular quantum numbers. Other fits were satisfactory for nonpolar buffers, but fit poorly for self-broadening In a recent paper (Aroui et al., 2009), the measured broadening coefficients of nine branches γ(m,K) = β0 + β 1 m + β2 K + β3 m2 + β4 K2+β5 mK (23) The fitted parameters β0, β1, β2, β3, β4 and β5 are in unit of cm-1 atm-1. They are given in Ref. (Aroui et al., 2009), along with the data points included in the fits. Most of the parameters Some authors have discussed (Markov et al., 1993; Pine & Markov, 2004; Nemtchinov et al., 2004, Brown & Peterson, 1994; Cottaz et al., 2001; Aroui et al., 2009) the vibrational dependence of broadening coefficients. In most cases the comparison of the measured results for different bands exhibits little vibrational dependence. Within experimental error, the broadening coefficients of ammonia, for example, are only slightly greater in the ν4 band than in the ν2 and 2ν2 bands. This is explained partially by the smaller splitting of the inversion doublets in the ν4 band than in the ν2 and 2ν2 ones. For example, when the ν2 and 2ν2 bands are compared, the differences between line broadening coefficients with similar However, for this molecule, Cottaz et al. (Cottaz et al., 2001) have highlighted a vibrational broadening effect when they compared their results for the R(3,1) line measured in six different vibrational bands. The present result for this line is slightly greater (3%) than that For OCS-N2 and OCS-O2, pressure broadening coefficients in previous works for different bands (Bouanich et al., 1986; Domenech et al., 2000; Koshelev et al., 2006, 2009; Mouchet et al., 1985; Galalou et al., 2011), illustrate that no vibrational effect can be clearly identified. To clarify this effect, Ref. (Galalou et al., 2011) presents data of pressure broadening coefficients reported in earlier works. The observation of these results does not illustrate any clear Line-mixing effects have received much less interest than line intensities and line broadening. This is mainly because they are rather small and their measurements are therefore difficult. However these spectroscopic parameters are necessary to detect molecules in tropospheric and planetary spectra, and to determine vertical concentration profiles. Moreover, even when laboratory measurements of these parameters are available, effects such as line-mixing may still require a sophisticated theoretical model in order to unravel observed spectra. When these effects are not negligible, the observed lineshapes exhibit significant deviations from the conventional Voigt profile, which may be attributed γν γ ν( ) (2 ) = 1.031 . quantum numbers do not exceed 4% with a weighted average ratio 2 2 in the ν2, 2ν2 and ν4 bands of NH3 are fitted using the following equation. are statistically well-determined and have remarkable regularities. 3.5.3 Vibrational dependence of pressure broadening at low J quantum number. obtained by these authors. differences between the bands. 3.6 Line-mixing effect to line-mixing. partners (B1 = B2 for CH3Br auto-perturbed). The spherical harmonic orders i A depend on the nature of the interaction. Therefore for the self- broadening dominated by the dipoledipole interaction ( A <sup>1</sup> = A <sup>2</sup> = l), the maximum of collision effectiveness is around Jmax = 18 in reasonable agreement with the experimental observation in Fig. 11. Fig. 11. Experimental self-broadening coefficients γ as function of the rotational quantum number J for the ν2 band PQ branch of 12CH379Br and 12CH381Br for K=0 to 9. For a given J value, the symbols indicate the different K-lines for which measurements are performed #### 3.5.2 Polynomial fit of broadening coefficients A constant need exists to examine the accuracy of the spectral line parameters listed in databases (Jacquinet-Husson et al., 2005; Perrin et al., 2005; Pickett et al., 1998; Rothman et al., 2009). For example, the NH3 dataset in these databases has been revised by the inclusion of recent results (Brown & Peterson, 1994; Kleiner et al., 1999, 2003; Nemtchinov et al., 2004) but still needs to be improved and purged from possible errors in line parameters. However, generating the spectroscopic parameters represents a lot of work. This is why sometimes, due to the absence of the necessary information, the spectroscopic parameters obtained for lines of a given vibrational band are attributed to the corresponding lines of another band. For example, in the Hitran database, the air-broadening coefficients of OCS lines of the excited vibrational states are based on the ν1 band measurement. In this context, in order to provide empirical interpolation and extrapolation models for all spectral lines, some authors used empirical polynomial equations (Brown & Peterson, 1994; Aroui et al., 2009; Devis et al., 2002; Fabian & Yamada, 1999; Jacquemart et al., 2007) to fit the J and K dependencies of their pressure broadening coefficients. Some least squares regressions fail to fit the observed values for particular quantum numbers. Other fits were satisfactory for nonpolar buffers, but fit poorly for self-broadening at low J quantum number. In a recent paper (Aroui et al., 2009), the measured broadening coefficients of nine branches in the ν2, 2ν2 and ν4 bands of NH3 are fitted using the following equation. $$\gamma(\mathbf{m}, \mathbf{K}) = \beta\_0 + \beta\_1 \,\mathbf{m} + \beta\_2 \,\mathbf{K} + \beta\_3 \,\mathbf{m}^2 + \beta\_4 \,\mathbf{K}^2 + \beta\_5 \,\mathbf{m} \,\mathbf{K} \tag{23}$$ The fitted parameters β0, β1, β2, β3, β4 and β5 are in unit of cm-1 atm-1. They are given in Ref. (Aroui et al., 2009), along with the data points included in the fits. Most of the parameters are statistically well-determined and have remarkable regularities. ## 3.5.3 Vibrational dependence of pressure broadening Some authors have discussed (Markov et al., 1993; Pine & Markov, 2004; Nemtchinov et al., 2004, Brown & Peterson, 1994; Cottaz et al., 2001; Aroui et al., 2009) the vibrational dependence of broadening coefficients. In most cases the comparison of the measured results for different bands exhibits little vibrational dependence. Within experimental error, the broadening coefficients of ammonia, for example, are only slightly greater in the ν4 band than in the ν2 and 2ν2 bands. This is explained partially by the smaller splitting of the inversion doublets in the ν4 band than in the ν2 and 2ν2 ones. For example, when the ν2 and 2ν2 bands are compared, the differences between line broadening coefficients with similar quantum numbers do not exceed 4% with a weighted average ratio 2 2 γν γ ν( ) (2 ) = 1.031 . However, for this molecule, Cottaz et al. (Cottaz et al., 2001) have highlighted a vibrational broadening effect when they compared their results for the R(3,1) line measured in six different vibrational bands. The present result for this line is slightly greater (3%) than that obtained by these authors. For OCS-N2 and OCS-O2, pressure broadening coefficients in previous works for different bands (Bouanich et al., 1986; Domenech et al., 2000; Koshelev et al., 2006, 2009; Mouchet et al., 1985; Galalou et al., 2011), illustrate that no vibrational effect can be clearly identified. To clarify this effect, Ref. (Galalou et al., 2011) presents data of pressure broadening coefficients reported in earlier works. The observation of these results does not illustrate any clear differences between the bands. ## 3.6 Line-mixing effect 90 Fourier Transform – Materials Analysis partners (B1 = B2 for CH3Br auto-perturbed). The spherical harmonic orders i A depend on the nature of the interaction. Therefore for the self- broadening dominated by the dipoledipole interaction ( A <sup>1</sup> = A <sup>2</sup> = l), the maximum of collision effectiveness is around Jmax = 18 in 0 10 20 30 40 50 60 J A constant need exists to examine the accuracy of the spectral line parameters listed in databases (Jacquinet-Husson et al., 2005; Perrin et al., 2005; Pickett et al., 1998; Rothman et al., 2009). For example, the NH3 dataset in these databases has been revised by the inclusion of recent results (Brown & Peterson, 1994; Kleiner et al., 1999, 2003; Nemtchinov et al., 2004) but still needs to be improved and purged from possible errors in line parameters. However, generating the spectroscopic parameters represents a lot of work. This is why sometimes, due to the absence of the necessary information, the spectroscopic parameters obtained for lines of a given vibrational band are attributed to the corresponding lines of another band. For example, in the Hitran database, the air-broadening coefficients of OCS lines of the In this context, in order to provide empirical interpolation and extrapolation models for all spectral lines, some authors used empirical polynomial equations (Brown & Peterson, 1994; Aroui et al., 2009; Devis et al., 2002; Fabian & Yamada, 1999; Jacquemart et al., 2007) to fit the Fig. 11. Experimental self-broadening coefficients γ as function of the rotational quantum number J for the ν2 band PQ branch of 12CH379Br and 12CH381Br for K=0 to 9. For a given J value, the symbols indicate the different K-lines for which measurements are performed reasonable agreement with the experimental observation in Fig. 11. 0,0 3.5.2 Polynomial fit of broadening coefficients excited vibrational states are based on the ν1 band measurement. J and K dependencies of their pressure broadening coefficients. 0,1 0,2 0,3 γ(cm-1atm-1 ) 0,4 0,5 0,6 0,7 Line-mixing effects have received much less interest than line intensities and line broadening. This is mainly because they are rather small and their measurements are therefore difficult. However these spectroscopic parameters are necessary to detect molecules in tropospheric and planetary spectra, and to determine vertical concentration profiles. Moreover, even when laboratory measurements of these parameters are available, effects such as line-mixing may still require a sophisticated theoretical model in order to unravel observed spectra. When these effects are not negligible, the observed lineshapes exhibit significant deviations from the conventional Voigt profile, which may be attributed to line-mixing. Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 93 l k k d W <sup>Y</sup> <sup>≠</sup> d Δσ0=σ0<sup>l</sup> -σ0k where σ0k and σ0<sup>l</sup> are the unperturbed positions of the k and l lines respectively; However, Table 1 of Ref. (Aroui et al., 2009) presents exceptions to this behavior: the parameter Y of PQ branch lines decreases with quantum number K. The situation is different for RQ and RR branches where no systematic dependence of interference parameter with J Otherwise the Δσ0 splitting could be large enough to contain lines other than those pertaining to the doublet of interest. So the line-mixing effects can occur not only between the two components of the doublets but also between more than two transitions, provided On the other hand, the ν2 band has a large inversion splitting, 35.7 cm-1 for the vibrational quantum number v2 = 1. Thus the line-mixing is negligible and the inversion contribution to this effect should be smaller than for the ν3 and ν4 bands. In these bands (with very small inversion splitting, about 0.5 cm-1) the line-mixing is essentially dominated by collisional transitions across the inversion doublets with the selection rules ΔJ = 0, ΔK = 0 and a→s. These two bands are thus the more appropriate to study line-mixing effects in the infrared Measurements of line-shifts have been investigated by numerous authors for various molecules. For NH3, line shifts were measured by Dhib et al. in the ν4 and 2ν2 bands (Dhib et al., 2007), by Raynaud et al. in the ν2 and (2ν2-ν2) bands (Raynaud et al., 1994), by Baldacchini et al. in the ν2 band, (Baldacchini et al., 1982), and by Aroui et al. in ν2, 2ν2 and ν<sup>4</sup> For OCS, line shifts were measured by Domenech et al. (Domenech et al., 2000) for the 2ν<sup>3</sup> band, by Koshelev et al. in the ν3 band (Koshelev et al., 2006) and in the ground vibrational Fig. 13 shows typical plots of line shift σk-σ0k as function of NH3 pressure for the RasR(2,1) and PasP(4,4) lines of the ν4 band of NH3. The self-shift coefficients δ0 are determined as the As may be seen by Fig. 13 and as illustrated by the results listed in Ref. (Aroui et al., 2009), shift coefficients are both positive and negative. A sample of these results is given by Table 3 for the PP branch of the ν4 band. About two thirds of the lines have positive shifts, and the more bipolar shifts are obtained for the ν4 band. Several symmetric and asymmetric components of the isolated inversion doublets in this band have opposite sign for the shift coefficients, so they appear to be shifting towards each other. As expected this mutual lineattraction is negligible in the ν2 band, for which the splitting is very large compared to that state (Koshelev & Tretyakov, 2009), and by Babay (Babay, 1997) in the ν2 band. k dk and dl are their reduced matrix elements of the dipole moment. 2 l lk 0 <sup>=</sup> <sup>∑</sup> . (24) Δσ and K is observed. bands of NH3. that the selection rules are valid. 3.7 Pressure induced shift bands (Aroui et al., 2009). slopes of the best-fit lines. of the ν3 and ν4 bands. In the past decade, line-mixing effects have been examined for diverse molecules such as CO2 (Ozanne et al., 1999), CH4 (Pieroni et al., 1999), and OCS (Broquier & Picard-Bersellini, 1984). However for C3v molecules the number of works is less. Measurements of these effects have been reported for CH3F (Thibault et al., 1999), CH3Cl (Hartmann et al., 1995) and CH3Br (Tran et al., 2008). Otherwise, for NH3, which presents line doubling due to its inversion motion, line-mixing effects were the subject of numerous works (Aroui et al., 1998, 2009; Dhib et al., 2007; Hadded et al., 2001). These works have clearly demonstrated the necessity of taking into account line-mixing in the molecular spectra. We present below some line-mixing results for the NH3 molecule. Plots of PY deduced from least squares fits (Eq. (16)) as a function of the NH3 pressure P are shown in Fig. 12 for the components of the doublet, pertaining to the ν4 band. The line-mixing parameter Y (in atm-1) are determined as the slopes of the best-fit lines and are presented in Table 3 for the PP(5,K) and PP(6,K) manifolds of the PP branch of the ν4 band of NH3 at 295 K. The values given in parentheses correspond to the estimated errors corresponding to one standard deviation value. Fig. 12. Variation of PY as function of pressure P of NH3 for the components of PasP(5,4) doublet of the ν4 band of NH3 self-perturbed This table shows that symmetric (s) and asymmetric (a) components of inversion doublets have opposite sign of Y. For numerous lines, Y appears to increase with K quantum number for a given value of J. This behavior could be explained in part by the decrease of the frequency difference between the manifold transitions with K, and particularly by the decrease of the Δσ0 splitting inversion doublets components. This may be easily understood, since line-mixing parameter Y is inversely proportional to Δσ0. Indeed, for a line k, Yk is related to Δσ0 and the off-diagonal element Wlk (line-coupling coefficient) of the relaxation matrix by (Gentry & Larrabee Strow, 1997; Pine, 1997; Pieroni et al., 1999): $$Y\_k = \mathfrak{D} \sum\_{l \neq k} \frac{d\_l}{d\_k} \frac{\mathcal{W}\_{lk}}{\Delta \sigma\_0} \,. \tag{24}$$ Δσ0=σ0<sup>l</sup> -σ0k where σ0k and σ0<sup>l</sup> are the unperturbed positions of the k and l lines respectively; dk and dl are their reduced matrix elements of the dipole moment. However, Table 1 of Ref. (Aroui et al., 2009) presents exceptions to this behavior: the parameter Y of PQ branch lines decreases with quantum number K. The situation is different for RQ and RR branches where no systematic dependence of interference parameter with J and K is observed. Otherwise the Δσ0 splitting could be large enough to contain lines other than those pertaining to the doublet of interest. So the line-mixing effects can occur not only between the two components of the doublets but also between more than two transitions, provided that the selection rules are valid. On the other hand, the ν2 band has a large inversion splitting, 35.7 cm-1 for the vibrational quantum number v2 = 1. Thus the line-mixing is negligible and the inversion contribution to this effect should be smaller than for the ν3 and ν4 bands. In these bands (with very small inversion splitting, about 0.5 cm-1) the line-mixing is essentially dominated by collisional transitions across the inversion doublets with the selection rules ΔJ = 0, ΔK = 0 and a→s. These two bands are thus the more appropriate to study line-mixing effects in the infrared bands of NH3. #### 3.7 Pressure induced shift 92 Fourier Transform – Materials Analysis In the past decade, line-mixing effects have been examined for diverse molecules such as CO2 (Ozanne et al., 1999), CH4 (Pieroni et al., 1999), and OCS (Broquier & Picard-Bersellini, 1984). However for C3v molecules the number of works is less. Measurements of these effects have been reported for CH3F (Thibault et al., 1999), CH3Cl (Hartmann et al., 1995) and CH3Br (Tran et al., 2008). Otherwise, for NH3, which presents line doubling due to its inversion motion, line-mixing effects were the subject of numerous works (Aroui et al., 1998, 2009; Dhib et al., 2007; Hadded et al., 2001). These works have clearly demonstrated the We present below some line-mixing results for the NH3 molecule. Plots of PY deduced from least squares fits (Eq. (16)) as a function of the NH3 pressure P are shown in Fig. 12 for the components of the doublet, pertaining to the ν4 band. The line-mixing parameter Y (in atm-1) are determined as the slopes of the best-fit lines and are presented in Table 3 for the PP(5,K) and PP(6,K) manifolds of the PP branch of the ν4 band of NH3 at 295 K. The values given in parentheses correspond to the estimated errors corresponding to one standard deviation 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 PNH3 This table shows that symmetric (s) and asymmetric (a) components of inversion doublets have opposite sign of Y. For numerous lines, Y appears to increase with K quantum number for a given value of J. This behavior could be explained in part by the decrease of the frequency difference between the manifold transitions with K, and particularly by the decrease of the Δσ0 splitting inversion doublets components. This may be easily understood, since line-mixing parameter Y is inversely proportional to Δσ0. Indeed, for a line k, Yk is related to Δσ0 and the off-diagonal element Wlk (line-coupling coefficient) of the relaxation Fig. 12. Variation of PY as function of pressure P of NH3 for the components of PasP(5,4) matrix by (Gentry & Larrabee Strow, 1997; Pine, 1997; Pieroni et al., 1999): s P P(5,4) (atm) a P P(5,4) necessity of taking into account line-mixing in the molecular spectra. value. doublet of the ν4 band of NH3 self-perturbed 0,00 0,04 PY 0,08 0,12 Measurements of line-shifts have been investigated by numerous authors for various molecules. For NH3, line shifts were measured by Dhib et al. in the ν4 and 2ν2 bands (Dhib et al., 2007), by Raynaud et al. in the ν2 and (2ν2-ν2) bands (Raynaud et al., 1994), by Baldacchini et al. in the ν2 band, (Baldacchini et al., 1982), and by Aroui et al. in ν2, 2ν2 and ν<sup>4</sup> bands (Aroui et al., 2009). For OCS, line shifts were measured by Domenech et al. (Domenech et al., 2000) for the 2ν<sup>3</sup> band, by Koshelev et al. in the ν3 band (Koshelev et al., 2006) and in the ground vibrational state (Koshelev & Tretyakov, 2009), and by Babay (Babay, 1997) in the ν2 band. Fig. 13 shows typical plots of line shift σk-σ0k as function of NH3 pressure for the RasR(2,1) and PasP(4,4) lines of the ν4 band of NH3. The self-shift coefficients δ0 are determined as the slopes of the best-fit lines. As may be seen by Fig. 13 and as illustrated by the results listed in Ref. (Aroui et al., 2009), shift coefficients are both positive and negative. A sample of these results is given by Table 3 for the PP branch of the ν4 band. About two thirds of the lines have positive shifts, and the more bipolar shifts are obtained for the ν4 band. Several symmetric and asymmetric components of the isolated inversion doublets in this band have opposite sign for the shift coefficients, so they appear to be shifting towards each other. As expected this mutual lineattraction is negligible in the ν2 band, for which the splitting is very large compared to that of the ν3 and ν4 bands. Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 95 coefficient is δ0 = ± 0.38 10-3cm-1atm-1. This value is about 10 times smaller than those of the 4ν2 band. These reasons, and the fact that the line shift coefficients do not illustrate any systematic variation with m, leads one to conclude that they depend on the vibrational band Lines σ0(cm-1) δ0(10-3 cm-1 atm-1) Y(atm-1) PsP(5,1) 1536.2088 8.9 (5.3) 0.18 (0.02) PaP(5,1) 1538.0102 -10.7 (5.7) 0.44 (0.10) PsP(5,2) 1539.7596 10.7 (5.8) 0.43 (0.07) PaP(5,2) 1541.0041 9.0 (5.0) -0.25 (0.04) PsP(5,3) 1542.9798 12.3 (4.1) 0.45 (0.02) PaP(5,3) 1543.8550 -16.8 (6.6) -0.42 (0.04) PsP(5,4) 1545.8043 43.6 (5.6) 1.22 (0.06) PaP(5,4) 1546.3315 -35.6 (6.6) -1.10 (0.06) PsP(5,5) 1548.1839 55.0 (10.0) 4.00 (0.09) PaP(5,5) 1548.4290 -53.8 (10.9) -4.16 (0.16) PsP(6,1) 1519.6627 38.0 (6.3) -0.62 (0.03) PaP(6,1) 1522.3847 -13.5 (6.3) 0.74 (0.07) PsP(6,2) 1522.7763 40.8 (6.0) -0.81 (0.19) PaP(6,2) 1524.7088 21.9 (4.7) 0.32 (0.05) PsP(6,3) 1525.7625 47.3 (6.4) -0.33 (0.03) PaP(6,3) 1527.0615 18.4 (6.0) -0.41 (0.03) PsP(6,4) 1528.3774 18.6 (6.2) 0.74 (0.05) PaP(6,4) 1529.2897 8.6 (4.7) -0.67 (0.01) PsP(6,5) 1530.6141 53.4 (5.5) 1.11 (0.04) PaP(6,5) 1531.1591 -26.4 (7.3) -1.28 (0.02) PsP(6,6) 1532.4503 111.0 (9.4) 3.83 (0.11) PaP(6,6) 1532.6830 -100.8 (13.4) -4.03 (0.09) Table 3. Self shift coefficients δ0 (in 10-3 cm-1 atm-1) and self mixing parameters Y (in atm-1) for the PP branch of ν4 band of NH3 at 295 K. The values given in parentheses correspond to the estimated errors expressed as one time standard deviation It should be noted that one can determine the air-induced line shape parameters G (broadening coefficients, shift coefficient, or line-mixing parameter) for the collision between an active molecule A and air using the measured A-N2 and A-O2 parameters assuming binary collisions and a standard atmospheric composition of air (Tejwani & Varnassi, 1971): G A - Air = 0.79G A - N + 0.21G A - O ( ) ( 2 2 ) ( ) (25) but are rotationally independent. Fig. 13. Plots of the pressure-induced variation of σ-σ0 versus NH3 pressure P for the components of the doublets RasR(2,1) and PasP(4,4) of the ν4 band of NH3 self-perturbed The self-shift coefficients of ammonia are larger than those obtained for other perturbers like He, H2 and CO2 (Hadded et al., 2001, Dhib et al., 2006) since the collisions in NH3 are long range dipole-dipole collisions while the foreign gases collisions are shorter range collisions. For all rotational lines of the molecules considered in this work, the shift coefficients do not reveal any systematic dependencies on the J and K quantum numbers with the exception seen in Table 3 for the NH3 lines with K approaching J, for which the shift seems to increase with increasing K. For most of the lines of this molecule, the shifts are large, ranging from about -0.140 to +150 cm-1 atm-1. The smallest one is δ0 = 0.003 ± 0.002 cm-1 atm-1. These shifts are in excellent agreement with the submillimeter measurement (Belov et al., 1982) of the ground state of NH3 where δ0 = 0.153 ± 0.002 cm-1 atm-1, and somewhat larger than the ν<sup>2</sup> band diode laser measurement (Baldacchini et al., 1982) with δ0 = 0.113 ± 0.025 cm-1 atm-1. Similar large values of shifts are reported for other molecules such as CH3CN studied by Rinsland et al. (Rinsland et al., 2008) using a multi-spectrum fitting technique. For the R and P branches of the 4ν2 band of OCS molecule perturbed by O2 and N2, the shift coefficients are mostly positive. The largest one is δ0 = (17.91 ± 3.02)×10-3 cm-1atm-1, obtained for the R(51) line. These shifts are small compared with those of NH3 and CH3CN; this makes them difficult to measure as evidenced from their relatively large errors (Galalou et al., 2011). In the 2ν3 (Domenech et al., 2000), ν3 (Koshelev et al., 2006) and ν2 (Babay, 1997) bands of OCS molecule the shift coefficients were found negative for all studied lines. In the ground vibrational state (Koshelev & Tretyakov, 2009), the upper limits of the pressure shift 0,00 0,04 0,08 0,12 0,16 0,20 P/atm Fig. 13. Plots of the pressure-induced variation of σ-σ0 versus NH3 pressure P for the components of the doublets RasR(2,1) and PasP(4,4) of the ν4 band of NH3 self-perturbed Rinsland et al. (Rinsland et al., 2008) using a multi-spectrum fitting technique. The self-shift coefficients of ammonia are larger than those obtained for other perturbers like He, H2 and CO2 (Hadded et al., 2001, Dhib et al., 2006) since the collisions in NH3 are long range dipole-dipole collisions while the foreign gases collisions are shorter range collisions. For all rotational lines of the molecules considered in this work, the shift coefficients do not reveal any systematic dependencies on the J and K quantum numbers with the exception seen in Table 3 for the NH3 lines with K approaching J, for which the shift seems to increase with increasing K. For most of the lines of this molecule, the shifts are large, ranging from about -0.140 to +150 cm-1 atm-1. The smallest one is δ0 = 0.003 ± 0.002 cm-1 atm-1. These shifts are in excellent agreement with the submillimeter measurement (Belov et al., 1982) of the ground state of NH3 where δ0 = 0.153 ± 0.002 cm-1 atm-1, and somewhat larger than the ν<sup>2</sup> band diode laser measurement (Baldacchini et al., 1982) with δ0 = 0.113 ± 0.025 cm-1 atm-1. Similar large values of shifts are reported for other molecules such as CH3CN studied by For the R and P branches of the 4ν2 band of OCS molecule perturbed by O2 and N2, the shift coefficients are mostly positive. The largest one is δ0 = (17.91 ± 3.02)×10-3 cm-1atm-1, obtained for the R(51) line. These shifts are small compared with those of NH3 and CH3CN; this makes them difficult to measure as evidenced from their relatively large errors (Galalou et In the 2ν3 (Domenech et al., 2000), ν3 (Koshelev et al., 2006) and ν2 (Babay, 1997) bands of OCS molecule the shift coefficients were found negative for all studied lines. In the ground vibrational state (Koshelev & Tretyakov, 2009), the upper limits of the pressure shift R P R P R(2,1)s P(4,4)a R(2,1)a P(4,4)s σk - al., 2011). σ0k / cm-1 0,000 0,004 0,008 0,012 coefficient is δ0 = ± 0.38 10-3cm-1atm-1. This value is about 10 times smaller than those of the 4ν2 band. These reasons, and the fact that the line shift coefficients do not illustrate any systematic variation with m, leads one to conclude that they depend on the vibrational band but are rotationally independent. Table 3. Self shift coefficients δ0 (in 10-3 cm-1 atm-1) and self mixing parameters Y (in atm-1) for the PP branch of ν4 band of NH3 at 295 K. The values given in parentheses correspond to the estimated errors expressed as one time standard deviation It should be noted that one can determine the air-induced line shape parameters G (broadening coefficients, shift coefficient, or line-mixing parameter) for the collision between an active molecule A and air using the measured A-N2 and A-O2 parameters assuming binary collisions and a standard atmospheric composition of air (Tejwani & Varnassi, 1971): $$\text{G(A-Air)} = 0.79 \text{G(A-N}\_2) + 0.21 \text{G(A-O}\_2) \tag{25}$$ Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 97 Considering the unknown systematic uncertainties in sample pressure and temperature, and the absorption path length, the errors are often estimated as one or two standard deviations (1 or 2σ) derived from the linear fits, and vary widely depending on the quality of spectral lines, which is related to the overlapping of neighboring lines and the base line location. The experimental errors reported in Table 3 are estimated as one standard This table also reveals that the line-mixing parameters are more accurate than the shifts. The mean value of accuracy is about 15% for line-mixing parameters and 25% for the shift coefficients that can attain more than 50% or are of the same order of magnitude as the Today the achievable accuracies with FTIR are about 3% for line intensities, 5% for pressure broadening coefficients, and 15-20% for pressure-induced line shifts and line-mixing It should be noted that, in some instances, the broadening coefficient accuracies of some lines tends to have greater values, which can attain 10-15%. These errors can be explained by For the temperature dependence of the broadening coefficients, since the values of temperature coefficient n are determined from a power-law relation, the estimated errors in the derived values of n are larger than the corresponding errors in the measured broadening coefficients. Assuming an error of 5% for broadening measurements, the uncertainty of n The purpose of this paper was to highlight and demonstrate the ability of the Fourier Transform Infrared Spectrometer to measure spectroscopic parameters such as line intensities, line broadenings, line shifts and line-mixing parameters for a select number of Compared to the dispersive spectrometers, this technique has the advantage to measure Fourier transform spectroscopy was used here to study infrared spectra of three molecules: one linear, OCS, and two symmetric tops: NH3 and CH3Br which are of considerable interest These spectra were fitted using a multi-spectrum fitting technique and the first order model absorption coefficient taking into account line-mixing effects. In the considered pressure range, this model is adequate to extract with high enough accuracy line parameters for the studied molecules. For CH3Br a Voigt profile is used to extract the line parameters, indeed in this case no significant deviations are observed when the measured and calculated spectra The estimated errors vary widely depending on the quality of spectral lines with, in general, improving accuracies in the order: line intensities, line broadenings, line-mixing and line with high precision complete sets of line parameters over large spectral regions. for quantitative and qualitative studies of planetary and Earth's atmospheres. deviation as derived from the linear fit of line mixing and line shifts parameters. parameter themselves. This makes them negligible and difficult to measure reliably. the relatively low intensities and the overlaps with neighboring lines. 3.9 Line parameter accuracies parameters. values could be about 15%. 4. Conclusion small molecules. are compared. #### 3.8 Line-mixing and shift correlation It should be noted that for the NH3 molecule, within the measurement errors, almost all linemixing and -shifts vary linearly with the considered pressures. However for some lines with small rotational energy gaps, the observed shift- and mixing-coefficients exhibit quadratic dependencies on pressure. This situation is more remarkable for strongly overlapped inversion doublets of the ν4 band which have small inversion splitting Δσ0 compared to the rotational energy separation. This is illustrated by Fig. 14 for PasP(8,8) doublet with a splitting Δσ0=0.210 cm-1. This non linear dependence of line-mixing or line shift on pressure can be expressed as (Smith, 1981): $$ \sigma\_{\rm s/a} = \sigma\_{0\rm s/a} + \text{P}\mathbf{\hat{S}}\_{0\rm s/a} \pm \frac{\mathbf{S}\_{\rm s}}{\mathbf{S}\_{\rm a}} \frac{\Delta \sigma\_0}{4} \mathbf{P}^2 \mathbf{Y}\_{\rm s/a}^2,\tag{26} $$ where Ss/a, σ0s/a, δ0s/a and Ys/a are, respectively, the intensity, the unperturbed position, the shift coefficient and line-mixing parameter of the (s) or (a) components of the doublets. The sign (±) is considered for the (s) or (a) transitions of NH3. This equation illustrates a correlation between line-mixing and shift phenomena. This quadratic evolution of frequency shift with pressure has been also observed previously by Thibault et al. (Thibault et al., 1992) for the 0-1 and 0-2 bands of CO perturbed by He. Also, nonlinear NO-He and NO-Ar shifts have been observed by Vyrodov et al. using laser-induced fluorescence (Vyrodov et al., 1995). Fig. 14. Variation of PY as function of pressure P for the two components of PasP(8,8) doublet of the ν4 band of NH3 self-perturbed ## 3.9 Line parameter accuracies 96 Fourier Transform – Materials Analysis It should be noted that for the NH3 molecule, within the measurement errors, almost all linemixing and -shifts vary linearly with the considered pressures. However for some lines with small rotational energy gaps, the observed shift- and mixing-coefficients exhibit quadratic dependencies on pressure. This situation is more remarkable for strongly overlapped inversion doublets of the ν4 band which have small inversion splitting Δσ0 compared to the rotational energy separation. This is illustrated by Fig. 14 for PasP(8,8) doublet with a This non linear dependence of line-mixing or line shift on pressure can be expressed as s/a 0s/a 0s/a s/a where Ss/a, σ0s/a, δ0s/a and Ys/a are, respectively, the intensity, the unperturbed position, the shift coefficient and line-mixing parameter of the (s) or (a) components of the doublets. The sign (±) is considered for the (s) or (a) transitions of NH3. This equation illustrates a correlation between line-mixing and shift phenomena. This quadratic evolution of frequency shift with pressure has been also observed previously by Thibault et al. (Thibault et al., 1992) for the 0-1 and 0-2 bands of CO perturbed by He. Also, nonlinear NO-He and NO-Ar shifts have been observed by Vyrodov et al. using laser-induced fluorescence (Vyrodov et al., <sup>S</sup> Δσ = +P<sup>δ</sup> ± PY S 4 a 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 Fig. 14. Variation of PY as function of pressure P for the two components of PasP(8,8) doublet , (26) P s P/atm P a P(8,8) P(8,8) s 0 2 2 σ σ 3.8 Line-mixing and shift correlation splitting Δσ0=0.210 cm-1. (Smith, 1981): 1995). YP of the ν4 band of NH3 self-perturbed Considering the unknown systematic uncertainties in sample pressure and temperature, and the absorption path length, the errors are often estimated as one or two standard deviations (1 or 2σ) derived from the linear fits, and vary widely depending on the quality of spectral lines, which is related to the overlapping of neighboring lines and the base line location. The experimental errors reported in Table 3 are estimated as one standard deviation as derived from the linear fit of line mixing and line shifts parameters. This table also reveals that the line-mixing parameters are more accurate than the shifts. The mean value of accuracy is about 15% for line-mixing parameters and 25% for the shift coefficients that can attain more than 50% or are of the same order of magnitude as the parameter themselves. This makes them negligible and difficult to measure reliably. Today the achievable accuracies with FTIR are about 3% for line intensities, 5% for pressure broadening coefficients, and 15-20% for pressure-induced line shifts and line-mixing parameters. It should be noted that, in some instances, the broadening coefficient accuracies of some lines tends to have greater values, which can attain 10-15%. These errors can be explained by the relatively low intensities and the overlaps with neighboring lines. For the temperature dependence of the broadening coefficients, since the values of temperature coefficient n are determined from a power-law relation, the estimated errors in the derived values of n are larger than the corresponding errors in the measured broadening coefficients. Assuming an error of 5% for broadening measurements, the uncertainty of n values could be about 15%. ## 4. Conclusion The purpose of this paper was to highlight and demonstrate the ability of the Fourier Transform Infrared Spectrometer to measure spectroscopic parameters such as line intensities, line broadenings, line shifts and line-mixing parameters for a select number of small molecules. Compared to the dispersive spectrometers, this technique has the advantage to measure with high precision complete sets of line parameters over large spectral regions. Fourier transform spectroscopy was used here to study infrared spectra of three molecules: one linear, OCS, and two symmetric tops: NH3 and CH3Br which are of considerable interest for quantitative and qualitative studies of planetary and Earth's atmospheres. These spectra were fitted using a multi-spectrum fitting technique and the first order model absorption coefficient taking into account line-mixing effects. In the considered pressure range, this model is adequate to extract with high enough accuracy line parameters for the studied molecules. For CH3Br a Voigt profile is used to extract the line parameters, indeed in this case no significant deviations are observed when the measured and calculated spectra are compared. The estimated errors vary widely depending on the quality of spectral lines with, in general, improving accuracies in the order: line intensities, line broadenings, line-mixing and line Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 99 Brassewell, R., 1965. The Fourier Transform and its applications. McGraw-Hill Book Company, Brassington, D.J., 1988. In Proceedings of International Symposium on Monitoring of gaseous Broquier, M. & Picard-Bersellini, A., 1984. Overlapping effects and collisional narrowing in rotational doublets of OCS in the infrared. Chem. Phys. Lett., 111, 602-06. Brown, L.R. & Peterson, D.B., 1994. 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Quant. Spectrosc. Radiat. Transfer., mixing in the ν4 band of NH3 perturbed by N2. J. Quant. Spectrosc. Radiat. Transfer., in the ν4 band of NH3 perturbed by CO2 and He: experimental results and IRC2 and IRC7 sources: a new probe of physical conditions and abundances in N2 and O2 Pressure Broadening and Pressure Shift in the 4ν2 Band of 16O12C32S. de molecules linéaires à partir de l'élargissement des raies spectrales. C.R. Acad. Sci. Pollutant by Diode Laser, Freiburg, Germany, 17 October. Fourier series, Maths. Comput. 19, 297-301. regions. J. Mol. Spectrosc., 209, 30–49. from far-infrared measurements. J. Mol. Spectrosc., 168, 593–606. détection de polluants. C. R. Acad. Sci. Paris., t. 2, Série IV, 905-922. New York. press Inc. 72, 139-91. 107, 372-84. 198, 102-109. Paris., 272, 1252–55. shifts. Among these spectroscopic parameters, the line shifts are the smallest, so are the most difficult to measure with a high accuracy. More measurements were performed in the ν2, 2ν2 and ν4 bands of NH3 at room temperature and at low temperatures, as well as in the ν2 and ν6 bands of CH3Br and in the 4ν2 band of OCS. However, in the PP branch of the ν4 band of NH3, the shifts are correlated to the line-mixing parameters and exhibited significant non-linearity. The broadening coefficients do not show any pronounced vibrational effect. For NH3, the self-shift coefficients are rather large and more bipolar than foreign gas shifts with no significant branch or rotational dependencies. ## 5. Acknowledgments The authors are grateful to the "Ministry of Education and Scientific Research in Tunisia" who has supported this work. ## 6. References shifts. Among these spectroscopic parameters, the line shifts are the smallest, so are the most More measurements were performed in the ν2, 2ν2 and ν4 bands of NH3 at room temperature and at low temperatures, as well as in the ν2 and ν6 bands of CH3Br and in the 4ν2 band of OCS. However, in the PP branch of the ν4 band of NH3, the shifts are correlated to the line-mixing parameters and exhibited significant non-linearity. The broadening coefficients do not show any pronounced vibrational effect. For NH3, the self-shift coefficients are rather large and more bipolar than foreign gas shifts with no significant The authors are grateful to the "Ministry of Education and Scientific Research in Tunisia" Abrams, M.C., Toons, G.C. & Schindler, R.A., 1994. A practical example of the correction of Fourier transform spectra for detector nonlinearity. Appl. Opt., 33, 6307-6314. Aliev, M.R., Papousek, D. & Urban, S., 1987. Third-order theory of the line intensities in the Aroui, H. Broquier, M. Picard-Bersellini, A. Bouanich, J.P. Chevalier, M. & Gherissi, S., 1998. Aroui, H., Nouri, S. & Bouanich, J.P., 2003. NH3 self-broadening coefficients in the ν2 and ν<sup>4</sup> bands and line intensities in the ν2 band. J. Mol. Spectrosc., 220, 248-58. Auwera, J.V., 2004. 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Absorption intensities, pressure-broadening and line-mixing parameters of some lines of NH3 in the ν4 Band. J. Quant. Spectrosc. Radiat. Transfer., 60, 1011-1023. Aroui, H., Laribi, H., Orphal, J. & Chelin, P., 2009. Self-broadening, self-shift and self-mixing in the ν2, 2ν2 and ν4 bands of NH3. J. Quant. Spectrosc. Radiat. Transfer., 110, 2037- high resolution Fourier transform infrared spectroscopy. May, Université libre de theoretical investigations of self-broadening and self-shifting of ammonia M.Y., 1982. The study of microwave pressure lineshifts. J. Mol. Spectrosc., 94, 264-82. difficult to measure with a high accuracy. branch or rotational dependencies. 5. Acknowledgments 6. References 2059. Bruxelle. and London. who has supported this work. Spectrosc., 124, 285-305. Fourier Transform Infrared Spectroscopy for the Measurement of Spectral Line Profiles 101 Martin, S., Martin-Pintado, J., Mauersberger, R., Henkel C. & Garcia-Burillo, S., 2005. 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Global Ozone Research and 5 USA Chanel Fortier Fourier Transform Spectroscopy United States Department of Agriculture, Agricultural Research Service, The application of Fourier Transform (FT) spectroscopy instruments has broadened the knowledge and capabilities of textile researchers to study a host of projects including surface modifications of cotton fibers to impart flame retardant capabilities, monitoring aging effects on ancient cellulosic artifacts (Kavkler et al., 2011; Lojewski et al., 2010), determining the amount of dye fixation to fabrics (Choi, H-M, et al., 1994), and characterization of cotton byproducts during the processing of cotton (Cheng and Biswas, 2011), to name a few. As can be observed from the above examples, the power and In the textile community, it is very important to characterize cotton quality parameters since this directly affects its potential profitability. Specifically, cotton strength, length, micronaire, fineness, color, and trash amount are conventionally monitored using an Uster® High Volume Instrument (HVI). In regards to the characterization of trash comingled with the cotton fiber, the identity of trash components evaluated with the current HVI system is not possible. Recently, FT spectroscopy has been instrumental in identifying cotton trash components and foreign matter that are present with cotton fiber (Fortier et al., 2010). The ability to classify, quantify, and ultimately remove cotton trash and foreign matter present with cotton lint has the potential to increase the market value and durability of cotton from its conversion of fiber to yarn. When compared to dispersive instruments, some advantages of FT spectroscopy are higher signal-to-noise capabilities, higher throughput, negligible stray light, continuous spectra, and higher resolution. In addition, FT spectroscopy affords for frequencies in spectra to be measured all at once with more precise wavelength calibrations. FT spectroscopy clearly enhances the data garnered from near-infrared (NIR) and midinfrared (MIR) spectroscopy studies of cotton fiber and cotton trash components. Since Fourier Transform spectroscopy is a broad topic, a brief introduction of its application to FT-MIR spectroscopy, commonly known as Fourier Transform infrared (FT-IR) spectroscopy, is a branch of vibrational spectroscopy and is a primary method that yields structural versatility of analyzing textiles with FT spectroscopy is proven. mid-infrared and near infrared spectroscopy will be included. 1. Introduction 2. Theory of FT-MIR of Cotton and Cotton Trash Southern Regional Research Center, New Orleans, LA, ## Fourier Transform Spectroscopy of Cotton and Cotton Trash ## Chanel Fortier United States Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, New Orleans, LA, USA ## 1. Introduction 102 Fourier Transform – Materials Analysis Rosenkranz, P.W., 1975. Shape of the 5mm oxygen band in the atmosphere. IEEE Trans. Rothman, L.S., Gordon, I.E., Barbe, A., Benner, D.C., Bernath, P.F., Birk, M., Boudon, V., Smith, E.W., 1981. Absorption and dispersion in the O2 microwave spectrum at atmospheric Tejwani, G.D.T. & Varnassi, P., 1971. Theoretical line widths in N2O-N2O and N2O-air Thibault, F., Boissoles, B., Grigoriev, I.M., Filippov, N.N. & Tonkov, M.V., 1999. Line-mixing Thibault, F., Boissoles, J., Le Doucen, R., Farrenq, R., Morillon-Chapey, M. & Boulet, C., 1992. Thomas, V.M., Bedford, J.A. & Cicerone, R.J., 1997. Bromine emissions from leaded gasoline. Tran, H. Jacquemart, D., Mandin, J.Y. & Lacome, N., 2008. Line mixing in the ν6 Q branches U.S. Environmental Protection Agency, 2011. Gaseous Pollutants - Fourier Transform Vyrodov, A.O., Heinze, J. & Meier U.E., 1995. Collisional broadening of spectral lines in the Watson, J.K.G., 1992. Quadratic Herman-Wallis factors for symmetric- and asymmetric-top Watts, S.F., 2000. The mass budgets of carbonyl sulfide, dimethyl sulfide, carbon disulfide Woodney, L.M., McMullin, J. & Ahearn, M.F., 1997. Detection of OCS in comet Hyakutake, induced fluorescence. J. Quant. Spectrosc. Radiat. Transfer., 53, 277-87. Wartewig, S., 2003. IR and Raman spectroscopy: fundamental processing. Weinheim: Wiley- effects in the ν3 band of CH3F in helium: experimental band shapes and ECS Line by line measurements of interference parameters for the 0-1 and 0-2 bands of CO in He, and comparison with coupled-states calculations. J. Chem. Phys., 97, 4623- of self-and nitrogen-broadened methyl bromide. J. Quant. Spectrosc. Radiat. A-X system of NO by N2, Ar and He at elevated pressure measured by laser- Brown, L.R., Campargue, A., Champion, J.P., Chance, K., Coudert, L.H., Dana, V., Devi, V.M., Fally, S., Flaud, J-M., Gamache, R. R., Goldman, A., Jacquemart, D., Kleiner, I. et al., 2009. The HITRAN 2008 molecular spectroscopic database. Antennas Propag., 23, 498-506. J. Quant. Spectrosc. Radiat. Transfer. 110, 533-72. collisions. J. Quant. Spectrosc. Radiat. Transfer., 11, 1659-64. pressures. J. Chem. Phys., 74, 6658–6673. analysis. Eur. Phys. J. D., 6, 343-53. Geophys. Res. Lett., 24, 1371–1374. Infrared Spectroscopy (FTIR) molecules. J. Mol. Spectrosc., 153, 211-24. (C/1996 B2). Planet Space Sci., 45, 717-9. and hydrogen sulfide. Atmos Environ., 34, 761-79. Transfer., 109, 119-31. 32. VCH. The application of Fourier Transform (FT) spectroscopy instruments has broadened the knowledge and capabilities of textile researchers to study a host of projects including surface modifications of cotton fibers to impart flame retardant capabilities, monitoring aging effects on ancient cellulosic artifacts (Kavkler et al., 2011; Lojewski et al., 2010), determining the amount of dye fixation to fabrics (Choi, H-M, et al., 1994), and characterization of cotton byproducts during the processing of cotton (Cheng and Biswas, 2011), to name a few. As can be observed from the above examples, the power and versatility of analyzing textiles with FT spectroscopy is proven. In the textile community, it is very important to characterize cotton quality parameters since this directly affects its potential profitability. Specifically, cotton strength, length, micronaire, fineness, color, and trash amount are conventionally monitored using an Uster® High Volume Instrument (HVI). In regards to the characterization of trash comingled with the cotton fiber, the identity of trash components evaluated with the current HVI system is not possible. Recently, FT spectroscopy has been instrumental in identifying cotton trash components and foreign matter that are present with cotton fiber (Fortier et al., 2010). The ability to classify, quantify, and ultimately remove cotton trash and foreign matter present with cotton lint has the potential to increase the market value and durability of cotton from its conversion of fiber to yarn. When compared to dispersive instruments, some advantages of FT spectroscopy are higher signal-to-noise capabilities, higher throughput, negligible stray light, continuous spectra, and higher resolution. In addition, FT spectroscopy affords for frequencies in spectra to be measured all at once with more precise wavelength calibrations. FT spectroscopy clearly enhances the data garnered from near-infrared (NIR) and midinfrared (MIR) spectroscopy studies of cotton fiber and cotton trash components. Since Fourier Transform spectroscopy is a broad topic, a brief introduction of its application to mid-infrared and near infrared spectroscopy will be included. ## 2. Theory of FT-MIR FT-MIR spectroscopy, commonly known as Fourier Transform infrared (FT-IR) spectroscopy, is a branch of vibrational spectroscopy and is a primary method that yields structural Fourier Transform Spectroscopy of Cotton and Cotton Trash 105 software preferably over a wide range of samples. Chemometrics involves the application of statistics and mathematics to define chemical processes (Moros et al., 2010). With the evolution of chemometrics and high performance computing over the last 40 years, the popularity and use of FT-NIR spectroscopy has increased. Moros and co-workers defined three of the most traditional types of chemometric approaches to vibrational spectroscopy: Pre-processing techniques, classification methods, and regression methods. Pre-processing techniques encompasses pre-processing of the spectral data including derivative math, normalization, and baseline correction. Classification methods involve the application of pattern recognition, be it supervised or unsupervised. Multivariate regression methods include applications of principal component analysis (PCA) and partial least squares (PLS). A detailed description of a FT-NIR system has been previously reported (Griffiths & Haseth, 2007; Bell, 1972). Briefly, a typical FT-NIR spectrometer is composed of a source, interferometer, beamsplitter, laser, detector, and optical components. A halogen source is conventionally used. The interferometer includes a moving and stationary mirror perpendicular to each other which modulates the NIR source. A beamsplitter, made up of quartz, calcium fluoride or potassium bromide, splits the NIR signal between the moving and stationary mirror, then recombines the signal and directs it to the detector. The laser, commonly made of helium and neon, controls the moving mirror and aligns the interferometer. NIR spectrometers can be selected based on their response, speed, and detection capabilities. Other optical components, such as mirrors or lenses, can focus or FT-IR affords the use of fundamental frequencies to identify molecular functional groups in both solid and liquid media. Also, when used in conjunction with the ATR accessory, little or no sample preparation is required. However, FT-NIR spectroscopy offers distinct advantages, such as flexibility of multiple sampling systems (e.g. fiber optic probe, rotating sphere) and the option of analyzing powder-size, pepper-size, raw samples (e.g. "sticks"), and large cotton trash samples, which is particularly useful for analyzing a heterogeneous sample such as cotton. Like the ATR/FT-IR sampling system, the FT-NIR sampling system is also non-destructive, easy to use, and has a short analysis time. In addition, FT-NIR instruments are capable of carrying out measurements on samples with longer path lengths than that of FT-IR spectrometers (Perkampus, 1995). These advantages make the study of textiles using FT-NIR, specifically for cotton, very attractive (Rodgers, 2002; Thomasson and Shearer, 1995; Camjani and Muller, 1996; Thibodeaux, 1992; Rodgers and Beck, 2005; Cotton fiber is composed mostly of cellulose with a small percentage being made up of waxes, proteins, pectic substances, organic acids, ash, and sugars (Lewin, 1998). The percentage of each non-cellulosic content can vary based on the maturity, cotton variety, and environmental conditions. This desirable chemical makeup along with its physical Rodgers and Beck, 2009; Rodgers and Ghosh, 2008; Montalvo, et al., 1991). 5. Instrumentation of FT-NIR collimate the NIR signal (McCarthy & Kemeny, 2008). 6. Comparison of the FT-IR and FT-NIR 7. Cotton fibers information about the chemical makeup of a sample. Group absorption frequencies defined by wavelength regions over the range of 400-4000 cm-1 (25000-2500 nm) can be used to get qualitative information about the functional groups in a sample. Typically, organic functional groups are further defined by molecular vibrations, such as symmetric or antisymmetric stretching, which refer to an oscillation in bond length, or deformation, and bending, which describes an oscillation in bond angle (Pelletier et al., 2010). The atoms in molecules display three types of energy: translational, rotational, and vibrational. In terms of FT-IR spectroscopy, the degrees of freedom for linear and non-linear molecules are defined by 3n-5 and 3n-6, respectively. The 3n term is defined by the three dimensions of the Cartesian coordinates for both types of molecules. However, the linear molecule has 2 degrees associated with rotational energy and 3 degrees are associated with translational energy. For the non-linear molecules, 3 of the degrees are rotational and 3 are translational while the remaining corresponds to fundamental vibrations. Therefore, the net degrees of freedom for non-linear molecules are 3n-6, and for linear molecules it is 3n-5 (Jiskoot, & Crommelin, 2005). Recently, an attenuated total reflection FT-IR accessory has been widely used. The ATR accessory greatly simplifies or removes sample preparation compared to traditional FT-IR samples employing KBr pellets. Briefly, the ATR accessory operates on the principle that a highly reflective crystal such as diamond or germanium is used as the internal reflective element (IRE). This IRE has a higher index of refraction compared to the sample. The sample and IRE must be in close contact, usually accomplished with a high pressure clamp, to achieve best data acquisition results. This accessory also yields the advantage of the ability to study both solids and liquids (Mirabella, Jr., 1993). ## 3. Instrumentation for FT-IR Since a detailed description of an FT-IR instrument is beyond the focus of this chapter, the reader is referred to reference (Griffiths & de Haseth, 2007). Briefly, the FT-IR is based on the Michelson interferometer furnished with a stationary and moving mirror and beamsplitter angled by bisecting the planes of these two mirrors. There is also a source, sensitive detector, and computer (Colthup et al, 1975). The moving mirror is controlled by a transducer. In FT-IR spectroscopy, the interferogram in the time domain is converted to the frequency domain through a Fourier transformation, from which the IR spectrum is derived (Perkampus, 1995). ## 4. Theory of FT-NIR The NIR spectral region encompasses 700-2500 nm (4000-12000 cm-1) where the regions are defined as first, second, and third overtones or combination bands. In terms of the electromagnetic spectrum, it is situated between the visible and mid-infrared spectral regions. The primary absorbencies observed in the NIR spectral region are for the chemical species CHi, NHi, and OH. However, absorbencies for the carbonyl, sulfydryl, and amide groups can also be detected. In addition, the first, second, and third overtones in the NIR region can be related to fundamental frequencies in the FT-MIR region (Perkampus, 1995). The wide and overlapping combination and overtone spectral bands typically found using FT-NIR, can complicate the interpretation of its data. In addition, the non-fundamental spectra of FT-NIR, compared to FT-IR, require that calibrations are made using chemometric software preferably over a wide range of samples. Chemometrics involves the application of statistics and mathematics to define chemical processes (Moros et al., 2010). With the evolution of chemometrics and high performance computing over the last 40 years, the popularity and use of FT-NIR spectroscopy has increased. Moros and co-workers defined three of the most traditional types of chemometric approaches to vibrational spectroscopy: Pre-processing techniques, classification methods, and regression methods. Pre-processing techniques encompasses pre-processing of the spectral data including derivative math, normalization, and baseline correction. Classification methods involve the application of pattern recognition, be it supervised or unsupervised. Multivariate regression methods include applications of principal component analysis (PCA) and partial least squares (PLS). ## 5. Instrumentation of FT-NIR 104 Fourier Transform – Materials Analysis information about the chemical makeup of a sample. Group absorption frequencies defined by wavelength regions over the range of 400-4000 cm-1 (25000-2500 nm) can be used to get qualitative information about the functional groups in a sample. Typically, organic functional groups are further defined by molecular vibrations, such as symmetric or antisymmetric stretching, which refer to an oscillation in bond length, or deformation, and The atoms in molecules display three types of energy: translational, rotational, and vibrational. In terms of FT-IR spectroscopy, the degrees of freedom for linear and non-linear molecules are defined by 3n-5 and 3n-6, respectively. The 3n term is defined by the three dimensions of the Cartesian coordinates for both types of molecules. However, the linear molecule has 2 degrees associated with rotational energy and 3 degrees are associated with translational energy. For the non-linear molecules, 3 of the degrees are rotational and 3 are translational while the remaining corresponds to fundamental vibrations. Therefore, the net degrees of freedom for non-linear molecules are 3n-6, and for linear molecules it is 3n-5 (Jiskoot, & Crommelin, 2005). Recently, an attenuated total reflection FT-IR accessory has been widely used. The ATR accessory greatly simplifies or removes sample preparation compared to traditional FT-IR samples employing KBr pellets. Briefly, the ATR accessory operates on the principle that a highly reflective crystal such as diamond or germanium is used as the internal reflective element (IRE). This IRE has a higher index of refraction compared to the sample. The sample and IRE must be in close contact, usually accomplished with a high pressure clamp, to achieve best data acquisition results. This accessory also yields the advantage of the ability to study both solids and liquids (Mirabella, Jr., 1993). Since a detailed description of an FT-IR instrument is beyond the focus of this chapter, the reader is referred to reference (Griffiths & de Haseth, 2007). Briefly, the FT-IR is based on the Michelson interferometer furnished with a stationary and moving mirror and beamsplitter angled by bisecting the planes of these two mirrors. There is also a source, sensitive detector, and computer (Colthup et al, 1975). The moving mirror is controlled by a transducer. In FT-IR spectroscopy, the interferogram in the time domain is converted to the frequency domain through a Fourier transformation, from which the IR spectrum is derived (Perkampus, The NIR spectral region encompasses 700-2500 nm (4000-12000 cm-1) where the regions are defined as first, second, and third overtones or combination bands. In terms of the electromagnetic spectrum, it is situated between the visible and mid-infrared spectral regions. The primary absorbencies observed in the NIR spectral region are for the chemical species CHi, NHi, and OH. However, absorbencies for the carbonyl, sulfydryl, and amide groups can also be detected. In addition, the first, second, and third overtones in the NIR region can be related to fundamental frequencies in the FT-MIR region (Perkampus, 1995). The wide and overlapping combination and overtone spectral bands typically found using FT-NIR, can complicate the interpretation of its data. In addition, the non-fundamental spectra of FT-NIR, compared to FT-IR, require that calibrations are made using chemometric bending, which describes an oscillation in bond angle (Pelletier et al., 2010). 3. Instrumentation for FT-IR 1995). 4. Theory of FT-NIR A detailed description of a FT-NIR system has been previously reported (Griffiths & Haseth, 2007; Bell, 1972). Briefly, a typical FT-NIR spectrometer is composed of a source, interferometer, beamsplitter, laser, detector, and optical components. A halogen source is conventionally used. The interferometer includes a moving and stationary mirror perpendicular to each other which modulates the NIR source. A beamsplitter, made up of quartz, calcium fluoride or potassium bromide, splits the NIR signal between the moving and stationary mirror, then recombines the signal and directs it to the detector. The laser, commonly made of helium and neon, controls the moving mirror and aligns the interferometer. NIR spectrometers can be selected based on their response, speed, and detection capabilities. Other optical components, such as mirrors or lenses, can focus or collimate the NIR signal (McCarthy & Kemeny, 2008). ## 6. Comparison of the FT-IR and FT-NIR FT-IR affords the use of fundamental frequencies to identify molecular functional groups in both solid and liquid media. Also, when used in conjunction with the ATR accessory, little or no sample preparation is required. However, FT-NIR spectroscopy offers distinct advantages, such as flexibility of multiple sampling systems (e.g. fiber optic probe, rotating sphere) and the option of analyzing powder-size, pepper-size, raw samples (e.g. "sticks"), and large cotton trash samples, which is particularly useful for analyzing a heterogeneous sample such as cotton. Like the ATR/FT-IR sampling system, the FT-NIR sampling system is also non-destructive, easy to use, and has a short analysis time. In addition, FT-NIR instruments are capable of carrying out measurements on samples with longer path lengths than that of FT-IR spectrometers (Perkampus, 1995). These advantages make the study of textiles using FT-NIR, specifically for cotton, very attractive (Rodgers, 2002; Thomasson and Shearer, 1995; Camjani and Muller, 1996; Thibodeaux, 1992; Rodgers and Beck, 2005; Rodgers and Beck, 2009; Rodgers and Ghosh, 2008; Montalvo, et al., 1991). ## 7. Cotton fibers Cotton fiber is composed mostly of cellulose with a small percentage being made up of waxes, proteins, pectic substances, organic acids, ash, and sugars (Lewin, 1998). The percentage of each non-cellulosic content can vary based on the maturity, cotton variety, and environmental conditions. This desirable chemical makeup along with its physical Fourier Transform Spectroscopy of Cotton and Cotton Trash 107 present with the cotton lint. Thus, being able to accurately identify individual cotton trash components would lead to more efficient trash removal and favorable yarn and fabric Cotton trash has been previously analyzed by a variety of methods. A clustering analysis method was used to identify cotton trash components using sum of squares, fuzzy and neural networks (Xu et.al, 1999). The clustering approach was based on color, shape and size attributes. When comparing the different clustering approaches, the color features were more reliable than the shape and size parameters since trash particles can become smaller during the processing of cotton lint. The neural network clustering method yielded an accurate trash type classification greater than 95% of the time. However, a limitation of this Siddaiah and co-workers also classified cotton trash components based on geometry. Their approach involved defining simple geometric shapes (disks, points, arcs, and straight lines) to identify bark leaf, and pepper trash. This technique, which incorporated imaging and intelligent pattern recognition, was highly accurate with 98% correct classification of the A machine vision system comparing a camera- and scanner-based imaging technique was developed (Siddaiah et.al, 2006). The Cotton Trash Identification System (CTIS) was compared to conventional instrumentation such as the Uster® HVI, Advanced Fiber Information System (AFIS), and the Shirley Analyzer for measuring cotton trash. It was determined that surface based measurements (CTIS, HVI) had a higher correlation compared to volumetric measurement systems (AFIS and Shirley Analyzer). The CTIS system, calibrated using "classer" calls (human visual identification) was later found to accurately identify trash categories (bark/grass, stick, leaf, and pepper) 97% of the time. Fortier and co-workers investigated the capability of identifying botanical cotton trash types using UV-Vis spectroscopy (Fortier et al., 2011a). Figure 1 shows the result of applying first derivative math to classify cotton and some botanical trash types (hull, leaf, seed coat, and stem). The chemometric software package employed in their study did not allow the use of sub-libraries to distinctly identify the highly similar trash spectra. Thus, 67% of the 10. Applications of fourier transform spectroscopy to cotton and cotton trash Cotton trash has been reported to reduce the efficacy of textile processing (Bargeron et al. 1988, Verschraege, 1989). After cotton fiber is separated from the cottonseed, the fiber is dried, cleaned and converted into yarn through different spinning techniques. The three most commonly used spinning methods are ring, open-end rotor, and vortex spinning. Ring spinning is characterized by a twist inserted into a yarn by a circulating traveler with the individual trash samples were correctly identified as shown in Table 1. 9. Non-fourier transform techniques to analyze cotton trash production. 9.1 Geometric/imaging approaches trash types (Siddaiah, 1999). 9.2 UV-Vis spectroscopy 10.1 Trash effect on spinning efficiency method was the time required for computation. properties makes cotton a very important agricultural crop in both domestic and international markets. The popularity of this natural fiber for many years stems in part from its versatility (used in apparel, bedding, home furnishings, and industrial products), appearance, and performance (Wakelyn et al., 2007). ## 8. Cotton trash ## 8.1 Impact of cotton trash on the industry Cotton trash present with cotton fiber affects the quality, appearance, price paid by textile mills to producers, yarn and fabric properties, as well as the performance of ginning and spinning equipment (Himmelsbach et al., 2006). In addition, cotton trash identification is important to the textile industry because certain types of trash have different effects on the textile processing of cotton and the marketability of the final fabric. Previous reports have shown the deleterious effects of cotton trash on yarn quality. Yarn breakage during spinning increased nearly 60% with a 1% increase in bark content when cotton fiber was processed with standard cleaners and carding equipment (Brashears et al, 1992). Veit and co-workers found a correlation between yarn imperfections and seed coat content present with cotton fiber (Veit et al., 1996). It was also determined that seed coat trash could be implicated in the onset of yarn breaks, deposits in rotor spinning equipment, and nep formation (Frey & Schneider, 1989). Thus, identifying cotton trash components present with the fiber may lead to more effective instrumental techniques that could be designed to remove the trash content co-mingled with cotton fiber. #### 8.2 Cotton trash from field to fabric Initially, raw cotton has both fiber and trash material present. In the U.S., cotton is conventionally machine-stripped or machine-picked during harvesting. It is then sent to a gin where the cotton fiber is separated from the cottonseed. The cotton fiber separated from the cottonseed is referred to as the cotton lint. The cotton lint is then cleaned to remove the cotton trash. During the ginning and lint cleaning methods, cotton trash present with the lint tends to become smaller and smaller in size. Next, the cotton lint is classified by the Agricultural Marketing Service (AMS), and sold to textile mills where the yarn and fabric is developed. It is necessary to clean the raw cotton because the value of cotton lint is strongly influenced by the amount of cotton trash present. Samples from each bale of American cotton are visually examined. Human "classers" give each bale a leaf grade and extraneous matter description. The leaf grade goes from 1-8 with leaf grade 1 having the least amount of trash and leaf grade 8 having the most amount of trash. Obviously, this method's accuracy and precision along with being time-consuming and labor intensive is limited by the subjective evaluation of a human "classer". Thus, instrumental techniques which can reproducibly classify cotton and the trash found with it are desired. An instrumental method employing the Uster® High Volume Instrument (HVI) yields information on key quality parameters such as length, strength, color, micronaire, length uniformity, as well as trash content. The Shirley Analyzer is another conventional method to analyze cotton trash through and aero-mechanical technique which separate cotton trash from lint. However, these methods do not yield individual trash component identification present with the cotton lint. Thus, being able to accurately identify individual cotton trash components would lead to more efficient trash removal and favorable yarn and fabric production. ## 9. Non-fourier transform techniques to analyze cotton trash ## 9.1 Geometric/imaging approaches 106 Fourier Transform – Materials Analysis properties makes cotton a very important agricultural crop in both domestic and international markets. The popularity of this natural fiber for many years stems in part from its versatility (used in apparel, bedding, home furnishings, and industrial products), Cotton trash present with cotton fiber affects the quality, appearance, price paid by textile mills to producers, yarn and fabric properties, as well as the performance of ginning and spinning equipment (Himmelsbach et al., 2006). In addition, cotton trash identification is important to the textile industry because certain types of trash have different effects on the textile processing of cotton and the marketability of the final fabric. Previous reports have shown the deleterious effects of cotton trash on yarn quality. Yarn breakage during spinning increased nearly 60% with a 1% increase in bark content when cotton fiber was processed with standard cleaners and carding equipment (Brashears et al, 1992). Veit and co-workers found a correlation between yarn imperfections and seed coat content present with cotton fiber (Veit et al., 1996). It was also determined that seed coat trash could be implicated in the onset of yarn breaks, deposits in rotor spinning equipment, and nep formation (Frey & Schneider, 1989). Thus, identifying cotton trash components present with the fiber may lead to more effective instrumental techniques that could be designed to remove the trash Initially, raw cotton has both fiber and trash material present. In the U.S., cotton is conventionally machine-stripped or machine-picked during harvesting. It is then sent to a gin where the cotton fiber is separated from the cottonseed. The cotton fiber separated from the cottonseed is referred to as the cotton lint. The cotton lint is then cleaned to remove the cotton trash. During the ginning and lint cleaning methods, cotton trash present with the lint tends to become smaller and smaller in size. Next, the cotton lint is classified by the Agricultural Marketing Service (AMS), and sold to textile mills where the yarn and fabric is It is necessary to clean the raw cotton because the value of cotton lint is strongly influenced by the amount of cotton trash present. Samples from each bale of American cotton are visually examined. Human "classers" give each bale a leaf grade and extraneous matter description. The leaf grade goes from 1-8 with leaf grade 1 having the least amount of trash and leaf grade 8 having the most amount of trash. Obviously, this method's accuracy and precision along with being time-consuming and labor intensive is limited by the subjective evaluation of a human "classer". Thus, instrumental techniques which can reproducibly An instrumental method employing the Uster® High Volume Instrument (HVI) yields information on key quality parameters such as length, strength, color, micronaire, length uniformity, as well as trash content. The Shirley Analyzer is another conventional method to analyze cotton trash through and aero-mechanical technique which separate cotton trash from lint. However, these methods do not yield individual trash component identification appearance, and performance (Wakelyn et al., 2007). 8.1 Impact of cotton trash on the industry content co-mingled with cotton fiber. 8.2 Cotton trash from field to fabric classify cotton and the trash found with it are desired. developed. 8. Cotton trash Cotton trash has been previously analyzed by a variety of methods. A clustering analysis method was used to identify cotton trash components using sum of squares, fuzzy and neural networks (Xu et.al, 1999). The clustering approach was based on color, shape and size attributes. When comparing the different clustering approaches, the color features were more reliable than the shape and size parameters since trash particles can become smaller during the processing of cotton lint. The neural network clustering method yielded an accurate trash type classification greater than 95% of the time. However, a limitation of this method was the time required for computation. Siddaiah and co-workers also classified cotton trash components based on geometry. Their approach involved defining simple geometric shapes (disks, points, arcs, and straight lines) to identify bark leaf, and pepper trash. This technique, which incorporated imaging and intelligent pattern recognition, was highly accurate with 98% correct classification of the trash types (Siddaiah, 1999). A machine vision system comparing a camera- and scanner-based imaging technique was developed (Siddaiah et.al, 2006). The Cotton Trash Identification System (CTIS) was compared to conventional instrumentation such as the Uster® HVI, Advanced Fiber Information System (AFIS), and the Shirley Analyzer for measuring cotton trash. It was determined that surface based measurements (CTIS, HVI) had a higher correlation compared to volumetric measurement systems (AFIS and Shirley Analyzer). The CTIS system, calibrated using "classer" calls (human visual identification) was later found to accurately identify trash categories (bark/grass, stick, leaf, and pepper) 97% of the time. ## 9.2 UV-Vis spectroscopy Fortier and co-workers investigated the capability of identifying botanical cotton trash types using UV-Vis spectroscopy (Fortier et al., 2011a). Figure 1 shows the result of applying first derivative math to classify cotton and some botanical trash types (hull, leaf, seed coat, and stem). The chemometric software package employed in their study did not allow the use of sub-libraries to distinctly identify the highly similar trash spectra. Thus, 67% of the individual trash samples were correctly identified as shown in Table 1. ## 10. Applications of fourier transform spectroscopy to cotton and cotton trash ## 10.1 Trash effect on spinning efficiency Cotton trash has been reported to reduce the efficacy of textile processing (Bargeron et al. 1988, Verschraege, 1989). After cotton fiber is separated from the cottonseed, the fiber is dried, cleaned and converted into yarn through different spinning techniques. The three most commonly used spinning methods are ring, open-end rotor, and vortex spinning. Ring spinning is characterized by a twist inserted into a yarn by a circulating traveler with the Fourier Transform Spectroscopy of Cotton and Cotton Trash 109 using a diverse set of ginning methods. The objective of this study was to identify the type of cotton and trash type or types built up in the rotor groove. The rotor dust was collected and analyzed by FT-IR and compared to the spectral database (calibration set). In the spectral database, the top five matches were provided for each sample. Classification results were determined by counting the number spectral matches for a particular type of trash as shown in Figure 2. The trash collected in the rotor grove was determined to be largely composed of shale and hull trash types (Foulk, 2004). 85 Hull vein 57 Hill outside <sup>36</sup> <sup>24</sup> Hill inside Shale inside Hill stem Trash type Fig. 2. FT-IR spectra matched hits for open end spinning rotor dust. Source: Foulk, J. et al, Allen and co-workers conducted an ATR/FT-IR spectroscopy study to analyze botanical cotton trash (leaf, seed coat, stem, and hull) subjected to different temperatures and particle sizes typically observed in a ginning or textile mill environment (Allen et al., 2007). During ginning the cotton fiber temperature is raised to aid in the separation of cotton trash from the lint. Also, raw trash components become smaller and smaller due to mechanical treatment of the cotton fiber. This effect makes it very challenging to determine the origin of The cotton trash components were evaluated at elevated (149 °C) and standard room temperature conditions (22 °C). The FT-IR spectral data was obtained at 21 °C using a benchtop FT-IR instrument fitted with an ATR accessory. To determine the effects of the two temperature settings, the ratio of the area under the total spectrum to the area under each of the IR bands of interest were calculated. The assigned ATR/FT-IR bands included in this study were based on the O-H stretch at 3300 cm-1, the C-H asymmetric stretch at 2900 cm-1, the C-H symmetric stretch at 2850 cm-1, the C=O stretch of a carboxylic acid and ester at 1700 cm-1, and the C=O stretch of an acid salt at 1600 cm-1 (Chung et al., 2004; Silverstein and Webster, 1998). All heated samples were allowed to return to room temperature to observe any irreversible changes in the FT-IR spectra. No change in trash color was observed in <sup>9</sup> <sup>2</sup> <sup>1</sup> Grass Bract 166 Shale vein 2004, Journal of Cotton Science, Vol. 8, p.249 10.2 Size effects and heat treatment the trash particles. Hits Fig. 1. Average first derivative UV-Vis spectra of cotton, hull, leaf, seed coat and stem botanical trash. Source: Fortier, C., et al., 2011a, Journal of Cotton Science, Vol. 15, pp.174. Table 1. UV-Vis identification results of botanical trash types Source: Fortier, C., et al., 2011a, Journal of Cotton Science, Vol. 15, p.173 winding action made possible by a rotating spindle. In open-end rotor spinning, cotton fiber bundles from the sliver (loose piece of cotton fiber) are separated into individual fibers by a roller which opens and an air stream. Vortex spinning has become available commercially in recent years. This spinning type is characterized by cotton fibers being introduced into a spindle orifice by way of air jet technology. One of the three spinning types, open-end rotor spinning efficiency has been found to be greatly influenced by the presence of cotton trash (Baker, et al., 1994). The presence of cotton contamination in the rotor groove has been associated with the onset of yarn breakage, thick places in the yarn, and yarn entanglement (Thibodeaux & Baril, 1981; McCreight et al., 1997). Compounding this issue is the tendency for trash types to become smaller and smaller in size making it virtually impossible to visibly identify individual cotton trash components. Foulk and co-workers carried out an FT-IR classification study where cotton trash components (e.g. hull vein, hull outside, hull stem, hull inside, shale vein, shale) were evaluated based on color differences (Foulk et al., 2004). Pima and upland cotton varieties having widely different trash amounts and moisture levels were analyzed Wavelength, nm Fig. 1. Average first derivative UV-Vis spectra of cotton, hull, leaf, seed coat and stem botanical trash. Source: Fortier, C., et al., 2011a, Journal of Cotton Science, Vol. 15, pp.174. Trash Type No. of Samples No. Correct % Correct Hull 27 14 52% Leaf 30 16 53% Seed Coat 12 9 75% Stem 27 25 93% Total 96 64 67% Table 1. UV-Vis identification results of botanical trash types Source: Fortier, C., et al., 2011a, winding action made possible by a rotating spindle. In open-end rotor spinning, cotton fiber bundles from the sliver (loose piece of cotton fiber) are separated into individual fibers by a roller which opens and an air stream. Vortex spinning has become available commercially in recent years. This spinning type is characterized by cotton fibers being introduced into a spindle orifice by way of air jet technology. One of the three spinning types, open-end rotor spinning efficiency has been found to be greatly influenced by the presence of cotton trash The presence of cotton contamination in the rotor groove has been associated with the onset of yarn breakage, thick places in the yarn, and yarn entanglement (Thibodeaux & Baril, 1981; McCreight et al., 1997). Compounding this issue is the tendency for trash types to become smaller and smaller in size making it virtually impossible to visibly identify individual cotton trash components. Foulk and co-workers carried out an FT-IR classification study where cotton trash components (e.g. hull vein, hull outside, hull stem, hull inside, shale vein, shale) were evaluated based on color differences (Foulk et al., 2004). Pima and upland cotton varieties having widely different trash amounts and moisture levels were analyzed 250 270 290 310 Hull Leaf Seed Coat Stem Cotton Journal of Cotton Science, Vol. 15, p.173 (Baker, et al., 1994). 0.00E+00 5.00E-02 Intensity 1.00E-01 1.50E-01 2.00E-01 using a diverse set of ginning methods. The objective of this study was to identify the type of cotton and trash type or types built up in the rotor groove. The rotor dust was collected and analyzed by FT-IR and compared to the spectral database (calibration set). In the spectral database, the top five matches were provided for each sample. Classification results were determined by counting the number spectral matches for a particular type of trash as shown in Figure 2. The trash collected in the rotor grove was determined to be largely composed of shale and hull trash types (Foulk, 2004). Fig. 2. FT-IR spectra matched hits for open end spinning rotor dust. Source: Foulk, J. et al, 2004, Journal of Cotton Science, Vol. 8, p.249 ## 10.2 Size effects and heat treatment Allen and co-workers conducted an ATR/FT-IR spectroscopy study to analyze botanical cotton trash (leaf, seed coat, stem, and hull) subjected to different temperatures and particle sizes typically observed in a ginning or textile mill environment (Allen et al., 2007). During ginning the cotton fiber temperature is raised to aid in the separation of cotton trash from the lint. Also, raw trash components become smaller and smaller due to mechanical treatment of the cotton fiber. This effect makes it very challenging to determine the origin of the trash particles. The cotton trash components were evaluated at elevated (149 °C) and standard room temperature conditions (22 °C). The FT-IR spectral data was obtained at 21 °C using a benchtop FT-IR instrument fitted with an ATR accessory. To determine the effects of the two temperature settings, the ratio of the area under the total spectrum to the area under each of the IR bands of interest were calculated. The assigned ATR/FT-IR bands included in this study were based on the O-H stretch at 3300 cm-1, the C-H asymmetric stretch at 2900 cm-1, the C-H symmetric stretch at 2850 cm-1, the C=O stretch of a carboxylic acid and ester at 1700 cm-1, and the C=O stretch of an acid salt at 1600 cm-1 (Chung et al., 2004; Silverstein and Webster, 1998). All heated samples were allowed to return to room temperature to observe any irreversible changes in the FT-IR spectra. No change in trash color was observed in Fourier Transform Spectroscopy of Cotton and Cotton Trash 111 Absorbance 0.08 0.06 al., 2007, Journal of Cotton Science, Vol. 11, p.71 spectra accounting for these physical applications. 0.04 0.02 p.72 Absorbance 0.14 0.12 0.10 0.08 0.06 0.04 0.02 B C A 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 Wave numbers Fig. 5. The effect of heat treatment on the ATR/FT-IR spectra of the pepper-size seed coat samples. The blue curve represented the spectra at standard conditions and the red curve represents samples heated to 149 ºC for 20 minutes then allowed to return to standard conditions prior to analyzing. Source: Allen et al., 2007, Journal of Cotton Science, Vol. 11, In terms of the effect of particle size (powder, pepper, and raw), distinct spectral FT-IR differences over the spectral range of 3500 to 2800 cm-1 were observed for the stem, (Figure 6) leaf and hull samples (data not shown), but for the seed coat trash type (Figure 7) no observable differences were found. Pepper and powder–sized trash samples were generated using 20 (0.841 mm std. diameter) and 80 (0.177 mm std. diameter) meshes, respectively. > 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 Wave numbers Fig. 6. The effect of size reduction on the ATR/FT-IR spectra of the stem sample, including ground raw size (blue, A), pepper-size (black, B), and powder size (red, C) Source: Allen et Overall, it was concluded that size and heat treatment can affect the FT-IR spectra of cotton trash types. Thus, spectral databases used for classifying these components should include pepper-sized trash (0.841mm standard diameter). However, over the O-H and C-H stretching frequencies (2900 and 3300 cm-1), the stem and hull trash had an appreciable change (Figures 3 and 4) compared to the leaf and seed coat spectra (data not shown). For the IR bands representing the C=O stretching frequency (1600 and 1700 cm-1), the seed coat trash component has the most noticeable spectral difference (Figure 5) compared to the leaf, hull, and stem components. The relevance of this observation is that the seed coat may have volatile aliphatic esters which are generated with heat. Fig. 3. The effects of heat treatment on the ATR/FT-IR spectra of the pepper-size stem samples. The blue curve represented the spectra at standard conditions and the red curve represebts samples heated to 149 ºC for 20 minutes then allowed to return to standard conditions prior to analyzing. Source: Allen et al., 2007, Journal of Cotton Science, Vol. 11, p.72 Fig. 4. The effects of heat treatment on the ATR/FT-IR spectra of the pepper-size hull samples. The blue curve represented the spectra at standard conditions and the red curve represents samples heated to 149 ºC for 20 minutes then allowed to return to standard conditions prior to analyzing. Source: Allen et al., 2007, Journal of Cotton Science, Vol. 11, p.72 pepper-sized trash (0.841mm standard diameter). However, over the O-H and C-H stretching frequencies (2900 and 3300 cm-1), the stem and hull trash had an appreciable change (Figures 3 and 4) compared to the leaf and seed coat spectra (data not shown). For the IR bands representing the C=O stretching frequency (1600 and 1700 cm-1), the seed coat trash component has the most noticeable spectral difference (Figure 5) compared to the leaf, hull, and stem components. The relevance of this observation is that the seed coat may have > 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 Wave numbers 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 Wave numbers Fig. 4. The effects of heat treatment on the ATR/FT-IR spectra of the pepper-size hull samples. The blue curve represented the spectra at standard conditions and the red curve represents samples heated to 149 ºC for 20 minutes then allowed to return to standard conditions prior to analyzing. Source: Allen et al., 2007, Journal of Cotton Science, Vol. 11, Fig. 3. The effects of heat treatment on the ATR/FT-IR spectra of the pepper-size stem samples. The blue curve represented the spectra at standard conditions and the red curve represebts samples heated to 149 ºC for 20 minutes then allowed to return to standard conditions prior to analyzing. Source: Allen et al., 2007, Journal of Cotton Science, Vol. 11, volatile aliphatic esters which are generated with heat. 0.08 0.06 0.04 Absorbance p.72 0.15 0.10 0.05 Absorbance p.72 0.02 Fig. 5. The effect of heat treatment on the ATR/FT-IR spectra of the pepper-size seed coat samples. The blue curve represented the spectra at standard conditions and the red curve represents samples heated to 149 ºC for 20 minutes then allowed to return to standard conditions prior to analyzing. Source: Allen et al., 2007, Journal of Cotton Science, Vol. 11, p.72 In terms of the effect of particle size (powder, pepper, and raw), distinct spectral FT-IR differences over the spectral range of 3500 to 2800 cm-1 were observed for the stem, (Figure 6) leaf and hull samples (data not shown), but for the seed coat trash type (Figure 7) no observable differences were found. Pepper and powder–sized trash samples were generated using 20 (0.841 mm std. diameter) and 80 (0.177 mm std. diameter) meshes, respectively. Fig. 6. The effect of size reduction on the ATR/FT-IR spectra of the stem sample, including ground raw size (blue, A), pepper-size (black, B), and powder size (red, C) Source: Allen et al., 2007, Journal of Cotton Science, Vol. 11, p.71 Overall, it was concluded that size and heat treatment can affect the FT-IR spectra of cotton trash types. Thus, spectral databases used for classifying these components should include spectra accounting for these physical applications. Fourier Transform Spectroscopy of Cotton and Cotton Trash 113 Fig. 8. Average FT-MIR absorbance spectra of cotton and cotton trash components at a resolution of 8 cm-1 with 128 scans Source: Fortier et al., 2010, Textile Research Journal, Vol. of the pure botanical trash types in the prediction set. (full spectra range, and small spectral ranges) and pre-processing methods (vector normalization and derivative math) were investigated to optimize the correct identification Figure 9 show the results of comparing the average FT-NIR absorbance spectra for a clean cotton reference to pure botanical trash components. The "clean" cotton has specific bands represented at 1490 nm, 1930 nm, and 2100 nm. Over the spectral region of 1490 nm to 1600 nm which is referred to as the first overtone of the hydroxyl, the cotton band is more uniquely defined compared to the cotton trash. The band at 1450-1490 nm represents the OH stretch first overtone due to moisture and indicates the presence of OH groups in cotton. There was considerable overlap of cotton and cotton trash spectra at the very intense moisture band present at 1930 nm which is also the OH bend stretch and water deformation combination band. At 2100 nm, which is the OH bend/CO stretch combination, the band representing OH groups present in cotton, and to a lesser extent moisture, had considerable overlap with the cotton trash spectra even though the intensity of the cotton band is more pronounced. In terms of the cotton trash, there was considerable overlap throughout the entire spectral region with a defining peak over the spectral region of 1400 nm to 1500 nm which did not include a cotton peak. Even though cotton had distinctive spectral bands, the investigation was continued to gain non-overlapping spectral identity for all the trash components with a validation report showing no confused components (Fortier et al., 2010). 81, p. 233 Fig. 7. The effect of size reduction on the ATR/FT-IR spectra of the seed coat sample, including ground raw size (blue, A), pepper-size (black, B), and powder size (red, C) Source: Allen et al., 2007, Journal of Cotton Science, Vol. 11, p.71 ### 10.3 Botanical and synthetic cotton trash To expand on the proven utility of applying ATR/FTIR as a technique to specifically identify cotton trash, Himmelsbach and co-workers did an extensive study on botanical cotton trash and other foreign matter both organic (other fibers, yarns, etc.) and synthetic (plastic bags, film, etc.) that can become co-mingled with cotton lint during its harvesting, ginning, and processing (Himmelsbach et al., 2006). The development of an FT-IR spectral database yielded highly accuracte identification of cotton trash and foreign matter serving as a "proof of concept" of the utility of this instrumental method. Fortier and co-workers later compared their FT-IR results to that of this preliminary FT-IR study by Himmelsbach and co-workers (Fortier et al., 2010). The two studies yielded comparable results. Representative FT-IR absorbance spectra results of cotton, hull, leaf, seed coat, and stem are shown in Figure 8. The spectra are based on three replicates averaged with a resolution of 8 cm-1 and 128 scans. #### 10.4 Fourier-transform near-infrared spectroscopy of botanical cotton trash More recently, the application of FT-NIR spectroscopy has also been employed to identify individual botanical trash components (hull, leaf, seed coat, and stem) in a spectral library. Pepper-sized and powder-sized pure trash components were included in the calibration set. While pepper-sized, powder-sized and raw samples of different varieties were included in the prediction set. Specifically, the calibration set was different from the prediction set in that the corresponding powder and pepper samples in the calibration set were represented as the opposite powder and pepper samples in the prediction set. There were 9 different varieties of trash samples taken from Mississippi, New Mexico, and South Carolina. The cotton trash varieties are denoted by the first two letters from the state the samples were acquired in and the last letter as the sample variety as shown in Table 2. Sample measurements were taken by directly placing a solid probe in contact with the trash samples. When creating the calibration set (spectral library), multiple wavelength regions Absorbance 0.12 0.10 0.08 0.06 0.04 0.02 B C A Allen et al., 2007, Journal of Cotton Science, Vol. 11, p.71 10.3 Botanical and synthetic cotton trash cm-1 and 128 scans. 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 Wave numbers including ground raw size (blue, A), pepper-size (black, B), and powder size (red, C) Source: To expand on the proven utility of applying ATR/FTIR as a technique to specifically identify cotton trash, Himmelsbach and co-workers did an extensive study on botanical cotton trash and other foreign matter both organic (other fibers, yarns, etc.) and synthetic (plastic bags, film, etc.) that can become co-mingled with cotton lint during its harvesting, ginning, and processing (Himmelsbach et al., 2006). The development of an FT-IR spectral database yielded highly accuracte identification of cotton trash and foreign matter serving as a "proof of concept" of the utility of this instrumental method. Fortier and co-workers later compared their FT-IR results to that of this preliminary FT-IR study by Himmelsbach and co-workers (Fortier et al., 2010). The two studies yielded comparable results. Representative FT-IR absorbance spectra results of cotton, hull, leaf, seed coat, and stem are shown in Figure 8. The spectra are based on three replicates averaged with a resolution of 8 Fig. 7. The effect of size reduction on the ATR/FT-IR spectra of the seed coat sample, 10.4 Fourier-transform near-infrared spectroscopy of botanical cotton trash More recently, the application of FT-NIR spectroscopy has also been employed to identify individual botanical trash components (hull, leaf, seed coat, and stem) in a spectral library. Pepper-sized and powder-sized pure trash components were included in the calibration set. While pepper-sized, powder-sized and raw samples of different varieties were included in the prediction set. Specifically, the calibration set was different from the prediction set in that the corresponding powder and pepper samples in the calibration set were represented as the opposite powder and pepper samples in the prediction set. There were 9 different varieties of trash samples taken from Mississippi, New Mexico, and South Carolina. The cotton trash varieties are denoted by the first two letters from the state the samples were acquired in and the last letter as the sample variety as shown in Table 2. Sample measurements were taken by directly placing a solid probe in contact with the trash samples. When creating the calibration set (spectral library), multiple wavelength regions Fig. 8. Average FT-MIR absorbance spectra of cotton and cotton trash components at a resolution of 8 cm-1 with 128 scans Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 233 (full spectra range, and small spectral ranges) and pre-processing methods (vector normalization and derivative math) were investigated to optimize the correct identification of the pure botanical trash types in the prediction set. Figure 9 show the results of comparing the average FT-NIR absorbance spectra for a clean cotton reference to pure botanical trash components. The "clean" cotton has specific bands represented at 1490 nm, 1930 nm, and 2100 nm. Over the spectral region of 1490 nm to 1600 nm which is referred to as the first overtone of the hydroxyl, the cotton band is more uniquely defined compared to the cotton trash. The band at 1450-1490 nm represents the OH stretch first overtone due to moisture and indicates the presence of OH groups in cotton. There was considerable overlap of cotton and cotton trash spectra at the very intense moisture band present at 1930 nm which is also the OH bend stretch and water deformation combination band. At 2100 nm, which is the OH bend/CO stretch combination, the band representing OH groups present in cotton, and to a lesser extent moisture, had considerable overlap with the cotton trash spectra even though the intensity of the cotton band is more pronounced. In terms of the cotton trash, there was considerable overlap throughout the entire spectral region with a defining peak over the spectral region of 1400 nm to 1500 nm which did not include a cotton peak. Even though cotton had distinctive spectral bands, the investigation was continued to gain non-overlapping spectral identity for all the trash components with a validation report showing no confused components (Fortier et al., 2010). Fourier Transform Spectroscopy of Cotton and Cotton Trash 115 Fig. 11. Average first derivative spectra from sub-library for hull and seed coat. Source: MSA (powder) Hull Leaf Seed Coat Stem NMA (pepper) Hull Leaf NS Stem NMB (powder) Hull Leaf Seed Coat Stem NMC (pepper) Hull Leaf Seed Coat Stem SCA (pepper) Hull Leaf Seed Coat Stem SCB (powder) Hull Leaf Seed Coat Stem SCC (pepper) Hull NS Seed Coat Stem SCD (pepper) Hull Leaf Seed Coat Stem SCE (powder) Hull Leaf Seed Coat Stem Cotton Trash Variety and Size Trash Types MSA, NMB,NMA,NMC,SCA Raw seed meat FM989, SCE = South Carolina PM1218). Journal, Vol. 81, p. 231 NS = no sample. (The cotton trash varieties are denoted by the first two letters from the state the samples were acquired in and the last letter as the sample variety (MSA = Mississippi DP555, NMA = New Mexico DP555, NMB = New Mexico AC151799, NMC = New Mexico Unknown, SCA = South Carolina DP458, SCB = South Carolina DP555, SCC = South Carolina DP555a, SCD = South Carolina Table 2. Calibration set of cotton trash samples. Source: Fortier et al., 2010, Textile Research %Correct Number of samples Number Correct Table 3. NIR Identification by Cotton Trash Type for Powder and Pepper samples. Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 236 Prediction Set Individual Powder and Pepper Samplesa Hull 100% 27 27 Leaf 100% 27 27 Seed Coat 91.7% 24 22 Stem 100% 27 27 Total 98.1% 105 103 Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 236 Thus, the investigation was expanded to include different wavelength ranges and other preprocessing methods. First derivative math was then employed to uniquely identify the botanical trash types, and cotton as shown in Figure 10. This pre-processing was able to distinctly identify cotton, leaf, and stem. However, a sub-library consisting of hull and seed coat was necessary to distinguish these trash types due to their very similar spectra as depicted in Figure 11. Overall, result in Table 3 reveals the trash types were correctly identified 98% of the time. The 2 misidentifications for the seed coat was a direct effect due to the spectral similarity of the hull and seed coat trash types. Raw samples of each botanical trash type was also investigated to determine the sample-size effect on correct identification of hull, leaf, seed coat, and stem pure trash types. Table 4 reveals the results of this experiment. The result for the raw stem trash misidentifications could be explained by the irregular shape of the stems during sampling with the solid fiber optic probe. Fig. 9. Average FT-NIR absorbance spectra for "clean" cotton and cotton trash samples over entire spectral range 1100-2400 nm where cotton can be identified but cotton trash components are overlapping. Vector normalization and standard method pre-processing was applied Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 234 Fig. 10. Average FT-NIR spectra for cotton and cotton trash over the narrow spectral range of (1427-1867 nm) where cotton and cotton trash components are uniquely identified except for hull and seed coat. A factorization and first derivative pre-processing method was applied Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 236 Thus, the investigation was expanded to include different wavelength ranges and other preprocessing methods. First derivative math was then employed to uniquely identify the botanical trash types, and cotton as shown in Figure 10. This pre-processing was able to distinctly identify cotton, leaf, and stem. However, a sub-library consisting of hull and seed coat was necessary to distinguish these trash types due to their very similar spectra as depicted in Figure 11. Overall, result in Table 3 reveals the trash types were correctly identified 98% of the time. The 2 misidentifications for the seed coat was a direct effect due to the spectral similarity of the hull and seed coat trash types. Raw samples of each botanical trash type was also investigated to determine the sample-size effect on correct identification of hull, leaf, seed coat, and stem pure trash types. Table 4 reveals the results of this experiment. The result for the raw stem trash misidentifications could be explained by the Fig. 9. Average FT-NIR absorbance spectra for "clean" cotton and cotton trash samples over Fig. 10. Average FT-NIR spectra for cotton and cotton trash over the narrow spectral range of (1427-1867 nm) where cotton and cotton trash components are uniquely identified except for hull and seed coat. A factorization and first derivative pre-processing method was applied Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 236 entire spectral range 1100-2400 nm where cotton can be identified but cotton trash components are overlapping. Vector normalization and standard method pre-processing was applied Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 234 irregular shape of the stems during sampling with the solid fiber optic probe. Fig. 11. Average first derivative spectra from sub-library for hull and seed coat. Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 236 NS = no sample. (The cotton trash varieties are denoted by the first two letters from the state the samples were acquired in and the last letter as the sample variety (MSA = Mississippi DP555, NMA = New Mexico DP555, NMB = New Mexico AC151799, NMC = New Mexico Unknown, SCA = South Carolina DP458, SCB = South Carolina DP555, SCC = South Carolina DP555a, SCD = South Carolina FM989, SCE = South Carolina PM1218). Table 2. Calibration set of cotton trash samples. Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 231 Table 3. NIR Identification by Cotton Trash Type for Powder and Pepper samples. Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 236 Fourier Transform Spectroscopy of Cotton and Cotton Trash 117 Field trash refers to any trash found in a cotton field which is not originating from the cotton plant. Recently, FT-NIR spectroscopy was used to identify field trash samples including a grocery bag, black plastic bag, blue module cover, clear plastic bag, module cover strap, twine, and white module cover (Fortier et al., 2011b). Three replicates were acquired for each trash type. Figure 12 shows the average first derivative spectra of cotton and field trash types. Applying a "top down" approach, Fortier and co-workers added field trash samples, to the FT-NIR spectral library (Fortier et al., 2011b). First, the seed meat and field trash were isolated from the rest of the botanical trash library. Next, a sub-library of the cotton and botanical trash types was designed. Finally a second sub-library to separate hull and seed coat was developed. The identification results can be found in Table 5. Overall, greater than Prediction Set Individual Powder, Pepper, and Field Trash Samples Trash Type %Correct Number of Samples Number Correct This chapter has summarized the application of FT spectroscopy towards the identification and classification of cotton trash components which are found present with cotton lint. The successes reported herein were not possible with conventional techniques such as using the HVI and Shirley Analyzer. In the future, the feasibility of using FT techniques can benefit those in the textile industry such as farmers, ginners, spinners and merchants by providing a well-defined cotton product which has a minimal amount of Hull 100% 27 27 Leaf 100% 27 27 Seed Coat 95.24% 21 20 Seed Meat 91.67% 12 11 Stem 100% 27 27 Total Botanical Trash 98.25% 114 112 Grocery Bag 100% 2 2 Black Plastic Bag 100% 2 2 Blue Module Cover 100% 2 2 Clear Plastic Bag 100% 2 2 Module Cover Strap 100% 2 2 Twine 100% 2 2 White Module Cover 100% 2 2 Total Field Trash 100% 14 14 Overall Total Trash 98.44% 128 126 Table 5. NIR Identification by Cotton Trash Type for Individual Powder and Pepper samples adding seed meat and field trash. Source: Fortier et al., 2011b, Journal of Cotton 10.6 Field trash addition to FT-NIR spectral library 98% correct identifications were achieved. Science, in press 11. Summary trash and a high market value. Table 4. NIR Identification by Cotton Trash Type for Raw samples. Source: Fortier et al., 2010, Textile Research Journal, Vol. 81, p. 236 #### 10.5 Seed meat addition to botanical trash library Fortier and co-workers added seed meat to the FT-NIR spectral library to make it more robust (Fortier et al., 2011b). The seed meat spectra (data not shown) was quite different from the original trash components in the FT-NIR spectral library (hull, leaf, seed coat, and stem) Thus, simply adding the seed meat spectra to the existing library caused some misidentifications by making the other botanical trash components' thresholds overlap. As a result, a different approach was necessary to improve the number of correct identifications in the prediction set. A "top down" approach was employed where the seed meat was isolated first in the library. Next, the cotton and botanical trash types were included in the spectral library, and a second sub-library was made to separate the hull and seed coat, due to their highly similar spectra. The end result was that 100% of the trash types were identified correctly in the prediction set (data not shown). Fig. 12. Average first derivative spectra of field trash and cotton. Source: Fortier et al., 2011b, Journal of Cotton Science, in press %Correct Number of samples Number Correct Fortier and co-workers added seed meat to the FT-NIR spectral library to make it more robust (Fortier et al., 2011b). The seed meat spectra (data not shown) was quite different from the original trash components in the FT-NIR spectral library (hull, leaf, seed coat, and stem) Thus, simply adding the seed meat spectra to the existing library caused some misidentifications by making the other botanical trash components' thresholds overlap. As a result, a different approach was necessary to improve the number of correct identifications in the prediction set. A "top down" approach was employed where the seed meat was isolated first in the library. Next, the cotton and botanical trash types were included in the spectral library, and a second sub-library was made to separate the hull and seed coat, due to their highly similar spectra. The end result was that 100% of the trash types were > 1200 1400 1600 1800 2000 2200 2400 Wavelength (nm) Fig. 12. Average first derivative spectra of field trash and cotton. Source: Fortier et al., 2011b, Hull 100% 9 9 Leaf 100% 9 9 Seed Coat 100% 8 8 Stem 77.78% 9 7 Total 94.3% 35 33 Table 4. NIR Identification by Cotton Trash Type for Raw samples. Source: Fortier et al., Prediction Set Average Raw Samplesa 2010, Textile Research Journal, Vol. 81, p. 236 10.5 Seed meat addition to botanical trash library identified correctly in the prediction set (data not shown). 0.02 0.01 0.00 Grocery bag Black Plastic bag Blue Module Cover Clear Plastic bag Module Cover Strap Twine Cotton Journal of Cotton Science, in press White Module Cover ## 10.6 Field trash addition to FT-NIR spectral library Field trash refers to any trash found in a cotton field which is not originating from the cotton plant. Recently, FT-NIR spectroscopy was used to identify field trash samples including a grocery bag, black plastic bag, blue module cover, clear plastic bag, module cover strap, twine, and white module cover (Fortier et al., 2011b). Three replicates were acquired for each trash type. Figure 12 shows the average first derivative spectra of cotton and field trash types. Applying a "top down" approach, Fortier and co-workers added field trash samples, to the FT-NIR spectral library (Fortier et al., 2011b). First, the seed meat and field trash were isolated from the rest of the botanical trash library. Next, a sub-library of the cotton and botanical trash types was designed. Finally a second sub-library to separate hull and seed coat was developed. The identification results can be found in Table 5. Overall, greater than 98% correct identifications were achieved. Table 5. NIR Identification by Cotton Trash Type for Individual Powder and Pepper samples adding seed meat and field trash. Source: Fortier et al., 2011b, Journal of Cotton Science, in press ## 11. Summary This chapter has summarized the application of FT spectroscopy towards the identification and classification of cotton trash components which are found present with cotton lint. The successes reported herein were not possible with conventional techniques such as using the HVI and Shirley Analyzer. In the future, the feasibility of using FT techniques can benefit those in the textile industry such as farmers, ginners, spinners and merchants by providing a well-defined cotton product which has a minimal amount of trash and a high market value. Fourier Transform Spectroscopy of Cotton and Cotton Trash 119 Himmelsbach, D., J. Hellgeth, D. McAlister. 2006. Development and Use of an Attenuated Jiskoot, W., Daan, J., & Crommelin, A. 2005. Methods for structural analysis of protein pharmaceuticals, Library of Congress, ISBN 0-9711767-2-8, Arlington, VA. Kavkler, K., Smit, Z., Jezersek, D., Eichert, D., & Demsar, A. 2011. Investigation of Lewin, M. & Pearce, E. 1998. Handbook of Fiber Chemistry, 2nd edition, Marcel Dekker, Inc., Lojewski, T., Miskowiec, P., Missori, M., Lubanska, A., Proniewicz, L., & Lojewska, J. 2010. McCarthy, W. & Kemeny, G. 2008. Fourier Transform Spectrophotometers in the Near- McCreight, D., Feil, R., Booterbaugh, J., & Backe, E. 1997, Short Staple Yarn Manufacturing, Mirabella, Jr., F. M. 1993. Internal Reflection Spectroscopy, Marcel Dekker, ISBN 0-8247-8730-7, Montalvo, J., Faught, S., & Buco, S. A 1991. Comparative Study of NIR Diffuse Reflectance of Pelletier, M., & Pelletier, C.C. 2010. Spectroscopic Theory for Chemical Imaging, In "Raman, Perkampus, H.-H. 1995. Encyclopedia of Spectroscopy, VCH Verlagsgesellschaft mbH, ISBN 3- Rodgers, J. 2002. Influences of Carpet and Instrumental Parameters on the Identification of Rodgers, J. E. & Beck, K. 2005. Rapid Determination by NIR of the Cotton Content of Blend Rodgers, J. & Beck, K., NIR 2009. Characterization and Measurement of the Cotton Content Rodgers, J. & Ghosh, S., 2008. NIR Analysis of Textiles, In: Handbook of Near-Infrared Analysis, Wiley and Sons, ISBN 978-0-470-38204-2, Hoboken, New Jersey. pp. 79-91CRC Press, ISBN 978-0-8493-7393-0, Boca Raton, Florida. Carolina Academic Press, ISBN 9780890898536, Durham, NC. 7412, ISSN 0021-8561. ISBN 0-8247-9471-0, New York, New York. pp.370-375, ISSN 0144-8617. New York, New York. No.7, pp. 578-591, ISSN 0165-9936. 527-29281-0, Weinheim, Germany. Review, pp. 27-32, ISSN 1532-8813. 0040-5175. 0, Boca Raton, Florida. New Orleans, LA,January 2005, pp. 2731-2736. 0141-3910. Total Reflectance/Fourier Infrared (ATR/FT-IR) Spectral Database to Identify Foreign Matter in Cotton. Journal of Agricultural and Food Chemistry, Vol.54, pp.7405- Biodeteriorated Historical Textiles by Conventional and Synchrotron Radiation FTIR spectroscopy. Polymer Degradation & Stability, Vol. 96, pp.1081-1086, ISSN FTIR and UV/Vis as Methods for Evaluation of Oxidative Degradation of Model Paper: DFT Approach for Carbonyl Vibrations. Carbohydrate Polymers, Vol. 82, Infrared, In: Handbook of Near-Infrared Analysis, 3rd Edition, Burns, D., Ciurczak, E., Cottons Grouped According to Fiber Cross-Sectional Dimensions, Part III. Experimental. Applied Spectroscopy, Vol. 45, No.5, pp.795-807, ISSN 0570-4928. Moros, J., Garrigues, S. & de la Guardia, M. 2010. Trends in Analytical Chemistry, Vol.29, Infrared, and Near-Infrared Chemical Imaging", Šašić, S., & Ozaki, Y., pp.1-20, John Carpet Face Fiber by NIR. American Association of Textile Chemists and Colorists Fabrics after Dyeing, Proceedings of the Beltwide Cotton Conference, ISSN 10592644, of Dyed Blend Fabrics. Textile. Research Journal, Vol. 79, No.8, pp.675-686, ISSN 3rd edition, Burns, D. & Ciurczak, E. pp 485-520.; CRC Press, ISBN 978-0-8493-7393- ### 12. References Allen, A., Foulk, J. & Gamble, G. 2007. Preliminary Fourier-Transform Infrared Spectroscopy Baker, R., Price, J., & Robert, K. 1994, Gin and mill cleaning for rotor spinning, Transactions of Bargeron, J., Rayburn, & S. Griffith, S. 1988. Effects of grass contaminated with cotton on Bell, R.J. 1972. Introductory Fourier Transform Spectroscopy, Academic, ISBN 0120851504, New Brashears, A., Baker, R., Bragg, C., & Simpson, C., 1992. Effect of bark on spinning efficiency Camajani, N., & Muller, D., 1996. Textile Analysis by FT-NIR: Fourier Transform Near Cheng, H. & Biswas, A. 2011. Chemical modification of Cotton-Based Natural Materials: Choi, H-M., Srinivasan, M., & Morris, N. 1994. Single-Step Dyeing and Finishing Treatment Chung, C., Myunghee, L. & Choe, E. 2004. Characterization of cotton fabrics scouring by Colthup, N., Daly, L., & Wiberley, S. 1975. Introduction to Infrared and Raman spectroscopy, Fortier, C., Rodgers, J., Cintron, M. S., Cui, X. & Foulk, J. 2010. Identification of Cotton and Fortier, C., Rodgers, J., & Foulk, J. 2011a. Investigation of the Impact of Instrumental and Fortier, C., Rodgers, J., Foulk, J., & Whitelock, D. 2011b. Near-Infrared classification of cotton lint, botanical and field, Journal of Cotton Science, in press, ISSN 15236919. Foulk, J., McAlister, D., Himmelsbach, D. & Hughs, E. 2004. Mid-Infrared Spectroscopy of Frey, M., & Schneider, U. 1989. Possibilities of Removing Seed Coat Fragments and Attached Griffiths, P., De Haseth, J. 2007. Fourier Transform Spectrometry, 2nd edition, John Wiley and Academic Press, ISBN 0-12-182552-3, New York, New York. Textile Research Journal, Vol.81, pp. 230-238, ISSN 0040-5175. H., & Hunter, W., pp. 1-12, Asheville, North Carolina. Analysis of Cotton Trash, Journal of Cotton Science, Vol.11, pp.68-74, ISSN 15236919. the American Society of Agricultural Engineers, Vol. 37, No.4, pp. 1077-1082, ISSN yarn manufacturing, Transactions of the American Society of Agricultural Engineers, of cotton, Proceedings of Beltwide Cotton Conference, ISSN 10592644, Nashville, TN, Infrared Spectroscopy, In: Textile Applications of Near Infrared Technology: American Association of Textile Chemists and Colorists Symposium, Rodgers, J., Beck, K., Howell, Products from Carboxymethylation. Carbohydrate Polymers, Vol 84, No. 3, pp.1004- of Cotton with 1,2,3,4-Butanetetracarboxylic acid. Journal of Applied Polymer Science, FT-IR ATR spectroscopy. Carbohydrate Polymers, Vol. 58, No.4, pp.417-420, ISSN Cotton Trash Components by Fourier Transform Near-Infrared Spectroscopy. Software Applications on Cotton and Botanical Trash Identification by Ultraviolet-Visible and Near-Infrared Spectroscopy, Journal of Cotton Science, Vol. 15, pp.170- Trash in Cotton Rotor Dust. Journal of Cotton Science, Vol.8, pp.243-253, ISSN Fibers in the Spinning Process, Melliand Textilberichte International Textile Reports, 12. References 0001-2351. York, New York. January 1992, pp.1218-1219. 1010, ISSN 01448617. 178, ISSN 15236919. 15236919. 0144-8617. Vol.31, No.1, pp.2-4, ISSN 0001-2351. Vol. 54, pp.2107-2118, ISSN 00218995. Vol. 70, No. 5, pp. 315-317, ISSN 0341-0781. Sons, ISBN 978-0-471-19404-0, Hoboken, New Jersey. 1. Introduction Jones (1982)]. Experimental methods based on magnetic resonance are among the most used techniques for investigating molecular and electronic structure. Nuclear magnetic resonance (NMR) is mostly applied to closed-shell molecules and can be used for structural research of matter in solid, liquid and gaseous form. The computation of NMR parameters, which are of a great interest not only in chemistry but also in biology and solid-state physics, presents severe analytical and numerical difficulties [Dickson & Ziegler (1996); Ditchfield (1974); Fukui et al. (2004); Helgaker et al. (1999); Ishida (2003); London (1937); Pople et al. (1968); Pyykkö (1988); Schreckenbach & Ziegler (1995); Vaara (2007); Watson et al. (2004)]. The computation of NMR parameters for any of the standard models of quantum chemistry constitute an important challenge [Helgaker et al. (1999)]. Calculations involving a magnetic field should preserve gauge invariance. This is conveniently accomplished by using a gauge including atomic orbitals (GIAO) [London (1937)], which is based on atom-centered basis functions with an explicit field dependence. Magnetic properties are sensitive to the quality of the basis sets due to many contributing physical phenomena arising from both the vicinity of the nucleus and from the valence region. A good atomic orbital basis should decay exponentially for large distances [Agmon (1985)] and should also satisfy Kato's conditions for exact solutions of the appropriate Schrödinger equation [Kato (1957)]. Exponential type functions (ETFs) are better suited than Gaussian functions (GTFs) [Boys (1950a;b)] to represent electron wave functions near the nucleus and at long range. Among the ETFs, Slater type functions (STFs) [Slater (1932)], have a dominating position due to their simple analytic expression, but their multi-center integrals are extremely difficult to evaluate for polyatomic molecules, particularly bi-electronic terms. We note that many researchers hope that the next generation of ab initio programs will be based on the usage of ETFs. Indeed much effort is being made to develop efficient molecular algorithms for integrals over conventional ETFs (STFs or B functions) [Barnett (1990); Fernández et al. (2001); Kutzelnigg (1988); Niehaus et al. (2008); Ozdogan & (Editors); Rico et al. (1998; 1999; 2001); Steinborn et al. (2000); Weatherford & Fourier Transformation Method for Computing Hassan Safouhi Canada 6 University of Alberta, Alberta Mathematical Section, Campus Saint-Jean, NMR Integrals over Exponential Type Functions Various studies focussed on the use of B functions. The use of B functions was proposed by Shavitt [Shavitt (1963)], since reduced Bessel functions possess a representation in terms of a remarkably simple Gauss transform. Detailed discussions of the mathematical properties of ## Fourier Transformation Method for Computing NMR Integrals over Exponential Type Functions ## Hassan Safouhi Mathematical Section, Campus Saint-Jean, University of Alberta, Alberta Canada #### 1. Introduction 120 Fourier Transform – Materials Analysis Siddaiah, M., Lieberman, M., Prasad, N., & Kreinovich, V. 1999. A Geometric Approach to Siddaiah, M., Hughs, E., Lieberman, M., & Foulk, J. 2006. Proceedings of the Beltwide Cotton Conference, ISSN 10592644, San Antonio, TX, January 2006, pp.1926-1937. Silverstein, R. & Webster, F. 1998. Spectrometric identification of organic compounds, John Wiley Thibideaux, D., & Baril, A. 1981. Laboratory techniques for predicting cotton dust residues Thibodeaux, D. 1992. NIR as a Tool for Measuring Cotton Quality., International Cotton Thomasson, J. & Shearer, S. 1995. Correlation of NIR Data with Cotton Quality Veit, D., Hormes, I., Bergmann, J., & Wulfhorst, B., 1996. Image Processing as a Tool to Verschaege, L. 1989. Cotton Fibre Impurities: Neps, Motes, and Seed Coat Fragments, Centre Wakelyn, P., Bertoniere, N., French, A., Thibodeaux, D., Triplett, B., Rousselle, M-A., Jiskoot, W., Daan, J., & Crommelin, A. 2005. Methods for structural analysis of protein pharmaceuticals, Library of Congress, ISBN 0-9711767-2-8, Arlington, VA. Xu, B., Fang, C. & Watson, M. 1999. Clustering Analysis for Cotton Trash Classification, Science and Technology, Vol. 8, No. 1-2, pp. 66-72, ISSN 0955-6222. Textile Research Journal, Vol.69, No.9, pp.656-662, ISSN 0040-5175. & Sons Ltd., ISBN 0-471-39362-2, Toronto, Canada. Conference, Bremen, Germany, March 1992, pp. 27-38. No. 4, pp. 1005-1010 ISSN 0001-2351. 581, pp. 77-82. Wallingford, UK. 5175. Classification of Trash in Ginned Cotton. Departmental Technical Reports, Paper in open-end spinning. Textile Research Journal, Vol.51, No.11, pp.688-695, ISSN 0040- Characteristics. Transactions of the American Society of Agricultural Engineers, Vol. 38, Improve Machine Performance and Process Control, International Journal of Clothing for Agricultural Bioscience International: International Conference on Analytical Chemistry: Review Articles on Cotton Production Research No. 1. ISBN 08-5198 633-1, Goynes, Jr., W., Edwards, J., Hunter, L., McAlister, D., & Gamble, G. 2007. Cotton Fiber Chemistry and Technology, CRC Press, ISBN -10: 1-4200-4587-3, Boca Raton, FL. Experimental methods based on magnetic resonance are among the most used techniques for investigating molecular and electronic structure. Nuclear magnetic resonance (NMR) is mostly applied to closed-shell molecules and can be used for structural research of matter in solid, liquid and gaseous form. The computation of NMR parameters, which are of a great interest not only in chemistry but also in biology and solid-state physics, presents severe analytical and numerical difficulties [Dickson & Ziegler (1996); Ditchfield (1974); Fukui et al. (2004); Helgaker et al. (1999); Ishida (2003); London (1937); Pople et al. (1968); Pyykkö (1988); Schreckenbach & Ziegler (1995); Vaara (2007); Watson et al. (2004)]. The computation of NMR parameters for any of the standard models of quantum chemistry constitute an important challenge [Helgaker et al. (1999)]. Calculations involving a magnetic field should preserve gauge invariance. This is conveniently accomplished by using a gauge including atomic orbitals (GIAO) [London (1937)], which is based on atom-centered basis functions with an explicit field dependence. Magnetic properties are sensitive to the quality of the basis sets due to many contributing physical phenomena arising from both the vicinity of the nucleus and from the valence region. A good atomic orbital basis should decay exponentially for large distances [Agmon (1985)] and should also satisfy Kato's conditions for exact solutions of the appropriate Schrödinger equation [Kato (1957)]. Exponential type functions (ETFs) are better suited than Gaussian functions (GTFs) [Boys (1950a;b)] to represent electron wave functions near the nucleus and at long range. Among the ETFs, Slater type functions (STFs) [Slater (1932)], have a dominating position due to their simple analytic expression, but their multi-center integrals are extremely difficult to evaluate for polyatomic molecules, particularly bi-electronic terms. We note that many researchers hope that the next generation of ab initio programs will be based on the usage of ETFs. Indeed much effort is being made to develop efficient molecular algorithms for integrals over conventional ETFs (STFs or B functions) [Barnett (1990); Fernández et al. (2001); Kutzelnigg (1988); Niehaus et al. (2008); Ozdogan & (Editors); Rico et al. (1998; 1999; 2001); Steinborn et al. (2000); Weatherford & Jones (1982)]. Various studies focussed on the use of B functions. The use of B functions was proposed by Shavitt [Shavitt (1963)], since reduced Bessel functions possess a representation in terms of a remarkably simple Gauss transform. Detailed discussions of the mathematical properties of <sup>H</sup><sup>e</sup> <sup>=</sup> <sup>−</sup><sup>1</sup> 2 ne ∑ j=1 ∇2 j − ne ∑ j=1 • rij is the distance that separates the i • ZK is the atomic number of the Kth nucleus whose mass is MK. • rjK is the distance that separates the Kth nucleus from the j <sup>j</sup> is the Laplacian operators for the coordinates of electron j. The stationary Schrödinger equation that needs to be solved is: orbitals. Unormalized STFs are given by [Slater (1932)]: Bm n,l > ˆ kn<sup>+</sup> <sup>1</sup> 2 (ζ,�r) = <sup>1</sup> ζn−<sup>1</sup> <sup>2</sup> if <sup>n</sup> <sup>−</sup> <sup>l</sup> is even or <sup>p</sup>˜ <sup>=</sup> <sup>n</sup> <sup>−</sup> <sup>l</sup> <sup>+</sup> <sup>1</sup> χm n,l where: • <sup>∇</sup><sup>2</sup> where Y<sup>m</sup> where ˆ kn<sup>−</sup> <sup>1</sup> 2 where <sup>p</sup>˜ <sup>=</sup> <sup>n</sup> <sup>−</sup> <sup>l</sup> magnetic quantum number. (1963); Steinborn & Filter (1975)]: χm n,l N ∑ K=1 Fourier Transformation Method for Computing NMR Integrals over Exponential Type Functions 123 th electron from the j H<sup>e</sup> Ψ(r, R) = Ee Ψ(r, R). The above Schrödinger equation is solved only in the case of hydrogen-like atom. The solutions are one-electron functions and are referred to as hydrogen-like atomic orbitals. These atomic orbitals form a complete and orthonormal basis. The use of hydrogen-like atomic orbitals was prevented due to the fact that their molecular multi-center integrals are extremely difficult to evaluate analytically and numerically. Linear combinations of the hydrogen-like atomic orbitals lead to STFs, which form the most popular basis set of atomic (ζ,�r) = rn−<sup>1</sup> e−<sup>ζ</sup> <sup>r</sup> Y<sup>m</sup> The B functions are given by [Filter & Steinborn (1978a); Steinborn & Filter (1975)]: 2n+l(n + l)! <sup>−</sup><sup>z</sup> <sup>n</sup> ∑ j=0 STFs can be expressed as finite linear combinations of B functions [Filter & Steinborn (1978a)]: (ζ,�r) = (ζr)<sup>l</sup> (z) = z<sup>n</sup> e n−l ∑ p=p˜ is the principal quantum number, l is the orbital angular momentum number and m is the <sup>l</sup> (θ, ϕ) is the surface spherical harmonic [Condon & Shortley (1951)] and where n ˆ kn<sup>−</sup> <sup>1</sup> 2 (ζ r) stands for the reduced spherical Bessel function of the second kind [Shavitt (n + j)! j!(n − j)! (−1)n−l−<sup>p</sup> <sup>2</sup>2p+2l−<sup>n</sup> (<sup>l</sup> <sup>+</sup> <sup>p</sup>)! (2<sup>p</sup> <sup>−</sup> <sup>n</sup> <sup>+</sup> <sup>l</sup>)! (<sup>n</sup> <sup>−</sup> <sup>l</sup> <sup>−</sup> <sup>p</sup>)! <sup>B</sup><sup>m</sup> <sup>2</sup> if <sup>n</sup> <sup>−</sup> <sup>l</sup> is odd. (ζr) Y<sup>m</sup> 1 (2 z)<sup>j</sup> ZK rjK + ne ∑ i=1 ∑ j>i 1 rij th electron. <sup>l</sup> (θ�r, ϕ�r), (2) <sup>l</sup> (θ�r, ϕ�r), (3) . (4) (ζ,�r), (5) p,l th electron. , (1) reduced Bessel functions and of their anisotropic generalizations can be found in [Weniger (1982)]. Furthermore, B functions have much more appealing properties applicable to multi-center integral problems, compared to other exponentially decaying functions [Filter & Steinborn (1978a;b); Steinborn & Filter (1975); Weniger (2005); Weniger & Steinborn (1983a)]. The multi-center molecular integrals over B functions can be computed much more easily than the corresponding integrals of other exponentially decaying functions. This can be explained in terms of the Fourier transform of B functions, which is of exceptional simplicity among exponentially decaying functions [Niukkanen (1984); Weniger (1982); Weniger & Steinborn (1983b)]. Moreover, the Fourier transforms of STFs, of hydrogen eigenfunctions, or of other functions based on the generalized Laguerre polynomials can all be expressed as finite linear combinations of Fourier transforms of B functions [Weniger (1985); Weniger & Steinborn (1983b)]. The basis set of B functions is well adapted to the Fourier transform method [Geller (1962); Grotendorst & Steinborn (1988); Prosser & Blanchard (1962); Trivedi & Steinborn (1983)], which allowed analytic expressions to be developed for molecular multi-center integrals over B functions [Grotendorst & Steinborn (1988); Trivedi & Steinborn (1983)]. Of the NMR parameters, the nuclear shielding tensor is of a great importance. The computation of the shielding tensor presents severe analytical and numerical difficulties especially when using ETFs as a basis set of atomic orbitals. The main difficulty arises from the operators associated with these parameters. An example of such operators is 3 rjN,<sup>β</sup> rjN ·σ(j) /r<sup>5</sup> jN, where β represents a cartesian coordinates, rjN is the vector separating the j th electron and the Nth nuclei and σ stands for Pauli spin matrix. These operators lead to extremely complicated integrals. Analytic treatment of the NMR parameters over GTFs was a subject of many articles (see for example [Ishida (2003)] and references therein). Although, the interest of using ETFs in the computation of NMR parameters is increasing [see the pioneer work by Dickson & Ziegler (1996); Schreckenbach & Ziegler (1995)] and Watson et al. (2004)], no effort was dedicated to their analytic treatment over ETFs. Straightforward numerical integration was used for the computation of integrals associated with these parameters. The analytical development of NMR integrals can be obtained using the Fourier transform method combined with B functions as a basis set of atomic orbitals [Berlu & Safouhi (2008); Safouhi (2010b); Slevinsky et al. (2010)]. The obtained analytic expressions turned out to be similar to those obtained for the so-called three-center nuclear attraction integrals (zeroth order integrals). The latter were the subject of significant research [Berlu & Safouhi (2003); Duret & Safouhi (2007); Fernández et al. (2001); Grotendorst & Steinborn (1988); Homeier & Steinborn (1993); Niehaus et al. (2008); Rico et al. (1998; 1999); Safouhi (2001b; 2004); Slevinsky & Safouhi (2009)]. In our research, we used techniques based on extrapolation methods combined with numerical quadratures to compute the analytic expressions of the NMR integrals. Numerical tables are listed and we refer the interested reader to [Safouhi (2010b); Slevinsky et al. (2010)] for an extensive list of numerical tables as well as detailed numerical discussions. #### 2. Molecular integrals in the absence of magnetic fields In the absence of magnetic fields, the molecular electronic Hamiltonian operator H<sup>e</sup> corresponding to total energy E<sup>e</sup> for a molecule of N nuclei and ne electron is given by: $$\mathcal{H}\_{\text{e}} = -\frac{1}{2} \sum\_{j=1}^{n\_{\text{e}}} \nabla\_{j}^{2} - \sum\_{j=1}^{n\_{\text{e}}} \sum\_{K=1}^{N} \frac{Z\_{K}}{r\_{jK}} + \sum\_{i=1}^{n\_{\text{e}}} \sum\_{j>i} \frac{1}{r\_{ij}},\tag{1}$$ where: 2 Will-be-set-by-IN-TECH reduced Bessel functions and of their anisotropic generalizations can be found in [Weniger (1982)]. Furthermore, B functions have much more appealing properties applicable to multi-center integral problems, compared to other exponentially decaying functions [Filter & Steinborn (1978a;b); Steinborn & Filter (1975); Weniger (2005); Weniger & Steinborn (1983a)]. The multi-center molecular integrals over B functions can be computed much more easily than the corresponding integrals of other exponentially decaying functions. This can be explained in terms of the Fourier transform of B functions, which is of exceptional simplicity among exponentially decaying functions [Niukkanen (1984); Weniger (1982); Weniger & Steinborn (1983b)]. Moreover, the Fourier transforms of STFs, of hydrogen eigenfunctions, or of other functions based on the generalized Laguerre polynomials can all be expressed as finite linear combinations of Fourier transforms of B functions [Weniger (1985); Weniger & Steinborn (1983b)]. The basis set of B functions is well adapted to the Fourier transform method [Geller (1962); Grotendorst & Steinborn (1988); Prosser & Blanchard (1962); Trivedi & Steinborn (1983)], which allowed analytic expressions to be developed for molecular multi-center integrals over B functions [Grotendorst & Steinborn (1988); Trivedi & Steinborn (1983)]. Of the NMR parameters, the nuclear shielding tensor is of a great importance. The computation of the shielding tensor presents severe analytical and numerical difficulties especially when using ETFs as a basis set of atomic orbitals. The main difficulty arises from the operators associated with these parameters. An example of such operators operators lead to extremely complicated integrals. Analytic treatment of the NMR parameters over GTFs was a subject of many articles (see for example [Ishida (2003)] and references therein). Although, the interest of using ETFs in the computation of NMR parameters is increasing [see the pioneer work by Dickson & Ziegler (1996); Schreckenbach & Ziegler (1995)] and Watson et al. (2004)], no effort was dedicated to their analytic treatment over ETFs. Straightforward numerical integration was used for the computation of integrals associated The analytical development of NMR integrals can be obtained using the Fourier transform method combined with B functions as a basis set of atomic orbitals [Berlu & Safouhi (2008); Safouhi (2010b); Slevinsky et al. (2010)]. The obtained analytic expressions turned out to be similar to those obtained for the so-called three-center nuclear attraction integrals (zeroth order integrals). The latter were the subject of significant research [Berlu & Safouhi (2003); Duret & Safouhi (2007); Fernández et al. (2001); Grotendorst & Steinborn (1988); Homeier & Steinborn (1993); Niehaus et al. (2008); Rico et al. (1998; 1999); Safouhi (2001b; 2004); Slevinsky & Safouhi (2009)]. In our research, we used techniques based on extrapolation methods combined with numerical quadratures to compute the analytic expressions of the NMR integrals. Numerical tables are listed and we refer the interested reader to [Safouhi (2010b); Slevinsky et al. (2010)] for an extensive list of numerical tables as well as detailed In the absence of magnetic fields, the molecular electronic Hamiltonian operator H<sup>e</sup> corresponding to total energy E<sup>e</sup> for a molecule of N nuclei and ne electron is given by: jN, where β represents a cartesian coordinates, rjN is the vector th electron and the Nth nuclei and σ stands for Pauli spin matrix. These is 3 rjN,<sup>β</sup> with these parameters. numerical discussions. 2. Molecular integrals in the absence of magnetic fields separating the j rjN ·σ(j) /r<sup>5</sup> The stationary Schrödinger equation that needs to be solved is: $$\mathcal{H}\_{\mathbf{e}}\,\Psi(\mathbf{r},\mathbf{R}) = E\_{\mathbf{e}}\,\Psi(\mathbf{r},\mathbf{R}).$$ The above Schrödinger equation is solved only in the case of hydrogen-like atom. The solutions are one-electron functions and are referred to as hydrogen-like atomic orbitals. These atomic orbitals form a complete and orthonormal basis. The use of hydrogen-like atomic orbitals was prevented due to the fact that their molecular multi-center integrals are extremely difficult to evaluate analytically and numerically. Linear combinations of the hydrogen-like atomic orbitals lead to STFs, which form the most popular basis set of atomic orbitals. Unormalized STFs are given by [Slater (1932)]: $$ \chi^{m}\_{n,l}(\zeta,\vec{r}) = r^{n-1} \mathbf{e}^{-\zeta \cdot r} Y^{m}\_{l}(\theta\_{\vec{r}\prime} \,\varphi\_{\vec{r}}),\tag{2} $$ where Y<sup>m</sup> <sup>l</sup> (θ, ϕ) is the surface spherical harmonic [Condon & Shortley (1951)] and where n is the principal quantum number, l is the orbital angular momentum number and m is the magnetic quantum number. The B functions are given by [Filter & Steinborn (1978a); Steinborn & Filter (1975)]: $$B\_{n,l}^{m}(\zeta,\vec{r}) = \frac{(\zeta r)^{l}}{2^{n+l}(n+l)!} \hat{k}\_{n-\frac{1}{2}}(\zeta r) \, \mathcal{Y}\_{l}^{m}(\theta\_{\vec{r}\prime} \, \varphi\_{\vec{r}}),\tag{3}$$ where ˆ kn<sup>−</sup> <sup>1</sup> 2 (ζ r) stands for the reduced spherical Bessel function of the second kind [Shavitt (1963); Steinborn & Filter (1975)]: $$\hat{k}\_{n+\frac{1}{2}}(z) = z^n e^{-z} \sum\_{j=0}^n \frac{(n+j)!}{j! \left(n-j\right)!} \frac{1}{(2\cdot z)^j}.\tag{4}$$ STFs can be expressed as finite linear combinations of B functions [Filter & Steinborn (1978a)]: $$\chi\_{n,l}^{m}(\zeta,\vec{r}) = \frac{1}{\zeta^{n-1}} \sum\_{p=\beta}^{n-l} \frac{(-1)^{n-l-p} \ 2^{2p+2l-n} \ (l+p)!}{(2p-n+l)!} B\_{p,l}^{m}(\zeta,\vec{r}),\tag{5}$$ where <sup>p</sup>˜ <sup>=</sup> <sup>n</sup> <sup>−</sup> <sup>l</sup> <sup>2</sup> if <sup>n</sup> <sup>−</sup> <sup>l</sup> is even or <sup>p</sup>˜ <sup>=</sup> <sup>n</sup> <sup>−</sup> <sup>l</sup> <sup>+</sup> <sup>1</sup> <sup>2</sup> if <sup>n</sup> <sup>−</sup> <sup>l</sup> is odd. × Δl ∑ j=0 × � 1 s=0 s where: (1983)]. (−2)<sup>j</sup> ( Δl j ) <sup>n</sup><sup>22</sup> (<sup>1</sup> <sup>−</sup> <sup>s</sup>)n<sup>11</sup> <sup>k</sup>l1−<sup>l</sup> (n<sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l<sup>2</sup> − j + 1)! <sup>γ</sup>(s, <sup>k</sup>) = � m<sup>12</sup> = (m<sup>2</sup> − m� Gaunt coefficients �l1m1\|l2m2\|l3m3� are defined by [Gaunt (1929)]: � π θ=0 3. Relativistic formulation of NMR shielding tensor where the electronic impulsion �pj is given by: <sup>−</sup><sup>i</sup> <sup>∇</sup>� <sup>i</sup> <sup>+</sup> <sup>e</sup> <sup>A</sup>� <sup>i</sup> In the presence of an external uniform magnetic field � � H = <sup>H</sup> <sup>=</sup> <sup>H</sup>(0) <sup>+</sup> <sup>H</sup>(r) <sup>+</sup> <sup>μ</sup>N,αH(0,1) <sup>α</sup> <sup>+</sup> <sup>B</sup>0,βH(1,0) n ∑ i=1 ⎡ ⎣ 1 2 �p 2 where <sup>A</sup>� <sup>i</sup> <sup>=</sup> <sup>1</sup> where A� <sup>i</sup> stands for the vector potential induced by the nuclear moments �μ<sup>N</sup> and the external �rij separating the electrons i and j.�riN is the vector separating the electron i and the nuclei N. The relativistic effects are important for the fourth and fifth rows in the periodic table and for transitions metals [Pyykkö (1988)]. In terms of perturbations with respect to μN,<sup>α</sup> and B0,<sup>β</sup> where α and β stand for cartesian coordinates (α, β ∈ (x, y, z)), the electronic relativistic Δl = <sup>l</sup> � <sup>1</sup>+l � <sup>2</sup>−l � <sup>2</sup> , �l1m1\|l2m2\|l3m3� = Hamiltonian is given by: �pi = � uniform magnetic field � Hamiltonian is given by: � <sup>1</sup>+l2−l � 2 ˆ ν = n<sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l<sup>2</sup> − l n<sup>11</sup> = n<sup>1</sup> + l<sup>1</sup> + l<sup>2</sup> − l n<sup>22</sup> = n<sup>2</sup> + l<sup>2</sup> + l<sup>1</sup> − l � 2π ϕ=0 [Ym<sup>1</sup> n<sup>γ</sup> = 2(n<sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l2) − (l (<sup>1</sup> <sup>−</sup> <sup>s</sup>) <sup>ζ</sup><sup>2</sup> k<sup>ν</sup> [R<sup>2</sup> γ(s, k)] [γ(s, <sup>k</sup>)]n<sup>γ</sup> <sup>e</sup> Fourier Transformation Method for Computing NMR Integrals over Exponential Type Functions 125 <sup>1</sup> <sup>+</sup> <sup>s</sup> <sup>ζ</sup><sup>2</sup> <sup>2</sup>) − (m<sup>1</sup> − m� � 2 � 1 <sup>l</sup><sup>1</sup> (θ, <sup>ϕ</sup>)]<sup>∗</sup> <sup>Y</sup>m<sup>2</sup> Equation (11) led to analytical expressions for all molecular multi-center integrals over B functions or STFs [Grotendorst & Steinborn (1988); Safouhi (2001a); Trivedi & Steinborn <sup>i</sup> + V(i) + 2 � � B<sup>0</sup> ∧�ri<sup>0</sup> � <sup>+</sup> <sup>μ</sup><sup>0</sup> <sup>4</sup> <sup>π</sup> ∑ N n ∑ i<j 1 rij B0. μ<sup>0</sup> stands for dielectric permittivity. rij is the modulus of the vector <sup>β</sup> <sup>+</sup> <sup>μ</sup>N,αB0,βH(1,1) ⎤ � <sup>1</sup> + l � <sup>2</sup> + l � ) + 1 � <sup>−</sup> <sup>j</sup> <sup>+</sup> <sup>1</sup> 2 1) <sup>2</sup> + <sup>s</sup>(<sup>1</sup> − <sup>s</sup>) <sup>k</sup><sup>2</sup> <sup>l</sup><sup>2</sup> (θ, <sup>ϕ</sup>) <sup>Y</sup>m<sup>3</sup> −i(1−s) �k· #—<sup>R</sup> <sup>2</sup> <sup>d</sup>s, (11) <sup>l</sup><sup>3</sup> (θ, ϕ) sin (θ) dθ dϕ. (13) B0, the electronic non-relativistic ⎦ , (14) �μ<sup>N</sup> ∧�riN r3 iN αβ + ··· , (16) , (15) (12) #### 2.1 Fourier transform in molecular multi-center integrals calculation The Fourier transform of B functions, which is of exceptional simplicity among exponentially decaying functions, is given by [Niukkanen (1984); Weniger (1982); Weniger & Steinborn (1983b)]: $$\mathcal{B}\_{n,l}^{m}(\zeta,\vec{p}) = \sqrt{\frac{2}{\pi}} \,\zeta^{2n+l-1} \frac{(-i\|p\|)^{l}}{(\zeta^{2}+\|p\|^{2})^{n+l+1}} \, Y\_{l}^{m}(\theta\_{\vec{p}\prime}\,\varphi\_{\vec{p}}).\tag{6}$$ In [Trivedi & Steinborn (1983)], the Fourier transform method is used in combination with equation (6) to derive analytic expressions for the following integrals: $$\mathcal{T} = \int\_{\vec{r}} \left[ B^{m\_1}\_{n\_1, l\_1} (\zeta\_1, \vec{r}) \right]^\* e^{-i \vec{k} \cdot \vec{r}} B^{m\_2}\_{n\_2, l\_2} (\zeta\_2, \vec{r} - \vec{R}\_2) \,\mathrm{d}\vec{r} \,. \tag{7}$$ The main idea of the Fourier integral transformation is given by: $$\int \left[f(\vec{r})\right]^\* e^{-i\vec{\mathcal{K}}\vec{\mathcal{F}}} g(\vec{r} - \vec{\mathcal{R}}) \,\mathrm{d}\vec{r} = (2\pi)^{-3/2} \int \left[\int \left[\vec{f}(\vec{q})\right]^\* e^{-i\vec{\mathcal{q}}\vec{\mathcal{F}}} e^{-i\vec{\mathcal{K}}\vec{\mathcal{F}}} g(\vec{r} - \vec{\mathcal{R}}) \,\mathrm{d}\vec{q}\right] \,\mathrm{d}\vec{r}$$ $$= e^{-i\vec{\mathcal{K}}\vec{\mathcal{R}}} \int \left[\vec{f}(\vec{q})\right]^\* e^{-i\vec{\mathcal{q}}\vec{\mathcal{R}}} (2\pi)^{-3/2} \left[\int e^{-i(\vec{\mathcal{q}} + \vec{\mathcal{r}}). (\vec{r} - \vec{\mathcal{R}})} g(\vec{r} - \vec{\mathcal{R}}) \,\mathrm{d}\vec{r}\right] \,\mathrm{d}\vec{q}$$ $$= e^{-i\vec{\mathcal{K}}\vec{\mathcal{R}}} \int \vec{f}^\(\vec{q}) \, e^{-i\vec{\mathcal{q}}\vec{\mathcal{R}}} \vec{g}(\vec{q} + \vec{\mathcal{x}}) \,\mathrm{d}\vec{q}\,\,\tag{8}$$ where ¯ f( k) stands for the Fourier transform of f(r). The function f(r) and its Fourier transform ¯ f( k) are connected by the symmetric relationships: $$f(\vec{k}) = (2\pi)^{-3/2} \int\_{\vec{r}} e^{-i\vec{k}\cdot\vec{r}} f(\vec{r}) \,\mathrm{d}\vec{r} \qquad \text{and} \qquad f(\vec{r}) = (2\pi)^{-3/2} \int\_{\vec{k}} e^{i\vec{k}\cdot\vec{r}} f(\vec{k}) \,\mathrm{d}\vec{k}.\tag{9}$$ Replacing f* by Bm<sup>1</sup> n1,l<sup>1</sup> (ζ1,<sup>r</sup>) and <sup>g</sup> by <sup>B</sup>m<sup>2</sup> n2,l<sup>2</sup> (ζ2,<sup>r</sup> <sup>−</sup> R) we obtain: $$\int \left[ B\_{\eta\_1, l\_1}^{\mathfrak{m}\_1} (\zeta\_1, \vec{r}) \right]^\* e^{-i\vec{\mathcal{K}} \cdot \vec{r}} B\_{\eta\_2, l\_2}^{\mathfrak{m}\_2} (\zeta\_2, \vec{r} - \vec{\mathcal{R}}) \, \mathrm{d}\vec{r} = e^{-i\vec{\mathcal{K}} \cdot \vec{\mathcal{R}}} \int \left[ \vec{B}\_{\eta\_1, l\_1}^{\mathfrak{m}\_1} (\zeta\_1, \vec{q}) \right]^\* e^{-i\vec{\mathcal{J}} \cdot \vec{\mathcal{R}}} \vec{B}\_{\eta\_2, l\_2}^{\mathfrak{m}\_2} (\zeta\_2, \vec{q} + \vec{\mathcal{x}}) \, \mathrm{d}\vec{\mathcal{q}}.\tag{10}$$ Equations (10) and (6) led to an analytic expression for the integral T in equation (7). This analytic expression is given by [Trivedi & Steinborn (1983)]: <sup>T</sup> <sup>=</sup> (4π)<sup>3</sup> (2l<sup>1</sup> <sup>+</sup> <sup>1</sup>)!!(2l<sup>2</sup> <sup>+</sup> <sup>1</sup>)!!(n<sup>1</sup> <sup>+</sup> <sup>l</sup><sup>1</sup> <sup>+</sup> <sup>n</sup><sup>2</sup> <sup>+</sup> <sup>l</sup><sup>2</sup> <sup>+</sup> <sup>1</sup>)! <sup>ζ</sup>2n1+l1−<sup>1</sup> <sup>1</sup> <sup>ζ</sup>2n2+l2−<sup>1</sup> 2 (n<sup>1</sup> + l1)!(n<sup>2</sup> + l2)! 2n1+n2+l1+l2+<sup>1</sup> × l1 ∑ l � <sup>1</sup>=0 (−i)l1+<sup>l</sup> � 1 l � 1 ∑ m� <sup>1</sup>=−l � 1 l1m<sup>1</sup> l � 1m� 1 l<sup>1</sup> − l � <sup>1</sup>m<sup>1</sup> − m� 1 (2l � <sup>1</sup> + 1)!![2(l<sup>1</sup> − l � <sup>1</sup>) + 1]!! × l2 ∑ l � <sup>2</sup>=0 (−i)l2+<sup>l</sup> � 2 l � 2 ∑ m� <sup>2</sup>=−l � 2 l2m<sup>2</sup> l � 2m� 2 l<sup>2</sup> − l � <sup>2</sup>m<sup>2</sup> − m� 2 (2l � <sup>2</sup> + 1)!![2(l<sup>2</sup> − l � <sup>2</sup>) + 1]!! × l � <sup>1</sup>+l � 2 ∑ l�=l � min,2 (−1)<sup>l</sup> � 1 l � 2m� 2 l � 1m� 1 l � m� <sup>2</sup> − m� 1 R<sup>l</sup> � <sup>2</sup> <sup>Y</sup>m� 2−m� 1 <sup>l</sup>� (<sup>θ</sup> #—<sup>R</sup> <sup>2</sup> , <sup>ϕ</sup>#—<sup>R</sup> <sup>2</sup> ) × l1−l � <sup>1</sup>+l2−l � 2 ∑ l12=l12min,2 l<sup>2</sup> − l � <sup>2</sup>m<sup>2</sup> − m� 2 l<sup>1</sup> − l � <sup>1</sup>m<sup>1</sup> − m� 1 <sup>l</sup>12m12 <sup>Y</sup>m<sup>12</sup> <sup>l</sup><sup>12</sup> (θ<sup>k</sup> , ϕ<sup>k</sup>) $$\begin{aligned} &\times \sum\_{j=0}^{\Delta l} \frac{(-2)^j \binom{\Delta l}{j}}{(n\_1 + n\_2 + l\_1 + l\_2 - j + 1)!} \\ &\times \int\_{s=0}^1 s^{n\_{22}} \left(1 - s\right)^{n\_{11}} k^{l\_1 - l\_1' + l\_2 - l\_2'} \frac{\pounds\_{\V} \left[R\_2 \ \gamma(s, k)\right]}{[\gamma(s, k)]^{n\_{\V}}} e^{-i(1 - s)\vec{k} \cdot \overrightarrow{R}\_2} \, \mathrm{d}s, \end{aligned} \tag{11}$$ where: 4 Will-be-set-by-IN-TECH The Fourier transform of B functions, which is of exceptional simplicity among exponentially decaying functions, is given by [Niukkanen (1984); Weniger (1982); Weniger & Steinborn <sup>π</sup> <sup>ζ</sup>2n+l−<sup>1</sup> (−<sup>i</sup> <sup>\|</sup>p\|)<sup>l</sup> In [Trivedi & Steinborn (1983)], the Fourier transform method is used in combination with <sup>−</sup><sup>i</sup><sup>k</sup> ·<sup>r</sup> <sup>B</sup>m<sup>2</sup> n2,l<sup>2</sup> ¯ k) stands for the Fourier transform of f(r). The function f(r) and its Fourier <sup>k</sup>·<sup>r</sup> <sup>f</sup>(<sup>r</sup>) <sup>d</sup><sup>r</sup> and <sup>f</sup>(<sup>r</sup>)=(2π)−3/2 (ζ2,<sup>r</sup> <sup>−</sup> <sup>−</sup><sup>i</sup><sup>x</sup>. R B¯m<sup>1</sup> n1,l<sup>1</sup> Equations (10) and (6) led to an analytic expression for the integral T in equation (7). This <sup>1</sup> + 1)!![2(l<sup>1</sup> − l <sup>2</sup> + 1)!![2(l<sup>2</sup> − l (n<sup>1</sup> + l1)!(n<sup>2</sup> + l2)! 2n1+n2+l1+l2+<sup>1</sup> � <sup>1</sup>) + 1]!! � <sup>2</sup>) + 1]!! <sup>R</sup> (2π)−3/2 (ζ<sup>2</sup> <sup>+</sup> <sup>\|</sup>p\|2)n+l+<sup>1</sup> <sup>Y</sup><sup>m</sup> f(q) <sup>∗</sup> e <sup>−</sup><sup>i</sup><sup>q</sup>.<sup>r</sup> e e R) we obtain: (ζ1,q) ∗ e <sup>−</sup><sup>i</sup><sup>q</sup>. <sup>R</sup> B¯m<sup>2</sup> n2,l<sup>2</sup> (ζ2,<sup>r</sup> <sup>−</sup> <sup>−</sup>i(<sup>q</sup><sup>+</sup><sup>x</sup>).(<sup>r</sup><sup>−</sup> <sup>R</sup> g¯(q +x) dq, (8) k e i <sup>k</sup>·<sup>r</sup> ¯ f( <sup>1</sup> <sup>ζ</sup>2n2+l2−<sup>1</sup> 2 , ϕ<sup>k</sup>) <sup>l</sup> (θ<sup>p</sup>, ϕ<sup>p</sup>). (6) R2) dr. (7) R) dq dr R) dr dq k) dk. (9) (ζ2,q +x) dq. (10) <sup>−</sup><sup>i</sup><sup>x</sup>.<sup>r</sup> <sup>g</sup>(<sup>r</sup> <sup>−</sup> <sup>R</sup>) <sup>g</sup>(<sup>r</sup> <sup>−</sup> 2.1 Fourier transform in molecular multi-center integrals calculation 2 equation (6) to derive analytic expressions for the following integrals: The main idea of the Fourier integral transformation is given by: (ζ1,r) ∗ e <sup>−</sup><sup>i</sup><sup>q</sup>. n2,l<sup>2</sup> R) dr = e <sup>T</sup> <sup>=</sup> (4π)<sup>3</sup> (2l<sup>1</sup> <sup>+</sup> <sup>1</sup>)!!(2l<sup>2</sup> <sup>+</sup> <sup>1</sup>)!!(n<sup>1</sup> <sup>+</sup> <sup>l</sup><sup>1</sup> <sup>+</sup> <sup>n</sup><sup>2</sup> <sup>+</sup> <sup>l</sup><sup>2</sup> <sup>+</sup> <sup>1</sup>)! <sup>ζ</sup>2n1+l1−<sup>1</sup> k) are connected by the symmetric relationships: R) dr = (2π)−3/2 B¯m n,l (ζ,p) = T = r Bm<sup>1</sup> n1,l<sup>1</sup> <sup>−</sup><sup>i</sup><sup>x</sup>.<sup>r</sup> <sup>g</sup>(<sup>r</sup> <sup>−</sup> r e −i (ζ1,<sup>r</sup>) and <sup>g</sup> by <sup>B</sup>m<sup>2</sup> analytic expression is given by [Trivedi & Steinborn (1983)]: l � 1 ∑ m� <sup>1</sup>=−l � 1 l � 2 ∑ m� <sup>2</sup>=−l � 2 l1m<sup>1</sup> l � 1m� 1 l<sup>1</sup> − l � <sup>1</sup>m<sup>1</sup> − m� 1 l2m<sup>2</sup> l � 2m� 2 l<sup>2</sup> − l � <sup>2</sup>m<sup>2</sup> − m� 2 (2l � (2l � (ζ2,<sup>r</sup> <sup>−</sup> = e <sup>−</sup><sup>i</sup><sup>x</sup>. R ¯ f(q) <sup>∗</sup> e <sup>−</sup><sup>i</sup><sup>q</sup>. = e <sup>−</sup><sup>i</sup><sup>x</sup>. R ¯ f ∗(q)e k)=(2π)−3/2 n1,l<sup>1</sup> (−i)l1+<sup>l</sup> � 1 (−i)l2+<sup>l</sup> � 2 ∑ l12=l12min,2 (−1)<sup>l</sup> � 1 l � 2m� 2 l � 1m� 1 l � m� <sup>2</sup> − m� 1 R<sup>l</sup> � <sup>2</sup> <sup>Y</sup>m� 2−m� 1 <sup>l</sup>� (<sup>θ</sup> #— R <sup>2</sup> , ϕ#— R <sup>2</sup> ) l<sup>2</sup> − l � <sup>2</sup>m<sup>2</sup> − m� 2 l<sup>1</sup> − l � <sup>1</sup>m<sup>1</sup> − m� 1 l12m<sup>12</sup> Ym<sup>12</sup> <sup>l</sup><sup>12</sup> (θ<sup>k</sup> (1983b)]: where ¯ f( Bm<sup>1</sup> n1,l<sup>1</sup> transform ¯ [ f(r)] ∗ e f( ¯ f( Replacing f by Bm<sup>1</sup> (ζ1,r) ∗ e <sup>−</sup><sup>i</sup><sup>x</sup>.<sup>r</sup> Bm<sup>2</sup> n2,l<sup>2</sup> > × l1 ∑ l � <sup>1</sup>=0 > × l2 ∑ l � <sup>2</sup>=0 × l � <sup>1</sup>+l � 2 ∑ l�=l � min,2 × l1−l � <sup>1</sup>+l2−l � 2 $$\begin{aligned} n\_{\uparrow} &= 2(n\_1 + n\_2 + l\_1 + l\_2) - (l\_1' + l\_2' + l') + 1 \\ \gamma(s, k) &= \sqrt{(1 - s)\xi\_1^2 + s\xi\_2^2 + s\left(1 - s\right)k^2} \\ \nu &= n\_1 + n\_2 + l\_1 + l\_2 - l' - j + \frac{1}{2} \\ m\_{12} &= (m\_2 - m\_2') - (m\_1 - m\_1') \\ n\_{11} &= n\_1 + l\_1 + l\_2 - l\_2' \\ m\_{22} &= n\_2 + l\_2 + l\_1 - l\_1' \\ \Delta l &= \frac{l\_1' + l\_2' - l'}{2}, \end{aligned} \tag{12}$$ Gaunt coefficients �l1m1\|l2m2\|l3m3� are defined by [Gaunt (1929)]: $$\langle l\_1 m\_1 \| l\_2 m\_2 \| l\_3 m\_3 \rangle = \int\_{\theta=0}^{\pi} \int\_{\varphi=0}^{2\pi} [Y\_{l\_1}^{\mathfrak{m}\_1}(\theta, \varphi)]^\* Y\_{l\_2}^{\mathfrak{m}\_2}(\theta, \varphi) \, Y\_{l\_3}^{\mathfrak{m}\_3}(\theta, \varphi) \sin \left(\theta \right) \, \mathrm{d}\theta \, \mathrm{d}\varphi. \tag{13}$$ Equation (11) led to analytical expressions for all molecular multi-center integrals over B functions or STFs [Grotendorst & Steinborn (1988); Safouhi (2001a); Trivedi & Steinborn (1983)]. #### 3. Relativistic formulation of NMR shielding tensor In the presence of an external uniform magnetic field � B0, the electronic non-relativistic Hamiltonian is given by: $$\mathcal{H} = \sum\_{i=1}^{n} \left[ \frac{1}{2} \vec{p}\_i^2 + V(i) + \sum\_{i$$ where the electronic impulsion �pj is given by: $$ \vec{p}\_i = \left[ -i\vec{\nabla}\_i + e\vec{A}\_i \right] \qquad \text{where} \qquad \vec{A}\_i = \frac{1}{2} \left( \vec{B}\_0 \wedge \vec{r}\_{i0} \right) + \frac{\mu\_0}{4\pi} \sum\_N \frac{\vec{\mu}\_N \wedge \vec{r}\_{iN}}{r\_{iN}^3}, \tag{15} $$ where A� <sup>i</sup> stands for the vector potential induced by the nuclear moments �μ<sup>N</sup> and the external uniform magnetic field � B0. μ<sup>0</sup> stands for dielectric permittivity. rij is the modulus of the vector �rij separating the electrons i and j.�riN is the vector separating the electron i and the nuclei N. The relativistic effects are important for the fourth and fifth rows in the periodic table and for transitions metals [Pyykkö (1988)]. In terms of perturbations with respect to μN,<sup>α</sup> and B0,<sup>β</sup> where α and β stand for cartesian coordinates (α, β ∈ (x, y, z)), the electronic relativistic Hamiltonian is given by: $$\mathcal{H} = \mathcal{H}^{(0)} + \mathcal{H}^{(r)} + \mu\_{\text{N},\mathfrak{a}} \mathcal{H}\_{\mathfrak{a}}^{(0,1)} + \mathcal{B}\_{0,\mathfrak{f}} \mathcal{H}\_{\mathfrak{f}}^{(1,0)} + \mu\_{\text{N},\mathfrak{a}} \mathcal{B}\_{0,\mathfrak{f}} \mathcal{H}\_{\mathfrak{a}\mathfrak{f}}^{(1,1)} + \cdots \, , \tag{16}$$ I<sup>23</sup> = rj χm<sup>1</sup> n1,l<sup>1</sup> <sup>I</sup><sup>13</sup> <sup>=</sup> <sup>−</sup> <sup>∑</sup><sup>α</sup> equation (5), the integrals <sup>I</sup>(α,β) B functions which are given by: BI(α,β) <sup>13</sup> = <sup>13</sup> = (<sup>2</sup> <sup>π</sup>)−3/2 BI(α,β) × l1 ∑ l � <sup>1</sup>=0 σj,<sup>α</sup> rj χm<sup>1</sup> n1,l<sup>1</sup> rj Bm<sup>1</sup> n1,l<sup>1</sup> Using the Fourier transform method, we obtain: k rjN,<sup>β</sup> of the operator is given by [Safouhi (2010b)]: of the operator in the generalized function sense. (−i)l1+<sup>l</sup> � 1 4.1 First order integrals Let <sup>I</sup>(α,β) BI(α,β) (ζ1,rjA) of the Euclidean space and 0 is the origin of the fixed coordinate system. After expanding the operator in the integrals (21), we can write I<sup>13</sup> as follows: (ζ1,rjA) (ζ1,rjA) 1 r3 jN rj Bm<sup>1</sup> n1,l<sup>1</sup> rβ ∂ ∂r<sup>α</sup> one can derive the following analytic expression for the integrals <sup>B</sup>I(α,β) l � 1 ∑ m� <sup>1</sup>=−l � 1 l1m<sup>1</sup> l � 1m� 1 l<sup>1</sup> − l � <sup>1</sup>m<sup>1</sup> − m� 1 (2l � ∂ ∂rjN,<sup>α</sup> ∗ rjN,<sup>β</sup> 1 r3 = 2 π In the case where α and β represent two different cartesian coordinates, the Fourier transform In the case where α and β represent the same cartesian coordinate, the calculations leads to the potential 1/r3, which poses serious difficulties because of the singularity and its Fourier transform does not exist in a sense of classical analysis. This case is a part of ongoing research where the theory of generalized functions will be used in order to derive the Fourier transform Using the analytic expression (11) obtained by Trivedi and Steinborn [Trivedi & Steinborn (1983)] for the integrals overrj involved in equation (25) and with the help of equation (26), (n<sup>1</sup> + l1)!(n<sup>2</sup> + l2)! 2n1+n2+l1+l2+<sup>2</sup> � <sup>1</sup>) + 1]!! <sup>1</sup> + 1)!![2(l<sup>1</sup> − l <sup>13</sup> <sup>=</sup> <sup>43</sup> <sup>π</sup> (2l<sup>1</sup> <sup>+</sup> <sup>1</sup>)!!(2l<sup>2</sup> <sup>+</sup> <sup>1</sup>)!!(n<sup>1</sup> <sup>+</sup> <sup>l</sup><sup>1</sup> <sup>+</sup> <sup>n</sup><sup>2</sup> <sup>+</sup> <sup>l</sup><sup>2</sup> <sup>+</sup> <sup>1</sup>)! <sup>ζ</sup>2n1+l1−<sup>1</sup> ∗ rjN,<sup>β</sup> <sup>13</sup> represent the integrals in summation of the RHS of the above equation. Using ∂ ∂rjN,<sup>α</sup> ∂ ∂rjN,<sup>α</sup> 1 r3 jN χm<sup>2</sup> n2,l<sup>2</sup> <sup>13</sup> are expressed as linear combinations of integrals <sup>B</sup>I(α,β) 1 r3 jN Bm<sup>2</sup> n2,l<sup>2</sup> (ζ1,rjA) k<sup>α</sup> k<sup>β</sup> ∗ e <sup>−</sup><sup>i</sup><sup>k</sup> ·rjN <sup>B</sup>m<sup>2</sup> n2,l<sup>2</sup> <sup>k</sup><sup>2</sup> . (26) <sup>13</sup> [Safouhi (2010b)]: <sup>1</sup> <sup>ζ</sup>2n2+l2−<sup>1</sup> 2 <sup>∗</sup> rj ·rjNδαβ − rjN,<sup>α</sup> rj,<sup>β</sup> r3 jN whererjA <sup>=</sup>rj <sup>−</sup> # — OA,rjB <sup>=</sup>rj <sup>−</sup> # — OB andrjN <sup>=</sup>rj <sup>−</sup> # — ON. <sup>A</sup>, <sup>B</sup> and <sup>N</sup> are three arbitrary points Fourier Transformation Method for Computing NMR Integrals over Exponential Type Functions 127 χm<sup>2</sup> n2,l<sup>2</sup> (ζ2,rjB) drj, (22) (ζ2,rjB)drj. (23) (ζ2,rjB)drj. (24) (ζ2,rjB) drj <sup>13</sup> over dk. (25) where <sup>H</sup>(0) is the zeroth-order hamiltonian (1) and <sup>H</sup>(r) is the relativistic perturbation term, which is independent of the magnetic perturbations and is given by [Fukui & Baba (1998)]: $$\mathcal{H}^{(r)} = \sum\_{j$$ which include the contributions; <sup>H</sup>(r) 1,<sup>j</sup> : two-electron Darwin term, <sup>H</sup>(r) 2,<sup>j</sup> : two-electron spin-orbit term, <sup>H</sup>(r) 3,<sup>j</sup> : the retarded orbit-orbit term, <sup>H</sup>(r) 4,<sup>j</sup> : spin-other-orbit term and H(r) 5,<sup>j</sup> : spin-spin term. The perturbations <sup>H</sup>(0,1) <sup>α</sup> <sup>=</sup> <sup>∂</sup><sup>H</sup> ∂μN,<sup>α</sup> {�μ<sup>N</sup> <sup>=</sup>�0,� <sup>B</sup>0=�0} , <sup>H</sup>(1,0) <sup>β</sup> = <sup>∂</sup><sup>H</sup> ∂B0,<sup>β</sup> {�μ<sup>N</sup> <sup>=</sup>�0,� <sup>B</sup>0=�0} and <sup>H</sup>(1,1) αβ = <sup>∂</sup><sup>2</sup><sup>H</sup> ∂μN,α∂B0,<sup>β</sup> {�μ<sup>N</sup> <sup>=</sup>�0,� <sup>B</sup>0=�0} are given by [Fukui & Baba (1998)]: $$\mathcal{H}\_{a}^{(0,1)} = \frac{\mu\_{0}}{2\pi} \sum\_{j=1}^{n} \frac{\vec{l}\_{jN,\notin}}{r\_{jN}^{3}} + \frac{\mu\_{0}}{4\pi} \sum\_{j=1}^{n} \left[ \frac{8\pi}{3} \delta(\vec{r}\_{jN}) \sigma\_{\notin}(j) - \frac{\sigma\_{\notin}(j)}{r\_{jN}^{3}} + 3\,\vec{r}\_{jN,\notin} \frac{\vec{r}\_{jN} \cdot \vec{\sigma}(j)}{r\_{jN}^{5}} \right] \tag{18}$$ $$\mathcal{H}\_{\mathfrak{F}}^{(1,0)} = \frac{1}{2} \sum\_{j=1}^{n} \vec{l}\_{j,\mathfrak{F}} + \frac{1}{2} \sum\_{j=1}^{n} \sigma\_{\mathfrak{F}}(j) \tag{19}$$ $$\mathcal{H}\_{a\beta}^{(1,1)} = \frac{\mu\_0}{8\pi} \sum\_{j=1}^n \frac{\vec{r}\_j \cdot \vec{r}\_{\text{jN}} \delta\_{a\beta} - r\_{\text{jN},a} r\_{\text{j},\beta}}{r\_{\text{jN}}^3},\tag{20}$$ where�ljX <sup>=</sup> <sup>−</sup><sup>i</sup> �rjX <sup>∧</sup> <sup>∇</sup>� <sup>j</sup> , �lj <sup>=</sup> <sup>−</sup><sup>i</sup> �rj <sup>∧</sup> <sup>∇</sup>� <sup>j</sup> and �σ<sup>j</sup> stands for the Pauli spin matrix of the electron j and its cartesian coordinates are given by: $$ \sigma\_{\mathbf{j}, \mathbf{x}} = \begin{pmatrix} 0 \ 1 \\ 1 \ 0 \end{pmatrix}, \quad \sigma\_{\mathbf{j}, \mathbf{y}} = \begin{pmatrix} 0 \ i \\ i \ 0 \end{pmatrix} \quad \text{and} \quad \sigma\_{\mathbf{j}, \mathbf{z}} = \begin{pmatrix} 1 & 0 \\ 0 & -1 \end{pmatrix}. $$ #### 4. Fourier transformation for the analytic development of NMR integrals The operators involved in equations (18), (19) and (20) lead to very complicated integrals. The analytic development of the these NMR integrals is difficult due to the presence of the operator involving 1/rn, which is not the case of the usual three-center molecular integrals (zeroth order molecular integrals) where the Coulomb operator 1/r is involved. In this review, we present the method based on Fourier transform that led to analytic expressions for first and second order integrals of the shielding tensor. Among the operators involved in integrals of the shielding tensors are �rj ·�rjNδαβ <sup>−</sup> rjN,<sup>α</sup> rj,<sup>β</sup> r3 jN in equation (20) for the second order terms and 3rjN,<sup>β</sup> �rjN ·�σ<sup>j</sup> r5 jN in equation (18) in the case of first order relativistic terms. The integrals induced by the above operators are given by: $$\mathcal{Z}\_{13} = \int\_{\vec{r}\_{\dot{j}}} \left[ \chi\_{n\_1, l\_1}^{m\_1} (\zeta\_{1}, \vec{r}\_{\dot{j}A}) \right]^\* \, \mathfrak{Z} r\_{\dot{j}N, \theta} \, \frac{\vec{r}\_{\dot{j}N} \cdot \vec{\sigma}\_{\dot{j}}}{r\_{\dot{j}N}^5} \, \chi\_{n\_2, l\_2}^{m\_2} (\zeta\_{2}, \vec{r}\_{\dot{j}B}) \, \mathrm{d}\vec{r}\_{\dot{j}} \tag{21}$$ $$\mathcal{Z}\_{23} = \int\_{\vec{\tau}\_{\dot{\jmath}}} \left[ \chi\_{n\_1 l\_1}^{m\_1} (\zeta\_1, \vec{r}\_{\dot{\jmath}A}) \right]^\* \xrightarrow{\vec{\tau}\_{\dot{\jmath}} \cdot \vec{r}\_{\dot{\jmath}N}} \frac{\vec{r}\_{\dot{\jmath}N} \delta\_{\text{a}\beta} - r\_{\dot{\jmath}N,a} r\_{\dot{\jmath},\beta}}{r\_{\dot{\jmath}N}^3} \chi\_{n\_2 l\_2}^{m\_2} (\zeta\_2, \vec{r}\_{\dot{\jmath}B}) \,\text{d}\vec{r}\_{\dot{\jmath}\nu} \tag{22}$$ whererjA <sup>=</sup>rj <sup>−</sup> # — OA,rjB <sup>=</sup>rj <sup>−</sup> # — OB andrjN <sup>=</sup>rj <sup>−</sup> # — ON. <sup>A</sup>, <sup>B</sup> and <sup>N</sup> are three arbitrary points of the Euclidean space and 0 is the origin of the fixed coordinate system. #### 4.1 First order integrals 6 Will-be-set-by-IN-TECH where <sup>H</sup>(0) is the zeroth-order hamiltonian (1) and <sup>H</sup>(r) is the relativistic perturbation term, which is independent of the magnetic perturbations and is given by [Fukui & Baba (1998)]: 3,<sup>j</sup> : the retarded orbit-orbit term, <sup>H</sup>(r) {�μ<sup>N</sup> <sup>=</sup>�0,� <sup>B</sup>0=�0} are given by [Fukui & Baba (1998)]: n ∑ j=1 8 π <sup>B</sup>0=�0} , <sup>H</sup>(1,0) <sup>β</sup> = <sup>3</sup> <sup>δ</sup>(�rjN)σβ(j) <sup>−</sup> σβ(j) and σj,<sup>z</sup> = r3 jN > χm<sup>2</sup> n2,l<sup>2</sup> in equation (18) in the case of first order relativistic 2,<sup>j</sup> <sup>+</sup> <sup>H</sup>(r) 3,<sup>j</sup> <sup>+</sup> <sup>H</sup>(r) 1,<sup>j</sup> : two-electron Darwin term, <sup>H</sup>(r) 4,<sup>j</sup> <sup>+</sup> <sup>H</sup>(r) 5,j <sup>∂</sup><sup>H</sup> ∂B0,<sup>β</sup> > r3 jN σβ(j) (19) 1 0 0 −1 , (17) 4,<sup>j</sup> : spin-other-orbit term and <sup>B</sup>0=�0} �rjN ·�σ(j) r5 jN in equation (20) for the (ζ2,�rjB) d�rj, (21) {�μ<sup>N</sup> <sup>=</sup>�0,� + 3 rjN,<sup>β</sup> , (20) and �σ<sup>j</sup> stands for the Pauli spin matrix of the . 2,<sup>j</sup> : two-electron and <sup>H</sup>(1,1) αβ = (18) <sup>H</sup>(r) <sup>=</sup> which include the contributions; <sup>H</sup>(r) spin-orbit term, <sup>H</sup>(r) 5,<sup>j</sup> : spin-spin term. H(1,0) <sup>β</sup> <sup>=</sup> <sup>1</sup> 2 H(1,1) αβ <sup>=</sup> <sup>μ</sup><sup>0</sup> 8π where�ljX <sup>=</sup> <sup>−</sup><sup>i</sup> The perturbations <sup>H</sup>(0,1) <sup>α</sup> <sup>=</sup> {�μ<sup>N</sup> <sup>=</sup>�0,� 2 π second order terms and 3rjN,<sup>β</sup> I<sup>13</sup> = �rj χm<sup>1</sup> n1,l<sup>1</sup> terms. n ∑ j=1 > n ∑ j=1 �rjX <sup>∧</sup> <sup>∇</sup>� <sup>j</sup> σj,<sup>x</sup> = n ∑ j=1 �ljN,<sup>β</sup> r3 jN �lj,<sup>β</sup> + 1 2 0 1 1 0 electron j and its cartesian coordinates are given by: <sup>H</sup>(0,1) <sup>α</sup> <sup>=</sup> <sup>μ</sup><sup>0</sup> H(r) <sup>∂</sup><sup>2</sup><sup>H</sup> ∂μN,α∂B0,<sup>β</sup> n ∑ j<k H(r) 1,<sup>j</sup> <sup>+</sup> <sup>H</sup>(r) <sup>∂</sup><sup>H</sup> ∂μN,<sup>α</sup> > <sup>+</sup> <sup>μ</sup><sup>0</sup> 4 π > > n ∑ j=1 , �lj <sup>=</sup> <sup>−</sup><sup>i</sup> �rj ·�rjNδαβ − rjN,<sup>α</sup> rj,<sup>β</sup> r3 jN > �rj <sup>∧</sup> <sup>∇</sup>� <sup>j</sup> , σj,<sup>y</sup> = 4. Fourier transformation for the analytic development of NMR integrals (zeroth order molecular integrals) where the Coulomb operator 1/r is involved. (ζ1,�rjA) ∗ 3 rjN,<sup>β</sup> �rjN ·�σ<sup>j</sup> r5 jN involved in integrals of the shielding tensors are �rj ·�rjNδαβ <sup>−</sup> rjN,<sup>α</sup> rj,<sup>β</sup> �rjN ·�σ<sup>j</sup> r5 jN The integrals induced by the above operators are given by: 0 i i 0 The operators involved in equations (18), (19) and (20) lead to very complicated integrals. The analytic development of the these NMR integrals is difficult due to the presence of the operator involving 1/rn, which is not the case of the usual three-center molecular integrals In this review, we present the method based on Fourier transform that led to analytic expressions for first and second order integrals of the shielding tensor. Among the operators After expanding the operator in the integrals (21), we can write I<sup>13</sup> as follows: $$\mathcal{L}\_{13} = -\sum\_{\mathbf{a}} \sigma\_{\mathbf{j},\mathbf{a}} \int\_{\vec{\tau}\_{\mathbf{j}}} \left[ \chi\_{\mathbf{n}\_1,\mathbf{l}\_1}^{\mathcal{m}\_1} (\zeta\_1, \vec{r}\_{\mathbf{j}A}) \right]^\* r\_{\mathbf{j}N,\mathbf{\mathcal{S}}} \frac{\partial}{\partial r\_{\mathbf{j}N,\mathbf{a}}} \left( \frac{1}{r\_{\mathbf{j}N}^3} \right) \chi\_{\mathbf{n}\_2,\mathbf{l}\_2}^{\mathcal{m}\_2} (\zeta\_2, \vec{r}\_{\mathbf{j}B}) \mathrm{d}\vec{r}\_{\mathbf{j}}.\tag{23}$$ Let <sup>I</sup>(α,β) <sup>13</sup> represent the integrals in summation of the RHS of the above equation. Using equation (5), the integrals <sup>I</sup>(α,β) <sup>13</sup> are expressed as linear combinations of integrals <sup>B</sup>I(α,β) <sup>13</sup> over B functions which are given by: $$\prescript{}{B}{\mathcal{Z}}\_{13}^{(a,\beta)} = \int\_{\vec{\tau}\_{j}} \left[ \prescript{}{B}{\mathcal{Z}}\_{n\_{1}l\_{1}}^{m\_{1}}(\zeta\_{1}, \vec{\tau}\_{jA}) \right]^{\} r\_{\vec{j}N,\theta} \frac{\partial}{\partial r\_{\vec{j}N,\theta}} \left( \frac{1}{r\_{\vec{j}N}^{3}} \right) \left. B^{m\_{2}}\_{n\_{2}l\_{2}}(\zeta\_{2}, \vec{\tau}\_{\vec{j}B}) \right\\| \vec{\tau}\_{\vec{j}}.\tag{24}$$ Using the Fourier transform method, we obtain: $$\mathbf{T}\_{\rm B} \mathbf{Z}\_{13}^{(\mathbf{a},\boldsymbol{\theta})} = (2\,\pi)^{-3/2} \int\_{\tilde{\mathbf{K}}} r\_{\rm jN,\theta} \,\frac{\partial}{\partial r\_{\rm jN,\theta}} \left(\frac{1}{r\_{\rm jN}^3}\right) \left[\int\_{\tilde{\mathbf{r}}\_{\tilde{\boldsymbol{\cdot}}}} \left[\mathcal{B}\_{\boldsymbol{n}\_1,\boldsymbol{\mathsf{i}}\_1}^{\boldsymbol{m}\_1}(\boldsymbol{\zeta}\_1,\boldsymbol{\vec{r}}\_{\boldsymbol{j}A})\right]^\ e^{-\mathbf{i}\cdot\boldsymbol{\overleftarrow{\boldsymbol{k}}} \cdot \vec{r}\_{\rm jN}} \,\mathcal{B}\_{\boldsymbol{m}\_2\boldsymbol{\sharp}\_2}^{\boldsymbol{m}\_2}(\boldsymbol{\zeta}\_2,\boldsymbol{\vec{r}}\_{\boldsymbol{j}B}) \,\mathrm{d}\mathbf{r}\_{\rm j}^\\right] \,\mathrm{d}\mathbf{\tilde{k}}.\tag{25}$$ In the case where α* and β represent two different cartesian coordinates, the Fourier transform of the operator is given by [Safouhi (2010b)]: $$ \overline{r\_{\beta} \frac{\partial}{\partial r\_{a}} \left( \frac{1}{r^{3}} \right)} = \sqrt{\frac{2}{\pi}} \frac{k\_{a}}{k^{2}} \frac{k\_{\beta}}{k^{2}}.\tag{26} $$ In the case where α and β represent the same cartesian coordinate, the calculations leads to the potential 1/r3, which poses serious difficulties because of the singularity and its Fourier transform does not exist in a sense of classical analysis. This case is a part of ongoing research where the theory of generalized functions will be used in order to derive the Fourier transform of the operator in the generalized function sense. Using the analytic expression (11) obtained by Trivedi and Steinborn [Trivedi & Steinborn (1983)] for the integrals overrj involved in equation (25) and with the help of equation (26), one can derive the following analytic expression for the integrals <sup>B</sup>I(α,β) <sup>13</sup> [Safouhi (2010b)]: $$\begin{split} \, \_B\mathcal{T}\_{13}^{(a,\beta)} &= \frac{4^3 \, \_1\pi \, (2l\_1+1)!! \, (2l\_2+1)!! \, (n\_1+l\_1+n\_2+l\_2+1) ! \, \zeta\_1^{2n\_1+l\_1-1} \, \zeta\_2^{2n\_2+l\_2-1}}{(n\_1+l\_1)! \, (n\_2+l\_2)! \, 2^{n\_1+n\_2+l\_1+l\_2+2}} \\ & \times \sum\_{l\_1'=0}^{l\_1} (-i)^{l\_1+l\_1'} \sum\_{m\_1'=-l\_1'}^{l\_1'} \frac{\langle l\_1 m\_1 \, \left\| \, l\_1' m\_1' \right\| \, l\_1-l\_1' m\_1-m\_1' \rangle}{(2l\_1'+1)!! [2(l\_1-l\_1')+1]!!} \end{split}$$ × × × η� ∑ r=0 where: BI(α,β) <sup>12</sup> = × × × × η� ∑ r=0 2 ∑ l=lmin,2 n1+n<sup>2</sup> ∑ τ=2 τ2 ∑ ς=τ<sup>1</sup> ( η <sup>2</sup> )<sup>r</sup> ( <sup>η</sup>+<sup>1</sup> 2<sup>τ</sup> ζ ς−1 <sup>1</sup> ζ τ−ς−1 2 ζτ s <sup>2</sup> )l1+l2+τ−<sup>1</sup> <sup>2</sup> )<sup>r</sup> (−<sup>r</sup> <sup>−</sup> <sup>l</sup>+<sup>1</sup> (l + <sup>3</sup> τ<sup>1</sup> = max(1, τ − n2) τ<sup>2</sup> = min(n1, τ − 1) η = l − τ − l<sup>1</sup> − l<sup>2</sup> + 1 ζ<sup>s</sup> = ζ<sup>1</sup> + ζ<sup>2</sup> <sup>2</sup> <sup>ζ</sup>l1+l2−<sup>1</sup> <sup>s</sup> <sup>R</sup>2l1+2l2−<sup>3</sup> (−1)m<sup>3</sup> <sup>c</sup>α,m<sup>3</sup> <sup>c</sup>β,m<sup>4</sup> l � r+ <sup>l</sup>−<sup>λ</sup> 2 ∑ s=0 <sup>η</sup>� <sup>=</sup> <sup>−</sup><sup>η</sup> 4n1+l1+n2+l<sup>2</sup> (n<sup>1</sup> + l1)!(n<sup>2</sup> + l2)! For the two-center integrals (A = B �= N): 1 ∑ m4=−1 (R ζs)−<sup>λ</sup> � <sup>√</sup>π ζl<sup>1</sup> <sup>1</sup> <sup>ζ</sup>l<sup>2</sup> 1 ∑ m3=−1 l+l � ∑ λ=λmin,2 n1+n<sup>2</sup> ∑ τ=2 τ2 ∑ ς=τ<sup>1</sup> (l + <sup>3</sup> <sup>2</sup> )<sup>r</sup> r! we assume A = O in equation (25). The operator involved in (22) is given by: = Iαβ <sup>23</sup> = � rj � χm<sup>1</sup> n1,l<sup>1</sup> ⎧ ⎪⎪⎪⎨ −rjN,<sup>α</sup> rj,<sup>β</sup> r3 jN rjN,<sup>u</sup> rj,<sup>u</sup> r3 jN + (ζ1,rj) ⎪⎪⎪⎩ ( η <sup>2</sup> )<sup>r</sup> ( <sup>η</sup>+<sup>1</sup> <sup>2</sup> )<sup>r</sup> 4.2 Second order integrals rj ·rjNδαβ − rjN,<sup>α</sup> rj,<sup>β</sup> r3 jN integrals of the form: <sup>2</sup>−<sup>l</sup> �<sup>1</sup> <sup>m</sup>4\|<sup>1</sup> <sup>m</sup>3\|l m<sup>4</sup> <sup>−</sup> <sup>m</sup>3� �l1m1\|l2m2\|lm<sup>1</sup> <sup>−</sup> <sup>m</sup>2� <sup>δ</sup>m1−m2,m4−m<sup>3</sup> Fourier Transformation Method for Computing NMR Integrals over Exponential Type Functions 129 <sup>2</sup> if <sup>η</sup> is even otherwise <sup>η</sup>� <sup>=</sup> <sup>−</sup>η+<sup>1</sup> m<sup>4</sup> − m3\|lm<sup>1</sup> − m2\|λm<sup>4</sup> − m<sup>3</sup> − m<sup>1</sup> + m<sup>2</sup> 1 m4\|1 m3\|l (<sup>ς</sup> <sup>−</sup> <sup>1</sup>)!(n<sup>1</sup> <sup>−</sup> <sup>ς</sup>)!(<sup>τ</sup> <sup>−</sup> <sup>ς</sup> <sup>−</sup> <sup>1</sup>)!(n<sup>2</sup> <sup>−</sup> <sup>τ</sup> <sup>+</sup> <sup>ς</sup>)! <sup>Γ</sup>(<sup>l</sup> <sup>+</sup> <sup>3</sup> (<sup>R</sup> <sup>ζ</sup>s)2<sup>s</sup> <sup>ˆ</sup> when α �= β χm<sup>2</sup> n2,l<sup>2</sup> �2<sup>s</sup> (<sup>1</sup> <sup>−</sup> <sup>l</sup><sup>1</sup> <sup>−</sup> <sup>l</sup><sup>2</sup> <sup>−</sup> <sup>τ</sup>)<sup>s</sup> The second order integrals I<sup>23</sup> are given by (22). For simplicity and without loss of generality, rjN,<sup>v</sup> rj,<sup>v</sup> r3 jN From the above equation, it is obvious that the integrals I<sup>23</sup> can be expressed in terms of �<sup>∗</sup> rjN,<sup>α</sup> rj,<sup>β</sup> r3 jN l1+l<sup>2</sup> ∑ l=lmin,2 2 ∑ l�=l� min,2 � <sup>R</sup>2<sup>τ</sup> (2n<sup>1</sup> <sup>−</sup> <sup>ς</sup> <sup>−</sup> <sup>1</sup>)!(2n<sup>2</sup> <sup>−</sup> <sup>τ</sup> <sup>+</sup> <sup>ς</sup> <sup>−</sup> <sup>1</sup>)! <sup>ζ</sup> �r + <sup>l</sup>−<sup>λ</sup> 2 s (2n<sup>1</sup> − ς − 1)!(2n<sup>2</sup> − τ + ς − 1)!(τ + l<sup>1</sup> + l2)l+<sup>1</sup> (<sup>ς</sup> <sup>−</sup> <sup>1</sup>)!(n<sup>1</sup> <sup>−</sup> <sup>ς</sup>)!(<sup>τ</sup> <sup>−</sup> <sup>ς</sup> <sup>−</sup> <sup>1</sup>)!(n<sup>2</sup> <sup>−</sup> <sup>τ</sup> <sup>+</sup> <sup>ς</sup>)! <sup>Γ</sup>(<sup>l</sup> <sup>+</sup> <sup>3</sup> <sup>2</sup> )<sup>r</sup> <sup>r</sup>! , (29) <sup>2</sup>−<sup>l</sup> �l1m1\|l2m2\|lm<sup>1</sup> <sup>−</sup> <sup>m</sup>2� ς−1 <sup>1</sup> ζ � m<sup>4</sup> − m<sup>3</sup> τ−ς−1 <sup>2</sup> <sup>ζ</sup><sup>τ</sup> <sup>2</sup> . � � <sup>k</sup>λ−l1−l2−τ+s<sup>+</sup> <sup>3</sup> when α = β and u, v �= α ∈ {x, y, z}. Ym4−m3−m1+m<sup>2</sup> <sup>λ</sup> (θ <sup>s</sup> (τ + l<sup>1</sup> + l2)l+<sup>1</sup> 2 ) 2 (ζ2,rjB) drj. (33) 2 ) (30) <sup>R</sup>, ϕ R) (32) (R ζs). (31) × l2 ∑ l � <sup>2</sup>=0 (−i)l2+<sup>l</sup> � 2 l � 2 ∑ m� <sup>2</sup>=−l � 2 l2m<sup>2</sup> l � 2m� 2 l<sup>2</sup> − l � <sup>2</sup>m<sup>2</sup> − m� 2 (2l � <sup>2</sup> + 1)!![2(l<sup>2</sup> − l � <sup>2</sup>) + 1]!! × l � <sup>1</sup>+l � 2 ∑ l�=l � min,2 (−1)<sup>l</sup> � 1 l � 2m� 2 l � 1m� 1 l � m� <sup>2</sup> − m� 1 R<sup>l</sup> � <sup>2</sup> <sup>Y</sup>m� 2−m� 1 <sup>l</sup>� (θ� R2 , ϕ� R2 ) × l1−l � <sup>1</sup>+l2−l � 2 ∑ l12=l12min,2 l<sup>2</sup> − l � <sup>2</sup>m<sup>2</sup> − m� 2 l<sup>1</sup> − l � <sup>1</sup>m<sup>1</sup> − m� 1 <sup>l</sup>12m<sup>12</sup> × 1 ∑ m3=−1 1 ∑ m4=−1 (−1)m<sup>4</sup> <sup>c</sup>α,m<sup>3</sup> <sup>c</sup>β,m<sup>4</sup> 2 ∑ l��=l��min,2 1 m4\|1 m3\|l �� m<sup>4</sup> − m<sup>3</sup> × l ��+l<sup>12</sup> ∑ λ=λmin,2 (−i)<sup>λ</sup> <sup>&</sup>lt; <sup>l</sup>12m12\|<sup>l</sup> ��m<sup>3</sup> − m4\|λ μ > × Δl ∑ j=0 (−2)<sup>j</sup> ( Δl j ) (n<sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l<sup>2</sup> − j + 1)! × 1 s=0 s <sup>n</sup><sup>22</sup> (<sup>1</sup> <sup>−</sup> <sup>s</sup>)n<sup>11</sup> <sup>Y</sup><sup>μ</sup> <sup>λ</sup> (θ�v, ϕ�v) × <sup>+</sup><sup>∞</sup> x=0 xnx ˆ k<sup>ν</sup> [R<sup>2</sup> γ(s, x)] [γ(s, <sup>x</sup>)]n<sup>γ</sup> <sup>j</sup>λ(v x) <sup>d</sup><sup>x</sup> ds, (27) where: � $$\begin{aligned} \vec{R}\_1 &= \overrightarrow{AA} \,\vec{R}\_2 = \overrightarrow{AB} \quad \text{and} \quad \vec{v} = (1-s)\vec{R}\_2 - \vec{R}\_1 \\ \gamma(s,x) &= \sqrt{(1-s)\xi\_1^2 + s\xi\_2^2 + s(1-s)x^2} \\ m\_\gamma &= 2(n\_1 + n\_2 + l\_1 + l\_2) - (l\_1' + l\_2' + l') + 1 \\ m\_1 &= n\_1 + l\_1 + l\_2 - l\_2', \eta\_{22} = n\_2 + l\_2 + l\_1 - l\_1' \\ \nu &= n\_1 + n\_2 + l\_1 + l\_2 - l' - j + \frac{1}{2} \\ m\_{12} &= (m\_2 - m\_2') - (m\_1 - m\_1') \\ n\_k &= l\_1 - l\_1' + l\_2 - l\_2' + 2 \\ \mu &= m\_{12} - m\_3 + m\_4 \\ \Delta l &= \frac{l\_1' + l\_2' - l'}{2}. \end{aligned} \tag{28}$$ In the case of one- and two-center integrals <sup>B</sup>I(α,β) <sup>11</sup> and <sup>B</sup>I(α,β) <sup>12</sup> , corresponding to A = B = N and A = B �= N respectively, we derived analytical expressions in [Slevinsky et al. (2010)]. For the one-center integrals (A = B = N): $$\begin{split} \, \_B T\_{11}^{(\alpha,\beta)} &= -\frac{\mathcal{Z}\_1^{l\_1} \mathcal{Z}\_2^{l\_2} \, \_2 \mathbb{Z}\_s^{2-l\_1-l\_2}}{\sqrt{\pi} \, 2^{2 \, n\_1+l\_1+2 \, n\_2+l\_2+3} \, (n\_1+l\_1)! \, (n\_2+l\_2)!} \\ &\times \sum\_{m\_3=-1 \, m\_4=-1}^{1} \sum\_{m\_4=-1}^{1} (-1)^{m\_3} \, \mathcal{L}\_{\alpha,m\_3} \, \mathcal{L}\_{\beta,m\_4} \end{split}$$ $$\begin{split} &\times \quad \sum\_{l=l\_{\min},2}^{2} 2^{-l} \left< 1 \, m\_{4} \vert 1 \, m\_{3} \vert l \, m\_{4} - m\_{3} \right> \left< l\_{1}m\_{1} \vert l\_{2}m\_{2} \vert l m\_{1} - m\_{2} \right> \delta\_{m\_{1} - m\_{2}, m\_{4} - m\_{3}} \\ &\times \sum\_{r=2}^{m\_{1} + m\_{2}} \sum\_{\xi=r\_{1}}^{2} \frac{2^{\tau} \, \xi\_{1}^{\xi - 1} \, \xi\_{2}^{\tau - \xi - 1}}{\xi\_{\overline{s}}^{\tau}} \frac{(2n\_{1} - \zeta - 1)! \left< 2n\_{2} - \tau + \zeta - 1 \right> (\tau + l\_{1} + l\_{2})\_{l+1}}{(\zeta - 1)! \left< n\_{1} - \zeta \right> (\tau - \zeta - 1)! \left< n\_{2} - \tau + \zeta \right> \Gamma (l + \frac{3}{2})} \\ &\times \sum\_{r=0}^{\eta^{\prime}} \frac{(\frac{\eta}{2})\_{r} \left( \frac{\eta + 1}{2} \right)\_{l} \left( -r - \frac{l + 1}{2} \right)\_{l\_{1} + l\_{2} + \tau - 1}}{(l + \frac{3}{2})\_{r} \tau!} \,, \tag{29} \end{split} \tag{29}$$ where: 8 Will-be-set-by-IN-TECH <sup>2</sup> + 1)!![2(l<sup>2</sup> − l 2 ∑ l��=l��min,2 ��m<sup>3</sup> − m4\|λ μ > AB and �<sup>v</sup> = (<sup>1</sup> <sup>−</sup> <sup>s</sup>) � � <sup>1</sup> + l � <sup>2</sup> + l � ) + 1 � <sup>−</sup> <sup>j</sup> <sup>+</sup> <sup>1</sup> 2 1) <sup>2</sup> + <sup>s</sup>(<sup>1</sup> − <sup>s</sup>) <sup>x</sup><sup>2</sup> <sup>2</sup>, n<sup>22</sup> = n<sup>2</sup> + l<sup>2</sup> + l<sup>1</sup> − l <sup>11</sup> and <sup>B</sup>I(α,β) <sup>1</sup> <sup>+</sup> <sup>s</sup> <sup>ζ</sup><sup>2</sup> � <sup>2</sup>) − (m<sup>1</sup> − m� and A = B �= N respectively, we derived analytical expressions in [Slevinsky et al. (2010)]. � <sup>2</sup> + 2 1 m4\|1 m3\|l �� m<sup>4</sup> − m<sup>3</sup> ds, (27) <sup>12</sup> , corresponding to A = B = N (28) <sup>R</sup><sup>2</sup> <sup>−</sup> � R1 > � 1 � <sup>2</sup>) + 1]!! × l2 ∑ l � <sup>2</sup>=0 × l � <sup>1</sup>+l � 2 ∑ l�=l � min,2 × l1−l � <sup>1</sup>+l2−l � 2 × × l ��+l<sup>12</sup> ∑ λ=λmin,2 × Δl ∑ j=0 × 1 s=0 s × where: � <sup>+</sup><sup>∞</sup> x=0 xnx ˆ <sup>R</sup><sup>1</sup> <sup>=</sup> # — AN, � γ(s, x) = (−i)l2+<sup>l</sup> � 2 ∑ l12=l12min,2 1 ∑ m3=−1 (−1)<sup>l</sup> � 1 l � 2m� 2 l � 1m� 1 l � m� <sup>2</sup> − m� 1 R<sup>l</sup> � <sup>2</sup> <sup>Y</sup>m� 2−m� 1 <sup>l</sup>� (θ� R2 , ϕ� R2 ) l<sup>2</sup> − l � <sup>2</sup>m<sup>2</sup> − m� 2 l<sup>1</sup> − l � <sup>1</sup>m<sup>1</sup> − m� 1 l12m<sup>12</sup> 1 ∑ m4=−1 l � 2 ∑ m� <sup>2</sup>=−l � 2 l2m<sup>2</sup> l � 2m� 2 l<sup>2</sup> − l � <sup>2</sup>m<sup>2</sup> − m� 2 (−1)m<sup>4</sup> <sup>c</sup>α,m<sup>3</sup> <sup>c</sup>β,m<sup>4</sup> <sup>λ</sup> (θ�v, ϕ�v) [γ(s, <sup>x</sup>)]n<sup>γ</sup> <sup>j</sup>λ(v x) <sup>d</sup><sup>x</sup> (−i)<sup>λ</sup> <sup>&</sup>lt; <sup>l</sup>12m12\|<sup>l</sup> (−2)<sup>j</sup> ( Δl j ) <sup>n</sup><sup>22</sup> (<sup>1</sup> <sup>−</sup> <sup>s</sup>)n<sup>11</sup> <sup>Y</sup><sup>μ</sup> (n<sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l<sup>2</sup> − j + 1)! k<sup>ν</sup> [R<sup>2</sup> γ(s, x)] <sup>R</sup><sup>2</sup> <sup>=</sup> # — n<sup>γ</sup> = 2(n<sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l2) − (l (<sup>1</sup> <sup>−</sup> <sup>s</sup>) <sup>ζ</sup><sup>2</sup> n<sup>11</sup> = n<sup>1</sup> + l<sup>1</sup> + l<sup>2</sup> − l m<sup>12</sup> = (m<sup>2</sup> − m� nk = l<sup>1</sup> − l Δl = <sup>l</sup> � <sup>1</sup>+l � <sup>2</sup>−l � <sup>2</sup> . In the case of one- and two-center integrals <sup>B</sup>I(α,β) For the one-center integrals (A = B = N): 1 ∑ m4=−1 <sup>11</sup> <sup>=</sup> <sup>−</sup> <sup>ζ</sup>l<sup>1</sup> 1 ∑ m3=−1 BI(α,β) × ν = n<sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l<sup>2</sup> − l � <sup>1</sup> + l<sup>2</sup> − l μ = m<sup>12</sup> − m<sup>3</sup> + m<sup>4</sup> <sup>1</sup> <sup>ζ</sup>l<sup>2</sup> <sup>2</sup> <sup>ζ</sup>2−l1−l<sup>2</sup> <sup>s</sup> <sup>√</sup><sup>π</sup> <sup>2</sup><sup>2</sup> <sup>n</sup>1+l1+<sup>2</sup> <sup>n</sup>2+l2+<sup>3</sup> (n<sup>1</sup> <sup>+</sup> <sup>l</sup>1)!(n<sup>2</sup> <sup>+</sup> <sup>l</sup>2)! (−1)m<sup>3</sup> <sup>c</sup>α,m<sup>3</sup> <sup>c</sup>β,m<sup>4</sup> (2l � $$\begin{aligned} \tau\_1 &= \max(1, \tau - \eta\_2) \\ \tau\_2 &= \min(\eta\_1, \tau - 1) \\ \zeta\_s &= \zeta\_1 + \zeta\_2 \\ \eta &= l - \tau - l\_1 - l\_2 + 1 \end{aligned} \tag{30}$$ <sup>η</sup>� <sup>=</sup> <sup>−</sup><sup>η</sup> <sup>2</sup> if <sup>η</sup> is even otherwise <sup>η</sup>� <sup>=</sup> <sup>−</sup>η+<sup>1</sup> <sup>2</sup> . For the two-center integrals (A = B �= N): $$\begin{split} \operatorname{rg}\_{12}^{(a,\beta)} &= \frac{\sqrt{\pi}c\_{11}^{l\_1}}{4^{m\_1+l\_1+m\_2+l\_2}} \frac{l\_1^{l\_1}l\_2^{l\_2}}{(n\_1+l\_1)!(n\_2+l\_2)!} \sum\_{l=l\_{\min}/2}^{l\_1+l\_2} 2^{-l} \left< l\_1m\_1 \middle\| 2m\_2 \middle\| lm\_1 - m\_2 \right> \\ & \times \sum\_{m\_3=-1}^1 \sum\_{m\_4=-1}^1 (-1)^{m\_3} c\_{a,m\_3} c\_{\beta,m\_4} \sum\_{l'=l\_{\min}/2}^2 \left< l \middle\| m\_4 \middle\| 1 \middle\| m\_3 \middle\| l'm\_4 - m\_3 \right> \\ & \times \sum\_{\lambda=-\lambda\_{\min}/2}^{l+l'} (R\not\leq s)^{-\lambda} \left< l'm\_4 - m\_3 \middle\| lm\_1 - m\_2 \middle\| \lambda m\_4 - m\_3 - m\_1 + m\_2 \right> Y\_{\lambda}^{m\_4 - m\_3 - m\_1 + m\_2}\_{\lambda} (\theta\_{\tilde{R}}, \theta\_{\tilde{R}}) \\ & \times \sum\_{\tau=2}^{n\_1+\eta\_2} \sum\_{\xi=\eta\_1}^{\tau\_2} \frac{\mathscr{R}^{2\tau} (2n\_1 - \xi - 1)! \left< 2n\_2 - \tau + \xi - 1 \right> t\_1^{\xi-1} \xi^{\tau-1} \xi^{\tau-1} \zeta\_{\xi}^{\tau-1} \zeta\_{\xi}^{\tau} \left< \tau + l\_1 + l\_2 \right>\_{l+1} \\ & \times \sum\_{\tau=0}^{\eta} \frac{(\mathscr{R})^{\tau} \left( \frac{\eta+1}{2} \right) r}{(l+\frac{\lambda}{2})^2 r\_1 r\_1} \sum\_{s=0}^{l+\frac{$$ #### 4.2 Second order integrals The second order integrals I<sup>23</sup> are given by (22). For simplicity and without loss of generality, we assume A = O in equation (25). The operator involved in (22) is given by: $$\frac{\vec{r}\_{j} \cdot \vec{r}\_{jN} \delta\_{a\hat{\boldsymbol{\beta}}} - r\_{jN,a} r\_{j,\boldsymbol{\beta}}}{r\_{jN}^3} = \begin{cases} \frac{-r\_{jN,a} r\_{j,\boldsymbol{\beta}}}{r\_{jN}^3} & \text{when } a \neq \boldsymbol{\beta} \\\\ \frac{r\_{jN,a} r\_{j,\boldsymbol{\mu}}}{r\_{jN}^3} + \frac{r\_{jN,\boldsymbol{\nu}} r\_{j,\boldsymbol{\nu}}}{r\_{jN}^3} & \text{when } a = \boldsymbol{\beta} \quad \text{and} \quad \boldsymbol{u}, v \neq a \in \{x, y, z\}. \end{cases} \tag{32}$$ From the above equation, it is obvious that the integrals I<sup>23</sup> can be expressed in terms of integrals of the form: $$\mathcal{L}\_{23}^{a\mathcal{B}} = \int\_{\vec{r}\_{\vec{j}}} \left[ \chi\_{n\_1,l\_1}^{m\_1} (\zeta\_1, \vec{r}\_{\vec{j}}) \right]^\* \frac{r\_{\vec{j}N,a} \, r\_{\vec{j},\vec{\theta}}}{r\_{\vec{j}N}^3} \chi\_{n\_2,l\_2}^{m\_2} (\zeta\_2, \vec{r}\_{\vec{j}B}) \, d\vec{r}\_{\vec{j}}.\tag{33}$$ Let <sup>I</sup>˜ αβ form: <sup>B</sup>I<sup>ˆ</sup> αβ Safouhi (2008)]: <sup>B</sup>I<sup>ˆ</sup> αβ × l1 ∑ l � <sup>1</sup>=0 × l2 ∑ l � <sup>2</sup>=0 × l � <sup>1</sup>+l � 2 ∑ l�=l � min,2 × l1−l � <sup>1</sup>+l2−l � 2 × × Δl ∑ j=0 × 1 s=0 s × <sup>23</sup> represent the integrals involved in the above equation. Using equation (5), these Fourier Transformation Method for Computing NMR Integrals over Exponential Type Functions 131 <sup>1</sup> (θrjN , φrjN ) r2 jN > (ζ1,rj) ∗ e > > 2 π (n˜ <sup>1</sup> + l1)! (n<sup>2</sup> + l2)! � <sup>1</sup>) + 1]!! � <sup>2</sup>) + 1]!! <sup>1</sup>, m˜ <sup>1</sup> − m� <sup>λ</sup> (θ−→<sup>v</sup> , ϕ−→<sup>v</sup> ) 1 <sup>l</sup>12, <sup>m</sup>12 <sup>1</sup>, m˜ <sup>1</sup> − m� <sup>2</sup>, m<sup>2</sup> − m� l� Yμ <sup>1</sup> (θ<sup>k</sup> , φ<sup>k</sup>) The Fourier transform of the operator involved in the above integrals is given by [Berlu & Using the analytic expression (11) obtained by Trivedi and Steinborn [Trivedi & Steinborn (1983)] for the integrals overrj involved in equation (42) and equation (43), one can derive the <sup>1</sup> + 1)!![2(l<sup>1</sup> − l <sup>2</sup> + 1)!![2(l<sup>2</sup> − l � <sup>2</sup>Y<sup>μ</sup> Bm<sup>2</sup> n2,l<sup>2</sup> <sup>−</sup><sup>i</sup><sup>k</sup> ·rjN <sup>B</sup>m<sup>2</sup> <sup>23</sup> [Berlu & Safouhi (2008)]: 1 2 <sup>θ</sup> # —OB, <sup>ϕ</sup># —OB ds, (44) n2,l<sup>2</sup> <sup>∗</sup> <sup>Y</sup>μ<sup>1</sup> <sup>23</sup> over B functions of the (ζ2,rjB) drj, (41) (ζ2,rjB) drj <sup>k</sup> . (43) <sup>1</sup> <sup>ζ</sup>2n2+l2−<sup>1</sup> 2 2n˜ <sup>1</sup>+n2+l1+l2+<sup>1</sup> ζ2n˜ <sup>1</sup>+l1−<sup>1</sup> dk. (42) integrals can be expressed as linear combinations of integrals <sup>B</sup>I<sup>ˆ</sup> αβ (ζ1,rj) rj Bm˜ <sup>1</sup> n˜ 1,l <sup>B</sup>I<sup>ˆ</sup> αβ <sup>23</sup> = rj Bm˜ <sup>1</sup> n˜ 1,l where m˜ <sup>1</sup> = m<sup>1</sup> − μ<sup>2</sup> and n˜ <sup>1</sup> = n<sup>1</sup> + 1. <sup>23</sup> = (<sup>2</sup> <sup>π</sup>)−3/2 Using the Fourier transform method, we obtain: Yμ<sup>1</sup> Y<sup>μ</sup> following analytic expression for the integrals <sup>B</sup>I<sup>ˆ</sup> αβ (−i)l1+<sup>l</sup> � 1 l1, m˜ <sup>1</sup> l � <sup>1</sup>, m� 1 l<sup>1</sup> − l � (−i)l2+<sup>l</sup> � 2 l2, m<sup>2</sup> l � <sup>2</sup>, m� 2 l<sup>2</sup> − l � l � 1 ∑ m� <sup>1</sup>=−l � 1 l � 2 ∑ m� <sup>2</sup>=−l � 2 ∑ l12=l12 min,2 > Δl j <sup>+</sup><sup>∞</sup> x=0 1+l<sup>12</sup> ∑ λ=λmin,2 (−1)<sup>l</sup> � 1 l � 2, m� 2 l � <sup>1</sup>, m� 1 l � m� 2, −m� 1 R<sup>l</sup> � <sup>2</sup> <sup>Y</sup>m� 2−m� 1 l<sup>2</sup> − l � n2+l2+l1−l xnx ˆ <sup>1</sup> (θrjN , φrjN ) r2 jN > <sup>1</sup> (θrjN , φrjN ) r2 jN <sup>23</sup> <sup>=</sup> <sup>8</sup> (4π)<sup>2</sup> (2l<sup>1</sup> <sup>+</sup> <sup>1</sup>)!! (2l<sup>2</sup> <sup>+</sup> <sup>1</sup>)!!(n˜ <sup>1</sup> <sup>+</sup> <sup>n</sup><sup>2</sup> <sup>+</sup> <sup>l</sup><sup>1</sup> <sup>+</sup> <sup>l</sup><sup>2</sup> <sup>+</sup> <sup>1</sup>)! (2l � (2l � <sup>2</sup>, m<sup>2</sup> − m� (−i)λ+<sup>1</sup> �λ, <sup>μ</sup> <sup>\|</sup>l12, <sup>m</sup>12<sup>\|</sup> 1, <sup>μ</sup>1� (n˜ <sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l<sup>2</sup> − j + 1)! <sup>1</sup> (<sup>1</sup> <sup>−</sup> <sup>s</sup>)n˜ <sup>1</sup>+l1+l2−<sup>l</sup> <sup>γ</sup>(s, <sup>x</sup>)n<sup>γ</sup> <sup>j</sup>λ(vx) <sup>d</sup><sup>x</sup> (−2)<sup>j</sup> � kν[γ(s, x) R2] 2 l<sup>1</sup> − l � ( <sup>k</sup>) = <sup>−</sup><sup>i</sup> k The cartesian coordinate rjN,<sup>α</sup> and rj,<sup>β</sup> can be expressed in terms of spherical harmonics and rjN and rj respectively as follows: $$r\_{\mathbf{j}\mathbf{N},\mathbf{a}} = r\_{\mathbf{j}\mathbf{N}} \sum\_{\mu\_{\mathbf{l}}=-1}^{1} c\_{\mathbf{a},\mu\_{\mathbf{l}}} \, \mathbf{Y}\_{\mathbf{l}}^{\mu\_{\mathbf{l}}} (\theta\_{\vec{r}\_{\mathbf{j}\mathbf{N}}}, \phi\_{\vec{r}\_{\mathbf{N}}}) \quad \text{and} \quad r\_{\mathbf{j},\mathbf{\tilde{\theta}}} = r\_{\mathbf{j}} \sum\_{\mu\_{\mathbf{2}}=-1}^{1} c\_{\mathbf{\tilde{\theta}},\mu\_{\mathbf{2}}} \, \mathbf{Y}\_{\mathbf{1}}^{\mu\_{\mathbf{2}}} (\theta\_{\vec{r}\_{\mathbf{l}}}, \phi\_{\vec{r}\_{\mathbf{l}}}), \tag{34}$$ where the coefficients cα,<sup>μ</sup> are given as follows: $$\begin{cases} \mathbf{c}\_{\mathbf{x},-1} = \sqrt{\frac{2\pi}{3}}, & \mathbf{c}\_{\mathbf{y},-1} = i\sqrt{\frac{2\pi}{3}} \quad \text{and } \mathbf{c}\_{\mathbf{z},-1} = 0\\ \mathbf{c}\_{\mathbf{x},0} = \mathbf{0}, & \mathbf{c}\_{\mathbf{y},0} = 0 \quad \text{and } \mathbf{c}\_{\mathbf{z},0} = \sqrt{\frac{4\pi}{3}}\\ \mathbf{c}\_{\mathbf{x},1} = -\sqrt{\frac{2\pi}{3}}, & \mathbf{c}\_{\mathbf{y},1} = i\sqrt{\frac{2\pi}{3}} \quad \text{and } \mathbf{c}\_{\mathbf{z},1} = \mathbf{0}. \end{cases} \tag{35}$$ Using the analytic expression of the Unormalized STFs (2), one can obtain: $$r\_{\vec{\rho},\vec{\rho}}\left[\boldsymbol{\chi}\_{n\_{1},l\_{1}}^{m\_{1}}(\boldsymbol{\xi}\_{1},\boldsymbol{\vec{r}}\_{\vec{\rho}})\right]^{\} = \left[r\_{\vec{\rho}}\sum\_{\mu\_{2}=-1}^{1}c\_{\vec{\rho},\mu\_{2}}\,\boldsymbol{Y}\_{1}^{\mu\_{2}}(\boldsymbol{\theta}\_{\vec{\tau}\_{\vec{\rho}}},\boldsymbol{\phi}\_{\vec{\tau}\_{\vec{\rho}}})\right]r\_{\vec{\rho}}^{n\_{1}-1}\,\boldsymbol{e}^{-\boldsymbol{\xi}\_{1}\cdot\boldsymbol{r}\_{\vec{\rho}}}\left[\boldsymbol{Y}\_{l\_{1}}^{m\_{1}}(\boldsymbol{\theta}\_{\vec{\tau}\_{\vec{\rho}}},\boldsymbol{\rho}\_{\vec{\tau}\_{\vec{\rho}}})\right]^{\}$$ $$=\sum\_{\mu\_{2}=-1}^{1}c\_{\vec{\rho},\mu\_{2}}\,\boldsymbol{r}\_{\vec{\rho}}^{\mu\_{1}}\,\boldsymbol{e}^{-\boldsymbol{\xi}\_{1}\cdot\boldsymbol{r}\_{\vec{\rho}}}\left[\boldsymbol{Y}\_{l\_{1}}^{m\_{1}}(\boldsymbol{\theta}\_{\vec{\tau}\_{\vec{\rho}}},\boldsymbol{\rho}\_{\vec{\tau}\_{\vec{\rho}}})\right]^{\}\,\boldsymbol{Y}\_{1}^{\mu\_{2}}(\boldsymbol{\theta}\_{\vec{\tau}\_{\vec{\rho}}},\boldsymbol{\phi}\_{\vec{\tau}\_{\vec{\rho}}}).\tag{36}$$ The product of two spherical harmonics can be linearized by Gaunt coefficients as follows: $$\left[Y\_{l\_1}^{\mathfrak{M}\_1}(\theta,\varphi)\right]^\ Y\_{l\_2}^{\mathfrak{M}\_2}(\theta,\varphi) = \sum\_{l=l\_{\min},2}^{l\_1+l\_2} \left Y\_{l}^{\mathfrak{M}\_2-\mathfrak{M}\_1}(\theta,\varphi),\tag{37}$$ where the summation index l runs in steps of 2 from lmin to l<sup>1</sup> + l2. The constant lmin is given by [Weniger & Steinborn (1982)]: $$l\_{\min} = \begin{cases} \max(\|l\_1 - l\_2\|, \|m\_2 - m\_1\|) & \text{if } l\_1 + l\_2 + \max(\|l\_1 - l\_2\|, \|m\_2 - m\_1\|) \text{ is even} \\\\ \max(\|l\_1 - l\_2\|, \|m\_2 - m\_1\|) + 1 \text{ if } l\_1 + l\_2 + \max(\|l\_1 - l\_2\|, \|m\_2 - m\_1\|) \text{ is odd} \end{cases} \tag{38}$$ From equation (37), it follows that: $$\begin{split} \left[r\_{\vec{\rho},\vec{\rho}}\left[\boldsymbol{\chi}\_{n\_{1}l\_{1}}^{m\_{1}}(\boldsymbol{\xi}\_{1},\boldsymbol{\tilde{\tau}}\_{\vec{\rho}})\right]^{\ast} = \sum\_{\mu\_{2}=-1}^{1} c\_{\vec{\rho},\mu\_{2}} r\_{\vec{\rho}}^{n} \, \mathrm{e}^{-\mathsf{f}\_{\vec{\rho}}r\_{\vec{\rho}}} \sum\_{l=l\_{\min},2}^{l\_{1}+1} \left<\boldsymbol{1}\,\mu\_{2}\|l\_{1}\,m\_{1}\|l\,\mu\_{2}-m\_{1}\right> \, Y\_{l}^{\mu\_{2}-m\_{1}}(\boldsymbol{\theta}\_{\vec{\tau}\_{\vec{\rho}}} \, \boldsymbol{\rho}\_{\vec{\tau}\_{\vec{\rho}}}) \\ = \sum\_{\mu\_{2}=-1}^{1} \sum\_{l=l\_{\min},2}^{l\_{1}+1} c\_{\vec{\rho},\mu\_{2}} \, \left<\boldsymbol{1}\,\mu\_{2}\|l\_{1}\,m\_{1}\|l\,\mu\_{2}-m\_{1}\right> \, (-1)^{\mu\_{2}-m\_{1}} \, \left[\boldsymbol{\chi}\_{n\_{1}+1,l}^{m\_{1}-\mu\_{2}}(\boldsymbol{\xi}\_{1},\boldsymbol{\vec{\rho}}\_{\vec{\rho}})\right]^{\ast}. \end{split} \tag{39}$$ Using equations (34) and (39), we obtain the following relation: $$\begin{split} \mathcal{I}\_{23}^{\mathbf{a}\boldsymbol{\beta}} &= \sum\_{\mu\_{2}=-1}^{1} \sum\_{l=l\_{\text{min}}}^{l\_{1}+1} \sum\_{\mu\_{1}=-1}^{1} (-1)^{\mu\_{2}-m\_{1}} c\_{\vec{\rho},\mu\_{2}} c\_{\vec{\kappa},\mu\_{1}} \left< 1 \,\mu\_{2} \,\|l\_{1} \, m\_{1} \,\|\, l \, \mu\_{2} - m\_{1} \right> \\ & \times \int\_{\vec{\tau}\_{j}} \left[ \chi\_{n\_{1}+1,l}^{m\_{1}-\mu\_{2}} (\zeta\_{1},\vec{\tau}\_{j}) \right]^{\} \frac{Y\_{1}^{\mu\_{1}} (\theta\_{\vec{\tau}\_{jN}}, \phi\_{\vec{\tau}\_{jN}})}{r\_{jN}^{2}} \chi\_{n\_{2},l\_{2}}^{m\_{2}} (\zeta\_{2},\vec{\tau}\_{jB}) \,\mathrm{d}\vec{\tau}\_{j} . \end{split} \tag{40}$$ Let <sup>I</sup>˜ αβ* <sup>23</sup> represent the integrals involved in the above equation. Using equation (5), these integrals can be expressed as linear combinations of integrals <sup>B</sup>I<sup>ˆ</sup> αβ <sup>23</sup> over B functions of the form: $$\prescript{}{B}{\mathcal{Z}}\_{23}^{a\beta} = \int\_{\vec{r}\_{\vec{I}}} \left[ \mathcal{B}\_{\vec{n}\_1,l}^{\mathfrak{M}\_1}(\zeta\_1, \vec{r}\_{\vec{I}}) \right]^\* \frac{Y\_1^{\mathcal{U}\_1}(\theta\_{\vec{r}\_{\vec{N}},l}, \phi\_{\vec{r}\_{\vec{N}}})}{r\_{\vec{j}N}^2} \mathcal{B}\_{\mathfrak{n}\_2,l\_2}^{\mathfrak{m}\_2}(\zeta\_2, \vec{r}\_{\vec{j}B}) \, d\vec{r}\_{\vec{j}\prime} \tag{41}$$ where m˜ <sup>1</sup> = m<sup>1</sup> − μ<sup>2</sup> and n˜ <sup>1</sup> = n<sup>1</sup> + 1. 10 Will-be-set-by-IN-TECH The cartesian coordinate rjN,<sup>α</sup> and rj,<sup>β</sup> can be expressed in terms of spherical harmonics and <sup>1</sup> (θrjN , φrjN ) and rj,<sup>β</sup> = rj �2<sup>π</sup> �2<sup>π</sup> � Ym<sup>1</sup> <sup>l</sup><sup>1</sup> (θrj 1 ∑ μ2=−1 <sup>3</sup> and cz,−<sup>1</sup> = <sup>0</sup> <sup>3</sup> and cz,1 = 0. , ϕrj ) �∗ Yμ<sup>2</sup> <sup>1</sup> (θrj , φrj �l<sup>2</sup> <sup>m</sup>2\|l<sup>1</sup> <sup>m</sup>1\|l m<sup>2</sup> <sup>−</sup> <sup>m</sup>1� <sup>Y</sup>m2−m<sup>1</sup> �<sup>1</sup> <sup>μ</sup>2\|l<sup>1</sup> <sup>m</sup>1\|<sup>l</sup> <sup>μ</sup><sup>2</sup> <sup>−</sup> <sup>m</sup>1� <sup>Y</sup>μ2−m<sup>1</sup> � <sup>χ</sup>m1−μ<sup>2</sup> <sup>n</sup>1+1,<sup>l</sup> (ζ1,rj) (−1)μ2−m<sup>1</sup> <sup>c</sup>β,μ<sup>2</sup> <sup>c</sup>α,μ<sup>1</sup> �<sup>1</sup> <sup>μ</sup>2\|l<sup>1</sup> <sup>m</sup>1\|<sup>l</sup> <sup>μ</sup><sup>2</sup> <sup>−</sup> <sup>m</sup>1� χm<sup>2</sup> n2,l<sup>2</sup> <sup>c</sup>β,μ<sup>2</sup> <sup>Y</sup>μ<sup>2</sup> <sup>1</sup> (θrj , φrj �4<sup>π</sup> 3 > � Ym<sup>1</sup> <sup>l</sup><sup>1</sup> (θrj , ϕrj ) �∗ <sup>l</sup> (θ, ϕ), (37) <sup>l</sup> (θrj (ζ2,rjB) drj. (40) �∗ , ϕrj ) . (39) ). (36) ), (34) (35) (38) rjN and rj respectively as follows: 1 ∑ μ1=−1 where the coefficients cα,<sup>μ</sup> are given as follows: cx,−<sup>1</sup> = cx,1 = − = �∗ Ym<sup>2</sup> ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ (ζ1,rj) �∗ = � rj <sup>c</sup>α,μ<sup>1</sup> <sup>Y</sup>μ<sup>1</sup> �2<sup>π</sup> �2<sup>π</sup> 1 ∑ μ2=−1 <sup>l</sup><sup>2</sup> (θ, ϕ) = 1 ∑ μ2=−1 Using equations (34) and (39), we obtain the following relation: 1 ∑ μ1=−1 l1+1 ∑ l=lmin,2 <sup>c</sup>β,μ<sup>2</sup> <sup>r</sup><sup>n</sup> <sup>j</sup> <sup>e</sup>−<sup>ζ</sup> rj �<sup>∗</sup> <sup>Y</sup>μ<sup>1</sup> <sup>3</sup> , cy,−<sup>1</sup> = <sup>i</sup> <sup>3</sup> , cy,1 = i Using the analytic expression of the Unormalized STFs (2), one can obtain: cβ,μ<sup>2</sup> r n1 <sup>j</sup> <sup>e</sup>−ζ<sup>1</sup> rj > l1+l<sup>2</sup> ∑ l=lmin,2 1 ∑ μ2=−1 cx,0 = 0, cy,0 = 0 and cz,0 = <sup>c</sup>β,μ<sup>2</sup> <sup>Y</sup>μ<sup>2</sup> <sup>1</sup> (θrj , φrj ) � r n1−1 <sup>j</sup> <sup>e</sup>−ζ<sup>1</sup> rj The product of two spherical harmonics can be linearized by Gaunt coefficients as follows: where the summation index l runs in steps of 2 from lmin to l<sup>1</sup> + l2. The constant lmin is given max(\|l<sup>1</sup> − l2\|, \|m<sup>2</sup> − m1\|) if l<sup>1</sup> + l<sup>2</sup> + max(\|l<sup>1</sup> − l2\|, \|m<sup>2</sup> − m1\|) is even max(\|l<sup>1</sup> − l2\|, \|m<sup>2</sup> − m1\|) + 1 if l<sup>1</sup> + l<sup>2</sup> + max(\|l<sup>1</sup> − l2\|, \|m<sup>2</sup> − m1\|) is odd. l1+1 ∑ l=lmin,2 <sup>c</sup>β,μ<sup>2</sup> �<sup>1</sup> <sup>μ</sup>2\|l<sup>1</sup> <sup>m</sup>1\|<sup>l</sup> <sup>μ</sup><sup>2</sup> <sup>−</sup> <sup>m</sup>1� (−1)μ2−m<sup>1</sup> <sup>1</sup> (θrjN , φrjN ) r2 jN rjN,<sup>α</sup> = rjN rj,<sup>β</sup> � χm<sup>1</sup> n1,l<sup>1</sup> > � Ym<sup>1</sup> <sup>l</sup><sup>1</sup> (θ, ϕ) ⎧ ⎨ ⎩ lmin = rj,<sup>β</sup> � χm<sup>1</sup> n1,l<sup>1</sup> by [Weniger & Steinborn (1982)]: From equation (37), it follows that: (ζ1,rj) �∗ = 1 ∑ μ2=−1 > × � rj � <sup>χ</sup>m1−μ<sup>2</sup> <sup>n</sup>1+1,<sup>l</sup> (ζ1,rj) l1+1 ∑ l=lmin,2 1 ∑ μ2=−1 = Iαβ <sup>23</sup> = Using the Fourier transform method, we obtain: $$\hat{\mathbf{z}}\_{\mathsf{B}}\hat{\mathbf{Z}}\_{\mathsf{23}}^{a\notin\mathsf{A}}=(\mathsf{2}\ \mathsf{π})^{-3/2}\int\_{\vec{k}}\frac{\overline{Y\_{1}^{\mathsf{M}}(\boldsymbol{\theta}\_{\vec{r}\_{\mathsf{IN}}},\boldsymbol{\phi}\_{\vec{r}\_{\mathsf{IN}}})}}{r\_{\vec{j}\mathsf{N}}^{2}}\left[\int\_{\vec{r}\_{\mathsf{I}}}\left[\mathcal{B}\_{\vec{m},\mathsf{I}}^{\mathsf{M}\_{1}}(\boldsymbol{\zeta}\_{1},\boldsymbol{\vec{r}}\_{\mathsf{I}})\right]^{\}e^{-i\boldsymbol{\vec{k}}\cdot\vec{\tau}\_{\mathsf{IN}}}\mathcal{B}\_{\mathsf{n}\_{2}\mathsf{I}\_{2}}^{\mathsf{M}\_{2}}(\boldsymbol{\zeta}\_{2},\boldsymbol{\vec{r}}\_{\mathsf{I}})\,\mathrm{d}\vec{r}\_{\mathsf{I}}\right]\,\mathrm{d}\vec{k}.\tag{42}$$ The Fourier transform of the operator involved in the above integrals is given by [Berlu & Safouhi (2008)]: $$\overline{\left(\frac{Y\_1^{\mu}(\theta\_{\vec{r}\_{\vec{N}'}},\phi\_{\vec{r}\_{\vec{N}}})}{r\_{jN}^2}\right)}(\vec{k}) = -i\sqrt{\frac{2}{\pi}}\,\frac{Y\_1^{\mu}(\theta\_{\vec{k}'}\phi\_{\vec{k}})}{k}.\tag{43}$$ Using the analytic expression (11) obtained by Trivedi and Steinborn [Trivedi & Steinborn (1983)] for the integrals overrj* involved in equation (42) and equation (43), one can derive the following analytic expression for the integrals <sup>B</sup>I<sup>ˆ</sup> αβ <sup>23</sup> [Berlu & Safouhi (2008)]: <sup>B</sup>I<sup>ˆ</sup> αβ <sup>23</sup> <sup>=</sup> <sup>8</sup> (4π)<sup>2</sup> (2l<sup>1</sup> <sup>+</sup> <sup>1</sup>)!! (2l<sup>2</sup> <sup>+</sup> <sup>1</sup>)!!(n˜ <sup>1</sup> <sup>+</sup> <sup>n</sup><sup>2</sup> <sup>+</sup> <sup>l</sup><sup>1</sup> <sup>+</sup> <sup>l</sup><sup>2</sup> <sup>+</sup> <sup>1</sup>)! (n˜ <sup>1</sup> + l1)! (n<sup>2</sup> + l2)! ζ2n˜ <sup>1</sup>+l1−<sup>1</sup> <sup>1</sup> <sup>ζ</sup>2n2+l2−<sup>1</sup> 2 2n˜ <sup>1</sup>+n2+l1+l2+<sup>1</sup> × l1 ∑ l � <sup>1</sup>=0 l � 1 ∑ m� <sup>1</sup>=−l � 1 (−i)l1+<sup>l</sup> � 1 l1, m˜ <sup>1</sup> l � <sup>1</sup>, m� 1 l<sup>1</sup> − l � <sup>1</sup>, m˜ <sup>1</sup> − m� 1 (2l � <sup>1</sup> + 1)!![2(l<sup>1</sup> − l � <sup>1</sup>) + 1]!! × l2 ∑ l � <sup>2</sup>=0 l � 2 ∑ m� <sup>2</sup>=−l � 2 (−i)l2+<sup>l</sup> � 2 l2, m<sup>2</sup> l � <sup>2</sup>, m� 2 l<sup>2</sup> − l � <sup>2</sup>, m<sup>2</sup> − m� 2 (2l � <sup>2</sup> + 1)!![2(l<sup>2</sup> − l � <sup>2</sup>) + 1]!! × l � <sup>1</sup>+l � 2 ∑ l�=l � min,2 (−1)<sup>l</sup> � 1 l � 2, m� 2 l � <sup>1</sup>, m� 1 l � m� 2, −m� 1 R<sup>l</sup> � <sup>2</sup> <sup>Y</sup>m� 2−m� 1 l� <sup>θ</sup> # —OB, <sup>ϕ</sup># —OB × l1−l � <sup>1</sup>+l2−l � 2 ∑ l12=l12 min,2 l<sup>2</sup> − l � <sup>2</sup>, m<sup>2</sup> − m� 2 l<sup>1</sup> − l � <sup>1</sup>, m˜ <sup>1</sup> − m� 1 <sup>l</sup>12, <sup>m</sup>12 × 1+l<sup>12</sup> ∑ λ=λmin,2 (−i)λ+<sup>1</sup> �λ, <sup>μ</sup> <sup>\|</sup>l12, <sup>m</sup>12<sup>\|</sup> 1, <sup>μ</sup>1� × Δl ∑ j=0 Δl j (−2)<sup>j</sup> (n˜ <sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l<sup>2</sup> − j + 1)! × 1 s=0 s n2+l2+l1−l � <sup>1</sup> (<sup>1</sup> <sup>−</sup> <sup>s</sup>)n˜ <sup>1</sup>+l1+l2−<sup>l</sup> � <sup>2</sup>Y<sup>μ</sup> <sup>λ</sup> (θ−→<sup>v</sup> , ϕ−→<sup>v</sup> ) × <sup>+</sup><sup>∞</sup> x=0 xnx ˆ kν[γ(s, x) R2] <sup>γ</sup>(s, <sup>x</sup>)n<sup>γ</sup> <sup>j</sup>λ(vx) <sup>d</sup><sup>x</sup> ds, (44) <sup>n</sup><sup>1</sup> <sup>l</sup><sup>1</sup> <sup>m</sup><sup>1</sup> <sup>ζ</sup><sup>1</sup> <sup>n</sup><sup>2</sup> <sup>l</sup><sup>2</sup> <sup>m</sup><sup>2</sup> <sup>ζ</sup><sup>2</sup> <sup>B</sup>I(x,z) Table 3. Evaluation of <sup>I</sup>(x,z) coordinates. et al. (2010)]. 7. References 6. Acknowledgment 5. Conclusion <sup>3</sup> (27). multi-center integrals and NMR multi-center integrals. known that these functions are better suited than GTFs. Engineering Research Council of Canada (NSERC). Safouhi (2007); Safouhi (2001b; 2004; 2010a); Slevinsky & Safouhi (2009)]. Graffi (editor), Schrödinger operators, Springer-Verlag, Berlin. 2 1 -1 2.0 2 1 1 1.0 .769304745002408(-4) 2 1 1 2.0 2 1 1 1.0 -.620498371396534(-4) 3 2 1 2.0 2 1 0 1.0 -.146444907098019(-5) 3 2 1 2.0 2 1 1 1.0 -.576608712417475(-5) 3 2 1 2.0 3 2 1 1.0 .137450124735083(-6) 3 2 1 2.0 3 2 -1 1.0 -.117106174581176(-6) 3 2 2 2.0 3 2 -2 1.0 .287818321676003(-6) 3 2 2 2.0 3 2 1 1.0 .143900856736229(-6) 3 2 2 2.0 3 2 2 1.0 -.121032718682770(-5) 4 2 -1 2.0 3 2 1 1.0 -.359169492307861(-6) 4 2 1 2.0 3 2 1 1.0 .343245699707943(-6) Fourier Transformation Method for Computing NMR Integrals over Exponential Type Functions 133 R<sup>1</sup> = (12.0, 90o, 0o) and In the present review, we showed how the Fourier transformation method allowed the derivation of compact formulae for one of the most challenging integrals, namely molecular Analytic expressions are obtained for integrals of the paramagnetic contribution in the relativistic calculation of the shielding tensor as well as integrals of second order in the non-relativistic calculation of the shielding tensor. The basis set of ETFs is used and it is well The obtained analytic expressions for the one- and two-center integrals can be computed easily and no quadrature rule is required. In the case of the three-center integrals, we need to compute semi-infinite integrals involving oscillatory functions. These oscillatory integrals can be computed to a high pre-determined accuracy using existing methods and algorithms based on extrapolation methods and numerical quadrature [Berlu & Safouhi (2003); Duret & Numerical tables for the NMR integrals of interest can be found in [Safouhi (2010b); Slevinsky The author acknowledges the financial support for this research by the Natural Sciences and Agmon, S. (1985). Bounds on exponential decay of eigenfunctions of Schrödinger operators, in S. Barnett, M. (1990). Molecular integrals over Slater orbitals, Chem. Phys. Lett. 166: 65–70. 3 R<sup>2</sup> = (2.0, 90o, 0o) in spherical where: $$\begin{aligned} m\_{12} &= (m\_2 - m\_2') - (\vec{m}\_1 - m\_1') \text{ and } \mu = \mu\_1 + m\_{12} \\ m\_{\gamma} &= 2(\vec{n}\_1 + n\_2 + l\_1 + l\_2) - (l\_1' + l\_2' + l') + 1 \\ \gamma(s, x) &= \sqrt{s\zeta\_2^2 + (1 - s)\zeta\_1^2 + s(1 - s)x^2} \\ \overrightarrow{\upsilon}^{\prime} &= (1 - s)\overrightarrow{OB} - \overrightarrow{ON} \quad \text{and} \quad \text{and } \upsilon = \|\vec{\upsilon}\| \\ \nu &= \ddot{\eta}\_1 + \eta\_2 + l\_1 + l\_2 - l' - j + \frac{1}{2} \\ m\_{\chi} &= l\_1 - l\_1' + l\_2 - l\_2' + 1 \\ \Delta l &= (l\_1' + l\_2' - l')/2. \end{aligned} \tag{45}$$ Table 1. Evaluation of <sup>B</sup>I(x,y) <sup>1</sup> (29). Table 2. Evaluation of <sup>B</sup>I(y,z) <sup>2</sup> (31). R = (1.5, 75o, 0o) in spherical coordinates. Table 3. Evaluation of <sup>I</sup>(x,z) <sup>3</sup> (27). R<sup>1</sup> = (12.0, 90o, 0o) and R<sup>2</sup> = (2.0, 90o, 0o) in spherical coordinates. ### 5. Conclusion 12 Will-be-set-by-IN-TECH <sup>2</sup>) − (m˜ <sup>1</sup> − m� <sup>2</sup> + (<sup>1</sup> <sup>−</sup> <sup>s</sup>)ζ<sup>2</sup> � <sup>2</sup> + 1 <sup>n</sup><sup>1</sup> <sup>l</sup><sup>1</sup> <sup>m</sup><sup>1</sup> <sup>ζ</sup><sup>1</sup> <sup>n</sup><sup>2</sup> <sup>l</sup><sup>2</sup> <sup>m</sup><sup>2</sup> <sup>ζ</sup><sup>2</sup> <sup>B</sup>I(x,y) <sup>n</sup><sup>1</sup> <sup>l</sup><sup>1</sup> <sup>m</sup><sup>1</sup> <sup>ζ</sup><sup>1</sup> <sup>n</sup><sup>2</sup> <sup>l</sup><sup>2</sup> <sup>m</sup><sup>2</sup> <sup>ζ</sup><sup>2</sup> <sup>B</sup>I(y,z) 2 1 1 1.0 2 1 -1 1.5 -.980852389239356(-3) 3 2 1 1.0 2 1 -1 1.5 -.319537014904337(-1) 3 2 1 1.0 2 1 1 1.5 .858668805557491(-2) 3 2 1 1.0 3 2 -1 1.5 -.219369423462985(-1) 3 2 2 1.0 3 2 -2 1.5 .391684472826858(-4) 3 2 2 1.0 3 2 -1 1.5 -.204743121872522(-1) 3 2 2 1.0 3 2 1 1.5 .163009587528461( 2) 4 2 1 1.0 2 1 -1 1.5 -.414819029773496(-1) 4 2 2 1.0 3 2 -2 1.5 .441086792217064(-4) 4 2 2 1.0 3 2 -1 1.5 -.298416140021138(-1) 4 2 2 1.0 3 2 1 1.5 .291083584089616( 2) R = (1.5, 75o, 0o) in spherical coordinates. 2 1 -1 2.0 2 1 -1 1.5 .844938515752529(-3) 2 1 0 2.0 2 1 0 1.5 .197895122059999(-3) 3 2 -1 2.0 2 1 -1 1.5 -.183961338177373(-4) 3 2 1 2.0 3 2 1 1.5 .314124180426832(-4) 3 2 2 2.0 3 2 2 1.5 .132485365811557(-3) 4 2 1 2.0 2 1 1 1.5 -.361569492726092(-4) 4 2 1 2.0 3 2 1 1.5 .764186724377223(-4) 4 2 2 2.0 3 2 2 1.5 .331525812542680(-3) 4 2 0 2.0 4 2 0 1.5 -.297315128733290(-4) 4 2 1 2.0 4 2 1 1.5 .215564810358993(-3) 4 2 2 2.0 4 2 2 1.5 .951453780055960(-3) OB <sup>−</sup> # — � <sup>1</sup> + l � <sup>2</sup> + l � ) + 1 � − j + <sup>1</sup> + <sup>s</sup>(<sup>1</sup> − <sup>s</sup>)x<sup>2</sup> 1 2 <sup>1</sup>) and μ = μ<sup>1</sup> + m<sup>12</sup> ON and and v = \|v\| (45) 2 2 m<sup>12</sup> = (m<sup>2</sup> − m� −→<sup>v</sup> = (<sup>1</sup> <sup>−</sup> <sup>s</sup>) # — sζ<sup>2</sup> ν = n˜ <sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l<sup>2</sup> − l � <sup>1</sup> + l<sup>2</sup> − l γ(s, x) = nx = l<sup>1</sup> − l <sup>1</sup> (29). <sup>2</sup> (31). Δl = (l � <sup>1</sup> + l � <sup>2</sup> − l � )/2. Table 1. Evaluation of <sup>B</sup>I(x,y) Table 2. Evaluation of <sup>B</sup>I(y,z) n<sup>γ</sup> = 2(n˜ <sup>1</sup> + n<sup>2</sup> + l<sup>1</sup> + l2) − (l where: In the present review, we showed how the Fourier transformation method allowed the derivation of compact formulae for one of the most challenging integrals, namely molecular multi-center integrals and NMR multi-center integrals. Analytic expressions are obtained for integrals of the paramagnetic contribution in the relativistic calculation of the shielding tensor as well as integrals of second order in the non-relativistic calculation of the shielding tensor. The basis set of ETFs is used and it is well known that these functions are better suited than GTFs. The obtained analytic expressions for the one- and two-center integrals can be computed easily and no quadrature rule is required. In the case of the three-center integrals, we need to compute semi-infinite integrals involving oscillatory functions. These oscillatory integrals can be computed to a high pre-determined accuracy using existing methods and algorithms based on extrapolation methods and numerical quadrature [Berlu & Safouhi (2003); Duret & Safouhi (2007); Safouhi (2001b; 2004; 2010a); Slevinsky & Safouhi (2009)]. Numerical tables for the NMR integrals of interest can be found in [Safouhi (2010b); Slevinsky et al. (2010)]. #### 6. Acknowledgment The author acknowledges the financial support for this research by the Natural Sciences and Engineering Research Council of Canada (NSERC). #### 7. References Agmon, S. (1985). Bounds on exponential decay of eigenfunctions of Schrödinger operators, in S. Graffi (editor), Schrödinger operators, Springer-Verlag, Berlin. Barnett, M. (1990). Molecular integrals over Slater orbitals, Chem. Phys. Lett. 166: 65–70. London, F. (1937). 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Brooks Discrete Fast Fourier Transform National Institutes of Health (NIH), Bethesda, MD 20892, Laboratory of Computational Biology, National Heart, Lung, and Blood Institute (NHLBI), Molecular simulation is widely used as a computer experiment to study molecular systems based on the first principle and accumulated knowledge. It extends our reach to the atomic level and to extreme conditions by providing information that is not available from experiment. As a complement to experimental studies, molecular simulation has played important roles in understanding the physics and chemistry of molecular systems. The rapid increase in computing power has facilitated extensive applications of molecular simulation in the study of complicated systems, such as ion channels, and difficult problems, such as Molecular simulations are often expensive, because the sizes of simulation systems and the time scales of simulations are many orders of magnitude smaller than studied in experiments. For example, many biological molecular assemblies have millions of atoms and take milliseconds or longer to function. These are beyond the reach of current simulations without size reduction, or the use of additional assumptions to simplify and reduce the scope of the problem. To be relevant to real experiments, simulators tend to maximize the sizes of their simulated systems and/or the time scales of their simulations to the limit they can afford with their available computing resources. Therefore, improving calculation Molecular interactions are the basis of all macroscopic properties. Accurate and efficient calculation of molecular interactions is the key for a successful simulation study. Long-range interactions, such as the electrostatic interaction and van der Waals interaction, play very important roles in the properties of molecular systems. However, they reach far beyond the size of a typical simulation system and are the most expensive part in molecular simulations. Improving the calculation efficiency for long-range interactions has long been the goal of method development. The Ewald sum (Ewald 1921) is a well known method for calculating electrostatic interactions without the need to deal with a vacuum boundary interface by approximating large systems as small systems with periodicity. Recently, a method called the isotropic periodic sum (IPS) was developed as a general approach to the calculation of long-range interactions of all types of potentials (Wu and Brooks 2005; Takahashi, Yasuoka et al. 2007; Wu and Brooks 2008; Wu and Brooks 2009; Takahashi, Narumi et al. 2010; Takahashi, Narumi et al. 2011; Takahashi, Narumi et al. 2011) and has been applied in many efficiency is always a focus of the development in molecular simulation methods. 1. Introduction protein folding. ## Molecular Simulation with Discrete Fast Fourier Transform Xiongwu Wu and Bernard R. Brooks Laboratory of Computational Biology, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA ## 1. Introduction 16 Will-be-set-by-IN-TECH 136 Fourier Transform – Materials Analysis Slevinsky, R., Temga, T., Mouattamid, M. & Safouhi, H. (2010). One- and two-Center Steinborn, E. & Filter, E. (1975). Translations of fields represented by spherical-harmonics Trivedi, H. & Steinborn, E. (1983). Fourier transform of a two-center product of Vaara, J. (2007). Theory and computation of nuclear magnetic resonance parameters, Phys. Watson, M., Handy, N., Cohen, A. & Helgaker, T. (2004). Density-functional Weatherford, C. & Jones, H. (1982). ETO Multicenter Molecular Integrals, Reidel, Dordrecht. Weniger, E. (1982). Reduzierte Bessel-Funktionen als LCAO-Basissatz: Analytische und numerische Weniger, E. (1985). Weakly convergent expansions of a plane wave and their use in Fourier Weniger, E. (2005). The spherical tensor gradient operator, Collect. Czech. Chem. Commun. Weniger, E. & Steinborn, E. (1982). Programs for the coupling of spherical harmonics, Comput. Weniger, E. & Steinborn, E. (1983a). Numerical properties of the convolution theorems of B Weniger, E. & Steinborn, E. (1983b). The Fourier transforms of some exponential-type functions and their relevance to multicenter problems, J. Chem. Phys. 78: 6121–6132. Untersuchungen, publisher = Ph.D.thesis, Universität Regensburg. Slater-type s-orbitals, and other functions, Theor. Chim. Acta. 38: 273–281. Steinborn, E., Homeier, H., Ema, I., López, R. & Ramírez, G. (2000). Molecular calculations with B functions, Int. J. Quantum Chem. 76: 244–251. Slater basis sets, J. Chem. Phys. 120: 7252–7261. Math. Theor. 43: 225202 (14pp). Phys. Rev. A. 27: 670–679. Chem. Chem. Phys. 9: 5399–5418. integrals, J. Math. Phys. 26: 276–291. functions, Phys. Rev. A. 28: 2026–2041. Phys.Commun. 25: 149–157. 70: 1125–1271. ETF-integrals of first order in relativistic calculation of NMR parameters, J. Phys. A: expansions for molecular calculations. III. Translations of reduced Bessel functions, exponential-type orbitals. Application to one- and two-electron multicenter integrals, generalized-gradient and hybrid calculations of electromagnetic properties using Molecular simulation is widely used as a computer experiment to study molecular systems based on the first principle and accumulated knowledge. It extends our reach to the atomic level and to extreme conditions by providing information that is not available from experiment. As a complement to experimental studies, molecular simulation has played important roles in understanding the physics and chemistry of molecular systems. The rapid increase in computing power has facilitated extensive applications of molecular simulation in the study of complicated systems, such as ion channels, and difficult problems, such as protein folding. Molecular simulations are often expensive, because the sizes of simulation systems and the time scales of simulations are many orders of magnitude smaller than studied in experiments. For example, many biological molecular assemblies have millions of atoms and take milliseconds or longer to function. These are beyond the reach of current simulations without size reduction, or the use of additional assumptions to simplify and reduce the scope of the problem. To be relevant to real experiments, simulators tend to maximize the sizes of their simulated systems and/or the time scales of their simulations to the limit they can afford with their available computing resources. Therefore, improving calculation efficiency is always a focus of the development in molecular simulation methods. Molecular interactions are the basis of all macroscopic properties. Accurate and efficient calculation of molecular interactions is the key for a successful simulation study. Long-range interactions, such as the electrostatic interaction and van der Waals interaction, play very important roles in the properties of molecular systems. However, they reach far beyond the size of a typical simulation system and are the most expensive part in molecular simulations. Improving the calculation efficiency for long-range interactions has long been the goal of method development. The Ewald sum (Ewald 1921) is a well known method for calculating electrostatic interactions without the need to deal with a vacuum boundary interface by approximating large systems as small systems with periodicity. Recently, a method called the isotropic periodic sum (IPS) was developed as a general approach to the calculation of long-range interactions of all types of potentials (Wu and Brooks 2005; Takahashi, Yasuoka et al. 2007; Wu and Brooks 2008; Wu and Brooks 2009; Takahashi, Narumi et al. 2010; Takahashi, Narumi et al. 2011; Takahashi, Narumi et al. 2011) and has been applied in many Molecular Simulation with Discrete Fast Fourier Transform 139 )( exp()( kT Here, k is the Boltzmann constant and T is temperature. A transition from conformation Ω<sup>m</sup> )()( exp( )( Ω−Ω −= <sup>Ω</sup> The commonly used Metropolis Monte Carlo method (Metropolis and Ulam 1949) propagates conformations according to eq. (2) to sample the canonical ensemble distribution. As can be seen from eq. (2), Monte Carlo simulations need calculate only energies. Further details of the Metropolis Monte Carlo method can be found elsewhere Molecular dynamics generates trajectories of particles by numerically solving the equation of motion (Allen and Tildesley 1987; Brooks, Brooks et al. 2009). The motion of simulation systems produces trajectories in the conformational space so that the ensemble properties as well as dynamics properties can be studied. Particles of a molecular system move according Here, <sup>i</sup> f is the net force acting on particle i. The vectors, pi and p<sup>i</sup> , are the momentum and its time derivative of particle i. As can be seen from eq. (3), molecular dynamics simulations rely on forces to generate trajectories. Molecular dynamics simulations can be performed in Many other methods have been developed to sample the conformational space, such as the genetic algorithm (Dandekar and Argos 1992; Le Grand and Merz 1994; Ogata, Akiyama et al. 1995; Beckers, Buydens et al. 1997; Jones, Willet et al. 1997). In addition, there are many improved methods based on above mentioned methods or their combinations. For example, the self-guided molecular dynamics (SGMD) and self-guided Langevin dynamics (SGLD) were developed based on molecular dynamics or Langevin dynamics. they accelerate conformational search through enhancing low frequency motions of simulation systems (Wu and Wang 1998; Wu and Wang 1999; Wu and Brooks 2003; Wu and Brooks 2011a; Wu and Brooks 2011b) and has been applied in many simulation studies such as protein folding (Wu and Sung 1999; Wu and Wang 2000; Wu and Wang 2001; Wu, Wang et al. 2002; Wen, Hsieh et al. 2004; Wen and Luo 2004; Wu and Brooks 2004; Lee and Chang 2010; Lee and Olson 2010), ligand binding (Varady, Wu et al. 2002), docking (Chandrasekaran, Lee et al. 2009), conformational transition (Damjanovic, Miller et al. 2008; Damjanovic, Wu et al. 2008; Damjanovic, Garcia-Moreno E et al. 2009; Pendse, Brooks et al. 2010), and surface absorption (Abe and Jitsukawa 2009; Sheng, Wang et al. 2010; Sheng, Wang et al. 2010; Tsuru, Yosuke et ρ )( <sup>Ω</sup> <sup>=</sup> ρ ρ mn π m n to conformation Ωn has a probability of (Allen and Tildesley 1987). 2.1.2 Molecular dynamics to the Newtonian equation of motion: 2.1.3 Other sampling methods al. 2010). many types of ensembles (Allen and Tildesley 1987). ) kT <sup>n</sup> EE <sup>m</sup> −∝Ω (1) ) ii = fp (3) (2) E Ω simulation studies (Lim, Rogaski, & Klauda, 2011; Venable, Chen, & Pastor, 2009; Wang, Zhu, Li, & Hansmann, 2011). The Ewald sum is a method of order N2 and is expensive for large systems. To improve the calculation efficiency, the discrete fast Fourier transform (DFFT) technique is utilized to calculate the reciprocal part of the Ewald sum, that leads to the particle mesh Ewald (PME) (Darden 1993; York, Wlodawer et al. 1994; Essmann, Perera et al. 1995). The PME method is of order of Nlog (N) and is suitable for large systems. The IPS method is of order N and is very efficient for homogeneous systems. It requires a local region to include all heterogeneous features. For homogeneous systems, a small local region can be used and the calculation is as efficient as the cutoff methods. For heterogeneous systems, a large local region up to the size of a simulation system is required. The IPS/DFFT method utilizes DFFT to speed up calculations with large local regions and can efficiently calculate longrange interactions for heterogeneous systems (Wu and Brooks 2008). This chapter reviews the application of DFFT in molecular simulation, specifically, in the calculation of long-range interactions. Section 2 describes major molecular simulation methods, which include conformational sampling and molecular interactions. Section 3 introduces methods for the calculation of long-range interactions. As a recently developed method, the IPS method is explained in more detail. The focus will be on the PME and IPS/DFFT methods (section 4) and their applications to demonstrate the benefits of DFFT (section 5). ## 2. Molecular simulation The main purpose of molecular simulation is to produce data points in the phase space of interested systems so that the structural, dynamics, and thermodynamic properties can be examined and studied. Molecular simulation contains two major components, molecular interaction and conformational sampling. An efficient molecular simulation requires an efficient calculation of molecular interactions and an efficient way to sample conformations so that a simulation can explore more conformations and larger conformational spaces while maintaining a correction ensemble distribution. ## 2.1 Conformational sampling methods Conformational sampling methods specify how the phase space (or conformational space) is explored or how conformations of a simulation system are propagated. There are many ways to perform molecular simulations. For example, the Monte Carlo method (Metropolis and Ulam 1949; Hoffman, Metropolis et al. 1955; Gardiner, Hoffman et al. 1956) is a mathematic approach, the molecular dynamics method (Allen and Tildesley 1987; Brooks, Brooks et al. 2009) is a physical approach, and the genetic algorithm (Dandekar and Argos 1992; Le Grand and Merz 1994; Ogata, Akiyama et al. 1995; Beckers, Buydens et al. 1997; Jones, Willet et al. 1997) is a genetic approach. Many more methods have been developed from combinations of these approaches to better sample the phase space. #### 2.1.1 Metropolis Monte Carlo method Monte Carlo simulation samples the conformational space according to the ensemble distribution probabilities (Metropolis and Ulam 1949; Allen and Tildesley 1987). In a canonical ensemble, a conformation, Ω, of energy E (Ω) has a probability of $$ \rho \rho(\Omega) \propto \exp(-\frac{E(\Omega)}{kT}) \tag{1} $$ Here, k is the Boltzmann constant and T is temperature. A transition from conformation Ω<sup>m</sup> to conformation Ωn has a probability of $$\pi\_{sm} = \frac{\rho(\Omega\_s)}{\rho(\Omega\_m)} = \exp(-\frac{E(\Omega\_s) - E(\Omega\_m)}{kT}) \tag{2}$$ The commonly used Metropolis Monte Carlo method (Metropolis and Ulam 1949) propagates conformations according to eq. (2) to sample the canonical ensemble distribution. As can be seen from eq. (2), Monte Carlo simulations need calculate only energies. Further details of the Metropolis Monte Carlo method can be found elsewhere (Allen and Tildesley 1987). #### 2.1.2 Molecular dynamics 138 Fourier Transform – Materials Analysis simulation studies (Lim, Rogaski, & Klauda, 2011; Venable, Chen, & Pastor, 2009; Wang, The Ewald sum is a method of order N2 and is expensive for large systems. To improve the calculation efficiency, the discrete fast Fourier transform (DFFT) technique is utilized to calculate the reciprocal part of the Ewald sum, that leads to the particle mesh Ewald (PME) (Darden 1993; York, Wlodawer et al. 1994; Essmann, Perera et al. 1995). The PME method is of order of Nlog (N) and is suitable for large systems. The IPS method is of order N and is very efficient for homogeneous systems. It requires a local region to include all heterogeneous features. For homogeneous systems, a small local region can be used and the calculation is as efficient as the cutoff methods. For heterogeneous systems, a large local region up to the size of a simulation system is required. The IPS/DFFT method utilizes DFFT to speed up calculations with large local regions and can efficiently calculate long- This chapter reviews the application of DFFT in molecular simulation, specifically, in the calculation of long-range interactions. Section 2 describes major molecular simulation methods, which include conformational sampling and molecular interactions. Section 3 introduces methods for the calculation of long-range interactions. As a recently developed method, the IPS method is explained in more detail. The focus will be on the PME and IPS/DFFT methods (section 4) and their applications to demonstrate the benefits of DFFT The main purpose of molecular simulation is to produce data points in the phase space of interested systems so that the structural, dynamics, and thermodynamic properties can be examined and studied. Molecular simulation contains two major components, molecular interaction and conformational sampling. An efficient molecular simulation requires an efficient calculation of molecular interactions and an efficient way to sample conformations so that a simulation can explore more conformations and larger conformational spaces while Conformational sampling methods specify how the phase space (or conformational space) is explored or how conformations of a simulation system are propagated. There are many ways to perform molecular simulations. For example, the Monte Carlo method (Metropolis and Ulam 1949; Hoffman, Metropolis et al. 1955; Gardiner, Hoffman et al. 1956) is a mathematic approach, the molecular dynamics method (Allen and Tildesley 1987; Brooks, Brooks et al. 2009) is a physical approach, and the genetic algorithm (Dandekar and Argos 1992; Le Grand and Merz 1994; Ogata, Akiyama et al. 1995; Beckers, Buydens et al. 1997; Jones, Willet et al. 1997) is a genetic approach. Many more methods have been developed Monte Carlo simulation samples the conformational space according to the ensemble distribution probabilities (Metropolis and Ulam 1949; Allen and Tildesley 1987). In a from combinations of these approaches to better sample the phase space. canonical ensemble, a conformation, Ω, of energy E (Ω) has a probability of range interactions for heterogeneous systems (Wu and Brooks 2008). Zhu, Li, & Hansmann, 2011). (section 5). 2. Molecular simulation maintaining a correction ensemble distribution. 2.1 Conformational sampling methods 2.1.1 Metropolis Monte Carlo method Molecular dynamics generates trajectories of particles by numerically solving the equation of motion (Allen and Tildesley 1987; Brooks, Brooks et al. 2009). The motion of simulation systems produces trajectories in the conformational space so that the ensemble properties as well as dynamics properties can be studied. Particles of a molecular system move according to the Newtonian equation of motion: $$ \dot{\mathbf{p}}\_i = \mathbf{f}\_i \tag{3} $$ Here, <sup>i</sup> f is the net force acting on particle i. The vectors, pi and p<sup>i</sup> , are the momentum and its time derivative of particle i. As can be seen from eq. (3), molecular dynamics simulations rely on forces to generate trajectories. Molecular dynamics simulations can be performed in many types of ensembles (Allen and Tildesley 1987). #### 2.1.3 Other sampling methods Many other methods have been developed to sample the conformational space, such as the genetic algorithm (Dandekar and Argos 1992; Le Grand and Merz 1994; Ogata, Akiyama et al. 1995; Beckers, Buydens et al. 1997; Jones, Willet et al. 1997). In addition, there are many improved methods based on above mentioned methods or their combinations. For example, the self-guided molecular dynamics (SGMD) and self-guided Langevin dynamics (SGLD) were developed based on molecular dynamics or Langevin dynamics. they accelerate conformational search through enhancing low frequency motions of simulation systems (Wu and Wang 1998; Wu and Wang 1999; Wu and Brooks 2003; Wu and Brooks 2011a; Wu and Brooks 2011b) and has been applied in many simulation studies such as protein folding (Wu and Sung 1999; Wu and Wang 2000; Wu and Wang 2001; Wu, Wang et al. 2002; Wen, Hsieh et al. 2004; Wen and Luo 2004; Wu and Brooks 2004; Lee and Chang 2010; Lee and Olson 2010), ligand binding (Varady, Wu et al. 2002), docking (Chandrasekaran, Lee et al. 2009), conformational transition (Damjanovic, Miller et al. 2008; Damjanovic, Wu et al. 2008; Damjanovic, Garcia-Moreno E et al. 2009; Pendse, Brooks et al. 2010), and surface absorption (Abe and Jitsukawa 2009; Sheng, Wang et al. 2010; Sheng, Wang et al. 2010; Tsuru, Yosuke et al. 2010). Molecular Simulation with Discrete Fast Fourier Transform 141 Many methods have been developed for the calculation of long-range interactions. In the early stage of molecular simulations, due to the limit in computing resources, molecular interactions are limited to a certain range by methods like the minimum image method or the cutoff based methods. When accurate long-range interactions are desired, typically for electrostatic interactions, more sophisticated methods are developed. Below we briefly describe four typical methods for the calculation of long-range interactions. Fig.1 illustrates (a) (b) <sup>+</sup> - r rc + - + r 2Rc Rc 4Rc ε + 3. Methods to calculate long-range interactions <sup>+</sup> - rc r (c) (d) Fig. 1. Calculation methods for long-range interactions. (a) the cutoff methods: interactions with particle within a cutoff distance; (b) the reaction field method: in addition to the cutoff interaction, the environment has an induced field on the cutoff region; (c) the lattice sum (the Ewald sum): interactions with all lattice images; (d) the isotropic periodic sum: Cutoff based methods consider only interactions within a certain distance range. Fig.1 (a) illustrates this concept. The cutoff based methods assume that a particle interacts with only the particles or their images within a certain distance (Steinbach and Brooks 1994). This distance is called the cutoff distance. To avoid discontinuities in energy and/or forces in the + + - r cutoff region, interaction potentials are shifted or switched as below: interactions with all isotropic periodic images 3.1 Cutoff methods the concepts of these methods. #### 2.2 Molecular interactions Molecular interactions are essential for all microscopic and macroscopic properties. The energy and forces of molecular interactions are the basis for conformational searching and sampling. In molecular simulations, molecular interactions are represented by force fields, which describe quantitatively relations of the interaction energy with system conformations. For computational feasibility, molecular interactions are often described as pairwise terms and total interactions are the sum of all pair interactions. For example, the CHARMM force field takes the following form (Brooks, Brooks et al. 2009): $$\begin{aligned} U(R) &= \frac{1}{2} \sum\_{\text{bonds}} K\_s (b - b\_0)^2 + \frac{1}{2} \sum\_{\text{angles}} K\_\theta (\theta - \theta\_0)^2 + \frac{1}{2} \sum\_{\text{Uky-Bulay}} K\_{\text{tBs}} (S - S\_0)^2 + \\ &+ \frac{1}{2} \sum\_{\text{dihedrals}} K\_\phi (1 - \cos(n\varphi - \delta)) + \frac{1}{2} \sum\_{\text{inuprocess}} K\_\alpha (\alpha - \alpha\_0)^2 + \sum\_{\text{residual}} U\_{\text{CMAP}} (\phi, \mathcal{W}) \\ &\sum\_{\text{non-bonded}} \left\{ \mathcal{E}\_{\tilde{y}}^{\text{min}} \left[ \left( \frac{R\_{\tilde{y}}^{\text{min}}}{r\_{\tilde{y}}} \right)^2 - 2 \left( \frac{R\_{\tilde{y}}^{\text{min}}}{r\_{\tilde{y}}} \right)^6 \right] + \frac{q\_i q\_j}{4 \pi \varepsilon\_0 \alpha\_{\tilde{y}}} \right\} \end{aligned} \tag{4}$$ The potential energy is a sum of individual terms representing the internal and nonbonded contributions. The internal terms include contributions from bond (b), valence angle (θ), Urey-Bradley (UB, S), dihedral angle (ϕ), improper dihedral angle (ω), and backbone torsional correction (CMAP, φ, ψ). The parameters <sup>b</sup> k , θ k , UB k , ϕ k , and ω k are the respective force constants and the variables with the subscript "0" are the respective equilibrium values. The nonbonded terms include Coulombic interactions between the point charges ( <sup>i</sup> q and <sup>j</sup> q ) and the Lennard-Jones (LJ) 6-12 term, which is used for the treatment of the corecore repulsion and the attractive van der Waals dispersion interaction. To further improve the accuracy, additional terms can be added. Polarizable force fields have conformational dependent atomic properties such as charges or dipole moments. In large molecule simulations, implicit solvation terms can be used to replace solvent molecules. There are also many attempts to directly calculate molecular interactions using quantum mechamics. #### 2.2.1 Short-range interactions Interactions with limited ranges are categorized into short-range interactions. All internal terms shown in eq. (4) are limited within a molecule and are typical short-range interactions. Some non-bonded interactions, such as the repulsion between atoms, are also considered to be in this category. Short-range interactions occur within a short distance range and the number of interactions is very small as compared to long-range interactions. Therefore, the computing cost for short-range interactions is relatively low and no special treatment is usually needed. #### 2.2.2 Long-range interactions Long-range interactions are interactions that can reach far beyond molecular sizes. Typically, long-range interactions have infinite interaction ranges. In molecular simulation, electrostatic and van der Waals interactions are two major long-range interactions. Because long-range interactions play a crucial role in system properties and are expensive to calculate, their accurate and efficient calculation is the focus of many method developments. Molecular interactions are essential for all microscopic and macroscopic properties. The energy and forces of molecular interactions are the basis for conformational searching and sampling. In molecular simulations, molecular interactions are represented by force fields, which describe quantitatively relations of the interaction energy with system conformations. For computational feasibility, molecular interactions are often described as pairwise terms and total interactions are the sum of all pair interactions. For example, the CHARMM force ∑ ∑ ∑ +−− +− nK K U angles 2 1 ⎜ ⎜ ⎝ <sup>⎛</sup> <sup>−</sup> <sup>⎟</sup> ⎟ ⎠ ⎞ <sup>6</sup> min <sup>12</sup> min 2 2 non bonded 0 core repulsion and the attractive van der Waals dispersion interaction. δϕ ij ij r R ∑ ∑ ∑ bbKRU K SSK )()( )( )( θ impropers 2 1 > + ⎥ ⎥ ⎦ 4 = − + − + − + b UB θ θ cos(1( )) )( ),( ω ⎤ ⎟ ⎟ ⎠ ⎞ The potential energy is a sum of individual terms representing the internal and nonbonded contributions. The internal terms include contributions from bond (b), valence angle (θ), Urey-Bradley (UB, S), dihedral angle (ϕ), improper dihedral angle (ω), and backbone force constants and the variables with the subscript "0" are the respective equilibrium values. The nonbonded terms include Coulombic interactions between the point charges ( <sup>i</sup> q and <sup>j</sup> q ) and the Lennard-Jones (LJ) 6-12 term, which is used for the treatment of the core- To further improve the accuracy, additional terms can be added. Polarizable force fields have conformational dependent atomic properties such as charges or dipole moments. In large molecule simulations, implicit solvation terms can be used to replace solvent molecules. There are also many attempts to directly calculate molecular interactions using Interactions with limited ranges are categorized into short-range interactions. All internal terms shown in eq. (4) are limited within a molecule and are typical short-range interactions. Some non-bonded interactions, such as the repulsion between atoms, are also considered to be in this category. Short-range interactions occur within a short distance range and the number of interactions is very small as compared to long-range interactions. Therefore, the computing cost for short-range interactions is relatively low and no special treatment is Long-range interactions are interactions that can reach far beyond molecular sizes. Typically, long-range interactions have infinite interaction ranges. In molecular simulation, electrostatic and van der Waals interactions are two major long-range interactions. Because long-range interactions play a crucial role in system properties and are expensive to calculate, their accurate and efficient calculation is the focus of many method developments. ij ij ). The parameters <sup>b</sup> k , r R − Urey Bradley 2 0 ϕk , and CMAP ψφ 2 ω (4) k are the respective residues ⎪ ⎭ ij ji r qq > θk , UB k , επε ωω > ⎪ ⎬ ⎫ 2 0 2 1 0 2.2 Molecular interactions ∑ dihedrals 2 1 > ⎪ ⎩ ⎢ ⎢ ⎣ ⎜ ⎜ ⎝ ⎛ > φ, ψ ⎡ min ij ε bonds ⎪ ⎨ ⎧ ϕ 1 − pairs torsional correction (CMAP, quantum mechamics. usually needed. 2.2.1 Short-range interactions 2.2.2 Long-range interactions field takes the following form (Brooks, Brooks et al. 2009): 2 0 ## 3. Methods to calculate long-range interactions Many methods have been developed for the calculation of long-range interactions. In the early stage of molecular simulations, due to the limit in computing resources, molecular interactions are limited to a certain range by methods like the minimum image method or the cutoff based methods. When accurate long-range interactions are desired, typically for electrostatic interactions, more sophisticated methods are developed. Below we briefly describe four typical methods for the calculation of long-range interactions. Fig.1 illustrates the concepts of these methods. Fig. 1. Calculation methods for long-range interactions. (a) the cutoff methods: interactions with particle within a cutoff distance; (b) the reaction field method: in addition to the cutoff interaction, the environment has an induced field on the cutoff region; (c) the lattice sum (the Ewald sum): interactions with all lattice images; (d) the isotropic periodic sum: interactions with all isotropic periodic images ## 3.1 Cutoff methods Cutoff based methods consider only interactions within a certain distance range. Fig.1 (a) illustrates this concept. The cutoff based methods assume that a particle interacts with only the particles or their images within a certain distance (Steinbach and Brooks 1994). This distance is called the cutoff distance. To avoid discontinuities in energy and/or forces in the cutoff region, interaction potentials are shifted or switched as below: Molecular Simulation with Discrete Fast Fourier Transform 143 from the real space, the first term, and the reciprocal space, the second term. The ∫ ∞ = − x <sup>x</sup> )exp( dtt <sup>2</sup> )(erfc <sup>2</sup> π The IPS method was developed to overcome some artefacts of the lattice sum methods. The lattice sum methods, like the Ewald sum, assume that the remote regions can be represented by images created by periodic boundary conditions (PBC). The PBC images distribute discretely in lattice points throughout the space and are anisotropic in nature, as shown in Fig.1 (c). When the PBC size is comparable to macromolecules, which is often the case due to the consideration of computing cost, macromolecules have detectable orientation bias due to their interaction with their images (Wu and Brooks 2005). The calculation of lattice sums is expensive and special techniques like the Ewald method and the particle meshed Ewald method are needed to save computing time. As shown in Fig. 1 (d), the IPS long-range interactions are isotropic so that the orientation bias caused by image interactions can be overcome. The IPS long-range interactions are represented by equivalent short-range functions, which can be calculated as efficiently as the cut-off based methods and the The concept of the IPS method is using isotropic distributed images of a local region to represent remote environment to calculate long-range interactions. The difference between the IPS method and the lattice sum methods such as the Ewald sum lies in the shape and distribution of remote images. The images for the lattice sum are generated from the periodic boundary conditions and are discretely and anisotropically positioned at the lattice points in space. The images for the IPS calculation are imaginary, which means they do not explicitly exist in a simulation system, and are distributed in an isotropic and periodic way around each particle. The IPS images are distributed equally in all homogeneous dimensions. Analytic solutions for IPS are available for many potential types and the Fig. 2 illustrates the definition of the local region and its isotropic periodic images in a square periodic boundary system. The local region of particle 1 is enclosed by a dashed circle of radius Rc. The isotropic periodic images of the local region and their particles are shown as dotted circles and dotted particles labeled correspondingly. There are an infinite number of image shells around the local region. The image regions of the first layer are bounded with the local region and occupy the area with a radius from Rc to 3Rc. In this layer, the isotropic periodic images of particle 1 are distributed on image shells with a radius of 2Rc. The image regions on an image shell are statistical representation of conformations around this image shell and can overlap with each other. Because the image regions are translation of the local region, the images of each particle will distribute on its own image shells centered at this particle. As shown in Fig. 2, the isotropic periodic images of particle 2 distribute on image shells centered at particle 2. Particle 1 only interacts with particles β controls the relative contributions Here, V is the volume of the PBC box. The parameter calculation is especially efficient for parallel computing. calculation of the IPS potentials is straightforward and very efficient. 3.4 The Isotropic Periodic Sum complementary error function, erfc (x), takes the following form: The Ewald sum is a method of order N2 and is expensive for large systems. $$\mathcal{L}(r, r\_{\mathfrak{c}}) = \begin{cases} \mathcal{S}(r) + S\_{\text{cut}}(r, r\_{\mathfrak{c}}) & r \le r\_{\mathfrak{c}} \\ 0 & r > r\_{\mathfrak{c}} \end{cases} \tag{5}$$ Here, rc is the cutoff distance, and ),( ccut rrS represents a shift or switch function. Under periodic boundary conditions (PBC), the minimum image convention must be used when applying the cutoff based methods. A pair of particles can have an infinite number of interaction pairs between the two particles or their images. The minimum image convention considers only the pair with the minimum distances for each pair of particles. The cutoff based methods save computing cost by neglecting interacting pairs with distances larger than rc. However, this approximation often causes large errors in simulation results, especially for electrostatic interactions. #### 3.2 Reaction field method The reaction field method describes a simulation system with a local cavity and a long-range reaction field, as illustrated in Fig. 1 (b). For each atom, there is a local spherical cavity centred on it. The central particle interacts directly with all particles within the cavity. Interactions with particles beyond the cavity are replaced by a reaction field. The reaction field acting on the particle is proportional to the moment of the cavity surrounding it: $$ \omega\_{\varepsilon} = \frac{2(\varepsilon\_s - 1)}{2\varepsilon\_s + 1} \frac{1}{r\_{\varepsilon}^3} \sum\_{r\_{\emptyset} \in r\_{\varepsilon}} \mu\_{\beta} \tag{6} $$ Here, <sup>s</sup> ε is the dielectric constant of the surrounding environment and μ <sup>j</sup> is the dipole moment of molecule j. This method acts like a cutoff method but retains a certain physical basis. Its use requires an artificial switching function to overcome the discontinuity crossing the cavity boundary. #### 3.3 The Ewald sum The Ewald sum (Ewald 1921) is an efficient method for the calculation of interactions with the lattice images created by the periodic boundary conditions (PBC), as shown in Fig.1 (c). For electrostatic interactions, the total energy of a system of N particles is: $$E = \sum\_{i$$ Here, qi is the charge of particle i. The first summation runs over all cell vectors, <sup>332211</sup> ++= nnn aaan . Here, <sup>21</sup> 3 and , , aaa are the lattice vectors of the PBC. The second summation runs over all reciprocal vectors, \* 33 \* 22 \* <sup>11</sup> ++= kkk aaak . Here, \* 3 \* 2 \* 1 and , , aaa are the conjugate reciprocal lattice vectors. The third term is a self-energy correction term. The direct potential and reciprocal potential have the following form: $$\text{hyp}\_{\text{dir}}(\mathbf{r}\_{\circ}, \beta) = \sum\_{\mathbf{n}} \frac{\text{erfc}(\beta \mid \mathbf{r}\_{\circ} - \mathbf{n} \mid)}{\|\mathbf{r}\_{\circ} - \mathbf{n} \mid} \tag{8}$$ $$\Psi\_{\rm nc}(\mathbf{r}\_{\circ}, \boldsymbol{\beta}) = \frac{1}{\pi V} \sum\_{\mathbf{k} \neq 0} \frac{\exp(-\pi^{2} \mathbf{k}^{2} / \boldsymbol{\beta}^{2})}{\mathbf{k}^{2}} \exp(2\pi \mathbf{k} \cdot \mathbf{r}\_{\circ}) \tag{9}$$ Here, V is the volume of the PBC box. The parameter β controls the relative contributions from the real space, the first term, and the reciprocal space, the second term. The complementary error function, erfc (x), takes the following form: $$\text{erfc}(x) = \frac{2}{\sqrt{\pi}} \int\_{x}^{\circ} \exp(-t^{\circ}) dt$$ The Ewald sum is a method of order N2 and is expensive for large systems. ## 3.4 The Isotropic Periodic Sum 142 Fourier Transform – Materials Analysis ),()( ),( rr rrSr rr rr Here, rc is the cutoff distance, and ),( ccut rrS represents a shift or switch function. Under periodic boundary conditions (PBC), the minimum image convention must be used when applying the cutoff based methods. A pair of particles can have an infinite number of interaction pairs between the two particles or their images. The minimum image convention considers only the pair with the minimum distances for each pair of particles. The cutoff based methods save computing cost by neglecting interacting pairs with distances larger than rc. However, this approximation often causes large errors in simulation results, The reaction field method describes a simulation system with a local cavity and a long-range reaction field, as illustrated in Fig. 1 (b). For each atom, there is a local spherical cavity centred on it. The central particle interacts directly with all particles within the cavity. Interactions with particles beyond the cavity are replaced by a reaction field. The reaction field acting on the particle is proportional to the moment of the cavity surrounding it: ∑ <sup>+</sup> <sup>∈</sup> i j ij r moment of molecule j. This method acts like a cutoff method but retains a certain physical basis. Its use requires an artificial switching function to overcome the discontinuity crossing The Ewald sum (Ewald 1921) is an efficient method for the calculation of interactions with the lattice images created by the periodic boundary conditions (PBC), as shown in Fig.1 (c). =∑ <sup>+</sup> ∑ <sup>−</sup> ∑ <sup>&</sup>lt; <sup>i</sup> jiqqE qq q<sup>2</sup> , dir rec ),( <sup>2</sup> r r βψ \* ji ij ijji Here, qi is the charge of particle i. The first summation runs over all cell vectors, <sup>332211</sup> ++= nnn aaan . Here, <sup>21</sup> 3 and , , aaa are the lattice vectors of the PBC. The second conjugate reciprocal lattice vectors. The third term is a self-energy correction term. The rec )2exp( )/(exp1 ),( ∑ <sup>−</sup> <sup>−</sup> <sup>=</sup> <sup>n</sup> nr <sup>⋅</sup> <sup>−</sup> <sup>=</sup> βπ 2 222 k k rij ij i c 3 cs <sup>s</sup> 1 12 )1(2 μ (6) i π 33 <sup>11</sup> ++= kkk aaak . Here, \* rk π \* 22 nr ij ij β β rr <sup>−</sup> <sup>=</sup> is the dielectric constant of the surrounding environment and ε For electrostatic interactions, the total energy of a system of N particles is: βψ summation runs over all reciprocal vectors, \* direct potential and reciprocal potential have the following form: ψ β r ij β ∑≠ k V π 0 <sup>1</sup> ),( ji ψ ε ε <sup>+</sup> <sup>≤</sup> <sup>=</sup> > ccut c c (5) μ <sup>j</sup> is the dipole (7) \* 2 \* (8) (9) 3 1 and , , aaa are the ⎩ ⎨ ⎧ ε especially for electrostatic interactions. 3.2 Reaction field method Here, <sup>s</sup> ε the cavity boundary. 3.3 The Ewald sum <sup>c</sup> 0 ε The IPS method was developed to overcome some artefacts of the lattice sum methods. The lattice sum methods, like the Ewald sum, assume that the remote regions can be represented by images created by periodic boundary conditions (PBC). The PBC images distribute discretely in lattice points throughout the space and are anisotropic in nature, as shown in Fig.1 (c). When the PBC size is comparable to macromolecules, which is often the case due to the consideration of computing cost, macromolecules have detectable orientation bias due to their interaction with their images (Wu and Brooks 2005). The calculation of lattice sums is expensive and special techniques like the Ewald method and the particle meshed Ewald method are needed to save computing time. As shown in Fig. 1 (d), the IPS long-range interactions are isotropic so that the orientation bias caused by image interactions can be overcome. The IPS long-range interactions are represented by equivalent short-range functions, which can be calculated as efficiently as the cut-off based methods and the calculation is especially efficient for parallel computing. The concept of the IPS method is using isotropic distributed images of a local region to represent remote environment to calculate long-range interactions. The difference between the IPS method and the lattice sum methods such as the Ewald sum lies in the shape and distribution of remote images. The images for the lattice sum are generated from the periodic boundary conditions and are discretely and anisotropically positioned at the lattice points in space. The images for the IPS calculation are imaginary, which means they do not explicitly exist in a simulation system, and are distributed in an isotropic and periodic way around each particle. The IPS images are distributed equally in all homogeneous dimensions. Analytic solutions for IPS are available for many potential types and the calculation of the IPS potentials is straightforward and very efficient. Fig. 2 illustrates the definition of the local region and its isotropic periodic images in a square periodic boundary system. The local region of particle 1 is enclosed by a dashed circle of radius Rc. The isotropic periodic images of the local region and their particles are shown as dotted circles and dotted particles labeled correspondingly. There are an infinite number of image shells around the local region. The image regions of the first layer are bounded with the local region and occupy the area with a radius from Rc to 3Rc. In this layer, the isotropic periodic images of particle 1 are distributed on image shells with a radius of 2Rc. The image regions on an image shell are statistical representation of conformations around this image shell and can overlap with each other. Because the image regions are translation of the local region, the images of each particle will distribute on its own image shells centered at this particle. As shown in Fig. 2, the isotropic periodic images of particle 2 distribute on image shells centered at particle 2. Particle 1 only interacts with particles Molecular Simulation with Discrete Fast Fourier Transform 145 <sup>+</sup> <sup>≤</sup> <sup>=</sup> r qq <sup>r</sup> <sup>21</sup> c c c R r ⎟ ≈ ⎠ , and ψ <sup>+</sup> <sup>⎜</sup> <sup>+</sup> <sup>⎜</sup> ⎜ ⎜ ⎝ ⎛ R r 16 26 1 + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ij ij ij Rrr Rr 0 ),()( ),( Rr φε The IPS potentials have analytic forms for many commonly used potentials (Wu and Brooks where <sup>1</sup> q and <sup>2</sup> q are the charges of the two interacting particles, its IPS image interaction is <sup>⎛</sup> −= ++−+ ) <sup>2</sup> 1() <sup>2</sup> 1(2 <sup>2</sup> ),( <sup>⎞</sup> <sup>⎜</sup> The IPS analytic solutions are often very complicated and are time consuming to compute directly. Instead, we use numerical functions that fit these analytic solutions for efficient calculations in simulations. To avoid numerical mistakes in implementation, we use the > ⎜ ⎝ ji <sup>c</sup> <sup>c</sup> R r qq ji For polar systems, the opposite charges play a screening effect in charge-charge interactions. After considering the opposite charge screening effect, we obtain the following expression > ⎜ ⎝ ji <sup>c</sup> <sup>c</sup> R r qq ⎛ 13 4 R r ele 26 c ele 16 R ⎛ <sup>m</sup> <sup>k</sup> cele R 1 m ⎝ <sup>⎛</sup> <sup>=</sup> ∑ <sup>−</sup> <sup>=</sup> ∞→ <sup>k</sup> <sup>m</sup> ψγ φ ⎩ ⎨ ⎧ ele ε)( = > ⎜ ⎜ ⎝ 21 R qq where γ is the Euler's constant, 577216.0log <sup>1</sup> lim following simplified polynomial with rational coefficients: ele ele φεRrrRr γ ∞ −− =Γ 0 <sup>1</sup> )( dtetz tz . += +≈ += += IPSp 35 1),()(),( ele ele φεRrrRr IPSn 1),()(),( c Rr ij , runs over all particles, including any PBC image particles, that > c c ij c ⎟ ⎟ ⎠ ⎞ ψ r Rr cij represents the long-range contribution as (11) (12) (13) (z) is the digamma ⎟ ⎟ ⎠ ⎟ ⎟ ⎠ ⎜ ⎜ ⎝ ⎛ r 5 ⎟ ⎟ ⎠ ⎞ c c c R r 3 5 7 ⎜ ⎜ ⎝ <sup>⎛</sup> <sup>−</sup> <sup>⎟</sup> ⎟ ⎠ ⎞ 16 21 ⎞ ⎟ ⎟ ⎠ ⎞ (14) (15) ⎜ ⎜ ⎝ ⎛ r 1 ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ c c c R r qq<sup>4</sup> 107.7 <sup>−</sup> <sup>×</sup> from eq. (13). We call eq. (13) or eq. (14) the 3 5 7 ⎞ ⎟ ⎟ ⎠ ⎞ is called the IPS potential, which is the sum of the pair Here, the summation, ∑≤Rr cij a function of ij r and Rc . ),( <sup>c</sup> 2005). For electrostatic interaction, function: ψ IPSn Eq. (14) has an average deviation of non-polar IPS electrostatic potential. for the polar IPS electrostatic potential: ε IPSp ε φRr )( )( )( <sup>z</sup> <sup>z</sup> <sup>z</sup> <sup>Γ</sup> Γ′ <sup>=</sup> , and ∫ are within the range of Rc from particle i. ),( IPS Rrij ε interactions within the local region and the image interactions: IPS ε within its local region, i.e., particles 2, 3, and 4, and their isotropic periodic images, including itself. Similarly, all particles in the local region will interact with the isotropic periodic images of particle 1. All other particles, such as, particles 5, 6, 7, and all images generated by the periodic boundary condition that are outside the local region are not seen by particle 1 and are represented by the isotropic periodic images of the local particles in the calculation of long-range energies. Particle 4 is at the boundary of the local region of particle 1 and has the same distance, Rc, to particle 1 and its nearest isotropic periodic image on the first image shell. Due to the periodicity, the total force on particle 4 from particle 1 and its images is zero. Please note that the total interaction between particle 1 and all images of particle 2 will be the same as that between particle 2 and all images of particle 1. Fig. 2. The local region and the isotropic periodic images in a square periodic boundary system. The local region of particle 1 is enclosed by the dashed circle. The image shells of particles 1 and 2 are shown as dotted-dashed circles around particles 1 and 2, respectively. The image shells of other particles are not shown for clarity. The isotropic periodic images of the local region shown as dotted circles distribute around the local region and can overlap with each other. Particle 1 interacts with particles 2, 3, and 4 in its local region and the isotropic periodic image particles, shown as dotted particles. Particle 4 is at the boundary of the local region and has the same distance to particle 1 and the nearest image of particle 1 If we assume that the structure beyond the local region can be represented by the isotropic periodic images of the local region, the summation over all particles beyond the local region becomes a function of the local region structure: $$E\_i = \frac{1}{2} \sum\_{r\_{\vec{\eta}} \le R\_{\vec{\iota}}} \left( \varepsilon(r\_{\vec{\eta}}) + \phi(r\_{\vec{\eta}}, R\_{\vec{\iota}}) \right) = \frac{1}{2} \sum\_{r\_{\vec{\eta}} \le R\_{\vec{\iota}}} \varepsilon^{\text{prs}}(r\_{\vec{\eta}}, R\_{\vec{\iota}}) \tag{10}$$ within its local region, i.e., particles 2, 3, and 4, and their isotropic periodic images, including itself. Similarly, all particles in the local region will interact with the isotropic periodic images of particle 1. All other particles, such as, particles 5, 6, 7, and all images generated by the periodic boundary condition that are outside the local region are not seen by particle 1 and are represented by the isotropic periodic images of the local particles in the calculation of long-range energies. Particle 4 is at the boundary of the local region of particle 1 and has the same distance, Rc, to particle 1 and its nearest isotropic periodic image on the first image shell. Due to the periodicity, the total force on particle 4 from particle 1 and its images is zero. Please note that the total interaction between particle 1 and all images of particle 2 will be the same as that between particle 2 and all images of particle 1. Local region of particle 1 2 7 4 Image shell of particle 2 4 1 6 Isotropic periodic images 3 5 becomes a function of the local region structure: Rr 3 2 1 1 6 5 3 4 7 2 1 1 Fig. 2. The local region and the isotropic periodic images in a square periodic boundary system. The local region of particle 1 is enclosed by the dashed circle. The image shells of particles 1 and 2 are shown as dotted-dashed circles around particles 1 and 2, respectively. The image shells of other particles are not shown for clarity. The isotropic periodic images of the local region shown as dotted circles distribute around the local region and can overlap with each other. Particle 1 interacts with particles 2, 3, and 4 in its local region and the isotropic periodic image particles, shown as dotted particles. Particle 4 is at the boundary of the local region and has the same distance to particle 1 and the nearest image of particle 1 If we assume that the structure beyond the local region can be represented by the isotropic periodic images of the local region, the summation over all particles beyond the local region > ∑( ) ∑ <sup>≤</sup> <sup>≤</sup> = + = φε Ei ij Rrr cij Rr ),( <sup>2</sup> <sup>1</sup> ),()( <sup>2</sup> 1 IPS cij Rr cij 4 6 1 <sup>R</sup><sup>c</sup> <sup>2</sup> 2R<sup>c</sup> 3 5 3 Rc 4 5 1 2 4 7 3 7 3 4 2 cij ε 2 1 Image shells of particle 1 4R<sup>c</sup> 2 1 1 4 3 (10) 3 3 1 4 6 2 Here, the summation, ∑≤Rr cij , runs over all particles, including any PBC image particles, that are within the range of Rc from particle i. ),( φ Rr cij represents the long-range contribution as a function of ij r and Rc . ),( <sup>c</sup> IPS Rrij ε is called the IPS potential, which is the sum of the pair interactions within the local region and the image interactions: $$\varepsilon^{\rm IPS}(r\_{\circ}, R\_{\circ}) = \begin{cases} \varepsilon(r\_{\circ}) + \phi(r\_{\circ}, R\_{\circ}) & r\_{\circ} \le R\_{\circ} \\ 0 & r\_{\circ} > R\_{\circ} \end{cases} \tag{11}$$ The IPS potentials have analytic forms for many commonly used potentials (Wu and Brooks 2005). For electrostatic interaction, $$ \varepsilon\_{\rm ele}(r) = \frac{q\_1 q\_2}{r} \tag{12} $$ where <sup>1</sup> q and <sup>2</sup> q are the charges of the two interacting particles, its IPS image interaction is $$\phi\_{\rm ele}(r, R\_{\rm c}) = -\frac{q\_1 q\_2}{2R\_{\rm c}} \left( 2\gamma + \wp(\mathbf{l} - \frac{r}{2R\_{\rm c}}) + \wp(\mathbf{l} + \frac{r}{2R\_{\rm c}}) \right) \tag{13}$$ where γ is the Euler's constant, 577216.0log <sup>1</sup> lim 1 ⎟ ≈ ⎠ <sup>⎞</sup> <sup>⎜</sup> ⎝ <sup>⎛</sup> <sup>=</sup> ∑ <sup>−</sup> <sup>=</sup> ∞→ <sup>m</sup> <sup>k</sup> m <sup>k</sup> <sup>m</sup> γ , and ψ(z) is the digamma function: )( )( )( <sup>z</sup> <sup>z</sup> <sup>z</sup> <sup>Γ</sup> Γ′ ψ <sup>=</sup> , and ∫ ∞ −− =Γ 0 <sup>1</sup> )( dtetz tz . The IPS analytic solutions are often very complicated and are time consuming to compute directly. Instead, we use numerical functions that fit these analytic solutions for efficient calculations in simulations. To avoid numerical mistakes in implementation, we use the following simplified polynomial with rational coefficients: $$\mathcal{L}\_{\rm ek}^{\rm IPSa}(r, R\_c) = \mathcal{x}\_{\rm ek}(r) + \phi\_{\rm ek}^{\rm IPSa}(r, R\_c) \approx \frac{q\_i q\_j}{r} \left( 1 + \frac{4}{13} \left( \frac{r}{R\_c} \right)^3 + \frac{1}{26} \left( \frac{r}{R\_c} \right)^5 + \frac{1}{26} \left( \frac{r}{R\_c} \right)^7 \right) \tag{14}$$ Eq. (14) has an average deviation of c ji R qq<sup>4</sup> 107.7 <sup>−</sup> <sup>×</sup> from eq. (13). We call eq. (13) or eq. (14) the non-polar IPS electrostatic potential. For polar systems, the opposite charges play a screening effect in charge-charge interactions. After considering the opposite charge screening effect, we obtain the following expression for the polar IPS electrostatic potential: $$\varepsilon\_{\rm elec}^{\rm IPSp}(r, R\_c) = \varepsilon\_{\rm elec}(r) + \phi\_{\rm elec}^{\rm IPSp}(r, R\_c) = \frac{q\_i q\_j}{r} \left( 1 + \frac{3\mathfrak{S}}{16} \left( \frac{r}{R\_c} \right)^3 - \frac{21}{16} \left( \frac{r}{R\_c} \right)^5 + \frac{\mathfrak{S}}{16} \left( \frac{r}{R\_c} \right)^7 \right) \tag{15}$$ Molecular Simulation with Discrete Fast Fourier Transform 147 After separating direct interactions, the electrostatic interaction energy can be written as: = ⋅+ −− ele dir ))()(()( <sup>2</sup> 1 > ⎩ ⎨ ⎧ u 0 \|1\|1 )( <sup>2</sup> uu <sup>u</sup> <sup>u</sup> uM <sup>n</sup> uM <sup>n</sup> <sup>n</sup> ijji qqE FQFFQ q <sup>−</sup> <sup>−</sup> such as the Lennard-Jones energy can also be treated like eq. (21). ψr )( <sup>1</sup> <sup>1</sup> = > 1 N ij N i 4.1 Particle Mesh Ewald (PME) function of the n-th order, we have: 1 , , 321 1111 321 i n nn N 321 <sup>1</sup> <sup>1</sup> The structure factor can be expressed as: 1 N j mmmAF ≈ m − ∑ ∑ ( ) ∑= Eq. (21) separates potentials to a short-range part calculated by a direct pairwise summation and a remaining long-range part calculated as a convolution. Other long-range interactions The PME method assigns the charge density to a finely spaced mesh in a simulation box so that the reciprocal part of the Ewald sum can be calculated as convolutions that can be accelerated with DFFT. Here we describe a smooth particle mesh Ewald method based on the b-spline interpolation (Essmann, Perera et al. 1995). If uM )( <sup>n</sup> represents the b-spline ≤≤−− <sup>=</sup> 2or 0 )1( <sup>1</sup> )( <sup>1</sup> )( <sup>1</sup> <sup>1</sup> <sup>−</sup> <sup>−</sup> <sup>−</sup> <sup>+</sup> <sup>−</sup> <sup>=</sup> <sup>−</sup> <sup>−</sup> uM <sup>n</sup> un uM <sup>n</sup> Here u is the coordinate to be interpolated. In a simulation box, atomic charges can be distributed over a set of predefined grid points, (k1, k2, k3), where k1 =1,2,…, K1, k2=1,2,.., K2, k3=1,2,…,K3. Assume the charges of a simulation system of N particles are { }<sup>N</sup> ,...,, qqq q = <sup>21</sup> . After spreading q on the grid points we have a distribution, Q, (Essmann, Perera et al. 1995): ),,( ( () () ) <sup>2222</sup> <sup>3333</sup> <sup>=</sup> ∑ ∑ ini −− −− −− <sup>=</sup> <sup>⎛</sup> <sup>=</sup> ++ <sup>⎛</sup> <sup>=</sup> <sup>−</sup> ++ 321 ikkkA ⎣ ⎡ ⎜ ⎜ ⎝ > ⎢ ⎢ ⎣ ⎡ 1 3 K ⎢ ⎢ ⎣ ⎡ π ⎜ ⎜ ⎝ 1 11 lm lllA <sup>i</sup> 1 11 > ⎜ ⎜ ⎝ 0 3 π <sup>l</sup> K K 0 3 <sup>k</sup> K π kkkQ KnkuMKnkuMKnkuMq in in We use AF )( to represent the discrete Fourier transform of an array ),,( <sup>321</sup> kkkA : ∑∑∑ − = 2 2 321 321 1 1 1 0 and )(1 AF <sup>−</sup> to represent the inverse discrete Fourier transform: 321 ),,)(()()()( 111111 321 mmmQFmbmbmb j j πrm )( )2exp( 2exp KKK K k − = 1 0 K − = 3 3 ),,)(( 2exp),,( K k km mmmAF 1 ∑∑∑ − = 2 2 1 0 K l 1 km iqS iq 2exp),,( <sup>1</sup> ),,)(( K l − = 1 0 K − = 3 3 N j j <sup>⎛</sup> <sup>=</sup> <sup>∑</sup> =⋅ <sup>∑</sup> ++ <sup>=</sup> <sup>=</sup> 1 <sup>⊗</sup> <sup>=</sup> BFAFBAF )()()( (20) dir ψψrr >< <sup>⎤</sup> <sup>⎢</sup> K km > 1 11 K 2 22 K km 2 22 20 N i i 1 self 2 (21) (22) (23) (24) (25) (26) (27) ⎥ ⎦ 33 lm ⎥ ⎥ ⎦ ⎤ ⎟ ⎟ ⎠ ⎞ ⎟ ⎟ ⎠ ⎞ 33 km 2 22 33 ⎥ ⎥ ⎦ ⎤ ⎟ ⎟ ⎠ ⎞ K km K lm ψ The Lernnard-Jones potential can be separated into repulsion and dispersion terms: $$\mathcal{E}\_{L-J}(r) = \mathcal{E}\_0 \left( \frac{r^\ast}{r} \right)^{12} - 2(\frac{r^\ast}{r})^6 \Big\| = \frac{A}{r^{12}} - \frac{C}{r^6} = \mathcal{E}\_{\text{rep}}(r) + \mathcal{E}\_{\text{disp}}(r) \tag{16}$$ where, <sup>0</sup> ε and \* r are the energy minimum and the minimum distance. <sup>12</sup> \* <sup>0</sup> = ε rA and 6\* <sup>2</sup> <sup>0</sup> <sup>=</sup> ε rC are the constants for repulsion and dispersion interactions, respectively. We obtained the following polynomials with rational coefficients by fitting into the analytic solutions of the dispersion and repulsion IPS potentials (Wu and Brooks 2005; Wu and Brooks 2009): $$\mathcal{E}\_{\text{disp}}^{\text{3D-IPS}}(r, R\_c) \approx -\frac{C}{r^6} \left( 1 + \frac{7}{16} \left( \frac{r}{R\_c} \right)^6 + \frac{9}{14} \left( \frac{r}{R\_c} \right)^8 - \frac{3}{28} \left( \frac{r}{R\_c} \right)^{10} + \frac{6}{7} \left( \frac{r}{R\_c} \right)^{12} \right) \tag{17}$$ $$\mathcal{L}\_{\text{sup}}^{\text{3D-PS}}(r, R\_c) \approx \frac{A}{r^{12}} \left( 1 + \frac{\mathfrak{F}}{787} \left( \frac{r}{R\_c} \right)^{12} + \frac{9}{26} \left( \frac{r}{R\_c} \right)^{16} - \frac{3}{13} \left( \frac{r}{R\_c} \right)^{20} + \frac{27}{26} \left( \frac{r}{R\_c} \right)^{24} \right) \tag{18}$$ The average deviations from analytic solutions are <sup>6</sup> <sup>3</sup> 102.6 Rc <sup>C</sup><sup>−</sup> <sup>×</sup> and <sup>12</sup> <sup>3</sup> 107.3 Rc <sup>A</sup> <sup>−</sup> <sup>×</sup> for dispersion and repulsion interactions, respectively. With eqs. (14), (15), (17), and (18), the IPS method calculates long-range interactions as the short-range functions with < Rr <sup>c</sup> . Obviously, the computating cost is comparable to the cutoff method with = Rr cc . Therefore, the IPS method is of order N, and can scale very well for parallel computing. The IPS method described above assumes that a system is fully homogeneous in all dimensions and is called the three-dimensional (3D) IPS method. For partial homogeneous systems like membrane or fibril systems, two-dimensional (2D) and one dimensional (1D) IPS methods have been developed. Please refer to the original paper (Wu and Brooks 2005) for details about the 2D-IPS and 1D-IPS methods. #### 4. DFFT for long-range interactions For pairwise molecular interactions, the interaction energy can be expressed as a convolution of a momentum distribution with potential functions. For example, electrostatic interaction energy can be written as the charge distribution convolved with the electrostatic potential: $$E\_{\rm ele} = \sum\_{i=1}^{N-1} q\_i \sum\_{j>i}^{N} q\_j \wp(\mathbf{r}\_{ij}) = \frac{1}{2} \underline{Q} \cdot \left( \underline{Q} \otimes \wp(\mathbf{r}) \right) - \sum\_{i=1}^{N} q\_i^2 \wp\_{\rm sefl} \tag{19}$$ Here, Q is the charge distribution. ψ r)( is the potential function at position r from a unit charge at origin and its all images. ψ self is the self-potential to correct the self-interaction included in the convolution. Fourier transform provides a convenient way to deal with such convolution operations: $$F(A \otimes B) = F(A)F(B) \tag{20}$$ After separating direct interactions, the electrostatic interaction energy can be written as: $$E\_{\rm clc} = \sum\_{i=1}^{N-1} q\_i \sum\_{j>i}^{N} q\_j \boldsymbol{\nu}\_{\rm dir}(\mathbf{r}\_{ij}) + \frac{1}{2} \boldsymbol{Q} \cdot \boldsymbol{F}^{-1} \Big( \boldsymbol{F}(\boldsymbol{Q}) \boldsymbol{F}(\boldsymbol{\nu}(\mathbf{r}) - \boldsymbol{\nu}\_{\rm dir}(\mathbf{r})) \Big) - \sum\_{i=1}^{N} q\_i^2 \boldsymbol{\nu}\_{\rm sat} \tag{21}$$ Eq. (21) separates potentials to a short-range part calculated by a direct pairwise summation and a remaining long-range part calculated as a convolution. Other long-range interactions such as the Lennard-Jones energy can also be treated like eq. (21). #### 4.1 Particle Mesh Ewald (PME) 146 Fourier Transform – Materials Analysis )(2)()( )()( <sup>612</sup> rep disp 6 \* ⎟ ⎠ ⎞ rC are the constants for repulsion and dispersion interactions, respectively. We obtained the following polynomials with rational coefficients by fitting into the analytic solutions of the dispersion and repulsion IPS potentials (Wu and Brooks 2005; Wu and > + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ rep R 12 14 9 26 9 With eqs. (14), (15), (17), and (18), the IPS method calculates long-range interactions as the short-range functions with < Rr <sup>c</sup> . Obviously, the computating cost is comparable to the cutoff method with = Rr cc . Therefore, the IPS method is of order N, and can scale very well The IPS method described above assumes that a system is fully homogeneous in all dimensions and is called the three-dimensional (3D) IPS method. For partial homogeneous systems like membrane or fibril systems, two-dimensional (2D) and one dimensional (1D) IPS methods have been developed. Please refer to the original paper (Wu and Brooks 2005) For pairwise molecular interactions, the interaction energy can be expressed as a convolution of a momentum distribution with potential functions. For example, electrostatic interaction energy can be written as the charge distribution convolved with the electrostatic ∑ ∑ ( ) ∑= included in the convolution. Fourier transform provides a convenient way to deal with such 1 <sup>2</sup> <sup>1</sup> ψr = −⊗⋅= ele )( <sup>2</sup> ψr )( ψ ψ ijji qqE QQ q <sup>7</sup> 1 ),( <sup>R</sup> c R r c R r 16 787 disp 7 6 <sup>0</sup> rr <sup>r</sup> ⎟ r A and \* r are the energy minimum and the minimum distance. <sup>12</sup> \* C 8 16 c R r c R r εε + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ <sup>⎛</sup> <sup>−</sup> <sup>⎟</sup> ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ <sup>⎛</sup> <sup>−</sup> <sup>⎟</sup> ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ <sup>C</sup><sup>−</sup> <sup>×</sup> and <sup>12</sup> 20 6 26 27 c R r c N i i 1 r)( is the potential function at position r from a unit self is the self-potential to correct the self-interaction self (19) ψ R r 28 3 13 3 <sup>3</sup> 102.6 Rc 10 ⎟ ⎟ ⎠ ⎟ ⎟ ⎠ <sup>A</sup> <sup>−</sup> <sup>×</sup> for dispersion ⎞ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ 24 ⎞ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ 12 c c <sup>3</sup> 107.3 Rc r r <sup>0</sup> = εrA and (16) (17) (18) The Lernnard-Jones potential can be separated into repulsion and dispersion terms: r r +=−= 12 \* r ⎜ ⎜ ⎝ ⎛ ⎜ ⎜ ⎝ ⎛ <sup>−</sup> = − <sup>r</sup> <sup>r</sup> JL εε +−≈ <sup>−</sup> +≈ <sup>−</sup> for details about the 2D-IPS and 1D-IPS methods. − 1 N i = > N ij 4. DFFT for long-range interactions Here, Q is the charge distribution. charge at origin and its all images. convolution operations: c 12 IPS3D IPS3D and repulsion interactions, respectively. εRr εRr for parallel computing. potential: c 6 5 1 ),( r A The average deviations from analytic solutions are <sup>6</sup> r C > ⎜ ⎜ ⎝ > ⎛ where, <sup>0</sup> ε > 6\* <sup>2</sup> <sup>0</sup> <sup>=</sup> ε Brooks 2009): The PME method assigns the charge density to a finely spaced mesh in a simulation box so that the reciprocal part of the Ewald sum can be calculated as convolutions that can be accelerated with DFFT. Here we describe a smooth particle mesh Ewald method based on the b-spline interpolation (Essmann, Perera et al. 1995). If uM )( <sup>n</sup> represents the b-spline function of the n-th order, we have: $$M\_{\,\,2}(u) = \begin{cases} 1 - \|u - 1\| & 0 \le u \le 2 \\ 0 & u < 0 \text{ or } u > 2 \end{cases} \tag{22}$$ $$M\_n(\mu) = \frac{u}{n-1} M\_{n-1}(\mu) + \frac{n-u}{n-1} M\_{n-1}(\mu - \mathbf{l}) \tag{23}$$ Here u is the coordinate to be interpolated. In a simulation box, atomic charges can be distributed over a set of predefined grid points, (k1, k2, k3), where k1 =1,2,…, K1, k2=1,2,.., K2, k3=1,2,…,K3. Assume the charges of a simulation system of N particles are { }<sup>N</sup> ,...,, qqq q = <sup>21</sup> . After spreading q on the grid points we have a distribution, Q, (Essmann, Perera et al. 1995): $$Q(k\_1, k\_2, k\_3) = \sum\_{l=1}^{N} \sum\_{n\_1, n\_2, n\_3} q\_i M\_{\ n} (\mathfrak{u}\_{1i} - k\_1 - n\_1 K\_1) M\_{\ n} (\mathfrak{u}\_{2i} - k\_2 - n\_2 K\_2) M\_{\ n} (\mathfrak{u}\_{3i} - k\_3 - n\_3 K\_3) \tag{24}$$ We use AF )( to represent the discrete Fourier transform of an array ),,( <sup>321</sup> kkkA : $$F(A)(m\_1, m\_2, m\_3) = \sum\_{k\_1=0}^{K\_1-1} \sum\_{k\_2=0}^{K\_2-1} \sum\_{k\_3=0}^{K\_3-1} A(k\_1, k\_2, k\_3) \exp\left[2\pi i \left(\frac{m\_1 k\_1}{K\_1} + \frac{m\_2 k\_2}{K\_2} + \frac{m\_3 k\_3}{K\_3}\right)\right] \tag{25}$$ and )(1 AF <sup>−</sup> to represent the inverse discrete Fourier transform: $$F^{-1}(A)(m\_1, m\_2, m\_3) = \frac{1}{K\_1 K\_2 K\_3} \sum\_{l\_1=0}^{K\_1 - 1} \sum\_{l\_2=0}^{K\_2 - 1} \sum\_{l\_3=0}^{K\_3 - 1} A(l\_1, l\_2, l\_3) \exp\left[ -2\pi \left( \frac{m\_1 l\_1}{K\_1} + \frac{m\_2 l\_2}{K\_2} + \frac{m\_3 l\_3}{K\_3} \right) \right] \tag{26}$$ The structure factor can be expressed as: $$\begin{split} S(\mathbf{m}) &= \sum\_{j=1}^{N} q\_{j} \exp(2\pi i \mathbf{m} \cdot \mathbf{r}\_{j}) = \sum\_{j=1}^{N} q\_{j} \exp 2\pi i \left[ \left( \frac{m\_{1}k\_{1}}{K\_{1}} + \frac{m\_{2}k\_{2}}{K\_{2}} + \frac{m\_{3}k\_{3}}{K\_{3}} \right) \right] \\ &\approx b\_{1}(m\_{1})b\_{1}(m\_{1})b\_{1}(m\_{1})F(Q)(m\_{1}, m\_{2}, m\_{3}) \end{split} \tag{27}$$ Molecular Simulation with Discrete Fast Fourier Transform 149 such a region is hard to be representative of the system. In other words, this system is hetereougeneous in a size scale of rc. If we look at the system with the images created by the PBC, we can see that the PBC forces the system to be repeated throughout the space. With a local region radius, Rc, larger than the PBC size, a local region has a reasonable number of particles and can be a good representative of the system. That is, the system is homogeneous in a size scale of Rc. With such a large local region radius, IPS energies can well approximate the long range interactions. Therefore, to use IPS to describe this system, it is desirable to use a local region (defined by Rc) larger than the cutoff regions (defined by rc). When Rc is larger than the PBC size, the lattice symmetry from the PBC will be imposed into long-range interactions. Obviously, the IPS energies will approach that of the lattice sum when R<sup>c</sup> becomes infinitely large. + + + + + r + + + Fig. 3. A two-ion periodic system with a square periodic boundary condition. A region within a small radius of rc is highly heterogeneous. However, a region within a large radius > C ε C ⎩ ⎨ ⎧ The long-range part is the difference between the IPS energy and the cutoff part, eq. (34). + − φε 0 ),()( ),(),( Rrr rrrr ϑ ),,(),(),( cc <sup>+</sup> <sup>≤</sup> <sup>=</sup> c c c 0 ),()( ),( rr rrr rr rr ϑε c εε rc + Rc + + + LR + + + + ϑ RrrrrRr (33) > > ≤< ≤ Rr Rrr rr c c c c c c c rr , and the long-range (LR) part, ),,( cc rr to split the IPS potential LR ε Rrr . (34) (35) of Rc would be much closer to a homogeneous system due to the PBC c c ⎪ ⎩ ⎪ ⎨ ⎧ φ = += To work with a large Rc , we define a smoothing function, ),( <sup>c</sup> IPS ε C cc ),,( Rrr ε into two smooth parts, the cutoff (C) part, ),( <sup>c</sup> The cutoff part goes to zero at the cutoff distance, rc. LR ε Where $$b\_i(m\_i) = \frac{\exp(2\pi i (n-1)k\_i / K\_i)}{\sum\_{k=0}^{n-2} M\_n(k+1) \exp(2\pi i m\_i k / K\_i)}\tag{28}$$ The electrostatic long-range sum can be calculated in the following way: $$\begin{split} E\_{\text{nc}} &= \frac{1}{2\pi V} \sum\_{m=0}^{} \frac{\exp(-\pi^{2}\mathbf{m}^{2}/\beta^{2})}{\mathbf{m}^{2}} B(m\_{1}, m\_{2}, m\_{3}) F(\underline{Q})(m\_{1}, m\_{2}, m\_{3}) F(\underline{Q})(-m\_{1}, -m\_{2}, -m\_{3}) \\ &= \frac{1}{2} \sum\_{m\_{1}=0}^{K\_{1}-1} \sum\_{m\_{1}=0}^{K\_{1}-1} \sum\_{m\_{3}=0}^{K\_{1}-1} \underline{Q}(m\_{1}, m\_{2}, m\_{3}) \cdot (F(B \cdot C) \otimes \underline{Q})(m\_{1}, m\_{2}, m\_{3}) \end{split} \tag{29}$$ Here, $$B(m\_1, m\_2, m\_3) = \left\| b\_1(m\_1) \right\|^2 \left\| b\_2(m\_2) \right\|^2 \left\| b\_3(m\_3) \right\|^2 \tag{30}$$ and $$C(m\_1, m\_2, m\_3) = \begin{cases} \frac{1}{\pi V} \frac{\exp(-\pi^2 \mathbf{m}^2 / \beta^2)}{\mathbf{m}^2} & \mathbf{m} = 0\\ 0 & \mathbf{m} \neq 0 \end{cases} \tag{31}$$ The force can be calculated by $$f\_{ai}^{\text{new}} = -\frac{\partial E\_{\text{rec}}}{\partial \mathbf{r}\_{ai}} = \sum\_{m\_1=0}^{K\_1=1} \sum\_{m\_2=0}^{K\_2=1} \sum\_{\mathcal{O}}^{K\_3=1} \frac{\partial}{\partial \mathbf{r}\_{ai}} Q(m\_1, m\_2, m\_3) \cdot (F(\mathcal{B} \cdot \mathcal{C}) \otimes Q)(m\_1, m\_2, m\_3) \tag{32}$$ The PME algorithm is of order Nln (N). As compared with the Ewald sum of order N2, PME is more suitable for large systems. #### 4.2 IPS/DFFT method The underlying assumption of the IPS method is that the simulation system is homogenous and isotropic over the size of the local region (defined by the local region radius, Rc), and for convenience the local region in a homogeneous system is defined to be the same as the region within a cutoff distance, rc., i.e., Rc= rc. However, in many cases a simulation system is not homogenous in such a length scale (rc ~ 10 Å). To accurately describe the long-range interaction of heterogeneous systems, IPS need use a local region large enough to cover the heterogeneous range, up to the size of the simulation system or the periodic boundary box. Obviously, it is highly time consuming to do direct pair-wise calculation with such a large cutoff distance. To efficiently calculate interactions within such a large local region, the IPS/DFFT method split long-range interactions into two parts, a cutoff part and a longrange part. The cutoff part is calculated by summing atom pairs within a cutoff range (about 10 Å), and the long-range part is treated as convolutions and is calculated efficiently using DFFT. A simple case of a heterogeneous system is a two-ion periodic system as shown in Fig. 3. With a small cutoff distance of rc, one ion often falls out of the sight of the other, therefore, + <sup>−</sup> <sup>=</sup> <sup>2</sup> −−− <sup>−</sup> <sup>=</sup> mmmQFmmmQFmmmB <sup>V</sup> π /)1(2exp )( <sup>n</sup> ni <sup>k</sup> <sup>K</sup> mb = k The electrostatic long-range sum can be calculated in the following way: ii 222 ),,)()((),,( <sup>2</sup> β 0 321 321 ⎪⎩ ⎪ ⎨ ⎧ πV ),,( <sup>2</sup> mmmQCBFmmmQ ( ) ∑ ( ) <sup>−</sup> ii 2 321 321 321 2 33 2 22 2 ≠ m 0 <sup>11321</sup> <sup>=</sup> mbmbmbmmmB )()()(),,( (30) ⊗⋅⋅ 321 321 ),,)()((),,( = − 222 <sup>=</sup> <sup>0</sup> m m βπ 0 <sup>321</sup> m <sup>i</sup> mmmQCBFmmmQ <sup>E</sup> <sup>f</sup> <sup>r</sup> <sup>r</sup> The PME algorithm is of order Nln (N). As compared with the Ewald sum of order N2, PME The underlying assumption of the IPS method is that the simulation system is homogenous and isotropic over the size of the local region (defined by the local region radius, Rc), and for convenience the local region in a homogeneous system is defined to be the same as the region within a cutoff distance, rc., i.e., Rc= rc. However, in many cases a simulation system is not homogenous in such a length scale (rc ~ 10 Å). To accurately describe the long-range interaction of heterogeneous systems, IPS need use a local region large enough to cover the heterogeneous range, up to the size of the simulation system or the periodic boundary box. Obviously, it is highly time consuming to do direct pair-wise calculation with such a large cutoff distance. To efficiently calculate interactions within such a large local region, the IPS/DFFT method split long-range interactions into two parts, a cutoff part and a longrange part. The cutoff part is calculated by summing atom pairs within a cutoff range (about 10 Å), and the long-range part is treated as convolutions and is calculated efficiently using A simple case of a heterogeneous system is a two-ion periodic system as shown in Fig. 3. With a small cutoff distance of rc, one ion often falls out of the sight of the other, therefore, exp(1 )/ (28) (29) (31) (32) /2exp)1( kM Kkim n ii ),,)((),,)((),,( exp( )/ π Where Here, and ∑∑∑ 2 2 π K m The force can be calculated by α 4.2 IPS/DFFT method DFFT. <sup>∂</sup> −= is more suitable for large systems. rec rec <sup>1</sup> α − = 1 0 − = 1 1 K m rec E 1 1 0 2 1 ∑ − = 3 3 m K m 1 0 0 = ⊗⋅⋅ m πm mmmC ∑∑∑ − = i m i 2 2 K m <sup>∂</sup> <sup>=</sup> <sup>∂</sup> 1 K m 1 0 − = 1 0 − = 3 3 K 1 0 ∂ α ≠ such a region is hard to be representative of the system. In other words, this system is hetereougeneous in a size scale of rc. If we look at the system with the images created by the PBC, we can see that the PBC forces the system to be repeated throughout the space. With a local region radius, Rc, larger than the PBC size, a local region has a reasonable number of particles and can be a good representative of the system. That is, the system is homogeneous in a size scale of Rc. With such a large local region radius, IPS energies can well approximate the long range interactions. Therefore, to use IPS to describe this system, it is desirable to use a local region (defined by Rc) larger than the cutoff regions (defined by rc). When Rc is larger than the PBC size, the lattice symmetry from the PBC will be imposed into long-range interactions. Obviously, the IPS energies will approach that of the lattice sum when R<sup>c</sup> becomes infinitely large. Fig. 3. A two-ion periodic system with a square periodic boundary condition. A region within a small radius of rc is highly heterogeneous. However, a region within a large radius of Rc would be much closer to a homogeneous system due to the PBC To work with a large Rc , we define a smoothing function, ),( <sup>c</sup> ϑ rr to split the IPS potential into two smooth parts, the cutoff (C) part, ),( <sup>c</sup> C ε rr , and the long-range (LR) part, ),,( cc LR εRrr . $$ \varepsilon^{\rm lPS}(r, R\_{\varepsilon}) = \varepsilon^{\rm C}(r, r\_{\varepsilon}) + \varepsilon^{\rm LR}(r, r\_{\varepsilon}, R\_{\varepsilon}) \tag{33} $$ The cutoff part goes to zero at the cutoff distance, rc. $$ \varepsilon^{\mathbb{C}}(r, r\_{\mathbb{c}}) = \begin{cases} \varepsilon(r) + \mathcal{G}(r, r\_{\mathbb{c}}) & r \le r\_{\mathbb{c}} \\ 0 & r > r\_{\mathbb{c}} \end{cases} \tag{34} $$ The long-range part is the difference between the IPS energy and the cutoff part, eq. (34). $$ \varepsilon^{\rm LR}(r, r\_{\varepsilon}, R\_{\varepsilon}) = \begin{cases} \phi(r, r\_{\varepsilon}) - \mathcal{G}(r, r\_{\varepsilon}) & r \le r\_{\varepsilon} \\ \varepsilon(r) + \phi(r, R\_{\varepsilon}) & r\_{\varepsilon} < r \le R\_{\varepsilon} \\ & 0 & r > R\_{\varepsilon} \end{cases} \tag{35} $$ Molecular Simulation with Discrete Fast Fourier Transform 151 Again, the smoothing function can be the IPS potential, eq. (17), with a local region radius of IPS LR Δε For the long-range sum, because the dispersion term is atom type dependent, to simplify the calculation, we transform the atom pair dependent dispersion parameters, Cij, to a transferable quantity, di, which is defined as the dispersion momentum to measure the ∑∑ C i N j ij In a simulation box, like atomic charges, atomic dispersion momentums are distributed over a set of predefined grid points, (k1, k2, k3), where k1 =1,2,…, K1, k2=1,2,.., K2, k3=1,2,…,K3. Assume the dispersion momentums of a simulation system of N particles are },...,,{ <sup>11</sup> <sup>N</sup> d = ddd . After spreading q and d on the grid points we have grid distributions Q and D. Like the charge spreading in eq. (24), we again use Cardinal b-spline to do the d ),,( ( () () ) <sup>2222</sup> <sup>3333</sup> <sup>=</sup> ∑ ∑ ini −− −− −− <sup>=</sup> ele 333222111 ),,)(()()()(),,)((<sup>q</sup> <sup>=</sup> <sup>332211321</sup> <sup>Q</sup> mmmFmbmbmbmmmF <sup>321</sup> (48) ),,)(()()()(),,)((<sup>d</sup> <sup>=</sup> <sup>332211321</sup> <sup>D</sup> mmmFmbmbmbmmmF <sup>321</sup> (49) r r q ε q ε 1 LR 1 i i kkkD KnkuMKnkuMKnkuMd in in 321 <sup>Δ</sup> ∑Δ= (( −+++ ) ) KnkKnkKnk Rr cc ε kkk The energy type subscript is dropped here to indicate that eq. (45) applies to all energy ),,)((),,)((),,(),,)(()( <sup>321</sup> <sup>321</sup> <sup>321</sup> <sup>321</sup> <sup>1</sup> <sup>=</sup> −−− mmmFmmmFmmmBmmmFF <sup>−</sup> qq <sup>Q</sup> <sup>Q</sup> (50) LR ),,( ,,)1,1,1(),,( E qq q FFFq =Δ =Δ =Δ⊗ Δ i i ∑ ∑ ∑ ∑ k ∑ <sup>=</sup> <sup>N</sup> C N j kj ε− cdisp disp ϑ= By doing so, the long-range part, ),,( cc hetereogeneity of a simulation system in a length scale of rc. contribution of each atom to long range dispersion interactions. spreading (Essmann, Perera et al. 1995). 1 Here, ),,( <sup>321</sup> LR ele 1 , , 321 1111 321 The electrostatic long-range sum can be calculated in the following way: LR ele ε mmmFFF 2 1 LR ele ),,()()()( <sup>2</sup> LR ε types. The Fourier transform of q and d can be approximated by qq ε <sup>321</sup> ,, nnn i n nn N 2 1 1 − LR Δε kkk is the energy array = Δ i j ji ,, ∑ mmm 321 Where )(mb ii is defined by eq. (28). We have 321 ),(),(),( cc IPS c disp rrrrrr (43) Rrr , measures the Lennard-Jones d (44) ( ) LR ele )()( <sup>2</sup> − (45) (46) (47) ε rc: Because IPS potentials have a non-zero boundary energy, to avoid energy discontinuities when particles move across the boundary, boundary energies are subtracted from the IPS potentials and calculated separately. Therefore, the actual long-range part used in the calculation is: $$ \Delta \boldsymbol{\varepsilon}^{\rm LR}(\boldsymbol{r}, \boldsymbol{r}\_{\rm c}, \boldsymbol{R}\_{\rm c}) = \boldsymbol{\varepsilon}^{\rm LR}(\boldsymbol{r}, \boldsymbol{r}\_{\rm c}, \boldsymbol{R}\_{\rm c}) - \boldsymbol{\varepsilon}^{\rm B}(\boldsymbol{r}, \boldsymbol{R}\_{\rm c}) \tag{36} $$ And the boundary energy is defined as: $$\mathcal{E}^{\rm B}(r, R\_{\varepsilon}) = \begin{cases} \mathcal{E}(R\_{\varepsilon}) + \phi(R\_{\varepsilon}, R\_{\varepsilon}) & r \le R\_{\varepsilon} \\ 0 & r > R\_{\varepsilon} \end{cases} \tag{37}$$ The boundary energies are summed separately in a simulation: $$E^{\rm B} = \frac{1}{2} \sum\_{l}^{N} \sum\_{j}^{N+M} \varepsilon\_{\rm ij}^{\rm B}(r\_{\rm ij}, R\_{\rm c}) = \frac{2\pi R\_{\rm c}^{\rm B}}{3V} \sum\_{l,j}^{N} \varepsilon\_{\rm ij}^{\rm B}(R\_{\rm c}, R\_{\rm c}) \tag{38}$$ Here N is the number of particles and M is the number of PBC image particles. V is the volume of the simulation system. The total IPS energy is a sum over all particle pairs within Rc: $$\begin{split} E^{\rm IPS} &= \frac{1}{2} \sum\_{i}^{N} \sum\_{j}^{N\M} \boldsymbol{\varepsilon}^{\rm IPS}(\boldsymbol{r}\_{\circ}, \boldsymbol{R}\_{\circ}) = \frac{1}{2} \sum\_{i}^{N} \sum\_{j}^{N\M} \left( \boldsymbol{\varepsilon}^{\rm C}(\boldsymbol{r}\_{\circ}, \boldsymbol{r}\_{\circ}) + \boldsymbol{\varepsilon}^{\rm IR}(\boldsymbol{r}\_{\circ}, \boldsymbol{r}\_{\circ}, \boldsymbol{R}\_{\circ}) \right) \\ &= \frac{1}{2} \sum\_{i}^{N} \sum\_{j}^{N\M} \left( \boldsymbol{\varepsilon}^{\rm C}(\boldsymbol{r}\_{\circ}, \boldsymbol{r}\_{\circ}) + \Delta \boldsymbol{\varepsilon}^{\rm IR}(\boldsymbol{r}\_{\circ}, \boldsymbol{r}\_{\circ}, \boldsymbol{R}\_{\circ}) + \boldsymbol{\varepsilon}^{\rm B}(\boldsymbol{r}\_{\circ}, \boldsymbol{R}\_{\circ}) \right) = \boldsymbol{E}^{\rm C} + \Delta \boldsymbol{E}^{\rm IR} + \boldsymbol{E}^{\rm B} \end{split} \tag{39}$$ The cutoff sum, <sup>C</sup> E* , can be calculated in a pair wise way like the cutoff methods, and the long range sum, LR ΔE , can be calculated using the DFFT technique. For electrostatic potential, eq. (9), we use the following smoothing function: $$\mathcal{G}\_{\rm alc}(r, r\_{\rm c}) = -\frac{q\_1 q\_2}{8r\_{\rm c}} \left( 15 - 10 \left( \frac{r}{r\_{\rm c}} \right)^2 + 3 \left( \frac{r}{r\_{\rm c}} \right)^4 \right) \tag{40}$$ Alternatively, the polar IPS potential, eq. (15), can be used as the smooththing function: $$\varepsilon \mathcal{G}\_{\rm elc}(r, r\_{\rm c}) = \varepsilon\_{\rm elc}^{\rm lPS}(r, r\_{\rm c}) - \varepsilon\_{\rm elc}^{\rm lPS}(r\_{\rm c}, r\_{\rm c}) \tag{41}$$ The advantage to use eq. (41) is that the long-range part, eq. (36), becomes a measure of the electrostatic hetereogeneity of a simulation system in a length scale of rc. If a simulation system is homogeneous in the length scale of rc, ),,( cc LR ΔεRrr should approaches zero. For the Lernnard-Jones potential, eq. (16), the repulsion part is short-ranged and its IPS is calculated only within the cutoff distance, cr , using eq. (18) with cc = rR . For the dispersion part, the smoothing function is: $$\mathcal{A}\_{dip}(r, r\_c) = \frac{C}{r\_c^6} \left( 10 - 15 \left( \frac{r}{r\_c} \right)^2 + 6 \left( \frac{r}{r\_c} \right)^4 \right) \tag{42}$$ Because IPS potentials have a non-zero boundary energy, to avoid energy discontinuities when particles move across the boundary, boundary energies are subtracted from the IPS potentials and calculated separately. Therefore, the actual long-range part used in the ),(),,(),,( <sup>c</sup> <sup>+</sup> <sup>≤</sup> <sup>=</sup> 0 ),()( ),( Rr <sup>B</sup> ),( <sup>3</sup> Here N is the number of particles and M is the number of PBC image particles. V is the volume of the simulation system. The total IPS energy is a sum over all particle pairs within φ − LR ε <sup>=</sup> ∑ ∑ <sup>=</sup> ∑ <sup>+</sup> <sup>N</sup> cij cc ⎩ ⎨ ⎧ ε > B ij ε <sup>2</sup> ),( <sup>2</sup> LR E Rr Rrrrr N i ),(),,(),( <sup>2</sup> ε For electrostatic potential, eq. (9), we use the following smoothing function: cele ele = c Rr The boundary energies are summed separately in a simulation: N 1 MN E Rr j i c long range sum, LR ΔE , can be calculated using the DFFT technique. <sup>8</sup> ),( rr ϑ= system is homogeneous in the length scale of rc, ),,( cc ϑdisp ϑele ∑ ∑ ∑ ∑ c + + = = + C ε ε <sup>1</sup> ),( <sup>2</sup> ij IPS IPS N i N i MN j + ∑ ∑ MN j 1 part, the smoothing function is: 1 LR Δε B ε And the boundary energy is defined as: cc c cc c V R ( ) <sup>C</sup> LR <sup>B</sup> cc = Δ+ + +Δ+= The cutoff sum, <sup>C</sup> E , can be calculated in a pair wise way like the cutoff methods, and the ij ij ij ⎜ ⎜ ⎝ Alternatively, the polar IPS potential, eq. (15), can be used as the smooththing function: IPSp ε c c ),(),(),( cc − The advantage to use eq. (41) is that the long-range part, eq. (36), becomes a measure of the electrostatic hetereogeneity of a simulation system in a length scale of rc. If a simulation For the Lernnard-Jones potential, eq. (16), the repulsion part is short-ranged and its IPS is calculated only within the cutoff distance, cr , using eq. (18) with cc = rR . For the dispersion > ⎜ ⎜ ⎝ r <sup>C</sup> rr c ⎛ <sup>⎛</sup> −= <sup>c</sup> <sup>6</sup> 61510),( <sup>r</sup> <sup>c</sup> 31015 ⎛ <sup>⎛</sup> −−= 21 r qq MN j C B 2 IPSp elec ε LR Δε 2 c r r r r EEERrRrrrr ε c εε ),,(),( <sup>2</sup> π 3 RRR Rr B εRrRrrRrr (36) > B ij ε ( ) ij ij LR c ji c , c RR cc cc ⎟ ⎟ ⎠ Rrr should approaches zero. ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ c rrrrrr (41) ⎟ ⎟ ⎠ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ c r ⎞ 4 r r ⎞ 4 (37) (38) (39) (40) (42) calculation is: Rc: Again, the smoothing function can be the IPS potential, eq. (17), with a local region radius of rc: $$ \varepsilon \mathcal{G}\_{\rm disp}(r, r\_{\rm c}) = \varepsilon\_{\rm disp}^{\rm lPS}(r, r\_{\rm c}) - \varepsilon\_{\rm disp}^{\rm lPS}(r\_{\rm c}, r\_{\rm c}) \tag{43} $$ By doing so, the long-range part, ),,( cc LR Δε Rrr , measures the Lennard-Jones hetereogeneity of a simulation system in a length scale of rc. For the long-range sum, because the dispersion term is atom type dependent, to simplify the calculation, we transform the atom pair dependent dispersion parameters, Cij, to a transferable quantity, di, which is defined as the dispersion momentum to measure the contribution of each atom to long range dispersion interactions. $$d\_k = \frac{\sum\_{j}^{N} C\_{y}}{\sqrt{\sum\_{i}^{N} \sum\_{j}^{N} C\_{y}}} \tag{44}$$ In a simulation box, like atomic charges, atomic dispersion momentums are distributed over a set of predefined grid points, (k1, k2, k3), where k1 =1,2,…, K1, k2=1,2,.., K2, k3=1,2,…,K3. Assume the dispersion momentums of a simulation system of N particles are },...,,{ <sup>11</sup> <sup>N</sup> d = ddd . After spreading q and d on the grid points we have grid distributions Q and D. Like the charge spreading in eq. (24), we again use Cardinal b-spline to do the d spreading (Essmann, Perera et al. 1995). $$D(k\_1, k\_2, k\_3) = \sum\_{i=1}^{N} \sum\_{n\_i, n\_2, n\_3} d\_i M\_n(\mu\_{1i} - k\_1 - n\_1 K\_1) M\_n(\mu\_{2i} - k\_2 - n\_2 K\_2) M\_n(\mu\_{3i} - k\_3 - n\_3 K\_3) \tag{45}$$ The electrostatic long-range sum can be calculated in the following way: $$\begin{split} \Delta E\_{\rm elec}^{\rm LR} &= \frac{1}{2} \sum\_{i} q\_{i} \sum\_{j} q\_{j} \Delta \mathbf{c}\_{\rm elec}^{\rm LR} = \frac{1}{2} \sum\_{i} q\_{i} \mathbf{q} \otimes \Delta \mathbf{c}\_{\rm elec}^{\rm LR} = \frac{1}{2} \sum\_{i} q\_{i} F^{-1} \Big( F(\mathbf{q}) F(\Delta \mathbf{c}\_{\rm elec}^{\rm LR}) \Big) \\ &= \frac{1}{2} \sum\_{m\_{1}, m\_{2}, m\_{3}} F^{-1} (\mathbf{q}) F(\mathbf{q}) F(\Delta \mathbf{c}\_{\rm elec}^{\rm LR}) (m\_{1}, m\_{2}, m\_{3}) \end{split} \tag{46}$$ Here, ),,( <sup>321</sup> LR Δε kkk is the energy array $$\Delta \mathfrak{L}^{\text{LR}}(k\_1, k\_2, k\_3) = \sum\_{n\_1, n\_2, n\_3} \Delta \mathfrak{x}^{\text{LR}} \left( \left( \mathbf{r}(k\_1 + n\_1 K\_1, k\_2 + n\_2 K\_2, k\_3 + n\_3 K\_3) - \mathbf{r}(1, 1, 1) \right) r\_\varepsilon, R\_\varepsilon \right) \tag{47}$$ The energy type subscript is dropped here to indicate that eq. (45) applies to all energy types. The Fourier transform of q and d can be approximated by $$F(\mathbf{q})(m\_1, m\_2, m\_3) = b\_1(m\_1)b\_2(m\_2)b\_3(m\_3)F(\mathbf{Q})(m\_1, m\_2, m\_3) \tag{48}$$ $$F(\mathbf{d})(m\_1, m\_2, m\_3) = b\_1(m\_1)b\_2(m\_2)b\_3(m\_3)F(\mathbf{D})(m\_1, m\_2, m\_3) \tag{49}$$ Where )(mb ii is defined by eq. (28). We have $$F^{-l}(\mathbf{q})F(\mathbf{q})(m\_1, m\_2, m\_3) = B(m\_1, m\_2, m\_3)F(\mathbf{Q})(m\_1, m\_2, m\_3)F(\mathbf{Q})(-m\_1, -m\_2, -m\_3) \tag{50}$$ Molecular Simulation with Discrete Fast Fourier Transform 153 L zn L yn L xn )z-z(2 )y-y(2 )x-x(2 = Δ+ = Δ+ = + Δ (56) (57) max min max min max min Here n is the order of the b-spline and Δx , Δy , and Δz are the grid sizes in x, y, and z directions, respectively. The charges and dispersion momentums are spread over the grid points in the PBC box according to eqs. (24) and (45). The second modification is that the local region radius will be infinity, R<sup>c</sup> = ∞ . The 3D IPS interaction with an infinity local interaction with images created by the virtual PBC, the long-range sum must be limited to a and <sup>L</sup> <sup>x</sup> xmax ymax Lx =2xmax+nΔx Fig. 4. The virtual periodic boundary for a finite system as defined by eq. (54). Ly=2ymax+nΔy ),,( ij ij ij > c LR x y z rr <<< Here, xij, yij, and zij are the differences of coordinates in x, y, and z directions, respectively. Fig.4 illustrates the virtual PBC box defined for a protein in vacuum. With these modifications, we can get rid of the artificial image interactions due to the virtual periodic boundary condition while taking advantage of DFFT, and a finite system can be treated in IPS ε otherwise <sup>2</sup> , <sup>2</sup> , <sup>2</sup> rr ≤ y c Rr =∞→ <sup>L</sup> <sup>z</sup> <sup>L</sup> ε r . To avoid z y x region radius becomes purely the original pair potential: )(),( <sup>c</sup> 0 )( ),( ε ϑ ⎪ ⎩ the same way as a periodic boundary system described above. ⎪ ⎨ ⎧− =∞ c rr rrr half of the box size in each dimension: ε c Here, ),,( mmmB <sup>321</sup> is calculated with eq. (30). The long-range sum of electrostatic interaction, eq. (32), can be rewritten to: $$ \Delta E\_{\rm ek}^{\rm LR} = \frac{1}{2} \sum\_{m\_1, m\_2, m\_i} \mathbf{B} F^{-l}(\mathbf{Q}) F(\mathbf{Q}) F(\Delta \mathbf{c}\_{\rm ek}^{\rm LR}) = \frac{1}{2} \sum\_{k\_1, k\_2, k\_3}^{K\_1, K\_2, K\_3} \mathbf{Q} F^{-l} \left( \mathbf{B} F(\mathbf{Q}) F(\Delta \mathbf{c}\_{\rm ek}^{\rm LR}) \right) \tag{51} $$ Similarly, the dispersion long-range sum is: $$\Delta E\_{\rm disp}^{\rm LR} = \frac{1}{2} \sum\_{m\_1, m\_2, m\_3} \mathbf{B} F^{-l}(\mathbf{D}) F(\mathbf{D}) F(\Delta \mathbf{z}\_{\rm disp}^{\rm LR}) = \frac{1}{2} \sum\_{k\_1, k\_2, k\_3}^{k\_1, k\_2, k\_3} \mathbf{D} F^{-l}(\mathbf{B} F(\mathbf{D}) F(\Delta \mathbf{z}\_{\rm disp}^{\rm LR})) \tag{52}$$ Pressure tensors are important quantities in molecular simulation. The contributions from these long-range sums are calculated directly as a summation in the Fourier space: $$P\_{a\beta}^{\text{LR}}V = \frac{1}{2} \sum\_{m\_1, m\_2, m\_3} \mathbf{B} F^{-1}(\mathbf{Q}) F(\mathbf{Q}) F(\mathbf{p}\_{\text{el} \, a\beta}^{\text{LR}}) + \frac{1}{2} \sum\_{m\_1, m\_2, m\_3} \mathbf{B} F^{-1}(\mathbf{D}) F(\mathbf{D}) F(\mathbf{p}\_{\text{dip} \, a\beta}^{\text{LR}}) \tag{53}$$ Here, α, β stand for either x, y, or z, and α ε αβ β ∂ Δ∂ −= LR LR p . The forces acting on each particle can be derived from the long-range energies, eqs. (51) and (52). $$\begin{split} \int f\_a^{1\mathbb{R}} &= -\frac{\hat{\mathcal{O}}}{\hat{\mathcal{O}}\alpha} (\Delta E\_{da}^{1\mathbb{R}} + \Delta E\_{dap}^{1\mathbb{R}}) \\ &= -\sum\_{k\_1,k\_2,k\_3} \frac{\hat{\mathcal{O}}\mathcal{Q}}{\hat{\mathcal{O}}\alpha} F^{-1} \Big( \mathbf{B}F(\mathbf{Q})F(\Delta \mathbf{z}\_{da}^{1\mathbb{R}}) \Big) - \sum\_{k\_1,k\_2,k\_3} \frac{\hat{\mathcal{O}}D}{\hat{\mathcal{O}}\alpha} F^{-1} \Big( \mathbf{B}F(\mathbf{D})F(\Delta \mathbf{z}\_{dap}^{1\mathbb{R}}) \Big) \end{split} \tag{54}$$ ∂α <sup>∂</sup>Q and ∂α <sup>∂</sup>D are calculated based on eqs. (24) and (43) from the property of the b-spline functions $$\frac{\partial M\_{\boldsymbol{u}}(\boldsymbol{u})}{\partial \boldsymbol{u}} = M\_{\boldsymbol{u}-1}(\boldsymbol{u}) - M\_{\boldsymbol{u}-1}(\boldsymbol{u}-1) \tag{55}$$ In summary, the IPS/DFFT method uses eq. (34) to calculate the cutoff part directly for all atom pairs within the cutoff distance, cr , and uses eq. (51) and (52) to calculate the longrange part through DFFT. The total interaction is a sum of these two parts as shown in eq. (33). #### 4.3 IPS/DFFT for finite systems Finite systems like proteins in vacuum are often the objects of simulation studies. They are hetereogeneous in nature. With some modifications, the IPS/DFFT method can be extended to finite systems. The first modification is to create a virtual periodic boundary for DFFT. The size of the boundary box is twice the maximum dimensions of the actual system plus b-spline widths. =Δ ∑ =Δ ∑ ( ) <sup>Δ</sup> <sup>−</sup> <sup>−</sup> <sup>321</sup> =Δ ∑ =Δ ∑ ( ) <sup>Δ</sup> <sup>−</sup> <sup>−</sup> <sup>321</sup> Pressure tensors are important quantities in molecular simulation. The contributions from LR ele LR <sup>1</sup> )()()( <sup>2</sup> αβ The forces acting on each particle can be derived from the long-range energies, eqs. (51) and β ∂ Δ∂ −= LR p . ∑ ( ) ∑ ( ) <sup>Δ</sup> <sup>∂</sup> −Δ FFF <sup>D</sup> FFF <sup>Q</sup> − − )1()( )( <sup>1</sup> <sup>1</sup> −−= <sup>∂</sup> In summary, the IPS/DFFT method uses eq. (34) to calculate the cutoff part directly for all atom pairs within the cutoff distance, cr , and uses eq. (51) and (52) to calculate the longrange part through DFFT. The total interaction is a sum of these two parts as shown in eq. Finite systems like proteins in vacuum are often the objects of simulation studies. They are hetereogeneous in nature. With some modifications, the IPS/DFFT method can be extended The first modification is to create a virtual periodic boundary for DFFT. The size of the boundary box is twice the maximum dimensions of the actual system plus b-spline <sup>−</sup> <sup>−</sup> uMuM n n LR ele α ε LR ∂ QB ε DB ε <sup>∂</sup>D are calculated based on eqs. (24) and (43) from the property of the b-spline )()( )()( α 1 E DDB FFF ε DBD FFF ε LR disp disp )()( <sup>2</sup> LR ele ele )()( <sup>2</sup> ,, ,, ,, ,, 1 1 1 FFF pDDB LR ele LR disp LR disp LR disp (55) αβ (51) (52) (53) (54) 321 321 321 321 these long-range sums are calculated directly as a summation in the Fourier space: ∑ ∑ <sup>−</sup> <sup>−</sup> <sup>=</sup> <sup>+</sup> <sup>321</sup> <sup>321</sup> ,, mmm mmm <sup>321</sup> <sup>321</sup> ,, kkk kkk LR disp αβ <sup>1</sup> )()()( <sup>2</sup> The long-range sum of electrostatic interaction, eq. (32), can be rewritten to: <sup>1</sup> )()()( <sup>2</sup> 1 KKK mmm kkk E QQB FFF ε QBQ FFF ε <sup>1</sup> )()()( <sup>2</sup> 1 KKK mmm kkk Here, ),,( mmmB <sup>321</sup> is calculated with eq. (30). Similarly, the dispersion long-range sum is: ,, LR 1 ,, ,, VP FFF pQQB ∂ <sup>∂</sup> −= α ∂ u uM n ∂ <sup>∂</sup> −= α f EE ,, Δ+Δ LR ele 1 ( ) 1 Here, α, β stand for either x, y, or z, and LR α αβ (52). ∂α (33). to finite systems. widths. <sup>∂</sup>Q and functions ∂α 4.3 IPS/DFFT for finite systems LR 1 $$\begin{aligned} L\_{\pm} &= \mathbf{2}(\mathbf{x}\_{\text{max}} \cdot \mathbf{z}\_{\text{min}}) + n\Delta\mathbf{x} \\ L\_{\text{y}} &= \mathbf{2}(\mathbf{y}\_{\text{max}} \cdot \mathbf{y}\_{\text{min}}) + n\Delta\mathbf{y} \\ L\_{\pm} &= \mathbf{2}(\mathbf{z}\_{\text{max}} \cdot \mathbf{z}\_{\text{min}}) + n\Delta\mathbf{z} \end{aligned} \tag{56}$$ Here n is the order of the b-spline and Δx , Δy , and Δz are the grid sizes in x, y, and z directions, respectively. The charges and dispersion momentums are spread over the grid points in the PBC box according to eqs. (24) and (45). The second modification is that the local region radius will be infinity, R<sup>c</sup> = ∞ . The 3D IPS interaction with an infinity local region radius becomes purely the original pair potential: )(),( <sup>c</sup> IPS ε Rr =∞→ ε r . To avoid interaction with images created by the virtual PBC, the long-range sum must be limited to a half of the box size in each dimension: $$\varepsilon^{\rm LR}(r, r\_{\varepsilon}, \infty) = \begin{cases} -\mathcal{G}(r, r\_{\varepsilon}) & r \le r\_{\varepsilon} \\ \varepsilon(r) & r > r\_{\varepsilon} \text{ and } \left\| x\_{\parallel} \right\| < \frac{L\_{\varepsilon}}{2}, \left\| \nu\_{\parallel} \right\| < \frac{L\_{\varepsilon}}{2}, \left\| z\_{\parallel} \right\| < \frac{L\_{\varepsilon}}{2} \\ 0 & \text{otherwise} \end{cases} \tag{57}$$ Here, xij, yij, and zij are the differences of coordinates in x, y, and z directions, respectively. Fig.4 illustrates the virtual PBC box defined for a protein in vacuum. With these modifications, we can get rid of the artificial image interactions due to the virtual periodic boundary condition while taking advantage of DFFT, and a finite system can be treated in the same way as a periodic boundary system described above. Fig. 4. The virtual periodic boundary for a finite system as defined by eq. (54). Molecular Simulation with Discrete Fast Fourier Transform 155 Fig. 6. The surface tension of the water interface calculated from simulations with different 10 12 14 16 18 20 Cutoff distance, Å PME 3D IPS 3D IPS/DFFT 2D IPS Pressure tensors are sensitive to long-range structures. In the case that the box size is small as compared to the homogeneity scale, i.e., the thickness of the water layer in this case, it is recommended to set the Rc to twice of the longest box side or larger to equally consider all images in every direction. Fig. 6 shows the surface tension results from different methods. As can be seen, the results from PME and 3D IPS strongly depend on the cutoff distance, while 2D IPS and the IPS/DFFT method produce results showing little dependence on the cutoff distance. Obviously, as the cutoff distance increases, the results from PME and 3D IPS approach that from 2D IPS and the IPS/DFFT methods. We can see that to calculate surface tension, the IPS/DFFT method with a normal cutoff distance, rc=10 Å, is more efficient in Another property that is sensitive to the long-range interaction is the electrostatic potential profile across the layer, which is calculated by a double integration of the Poisson's > () () ( ) ∫ ∫ ′ −=− ′′′′′ z z <sup>z</sup> zz 0 0 <sup>c</sup> 0 ψ 48 50 52 Surface tension, dyn/cm 54 56 58 ψ IPS/DFFT, as well as the PME method, produce almost identical results. atom pairs than the cylinder cutoff in the 2D IPS method (Wu and Brooks 2005). ε π Fig. 7 shows the electrostatic potential profiles calculated from simulations using the PME, 3D IPS, 2D IPS, and the IPS/DFFT methods. Clearly, 3D IPS cannot produce correct electrostatic potential profiles with small cutoff distances. However, as the cutoff distance increases, the result from 3D IPS becomes closer to the PME result. The 2D IPS, the These results indicate that both the IPS/DFFT method and the 2D IPS method can accurately describe the long-range interactions of this interface system. The IPS/DFFT method is much faster than the 2D IPS because its short-range cutoff contains much fewer ddz <sup>4</sup> <sup>0</sup> ρ z)( are the charge density and electrostatic potential along the z (59) methods equation, Where )( <sup>c</sup> ρ the interface system simulation. z and direction, respectively. ψ ## 5. Applications Due to efficient calculation of convolutions through DFFT, PME and IPS/DFFT can efficiently and accurately calculate long-range electrostatic interactions for large systems. In addition, IPS/DFFT can be applied to all types of potentials, including the Lennard-Jones potential. Here we present several examples to demonstrate the application of these methods. ## 5.1 Water interfaces A water interface system is created by enlarging a cubic PBC box length along the z-axis, so that a gas phase is produced above and below the water liquid. There are 2180 TIP3P water molecules in the 40×40×80 Å3 orthorhombic periodic boundary box. Fig. 5 shows a snapshot of this system. This is a typical heterogeneous system involving phase equilibrium. In this system, 2 ns MD simulations at constant temperature (300K), constant volume (40×40×80 Å3) are performed with PME, 3D IPS, 2D IPS, and the IPS/DFFT method. Here, 2D IPS is a method designed specifically for two dimensional partial homogenous systems (Wu and Brooks 2005; Klauda, Wu et al. 2007). An important property of interface systems is the surface tension. Because the surface tension is very sensitive to long range interactions, an accurate calculation of surface tension is often time consuming. The surface tension is evaluated from, $$\mathcal{Y} = 0.5 \{ L\_z \left[ P\_{zz} - 0.5 (P\_{\infty} + P\_{\mathcal{Y}}) \right] \}\tag{58}$$ where Lz is the size of the simulation box normal to the interface, Pzz is the normal component of the internal pressure tensor and Pxx and Pyy are the tangential components. Since the MD simulations here contain two interfaces (see Fig. 5), the prefactor 0.5 is required to obtain γon a per interface basis. Fig. 5. A water interface system with 2108 TIP3P water molecules in a 40×40×80 Å3 orthorhombic boundary box Due to efficient calculation of convolutions through DFFT, PME and IPS/DFFT can efficiently and accurately calculate long-range electrostatic interactions for large systems. In addition, IPS/DFFT can be applied to all types of potentials, including the Lennard-Jones potential. Here we present several examples to demonstrate the application of these A water interface system is created by enlarging a cubic PBC box length along the z-axis, so that a gas phase is produced above and below the water liquid. There are 2180 TIP3P water molecules in the 40×40×80 Å3 orthorhombic periodic boundary box. Fig. 5 shows a snapshot of this system. This is a typical heterogeneous system involving phase equilibrium. In this system, 2 ns MD simulations at constant temperature (300K), constant volume (40×40×80 Å3) are performed with PME, 3D IPS, 2D IPS, and the IPS/DFFT method. Here, 2D IPS is a method designed specifically for two dimensional partial homogenous systems (Wu and An important property of interface systems is the surface tension. Because the surface tension is very sensitive to long range interactions, an accurate calculation of surface tension [ ( )] = zzz +− PPPL yyxx where Lz is the size of the simulation box normal to the interface, Pzz is the normal component of the internal pressure tensor and Pxx and Pyy are the tangential components. Since the MD simulations here contain two interfaces (see Fig. 5), the prefactor 0.5 is 40 Å Fig. 5. A water interface system with 2108 TIP3P water molecules in a 40×40×80 Å3 5.05.0 (58) 80 Å 5. Applications 5.1 Water interfaces required to obtain Brooks 2005; Klauda, Wu et al. 2007). γ orthorhombic boundary box is often time consuming. The surface tension is evaluated from, γ on a per interface basis. methods. Fig. 6. The surface tension of the water interface calculated from simulations with different methods Pressure tensors are sensitive to long-range structures. In the case that the box size is small as compared to the homogeneity scale, i.e., the thickness of the water layer in this case, it is recommended to set the Rc to twice of the longest box side or larger to equally consider all images in every direction. Fig. 6 shows the surface tension results from different methods. As can be seen, the results from PME and 3D IPS strongly depend on the cutoff distance, while 2D IPS and the IPS/DFFT method produce results showing little dependence on the cutoff distance. Obviously, as the cutoff distance increases, the results from PME and 3D IPS approach that from 2D IPS and the IPS/DFFT methods. We can see that to calculate surface tension, the IPS/DFFT method with a normal cutoff distance, rc=10 Å, is more efficient in the interface system simulation. Another property that is sensitive to the long-range interaction is the electrostatic potential profile across the layer, which is calculated by a double integration of the Poisson's equation, $$ \psi(z) - \psi(0) = -\frac{4\pi}{\mathcal{E}\_0} \int\_0^z \int\_0^{z'} \rho\_\varepsilon(\mathbf{z''}) d\mathbf{z''} d\mathbf{z'} \tag{59} $$ Where )( <sup>c</sup> ρ z and ψ z)( are the charge density and electrostatic potential along the z direction, respectively. Fig. 7 shows the electrostatic potential profiles calculated from simulations using the PME, 3D IPS, 2D IPS, and the IPS/DFFT methods. Clearly, 3D IPS cannot produce correct electrostatic potential profiles with small cutoff distances. However, as the cutoff distance increases, the result from 3D IPS becomes closer to the PME result. The 2D IPS, the IPS/DFFT, as well as the PME method, produce almost identical results. These results indicate that both the IPS/DFFT method and the 2D IPS method can accurately describe the long-range interactions of this interface system. The IPS/DFFT method is much faster than the 2D IPS because its short-range cutoff contains much fewer atom pairs than the cylinder cutoff in the 2D IPS method (Wu and Brooks 2005). Molecular Simulation with Discrete Fast Fourier Transform 157 Table 1 lists the electrostatic solvation energies from different simulations. As can be seen, 3D IPS underestimates the solvation energy by ca. 3 kcal/mol as compared to the PME result. This difference indicates that the homogenous approximation with a cutoff of 10 Å causes significant error for the solvation energy calculation. By contrast, the IPS/DFFT method produces results very close to the PME results, supporting the idea that with a cutoff larger than the homogeneity scale, 3D IPS can well approximate a heterogeneous system. Table 1 also lists the result of the IPS/DFFT method with different grid sizes. Clearly, the results are almost independent of grid sizes. The cpu times of the IPS/DFFT Systems without periodic boundary conditions are clearly heterogeneous by nature. By imposing a virtual periodic boundary and avoiding interactions with images, the IPS/DFFT We chose the x-ray structure of acetylcholine binding protein (ACHBP) (Brejc, van Dijk et al. 2001) (PDB code: i9b) to examine the energy calculation for non-periodic systems. We use its monomer and pentamer as examples of systems of small and large sizes. Fig. 8 shows the Pentamer top view 80Å δ f , of 0.019 method are comparable to those of the PME method. image of this protein in its monomer and pentamer form. Monomer side view Monomer top view 40Å Fig. 8. Acetylcholine binding protein (ACHBP) (PDB code: i9b) in its monomer and pentamer forms. The backbones are shown as ribbons. For clarity, atoms are not shown in Figs. 9 (a) and (b) show the force root-mean-square deviations and cpu times at different cutoff distances for the monomer and pentamer. Even though 3D IPS shows better results than the cutoff method at small cutoff distances, they both show significant force deviations with the cutoff distances up to 50 Å. By contrast, the IPS/DFFT result is better than these For the monomer, the IPS/DFFT costs more cpu time than the cutoff method at a given cutoff distance. However, at 10 Å, the IPS/DFFT method has a force deviation, method can be applied to such finite systems. 60Å two methods by an order of magnitude. 5.3 Proteins in vacuum the top views Fig. 7. The electrostatic potential profile cross the water interface system from simulations with different methods #### 5.2 A sodium aqueous system Solvation of an ion involves interactions far beyond the simulation box. The small number of ions in a simulation box makes the system highly heterogeneous. The solvation energy of an ionic solution can reflect the enthalpy effect as well as the entropy effect of charged ions onto the solvent, which provides a good case to examine the IPS/DFFT method. We performed MD simulations of a sodium aqueous solution in both charged and neutral states to examine the energy difference, defined as the electrostatic solvation energy: $$E\_{\rm valv} = E\_{\rm aq}(q=1) - E\_{\rm vac}(q=1) - \left\{ E\_{\rm aq}(q=0) - E\_{\rm vac}(q=0) \right\} \tag{60}$$ Here Eaq and Evac represent the average potential energies of the system with and without water in the same periodic boundary box, and q = 1 and q = 0 represent the charged and neutral states of the solvated ion, respectively. The system contains one sodium ion and 265 TIP3P water molecules. Simulations of 20 ns are performed at 300 K and in a 20×20×20 Å3 cubic periodic boundary box for both the charged and neutral states with PME, 3D IPS, and the IPS/DFFT method. A cutoff distance of 10 Å is used for all simulations. Table 1. Electrostatic solvation energies of a sodium ion calculated with different methods. K1, K2, and K3 are the grid numbers along the three sides of the 20×20×20 Å3 cubic box Table 1 lists the electrostatic solvation energies from different simulations. As can be seen, 3D IPS underestimates the solvation energy by ca. 3 kcal/mol as compared to the PME result. This difference indicates that the homogenous approximation with a cutoff of 10 Å causes significant error for the solvation energy calculation. By contrast, the IPS/DFFT method produces results very close to the PME results, supporting the idea that with a cutoff larger than the homogeneity scale, 3D IPS can well approximate a heterogeneous system. Table 1 also lists the result of the IPS/DFFT method with different grid sizes. Clearly, the results are almost independent of grid sizes. The cpu times of the IPS/DFFT method are comparable to those of the PME method. ## 5.3 Proteins in vacuum 156 Fourier Transform – Materials Analysis Fig. 7. The electrostatic potential profile cross the water interface system from simulations PME, rc =10 Å 3D IPS/DFFT, rc 2D IPS, rc 3D IPS, rc 3D IPS, rc 3D IPS, rc =10 Å =10 Å =10 Å =12 Å =20 Å z Solvation of an ion involves interactions far beyond the simulation box. The small number of ions in a simulation box makes the system highly heterogeneous. The solvation energy of an ionic solution can reflect the enthalpy effect as well as the entropy effect of charged ions We performed MD simulations of a sodium aqueous solution in both charged and neutral Here Eaq and Evac represent the average potential energies of the system with and without water in the same periodic boundary box, and q = 1 and q = 0 represent the charged and neutral states of the solvated ion, respectively. The system contains one sodium ion and 265 TIP3P water molecules. Simulations of 20 ns are performed at 300 K and in a 20×20×20 Å3 cubic periodic boundary box for both the charged and neutral states with PME, 3D IPS, and Methods K<sup>1</sup> K<sup>2</sup> K3 Esolv, kcal/mol Time, Hours Table 1. Electrostatic solvation energies of a sodium ion calculated with different methods. K1, K2, and K3 are the grid numbers along the three sides of the 20×20×20 Å3 cubic box PME 20 20 20 -67.17±0.19 28.86 3D IPS - - - -64.75±0.50 23.80 IPS/DFFT 6 6 6 -67.38±0.19 28.57 IPS/DFFT 8 8 8 -67.42±0.19 27.98 IPS/DFFT 12 12 12 -67.32±0.19 28.08 IPS/DFFT 20 20 20 -67.75±0.19 29.74 IPS/DFFT 6 12 20 -67.67±0.19 27.85 ( )0()0()1()1( ) solv = aq = − vac = − aq = − vac qEqEqEqEE = (60) onto the solvent, which provides a good case to examine the IPS/DFFT method. the IPS/DFFT method. A cutoff distance of 10 Å is used for all simulations. states to examine the energy difference, defined as the electrostatic solvation energy: with different methods 5.2 A sodium aqueous system 0 2 Electrostatic potential, kcal/mol 4 Systems without periodic boundary conditions are clearly heterogeneous by nature. By imposing a virtual periodic boundary and avoiding interactions with images, the IPS/DFFT method can be applied to such finite systems. We chose the x-ray structure of acetylcholine binding protein (ACHBP) (Brejc, van Dijk et al. 2001) (PDB code: i9b) to examine the energy calculation for non-periodic systems. We use its monomer and pentamer as examples of systems of small and large sizes. Fig. 8 shows the image of this protein in its monomer and pentamer form. Fig. 8. Acetylcholine binding protein (ACHBP) (PDB code: i9b) in its monomer and pentamer forms. The backbones are shown as ribbons. For clarity, atoms are not shown in the top views Figs. 9 (a) and (b) show the force root-mean-square deviations and cpu times at different cutoff distances for the monomer and pentamer. Even though 3D IPS shows better results than the cutoff method at small cutoff distances, they both show significant force deviations with the cutoff distances up to 50 Å. By contrast, the IPS/DFFT result is better than these two methods by an order of magnitude. For the monomer, the IPS/DFFT costs more cpu time than the cutoff method at a given cutoff distance. However, at 10 Å, the IPS/DFFT method has a force deviation, δf , of 0.019 Molecular Simulation with Discrete Fast Fourier Transform 159 Fig. 10 illustrates a lipid monolayer and periodic images with RC equal to twice the longest edge length. This picture shows that even highly heterogeneous systems such as monolayers can be accurately treated as homogenous when particles from many periodic replicates are included. With the IPS/DFFT method, interactions within a 10–12 Å cutoff distance rC are calculated directly, like the "real-space" part in PME. The remaining IPS interactions within RC are then evaluated by DFFT on a grid, and the direct interaction is subtracted to correct for overcounting. This is analogous to the splitting of real and k-space terms in PME that allows the Ewald equations to be solved in N ln N rather than N2 time, where N is the Fig. 10. The long (RC, equal to twice the longest edge length) and short (rC =10 Å) cutoffs of the 3D-IPS/DFFT method illustrated for a DMPC monolayer. Coloring is as follows: water, blue; hydrocarbon chains, grey; carbonyl oxygens, red; phosphate groups, green; quaternary amines, purple. The primary cell in the center is darker than the images, and the vapor phase between the chains is white. Top panel shows the substantial number of image atoms within RC (leading to homogeneity of the region) while bottom panel highlights the highly anisotropic distribution of atoms within rC (and why the longer cutoff is necessary) One important property for interfacial systems is the surface tension, which is very sensitive to long-range interactions, both electrostatic and Lennard-Jones interactions. Proper consideration of long-range interactions is essential to obtain accurate surface tension results. Table 2 compares the surface tensions for DMPC obtained with several methods. The most important and clear cut result is that γ for the monolayer for IPS/DFFT is number of particles. (Venable, Chen et al. 2009) kcal/mol·Å, while for the cutoff (force switch) method, δf = 1.92 kcal/mol·Å. Even with a cutoff distance of 50 Å, the cutoff method has δf = 0.022 kcal/mol·Å. Therefore, the IPS/DFFT method can reach a better accuracy with a 10 Å cutoff than the force switch method with a cutoff distance of 50 Å. In other words, to reach the same accuracy, the IPS/DFFT method needs less cpu time. This is obvious for large systems. For the pentamer, at a 10 Å cutoff, the IPS/DFFT method can reach δf = 0.022 kcal/mol·Å with 0.158 seconds of cpu time. The force switch method with a cutoff distance of 55 Å can only reach an accuracy of δf = 0.289 kcal/mol·Å, but uses 2.03 seconds of cpu time. Clearly, for small systems, accurate forces can be calculated by summing directly over all atom pairs, while for large systems, the IPS/DFFT method is a superior way to efficiently get accurate forces. Fig. 9. The root-mean-square deviations (rmsd) of the forces and cpu times from the cutoff method, 3D IPS, and the IPS/DFFT method with different cutoff distances. The rmsd is calculated against the forces calculated with no cutoff. (a) the ACHBP monomer, i9b; (b) the ACHBP pentamer, (i9b) ### 5.4 Lipid bilayer system Venable et al. applied IPS/DFFT and PME methods for simulations of lipid bilayers and monolayers (Venable, Chen et al. 2009). The method is demonstrated to be highly accurate for simple bulk fluids, liquid/liquid and liquid/vapor interfaces, and lipid bilayers and monolayers (Klauda, Wu et al. 2007). Values for rC (the cutoff distance for direct evaluation of pairs) and RC (the local region radius) equal to 10 Å and twice the longest edge of the periodic cell, respectively, provide excellent efficiency and accuracy. Dimyristoylphosphatidylcholine (DMPC) monolayers and bilayers are simulated with the CHARMM (Chemistry at HARvard Molecular Mechanics) C27r lipid parameter set using IPS/DFFT and PME. IPS/DFFT method can reach a better accuracy with a 10 Å cutoff than the force switch method with a cutoff distance of 50 Å. In other words, to reach the same accuracy, the IPS/DFFT method needs less cpu time. This is obvious for large systems. For the pentamer, of cpu time. The force switch method with a cutoff distance of 55 Å can only reach an systems, accurate forces can be calculated by summing directly over all atom pairs, while for large systems, the IPS/DFFT method is a superior way to efficiently get accurate forces. (a) (b) Fig. 9. The root-mean-square deviations (rmsd) of the forces and cpu times from the cutoff method, 3D IPS, and the IPS/DFFT method with different cutoff distances. The rmsd is calculated against the forces calculated with no cutoff. (a) the ACHBP monomer, i9b; (b) the 0 1 2 δf,kcal/mol.Å cpu time, seconds 3 4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Venable et al. applied IPS/DFFT and PME methods for simulations of lipid bilayers and monolayers (Venable, Chen et al. 2009). The method is demonstrated to be highly accurate for simple bulk fluids, liquid/liquid and liquid/vapor interfaces, and lipid bilayers and monolayers (Klauda, Wu et al. 2007). Values for rC (the cutoff distance for direct evaluation of pairs) and RC (the local region radius) equal to 10 Å and twice the longest edge of the periodic cell, respectively, provide excellent efficiency and accuracy. Dimyristoylphosphatidylcholine (DMPC) monolayers and bilayers are simulated with the CHARMM (Chemistry at HARvard Molecular Mechanics) C27r lipid parameter set using δ δ δ f = 0.289 kcal/mol·Å, but uses 2.03 seconds of cpu time. Clearly, for small f = 1.92 kcal/mol·Å. Even with a f = 0.022 kcal/mol·Å. Therefore, the f = 0.022 kcal/mol·Å with 0.158 seconds 0 10 20 30 40 50 60 Cutoff, Å Cutoff 3D IPS 3D IPS/DFFT Cutoff 3D IPS 3D IPS/DFFT kcal/mol·Å, while for the cutoff (force switch) method, cutoff distance of 50 Å, the cutoff method has at a 10 Å cutoff, the IPS/DFFT method can reach 0 10 20 30 40 50 60 Cutoff, Å Cutoff 3D IPS 3D IPS/DFFT Cutoff 3D IPS 3D IPS/DFFT accuracy of δ ACHBP pentamer, (i9b) 0 1 2 δf,kcal/mol.Å cpu time, seconds 3 4 0.0 0.1 0.2 0.3 0.4 0.5 5.4 Lipid bilayer system IPS/DFFT and PME. Fig. 10 illustrates a lipid monolayer and periodic images with RC equal to twice the longest edge length. This picture shows that even highly heterogeneous systems such as monolayers can be accurately treated as homogenous when particles from many periodic replicates are included. With the IPS/DFFT method, interactions within a 10–12 Å cutoff distance rC are calculated directly, like the "real-space" part in PME. The remaining IPS interactions within RC are then evaluated by DFFT on a grid, and the direct interaction is subtracted to correct for overcounting. This is analogous to the splitting of real and k-space terms in PME that allows the Ewald equations to be solved in N ln N rather than N2 time, where N is the number of particles. Fig. 10. The long (RC, equal to twice the longest edge length) and short (rC =10 Å) cutoffs of the 3D-IPS/DFFT method illustrated for a DMPC monolayer. Coloring is as follows: water, blue; hydrocarbon chains, grey; carbonyl oxygens, red; phosphate groups, green; quaternary amines, purple. The primary cell in the center is darker than the images, and the vapor phase between the chains is white. Top panel shows the substantial number of image atoms within RC (leading to homogeneity of the region) while bottom panel highlights the highly anisotropic distribution of atoms within rC (and why the longer cutoff is necessary) (Venable, Chen et al. 2009) One important property for interfacial systems is the surface tension, which is very sensitive to long-range interactions, both electrostatic and Lennard-Jones interactions. Proper consideration of long-range interactions is essential to obtain accurate surface tension results. Table 2 compares the surface tensions for DMPC obtained with several methods. The most important and clear cut result is that γ for the monolayer for IPS/DFFT is Molecular Simulation with Discrete Fast Fourier Transform 161 Long-range molecular interactions play a crucial role in molecular systems. Because longrange interactions require the most of the computing time in molecular simulations, improving their calculation efficiency is a major focus in molecular simulation method development. One approach to an efficient calculation of long-range interactions is treating long-range interactions as convolutions between related property distributions and potential functions so that the calculation can be efficiently handled with DFFT. Particle-mesh-Ewald (PME) is a widely used method that utilizes DFFT to perform Ewald summation for electrostatic interaction. IPS/DFFT is a recently developed method for the calculation of potentials of all kinds. For applications where additional long-range interactions, such as Lennard-Jones interactions, are crucial, IPS/DFFT is a better choice for calculating long- This work is supported by NIH/NHLBI intramural research program. We thank Eunice Wu Abe, Y. and S. Jitsukawa (2009). "Phase transformation of Cu precipitate in Fe-Cu alloy Allen, M. 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Information and Modeling 48 (10): 2021-2029. studied using self-guided molecular dynamics." Philosophical Magazine Letters 89 (9): conformational analysis of methylene-acetal-linked thymine dimers in DNA: dynamics study of the Michaelis complex of human protein Z-dependent protease inhibitor (ZPI) and factor Xa (FXa)." Journal of Molecular Modeling 15 (8): 897-911. Damjanovic, A., B. Garcia-Moreno E, et al. (2009). "Self-guided Langevin dynamics study of regulatory interactions in NtrC." Proteins: Structure, Function and Bioformatics 76 (4): conformation and water content in the interior of a protein." Journal of Chemical internal groups in proteins: A self-guided Langevin dynamics study." Biophysical 6. Conclusion range interactions. 8. References 7. Acknowledgment for editing the manuscript. 535-543. Press. 1007-1019. Journal 95 (9): 4091-4101. substantially, and statistically significantly higher than for PME. The difference is 10 dyn/cm for both cutoffs, and the trend holds for other surface areas of the DMPC isotherm (Figure 11), where the average difference is 8 dyn/cm. This parallels the results obtained for octane/vapor interfaces reported in the study, where very long cutoffs on the LJ interactions are required. In other words, the inclusion of long-range LJ interactions raises the surface tension. Consequently, the very good agreement of experimental monolayer surface tensions and those obtained from C27r simulated with PME arise from cancellation of errors. Fig. 11. Surface tension-surface area isotherm for a DMPC monolayer (Venable, Chen et al. 2009) Table 2. Surface tensions of bilayers (dyn/cm/leaflet) and monolayers (dyn/cm) of DMPC (T=310 K, area/per lipid = 59.6 Å2) simulated with the force field C27r, and of DPPC (T=323 K, area/per lipid = 64 Å2) with a modified version (Sonne, Jensen et al. 2007) of C27 (Venable, Chen et al. 2009). a(Lide 2000), b(Small 1986) ## 6. Conclusion 160 Fourier Transform – Materials Analysis substantially, and statistically significantly higher than for PME. The difference is 10 dyn/cm for both cutoffs, and the trend holds for other surface areas of the DMPC isotherm (Figure 11), where the average difference is 8 dyn/cm. This parallels the results obtained for octane/vapor interfaces reported in the study, where very long cutoffs on the LJ interactions are required. In other words, the inclusion of long-range LJ interactions raises the surface tension. Consequently, the very good agreement of experimental monolayer surface tensions and those obtained from C27r simulated with PME arise from cancellation of Fig. 11. Surface tension-surface area isotherm for a DMPC monolayer (Venable, Chen et al. Method rc (Å) DMPC (C27r) DPPC (modified C27) bilayer monolayer bilayer monolayer 3D-IPS/DFFT 15.5 54.8 −0.8 33.8 PME 12 13.0 44.7 −6.2 23.4 ave se 1.2 1.0 1.0 0.9 experiment 47.0a 41.5b Table 2. Surface tensions of bilayers (dyn/cm/leaflet) and monolayers (dyn/cm) of DMPC (T=310 K, area/per lipid = 59.6 Å2) simulated with the force field C27r, and of DPPC (T=323 K, area/per lipid = 64 Å2) with a modified version (Sonne, Jensen et al. 2007) of C27 3D-IPS/DFFT 20.0 51.1 PME 10 10.8 40.3 PME/IPS 16.2 53.0 PME/IPS 18.5 51.9 PME/p-LRC 12.3 PME/p-LRC 14.3 (Venable, Chen et al. 2009). a(Lide 2000), b(Small 1986) errors. 2009) Long-range molecular interactions play a crucial role in molecular systems. Because longrange interactions require the most of the computing time in molecular simulations, improving their calculation efficiency is a major focus in molecular simulation method development. One approach to an efficient calculation of long-range interactions is treating long-range interactions as convolutions between related property distributions and potential functions so that the calculation can be efficiently handled with DFFT. Particle-mesh-Ewald (PME) is a widely used method that utilizes DFFT to perform Ewald summation for electrostatic interaction. IPS/DFFT is a recently developed method for the calculation of potentials of all kinds. For applications where additional long-range interactions, such as Lennard-Jones interactions, are crucial, IPS/DFFT is a better choice for calculating longrange interactions. ## 7. Acknowledgment This work is supported by NIH/NHLBI intramural research program. We thank Eunice Wu for editing the manuscript. #### 8. References Molecular Simulation with Discrete Fast Fourier Transform 163 Takahashi, K., T. Narumi, et al. (2011). "Cot-off radius effect of the isotropic periodic sum method for polar molecules in a bulk water system." Molecular Simulation Online. Takahashi, K., K. Yasuoka, et al. (2007). "Cutoff radius effect of isotropic periodic sum Takahashi, K. Z., T. Narumi, et al. (2011). "Cutoff radius effect of the isotropic periodic sum Tsuru, T., A. B. E. Yosuke, et al. (2010). "Atomistic simulations of phase transformation of Varady, J., X. Wu, et al. (2002). "Competitive and reversible binding of a guest molecule to Wang, J., Zhu, W., Li, G., & Hansmann, U. H. E. (2011). Velocity-scaling optimized replica Wen, E. Z., M. J. Hsieh, et al. (2004). "Enhanced ab initio protein folding simulations in Wen, E. Z. and R. Luo (2004). "Interplay of secondary structures and side-chain contacts in the denatured state of BBA1." Journal of Chemical Physics 121 (5): 2412-2421. Wu, X.-W. and S.-S. Sung (1999). "Simulation of peptide folding with explicit water-a mean solvation mothod " PROTEINS: Structure, Function, and Genetics 34 (3): 295-302. Wu, X. and B. R. Brooks (2003). "Self-guided Langevin dynamics simulation method." Wu, X. and B. R. Brooks (2004). "Beta-hairpin folding mechanism of a nine-residue peptide Wu, X. and B. R. Brooks (2005). "Isotropic periodic sum: a method for the calculation of long- Wu, X. and B. R. Brooks (2008). "Using the isotropic periodic sum method to calculate longrange interactions of heterogeneous systems." J Chem Phys 129 (15): 154115. Wu, X. and B. R. Brooks (2009). "Isotropic periodic sum of electrostatic interactions for polar Wu, X. and B. R. Brooks (2011a). "Force-Momentum Based Self-Guided Langevin Dynamics: Wu, X. and B. R. Brooks (2011b). "Toward Canonical Ensemble Distribution from Self- Wu, X. and S. Wang (1998). "Self-guided molecular dynamics simulation for efficient conformational search." Journal of Physical Chemistry B 102 (37): 7238-7250. Wu, X. and S. Wang (1999). "Enhancing systematic motion in molecular dynamics Guided Langevin Dynamics Simulation." J Chem Phys 134: 134108. simulation." Journal of Chemical Physics 110 (19): 9401-9410. An Rapid Sampling Method that Approaches the Canonical Ensemble." J Chem Phys revealed from molecular dynamics simulations in explicit water." Biophys J 86 (4): the Society of Materials Science, Japan 59 (8): 583-588. monolayers." J Phys Chem B 113 (17): 5855-5862. Molecular Graphics and Modelling 22 (5): 415-424. Journal of Chemical Physics, 135(8), 084115. Chemical Physics Letters 381 (3-4): 512-518. range interactions." J Chem Phys 122 (4): 44107. systems." J Chem Phys 131 (2): 024107. 114511. 1946-1958. 135 (20): 204101. method for transport coefficients of Lennard-Jones liquid." J Chem Phys 127 (11): and Wolf method in liquid-vapor interfaces of water." J Chem Phys 134 (17): 174112. copper precipitation and its effect on obstacle strength in α-iron." Zairyo/Journal of its host in aqueous solution through molecular dynamics simulation: Benzyl alcohol/β-cyclodextrin system." Journal of Physical Chemistry B 106 (18): 4863-4872. Venable, R. M., L. E. Chen, et al. (2009). "Comparison of the extended isotropic periodic sum and particle mesh Ewald methods for simulations of lipid bilayers and exchange molecular dynamics of proteins in a hybrid explicit/implicit solvent. Poisson-Boltzmann molecular dynamics with self-guiding forces." Journal of Darden, T. (1993). "Particle mesh Ewald: An N?log (N) method for Ewald sums in large Essmann, U., L. Perera, et al. (1995). "A smooth particle mesh Ewald method." The Journal of Ewald, P. P. (1921). "Die Berechnung optischer und elektrostatischer Gitterpotentiale." Gardiner, V., J. G. Hoffman, et al. (1956). "Digital computer studies of cell multiplication by Hoffman, J. G., N. Metropolis, et al. (1955). "Study of tumor cell populations by Monte Carlo Jones, G., P. Willet, et al. (1997). "Development and Validation of a Genetic Algorithm for Klauda, J. B., X. Wu, et al. (2007). "Long-range Lennard-Jones and electrostatic interactions in Le Grand, S. M. and K. M. Merz, Jr. (1994). "The Genetic Algorithm and Protein Tertiary Lee, C. I. and N. Y. Chang (2010). "Characterizing the denatured state of human prion 121- Lee, M. S. and M. A. Olson (2010). "Protein Folding Simulations Combining Self-Guided Lim, J. B., Rogaski, B., & Klauda, J. B. (2011). Update of the Cholesterol Force Field Parameters in CHARMM. The Journal of Physical Chemistry B, 116(1), 203-210. Metropolis, N. and S. Ulam (1949). "The Monte Carlo method." J Am Stat Assoc 44 (247): 335- Ogata, H., Y. Akiyama, et al. (1995). "A genetic algorithm based molecular modeling technique for RNA stem-loop structures." Nucleic Acids Res 23 (3): 419-426. Pendse, P. Y., B. R. Brooks, et al. (2010). "Probing the periplasmic-open state of lactose Sheng, Y., W. Wang, et al. (2010). "Adsorption of an ionic complementary peptide on the hydrophobic graphite surface." Journal of Physical Chemistry C 114 (1): 454-459. Sheng, Y., W. Wang, et al. (2010). "Interaction of an ionic complementary peptide with a Sonne, J., M. Ø. Jensen, et al. (2007). "Reparameterization of All-Atom Steinbach, P. J. and B. R. Brooks (1994). "New Spherical-Cutoff Methods for Long-Range Takahashi, K., T. Narumi, et al. (2010). "Cutoff radius effect of the isotropic periodic sum method in homogeneous system. II. Water." J Chem Phys 133 (1): 014109. hydrophobic graphite surface." Protein Science 19 (9): 1639-1648. Bilayers at Zero Tension." Biophysical Journal 92 (12): 4157-4167. Small, D. M. (1986). Handbook of Lipid Research. New York, Plenum Press. interfaces: application of the isotropic periodic sum method." J Phys Chem B 111 Langevin Dynamics and Temperature-Based Replica Exchange." Journal of Chemical permease in response to sugar binding and proton translocation." Journal of Dipalmitoylphosphatidylcholine Lipid Parameters Enables Simulation of Fluid Forces in Macromolecular Simulation." Journal of Computational Chemistry 15 (No. 7): Monte Carlo methods." J Natl Cancer Inst 17 (2): 175-188. Structure Prediction." DON'T KNOW/ NOT SURE: 109-124. systems." J. Chem. Phys. 98 (12): 10089. Chemical Physics 103 (19): 8577-8593. methods." Science 122 (3167): 465-466. Flexible Docking." J.Mol.Biol.: 727-748. 230." Biophysical Chemistry 151 (1-2): 86-90. Theory and Computation 6 (8): 2477-2487. Lide, D. R. (2000). CRC handbook. Boca Raton, FL, CRC Press. Molecular Biology 404 (3): 506-521. Ann.Phys. 64: 253-287. (17): 4393-4400. 341. 667-683. 8 Bingzheng Li Aqueous solubility at 25ºC(g/L) P. R. China Taiyuan University of Science & Technology Charaterization of Pore Structure and Surface There is a rapid development in industries since industrial revolution began in the latter half of the 18th century, which not only significantly promotes development of economy all over the world and prosperity in societies, but also extremely improves our lives. However, a large amount of wastewaters including some toxic and hazardous materials such as some metals ions and some non-biodegraded organic compounds are usually produced in many factories during production process. These pollutants such as simple/complex aromatic compounds in wastewaters discharged from factories such as textiles, rubber, paper, plastic, explosive, pharmaceuticals, petroleum refining and cosmetics factories into ditches/rivers are stable to light and oxidation as resistant to aerobic digestion, causing damage to the aquatic life and food web (Kagalkar, et al., 2010, Saratale, et al., 2011, Tabrez&Ahmad, 2009). The basic properties of simple aromatic compounds are shown in Table 1. The compounds can also cause allergic dermatitis and skin irritation. Furthermore, Most of them have been reported to be carcinogenic and mutagenic for aquatic organisms (Lorenc-Grabowska&Gryglewicz, 2007), so discharging of the pollutants is becoming the most challenging threat to environment safety and even human beings' lives. The introduction of toxic and/or non-biodegradable compounds in the natural environment has been a world wide serious problem that has been paid more attention to by many environmentalist and 1. Introduction <sup>N</sup> <sup>H</sup> <sup>2</sup> O H research institutions from different countries. Simple aromatic compounds Boiling point (ºC) <sup>N</sup> 115.3 5.17 ∞ Table 1. Basic properties of some simple aromatic compounds (Gokel&Dean, 2004) pKa at 25ºC 80.4 – 1.79 184.4 4.60 34 182 9.89 93 Chemistry of Activated Carbons – A Review ## Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review Bingzheng Li Taiyuan University of Science & Technology P. R. China ## 1. Introduction 164 Fourier Transform – Materials Analysis Wu, X. and S. Wang (2000). "Folding studies of a linear pentamer peptide adopting a reverse Wu, X. and S. Wang (2001). "Helix folding of an alanine-based peptide in explicit water." Wu, X., S. Wang, et al. (2002). "Direct observation of the folding and unfolding of a beta- York, D. M., A. Wlodawer, et al. (1994). "Atomic-level accuracy in simulations of large protein crystals." Proc.Natl.Acad.Sci.U.S.A 91 (18): 8715-8718. Journal of Physical Chemistry B 104 (33): 8023-8034. Journal of Physical Chemistry B 105 (11): 2227-2235. 5282-5283. turn conformation in aqueous solution through molecular dynamics simulation." hairpin in explicit water through computer simulation." J Am Chem Soc 124 (19): There is a rapid development in industries since industrial revolution began in the latter half of the 18th century, which not only significantly promotes development of economy all over the world and prosperity in societies, but also extremely improves our lives. However, a large amount of wastewaters including some toxic and hazardous materials such as some metals ions and some non-biodegraded organic compounds are usually produced in many factories during production process. These pollutants such as simple/complex aromatic compounds in wastewaters discharged from factories such as textiles, rubber, paper, plastic, explosive, pharmaceuticals, petroleum refining and cosmetics factories into ditches/rivers are stable to light and oxidation as resistant to aerobic digestion, causing damage to the aquatic life and food web (Kagalkar, et al., 2010, Saratale, et al., 2011, Tabrez&Ahmad, 2009). The basic properties of simple aromatic compounds are shown in Table 1. The compounds can also cause allergic dermatitis and skin irritation. Furthermore, Most of them have been reported to be carcinogenic and mutagenic for aquatic organisms (Lorenc-Grabowska&Gryglewicz, 2007), so discharging of the pollutants is becoming the most challenging threat to environment safety and even human beings' lives. The introduction of toxic and/or non-biodegradable compounds in the natural environment has been a world wide serious problem that has been paid more attention to by many environmentalist and research institutions from different countries. Table 1. Basic properties of some simple aromatic compounds (Gokel&Dean, 2004) Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 167 extensive use in industries. To greatly decease the cost of AC, the various ACs prepared from non-conventional sources such as coir pith (Agrawal, et al., 2011), sawdust and rice husk (Chen, Y., et al., 2011), pinewood (Tseng, et al., 2003) applied in removal of some The reason why adsorption capacity of adsorbents is high or low is mainly due to pore development and surface groups of AC, which is closely associated with its preparation methods. Here, preparation process of AC should be discussed because it has an important effect on pore structures and surface groups of AC. Preparation of AC includes a primary process and a secondary one which are denoted as carbonization and activation, respectively. Carbonization is followed by activation in sequence. In carbonization process, a large amount of gases, mainly small low molecular weight hydrocarbons-containing molecules or fragments possibly with oxygen (O), nitrogen (N) and sulfur (S), are released from materials surface and adhesion agents from 380 °C to 550 °C in an inert atmosphere due to decomposition of some groups which are readily destroyed. The carbonized materials have primary development of pore structure. In contrary to inert atmosphere used in carbonization process, some activating agents are frequently used in activation process (the secondary process). Gas activating agents consist of vapour, carbon dioxide and air used to further/promote development of pore properties at 600 °C to 980 °C. It is noting that chemical activating agents including phosphorus acid (H3PO4), phosphorus pentoxide (P2O5) (Izquierdo, et al., 2011), zinc chloride (ZnCl2) (Cronje, et al., 2011), calcium chloride (CaCl2) (Hu&Srinivasan, 2001), sodium hydroxide (NaOH) (Vargas, et al., 2011), potassium hydroxide (KOH) (Król, et al., 2011) and potassium sulfide (K2S) (Huang, Z., 2006) seem to widely applied in preparation of wood-derived ACs which has only the carbonization process. The resulting AC after being cooled down to room temperature must be washed several times in order to eliminate residues of activating agents. After carbonization and activation, ACs have huge surface area, deep developed pore structures and plenty of different types of surface groups are produced. In recent years, combination of a chemical method and a physical one has been also introduced into the activation process and can compounds have also been investigated and better results have been attained. yield high surface area and developed pore structures (Huang, Z., 2006). It is well known that principal component of AC is carbon element and surface of AC itself is non-polar, and hence it is concluded that as a whole, AC has hydrophobic surface. Various activation conditions and more or less amount of oxygen AC contacts when it is stored/exposed in air will to some extent implant some/many oxygenated groups on AC surface. The surface groups will alter surface state of AC and subsequently has an influence on its surface chemical properties. This results in the difference in surface chemistry of ACs. When a significant effect of surface chemistry on adsorption and catalysis process is investigated, AC is frequently modified through different treatment methods in order to strengthen or attenuate the surface chemical properties, e.g. surface functional groups. Usually, the properties of the functional groups such as oxygen-containing groups and nitrogen-containing groups of AC surface can be modified by physical, chemical, and electrochemical treatment methods. These methods consist of gas phase oxidation via O2 or N2O at different temperatures, various contact times and partial/total pressures, liquid phase oxidation treatments through HNO3 or H2O2-containing aqueous solutions at various temperatures, concentrations, and reaction times and heat treatment at various high temperatures and in different gaseous environments (N2 and H2 and some high pure inert gases such as Ar and He) in order to selectively eliminate some of the functional groups. To decrease or even eliminate the concentration of the compounds in wastewaters before discharging into environment, the various treatment methods are used for the removal of the pollutants mentioned above from wastewaters, which usually consist of cloud point extraction (Silva, et al., 2009), coagulation using alum (Ghafari, et al., 2009), lime (Foley, et al., 2010), ferric sulphate (Matilainen, et al., 2010) and ferric chloride (Liang, Z., et al., 2009), chemical oxidation (Lee&Von Gunten, 2010) using Fenton reagents (ferrous ion and H2O2) (Padoley, et al., 2011), ozone (O3) (Bundschuh, et al., 2011), chlorine and air (Wang, X., et al., 2011), membrane separation process including nanofiltration (Zahrim, et al., 2011), adsorption (Dos Santos, et al., 2011, Lu, et al., 2011) and so on. Among these, adsorption appears to be one of the best potential and effective methods for removal of pollutants from wastewaters. The adsorbents can be classified into two types of adsorption materials (natural materials and synthetic ones). Natural adsorbents, usually non-conventional low cost adsorbents used for removal of compounds, include peat/sphagnum moss peat (Naumova, et al., 2011), red mud (oruh, et al., 2011), coir pith (Parab, et al., 2010), leaves (Chakravarty, et al., 2010), activated sludge (Bassin, et al., 2011, Zhang, C., et al., 2011a), waste organic peel (Feng, et al., 2011), tree fern (Liu, X. L., et al., 2011b), lignite (Al-Asheh, et al., 2003), sawdust (Mane&Babu, 2011), banana pith (Namasivayam, et al., 1998), peanut hull (Tanyildizi, 2011), modified chitosan beads (Liu, B., et al., 2011a), natural biopolymers (Vinod&Sashidhar, 2011), biosorption materials (Montazer-Rahmati, et al., 2011), and minerals such as activated ash/clay (Andersson, et al., 2011) and natural sepiolite (Sevim, et al., 2011). However, the adsorption capacity of the above adsorbents is relatively low. Usually, there are many types of synthetic or artificial adsorbents (convential adsorbents) such as activated carbon (AC) (Li, et al., 2010b), resin (Vinodh, et al., 2011), zeolite (Seifi, et al., 2011), and so on used in adsorption operation, but AC is the most widely used adsorbents for removal of inorganic and organic compounds from wastewaters or waste gases because they have excellent capacities for adsorption of compounds derived from their huge surface area, developed pore texture, as well as easy availability (Khan, et al., 1997a, Khan, et al., 1997b, Okolo, et al., 2000, Viraraghavan&De Maria Alfaro, 1998). According to its various appearance, AC is divided into cylindrical activated carbon (CAC) (Wang, J. C., et al., 2008), granular activated carbon (GAC) (Huang, L., et al., 2011, Saoudi&Hamdaoui, 2011), powder activated carbon (PAC) (Li, et al., 2009a, Li, et al., 2009b, Li, et al., 2010b) and acivated carbon fiber (ACF) (Li, et al., 2010a). In terms of extent of its pore development, AC is classified as microporous activated carbon/activated carbon fiber (Li, et al., 2009a, Li, et al., 2010a) and mesoporous activated carbon (Zhang, Y., et al., 2011b). In accordance with range of its use, AC is divided into gas activated carbon and water activated carbon, which are used in treatment of feed/waste gases and waters/wastewaters, respectively. Usually, AC is prepared from different raw materials such as coal, wood, and so on. It is well known that a type of AC is coal-derived AC. Another one is prepared from woods such as pine wood (Hadi, et al., 2011) and fir wood (Eletskii, et al., 2011), etc. Certainly, AC can be also prepared from various carbon-contained materials (principally plant wastes (Hameed&Daud, 2008, Kula, et al., 2008) in past decades. It should be noted that high initial cost and costly maintenance of AC including high regeneration temperature and tremendous carbon consumption have greatly restricted to its To decrease or even eliminate the concentration of the compounds in wastewaters before discharging into environment, the various treatment methods are used for the removal of the pollutants mentioned above from wastewaters, which usually consist of cloud point extraction (Silva, et al., 2009), coagulation using alum (Ghafari, et al., 2009), lime (Foley, et al., 2010), ferric sulphate (Matilainen, et al., 2010) and ferric chloride (Liang, Z., et al., 2009), chemical oxidation (Lee&Von Gunten, 2010) using Fenton reagents (ferrous ion and H2O2) (Padoley, et al., 2011), ozone (O3) (Bundschuh, et al., 2011), chlorine and air (Wang, X., et al., 2011), membrane separation process including nanofiltration (Zahrim, et al., 2011), adsorption (Dos Santos, et al., 2011, Lu, et al., 2011) and so on. Among these, adsorption appears to be one of the best potential and effective methods for removal of pollutants from The adsorbents can be classified into two types of adsorption materials (natural materials and synthetic ones). Natural adsorbents, usually non-conventional low cost adsorbents used for removal of compounds, include peat/sphagnum moss peat (Naumova, et al., 2011), red mud (oruh, et al., 2011), coir pith (Parab, et al., 2010), leaves (Chakravarty, et al., 2010), activated sludge (Bassin, et al., 2011, Zhang, C., et al., 2011a), waste organic peel (Feng, et al., 2011), tree fern (Liu, X. L., et al., 2011b), lignite (Al-Asheh, et al., 2003), sawdust (Mane&Babu, 2011), banana pith (Namasivayam, et al., 1998), peanut hull (Tanyildizi, 2011), modified chitosan beads (Liu, B., et al., 2011a), natural biopolymers (Vinod&Sashidhar, 2011), biosorption materials (Montazer-Rahmati, et al., 2011), and minerals such as activated ash/clay (Andersson, et al., 2011) and natural sepiolite (Sevim, et al., 2011). However, the Usually, there are many types of synthetic or artificial adsorbents (convential adsorbents) such as activated carbon (AC) (Li, et al., 2010b), resin (Vinodh, et al., 2011), zeolite (Seifi, et al., 2011), and so on used in adsorption operation, but AC is the most widely used adsorbents for removal of inorganic and organic compounds from wastewaters or waste gases because they have excellent capacities for adsorption of compounds derived from their huge surface area, developed pore texture, as well as easy availability (Khan, et al., According to its various appearance, AC is divided into cylindrical activated carbon (CAC) (Wang, J. C., et al., 2008), granular activated carbon (GAC) (Huang, L., et al., 2011, Saoudi&Hamdaoui, 2011), powder activated carbon (PAC) (Li, et al., 2009a, Li, et al., 2009b, Li, et al., 2010b) and acivated carbon fiber (ACF) (Li, et al., 2010a). In terms of extent of its pore development, AC is classified as microporous activated carbon/activated carbon fiber (Li, et al., 2009a, Li, et al., 2010a) and mesoporous activated carbon (Zhang, Y., et al., 2011b). In accordance with range of its use, AC is divided into gas activated carbon and water activated carbon, which are used in treatment of feed/waste gases and waters/wastewaters, Usually, AC is prepared from different raw materials such as coal, wood, and so on. It is well known that a type of AC is coal-derived AC. Another one is prepared from woods such as pine wood (Hadi, et al., 2011) and fir wood (Eletskii, et al., 2011), etc. Certainly, AC can be also prepared from various carbon-contained materials (principally plant wastes It should be noted that high initial cost and costly maintenance of AC including high regeneration temperature and tremendous carbon consumption have greatly restricted to its 1997a, Khan, et al., 1997b, Okolo, et al., 2000, Viraraghavan&De Maria Alfaro, 1998). adsorption capacity of the above adsorbents is relatively low. (Hameed&Daud, 2008, Kula, et al., 2008) in past decades. wastewaters. respectively. extensive use in industries. To greatly decease the cost of AC, the various ACs prepared from non-conventional sources such as coir pith (Agrawal, et al., 2011), sawdust and rice husk (Chen, Y., et al., 2011), pinewood (Tseng, et al., 2003) applied in removal of some compounds have also been investigated and better results have been attained. The reason why adsorption capacity of adsorbents is high or low is mainly due to pore development and surface groups of AC, which is closely associated with its preparation methods. Here, preparation process of AC should be discussed because it has an important effect on pore structures and surface groups of AC. Preparation of AC includes a primary process and a secondary one which are denoted as carbonization and activation, respectively. Carbonization is followed by activation in sequence. In carbonization process, a large amount of gases, mainly small low molecular weight hydrocarbons-containing molecules or fragments possibly with oxygen (O), nitrogen (N) and sulfur (S), are released from materials surface and adhesion agents from 380 °C to 550 °C in an inert atmosphere due to decomposition of some groups which are readily destroyed. The carbonized materials have primary development of pore structure. In contrary to inert atmosphere used in carbonization process, some activating agents are frequently used in activation process (the secondary process). Gas activating agents consist of vapour, carbon dioxide and air used to further/promote development of pore properties at 600 °C to 980 °C. It is noting that chemical activating agents including phosphorus acid (H3PO4), phosphorus pentoxide (P2O5) (Izquierdo, et al., 2011), zinc chloride (ZnCl2) (Cronje, et al., 2011), calcium chloride (CaCl2) (Hu&Srinivasan, 2001), sodium hydroxide (NaOH) (Vargas, et al., 2011), potassium hydroxide (KOH) (Król, et al., 2011) and potassium sulfide (K2S) (Huang, Z., 2006) seem to widely applied in preparation of wood-derived ACs which has only the carbonization process. The resulting AC after being cooled down to room temperature must be washed several times in order to eliminate residues of activating agents. After carbonization and activation, ACs have huge surface area, deep developed pore structures and plenty of different types of surface groups are produced. In recent years, combination of a chemical method and a physical one has been also introduced into the activation process and can yield high surface area and developed pore structures (Huang, Z., 2006). It is well known that principal component of AC is carbon element and surface of AC itself is non-polar, and hence it is concluded that as a whole, AC has hydrophobic surface. Various activation conditions and more or less amount of oxygen AC contacts when it is stored/exposed in air will to some extent implant some/many oxygenated groups on AC surface. The surface groups will alter surface state of AC and subsequently has an influence on its surface chemical properties. This results in the difference in surface chemistry of ACs. When a significant effect of surface chemistry on adsorption and catalysis process is investigated, AC is frequently modified through different treatment methods in order to strengthen or attenuate the surface chemical properties, e.g. surface functional groups. Usually, the properties of the functional groups such as oxygen-containing groups and nitrogen-containing groups of AC surface can be modified by physical, chemical, and electrochemical treatment methods. These methods consist of gas phase oxidation via O2 or N2O at different temperatures, various contact times and partial/total pressures, liquid phase oxidation treatments through HNO3 or H2O2-containing aqueous solutions at various temperatures, concentrations, and reaction times and heat treatment at various high temperatures and in different gaseous environments (N2 and H2 and some high pure inert gases such as Ar and He) in order to selectively eliminate some of the functional groups. Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 169 Here, physical structures of AC analyzed only by liquid Nitrogen adsorption will be introduced in this paper because Nitrogen adsorption are frequently used in investigating the texture of AC. Brunauer-Emmitt-Teller surface area (SBET), Langmuir surface area (Slangmuir), pore volume and pore distribution of the adsorbents were determined through liquid Nitrogen adsorption at 77 K on a Micromertics ASAP 2020 surface area analyzer (USA). Micropore and mesopore volumes and surface areas of the adsorbents were calculated using Density Functional Theory (DFT). The pore size distribution of adsorbents > Smes (m2/g) 1 10 100 Pore width (nm) Table 2 suggests that the difference in micro-pore properties, most relevant to adsorption of organic compounds, are small, with similar micro-pore volumes but different micro-pore surface areas, indicating the major difference between them is pore distribution within the micro-pore range as indicated by the mean pore diameter (Table 1). Among the four adsorbents, ACD contains more smaller micropores than the other adsorbents while AC contains less smaller pores in comparison to ACO1 and ACO2. This trend seems correlating well with the ash contents of the adsorbents (Table 4) and suggests that the acid leaching processes remove fine mineral matters from the adsorbents and result in formation of micropores, as depicted as Fig. 1. The meso-pore properties (Vmeso and Smeso) of AC, ACO1 and ACO2 are very similar, but that of ACD are significantly smaller than others. These r (nm) SBET (m2/g) AC ACD ACO1 ACO2 Slangmuir (m2/g) Vmes (cm3/g) AC 0.305 689 0.069 51 1.103 977 1231±8 ACO1 0.304 777 0.071 50 0.996 903 1135±8 ACO2 0.302 779 0.063 45 0.968 890 1121±8 ACD 0.306 805 0.028 21 0.847 847 1066±5 was determined from adsorption isotherm data using DFT. Smic (m2/g) Table 2. Textural characterization of the adsorbents (Li, et al., 2009a) Adsorbent Vmic Note: r is median pore radius (cm3/g) 0.000 Fig. 1. Pore size distribution of adsorbents (Li, et al., 2009a) 0.005 0.010 0.015 Pore volume(cm3/g) 0.020 0.025 0.030 Functional groups of AC surface may be acidic groups and basic ones because the surface chemistry of modified AC is closely associated to the types of heteroatoms (oxygen, hydrogen, nitrogen, et al) other than carbon atom within the carbon matrix (Wibowo, et al., 2007). Some common heteroatoms mainly including oxygen (O), nitrogen (N), phosphorus (P), hydrogen (H), chlorine (Cl) and sulfur (S) introduced in the modification process are bound to the edges of graphite-like lays, forming organic functional groups such as carboxyl groups, lactonic groups, phenolic groups, carbonyl groups, aldehydes, ethers, animo groups and other N-containng groups and phophates (Salame&Bandosz, 2001). The surface functional groups of ACs determines their moisture content, catalytic properties, acid/base character, and adsorption capacity (Salame&Bandosz, 2001). Therefore, it is important that the above groups are characterized. However, the surface chemistry characterization of ACs is a complex/difficult task. At present, the characterization methods are better established. In characterization of ACs, it is possible not only to identify the surface chemical groups but also to quantify them. The objective of this work was to investigate the pore structure and surface chemistry of activated carbons (ACs) due to its effect on adsorption and catalytic property. The present review compiles the work done over the last few decades on types, formation and characterization of surface functionalities of ACs. Special attention is paid to characterization of pore structure and surface groups on ACs by various technologies including N2 adsorption, scanning electron micrograph, elemental analysis, Boehm Titration, potentiometric titration, Fourier transform infra red/diffuse Fourier infrared transform spectroscopy, X-ray photoelectron spectroscopy, temperature programmed desorption, thermal gravimetric analysis/differential scanning calorimetry, which are illustrated by some examples. Moreover, the most important aspects referring to comparison of different characterization methods are also overviewed in this paper. The present work carried out indicates that various methods have their advantages and disadvantages in investigating the surface groups of ACs and the methods mentioned above must be used to complement each other. As so far, relatively little work has been published and there is considerable scope for more detailed studies on the characterization of surface groups of ACs. ## 2. Characterization Adsorption capacity of ACs is dependent on surface area, pore structure and surface groups, polarity, solubility and molecule size of adsorbates; solution pH and the presence of other ions in solution and so on (Radovic, et al., 2001). To investigate and obtain information pertaining to its physical structures and chemistry properties, AC needs to be characterized by various analysis apparatus. ## 2.1 Physical structure characterization ## 2.1.1 N2 adsorption Surface area and pore structure can be determined by N2 adsorption (Li, et al., 2010b), mercury porosimetry (García, et al., 2011), capillary condensation (CC) (Liu, Y., et al., 2011c), X-ray Diffraction (XRD) (Tongpoothorn, et al., 2011) and scanning/transmission electron microscope (SEM) (Chingombe, et al., 2005), small angle X-ray scattering (SAXS) (Bradley, et al., 2011). Here, physical structures of AC analyzed only by liquid Nitrogen adsorption will be introduced in this paper because Nitrogen adsorption are frequently used in investigating the texture of AC. Brunauer-Emmitt-Teller surface area (SBET), Langmuir surface area (Slangmuir), pore volume and pore distribution of the adsorbents were determined through liquid Nitrogen adsorption at 77 K on a Micromertics ASAP 2020 surface area analyzer (USA). Micropore and mesopore volumes and surface areas of the adsorbents were calculated using Density Functional Theory (DFT). The pore size distribution of adsorbents was determined from adsorption isotherm data using DFT. Note: r is median pore radius 168 Fourier Transform – Materials Analysis Functional groups of AC surface may be acidic groups and basic ones because the surface chemistry of modified AC is closely associated to the types of heteroatoms (oxygen, hydrogen, nitrogen, et al) other than carbon atom within the carbon matrix (Wibowo, et al., 2007). Some common heteroatoms mainly including oxygen (O), nitrogen (N), phosphorus (P), hydrogen (H), chlorine (Cl) and sulfur (S) introduced in the modification process are bound to the edges of graphite-like lays, forming organic functional groups such as carboxyl groups, lactonic groups, phenolic groups, carbonyl groups, aldehydes, ethers, animo groups and other N-containng groups and phophates (Salame&Bandosz, 2001). The surface functional groups of ACs determines their moisture content, catalytic properties, acid/base character, and adsorption capacity (Salame&Bandosz, 2001). Therefore, it is important that the above groups are characterized. However, the surface chemistry characterization of ACs is a complex/difficult task. At present, the characterization methods are better established. In characterization of ACs, it is possible not only to identify the surface chemical groups but The objective of this work was to investigate the pore structure and surface chemistry of activated carbons (ACs) due to its effect on adsorption and catalytic property. The present review compiles the work done over the last few decades on types, formation and characterization of surface functionalities of ACs. Special attention is paid to characterization of pore structure and surface groups on ACs by various technologies including N2 adsorption, scanning electron micrograph, elemental analysis, Boehm Titration, potentiometric titration, Fourier transform infra red/diffuse Fourier infrared transform spectroscopy, X-ray photoelectron spectroscopy, temperature programmed desorption, thermal gravimetric analysis/differential scanning calorimetry, which are illustrated by some examples. Moreover, the most important aspects referring to comparison of different characterization methods are also overviewed in this paper. The present work carried out indicates that various methods have their advantages and disadvantages in investigating the surface groups of ACs and the methods mentioned above must be used to complement each other. As so far, relatively little work has been published and there is considerable scope for more detailed studies on the characterization of surface Adsorption capacity of ACs is dependent on surface area, pore structure and surface groups, polarity, solubility and molecule size of adsorbates; solution pH and the presence of other ions in solution and so on (Radovic, et al., 2001). To investigate and obtain information pertaining to its physical structures and chemistry properties, AC needs to be characterized Surface area and pore structure can be determined by N2 adsorption (Li, et al., 2010b), mercury porosimetry (García, et al., 2011), capillary condensation (CC) (Liu, Y., et al., 2011c), X-ray Diffraction (XRD) (Tongpoothorn, et al., 2011) and scanning/transmission electron microscope (SEM) (Chingombe, et al., 2005), small angle X-ray scattering (SAXS) (Bradley, et also to quantify them. groups of ACs. 2. Characterization 2.1.1 N2 adsorption al., 2011). by various analysis apparatus. 2.1 Physical structure characterization Table 2. Textural characterization of the adsorbents (Li, et al., 2009a) Fig. 1. Pore size distribution of adsorbents (Li, et al., 2009a) Table 2 suggests that the difference in micro-pore properties, most relevant to adsorption of organic compounds, are small, with similar micro-pore volumes but different micro-pore surface areas, indicating the major difference between them is pore distribution within the micro-pore range as indicated by the mean pore diameter (Table 1). Among the four adsorbents, ACD contains more smaller micropores than the other adsorbents while AC contains less smaller pores in comparison to ACO1 and ACO2. This trend seems correlating well with the ash contents of the adsorbents (Table 4) and suggests that the acid leaching processes remove fine mineral matters from the adsorbents and result in formation of micropores, as depicted as Fig. 1. The meso-pore properties (Vmeso and Smeso) of AC, ACO1 and ACO2 are very similar, but that of ACD are significantly smaller than others. These Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 171 Prior to 1960s, researchers did not know how to characterize surface chemistry of AC. Fortunately, Boehm in 1966 began to make attempt to determinate surface chemistry of AC with acid/base titration theory/method, which makes researchers further analyze/ understand surface chemistry of AC. Thereafter, with the development of science and technologies, more and more characterization methods including elemental analysis, potentiometric titration, Fourier transform infrared/diffuse Fourier infrared transform spectroscopy, X-ray photoelectron spectroscopy, temperature programmed desorption, thermal gravimetric analysis/differential scanning calorimetry, X-ray diffraction (XRD), and Sometimes, contents of elements contained in raw material of AC, e.g. characteristics of raw tobacco residue are provided in the literature (Kilic, et al., 2011). The characteristics of raw tobacco residue were shown in Table 3. From Table 3, it is obviously seen that content of carbon is high and content ash is low, indicating that tobacco residue is suitable for AC so on have been proposed and utilized by researchers all over the world. Type Value Moisture 8.13 Ash 11.73 Volatiles 67.55 Fixed C 12.59 C 40.95 H 5.21 N 5.99 O\* 47.85 H/C 1.52 O/C 0.88 HHV (MJ/kg) 15.07 Lignin 8.75 Cellulose 42.30 Oil 3.30 Table 3. Characteristics of raw tobacco residue (Kilic, et al., 2011) was calculated by difference. Analytic results were shown in Table 4. Li, et al determined ash amount and ultimate analyses of the adsorbents in the literature (Li, et al., 2009a). Carbon (C), hydrogen (H), and nitrogen (N) contents of the adsorbents were determined by an element analyzer (Analysensysteme Gmbh Elementar Vario EL), sulfur (S) content was measured by a sulfur analyzer (SC-132, LECO, USA), and oxygen (O) content Ultimate analysis (%) (dry-ash basis) 2.2 Surface chemistry characterization 2.2.1 Element analyses Proximate analysis (%) Structural analysis (%) production. parameters again correlate well with ash content of the adsorbents, suggesting that there is removal of minerals from the mesopores, which converts them into macro-pores. ### 2.1.2 Scanning electron micrograph (SEM) ### Preparation (Chingombe, et al., 2005) The surface morphology of the carbons was analyzed using a Cambridge Instrument 360 scanning electron microscope at accelerating voltages of 10–20 kV. Prior to analysis, samples were dried at 373 K and stored in a desiccator overnight. The samples were then put on an aluminum platform for analysis. Fig. 2. SEM images of carbon samples before and after modification. (Chingombe, et al., 2005) Chingombe, et al described the SEM of the samples in Fig. 2 (Chingombe, et al., 2005). There is little difference in the surface morphology of the samples except for some apparent pore widening on AC1 that could have occurred from the oxidation process. It was noticed that the oxidized samples disintegrated to small particles when compared to F400 sample particles. This observation could be linked to the cleavage of C–O bridging bonds on the carbon surface during the oxidation process. The SEM images of AC2 and AC3 were similar in appearance to AC1 image. This implies that post-oxidation treatments of the carbon samples did not make any apparent change in the surface morphology of the adsorbents. The surface morphology for AC4 was compared to that of AC5 and it is readily found that modification of AC4 to produce AC5 had a visual impact on surface morphology. The surface looks spongy and to some extent eroded. ## 2.2 Surface chemistry characterization Prior to 1960s, researchers did not know how to characterize surface chemistry of AC. Fortunately, Boehm in 1966 began to make attempt to determinate surface chemistry of AC with acid/base titration theory/method, which makes researchers further analyze/ understand surface chemistry of AC. Thereafter, with the development of science and technologies, more and more characterization methods including elemental analysis, potentiometric titration, Fourier transform infrared/diffuse Fourier infrared transform spectroscopy, X-ray photoelectron spectroscopy, temperature programmed desorption, thermal gravimetric analysis/differential scanning calorimetry, X-ray diffraction (XRD), and so on have been proposed and utilized by researchers all over the world. ## 2.2.1 Element analyses 170 Fourier Transform – Materials Analysis parameters again correlate well with ash content of the adsorbents, suggesting that there is The surface morphology of the carbons was analyzed using a Cambridge Instrument 360 scanning electron microscope at accelerating voltages of 10–20 kV. Prior to analysis, samples were dried at 373 K and stored in a desiccator overnight. The samples were then put on an Fig. 2. SEM images of carbon samples before and after modification. (Chingombe, et al., Chingombe, et al described the SEM of the samples in Fig. 2 (Chingombe, et al., 2005). There is little difference in the surface morphology of the samples except for some apparent pore widening on AC1 that could have occurred from the oxidation process. It was noticed that the oxidized samples disintegrated to small particles when compared to F400 sample particles. This observation could be linked to the cleavage of C–O bridging bonds on the carbon surface during the oxidation process. The SEM images of AC2 and AC3 were similar in appearance to AC1 image. This implies that post-oxidation treatments of the carbon samples did not make any apparent change in the surface morphology of the adsorbents. The surface morphology for AC4 was compared to that of AC5 and it is readily found that modification of AC4 to produce AC5 had a visual impact on surface morphology. The removal of minerals from the mesopores, which converts them into macro-pores. 2.1.2 Scanning electron micrograph (SEM) surface looks spongy and to some extent eroded. Preparation (Chingombe, et al., 2005) aluminum platform for analysis. 2005) Sometimes, contents of elements contained in raw material of AC, e.g. characteristics of raw tobacco residue are provided in the literature (Kilic, et al., 2011). The characteristics of raw tobacco residue were shown in Table 3. From Table 3, it is obviously seen that content of carbon is high and content ash is low, indicating that tobacco residue is suitable for AC production. Table 3. Characteristics of raw tobacco residue (Kilic, et al., 2011) Li, et al determined ash amount and ultimate analyses of the adsorbents in the literature (Li, et al., 2009a). Carbon (C), hydrogen (H), and nitrogen (N) contents of the adsorbents were determined by an element analyzer (Analysensysteme Gmbh Elementar Vario EL), sulfur (S) content was measured by a sulfur analyzer (SC-132, LECO, USA), and oxygen (O) content was calculated by difference. Analytic results were shown in Table 4. Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 173 Hydrogen ion of carboxylic groups and phenolic groups is replaced by sodium cation of sodium hydroxide, forming sodium carboxylates and sodium phenolates when sodium hydroxide reacts with carboxylic groups, and phenolic groups of AC surface. In reaction of sodium hydroxide with lactonic groups, sodium carboxylates and phenolic groups are Owing to basic property of sodium carbonate poorer than that of sodium hydroxide, sodium carbonate centralizes only carboxyl groups and lactonic groups, of which reaction Sodium bicarbonate reacts only with carboxyl groups of AC surface, yielding sodium carboxylates due to its poorest basic property among the three bases mentioned above. The <sup>C</sup> <sup>O</sup> <sup>O</sup> <sup>H</sup> <sup>C</sup> <sup>O</sup> <sup>O</sup> <sup>N</sup> <sup>a</sup> Hydrochloric acid can centralize surface base of AC, which is shown in Equation 7. <sup>H</sup> <sup>+</sup> Cl- <sup>C</sup> <sup>O</sup> <sup>O</sup> <sup>H</sup> <sup>C</sup> <sup>O</sup> <sup>O</sup> <sup>N</sup> <sup>a</sup> 1 / 2 N a N a H C O 3 <sup>2</sup> <sup>1</sup> / <sup>2</sup> <sup>H</sup> <sup>2</sup> <sup>1</sup> / <sup>2</sup> <sup>N</sup> <sup>a</sup> <sup>O</sup> <sup>2</sup> <sup>C</sup> <sup>O</sup> <sup>3</sup> <sup>2</sup> <sup>C</sup> <sup>O</sup> <sup>3</sup> <sup>1</sup> / <sup>2</sup> <sup>H</sup> <sup>2</sup> <sup>O</sup> <sup>1</sup> / <sup>2</sup> <sup>C</sup> <sup>O</sup> <sup>2</sup> <sup>C</sup> <sup>O</sup> <sup>O</sup> <sup>N</sup> <sup>a</sup> <sup>O</sup> <sup>H</sup> H O + O H H <sup>2</sup> <sup>O</sup> 1 / 2 C O <sup>2</sup> <sup>O</sup> <sup>C</sup> <sup>O</sup> <sup>2</sup> (6) Cl- (7) (3) (4) (5) O N a N a O H O H processes are shown in Equations 4-5. <sup>C</sup> <sup>O</sup> Equation 6 describes the above reaction. O O O formed. Table 4. Ash amount and ultimate analyses of the adsorbents Table 4 shows the composition analysis of the four adsorbents. Among the four adsorbents, AC has the highest ash content at 8.32 wt%. The nitric acid treatment removed some of the ash and yielded adsorbents with medium ash contents, 7.11 wt.% for ACO1 and 7.04 wt.% for ACO2. The HCl/HF treatment removed most of the ash and yielded an adsorbent with the lowest ash content (ACD, 0.53 wt.%). Compared to that of the AC, the higher H, N and O contents for adsorbents ACO1 and ACO2 indicate that the main role of the nitric acid treatment is chemistry modification, with formation of many H, N and O containing groups on the surface; the slightly lower S contents for the acid treated adsorbents indicate that most of the sulfur in the adsorbents is organic. #### 2.2.2 Boehm titration (Ayranci&Duman, 2006, Boehm, 1966, Nevskaia, et al., 2004) Boehm Titration proposed by Boehm in 1966 is one of many methods to characterize surface chemistry of AC. As so far, the titration based on acid/base titration theory is widely used because it can effectively determine oxygen-containing functional groups of AC surface and obtain information of groups. Boehm titration is based on the theory that acid and base in aqueous solution react with various basic sites and acidic sites of AC which are derived from different oxygenated groups, respectively. Titration principle (Boehm, 1966) Sodium hydroxide can centralize carboxylic groups, lactonic groups and phenolic groups of AC surface. The reaction processes of sodium hydroxide with carboxyl groups, lactonic groups, phenolic groups are shown in Equations 1-3, respectively. S (wt.%) N (wt.%) O (wt.%) H <sup>2</sup> <sup>O</sup> (1) (2) C O O N a C O O N a O H H (wt.%) AC 8.32 88.47 0.50 0.46 0.43 1.82 ACO1 7.11 86.89 2.11 0.38 0.83 2.67 ACO2 7.04 86.36 2.04 0.36 0.77 3.49 ACD 0.53 93.40 1.76 0.42 0.58 1.95 Table 4 shows the composition analysis of the four adsorbents. Among the four adsorbents, AC has the highest ash content at 8.32 wt%. The nitric acid treatment removed some of the ash and yielded adsorbents with medium ash contents, 7.11 wt.% for ACO1 and 7.04 wt.% for ACO2. The HCl/HF treatment removed most of the ash and yielded an adsorbent with the lowest ash content (ACD, 0.53 wt.%). Compared to that of the AC, the higher H, N and O contents for adsorbents ACO1 and ACO2 indicate that the main role of the nitric acid treatment is chemistry modification, with formation of many H, N and O containing groups on the surface; the slightly lower S contents for the acid treated adsorbents indicate that 2.2.2 Boehm titration (Ayranci&Duman, 2006, Boehm, 1966, Nevskaia, et al., 2004) Boehm Titration proposed by Boehm in 1966 is one of many methods to characterize surface chemistry of AC. As so far, the titration based on acid/base titration theory is widely used because it can effectively determine oxygen-containing functional groups of AC surface and obtain information of groups. Boehm titration is based on the theory that acid and base in aqueous solution react with various basic sites and acidic sites of AC which are derived Sodium hydroxide can centralize carboxylic groups, lactonic groups and phenolic groups of AC surface. The reaction processes of sodium hydroxide with carboxyl groups, lactonic N a O H Adsorbent Ash (wt.%) most of the sulfur in the adsorbents is organic. from different oxygenated groups, respectively. C O O H <sup>C</sup> <sup>O</sup> O groups, phenolic groups are shown in Equations 1-3, respectively. N a O H Titration principle (Boehm, 1966) C (wt.%) Table 4. Ash amount and ultimate analyses of the adsorbents Hydrogen ion of carboxylic groups and phenolic groups is replaced by sodium cation of sodium hydroxide, forming sodium carboxylates and sodium phenolates when sodium hydroxide reacts with carboxylic groups, and phenolic groups of AC surface. In reaction of sodium hydroxide with lactonic groups, sodium carboxylates and phenolic groups are formed. Owing to basic property of sodium carbonate poorer than that of sodium hydroxide, sodium carbonate centralizes only carboxyl groups and lactonic groups, of which reaction processes are shown in Equations 4-5. Sodium bicarbonate reacts only with carboxyl groups of AC surface, yielding sodium carboxylates due to its poorest basic property among the three bases mentioned above. The Equation 6 describes the above reaction. Hydrochloric acid can centralize surface base of AC, which is shown in Equation 7. Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 175 including anhydrides, pyrones, benzoquinones and benzofurans and nitrogen-containing It comes to conclusion that the incomprehensive information, instead of total surface information, of oxygen-containing groups of AC can be discovered by results analyses of Boehm titration due to ability of the titration to qualitatively and quantitatively detecting only some surface acidic groups (carboxylic, lactonic, and phenolic groups) and the basic 2.2.3 Potentiometric titration (Zpc Titration) (Dabrowski, et al., 2005, Martin, et al., 2003, Potentiometric titration was proposed by Y.Matsura when the effect of surface charge of AC on its adsorption/catalysis abilities began to be concerned about by researchers. At the beginning of potentiometric titration proposed, acidic functional groups of carbon black were only approximately classified into strong acid type (pK<7) and weak acid one (7<pK<11) due to the researcher's poor understanding of acidic functional groups. Potentiometric titration to some extent has been developed since Y.Matsura,et al. proposed a novel analysis technology, potentiometric titration, to estimate the surface acidity of carbon black with in 1970s, but the potentiometric titration method has been greatly developed since constant distribution of AC surface was investigated by Teresa J., et al, in 1990s. At present, potentiometric titration has been a convenient analysis method of surface chemistry of AC through determining surface charge (Chen, J. P., et al., 2003, Owing to the amphoteric character of the carbon surface derived from the acidic and/or basic functional groups, the surface properties may be influenced by the pH value of the coexisting liquid bulk phase. The effects of surface functionalities on adsorption of organic electrolytes including weak electrolytes such as phenols are usually significantly more complex than porosity effects, and so is their assessment. The attention to the surface charge It is commonly assumed, that for pH < pKa adsorption of non-ionized organics does not depend on the surface charge of AC. However, for pH > pKa the phenolic compound is Potentiometric titration designed to determinate surface chemistry is based on the principle that surface charge of AC is a function of pH value of aqueous solutions. Potentiometric The pH mode of the titrator was set, then 0.1000 g of AC and 50ml of solution containing 0.1 M NaNO3 were placed in the reaction flask, and finally the mixture including AC and electrolyte was balanced in 298K for 24h. To eliminate the effect of CO2 in the air, the volume above liquid was protected with N2. In measurements with 0.1M NaOH or HCl standard solution, solution was titrated to pH = 3~10. The pH of the solution adjusted to the of the carbon as well as the extent of ionization of the solute should be paid. dissociated, and adsorption of its ionic form depends on the surface charge. titration measurements are carried out by an automatic titrator. corresponding potential of the solution is Zpc potential Determination of potentiometric titration (Dabrowski, et al., 2005) groups of adsorbents can not be determined by Boehm titration. groups. Ramrakhiani, et al., 2011) Noh&Schwarz, 1990). Principle ## Preparation and test (Li, et al., 2010a) Li, et al determined and analyzed the surface functional groups of activated carbon (AC) and activated carbon fiber (ACF). AC and ACF, used as the control adsorbents in the experiments, were commercial coal-based granular activated carbon from Xinhua Chemical Plant (Taiyuan, China) and novel cellulose activated carbon fiber from Shanxi Institute of Coal Chemistry (Taiyuan, China), respectively. AC was crushed into particles of 40–60 mesh size (0.3–0.45 mm) and the ACF was cut into pieces (40 mm × 40 mm); The carbon materials mentioned above were finally dried at 110 °C for 48 h. To obtain adsorbents with similar textural properties and different surface chemistry, the chemical treatment of ACF by HNO3 oxidation was carried out in a 500 mL Teflon bottle containing 16 g ACF and 200 mL 5 M nitric acid solution at 70 °C for 6 h. Once the oxidation was complete, the oxidized ACF was repeatedly filtered and washed with distilled water several times to remove the impurities. The oxidized and washed ACF was dried at 110 °C for 48 h. The resulting ACF was designated as ACFN. An exact amount of adsorbent (0.200 g) was placed in a series of 100 mL well-sealed Teflon bottles containing 25 mL of 0.1 M NaOH, Na2CO3, NaHCO3, and HCl solutions, respectively. After shaking at 150 rpm and 30 °C for 24 h in a thermostatic automatic shaker (HZQ-C, Haerbin, China), the adsorbent was separated from the solutions by filtration, and the filtrates were then titrated with a 0.1 M HCl or NaOH. The number of acidic groups was calculated based on the following assumptions: NaOH neutralizes carboxylic, lactonic, and phenolic groups; Na2CO3 neutralizes carboxylic and lactonic groups; and NaHCO3 neutralizes only carboxylic groups. The number of basic sites was determined from the amount of HCl that reacted with the adsorbents. Note: Surface Acidity and Surface Basicity are the Adsorbent's surface acidic groups and surface basic groups, respectively. Table 5. Surface chemistry of the adsorbents determined by Boehm titration (Li, et al., 2010a) Table 5 showed the acid/base properties of the adsorbents characterized by Boehm titration. From table 5, it is easily seen that the surface property of AC and ACF is totally basic, but that ACFN is acidic. The total surface acidic groups, carboxyl groups, lactonic groups, phenolic groups and basic groups in ACF are much higher than the corresponding parameters in AC, indicating the difference in surface chemistry between AC and ACF resulting from the different preparation of the carbon materials. In addition, contents of total surface acidic groups, carboxyl groups, lactonic groups, phenolic groups but basic groups in ACFN are much higher than those in ACF. It indicates that the HNO3 oxidation clearly increased the surface acidic groups (carboxylic, lactonic, and phenolic groups) and decreased the basic groups of ACF. It should be noted that other surface functional groups including anhydrides, pyrones, benzoquinones and benzofurans and nitrogen-containing groups of adsorbents can not be determined by Boehm titration. It comes to conclusion that the incomprehensive information, instead of total surface information, of oxygen-containing groups of AC can be discovered by results analyses of Boehm titration due to ability of the titration to qualitatively and quantitatively detecting only some surface acidic groups (carboxylic, lactonic, and phenolic groups) and the basic groups. ## 2.2.3 Potentiometric titration (Zpc Titration) (Dabrowski, et al., 2005, Martin, et al., 2003, Ramrakhiani, et al., 2011) Potentiometric titration was proposed by Y.Matsura when the effect of surface charge of AC on its adsorption/catalysis abilities began to be concerned about by researchers. At the beginning of potentiometric titration proposed, acidic functional groups of carbon black were only approximately classified into strong acid type (pK<7) and weak acid one (7<pK<11) due to the researcher's poor understanding of acidic functional groups. Potentiometric titration to some extent has been developed since Y.Matsura,et al. proposed a novel analysis technology, potentiometric titration, to estimate the surface acidity of carbon black with in 1970s, but the potentiometric titration method has been greatly developed since constant distribution of AC surface was investigated by Teresa J., et al, in 1990s. At present, potentiometric titration has been a convenient analysis method of surface chemistry of AC through determining surface charge (Chen, J. P., et al., 2003, Noh&Schwarz, 1990). Owing to the amphoteric character of the carbon surface derived from the acidic and/or basic functional groups, the surface properties may be influenced by the pH value of the coexisting liquid bulk phase. The effects of surface functionalities on adsorption of organic electrolytes including weak electrolytes such as phenols are usually significantly more complex than porosity effects, and so is their assessment. The attention to the surface charge of the carbon as well as the extent of ionization of the solute should be paid. It is commonly assumed, that for pH < pKa adsorption of non-ionized organics does not depend on the surface charge of AC. However, for pH > pKa the phenolic compound is dissociated, and adsorption of its ionic form depends on the surface charge. ## Principle 174 Fourier Transform – Materials Analysis Li, et al determined and analyzed the surface functional groups of activated carbon (AC) and activated carbon fiber (ACF). AC and ACF, used as the control adsorbents in the experiments, were commercial coal-based granular activated carbon from Xinhua Chemical Plant (Taiyuan, China) and novel cellulose activated carbon fiber from Shanxi Institute of Coal Chemistry (Taiyuan, China), respectively. AC was crushed into particles of 40–60 mesh size (0.3–0.45 mm) and the ACF was cut into pieces (40 mm × 40 mm); The carbon materials To obtain adsorbents with similar textural properties and different surface chemistry, the chemical treatment of ACF by HNO3 oxidation was carried out in a 500 mL Teflon bottle containing 16 g ACF and 200 mL 5 M nitric acid solution at 70 °C for 6 h. Once the oxidation was complete, the oxidized ACF was repeatedly filtered and washed with distilled water several times to remove the impurities. The oxidized and washed ACF was dried at 110 °C An exact amount of adsorbent (0.200 g) was placed in a series of 100 mL well-sealed Teflon bottles containing 25 mL of 0.1 M NaOH, Na2CO3, NaHCO3, and HCl solutions, respectively. After shaking at 150 rpm and 30 °C for 24 h in a thermostatic automatic shaker (HZQ-C, Haerbin, China), the adsorbent was separated from the solutions by filtration, and the filtrates were then titrated with a 0.1 M HCl or NaOH. The number of acidic groups was calculated based on the following assumptions: NaOH neutralizes carboxylic, lactonic, and phenolic groups; Na2CO3 neutralizes carboxylic and lactonic groups; and NaHCO3 neutralizes only carboxylic groups. The number of basic sites was determined from the > Lactonic (mmol/g) Phenolic (mmol/g) Surface Basicity (mmol/g) Carboxylic (mmol/g) AC 0.489 0.033 0.327 0.129 0.646 ACF 1.279 0.068 0.911 0.300 1.605 ACFN 3.724 1.409 1.224 1.091 1.383 Note: Surface Acidity and Surface Basicity are the Adsorbent's surface acidic groups and surface basic Table 5. Surface chemistry of the adsorbents determined by Boehm titration (Li, et al., 2010a) Table 5 showed the acid/base properties of the adsorbents characterized by Boehm titration. From table 5, it is easily seen that the surface property of AC and ACF is totally basic, but that ACFN is acidic. The total surface acidic groups, carboxyl groups, lactonic groups, phenolic groups and basic groups in ACF are much higher than the corresponding parameters in AC, indicating the difference in surface chemistry between AC and ACF resulting from the different preparation of the carbon materials. In addition, contents of total surface acidic groups, carboxyl groups, lactonic groups, phenolic groups but basic groups in ACFN are much higher than those in ACF. It indicates that the HNO3 oxidation clearly increased the surface acidic groups (carboxylic, lactonic, and phenolic groups) and decreased the basic groups of ACF. It should be noted that other surface functional groups Preparation and test (Li, et al., 2010a) mentioned above were finally dried at 110 °C for 48 h. for 48 h. The resulting ACF was designated as ACFN. amount of HCl that reacted with the adsorbents. Acidity (mmol/g) Adsorbent Surface groups, respectively. Potentiometric titration designed to determinate surface chemistry is based on the principle that surface charge of AC is a function of pH value of aqueous solutions. Potentiometric titration measurements are carried out by an automatic titrator. ## Determination of potentiometric titration (Dabrowski, et al., 2005) The pH mode of the titrator was set, then 0.1000 g of AC and 50ml of solution containing 0.1 M NaNO3 were placed in the reaction flask, and finally the mixture including AC and electrolyte was balanced in 298K for 24h. To eliminate the effect of CO2 in the air, the volume above liquid was protected with N2. In measurements with 0.1M NaOH or HCl standard solution, solution was titrated to pH = 3~10. The pH of the solution adjusted to the corresponding potential of the solution is Zpc potential Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 177 process on detecting chemistry, loose powder of some samples is also effectively analyzed. In addition, organic functional groups of AC surface not being detected by common FTIR due to special property of AC, which belongs to a type of blank body, can be determined by Usually, infrared absorption spectra of molecules are vibrational/rotational spectrums of molecules. Infrared absorption spectrums are based on the absorption resulted from transition of vibrational/rotational energy level of molecules. Compared with standard spectrums, information of functional groups of samples can be determined by frequency of F400 is a commercial granular coal-based AC from Chemviron (USA). In the surface modification, F400 was washed with distilled water, and dried at 383 K for 24 h, followed by various treatments including HNO3, amination and annealing in H2. All other reagents but The pre-treated F400 was stirringly oxidized by about 35 wt.% of HNO3 at 363 K for 6 h. After the oxidation, a fresh HNO3 solution at the same concentration oxidized the above treated sample for 3 h. The oxidized carbon was then washed with distilled water until no further change in pH could be detected. The resultant water-washed sample (after oxidation) was divided into three portions. The first portion was denoted as AC1, and this is a sample that was washed with water after the oxidation reaction. AC2 was the sample that was washed with 0.1 M NaOH to remove humus that is a byproduct of the oxidation process. The washing was continued until no further coloration from the humic substances could be detected. Reconditioning of the sample to the hydrogen form was performed using 0.1 M hydrochloric acid and further washing with distilled water was done until the pH of the supernatant solution stabilized to a pH of about 4.0. The third sample, denoted as AC3, was as a result of heating the oxidized sample at a temperature of 580 K under vacuum of 2 mm Hg for 12 h to remove humic substances. Prior to the annealing process the sample was conditioned in a flow of hydrogen (3.3×10-6 m3/s) for 45 min at ambient temperature. The furnace temperature was then raised at a rate of 10 °C /min until it reached 1173 K where it was maintained for 3 h. After the annealing cycle, the furnace was allowed to cool down and Chingombe P., et al. investigate surface groups of ACs through DRIFTS. Fourier Transmission infrared experiments, which were conducted on a Nicolet DXC20 FTIR spectrometer with liquid nitrogen-cooled mercury–cadmium–telluride detector and a Spectra Tech diffuse reflectance accessory (Chingombe, et al., 2005). The preparation process was drying of samples of particle size <45 μm for 24 h at a 383 K and mixture of the dried samples with fine KBr at a ratio of 1:100. In the preparation process, FTIR spectra of samples were recorded at a resolution of 4 cm-1 and with 200 scans and an aperture setting of 15. A previously recorded background spectra of water vapour was subtracted from the spectrum the flow of hydrogen was maintained even during the cooling process. DRIFTS (Brown, 1990, Liang, Xiaotian, 1988, Wang, K., et al., 2006, Zhong, 1984). Principle absorption band. HNO3 were analytically pure. Operation conditions of each sample (Dabrowski, et al., 2005). Sample preparation (Chingombe, et al., 2005) It should be noted that C1, C2 and C3 represents ACs with different surface chemistry. Fig. 3. the relation of surface potential with pH (hypothetical) ACs: C1, acidic carbon (pHzpc=8.0); C2, amphoteric carbon (pHzpc=6.5); C3, basic carbon (pHzpc=10.0) (Radovic, et al., 2001) Fig. 3 showed the dependence of surface potential on pH for hypothetical ACs supposed by Radovic. The pH value, at which the surface charge is zero, is called the point of zero charge (PZC). For typical amphoteric carbons, the surface is positively charged at pH < pHPZC and negatively charged at pH > pHPZC. The so-called isoelectric point, IEP, is denoted as the pH value of zero potential. In practice, pHIEP is usually close to pHPZC, but it is lower than pHPZC of ACs. Potential of C1 is less than zero, indicating the surface of C1 is acidic; Potential of C3 is more than zero, indicating the surface of C3 is basic. It is commonly assumed that for pH < pKa adsorption of non-ionized organics does not depend on the surface charge of AC. However, for pH > pKa the phenolic compounds is dissociated, and adsorption of its ionic form depends on the surface charge. In accordance with pKa, it is obtained that C1 surface has carboxylic groups and C3 has carbonyls and benzoquonones (Dabrowski, et al., 2005). From Fig. 3, various acidic or basic groups can not be differentiated by potentiometric titration and specific amount of groups such as carboxylic group, anhydrides, and so on, can not be given by potentiometric titration. #### 2.2.4 Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) (Azargohar&Dalai, 2011, Chingombe, et al., 2005, Li, et al., 2010a) The method pertaining to Fourier infrared transform spectroscope sprang up at the beginning of 20th century and was extremely developed due to the rapid development of technologies on computer and Fourier transform. It, combining Michelson interferometer with computer technology, has many advantages such as short time of measurement, high sensibility and resolution, broad range of measurement spectrum. In 1970s, Kortuum and GriffihsIn narrated the fundamental of principle of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). DRIFTS is applied in determining the samples with scattering property and strong adsorption capacity, especially AC. Owing to its ability of decreasing or even eliminating the effect of scattering factor derived from preforming process on detecting chemistry, loose powder of some samples is also effectively analyzed. In addition, organic functional groups of AC surface not being detected by common FTIR due to special property of AC, which belongs to a type of blank body, can be determined by DRIFTS (Brown, 1990, Liang, Xiaotian, 1988, Wang, K., et al., 2006, Zhong, 1984). ## Principle 176 Fourier Transform – Materials Analysis C2 3 4 5 6 7 8 9 10 Fig. 3 showed the dependence of surface potential on pH for hypothetical ACs supposed by Radovic. The pH value, at which the surface charge is zero, is called the point of zero charge (PZC). For typical amphoteric carbons, the surface is positively charged at pH < pHPZC and negatively charged at pH > pHPZC. The so-called isoelectric point, IEP, is denoted as the pH value of zero potential. In practice, pHIEP is usually close to pHPZC, but it is lower than pHPZC of ACs. Potential of C1 is less than zero, indicating the surface of C1 is acidic; Potential of C3 is more than zero, indicating the surface of C3 is basic. It is commonly assumed that for pH < pKa adsorption of non-ionized organics does not depend on the surface charge of AC. However, for pH > pKa the phenolic compounds is dissociated, and adsorption of its ionic form depends on the surface charge. In accordance with pKa, it is obtained that C1 surface has carboxylic groups and C3 has carbonyls and benzoquonones (Dabrowski, et al., 2005). From Fig. 3, various acidic or basic groups can not be differentiated by potentiometric titration and specific amount of groups such as carboxylic group, anhydrides, and so on, can Fig. 3. the relation of surface potential with pH (hypothetical) ACs: C1, acidic carbon (pHzpc=8.0); C2, amphoteric carbon (pHzpc=6.5); C3, basic carbon (pHzpc=10.0) (Radovic, et 2.2.4 Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) The method pertaining to Fourier infrared transform spectroscope sprang up at the beginning of 20th century and was extremely developed due to the rapid development of technologies on computer and Fourier transform. It, combining Michelson interferometer with computer technology, has many advantages such as short time of measurement, high sensibility and resolution, broad range of measurement spectrum. In 1970s, Kortuum and GriffihsIn narrated the fundamental of principle of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). DRIFTS is applied in determining the samples with scattering property and strong adsorption capacity, especially AC. Owing to its ability of decreasing or even eliminating the effect of scattering factor derived from preforming (Azargohar&Dalai, 2011, Chingombe, et al., 2005, Li, et al., 2010a) C3 It should be noted that C1, C2 and C3 represents ACs with different surface chemistry. Dimensionless potential (F /RT) not be given by potentiometric titration. al., 2001) φ<sup>s</sup> 4 2 0 C1 pH Usually, infrared absorption spectra of molecules are vibrational/rotational spectrums of molecules. Infrared absorption spectrums are based on the absorption resulted from transition of vibrational/rotational energy level of molecules. Compared with standard spectrums, information of functional groups of samples can be determined by frequency of absorption band. ## Sample preparation (Chingombe, et al., 2005) F400 is a commercial granular coal-based AC from Chemviron (USA). In the surface modification, F400 was washed with distilled water, and dried at 383 K for 24 h, followed by various treatments including HNO3, amination and annealing in H2. All other reagents but HNO3 were analytically pure. The pre-treated F400 was stirringly oxidized by about 35 wt.% of HNO3 at 363 K for 6 h. After the oxidation, a fresh HNO3 solution at the same concentration oxidized the above treated sample for 3 h. The oxidized carbon was then washed with distilled water until no further change in pH could be detected. The resultant water-washed sample (after oxidation) was divided into three portions. The first portion was denoted as AC1, and this is a sample that was washed with water after the oxidation reaction. AC2 was the sample that was washed with 0.1 M NaOH to remove humus that is a byproduct of the oxidation process. The washing was continued until no further coloration from the humic substances could be detected. Reconditioning of the sample to the hydrogen form was performed using 0.1 M hydrochloric acid and further washing with distilled water was done until the pH of the supernatant solution stabilized to a pH of about 4.0. The third sample, denoted as AC3, was as a result of heating the oxidized sample at a temperature of 580 K under vacuum of 2 mm Hg for 12 h to remove humic substances. Prior to the annealing process the sample was conditioned in a flow of hydrogen (3.3×10-6 m3/s) for 45 min at ambient temperature. The furnace temperature was then raised at a rate of 10 °C /min until it reached 1173 K where it was maintained for 3 h. After the annealing cycle, the furnace was allowed to cool down and the flow of hydrogen was maintained even during the cooling process. ## Operation conditions Chingombe P., et al. investigate surface groups of ACs through DRIFTS. Fourier Transmission infrared experiments, which were conducted on a Nicolet DXC20 FTIR spectrometer with liquid nitrogen-cooled mercury–cadmium–telluride detector and a Spectra Tech diffuse reflectance accessory (Chingombe, et al., 2005). The preparation process was drying of samples of particle size <45 μm for 24 h at a 383 K and mixture of the dried samples with fine KBr at a ratio of 1:100. In the preparation process, FTIR spectra of samples were recorded at a resolution of 4 cm-1 and with 200 scans and an aperture setting of 15. A previously recorded background spectra of water vapour was subtracted from the spectrum of each sample (Dabrowski, et al., 2005). Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 179 ACFN ACF 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 The DRIFTS spectra of the adsorbents were shown in Fig. 5. As can be seen, among the three adsorbents, the lowest intensity of bands is in AC, indicating that AC has fewer groups than both ACF and ACFN. Compared with ACF, ACFN has stretching vibrations similar to those of ACF, with the exception of the stronger intensity of bands of the surface functionalities of ACFN with OH of around 3384cm−1, C=O (carboxylic groups, anhydride, lactone and ketene) at 1674cm−1, C–O (lactonic groups, ether, phenol, etc.) including υas (C–O) at 1300cm−1, and υs (C–O) at 1110cm−1. This indicates that more oxygen-containing groups have been introduced on ACF surface after HNO3 oxidation. Although the DRIFTS spectra do not provide quantitative information of the carbon surface chemistry, it can provide other important results, such as nitrogen-containing groups, that could not be detected using the titration method due to their inability to dissociate. Clearly, ACFN has the strong band with the stretching vibrations around 1519cm−1, which can be ascribed to the presence From the analysis, DRIFTS does not accurately quantify oxygen-containing functional groups, but can acquire carboxyl groups, hydroxyl groups, carbonyls and nitro group produced from HNO3 oxidation. Furthermore, some absorption peaks can not be ascribed to The chemical shift effect of inner electron energy level originated from copper oxidation was discovered by Siegbahn K. in 1958, which represented the birth of XPS. XPS technology was fast developed during 1970s and 1980s. From then on, XPS has been not only an important measurement method in research, but also an effective means of analysis and test in Wave number (cm ) Fig. 5. DRIFTS curves of the adsorbents (Li, et al., 2010a) AC 60 50 40 30 K-M 20 10 of more nitro groups on the ACF surface. a specific group due to the overlapping peaks. controlling industrial quality. 2.2.5 X-ray photoelectron spectroscopy (XPS) #### Results (Chingombe, et al., 2005) DRIFTS spectra for a series of F400 and oxidized samples were shown in Fig. 4. It is seen that some characterization peaks were at wave numbers 1240, 1610 and 1750 cm-1. The spectra for AC1, AC2 and AC3 have some similar marked curves, indicating the three samples could possess similar surface groups. F400 does not have a characterization peak at 1750 cm-1, and its peak at 1240 cm-1 is also less pronounced than that for the other oxidized samples, suggesting that oxidation increase surface groups. It is usually difficult to ascribe the peak at 1240 cm-1 (a superimposed peak) to a specific groups because the peak of a certain bands is overlapped by that of other bands. The superimposed peaks can be resulted from ether, epi-oxide and phenolic structures in various chemical environments. Beniak et al. reported that tertiary C–N stretching vibration may also result in the contribution of the peak in the region of 1240 cm-1. The C–N contribution can probably explain the lack of a pronounced peak on F400 sample which did not undergo HNO3 oxidation. The peak at 1610 cm-1 can be ascribed to quinone-like structures, and this peak appears in all the samples. Sutherland et al. and Shim et al. have also observed such a peak. As mentioned earlier, the peak at 1750 cm-1 appears on the oxidized samples only, but its position on the spectra of the other samples makes it difficult for positive identification. However, Lopez et al. obtained peaks in this region when they modified carbon samples by air oxidation and they assigned it to free carboxyl groups, lactonic groups, esters and carbonyl groups near the hydroxyl groups. The most logical explanation for the peak at 1750 cm-1 would be the existence of carboxyl groups that are formed as a result of nitric acid oxidation. This is also supported by the high sodium capacity results on the oxidized samples as opposed to F400 sample. Fig. 4. DRIFTS curves for F400 with different treatments. (Chingombe, et al., 2005) Owing to the similarity of DRIFTS curves of samples in Fig. 4, another example pertaining to characterization of surface chemistry by DRIFTS below is given to better describe the role of DRIFTS. DRIFTS spectra for a series of F400 and oxidized samples were shown in Fig. 4. It is seen that some characterization peaks were at wave numbers 1240, 1610 and 1750 cm-1. The spectra for AC1, AC2 and AC3 have some similar marked curves, indicating the three samples could possess similar surface groups. F400 does not have a characterization peak at 1750 cm-1, and its peak at 1240 cm-1 is also less pronounced than that for the other oxidized It is usually difficult to ascribe the peak at 1240 cm-1 (a superimposed peak) to a specific groups because the peak of a certain bands is overlapped by that of other bands. The superimposed peaks can be resulted from ether, epi-oxide and phenolic structures in various chemical environments. Beniak et al. reported that tertiary C–N stretching vibration may also result in the contribution of the peak in the region of 1240 cm-1. The C–N contribution can probably explain the lack of a pronounced peak on F400 sample which did not undergo HNO3 oxidation. The peak at 1610 cm-1 can be ascribed to quinone-like structures, and this peak appears in all the samples. Sutherland et al. and Shim et al. have also observed such a peak. As mentioned earlier, the peak at 1750 cm-1 appears on the oxidized samples only, but its position on the spectra of the other samples makes it difficult for positive identification. However, Lopez et al. obtained peaks in this region when they modified carbon samples by air oxidation and they assigned it to free carboxyl groups, lactonic groups, esters and carbonyl groups near the hydroxyl groups. The most logical explanation for the peak at 1750 cm-1 would be the existence of carboxyl groups that are formed as a result of nitric acid oxidation. This is also supported by the high sodium > 500 1000 1500 2000 2500 Wave number (cm ) Owing to the similarity of DRIFTS curves of samples in Fig. 4, another example pertaining to characterization of surface chemistry by DRIFTS below is given to better describe the role Fig. 4. DRIFTS curves for F400 with different treatments. (Chingombe, et al., 2005) -1 1240 1610 1750 AC1 AC2 F400 AC3 Results (Chingombe, et al., 2005) Absorbance of DRIFTS. samples, suggesting that oxidation increase surface groups. capacity results on the oxidized samples as opposed to F400 sample. Fig. 5. DRIFTS curves of the adsorbents (Li, et al., 2010a) The DRIFTS spectra of the adsorbents were shown in Fig. 5. As can be seen, among the three adsorbents, the lowest intensity of bands is in AC, indicating that AC has fewer groups than both ACF and ACFN. Compared with ACF, ACFN has stretching vibrations similar to those of ACF, with the exception of the stronger intensity of bands of the surface functionalities of ACFN with OH of around 3384cm−1, C=O (carboxylic groups, anhydride, lactone and ketene) at 1674cm−1, C–O (lactonic groups, ether, phenol, etc.) including υas (C–O) at 1300cm−1, and υs (C–O) at 1110cm−1. This indicates that more oxygen-containing groups have been introduced on ACF surface after HNO3 oxidation. Although the DRIFTS spectra do not provide quantitative information of the carbon surface chemistry, it can provide other important results, such as nitrogen-containing groups, that could not be detected using the titration method due to their inability to dissociate. Clearly, ACFN has the strong band with the stretching vibrations around 1519cm−1, which can be ascribed to the presence of more nitro groups on the ACF surface. From the analysis, DRIFTS does not accurately quantify oxygen-containing functional groups, but can acquire carboxyl groups, hydroxyl groups, carbonyls and nitro group produced from HNO3 oxidation. Furthermore, some absorption peaks can not be ascribed to a specific group due to the overlapping peaks. #### 2.2.5 X-ray photoelectron spectroscopy (XPS) The chemical shift effect of inner electron energy level originated from copper oxidation was discovered by Siegbahn K. in 1958, which represented the birth of XPS. XPS technology was fast developed during 1970s and 1980s. From then on, XPS has been not only an important measurement method in research, but also an effective means of analysis and test in controlling industrial quality. Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 181 Wang used the three samples including AC (A commercial activated carbon), AC-HCl (AC treated with 2 M HCl at room temperature) and AC-HNO3 (2 M HNO3 for about 24 h at room temperature), and determined the surface chemistry of the above samples by The XPS measurements were carried out in PHI-560 ESCA system (Perkin Elmer), conditions of which were set at a basic pressure of 2×10-7 Torr with Mg Ka excitation at 15 It should be noted that in measuring functional groups, the content percent (%) of other elements of functional groups to determine can be identified by XPS if the elements (such as C or O) have been given. The distribution of C and O structures can be derived from C1s Fig. 6 showed the C1s signals of ACs. It is readily seen that C1s signals of samples are different. The maximum C1s signal on acid-treated carbons are shifted to higher binding energy due to an increase in oxidic species (alcohols, carbonates, or carboxylic groups) and ethers on the carbon surface after acid treatment. The resolution of peak indicates that three functional groups (C-C, C-O and C=O) can be obtained. For AC-HNO3, C-O and C=O functional groups are much higher than AC and AC-HCl and C=O on AC-HCl is also higher than AC (Wang, S.&Zhu, 2007). Therefore, XPS results confirm that more acidic groups such as carboxylic groups and ethers were produced by acid treatment (Wang, S.&Zhu, 2007). From analysis of Fig. 6, XPS can monitor oxygen-containing groups, but inaccurately TPD is one of temperature-programmed analysis technology to measure structures and properties of catalysts and interaction between reaction molecules. It has been frequently used to identify and quantify oxygen-containing groups on AC through chemical Surface oxygen-containing groups on carbon materials decompose upon heating, releasing CO2 and/or CO at different characteristic temperatures (Mahajan&Moreno-Castilla, 1980), so it is readily concluded that a certain functional group decomposes at its special/corresponding temperature. Usually, decomposition law in TPD is as follows. For example, the CO2 produced at temperatures lower than 400 °C may be ascribed to the decomposition of carboxylic acids, and at around 650 °C to the decomposition of lactones. CO2 and CO released at temperatures about 550 °C account for the decomposition of anhydrides; the CO released at around 700 °C can be attributed to the decomposition of phenolic groups, and beyond 850 °C to the decomposition of carbonyl groups and quinones. To obtain the surface chemistry of AC, samples can be also characterized by TPD experiments. Here, an example of TPD of the samples treated by different methods is given 2.2.6 Temperature programmed desorption (TPD) (Li, et al., 2009a, Li, et al., 2010a) kV and recorded in ΔE constant mode, pass energy 50 and 100 eV. resolution analysis of XPS peaks (Fig. 6). and XP O1s spectra. quantify the groups. Principle Experiments adsorption apparatus or mass spectrum. to describe the analysis process (Li, et al., 2009a). #### Principle (Wang, K., et al., 2006) When samples to determine is being exposed by X-ray with enough energy, an inner-shell electron will be bombarded from the sample atoms, producing an ion in an excited state (Equation 8) and XPS can detect the kinetic energy of the bombarded electron. $$S + l\nu\_1(Xray) \to S^{+\} + e^- \tag{8}$$ Kinetic energy of each electron is related with the orbital energy of emission electron, and orbital energy is characteristic of atoms or molecules, so the XPS can be used to qualitatively analyze samples. In a given experimental condition, number of emission electron is generally proportional to the concentration of emitters, so the XPS can also quantify the samples. The depth of samples studied in electron spectrum is less than 5 nm, so XPS is a surface analysis method. Specific binding energy of each electron corresponds to a Gaussian peak, representing a type of functional group. #### Experiments (Wang, S.&Zhu, 2007)* To obtain the surface chemistry of AC, samples can be also characterized by XPS. Here, an example of XPS of the samples done by Wang is given below. Fig. 6. XPS spectra of C1s on AC surface (Wang, S.&Zhu, 2007) Wang used the three samples including AC (A commercial activated carbon), AC-HCl (AC treated with 2 M HCl at room temperature) and AC-HNO3 (2 M HNO3 for about 24 h at room temperature), and determined the surface chemistry of the above samples by resolution analysis of XPS peaks (Fig. 6). The XPS measurements were carried out in PHI-560 ESCA system (Perkin Elmer), conditions of which were set at a basic pressure of 2×10-7 Torr with Mg Ka excitation at 15 kV and recorded in ΔE constant mode, pass energy 50 and 100 eV. It should be noted that in measuring functional groups, the content percent (%) of other elements of functional groups to determine can be identified by XPS if the elements (such as C or O) have been given. The distribution of C and O structures can be derived from C1s and XP O1s spectra. Fig. 6 showed the C1s signals of ACs. It is readily seen that C1s signals of samples are different. The maximum C1s signal on acid-treated carbons are shifted to higher binding energy due to an increase in oxidic species (alcohols, carbonates, or carboxylic groups) and ethers on the carbon surface after acid treatment. The resolution of peak indicates that three functional groups (C-C, C-O and C=O) can be obtained. For AC-HNO3, C-O and C=O functional groups are much higher than AC and AC-HCl and C=O on AC-HCl is also higher than AC (Wang, S.&Zhu, 2007). Therefore, XPS results confirm that more acidic groups such as carboxylic groups and ethers were produced by acid treatment (Wang, S.&Zhu, 2007). From analysis of Fig. 6, XPS can monitor oxygen-containing groups, but inaccurately quantify the groups. ## 2.2.6 Temperature programmed desorption (TPD) (Li, et al., 2009a, Li, et al., 2010a) TPD is one of temperature-programmed analysis technology to measure structures and properties of catalysts and interaction between reaction molecules. It has been frequently used to identify and quantify oxygen-containing groups on AC through chemical adsorption apparatus or mass spectrum. ## Principle 180 Fourier Transform – Materials Analysis When samples to determine is being exposed by X-ray with enough energy, an inner-shell electron will be bombarded from the sample atoms, producing an ion in an excited state Kinetic energy of each electron is related with the orbital energy of emission electron, and orbital energy is characteristic of atoms or molecules, so the XPS can be used to qualitatively analyze samples. In a given experimental condition, number of emission electron is generally proportional to the concentration of emitters, so the XPS can also quantify the samples. The depth of samples studied in electron spectrum is less than 5 nm, so XPS is a surface analysis method. Specific binding energy of each electron corresponds to a Gaussian To obtain the surface chemistry of AC, samples can be also characterized by XPS. Here, an \* ( ) y S e <sup>+</sup> <sup>−</sup> + → + (8) (Equation 8) and XPS can detect the kinetic energy of the bombarded electron. <sup>1</sup> S h Xra ν Principle (Wang, K., et al., 2006) peak, representing a type of functional group. example of XPS of the samples done by Wang is given below. Fig. 6. XPS spectra of C1s on AC surface (Wang, S.&Zhu, 2007) Experiments (Wang, S.&Zhu, 2007) Surface oxygen-containing groups on carbon materials decompose upon heating, releasing CO2 and/or CO at different characteristic temperatures (Mahajan&Moreno-Castilla, 1980), so it is readily concluded that a certain functional group decomposes at its special/corresponding temperature. Usually, decomposition law in TPD is as follows. For example, the CO2 produced at temperatures lower than 400 °C may be ascribed to the decomposition of carboxylic acids, and at around 650 °C to the decomposition of lactones. CO2 and CO released at temperatures about 550 °C account for the decomposition of anhydrides; the CO released at around 700 °C can be attributed to the decomposition of phenolic groups, and beyond 850 °C to the decomposition of carbonyl groups and quinones. ## Experiments To obtain the surface chemistry of AC, samples can be also characterized by TPD experiments. Here, an example of TPD of the samples treated by different methods is given to describe the analysis process (Li, et al., 2009a). Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 183 DSC (mW/mg) ACO1 ACD AC 0 50 100 150 200 250 300 350 Long process and high expense Inability of detecting pore Inability of specific functional Inability of indentifying anhydrides, benzoquinones pyrones and other groups, complex operation, long process. Sample was destroyed Inability of indentifying types of Inability of some absorption to be attributed and approximate Inner surface layer in depth less than 5 nm, approximate quantification, high expense and Medium expense, medium easy and Inability of quantification. Sample was destroyed distribution some groups, quantification easy operation. indirect results. Sample was destroyed Some peaks is difficult to disintegrate and fit. groups Time (min) (b) ACO2 0 50 100 150 200 250 300 350 Time (min) N2 adsorption Determination of micro/meso pore SEM Determination of Pore structure and morphology content direct results DRIFTS Part of functional groups, nitrogen- Element analysis Determinating element C, H, O, N, S, ash Boehm Titration Determination of acidic groups (carboxylic Pzc Titration less samples, low expense, process slow, indirect results ACD AC 40 o C (a) Type Advantages Disadvantages structure, surface area, average pore diameter and pore distribution groups, lactonic groups, anhydrides, phenolic groups and carbonyl groups) and the basic groups, accurate quantification, large amount of sample, low expense and containing groups to determine, less samples, medium expense, easy operation, at temperatures lower than 1100 °C, accurate quantitative, a small amount of sample Table 6. Comparisons of characterization methods of AC surface chemistry intact samples and direct results XPS Medium mass, more samples, high expense and direct results TPD Analysis of functional groups decomposing Fig. 8. Water adsorption on adsorbents (AC, ACD, ACO1 and ACO2) (Li, et al., 2009a) ACO1 103.0 ACO2 H2 O addition 99.5 100.0 100.5 101.0 101.5 102.0 102.5 TG (%) Fig. 7. CO2 (a) and CO (b) evolution during TPD of the four adsorbents (Li, et al., 2009a) Fig. 7 showed curves of CO2 and CO released from the adsorbents during TPD. In Fig. 7, The similar CO2 and CO release profiles for AC and ACD indicate that the HCl/HF treatment resulted in little change in acid and basic groups on the adsorbents' surface and the main groups on AC and ACD are basic, i.e. carbonyl and quinones. More CO2 released from ACO1 and ACO2 at temperatures lower than 400 °C and around 650 °C is higher than that AC, indicating carboxylic acids and lactones of AC surface are increased by HNO3 oxidationt; more CO2 and CO released from ACO1 and ACO2 around 550 °C account for decomposition of more anhydrides; more CO released from ACO1 and ACO2 than that AC at around 700 °C and beyond 850 °C shows that more phenolic groups, carbonyl groups and quinones of AC were formed by HNO3 treatment. Carboxylic acids, lactones, anhydrides, phenolic groups, carbonyl groups and quinones introduced by HNO3 treatment can be analyzed by TPD. It should be noted that some nitrogen-containing functional groups were also produced during HNO3 oxidationt. However, these nitrogen-containing groups can not be better monitored and analyzed due to disturbance of other gases produced in heating. For example, mass charge ratio of 46 can represent NO2 and CO2, so NO2 curve of AC is frequently disturbed by CO2 released in heating. Furthermore, the information of surface groups of samples obtained by TPD is an indirect analysis result because groups to determine are ascribed only to the CO2 and CO released from decomposition of groups at different temperature. Therefore, it draws a conclusion that part of surface groups (the acidic groups such as carboxylic, lactonic, phenolic groups and the basic groups) of AC are determined by TPD, but it does not directly reflect surface chemical properties of AC and only analyze the decomposition behavior of oxygen-containing functional groups. Characterization of surface chemistry through TPD method should be further investigated. #### 2.2.7 Water vapor adsorption by thermal gravimetric analysis/differential scanning calorimetry (Li, et al., 2009a) To identify hydrophilic properties of the adsorbents, water vapor adsorption of the adsorbents was measured by a thermal gravimetric analyzer (TGA 409 PC, Netzsch). The AC Fig. 7. CO2 (a) and CO (b) evolution during TPD of the four adsorbents (Li, et al., 2009a) Fig. 7 showed curves of CO2 and CO released from the adsorbents during TPD. In Fig. 7, The similar CO2 and CO release profiles for AC and ACD indicate that the HCl/HF treatment resulted in little change in acid and basic groups on the adsorbents' surface and the main groups on AC and ACD are basic, i.e. carbonyl and quinones. More CO2 released from ACO1 and ACO2 at temperatures lower than 400 °C and around 650 °C is higher than that AC, indicating carboxylic acids and lactones of AC surface are increased by HNO3 oxidationt; more CO2 and CO released from ACO1 and ACO2 around 550 °C account for decomposition of more anhydrides; more CO released from ACO1 and ACO2 than that AC at around 700 °C and beyond 850 °C shows that more phenolic groups, carbonyl groups and quinones of AC were formed by HNO3 treatment. Carboxylic acids, lactones, anhydrides, phenolic groups, carbonyl groups and quinones introduced by HNO3 treatment can be analyzed by TPD. It should be noted that some nitrogen-containing functional groups were also produced during HNO3 oxidationt. However, these nitrogen-containing groups can not be better monitored and analyzed due to disturbance of other gases produced in heating. For example, mass charge ratio of 46 can represent NO2 and CO2, so NO2 curve of AC is frequently disturbed by CO2 released in heating. Furthermore, the information of surface groups of samples obtained by TPD is an indirect analysis result because groups to determine are ascribed only to the CO2 and CO released from decomposition of groups at Therefore, it draws a conclusion that part of surface groups (the acidic groups such as carboxylic, lactonic, phenolic groups and the basic groups) of AC are determined by TPD, but it does not directly reflect surface chemical properties of AC and only analyze the decomposition behavior of oxygen-containing functional groups. Characterization of surface 2.2.7 Water vapor adsorption by thermal gravimetric analysis/differential scanning To identify hydrophilic properties of the adsorbents, water vapor adsorption of the adsorbents was measured by a thermal gravimetric analyzer (TGA 409 PC, Netzsch). The 0.00E+000 5.00E-009 1.00E-008 1.50E-008 2.00E-008 2.50E-008 3.00E-008 MS intensity of CO(a.u.) (b) CO (m/e=28) 0 100 200 300 400 500 600 700 800 900 1000 Temperature (<sup>o</sup> ACO2 ACO1 AC ACD C) 0 100 200 300 400 500 600 700 800 900 1000 C) chemistry through TPD method should be further investigated. Temperature (o ACD ACO2 ACO1 0.00E+000 different temperature. calorimetry (Li, et al., 2009a) 1.00E-009 2.00E-009 MS intensity of CO2(a.u.) 3.00E-009 4.00E-009 (a) CO2 (m/e=44) Fig. 8. Water adsorption on adsorbents (AC, ACD, ACO1 and ACO2) (Li, et al., 2009a) Table 6. Comparisons of characterization methods of AC surface chemistry Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 185 Al-Asheh, S., Banat, F. & Abu-Aitah, L. (2003). Adsorption of phenol using different types of activated bentonites. Sep. Purif. Technol., Vol. 33, No. 1, pp. (1-10), 1383-5866 Andersson, K. I., Eriksson, M. & Norgren, M. (2011). Removal of lignin in wastewater Ayranci, E. & Duman, O. (2006). Adsorption of aromatic organic acids onto high area Azargohar, R. & Dalai, A. K. (2011). The direct oxidation of hydrogen sulphide over Bassin, J. P., Pronk, M., Kraan, R., Kleerebezem, R. & van Loosdrecht, M. C. M. (2011). Boehm, H. P. (1966). Chemical identification of surface groups. Advances in catalysis, Vol. 16, Bradley, R. H., Smith, M. W., Andreu, A. & Falco, M. (2011). Surface studies of novel Bundschuh, M., Pierstorf, R., Schreiber, W. H. & Schulz, R. (2011). Positive Effects of Chakravarty, S., Mohanty, A., Sudha, T. N., Upadhyay, A. K., Konar, J., Sircar, J. K., Chen, J. P., Wu, S. & Chong, K. H. (2003). Surface modification of a granular activated Chen, Y., Zhu, Y., Wang, Z., Li, Y., Wang, L., Ding, L., Gao, X., Ma, Y. & Guo, Y. (2011). Chingombe, P., Saha, B. & Wakeman, R. J. (2005). Surface modification and characterisation Çoruh, S., Geyikçi, F. & Ergun, O. N. (2011). 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Desalination, Vol. 275, measurement started with flowing Ar (99.99%, 50 mL/min) over 8.0 mg adsorbent at 40 °C, followed by replacing the Ar stream by water vapour-containing Ar stream ( H O2 P of 7.381 kPa) at steady state. The weight increase in the adsorbent after the introduction of water vapour is attributed to water adsorption. The TG and DSC data in Fig. 8 all show that water adsorption of the adsorbents increases from AC to ACD and then to ACO1 and to ACO2. Clearly, this trend corresponds to the changes in surface acid/basic properties; a higher surface acidity of an adsorbent is consistent with a higher water adsorption capability. Adsorption heat also verifies the trend. ## 2.3 Comparison of characterization methods Form the discussion in many examples, characterization methods mentioned above can be used to analyze physical texture and surface chemistry of AC. To further understand the methods, it is important to investigate the advantages and disadvantages between the methods, which were listed below. Comparisons of characterization methods of AC surface chemistry were shown in Table 6. N2 adsorption and SEM can be used to better analyze physical structures of AC. It should be noted that in analyzing surface chemistry of AC by Boehm, Pzc Titration and TPD, carboxylic, anhydride, lactonic groups and phenolic groups can be determined, but ketones, aldehydes, ethers, esters, pyrones and other functional nitrogen-containing groups can not be detected, which can be characterized by DRIFTS and XPS. DRIFTS and XPS have the greater advantage of determining nitrogen-containing functional groups than other methods mentioned above. ## 3. Conclusion There is the difficulty in analyzing its chemical properties of surface derived from the special nature of activated carbon itself, which is a black body and has complex components on surface, so the different characterization methods are proposed and used. Given the above characterization methods are not perfect in characterizing activated carbon surface chemistry, a variety of characterization methods complement each other to acquire more accurate and more comprehensive surface information. It is predicted that with the development of science and technology, these methods will be continually developed and improved and the nature of surface chemistry of AC will be further understood. ## 4. Acknowledgment The author expresses his grateful appreciation for the financial support from the Doctorate Science Fund of Taiyuan University of Science & Technology (20102001) ## 5. References Agrawal, S. G., King, K. W., Fischer, E. N. & Woner, D. N. (2011). PO4 3- Removal by and Permeability of Industrial Byproducts and Minerals: Granulated Blast Furnace Slag, Cement Kiln Dust, Coconut Shell Activated Carbon, Silica Sand, and Zeolite. Water Air Soil Poll, Vol. 219, No. 1-4, pp. (91-101), 0049-6979 measurement started with flowing Ar (99.99%, 50 mL/min) over 8.0 mg adsorbent at 40 °C, followed by replacing the Ar stream by water vapour-containing Ar stream ( H O2 P of 7.381 kPa) at steady state. The weight increase in the adsorbent after the introduction of water The TG and DSC data in Fig. 8 all show that water adsorption of the adsorbents increases from AC to ACD and then to ACO1 and to ACO2. Clearly, this trend corresponds to the changes in surface acid/basic properties; a higher surface acidity of an adsorbent is consistent with a higher water adsorption capability. Adsorption heat also verifies the trend. Form the discussion in many examples, characterization methods mentioned above can be used to analyze physical texture and surface chemistry of AC. To further understand the methods, it is important to investigate the advantages and disadvantages between the Comparisons of characterization methods of AC surface chemistry were shown in Table 6. N2 adsorption and SEM can be used to better analyze physical structures of AC. It should be noted that in analyzing surface chemistry of AC by Boehm, Pzc Titration and TPD, carboxylic, anhydride, lactonic groups and phenolic groups can be determined, but ketones, aldehydes, ethers, esters, pyrones and other functional nitrogen-containing groups can not be detected, which can be characterized by DRIFTS and XPS. DRIFTS and XPS have the greater advantage of determining nitrogen-containing functional groups than other methods There is the difficulty in analyzing its chemical properties of surface derived from the special nature of activated carbon itself, which is a black body and has complex components on surface, so the different characterization methods are proposed and used. Given the above characterization methods are not perfect in characterizing activated carbon surface chemistry, a variety of characterization methods complement each other to acquire more accurate and more comprehensive surface information. It is predicted that with the development of science and technology, these methods will be continually developed and The author expresses his grateful appreciation for the financial support from the Doctorate Agrawal, S. G., King, K. W., Fischer, E. N. & Woner, D. N. (2011). PO43- Removal by and Permeability of Industrial Byproducts and Minerals: Granulated Blast Furnace Slag, Cement Kiln Dust, Coconut Shell Activated Carbon, Silica Sand, and Zeolite. Water improved and the nature of surface chemistry of AC will be further understood. Science Fund of Taiyuan University of Science & Technology (20102001) Air Soil Poll, Vol. 219, No. 1-4, pp. (91-101), 0049-6979 vapour is attributed to water adsorption. 2.3 Comparison of characterization methods methods, which were listed below. mentioned above. 3. Conclusion 4. Acknowledgment 5. References Charaterization of Pore Structure and Surface Chemistry of Activated Carbons – A Review 187 Khan, A. R., Al-Bahri, T. A. & Al-Haddad, A. (1997a). Adsorption of phenol based organic Kilic, M., Apaydin-Varol, E. & Pütün, A. E. (2011). Adsorptive removal of phenol from Król, M., Gryglewicz, G. & Machnikowski, J. (2011). KOH activation of pitch-derived Kula, I., Ugurlu, M., Karaoglu, H. & elik, A. (2008). 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Activated carbons developed Matilainen, A., Vepsäläinen, M. & Sillanpää, M. (2010). Natural organic matter removal by Montazer-Rahmati, M. M., Rabbani, P., Abdolali, A. & Keshtkar, A. R. (2011). Kinetics and Namasivayam, C., Prabha, D. & Kumutha, M. (1998). Removal of direct red and acid Naumova, L. B., Minakova, T. S., Chernov, E. B., Gorlenko, N. P. & Ekimova, I. A. (2011). Russian Journal of Applied Chemistry, Vol. 84, No. 5, pp. (792-797), 1070-4272 Nevskaia, D. M., Castillejos-Lopez, E., Guerrero-Ruiz, A. & Mu oz, V. (2004). Effects of the Noh, J. S. & Schwarz, J. A. (1990). Effect of HNO3 treatment on the surface acidity of Okolo, B., Park, C. & Keane, M. A. (2000). Interaction of phenol and chlorophenols with Padoley, K. V., Mudliar, S. N., Banerjee, S. K., Deshmukh, S. C. & Pandey, R. A. (2011). Parab, H., Joshi, S., Sudersanan, M., Shenoy, N., Lali, A. & Sarma, U. (2010). Removal and Radovic, L. R., Moreno-Castilla, C. & Rivera-Utrilla, J. (2001). 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A series of advances, Vol. 27, No. pp. (227-405), 9 Colombia Bioleaching of Galena (PbS) National University of Colombia, Medellín AA 1027, 2Solid State Group, University Research Centre Antioquia University, Medellín AA 1226, E. R. Mejía1, J. D. Ospina1, M. A. Márquez1 and A. L. Morales2 1Materials Engineering School, Applied Mineralogy and Bio-process Group (GMAB) Microbial leaching of metals from sulfide minerals has been widely studied (Marsden & House, 1992; Brierley e Luinstra, 1993; Partha & Nataraja, 2006; Watling, 2006; Al-Harahssheh et al., 2006). However, few studies have reported on the bacterial oxidation of galena (PbS) to insoluble lead sulfate (Santhiya et al., 2000; Da Silva, 2004a and b). Formation of lead sulfate prevents the recovery of lead from traditional solvent extraction via electrowinning routes Galena is a mineral of vast industrial importance, not only for being the world's main source of lead, but also for being a semiconducting material with a band gap around of 0.4 eV (Muscat et al., 2003). Sulfide materials are also of interest from an environmental perspective In contrast to studies on galena, great attention has been paid to the bioleaching of sphalerite ((Zn,Fe)S) (Muscat & Gale, 2003). This interest stems from the increasing need to process grade ores of mixed mineralogy (Da Silva, 2004b; Muscat & Gale, 2003). One particular problem is the common association of sphalerite with galena, especially at fine particle sizes, which could complicate the differential flotation of the two minerals (Da Silva, 2004; Liao & Deng, 2004; Bolorunduro et al., 2003). Although the kinetics and mechanism of sphalerite bioleaching are well known, they are not completely understood (Da Silva, 2004a,b; Boon et al., 1998; Paar et al., 1984; Rodrigez et al., 2003; Zapata et al., Two different minerals can be selectively bioleached with galvanic interactions where the mineral with lower rest potential is sacrificed and that with higher potential is passivated (Das et al., 1999; Suzuki, 2001; Da Silva, 2004a,b; Abraitis et al., 2004; Cruz et al., 2005; Urbano The mechanism of galena oxidation is important in flotation processes, where mineral oxidation, through the grinding/flotation circuit, can affect its hydrophobicity and, therefore, its interaction with surfactants (Da Silva, 2004; Jañezuk et al., 1993; Nowak et al., 2000; Peng et al., 2002). However, the pretreatment of refractory ores to recover metals from lower-grade sulfide ores or refractory minerals is unusual in Colombia (Muñoz et al., 2003). as they are a major cause of water system acidification in mining operations. 1. Introduction (Da Silva, 2004b). 2007). et al., 2007). ## Bioleaching of Galena (PbS) E. R. Mejía1, J. D. Ospina1, M. A. Márquez1 and A. L. Morales2 1Materials Engineering School, Applied Mineralogy and Bio-process Group (GMAB) National University of Colombia, Medellín AA 1027, 2Solid State Group, University Research Centre Antioquia University, Medellín AA 1226, Colombia ## 1. Introduction 190 Fourier Transform – Materials Analysis Vinodh, R., Padmavathi, R. & Sangeetha, D. (2011). Separation of heavy metals from water Viraraghavan, T. & de Maria Alfaro, F. (1998). Adsorption of phenol from wastewater by Wang, J. C., Liu, Q. Y., Liu, Z. Y. & Huang, Z. G. (2008). Heterogeneity of V2O5 supported Wang, K., Zhang, Y., Qi, R., Yang, M. & Deng, R. (2006). Effects of activated carbon surface Wang, S. & Zhu, Z. H. (2007). Effects of acidic treatment of activated carbons on dye Wang, X., Hu, X., Hu, C. & Wei, D. (2011). Sequential use of ultraviolet light and chlorine for Wibowo, N., Setyadhi, L., Wibowo, D., Setiawan, J. & Ismadji, S. (2007). Adsorption of Zahrim, A. Y., Tizaoui, C. & Hilal, N. (2011). Coagulation with polymers for nanofiltration Zhang, C., Tezel, U., Li, K., Liu, D., Ren, R., Du, J. & Pavlostathis, S. G. (2011a). Evaluation Zhang, Y., Xu, S., Luo, Y., Pan, S., Ding, H. & Li, G. (2011b). Synthesis of mesoporous carbon Chemical Industry and Engineering, Vol. 57, No. 007, pp. (1659-1663), adsorption. Dyes Pigments, Vol. 75, No. 2, pp. (306-314), 0143-7208 Hazardous Materials, Vol. 146, No. 1-2, pp. (237-242), 0304-3894 sludge. Water Res., Vol. 45, No. 3, pp. (1238-1246), 0043-1354 Zhong, H. (1984). Introduction to Infrared Spectroscopy Chemical Industry Press 267, No. 2, pp. (267-276), 0011-9164 Vol. 31, No. 7, pp. (1056-1061), 1521-4125 0742 pp. (1-16), 0011-9164 samples using anion exchange polymers by adsorption process. Desalination, Vol. peat, fly ash and bentonite. J. Hazard. Mater., Vol. 57, No. 1-3, pp. (59-70), 0304-3894 cylindrical activated coke used for SO2 removal from flue gas. Chem. Eng. Technol., chemistry and pore structure on adsorption of HAAs from water. Journal of reclaimed water disinfection. J Environ Sci, Vol. 23, No. 10, pp. (1605-1610), 1001- benzene and toluene from aqueous solutions onto activated carbon and its acid and heat treated forms: Influence of surface chemistry on adsorption. Journal of pre-treatment of highly concentrated dyes: A review. Desalination, Vol. 266, No. 1, and modeling of benzalkonium chloride inhibition and biodegradation in activated capsules encapsulated with magnetite nanoparticles and their application in wastewater treatment. J. Mater. Chem, Vol. 21, No. pp. (3664-3671), 0959-9428 Microbial leaching of metals from sulfide minerals has been widely studied (Marsden & House, 1992; Brierley e Luinstra, 1993; Partha & Nataraja, 2006; Watling, 2006; Al-Harahssheh et al., 2006). However, few studies have reported on the bacterial oxidation of galena (PbS) to insoluble lead sulfate (Santhiya et al., 2000; Da Silva, 2004a and b). Formation of lead sulfate prevents the recovery of lead from traditional solvent extraction via electrowinning routes (Da Silva, 2004b). Galena is a mineral of vast industrial importance, not only for being the world's main source of lead, but also for being a semiconducting material with a band gap around of 0.4 eV (Muscat et al., 2003). Sulfide materials are also of interest from an environmental perspective as they are a major cause of water system acidification in mining operations. In contrast to studies on galena, great attention has been paid to the bioleaching of sphalerite ((Zn,Fe)S) (Muscat & Gale, 2003). This interest stems from the increasing need to process grade ores of mixed mineralogy (Da Silva, 2004b; Muscat & Gale, 2003). One particular problem is the common association of sphalerite with galena, especially at fine particle sizes, which could complicate the differential flotation of the two minerals (Da Silva, 2004; Liao & Deng, 2004; Bolorunduro et al., 2003). Although the kinetics and mechanism of sphalerite bioleaching are well known, they are not completely understood (Da Silva, 2004a,b; Boon et al., 1998; Paar et al., 1984; Rodrigez et al., 2003; Zapata et al., 2007). Two different minerals can be selectively bioleached with galvanic interactions where the mineral with lower rest potential is sacrificed and that with higher potential is passivated (Das et al., 1999; Suzuki, 2001; Da Silva, 2004a,b; Abraitis et al., 2004; Cruz et al., 2005; Urbano et al., 2007). The mechanism of galena oxidation is important in flotation processes, where mineral oxidation, through the grinding/flotation circuit, can affect its hydrophobicity and, therefore, its interaction with surfactants (Da Silva, 2004; Jañezuk et al., 1993; Nowak et al., 2000; Peng et al., 2002). However, the pretreatment of refractory ores to recover metals from lower-grade sulfide ores or refractory minerals is unusual in Colombia (Muñoz et al., 2003). Bioleaching of Galena (PbS) 193 A. ferrooxidans- and Acidithiobacillus thiooxidans-like strains were used in the bioleaching experiments. The strains were isolated by Cardona (2008). The microorganisms were previously grown in T&K medium by successive replacement of the ferrous sulfate with galena. The medium was acidified to pH 1.8 using H2SO4. The flasks were sterilized by autoclaving for 20 min at 120°C and 18 psi. The experiments were inoculated with A. ferrooxidans 10% (v/v) for the single culture and A. ferrooxidans 5% (v/v) and A. thiooxidans 5%(v/v) for the mixed culture. The experiments were carried out for 30 days in 500 mL shake flasks containing 300 mL of medium with 10% (w/v) galena at 180 rpm and 30ºC. All Measurements of pH (HACH HQ40d multi PHC30103) and redox potential (Shot Handylab 1 Pt 6880) in situ (reference electrode Ag0/AgCl) were performed daily. Samples were aseptically withdrawn from the flasks after 24 h and then every five days. The samples were separated in a DIAMOND IEC DIVISION centrifuge for 15 min at 3000 rpm. Iron and sulfate concentrations were measured using an ultraviolet-visible spectrophotometer GENESYS™ 10. The methods employed included 3500-FeD (O-phenanthroline) for ferrous Combinations of analytical techniques were used in the mineralogical characterization of the samples. The FTIR spectra of the solid samples were recorded by an FTIR spectrophotometer (Shimadzu Advantage 8400) using KBr pellets in transmission mode. A sample KBr mixture at a ratio of 1:200 was used. The total number of scans was 20, and a spectral resolution of 4 The biooxidation samples were mounted in epoxy resin and polished with sequentially finer SiC grit paper followed by a final polish with 0.05-µm sized alumina powder. Analysis of the polished sections was performed with a JEOL JSM 5910 LV SEM in backscattering electron mode with an energy dispersive X-ray (EDX) detector (Oxford Instruments), using a beam voltage of 18kV. XRD analyses of the samples were conducted on a Bruker D8ADVANCE diffractometer with Cu λ= 1.5406 Å radiation generated at 35 kV and 30 mA. XRD data were obtained using computer-controlled X-ray Diffractometer Panalytical X'Pert Pro MPD. The initial characterizations of the mineral-polished sections were performed by Variation on pH and redox potential (Eh) values for the inoculated systems and the abiotic controls are presented in Fig. 2. In order to prevent inhibition of the bacteria, H2SO4 was added to maintain the pH values around 2.0 until day 15. The pH values first increased and then decreased over time to levels around 1.1. The pH values in the abiotic controls conditions were duplicated and the respective abiotic control was included. and total iron according to the standard methods for water analysis. cm−1, a range of 400–4000 cm-1, and Happ-Henzel correction were used. 2.2 Bioleaching experiments 2.3 Chemical analysis 2.4 Mineralogical analysis optical microscopy of reflected light. stabilized at around 2.0 after day 15. 3.1 Galena leaching experiments using A. ferrooxidans 3. Results This leads to economic losses in mining processes, especially in subsistence mining. Their implementations in mining and metallurgical industries are also very attractive (Flower et al., 1999; Rohwerder et al., 2003; Olson et al., 2003). In this work, biological oxidation of galena was performed using Acidithiobacillus ferrooxidans-like bacteria and a mixed culture. Characterization techniques such as scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) were used to follow the morphologic and chemical changes occurring during the process. ## 2. Materials and methods #### 2.1 Minerals All experiments were carried out using a galena sample from an El Silencio miner, property of Frontino Gold Mines Company (Segovia, Antioquia, Colombia). The mineral was subjected to crushing and milling processes followed by gravimetric separation in a Wilfley table. Manual concentration using stereographic microscopy was then performed. The mineralogical composition of the concentrate measured by countdown points was 93.3% galena (PbS), 6.2% sphalerite (ZnS), and 0.5% chalcopyrite (CuFeS2) for -200 Tyler and 90% galena (PbS), 7.5% sphalerite (ZnS), 0.7% chalcopyrite (CuFeS2), and 1.8% gangue (SiO2), for -325 Tyler. An agate mortar was used to obtain two particle sizes: a pass through 200 Tyler mesh (~75 μm) and a pass through 325 Tyler mesh (~45 μm). XRD results confirmed that galena was the principal mineral phase in both sizes (Fig. 1). The mineral was sterilized in a furnace at 80°C for 90 min. Fig. 1. X-ray spectra of the concentrates. Mineral pass through A) 200 and B) 325 Tyler mesh This leads to economic losses in mining processes, especially in subsistence mining. Their implementations in mining and metallurgical industries are also very attractive (Flower et In this work, biological oxidation of galena was performed using Acidithiobacillus ferrooxidans-like bacteria and a mixed culture. Characterization techniques such as scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) were used to follow the morphologic and chemical changes occurring All experiments were carried out using a galena sample from an El Silencio miner, property of Frontino Gold Mines Company (Segovia, Antioquia, Colombia). The mineral was subjected to crushing and milling processes followed by gravimetric separation in a Wilfley table. Manual concentration using stereographic microscopy was then performed. The mineralogical composition of the concentrate measured by countdown points was 93.3% galena (PbS), 6.2% sphalerite (ZnS), and 0.5% chalcopyrite (CuFeS2) for -200 Tyler and 90% galena (PbS), 7.5% sphalerite (ZnS), 0.7% chalcopyrite (CuFeS2), and 1.8% gangue (SiO2), for -325 Tyler. An agate mortar was used to obtain two particle sizes: a pass through 200 Tyler mesh (~75 μm) and a pass through 325 Tyler mesh (~45 μm). XRD results confirmed that galena was the principal mineral phase in both sizes (Fig. 1). The mineral was sterilized in a Gn Gn Qz 2-Theta - Scale 15 20 30 40 50 B. Galena 325 Gn SpySpy A. Galena 200 Gn Spy Lin (Counts) 50 Fig. 1. X-ray spectra of the concentrates. Mineral pass through A) 200 and B) 325 Tyler mesh 0 1000 2000 3000 4000 5000 6000 7000 8000 2-Theta - Scale Arg 16 20 30 40 Spy Gn Qz Arg Qz al., 1999; Rohwerder et al., 2003; Olson et al., 2003). during the process. 2.1 Minerals Lin (Counts) 2000 3000 4000 5000 0 1000 2. Materials and methods furnace at 80°C for 90 min. Gn ## 2.2 Bioleaching experiments A. ferrooxidans- and Acidithiobacillus thiooxidans-like strains were used in the bioleaching experiments. The strains were isolated by Cardona (2008). The microorganisms were previously grown in T&K medium by successive replacement of the ferrous sulfate with galena. The medium was acidified to pH 1.8 using H2SO4. The flasks were sterilized by autoclaving for 20 min at 120°C and 18 psi. The experiments were inoculated with A. ferrooxidans 10% (v/v) for the single culture and A. ferrooxidans 5% (v/v) and A. thiooxidans 5%(v/v) for the mixed culture. The experiments were carried out for 30 days in 500 mL shake flasks containing 300 mL of medium with 10% (w/v) galena at 180 rpm and 30ºC. All conditions were duplicated and the respective abiotic control was included. ## 2.3 Chemical analysis Measurements of pH (HACH HQ40d multi PHC30103) and redox potential (Shot Handylab 1 Pt 6880) in situ (reference electrode Ag0/AgCl) were performed daily. Samples were aseptically withdrawn from the flasks after 24 h and then every five days. The samples were separated in a DIAMOND IEC DIVISION centrifuge for 15 min at 3000 rpm. Iron and sulfate concentrations were measured using an ultraviolet-visible spectrophotometer GENESYS™ 10. The methods employed included 3500-FeD (O-phenanthroline) for ferrous and total iron according to the standard methods for water analysis. ## 2.4 Mineralogical analysis Combinations of analytical techniques were used in the mineralogical characterization of the samples. The FTIR spectra of the solid samples were recorded by an FTIR spectrophotometer (Shimadzu Advantage 8400) using KBr pellets in transmission mode. A sample KBr mixture at a ratio of 1:200 was used. The total number of scans was 20, and a spectral resolution of 4 cm−1, a range of 400–4000 cm-1, and Happ-Henzel correction were used. The biooxidation samples were mounted in epoxy resin and polished with sequentially finer SiC grit paper followed by a final polish with 0.05-µm sized alumina powder. Analysis of the polished sections was performed with a JEOL JSM 5910 LV SEM in backscattering electron mode with an energy dispersive X-ray (EDX) detector (Oxford Instruments), using a beam voltage of 18kV. XRD analyses of the samples were conducted on a Bruker D8ADVANCE diffractometer with Cu λ= 1.5406 Å radiation generated at 35 kV and 30 mA. XRD data were obtained using computer-controlled X-ray Diffractometer Panalytical X'Pert Pro MPD. The initial characterizations of the mineral-polished sections were performed by optical microscopy of reflected light. ## 3. Results ## 3.1 Galena leaching experiments using A. ferrooxidans Variation on pH and redox potential (Eh) values for the inoculated systems and the abiotic controls are presented in Fig. 2. In order to prevent inhibition of the bacteria, H2SO4 was added to maintain the pH values around 2.0 until day 15. The pH values first increased and then decreased over time to levels around 1.1. The pH values in the abiotic controls stabilized at around 2.0 after day 15. Bioleaching of Galena (PbS) 195 Fig. 4. Changes in Fe2- and Fe3+ during the bacterial oxidation process. Graph legend: 200F, test with Acidithiobacillus ferrooxidans and 200 Tyler mesh; 325F, test with A. ferrooxidans and 325 Tyler mesh; 200FT, test with consortium and 200 Tyler mesh; 325FT, test with consortium and 325 Tyler mesh; 200C, inoculate test with 200 Tyler mesh; and 325C, inoculate test with In the abiotic experiments, ferrous iron increased between day 5 and 10 and then remained stationary until the end of the process. The Fe3+ increased from day 10 to day 15, became stationary until day 25, and finally decreased sharply to about 1 ppm, where it remained until the end of the experiment. Lead extraction was around 57% for all texts. On the other Fig. 5. Galena oxidation during the bacterial oxidation process. Graph legend: 200F, test with Acidithiobacillus ferrooxidans and 200 Tyler mesh; 325F, test with A. ferrooxidans and 325 Tyler mesh; 200FT, test with consortium and 200 Tyler mesh; 325FT, test with consortium and 325 Tyler mesh; 200C, inoculate test with 200 Tyler mesh; and 325C, inoculate test with 325 Tyler hand, <5% of Pb was solubilized in the chemical controls (Fig. 5). 325 Tyler mesh mesh Fig. 2. Changes in redox potential and pH during the bacterial oxidation process. Graph legend: 200F, test with Acidithiobacillus ferrooxidans and 200 Tyler mesh; 325F, test with A. ferrooxidans and 325 Tyler mesh; 200FT, test with consortium and 200 Tyler mesh; 325FT, test with consortium and 325 Tyler mesh; 200C, inoculate test with 200 Tyler mesh; and 325C, inoculate test with 325 Tyler mesh The variation in redox potential in both cultures with different grain sizes presented a relatively low redox potential at the beginning of the test until day 11 followed by an increase and finally a stationary phase with a small decrease with time. Eh values in the abiotic controls were around 284 mV throughout the processes. The SO4 2- concentration in the solution as well as in the solid phase gradually increased with time. However, the increase of sulfate concentration was greater in the solid phase than in the solution (Fig. 3). Fig. 3. Changes in SO42- during the bacterial oxidation process. Graph legend: 200F, test with Acidithiobacillus ferrooxidans and 200 Tyler mesh; 325F, test with A. ferrooxidans and 325 Tyler mesh; 200FT, test with consortium and 200 Tyler mesh; 325FT, test with consortium and 325 Tyler mesh; 200C, inoculate test with 200 Tyler mesh; and 325C, inoculate test with 325 Tyler mesh The dissolution of Fe2+ increased and reached a maximum during days 6–15. It then decreased sharply, becoming somewhat stationary with a small decrease on day 25 (Fig. 4). Fig. 2. Changes in redox potential and pH during the bacterial oxidation process. Graph legend: 200F, test with Acidithiobacillus ferrooxidans and 200 Tyler mesh; 325F, test with A. ferrooxidans and 325 Tyler mesh; 200FT, test with consortium and 200 Tyler mesh; 325FT, test with consortium and 325 Tyler mesh; 200C, inoculate test with 200 Tyler mesh; and 325C, The variation in redox potential in both cultures with different grain sizes presented a relatively low redox potential at the beginning of the test until day 11 followed by an increase and finally a stationary phase with a small decrease with time. Eh values in the the solution as well as in the solid phase gradually increased with time. However, the increase of sulfate concentration was greater in the solid phase than in the solution (Fig. 3). Fig. 3. Changes in SO42- during the bacterial oxidation process. Graph legend: 200F, test with Acidithiobacillus ferrooxidans and 200 Tyler mesh; 325F, test with A. ferrooxidans and 325 Tyler mesh; 200FT, test with consortium and 200 Tyler mesh; 325FT, test with consortium and 325 Tyler mesh; 200C, inoculate test with 200 Tyler mesh; and 325C, inoculate test with 325 Tyler The dissolution of Fe2+ increased and reached a maximum during days 6–15. It then decreased sharply, becoming somewhat stationary with a small decrease on day 25 (Fig. 4). 2- concentration in abiotic controls were around 284 mV throughout the processes. The SO4 inoculate test with 325 Tyler mesh mesh Fig. 4. Changes in Fe2- and Fe3+ during the bacterial oxidation process. Graph legend: 200F, test with Acidithiobacillus ferrooxidans and 200 Tyler mesh; 325F, test with A. ferrooxidans and 325 Tyler mesh; 200FT, test with consortium and 200 Tyler mesh; 325FT, test with consortium and 325 Tyler mesh; 200C, inoculate test with 200 Tyler mesh; and 325C, inoculate test with 325 Tyler mesh In the abiotic experiments, ferrous iron increased between day 5 and 10 and then remained stationary until the end of the process. The Fe3+ increased from day 10 to day 15, became stationary until day 25, and finally decreased sharply to about 1 ppm, where it remained until the end of the experiment. Lead extraction was around 57% for all texts. On the other hand, <5% of Pb was solubilized in the chemical controls (Fig. 5). Fig. 5. Galena oxidation during the bacterial oxidation process. Graph legend: 200F, test with Acidithiobacillus ferrooxidans and 200 Tyler mesh; 325F, test with A. ferrooxidans and 325 Tyler mesh; 200FT, test with consortium and 200 Tyler mesh; 325FT, test with consortium and 325 Tyler mesh; 200C, inoculate test with 200 Tyler mesh; and 325C, inoculate test with 325 Tyler mesh Bioleaching of Galena (PbS) 197 SEM images of leached galena are shown in Figs. 7, 8, and 9. All of the samples had corrosion features such as pits and gulfs on the grain surfaces (Figs. 7H and 7I). Moreover, coarse particle size porous films coating grains (Figs. 7C, 7F, and 7L), and aciculate precipitates of anglesite (Figs. 7D and 7L) were observed. These characteristics became observable on day 7 of the process (Figs. 7A, 7B, 7G, and 7H) and were more evident with time. After day 15, the galena grains were coated with anglesite film with porous texture (Figs. 7C and 7K). The anglesite aciculate formations were more evident at the end of the process (Fig. 7L). It is important to note the passivating effect that galena has on sphalerite and pyrite in which the pyrite and sphalerite grains did not show evidence of oxidation Fig. 7. Scanning electron microscopy micrograph of the residues after galena bioleaching by passing through 200 (A, B, C, D, E, and F) and 325 (G, H, I, J, K, and L) Tyler mesh. Gn, galena; Ang, anglesite; Py, pyrite. A) Galena grain and aciculate anglesite precipitates (arrows indicate corrosion gulfs on day 5). B) Galena grain in an incipient oxidation state (day 5). C) Galena grain covered and coated with anglesite porous film and pyrite grain without apparent oxidation (day 15). D) Anglesite grains aciculate and anhedrals, quartz grains without apparent oxidation and pyrite grains in the insipient oxidation state (day 15). E) Galena grain oxidant along the cleavage plane (arrows indicate cleavage plane) and anglesite grain (day 30). F) Galena grain covered and coated with anglesite porous film and pyrite grain without apparent oxidation (day 30). G) Galena grain corroded in gulfs and cleavage plane (day 5). H) Galena grain in an incipient oxidation state showed gulfs of corrosion (day 5). I) Galena grain corroded with anglesite cavity formation (day 15). J) Galena grain covered and coated with anglesite porous film and pyrite grain without apparent oxidation (day 15). K) The anglesite was porous and had a remaining galena nucleus. L) Anglesite grains aciculate and anhedrals and galena grains coated with anglesite porous film and sphalerite grain without apparent oxidation 3.3 SEM/EDX (Figs. 7C, 7D, 7J, 7F, and 7L). ### 3.2 FTIR Results obtained by FTIR for the bioleached galena samples showed typical bands of anglesite, the main mineral product of the process, with absorption bands at 950–1000, 1165– 1765, 1115–1125, 1050–1060, and 592–620 cm-1 (Chernyshova, 2003). Scotlandite (PbSO3) was also identified by bands at 920, 870, 970, and 600–620 cm-1 (Paar et al., 1984; Chernyshova, 2003). The bands around 2935 cm-1 related to the total carbon present on the cell surface increased permanently (Naumann & Helm, 1995; Sharma & Hynumantha, 2005; Xia et al., 2008).The FTIR spectra showed at the beginning of the process, for all tests, an increased numbers of anglesite and scontlandite bands. The tests conducted using A. ferrooxidans with different grain sizes presented a continuous increase in the bands of the anglesite and scontlandite until day 10, followed the strong band increases. This was possibly due to the strong anglesite and scontlandite precipitate. It then increased continuously until day 20. Finally, the typical bands of anglesite and scontlandite showed a strong increase (Figs. 6a and 6b). However, the FTIR spectra for the test conducted using the mixed culture with different grain sizes presented continuous increases in the anglesite and scontlandite bands throughout the process (Figs. 6c and 6d). Fig. 6. Fourier transform infrared spectra of solid residues after galena bioleaching. OM, organic matter; SO42-: anglesite ## 3.3 SEM/EDX 196 Fourier Transform – Materials Analysis Results obtained by FTIR for the bioleached galena samples showed typical bands of anglesite, the main mineral product of the process, with absorption bands at 950–1000, 1165– 1765, 1115–1125, 1050–1060, and 592–620 cm-1 (Chernyshova, 2003). Scotlandite (PbSO3) was also identified by bands at 920, 870, 970, and 600–620 cm-1 (Paar et al., 1984; Chernyshova, 2003). The bands around 2935 cm-1 related to the total carbon present on the cell surface increased permanently (Naumann & Helm, 1995; Sharma & Hynumantha, 2005; Xia et al., 2008).The FTIR spectra showed at the beginning of the process, for all tests, an increased The tests conducted using A. ferrooxidans with different grain sizes presented a continuous increase in the bands of the anglesite and scontlandite until day 10, followed the strong band increases. This was possibly due to the strong anglesite and scontlandite precipitate. It then increased continuously until day 20. Finally, the typical bands of anglesite and scontlandite showed a strong increase (Figs. 6a and 6b). However, the FTIR spectra for the test conducted using the mixed culture with different grain sizes presented continuous increases in the anglesite and scontlandite bands throughout the process (Figs. 6c and 6d). Fig. 6. Fourier transform infrared spectra of solid residues after galena bioleaching. OM, 3.2 FTIR numbers of anglesite and scontlandite bands. organic matter; SO42-: anglesite SEM images of leached galena are shown in Figs. 7, 8, and 9. All of the samples had corrosion features such as pits and gulfs on the grain surfaces (Figs. 7H and 7I). Moreover, coarse particle size porous films coating grains (Figs. 7C, 7F, and 7L), and aciculate precipitates of anglesite (Figs. 7D and 7L) were observed. These characteristics became observable on day 7 of the process (Figs. 7A, 7B, 7G, and 7H) and were more evident with time. After day 15, the galena grains were coated with anglesite film with porous texture (Figs. 7C and 7K). The anglesite aciculate formations were more evident at the end of the process (Fig. 7L). It is important to note the passivating effect that galena has on sphalerite and pyrite in which the pyrite and sphalerite grains did not show evidence of oxidation (Figs. 7C, 7D, 7J, 7F, and 7L). Fig. 7. Scanning electron microscopy micrograph of the residues after galena bioleaching by passing through 200 (A, B, C, D, E, and F) and 325 (G, H, I, J, K, and L) Tyler mesh. Gn, galena; Ang, anglesite; Py, pyrite. A) Galena grain and aciculate anglesite precipitates (arrows indicate corrosion gulfs on day 5). B) Galena grain in an incipient oxidation state (day 5). C) Galena grain covered and coated with anglesite porous film and pyrite grain without apparent oxidation (day 15). D) Anglesite grains aciculate and anhedrals, quartz grains without apparent oxidation and pyrite grains in the insipient oxidation state (day 15). E) Galena grain oxidant along the cleavage plane (arrows indicate cleavage plane) and anglesite grain (day 30). F) Galena grain covered and coated with anglesite porous film and pyrite grain without apparent oxidation (day 30). G) Galena grain corroded in gulfs and cleavage plane (day 5). H) Galena grain in an incipient oxidation state showed gulfs of corrosion (day 5). I) Galena grain corroded with anglesite cavity formation (day 15). J) Galena grain covered and coated with anglesite porous film and pyrite grain without apparent oxidation (day 15). K) The anglesite was porous and had a remaining galena nucleus. L) Anglesite grains aciculate and anhedrals and galena grains coated with anglesite porous film and sphalerite grain without apparent oxidation Bioleaching of Galena (PbS) 199 Table 1. Energy dispersive spectrography (EDS) analysis of the residues after bioleaching of Initial XRD analyses revealed that galena was the main mineral phase present in the original samples, with small quantities of quartz (SiO2), sphalerite (ZnS), chalcopyrite (CuFeS2), and aragonite (Fig. 1). XRD spectra of bioleached samples are shown in Fig. 11. Mineralogical evolution of the mineral phases consists of a gradual reduction of galena peaks and the appearance of anglesite (PbSO4). Anglesite peaks were observed from day 5 onward for all samples. However, peak intensity was higher in concentrate passed through -325 Tyler mesh. The sphalerite peaks remained unchanged throughout the process. XRD for uninoculated controls showed minimal anglesite formation around day 30 (Fig. 10). (A) (B) galena; sph, sphalerite; and Ang, anglesite Fig. 10. X-ray diffractograms of inoculated samples after 30 days of the biooxidation process. A) Sample passed through -200 Tyler mesh; B) Sample passed through -325 Tyler mesh. Gn, galena 3.4 XRD analysis However, in some cases, we observed sphalerite and pyrite grains with oxidation grooves at the end of the process (Figs. 8A and 8B). Moreover, we observed remaining galena and anglesite nuclei with porous texture (Figs. 7K and 8B). On other hand, some galena grains showed oxidation along the cleavage planes (Figs. 7E and 7G). SEM images of the uninoculated samples for all tests (Figs. 8a and 8b) showed surfaces with a few alterations such as small oxidation in the cleavage planes. Fig. 8. Scanning electron microscopy micrograph of the residues after galena bioleaching. Gn, galena; Ang, anglesite; Py, pyrite; and Spy, sphalerite. A) Sphalerite and pyrite grains showed a typical corrosion groove and pits and porous anglesite. B) Pyrite grain with corrosion grooves, remaining galena nucleus, and porous anglesite Fig. 9. Scanning electron microscopy micrographs of uninoculated residues after galena bioleaching. A) Galena grain with insipient oxidation state along the cleavage plane (pass through 200 Tyler mesh) and anglesite aciculate on the galena surface. B) Galena grain with an incipient oxidation state along the cleavage plane (pass through 325 Tyler mesh), and pyrite without an oxidation state. Cpy, chalcopyrite; Qz: quartz EDX analysis of the grains showed the galena, anglesite, pyrite, and sphalerite stoichiometric composition (Table 1). However, in some cases, we observed sphalerite and pyrite grains with oxidation grooves at the end of the process (Figs. 8A and 8B). Moreover, we observed remaining galena and anglesite nuclei with porous texture (Figs. 7K and 8B). On other hand, some galena grains showed oxidation along the cleavage planes (Figs. 7E and 7G). SEM images of the uninoculated samples for all tests (Figs. 8a and 8b) showed surfaces with a few alterations Fig. 8. Scanning electron microscopy micrograph of the residues after galena bioleaching. Gn, galena; Ang, anglesite; Py, pyrite; and Spy, sphalerite. A) Sphalerite and pyrite grains showed a typical corrosion groove and pits and porous anglesite. B) Pyrite grain with Fig. 9. Scanning electron microscopy micrographs of uninoculated residues after galena bioleaching. A) Galena grain with insipient oxidation state along the cleavage plane (pass through 200 Tyler mesh) and anglesite aciculate on the galena surface. B) Galena grain with an incipient oxidation state along the cleavage plane (pass through 325 Tyler mesh), and EDX analysis of the grains showed the galena, anglesite, pyrite, and sphalerite corrosion grooves, remaining galena nucleus, and porous anglesite pyrite without an oxidation state. Cpy, chalcopyrite; Qz: quartz stoichiometric composition (Table 1). such as small oxidation in the cleavage planes. Table 1. Energy dispersive spectrography (EDS) analysis of the residues after bioleaching of galena ### 3.4 XRD analysis Initial XRD analyses revealed that galena was the main mineral phase present in the original samples, with small quantities of quartz (SiO2), sphalerite (ZnS), chalcopyrite (CuFeS2), and aragonite (Fig. 1). XRD spectra of bioleached samples are shown in Fig. 11. Mineralogical evolution of the mineral phases consists of a gradual reduction of galena peaks and the appearance of anglesite (PbSO4). Anglesite peaks were observed from day 5 onward for all samples. However, peak intensity was higher in concentrate passed through -325 Tyler mesh. The sphalerite peaks remained unchanged throughout the process. XRD for uninoculated controls showed minimal anglesite formation around day 30 (Fig. 10). Fig. 10. X-ray diffractograms of inoculated samples after 30 days of the biooxidation process. A) Sample passed through -200 Tyler mesh; B) Sample passed through -325 Tyler mesh. Gn, galena; sph, sphalerite; and Ang, anglesite Bioleaching of Galena (PbS) 201 2H2S + O2 bacteria→ 2S + 2H2O (2) Furthermore, this study suggests that galena biooxidation also produces anglesite (PbSO4) This finding is in agreement with the results obtained in this work in which Pb2+ was released from galena and precipitated as lead sulfate. Nevertheless, in accordance with the results obtained by FTIR, where there was evidence of the presence of scontlandite (PbSO3), it is possible that the anglesite was not the only sulfate mineral phase and was able to On other hand, the solubility of Pb2+ is very low, around 45 ppm (Mousavi et al., 2006), and the SO42- increased gradually in the solid and liquid phases (Fig. 3), being higher in solids (PbSO4) and scontlandite (PbSO3) as shown in equations 6 and 7. Moreover, anglesite (PbSO4) was detected in the residual solid and increased over time according to FTIR, XRD, Pb2+ + SO42- → PbSO4 (6) Pb2+ + SO32- → PbSO3 (7) However, elemental sulfur was not detected by SEM, XRD, or FTIR. Moreover, the pH (Fig. 2) decreased after six days around to 1.3 for all tests after it was 1.8, indicating that the increased H+ concentration was produced by bacterial activity. This behavior was due to the galena dissolution in an acid environment occurring as a result of the protonation of the mineral surface. The only protonation mechanism that has been proven to be energetically favorable in aqueous solution consists of the attachment of three H+ onto three surface S atoms surrounding a central Pb atom, which is then replaced by a fourth This work detected few changes in chemical controls in galena oxidation and SO42- in solid and liquid concentration at the beginning of the process, indicating that galena dissolution was favored in acid media via a purely chemical mechanism. This finding is in agreement The bacteria oxidize H2S, generating elemental sulfur and water (equation 1). Elemental 2H2S + O2 bacteria→ 2S + 2H2O (2) The H+ reattach to the mineral and, thus, generate a cycle. This resulted in great dissolution with those of Gerson & O'Deo (2003) and Acero et al. (2007). sulfur is also oxidized by bacteria producing sulfates and H+ (equation 2). in the inoculate tests (around 57%) compared with the uninoculated test. and SEM analysis. This phenomenon can be represented by the following equations: by reacting with sulfuric acid as shown in equation 1. generate other PbS mineral phases as accessories. (Fig. 4). This finding indicates that Pb2+ and SO4 H+ (equation 1). 2S + 2H2O + 3O2 bacteria → 2SO42- + 4H+ (3) PbS + H2SO4 +0.5O2 → PbSO4 + H2O + S0 (4) 2PbS + H2SO4 + 3/2O2 → 2PbSO3 + H2O + S0 (5) PbS + H2SO4→ PbSO4 +H2S (1) 2- or SO32- ions react to form anglesite Fig. 11. X-ray diffraction spectra for galena before the bioleaching process. A. Particle size using 200 Tyler mesh for pure culture. B. Particle size using 325 Tyler mesh for the pure culture. C. Particle size using 200 Tyler mesh for the mixed culture. D. Particle size using 325 Tyler mesh for the mixed culture. Gn, galena; sph, sphalerite; Qz, quartz; and Ang, anglesite ### 4. Discussion #### 4.1 Galena leaching experiment A. ferrooxidans-like bacteria showed good adaptation on galena with a high oxidative capacity (SEM, FTIR, XRD, and chemical data) since the microorganism was grown in a mineral concentrate that was the only source of energy. Jiang et al. (2008) stated that the bacteria may directly oxidize galena by taking energy from it. However, several authors suggest that A. ferrooxidans does not have a direct effect on galena oxidation; rather, it only indirectly acts on it via oxidizing hydrogen sulfide (H2S) and sulfur (Da Silva 2004; Muscat & Gale, 2003; Garcia et al., 1995). Thus, A. ferrooxidans utilizes H2S dissolved in the solution as an energy source (Dutrizac & Chen, 1995; Mizoguchi & Habashi, 1981) according to the following equations: $$\text{PbS} + \text{H}\_2\text{SO}\_4 \rightarrow \text{PbSO}\_4 + \text{H}\_2\text{S} \tag{1}$$ Fig. 11. X-ray diffraction spectra for galena before the bioleaching process. A. Particle size using 200 Tyler mesh for pure culture. B. Particle size using 325 Tyler mesh for the pure culture. C. Particle size using 200 Tyler mesh for the mixed culture. D. Particle size using 325 Tyler mesh for the mixed culture. Gn, galena; sph, sphalerite; Qz, quartz; and Ang, anglesite A. ferrooxidans-like bacteria showed good adaptation on galena with a high oxidative capacity (SEM, FTIR, XRD, and chemical data) since the microorganism was grown in a mineral concentrate that was the only source of energy. Jiang et al. (2008) stated that the bacteria may directly oxidize galena by taking energy from it. However, several authors suggest that A. ferrooxidans does not have a direct effect on galena oxidation; rather, it only indirectly acts on it via oxidizing hydrogen sulfide (H2S) and sulfur (Da Silva 2004; Muscat & Gale, 2003; Garcia et al., 1995). Thus, A. ferrooxidans utilizes H2S dissolved in the solution as an energy source (Dutrizac & Chen, 1995; Mizoguchi & Habashi, 1981) according to the PbS + H2SO4→ PbSO4 +H2S (1) 4. Discussion following equations: 4.1 Galena leaching experiment $$2\text{H}\_2\text{S} + \text{O}\_2\text{\text{\textquotedblleft}Cartan} \xrightarrow{\text{\textquotedblleft}Con\text{\textquotedblright}}2\text{S} + 2\text{H}\_2\text{O}\tag{2}$$ $$2\text{S} + 2\text{H}\_2\text{O} + 3\text{O}\_2 \text{ bacteria} \rightarrow 2\text{SO}\_4{}^{2-} + 4\text{H}^\* \tag{3}$$ Furthermore, this study suggests that galena biooxidation also produces anglesite (PbSO4) by reacting with sulfuric acid as shown in equation 1. This finding is in agreement with the results obtained in this work in which Pb2+ was released from galena and precipitated as lead sulfate. Nevertheless, in accordance with the results obtained by FTIR, where there was evidence of the presence of scontlandite (PbSO3), it is possible that the anglesite was not the only sulfate mineral phase and was able to generate other PbS mineral phases as accessories. On other hand, the solubility of Pb2+ is very low, around 45 ppm (Mousavi et al., 2006), and the SO4 2- increased gradually in the solid and liquid phases (Fig. 3), being higher in solids (Fig. 4). This finding indicates that Pb2+ and SO4 2- or SO3 2- ions react to form anglesite (PbSO4) and scontlandite (PbSO3) as shown in equations 6 and 7. Moreover, anglesite (PbSO4) was detected in the residual solid and increased over time according to FTIR, XRD, and SEM analysis. This phenomenon can be represented by the following equations: $$\text{PbS} + \text{H}\_2\text{SO}\_4 + 0.5\text{O}\_2 \rightarrow \text{PbSO}\_4 + \text{H}\_2\text{O} + \text{S}^\text{0} \tag{4}$$ $$2\text{PbS} + \text{H}\_2\text{SO}\_4 + 3/2\text{O}\_2 \to 2\text{PbSO}\_3 + \text{H}\_2\text{O} + \text{S}^\text{yl} \tag{5}$$ $$\text{Pb}^{2+} + \text{SO}\_4{}^{2-} \rightarrow \text{PbSO}\_4 \tag{6}$$ $$\text{Pb}^{2+} + \text{SO}\_3{2} \rightarrow \text{PbSO}\_3 \tag{7}$$ However, elemental sulfur was not detected by SEM, XRD, or FTIR. Moreover, the pH (Fig. 2) decreased after six days around to 1.3 for all tests after it was 1.8, indicating that the increased H+ concentration was produced by bacterial activity. This behavior was due to the galena dissolution in an acid environment occurring as a result of the protonation of the mineral surface. The only protonation mechanism that has been proven to be energetically favorable in aqueous solution consists of the attachment of three H+ onto three surface S atoms surrounding a central Pb atom, which is then replaced by a fourth H+ (equation 1). $$\text{PbS} + \text{H}\_2\text{SO}\_4 \rightarrow \text{PbSO}\_4 + \text{H}\_2\text{S} \tag{1}$$ This work detected few changes in chemical controls in galena oxidation and SO42- in solid and liquid concentration at the beginning of the process, indicating that galena dissolution was favored in acid media via a purely chemical mechanism. This finding is in agreement with those of Gerson & O'Deo (2003) and Acero et al. (2007). The bacteria oxidize H2S, generating elemental sulfur and water (equation 1). Elemental sulfur is also oxidized by bacteria producing sulfates and H+ (equation 2). $$2\text{H}\_2\text{S} + \text{O}\_2\text{\text{\textquotedblleft}Cartan} \rightarrow 2\text{S} + 2\text{H}\_2\text{O}\tag{2}$$ The H+ reattach to the mineral and, thus, generate a cycle. This resulted in great dissolution in the inoculate tests (around 57%) compared with the uninoculated test. Bioleaching of Galena (PbS) 203 Finally, mineralogical data showed the passivating effect of galena on pyrite and sphalerite (Figs. 7F and 7L, respectively), in which the latter, with a higher rest potential, is provided at the expense of galena oxidation, which acts as the sacrificial anode in agreement with earlier studies (Das et al., 1999; Suzuki, 2001; Da Silva 2004b; Abraitis et al., 2004; Cruz et al., 2005; Urbano et al., 2007). However, in some cases, dissolution was observed in pyrite and sphalerite grains (Figs. 8A and 8B). These data confirmed that Fe2+ lixiviation likely originates from the minor quantities of sphalerite and pyrite that are present in the sample concentrate. However, the rest potential of sphalerite is less than that of pyrite, which possibly indicates that iron leached into sphalerite, but high iron concentration in solution (Fig. 4) apparently indicates that the pyrite made an important contribution. Moreover, the iron content of sphalerite (around 8.6% weight) was smaller than that of pyrite (around 50.48% weight) (Table 1). ferrooxidans-like bacteria and mixed culture resulted in the following conclusions: 57%, whereas that of the control examination was only 6%. galena dissolution was favored. signal became more intense over time. molecular studies of the University of Antioquia. • The galena was initially dissolved by acidic medium. inside the grain. 6. Acknowledgement 7. References Examinations of the bioleaching of natural galena concentrate in T&K medium by A. • The bacteria have an impact on higher yield in the course of reaction of oxidizing PbS into PbSO4 in acid medium (1.8), and experiments were inoculated with Acidithiobacillus ferrooxidans 10(v/v)% and 5%(v/v) for the single culture and Acidithiobacillus thiooxidans 5%(v/v) with 10%(w/v) galena. Where there was released the lead from galena and precipitated as lead sulfate. The level of lixiviation after 30 days of bioleaching was • The Fe3+ favored the biolixiviation of galena because when its concentration increased, • The predominant new mineral phase was anglesite, a porous film that was formed on galena, but this film did not limit the access of the leaching agent and microorganisms • In the presence of bacteria, the XRD peaks corresponding to galena decreased and, at the same time, new peaks appeared, anglesite, during the bioleaching process. This • In both cultures, the microorganisms gradually modified the original galena surface, The authors would like to thank the biotechnology program of Colciencias, Colombia, the laboratory of biomineralogical of the National University of Colombia, Medellínd, Professor Diego Hernan Giraldo of the University of Antioquia, and the laboratory and group of Abraitis, P. K., Pattrick, R. A. D., Kelsall, G. H., Vaughan, D. J. 2004. Acid leaching and complex systems. Mineralogical Magazine. 68(2) 343–351 dissolution of major sulphide ore minerals: processes and galvanic effects in increasing the rest potential and the SO42- of the solid and liquid states. • The particle size was apparently not a determining factor in the process. 5. Conclusions On the other hand, on day 10, when the concentration of Fe3+ was high probably due to biooxidation of the minor quantities of sphalerite and pyrite (Fig. 4), galena oxidation was favored, possibly indicating that this ion contributes to the overall process efficiency. Jiang et al. (2007) found that ferric can oxidize galena and generate elemental sulfur according to the following reaction: $$\text{PbS} + 2\text{Fe}^{3+} \rightarrow \text{Pb}^{2+} + 2\text{ Fe}^{2+} + \text{S}^{0} \tag{8}$$ It is important to emphasize that the behaviors of the kinetic parameters analysis were similar for both culture types. This finding was probably due to the fact that the pH value was highly unstable at the beginning of the process, increasing to around 4.0, inhibiting A. thiooxidans. This suggested that this type of microorganism was unable to obtain energy from galena and required elemental sulfur addition as an additional energy source. On the other hand, the particle size used did not generate test differences. ## 4.2 Mineralogical analysis Mineralogical studies indicated that anglesite was the predominant mineral phase of galena biooxidation. The presence of anglesite was not clear, and evidence of passivation of the galena biooxidation process was seen in the galena oxidation curve, which was linear and increased throughout the process (Fig. 5), and the SEM images, where anglesite grains with remaining galena nuclei were observed on day 30 (Figs. 7K and 8B). The formation of this mineral phase was confirmed by the FTIR spectra, which showed increased numbers of anglesite and scontlandite bands at the beginning of the process for all tests, probably because anglesite and scontlandite present precipitate pulse (Fig. 6). This sharp increase was observed on days 10 and 20 for the tests using A. ferrooxidans, and its further slow increases on the other days of the process were consistent with sharp increases in the chemical sulfate data (Fig. 3). The FTIR spectra for the test conducted using the mixed culture presented continuously increased numbers of bands of anglesite and scontlandite throughout the process (Figs. 6C and 6D). On the other hand, the band increases at 2935 and 2847 cm-1 organic matter was possibly due to an increase in bacterial population, indicating bacterial activity (Naumann et al., 1995; Sharma et al., 2005; Xia et al., 2008). Furthermore, SEM analysis indicated that galena dissolution in the presence of the microorganisms occurred on the surface due to the presence of grain roughening, dissolution gulf formation (Figs. 7A and 7H), and preferential cleavage planes dissolution (Fig. 7E), all of which increase with time. The anglesite precipitation phenomenon occurs because the solubility limit of lead and sulfates in the medium is exceeded and could be mitigated by a sulfate concentration reduction in the medium. Moreover, SEM analysis revealed anglesite film precipitation on the galena grain from day 15 onward (Fig. 7C). The preferential oxidation of the galena cleavage planes was probably due to the fact that this region was more potentially favorable or more chemically reactive because this zone has higher surface energy and, therefore, was easily oxidized as demonstrated by Bennett and Tributsch (1978). XRD analysis (Fig. 10) indicated that anglesite (PbSO4) was formed at the expense of galena dissolution. In contrast, the galena in the control reaction system showed a few alterations. Nevertheless, dissolution of the minority phases as sphalerite can be observed. On the other hand, on day 10, when the concentration of Fe3+ was high probably due to biooxidation of the minor quantities of sphalerite and pyrite (Fig. 4), galena oxidation was favored, possibly indicating that this ion contributes to the overall process efficiency. Jiang et al. (2007) found that ferric can oxidize galena and generate elemental sulfur according to the It is important to emphasize that the behaviors of the kinetic parameters analysis were similar for both culture types. This finding was probably due to the fact that the pH value was highly unstable at the beginning of the process, increasing to around 4.0, inhibiting A. thiooxidans. This suggested that this type of microorganism was unable to obtain energy from galena and required elemental sulfur addition as an additional energy source. On the Mineralogical studies indicated that anglesite was the predominant mineral phase of galena biooxidation. The presence of anglesite was not clear, and evidence of passivation of the galena biooxidation process was seen in the galena oxidation curve, which was linear and increased throughout the process (Fig. 5), and the SEM images, where anglesite grains with remaining galena nuclei were observed on day 30 (Figs. 7K and 8B). The formation of this mineral phase was confirmed by the FTIR spectra, which showed increased numbers of anglesite and scontlandite bands at the beginning of the process for all tests, probably because anglesite and scontlandite present precipitate pulse (Fig. 6). This sharp increase was observed on days 10 and 20 for the tests using A. ferrooxidans, and its further slow increases on the other days of the process were consistent with sharp increases in the chemical sulfate data (Fig. 3). The FTIR spectra for the test conducted using the mixed culture presented continuously increased numbers of bands of anglesite and scontlandite throughout the On the other hand, the band increases at 2935 and 2847 cm-1 organic matter was possibly due to an increase in bacterial population, indicating bacterial activity (Naumann et al., 1995; Sharma et al., 2005; Xia et al., 2008). Furthermore, SEM analysis indicated that galena dissolution in the presence of the microorganisms occurred on the surface due to the presence of grain roughening, dissolution gulf formation (Figs. 7A and 7H), and preferential cleavage planes dissolution (Fig. 7E), all of which increase with time. The anglesite precipitation phenomenon occurs because the solubility limit of lead and sulfates in the medium is exceeded and could be mitigated by a sulfate concentration reduction in the medium. Moreover, SEM analysis revealed anglesite film precipitation on the galena grain The preferential oxidation of the galena cleavage planes was probably due to the fact that this region was more potentially favorable or more chemically reactive because this zone has higher surface energy and, therefore, was easily oxidized as demonstrated by Bennett and XRD analysis (Fig. 10) indicated that anglesite (PbSO4) was formed at the expense of galena dissolution. In contrast, the galena in the control reaction system showed a few alterations. Nevertheless, dissolution of the minority phases as sphalerite can be observed. other hand, the particle size used did not generate test differences. PbS + 2Fe3+ → Pb2+ + 2 Fe2+ + S0 (8) following reaction: 4.2 Mineralogical analysis process (Figs. 6C and 6D). from day 15 onward (Fig. 7C). Tributsch (1978). Finally, mineralogical data showed the passivating effect of galena on pyrite and sphalerite (Figs. 7F and 7L, respectively), in which the latter, with a higher rest potential, is provided at the expense of galena oxidation, which acts as the sacrificial anode in agreement with earlier studies (Das et al., 1999; Suzuki, 2001; Da Silva 2004b; Abraitis et al., 2004; Cruz et al., 2005; Urbano et al., 2007). However, in some cases, dissolution was observed in pyrite and sphalerite grains (Figs. 8A and 8B). These data confirmed that Fe2+ lixiviation likely originates from the minor quantities of sphalerite and pyrite that are present in the sample concentrate. However, the rest potential of sphalerite is less than that of pyrite, which possibly indicates that iron leached into sphalerite, but high iron concentration in solution (Fig. 4) apparently indicates that the pyrite made an important contribution. Moreover, the iron content of sphalerite (around 8.6% weight) was smaller than that of pyrite (around 50.48% weight) (Table 1). ## 5. Conclusions Examinations of the bioleaching of natural galena concentrate in T&K medium by A. ferrooxidans-like bacteria and mixed culture resulted in the following conclusions: ## 6. Acknowledgement The authors would like to thank the biotechnology program of Colciencias, Colombia, the laboratory of biomineralogical of the National University of Colombia, Medellínd, Professor Diego Hernan Giraldo of the University of Antioquia, and the laboratory and group of molecular studies of the University of Antioquia. ## 7. References Abraitis, P. K., Pattrick, R. A. D., Kelsall, G. H., Vaughan, D. J. 2004. Acid leaching and dissolution of major sulphide ore minerals: processes and galvanic effects in complex systems. Mineralogical Magazine. 68(2) 343–351 Bioleaching of Galena (PbS) 205 Marsden J. and House I. 1992. The chemistry of gold extraction. Ed. 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Minerals Engineering. 16 de la zona río Aguachinte – río Asnazú (valle del cauca y cauca). Tesis de Maestría. experimental strategy to determine galvanic interactions affecting the reactivity of bacterial and ferric sulphate leaching of zinc sulphide. Hydrometallurgy. Vol. 73. Chemical Geology. 245 219–229. Bacteriol. 134:310-317. Vol 558. Pp. 83-98. pp. 313 – 324. 2987-2993. 508 – 514. 80 2-4. 375 – 389. 10 Sei Ueda th order Bessel or F s( ) ν, of a Japan Osaka Institute of Technology ν is the ν > −1/2 , H fr [ ( )] ν ν Application of Hankel Transform for Solving a Fracture Problem of a Cracked Piezoelectric Strip Under Thermal Loading In this chapter, an example of the application of Hankel transform for solving a fracture problem will be explained. In discussing axisymmetric problems, it is advantageous to use polar coordinates, and the Hankel transform method is powerful to solve the general equations in polar coordinates. A brief account of the Hankel transform will be given. Here function of the first kind, and the nature of the transformation either by a suffix or by a characteristic new variable s . It will be assumed without comment that the integrals in question exist, and that, if necessary, the functions and their derivatives tend to zero as the > 0 H [ ( )] ( ) ( ) ( ) f r F s rJ sr f r dr > > <sup>0</sup> <sup>f</sup> () ( ) () r sJ sr F s ds ν The piezoelectric materials have attracted considerable attention recently. Owing to the coupling effect between the thermo-elastic and electric fields in piezoelectric materials, thermo-mechanical disturbances can be determined form measurement of the induced electric potential, and the ensuing response can be controlled through application of an appropriate electric field (Rao & Sunar, 1994). For successful and efficient utilization of ≡ = ∫ ∞ = ∫ 2 2 ⎡ ⎤ ν ⎢ ⎥ + − =− ⎣ ⎦ 2 2 <sup>1</sup> ( ) d f df H f <sup>s</sup> <sup>F</sup> <sup>s</sup> dr r r dr ν provided that rf r( ) and rdf r dr ( )/ tend to zero as 0 r → and as r → ∞ . ∞ ν ν 2 ν f is a function of r , its transform is indicated by a capital F , J νν variable tends to infinity. The Hankel transform of order 1. Introduction function f ( )r is defined as and its inversion formula is Also, integrating by parts twice gives and Acidithiobacillus thiooxidans. Trans. Nonferrous Met. Soc. China. 12, pp 190- 195. Zapata, D.M., Márquez M.A., Ossa, D.M. 2007. Sulphur product layer in sphalerite biooxidation: Evidences for a mechanism of formation. Advances Materials Research. 20-21 134 – 138. ## Application of Hankel Transform for Solving a Fracture Problem of a Cracked Piezoelectric Strip Under Thermal Loading Sei Ueda Osaka Institute of Technology Japan ## 1. Introduction 206 Fourier Transform – Materials Analysis Zapata, D.M., Márquez M.A., Ossa, D.M. 2007. Sulphur product layer in sphalerite 195. Research. 20-21 134 – 138. and Acidithiobacillus thiooxidans. Trans. Nonferrous Met. Soc. China. 12, pp 190- biooxidation: Evidences for a mechanism of formation. Advances Materials In this chapter, an example of the application of Hankel transform for solving a fracture problem will be explained. In discussing axisymmetric problems, it is advantageous to use polar coordinates, and the Hankel transform method is powerful to solve the general equations in polar coordinates. A brief account of the Hankel transform will be given. Here f is a function of r , its transform is indicated by a capital F , Jν is the ν th order Bessel function of the first kind, and the nature of the transformation either by a suffix or by a characteristic new variable s . It will be assumed without comment that the integrals in question exist, and that, if necessary, the functions and their derivatives tend to zero as the variable tends to infinity. The Hankel transform of order ν > −1/2 , H fr [ ( )] ν or F s( ) ν , of a function f ( )r is defined as $$H\_{\nu}[f(r)] \equiv F\_{\nu}(s) = \int\_{0}^{\infty} r f\_{\nu}(sr) f(r) dr$$ and its inversion formula is $$f(r) = \int\_0^\infty s f\_\nu(sr) F\_\nu(s) ds$$ Also, integrating by parts twice gives $$H\_{\nu} \left[ \frac{d^2 f}{dr^2} + \frac{1}{r} \frac{df}{dr} - \frac{\nu^2}{r^2} f \right] = -s^2 F\_{\nu}(s) f$$ provided that rf r( ) and rdf r dr ( )/ tend to zero as 0 r → and as r → ∞ . The piezoelectric materials have attracted considerable attention recently. Owing to the coupling effect between the thermo-elastic and electric fields in piezoelectric materials, thermo-mechanical disturbances can be determined form measurement of the induced electric potential, and the ensuing response can be controlled through application of an appropriate electric field (Rao & Sunar, 1994). For successful and efficient utilization of Application of Hankel Transform for Solving a Fracture θθ where ( ) T rz <sup>i</sup> , is the temperature, ( ) <sup>i</sup> displacement components, ( ) rri expressed as follows: where <sup>2</sup> κ κκ= / r z . σ σ σ zri σ ri 2 ( ) ( ) The boundary conditions can be written as 2 222 11 2 22 44 13 44 31 15 11 u uu u u <sup>T</sup> c c <sup>c</sup> <sup>c</sup> <sup>e</sup> <sup>e</sup> r rz r r rz rz r ⎛ ⎞ ∂ ∂∂ ∂ ∂ ∂ ∂ ⎜ ⎟ + − + ++ ++ = ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ⎝ ⎠ κ σ Problem of a Cracked Piezoelectric Strip Under Thermal Loading 209 , , , r z, is the electric potential, ( ) u rz ri , , ( ) u rz zi , are the r z, , ( )( 1 2) zri σ ( 1 2) ( 1 2) φ φ ε 2 33 λ (5) , ⎪⎭ ⎫ ⎪ ⎪ ⎪ ⎪ <sup>⎪</sup> <sup>∂</sup> <sup>=</sup> ⎪⎪ <sup>∂</sup> <sup>⎬</sup> = , ⎪ ⎪ i T z z i ( 1 2) i ⎬ = , rz i , = , are the stress (1) (2) (3) (4) 11 12 13 31 11 ∂ ∂∂ <sup>⎫</sup> = ++ + − <sup>⎪</sup> ∂ ∂∂ <sup>⎪</sup> ∂ ∂∂ <sup>⎪</sup> = ++ + − <sup>⎪</sup> ∂ ∂∂ <sup>⎪</sup> uu u c cc e T rr z z uu u c cc e T ri ri zi i rri i 12 11 13 31 11 ri ri zi i i i ri ri zi i zzi i 13 13 33 33 33 uu u c cc e T rr z z u u c e = ++ + − ⎪ ∂ ∂∂ ⎪ <sup>⎪</sup> ⎛ ⎞ ∂∂ ∂ = ++ <sup>⎪</sup> ⎜ ⎟ ⎝ ⎠ ∂∂ ∂ ⎪⎭ 44 15 φ u u D e r z, , ( ) <sup>i</sup> r z σθθ <sup>2</sup> − ≤≤ h z 0 , respectively. For the electric field, the constitutive relations are 15 11 ri ri zi i zi z i 31 31 33 33 where ( ) D rz ri , , ( )( 1 2) D rz i zi , = , are the electric displacement components. uu u De e e p T rr z z 2 2 2 2 r z r r 2 22 22 44 2 2 33 13 44 15 2 33 2 22 22 15 2 2 33 15 31 11 2 2 33 uu u uu <sup>T</sup> <sup>e</sup> e ee <sup>p</sup> r z r r rz r z r z r r <sup>z</sup> zi zi zi ri ri i i i 1 1 1 1 1 1 1 1 2 z ⎛ ⎞ ∂ ∂ ∂ ∂ ∂ ∂∂ ∂ ⎛ ⎞⎛ ⎞ ⎜ ⎟ + + ++ + + + + ⎜ ⎟⎜ ⎟ ∂ ∂ ∂ ∂ <sup>∂</sup> ∂ ∂ ∂ ∂ ⎝ ⎠ ⎝ ⎠⎝ ⎠ uu u uu c cc c ee r z r r rz r z <sup>r</sup> r r <sup>1</sup> , ri ri ri ri zi i i zi ri i rz r components. The subscript i = 1 2, denotes the thermo-electro-elastic fields in 1 0 ≤ ≤z h and zi ri i rz r <sup>⎪</sup> ∂ ∂∂ = ++ − + ⎪ ∂ ∂∂ ⎪⎭ The governing equations for the thermo-electro-elastic fields of the medium may be ⎛ ⎞ ∂ ∂∂ ⎜ ⎟ <sup>+</sup> + = =, ∂ ∂ <sup>∂</sup> ⎝ ⎠ ( ) ( ) zi zi zi ri ri i i <sup>i</sup> <sup>i</sup> <sup>z</sup> <sup>⎪</sup> ⎛ ⎞ ∂ ∂ ∂ ∂ ∂ ∂∂ ∂ ∂ ⎛ ⎞⎛ ⎞ + + + + + − + − =− <sup>⎪</sup> ⎜ ⎟ ⎜ ⎟⎜ ⎟ ∂ ∂ <sup>∂</sup> ∂∂ ∂ ∂ ∂ <sup>∂</sup> <sup>∂</sup> <sup>⎪</sup> ⎝ ⎠ ⎝ ⎠⎝ ⎠ <sup>⎪</sup> ε ( 0) 0 (0 ) ( 0) ( 0) ( ) T r r c <sup>∂</sup> <sup>⎫</sup> , = ≤< <sup>⎪</sup> <sup>∂</sup> <sup>⎬</sup> , = , ≤ <∞ ⎪ Tr Tr c r <sup>1</sup> 0 ( 1 2) TT T ii i <sup>i</sup> rr z z <sup>i</sup> <sup>⎬</sup> <sup>=</sup> , ∂ ∂∂ , , ( ) zzi σ ⎛ ⎞ ∂∂ ∂ ⎫ = +− ⎜ ⎟ <sup>⎪</sup> ⎝ ⎠ ∂∂ ∂ ⎪⎪ φ > φ φ φ , φ ε > φ λ φφ φφ ⎭ φ ε λ > λ λ piezoelectric as sensors and actuators in intelligent systems, several researches on piezothermo-elastic behavior have been reported (Tauchert, 1992). Moreover a better understanding of the mechanics of fracture in piezoelectric materials under thermal load conditions is needed for the requirements of reliability and lifetime of these systems. Using the Fourier transform, the present author studied the thermally induced fracture of a piezoelectric strip with a two-dimensional crack (Ueda, 2006a, 2006b). Here the mixed-mode thermo-electro-mechanical fracture problem for a piezoelectric material strip with a penny-shaped crack is considered. It is assumed that the strip is under the thermal loading. The crack faces are supposed to be insulated thermally and electrically. By using the Hankel transform (Sneddon & Lowengrub, 1969), the thermal and electromechanical problems are reduced to a singular integral equation and a system of singular integral equations (Erdogan & Wu, 1996), respectively, which are solved numerically (Sih, 1972). Numerical calculations are carried out, and detailed results are presented to illustrate the influence of the crack size and the crack location on the stress and electric displacement intensity factors. The temperature, stress and electric displacement distributions are also presented. ## 2. Formulation of the problem Fig. 1. Penny-shaped crack in a piezoelectric strip A penny-shaped crack of radius c is embedded in an infinite long piezoelectric strip of thickness 1 2 hh h = + as shown in Figure 1. The crack is located parallel to the boundaries and at an arbitrary position in the strip, and the crack faces are supposed to be insulated thermally and electrically. The cylindrical coordinate system is denoted by ( ) r z , , θ with its origin at the center of the crack face and the plane r −θ along the crack plane, where z is the poling axis. It is assumed that uniform temperatures T10 and T20 are maintained over the stress-free boundaries. In the following, the subscripts r z ,θ, will be used to refer to the direction of coordinates. The material properties, such as the elastic stiffness constants, the piezoelectric constants, the dielectric constants, the stress-temperature coefficients, the coefficients of heat conduction, and the pyroelectric constant, are denoted by kl c , kl e , kk ε , ( 1 2 3) kk λ k l,=,, , κ <sup>r</sup> , κ<sup>z</sup> , and <sup>z</sup> p , respectively. The constitutive equations for the elastic field are piezoelectric as sensors and actuators in intelligent systems, several researches on piezo- Moreover a better understanding of the mechanics of fracture in piezoelectric materials under thermal load conditions is needed for the requirements of reliability and lifetime of these systems. Using the Fourier transform, the present author studied the thermally induced fracture of a piezoelectric strip with a two-dimensional crack (Ueda, 2006a, 2006b). Here the mixed-mode thermo-electro-mechanical fracture problem for a piezoelectric material strip with a penny-shaped crack is considered. It is assumed that the strip is under the thermal loading. The crack faces are supposed to be insulated thermally and electrically. By using the Hankel transform (Sneddon & Lowengrub, 1969), the thermal and electromechanical problems are reduced to a singular integral equation and a system of singular integral equations (Erdogan & Wu, 1996), respectively, which are solved numerically (Sih, 1972). Numerical calculations are carried out, and detailed results are presented to illustrate the influence of the crack size and the crack location on the stress and electric displacement intensity factors. The temperature, stress and electric displacement distributions are also A penny-shaped crack of radius c is embedded in an infinite long piezoelectric strip of thickness 1 2 hh h = + as shown in Figure 1. The crack is located parallel to the boundaries and at an arbitrary position in the strip, and the crack faces are supposed to be insulated the poling axis. It is assumed that uniform temperatures T10 and T20 are maintained over direction of coordinates. The material properties, such as the elastic stiffness constants, the piezoelectric constants, the dielectric constants, the stress-temperature coefficients, the coefficients of heat conduction, and the pyroelectric constant, are denoted by kl c , kl e , kk θ θ θ along the crack plane, where z is , will be used to refer to the with its ε, thermally and electrically. The cylindrical coordinate system is denoted by ( ) r z , , thermo-elastic behavior have been reported (Tauchert, 1992). presented. ( 1 2 3) kk k l,=,, , κ <sup>r</sup> , κ λ 2. Formulation of the problem Fig. 1. Penny-shaped crack in a piezoelectric strip origin at the center of the crack face and the plane r − The constitutive equations for the elastic field are the stress-free boundaries. In the following, the subscripts r z , <sup>z</sup> , and <sup>z</sup> p , respectively. $$\begin{aligned} \sigma\_{rii} &= c\_{11} \frac{\partial u\_{ri}}{\partial r} + c\_{12} \frac{u\_{ri}}{r} + c\_{13} \frac{\partial u\_{zi}}{\partial z} + e\_{31} \frac{\partial \phi\_i}{\partial z} - \lambda\_{11} T\_{i\prime} \\ \sigma\_{\theta\theta i} &= c\_{12} \frac{\partial u\_{ri}}{\partial r} + c\_{11} \frac{u\_{ri}}{r} + c\_{13} \frac{\partial u\_{zi}}{\partial z} + e\_{31} \frac{\partial \phi\_i}{\partial z} - \lambda\_{11} T\_{i\prime} \\ \sigma\_{zzi} &= c\_{13} \frac{\partial u\_{ri}}{\partial r} + c\_{13} \frac{u\_{ri}}{r} + c\_{33} \frac{\partial u\_{zi}}{\partial z} + e\_{33} \frac{\partial \phi\_i}{\partial z} - \lambda\_{33} T\_{i\prime} \\ \sigma\_{zri} &= c\_{44} \left( \frac{\partial u\_{zi}}{\partial r} + \frac{\partial u\_{ri}}{\partial z} \right) + e\_{15} \frac{\partial \phi\_i}{\partial r} \end{aligned} \tag{1}$$ where ( ) T rz <sup>i</sup> , is the temperature, ( ) <sup>i</sup> φ r z, is the electric potential, ( ) u rz ri , , ( ) u rz zi , are the displacement components, ( ) rri σ r z, , ( ) <sup>i</sup> r z σθθ , , ( ) zzi σ r z, , ( )( 1 2) zri σ rz i , = , are the stress components. The subscript i = 1 2, denotes the thermo-electro-elastic fields in 1 0 ≤ ≤z h and <sup>2</sup> − ≤≤ h z 0 , respectively. For the electric field, the constitutive relations are $$D\_{ri} = e\_{15} \left( \frac{\partial u\_{zi}}{\partial r} + \frac{\partial u\_{ri}}{\partial z} \right) - \varepsilon\_{11} \frac{\partial \phi\_i}{\partial r},$$ $$D\_{zi} = e\_{31} \frac{\partial u\_{ri}}{\partial r} + e\_{31} \frac{u\_{ri}}{r} + e\_{33} \frac{\partial u\_{zi}}{\partial z} - \varepsilon\_{33} \frac{\partial \phi\_i}{\partial z} + p\_z T\_i \Bigg\| \tag{2}$$ where ( ) D rz ri , , ( )( 1 2) D rz i zi , = , are the electric displacement components. The governing equations for the thermo-electro-elastic fields of the medium may be expressed as follows: $$\kappa^2 \left( \frac{\partial^2 T\_i}{\partial r^2} + \frac{1}{r} \frac{\partial T\_i}{\partial r} \right) + \frac{\partial^2 T\_i}{\partial z^2} = 0 \quad \text{( $i = 1, 2$ )}\tag{3}$$ ( ) ( ) ( ) 2 222 11 2 22 44 13 44 31 15 11 2 22 22 44 2 2 33 13 44 15 2 33 <sup>1</sup> , 1 1 1 ri ri ri ri zi i i zi zi zi ri ri i i i u uu u u <sup>T</sup> c c <sup>c</sup> <sup>c</sup> <sup>e</sup> <sup>e</sup> r rz r r rz rz r uu u uu c cc c ee r z r r rz r z <sup>r</sup> r r φ λ φφ φ ⎛ ⎞ ∂ ∂∂ ∂ ∂ ∂ ∂ ⎜ ⎟ + − + ++ ++ = ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ⎝ ⎠ ⎛ ⎞ ∂ ∂ ∂ ∂ ∂ ∂∂ ∂ ⎛ ⎞⎛ ⎞ ⎜ ⎟ + + ++ + + + + ⎜ ⎟⎜ ⎟ ∂ ∂ ∂ ∂ <sup>∂</sup> ∂ ∂ ∂ ∂ ⎝ ⎠ ⎝ ⎠⎝ ⎠ ( ) 2 33 2 22 22 15 2 2 33 15 31 11 2 2 33 , ( 1 2) 1 1 1 i zi zi zi ri ri i i <sup>i</sup> <sup>i</sup> <sup>z</sup> T z z i uu u uu <sup>T</sup> <sup>e</sup> e ee <sup>p</sup> r z r r rz r z r z r r <sup>z</sup> λ φφ φ ε ε ⎫ ⎪ ⎪ ⎪ ⎪ <sup>⎪</sup> <sup>∂</sup> <sup>=</sup> ⎪⎪ <sup>∂</sup> <sup>⎬</sup> = , ⎪ ⎪ <sup>⎪</sup> ⎛ ⎞ ∂ ∂ ∂ ∂ ∂ ∂∂ ∂ ∂ ⎛ ⎞⎛ ⎞ + + + + + − + − =− <sup>⎪</sup> ⎜ ⎟ ⎜ ⎟⎜ ⎟ ∂ ∂ <sup>∂</sup> ∂∂ ∂ ∂ ∂ <sup>∂</sup> <sup>∂</sup> <sup>⎪</sup> ⎝ ⎠ ⎝ ⎠⎝ ⎠ <sup>⎪</sup> ⎪⎭ (4) where <sup>2</sup> κ κκ= / r z . The boundary conditions can be written as $$\begin{cases} \frac{\partial}{\partial z} T\_1(r, 0) = 0 & (0 \le r < \infty) \\ T\_1(r, 0) = T\_2(r, 0) & (c \le r < \infty) \end{cases} \tag{5}$$ Application of Hankel Transform for Solving a Fracture It is easy to find from Eqs.(12) and (13) that (2) 0 (1) 0 M tr In Eq.(21), the kernel functions (1) ( )( 0 1 2) <sup>k</sup> s k = ,, are given by ρ (Erdogan & Wu, 1996): Problem of a Cracked Piezoelectric Strip Under Thermal Loading 211 , = ⎫ Tr Tr r <sup>⎪</sup> ∂ ∂ <sup>⎪</sup> , = , ≤ <∞ <sup>⎬</sup> ∂ ∂ <sup>⎪</sup> ,− = ⎪ { } ( ) (1) ( ) ( ) ( )exp( ) ( 1 2) <sup>i</sup> ij ij <sup>∞</sup> <sup>1</sup> T z T T z Th Th ( ) h h = −++ By applying the Hankel transform to Eq.(14) (Sneddon & Lowengrub, 1969), we have T r z D s J sr s z ds i 11 22 12 21 τ τ κ { } (2) (2) 1 2 T r T r rc G r <sup>r</sup> ( 0) ( 0) (0 ) ( ) Making use of the first boundary condition (15) with Eqs.(16), we have the following <sup>2</sup> ( ) ( ) () (0 ) <sup>c</sup> T T t M t r M t r G t dt r c 2 2 <sup>0</sup> 0 1 <sup>0</sup> <sup>0</sup> 2 () () ( ) 1 ()() ( ) s s <sup>M</sup> tr s J sr J st ds s <sup>∞</sup> ⎧ ⎫ ⎪ ⎪ where K and E are complete elliptic integrals of the first and second kind, and κρ ρ ρ ( ) 2 1 ( ) ( ) , = <sup>⎨</sup> <sup>⎬</sup> <sup>⎪</sup> ⎧ ⎫ ⎪ ⎪ ⎛⎞ ⎛⎞ <sup>⎪</sup> ⎨ ⎬ + > ⎜⎟ ⎜⎟ <sup>⎪</sup> <sup>−</sup> <sup>⎪</sup> ⎪ ⎪ ⎩ ⎭ ⎝⎠ ⎝⎠ ⎩ ⎭ tt r r rt r t r t <sup>−</sup> , + , =− ≤ < <sup>⎧</sup> <sup>∂</sup> <sup>⎫</sup> <sup>⎪</sup> , − , ≤< <sup>⎪</sup> <sup>=</sup> ⎨∂ <sup>⎬</sup> <sup>⎪</sup> <sup>≤</sup> < ∞ <sup>⎪</sup> ⎩ ⎭ Taking the second boundary condition (15) into consideration, the problem may be reduced to a singular integral equation by defining the following new unknown function 0 G r( ) κ, = = − ⎫ <sup>⎬</sup> = = <sup>⎭</sup> <sup>0</sup> <sup>0</sup> <sup>1</sup> where ( )( 1 2) D s ij ij ,=, are unknown functions to be solved and ( 1 2) ij τ > τ singular integral equation for the determination of the unknown function 0 G t( ): { } (1) (2) 10 20 0 0 <sup>0</sup> <sup>0</sup> 1 2 <sup>0</sup> <sup>M</sup> ( ) t r, and (2) 2 2 π (2) 1 2 π ( 0) ( 0), (0 ) ⎭ 10 20 10 2 20 1 τ 0 () κh h ⎧ ⎛ ⎞ ⎫ <sup>&</sup>lt; <sup>⎪</sup> ⎜ ⎟ <sup>⎪</sup> <sup>⎪</sup> <sup>−</sup> ⎝ ⎠ <sup>⎪</sup> <sup>+</sup> ∫ (21) 2 1 ( ), rt t E K rt <sup>0</sup> M ( ) t r, are given by <sup>r</sup> E r <sup>t</sup> , =− ⎨ ⎬ <sup>+</sup> ⎪ ⎪ ⎩ ⎭ <sup>∫</sup> (23) c r , <sup>=</sup> ∑ <sup>=</sup> , ∫ (18) τ (16) (17) i j , = , are given by (20) (22) (19) ( ) 0, ( )0 (2) 1 1 (2) (2) 1 2 (2) 2 2 z z T rh 1 2 + 2 j = T rh $$\begin{aligned} T\_1(r, h\_1) &= T\_{10}, \\ \frac{\partial}{\partial z} T\_1(r, 0) &= \frac{\partial}{\partial z} T\_2(r, 0)\_r \\ T\_2(r, -h\_2) &= T\_{20} \end{aligned} \quad \text{(} 0 \le r < \infty\text{)} \tag{6}$$ for thermal loading conditions and $$\begin{aligned} \sigma\_{zz1}(r,0) &= 0 \quad & \quad (0 \le r < c) \\ \mu\_{z1}(r,0) &= \mu\_{z2}(r,0) \quad & \quad (c \le r < \infty) \end{aligned} \tag{7}$$ $$\begin{aligned} \sigma\_{zr1}(r,0) &= 0 & \quad \text{( $0 \le r < c$ )}\\ \mu\_{r1}(r,0) &= \mu\_{r2}(r,0) & \quad \text{( $c \le r < c$ )} \end{aligned} \tag{8}$$ $$\begin{aligned} D\_{z1}(r,0) &= 0 \quad & \quad (0 \le r < c) \\ \phi\_1(r,0) &= \phi\_2(r,0) \quad & \quad (c \le r < \infty) \end{aligned} \tag{9}$$ $$\begin{aligned} \sigma\_{zz1}(r,0) &= \sigma\_{zz2}(r,0), & \sigma\_{zz1}(r,h\_1) &= 0, & \sigma\_{zz2}(r,-h\_2) &= 0, \\ \sigma\_{zr1}(r,0) &= \sigma\_{zr2}(r,0), & \sigma\_{zr1}(r,h\_1) &= 0, & \sigma\_{zr2}(r,-h\_2) &= 0, \\ D\_{z1}(r,0) &= D\_{z2}(r,0), & D\_{z1}(r,h\_1) &= 0, & D\_{z2}(r,-h\_2) &= 0 \end{aligned} \tag{10}$$ for the electromechanical conditions. #### 3. Temperature field For the problem considered here, it is convenient to represent the temperature as the sum of two functions. $$T\_i(r, z) = T^{(1)}(z) + T\_i^{(2)}(r, z) \quad (i = 1, 2) \tag{11}$$ where (1) T z( ) satisfies the following equation and boundary conditions: $$\frac{d^2T^{(1)}}{dz^2} = 0\tag{12}$$ $$\begin{aligned} T^{(1)}(\hbar\_1) &= T\_{10\prime} \\ T^{(1)}(-\hbar\_2) &= T\_{20} \end{aligned} \tag{13}$$ and (2)( )( 1 2) T rz i <sup>i</sup> , =, is subjected to the relations: $$\kappa^2 \left( \frac{\partial^2 T\_i^{(2)}}{\partial r^2} + \frac{1}{r} \frac{\partial T\_i^{(2)}}{\partial r} \right) + \frac{\partial^2 T\_i^{(2)}}{\partial z^2} = 0 \quad \text{( $i = 1, 2$ )}\tag{14}$$ $$\begin{aligned} \frac{\partial}{\partial z} T\_1^{(2)}(r, 0) &= -\frac{d}{dz} T^{(1)}(0) \quad \text{ (\$0 \le r < c\$)}\\ T\_1^{(2)}(r, 0) &= T\_2^{(2)}(r, 0) \quad \text{ (\$c \le r < \infty)} \end{aligned} \tag{15}$$ $$\begin{aligned} T\_1^{(2)}(r, h\_1) &= 0, \\ \frac{\partial}{\partial z} T\_1^{(2)}(r, 0) &= \frac{\partial}{\partial z} T\_2^{(2)}(r, 0), \\ T\_2^{(2)}(r, -h\_2) &= 0 \end{aligned} \quad \text{( $0 \le r < \infty$ )}\tag{16}$$ It is easy to find from Eqs.(12) and (13) that 210 Fourier Transform – Materials Analysis ( 0) ( 0), (0 ) ( 0) 0 (0 ) ( 0) ( 0) ( ) r r c ( 0) 0 (0 ) ( 0) ( 0) ( ) r r c ( 0) 0 (0 ) ( 0) ( 0) ( ) D r <sup>z</sup> r c ( 0) ( 0), ( ) 0, ( ) 0, (0 ) <sup>⎪</sup> , = , , = ,− = ≤ < ∞ <sup>⎬</sup> r r rh r h r For the problem considered here, it is convenient to represent the temperature as the sum of 2 (1) <sup>2</sup> <sup>0</sup> d T ( ) Th T T hT 1 10 () , = − = 2 20 <sup>1</sup> 0 ( 1 2) TT T ii i <sup>i</sup> ( 0) (0) (0 ) ( 0) ( 0) ( ) <sup>⎪</sup> , = , ≤ <∞ <sup>⎭</sup> ⎜ ⎟ <sup>+</sup> + = =, ∂ ∂ <sup>∂</sup> ⎝ ⎠ <sup>d</sup> T r T rc ∂ ⎫ , =− ≤ < <sup>⎪</sup> <sup>∂</sup> <sup>⎬</sup> T r T r cr ⎫ ⎪ ⎬ ⎪ ⎭ (1) (1) 2 2 (2) (2) (2) ⎛ ⎞ ∂ ∂∂ (2) (2) 1 2 z dz 2 2 r z r r (2) (1) , = ≤< ⎫ <sup>⎬</sup> , = , ≤ <∞ <sup>⎭</sup> > σ > σ (1) (2) ( ) ( ) ( ) ( 1 2) T rz T z T rz i <sup>i</sup> <sup>i</sup> , = + , =, (11) dz <sup>=</sup> (12) (13) (14) (15) , = ≤< ⎫ <sup>⎬</sup> , = , ≤ <∞ <sup>⎭</sup> , = ≤< ⎫ <sup>⎬</sup> , = , ≤ <∞ <sup>⎭</sup> (6) (7) (8) (9) (10) Tr Tr r ,− = ⎪⎭ ur ur cr ur ur cr φr r cr ( 0) ( 0), ( ) 0, ( ) 0, , = , , = ,− = ⎫ <sup>⎪</sup> , = , , = ,− = <sup>⎭</sup> ( 0) ( 0), ( ) 0, ( ) 0 <sup>⎪</sup> ∂ ∂ <sup>⎪</sup> , = , ≤ <∞ <sup>⎬</sup> ∂ ∂ <sup>⎪</sup> , = ⎫ 1 1 10 T rh T () , 1 2 ( ) 1 1 2 1 1 2 1 1 2 1 2 11 2 2 1 2 11 2 2 1 2 11 2 2 D r D r D rh D r h where (1) T z( ) satisfies the following equation and boundary conditions: and (2)( )( 1 2) T rz i <sup>i</sup> , =, is subjected to the relations: 2 1 κ r r rh r h zr r r σ φ zz zz zz zz zr zr zr zr zz z z σ σ zz z z σ z z Tr h T for thermal loading conditions and σσ σσ for the electromechanical conditions. 3. Temperature field two functions. 2 2 20 $$T^{(1)}(z) = \frac{1}{h\_1 + h\_2} \left\{ (T\_{10} - T\_{20})z + T\_{10}h\_2 + T\_{20}h\_1 \right\} \tag{17}$$ By applying the Hankel transform to Eq.(14) (Sneddon & Lowengrub, 1969), we have $$T\_i^{(2)}(r,z) = \sum\_{j=1}^{2} \int\_0^r D\_{ij}(s) I\_0(sr) \exp(s\tau\_{ij}z) ds \quad (i=1,2) \tag{18}$$ where ( )( 1 2) D s ij ij ,=, are unknown functions to be solved and ( 1 2) ij τi j , = , are given by $$\begin{aligned} \tau\_{11} = \tau\_{22} = -\kappa \text{ .}\\ \tau\_{12} = \tau\_{21} = \kappa \text{ .} \end{aligned} \tag{19}$$ Taking the second boundary condition (15) into consideration, the problem may be reduced to a singular integral equation by defining the following new unknown function 0 G r( ) (Erdogan & Wu, 1996): $$G\_0(r) = \begin{cases} \frac{\partial}{\partial r} \left\{ T\_1^{(2)}(r, 0) - T\_2^{(2)}(r, 0) \right\} & \text{( $0 \le r < c$ )}\\ 0 & \text{( $c \le r < \infty$ )} \end{cases} \tag{20}$$ Making use of the first boundary condition (15) with Eqs.(16), we have the following singular integral equation for the determination of the unknown function 0 G t( ): $$\int\_{0}^{c} \left\{ M\_{0}^{(1)}(t,r) + M\_{0}^{(2)}(t,r) \right\} G\_{0}(t) dt = -\frac{2}{\kappa} \frac{T\_{10} - T\_{20}}{h\_{1} + h\_{2}} \qquad \text{(} 0 \le r < c \text{)}\tag{21}$$ In Eq.(21), the kernel functions (1) <sup>0</sup> <sup>M</sup> ( ) t r, and (2) <sup>0</sup> M ( ) t r, are given by $$\mathcal{M}\_0^{(1)}(t,r) = \begin{cases} \frac{2}{\pi} \frac{1}{t^2 - r^2} E\left(\frac{r}{t}\right) & (r < t)\_r\\ \frac{2}{\pi} \left\{ \frac{r}{t(t^2 - r^2)} E\left(\frac{t}{r}\right) + \frac{1}{rt} K\left(\frac{t}{r}\right) \right\} & (r > t) \end{cases} \tag{22}$$ $$M\_0^{(2)}(t,r) = -\int\_0^r s \left\{ \frac{2\,\rho\_1(s)\,\rho\_2(s)}{\kappa \rho\_0(s)} + 1 \right\} \mathbf{J}\_0(sr)\mathbf{J}\_1(st)ds\tag{23}$$ where K and E are complete elliptic integrals of the first and second kind, and ( )( 0 1 2) <sup>k</sup> ρs k = ,, are given by Application of Hankel Transform for Solving a Fracture ( ) zri σ 1969): ij γ can be obtained as follows: σ σ φ σ σ φ Problem of a Cracked Piezoelectric Strip Under Thermal Loading 213 homogeneous equations obtained by setting 0 ( 1 2) T i <sup>i</sup> = = , in Eqs.(4). In the following, the superscripts (1) and (2) indicate the particular and general solutions of Eqs.(4). Using the displacement potential function method (Ueda, 2006a), the particular solutions σ τ τ τ τ τ τ γ γ γ exp( ) , γ γ γ ij s z ds r z, , ( ) <sup>i</sup> r z σθθ ( 1 2) i ⎪ ⎪ ⎭ ( 1 2) i ⎪ ⎪ ⎭ ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ = , ⎪ ⎪ ⎪ ⎪ ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ = , ⎪ ⎪ ⎪ ⎪ , , ( ) zzi σr z, , (30) (31) Substituting Eqs.(29) into Eqs.(1) and (2), one obtains stress ( ) rri 2 (1) (1) ∑ ∫ ∞ ∞ ∞ 2 (1) (1) ∑ ∫ = j , = , = , = 2 (1) (1) ∑ ∫ = j 2 (1) ( j = ∞ <sup>1</sup> ( ) u rz p , = zi ij j = 2 (1) (1) ∑ ∫ j = 2 (1) (1) ∑ ∫ 6 (2) (2) ∑ ∫ ∞ ∞ ∞ 6 (2) (2) ∑ ∫ = j , = , = , = , = 6 (2) (2) ∑ ∫ = j 6 (2) (2) = zi ij ij j ∑ ∫ = 6 (2) (2) ∑ ∫ j = 6 (2) (2) ∑ ∫ j and (2) ( 1 2 1 2 6) kij p i jk = , , , = , ,..., are given in Appendix B. = j j = <sup>4</sup> <sup>0</sup> <sup>1</sup> ∞ ∑ ∫ 1) s s ∞ s r z, and electric displacement ( ) D rz ri , , ( )( 1 2) D rz i zi , = , expressions. <sup>1</sup> 0 0 <sup>0</sup> <sup>1</sup> zzi ij ij ij ( ) ( ) ( ) ( )exp( ) , r z p R s R s J sr s z ds ( ) ( ) ( ) ( )exp( ) , r z p R s R s J sr s z ds ( ) ( ) ( ) ( )exp( ) , <sup>1</sup> ( ) ( ) ( ) ( )exp( ) , , = ⎪ 0 0 ij ij R s R s J sr s z ds ( ) ( ) ( )exp( ) , <sup>2</sup> 0 1 <sup>0</sup> <sup>1</sup> zri ij ij ij <sup>3</sup> 0 0 <sup>0</sup> <sup>1</sup> <sup>5</sup> 0 1 <sup>0</sup> <sup>1</sup> ri ij ij ij u r z p R s R s J sr s z ds <sup>6</sup> 0 0 <sup>0</sup> <sup>1</sup> i ij ij ij <sup>1</sup> <sup>0</sup> <sup>0</sup> <sup>1</sup> zzi ij ij ij ( ) ( ) ( )exp( ) , r z sp A s J sr s z ds ( ) ( ) ( )exp( ) , r z sp A s J sr s z ds ( ) ( ) ( )exp( ) , ( ) ( ) ( )exp( ) , , = ⎪ <sup>2</sup> <sup>1</sup> <sup>0</sup> <sup>1</sup> zri ij ij ij <sup>3</sup> <sup>0</sup> <sup>0</sup> <sup>1</sup> zi ij ij ij D r z sp A s J sr s z ds <sup>4</sup> <sup>0</sup> <sup>0</sup> <sup>1</sup> <sup>5</sup> <sup>1</sup> <sup>0</sup> <sup>1</sup> ri ij ij ij u r z p A s J sr s z ds <sup>6</sup> <sup>0</sup> <sup>0</sup> <sup>1</sup> i ij ij ij ( ) ( ) ( )exp( ) r z p A s J sr s z ds <sup>⎪</sup> , =− <sup>⎪</sup> where ( ) ( 1 2 1 2 6) Asi j ij = , , = , ,..., are the unknown functions to be solved, and the constants ( ) () ( ) u r z p A s J sr ∞ ∞ ∞ <sup>1</sup> ( ) ( ) ( ) ( )exp( ) r z p R s R s J sr s z ds <sup>⎪</sup> , =− <sup>⎪</sup> where the constants (1) ( 1 2 1 2 6) kij p ij k , = , , = , ,..., are given in Appendix A. The general solutions are obtained by using the Hankel transform technique (Sneddon & Lowengrub, zi ij ij ij D r z p R s R s J sr s z ds $$\rho\_0(s) = \rho\_2(s)\left\{1 - \exp(-2s\kappa h\_1)\right\} - \rho\_1(s)\left\{1 - \exp(-2s\kappa h\_2)\right\},$$ $$\rho\_i(s) = \tau\_{i1} - \tau\_{i2}\exp(-2s\kappa h\_i) \quad (i = 1, 2)$$ Once 0 G t( ) is obtained from Eq.(21), the temperature field can be easily calculated as follows: $$T\_i^{(2)}(r, z) = \sum\_{j=1}^{2} T\_{ij}^{(2)}(r, z) \quad (i = 1, 2) \tag{25}$$ where $$T\_{ij}^{(2)}(r,z) = \int\_0^x R\_{ij}(s)R\_0(s)f\_0(sr) \exp(s\tau\_{ij}z)ds \quad \text{(i,j=1,2)}\tag{26}$$ with $$\begin{aligned} R\_{0}(\mathbf{s}) &= \int\_{0}^{c} t \mathbf{G}\_{0}(t) f\_{1}(\mathbf{s}t) dt, \\ R\_{11}(\mathbf{s}) &= -\frac{\rho\_{2}(\mathbf{s})}{\rho\_{0}(\mathbf{s})}, \\ R\_{21}(\mathbf{s}) &= -\frac{\rho\_{1}(\mathbf{s})}{\rho\_{0}(\mathbf{s})}, \qquad R\_{22}(\mathbf{s}) = \frac{\rho\_{1}(\mathbf{s})}{\rho\_{0}(\mathbf{s})} \exp(-2s\kappa l\_{2}) \end{aligned} \tag{27}$$ On the plane 0 <sup>z</sup> <sup>=</sup> , the temperatures (2)( 0) ( 1 2) Tr i <sup>i</sup> , = , are reduced to $$\, \, \, T\_i^{(2)}(r, 0) = \frac{(-1)^i}{2} \int\_r^c G\_0(t)dt + \int\_0^\infty \left\{ \sum\_{j=1}^2 R\_{ij}(s) - \frac{(-1)^i}{2} \right\} R\_0(s) f\_0(sr) ds \quad \text{( $i = 1, 2$ )}\tag{28}$$ #### 4. Thermally induced elastic and electric fields The non-disturbed temperature filed (1) T z( ) given by Eq.(17) does not induce the stress and electric displacement components, which affect the singular field. Thus, we consider the elastic and electric fields due to the disturbed temperature distribution (2)( )( 1 2) T rz i <sup>i</sup> , =, only. It is convenient to represent the solutions ( ) u rz zi , , ( ) u rz ri , and ( )( 1 2) <sup>i</sup> φ rz i , = , as the sum of two functions, respectively. $$u\_{zi}(r,z) = u\_{zi}^{(1)}(r,z) + u\_{zi}^{(2)}(r,z)\_{\prime} \Bigg\| \tag{29}$$ $$u\_{ri}(r,z) = u\_{ri}^{(1)}(r,z) + u\_{ri}^{(2)}(r,z)\_{\prime} \Bigg\| \tag{20}$$ $$\phi\_i(r,z) = \phi\_i^{(1)}(r,z) + \phi\_i^{(2)}(r,z)$$ where (1)( ) zi u rz, , (1)( ) ri u rz, , (1)( )( 1 2) <sup>i</sup> φ rz i , = , are the particular solutions of Eqs.(4) replaced Ti by (2) Ti , and (2)( ) zi u rz, , (2)( ) ri u rz, , (2)( ) ( 1 2) <sup>i</sup> φrz i , = , are the general solutions of 0 2 { 1 1 } { <sup>2</sup> } s s sh s sh ( ) exp( 2 ) ( 1 2) i ii i Once 0 G t( ) is obtained from Eq.(21), the temperature field can be easily calculated as 0 0 <sup>0</sup> ( ) ( ) ( ) ( )exp( ) ( 1 2) T r z R s R s J sr s z ds i j ij ij ij 2 2 11 12 1 0 0 1 1 21 22 2 0 0 2 T r G t dt R s R s J sr ds i <sup>∞</sup> = i i <sup>c</sup> s s R s R s s h s s s s R s R s s h s s ( ) ( ) ( ) , ( ) exp( 2 ), ( ) ( ) ⎪⎪ = − = − <sup>⎬</sup> <sup>⎪</sup> = − = − <sup>⎪</sup> = ⎪ ρ ρ > ρ ρ ⎪ ⎪ ⎩ ⎭ ∫ ∫ <sup>∑</sup> (28) ( ) ( ) ( ) , ( ) exp( 2 ) ( ) ( ) 0 0 <sup>0</sup> <sup>0</sup> <sup>1</sup> ( 1) ( 1) ( 0) ( ) ( ) ( ) ( ) ( 1 2) 2 2 The non-disturbed temperature filed (1) T z( ) given by Eq.(17) does not induce the stress and electric displacement components, which affect the singular field. Thus, we consider the elastic and electric fields due to the disturbed temperature distribution (2)( )( 1 2) T rz i <sup>i</sup> , =, only. It is convenient to represent the solutions ( ) u rz zi , , ( ) u rz ri , and ( )( 1 2) <sup>i</sup> (1) (2) ,= ,+ , ⎫ ( ) ( ) ( ), zi zi zi u rz u rz u rz ri ri ri i i i φφ φ Ti by (2) Ti , and (2)( ) zi u rz, , (2)( ) ri u rz, , (2)( ) ( 1 2) <sup>i</sup> (1) (2) u rz u rz u rz i ( ) ( ) ( ), ( 1 2) ,= ,+ , ⎬ = , ⎪ ⎪ ⎪ ⎪ ⎪ rz i , = , are the particular solutions of Eqs.(4) replaced rz i , = , are the general solutions of (1) (2) <sup>⎪</sup> ,= ,+ , <sup>⎭</sup> φ φ () () () rz rz rz ⎧ ⎫ − − ⎪ ⎪ , = +− = ⎨ ⎬ , s s h i κ 2 (2) (2) 1 ( ) ( ) ( 1 2) i ij <sup>∞</sup> j T rz T rz i = ( ) ( ) 1 exp( 2 ) ( ) 1 exp( 2 ) , ρ = −− − −− ⎫ κ <sup>⎪</sup> = − − =, <sup>⎭</sup> τ , <sup>=</sup> , <sup>=</sup> , ∫ (26) κ , = , =, ∑ (25) κ ⎫ ⎪ ⎪ ⎪⎭ φ κ ⎪ ⎬ (24) (27) rz i , = , as the (29) 1 2 ττ ρρ (2) (2) sum of two functions, respectively. where (1)( ) zi u rz, , (1)( ) ri u rz, , (1)( )( 1 2) <sup>i</sup> 0 01 <sup>0</sup> <sup>i</sup> ij <sup>r</sup> <sup>j</sup> 4. Thermally induced elastic and electric fields c R s tG t J st dt ∫ () () ( ) , ρ ρ ρ ρ On the plane 0 <sup>z</sup> <sup>=</sup> , the temperatures (2)( 0) ( 1 2) Tr i <sup>i</sup> , = , are reduced to follows: where with ρ homogeneous equations obtained by setting 0 ( 1 2) T i <sup>i</sup> = = , in Eqs.(4). In the following, the superscripts (1) and (2) indicate the particular and general solutions of Eqs.(4). Substituting Eqs.(29) into Eqs.(1) and (2), one obtains stress ( ) rri σ r z, , ( ) <sup>i</sup> r z σθθ , , ( ) zzi σ r z, , ( ) zri σr z, and electric displacement ( ) D rz ri , , ( )( 1 2) D rz i zi , = , expressions. Using the displacement potential function method (Ueda, 2006a), the particular solutions can be obtained as follows: 2 (1) (1) <sup>1</sup> 0 0 <sup>0</sup> <sup>1</sup> 2 (1) (1) <sup>2</sup> 0 1 <sup>0</sup> <sup>1</sup> 2 (1) (1) <sup>3</sup> 0 0 <sup>0</sup> <sup>1</sup> 2 (1) ( <sup>4</sup> <sup>0</sup> <sup>1</sup> ( ) ( ) ( ) ( )exp( ) , ( ) ( ) ( ) ( )exp( ) , ( ) ( ) ( ) ( )exp( ) , <sup>1</sup> ( ) zzi ij ij ij j zri ij ij ij j zi ij ij ij j zi ij j r z p R s R s J sr s z ds r z p R s R s J sr s z ds D r z p R s R s J sr s z ds u rz p s σ τ σ τ τ ∞ = ∞ = ∞ = ∞ = , = , = , = , = ∑ ∫ ∑ ∫ ∑ ∫ ∑ ∫ 1) 0 0 2 (1) (1) <sup>5</sup> 0 1 <sup>0</sup> <sup>1</sup> 2 (1) (1) <sup>6</sup> 0 0 <sup>0</sup> <sup>1</sup> ( 1 2) ( ) ( ) ( )exp( ) , <sup>1</sup> ( ) ( ) ( ) ( )exp( ) , <sup>1</sup> ( ) ( ) ( ) ( )exp( ) ij ij ri ij ij ij j i ij ij ij j i R s R s J sr s z ds u r z p R s R s J sr s z ds s r z p R s R s J sr s z ds s τ τ φ τ ∞ = ∞ = ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ = , ⎪ ⎪ ⎪ ⎪ , = ⎪ ⎪ ⎪ <sup>⎪</sup> , =− <sup>⎪</sup> ⎭ ∑ ∫ ∑ ∫ (30) where the constants (1) ( 1 2 1 2 6) kij p ij k , = , , = , ,..., are given in Appendix A. The general solutions are obtained by using the Hankel transform technique (Sneddon & Lowengrub, 1969): $$\begin{aligned} &\sigma\_{zz}^{(2)}(r,z) = \sum\_{j=1}^{6} \int\_{0}^{\infty} sp\_{1j}^{(2)} A\_{ij}(s) f\_{0}(sr) \exp(s\gamma\_{jz}) ds, \\ &\sigma\_{zz}^{(2)}(r,z) = \sum\_{j=1}^{6} \int\_{0}^{\infty} sp\_{2j}^{(2)} A\_{ij}(s) f\_{1}(sr) \exp(s\gamma\_{jz}) ds, \\ &D\_{zi}^{(2)}(r,z) = \sum\_{j=1}^{6} \int\_{0}^{\infty} sp\_{3j}^{(2)} A\_{ij}(s) f\_{0}(sr) \exp(s\gamma\_{jz}) ds, \\ &u\_{zi}^{(2)}(r,z) = \sum\_{j=1}^{6} \int\_{0}^{\infty} p\_{4j}^{(2)} A\_{ij}(s) f\_{0}(sr) \exp(s\gamma\_{jz}) ds, \\ &u\_{ri}^{(2)}(r,z) = \sum\_{j=1}^{6} \int\_{0}^{\infty} p\_{5j}^{(2)} A\_{ij}(s) f\_{1}(sr) \exp(s\gamma\_{jz}) ds, \\ &\phi\_{i}^{(2)}(r,z) = -\sum\_{j=1}^{6} \int\_{0}^{\infty} p\_{6j}^{(2)} A\_{ij}(s) f\_{0}(sr) \exp(s\gamma\_{jz}) ds \end{aligned} \tag{31}$$ where ( ) ( 1 2 1 2 6) Asi j ij = , , = , ,..., are the unknown functions to be solved, and the constants ij γand (2) ( 1 2 1 2 6) kij p i jk = , , , = , ,..., are given in Appendix B. Application of Hankel Transform for Solving a Fracture r , 0( ) zr σ The functions 0( ) zz σ σ σ where the functions 1 ( ) <sup>T</sup> may be defined and evaluated as: superposition. 6 (2) 1 1 1 the r -axis in the plate without crack, are obtained as follows: ∞ ∞ ∞ zz r c + π + → + → 5. Numerical results and discussion <sup>r</sup> = and 29.82[W/mK] κ 21.25[W/mK] κ <sup>z</sup> r c π → zr r c π σ Problem of a Cracked Piezoelectric Strip Under Thermal Loading 215 ( ) ( ), lim ( ) ( 1 2 3) kl k j jl kl kl <sup>s</sup> <sup>j</sup> displacement components induced by the disturbed temperature field (2)( )( 1 2) T rz i <sup>i</sup> , = , on 6 2 (2) (1) T ⎧ ⎫ ⎪ ⎪ <sup>=</sup> ⎨ ⎬ <sup>+</sup> = = () () () () ( ) , r R s p d s p R s J sr ds ⎪ ⎪ ⎩ ⎭ ⎪ ⎪ ⎩ ⎭ ∫ ∑ ∑ <sup>0</sup>J sr ds ( ) <sup>⎪</sup> <sup>⎫</sup> ⎪ ⎪ <sup>⎪</sup> ⎨ ⎬ <sup>⎪</sup> ⎪ ⎪ ⎩ ⎭ ⎪⎭ <sup>j</sup> d s ( 1 2 6) j = , ,..., are also given in Appendix C. These components () () () () ( ) , r R s p d s p R s J sr ds 0 0 1 10 <sup>11</sup> <sup>11</sup> <sup>0</sup> 1 1 6 2 (2) (1) zz j j j j j j ∫ ∑ ∑ zr j j j j j j ∫ ∑ ∑ 0 0 1 11 <sup>21</sup> <sup>21</sup> <sup>0</sup> 1 1 6 2 (2) (1) T ⎧ ⎫ ⎪ ⎪ <sup>=</sup> ⎨ ⎬ <sup>+</sup> = = T z j j j j j j = = are superficial quantities and have no physical meaning in this analysis. However, they are equivalent to the crack face tractions in solving the crack problem by a proper To solve the singular integral equations (21) and (35)- (37) by using the Gauss-Jacobi integration formula (Sih, 1972), we introduce the following functions ( ) ( 0 1 2 3) <sup>l</sup> > 1 2 ( ) ( ) ( 0 1 2 3) l l Φ Then the stress intensity factors K<sup>I</sup> , KII and the electric displacement intensity factor K<sup>D</sup> 1 2 1 2 = − ,= ⎬ / / ∞ ∞ / / ∞ / / ∞ ∞ lim {2 ( )} ( 0) ( ) ( ) ( ) , = − ,= + ⎪ lim {2 ( )} ( 0) ( ) ( ), lim {2 ( )} ( 0) ( ) ( ) ( ) For the numerical calculations, the thermo-electro-elastic properties of the plate are assumed to be ones of cadmium selenide with the following properties (Ashida & Tauchert, 1998). The values of the coefficients of heat conduction for cadmium selenide could not be found in the literature. Since the values of them for orthotropic Alumina (Al2O3) are assumed. To examine the effects of the normalized crack size c h/ and the normalized crack = − ,= + ⎪ 1 2 1 2 I 111 1 13 3 K cr r c Z c Z c II 1 22 2 1 2 1 2 D 131 1 33 3 σ K cr Dr c Z c Z c K cr r c Z c c t G t t l c t () () () () 00 1 1 <sup>31</sup> <sup>31</sup> <sup>0</sup> 1 1 D r Rs p d s p R s <sup>⎧</sup> <sup>=</sup> <sup>−</sup> <sup>=</sup> ∑ <sup>=</sup> , <sup>=</sup> , , (40) r and 0( ) D r <sup>z</sup> , which correspond to the stress and electric ⎫ ⎪ ⎪ ⎪ ⎪⎪ ⎬ ⎪ Φ / ⎛ ⎞ <sup>+</sup> <sup>=</sup> = ,,, ⎜ ⎟ ⎝ ⎠ <sup>−</sup> (42) { } πΦΦ πΦ πΦΦ <sup>z</sup> = (Dag, 2006), the value <sup>2</sup> 1 15 { } t l = ,,, : ⎫ ⎪⎪ (43) = r z / = /. is ⎪ ⎪⎭ κ κκ (41) Z s p d s Z Z s kl <sup>∞</sup> →∞ <sup>=</sup> In Eq.(40), the functions 1 ( ) ( 1 2 6 1 2 3) jl d sj l = , ,..., , = , , are given in Appendix C. Similar to the temperature analysis, the problem may be reduced to a system of singular integral equations by taking the second boundary conditions (7)-(9) into consideration and by defining the following new unknown functions ( ) ( 1 2 3) Gr l <sup>l</sup> = , , : $$G\_1(r) = \begin{cases} \frac{\partial}{\partial r} \left( u\_{z1}^{(2)}(r, 0) - u\_{z2}^{(2)}(r, 0) \right) & (0 \le r < c) \\\ 0 & (c \le r < \infty) \end{cases} \tag{32}$$ $$\mathbf{G}\_2(r) = \begin{cases} r \frac{\partial}{\partial r} \left[ \frac{1}{r} \{ \mu\_{r1}^{(2)}(r, 0) - \mu\_{r2}^{(2)}(r, 0) \} \right] & \text{(\$0 \le r < c\$)}\\ 0 & \text{(\$c \le r < \infty)} \end{cases} \tag{33}$$ $$G\_3(r) = \begin{cases} -\frac{\partial}{\partial r} \left\{ \phi\_1^{(2)}(r, 0) - \phi\_2^{(2)}(r, 0) \right\} & \text{( $0 \le r < c$ )}\\ 0 & \text{( $c \le r < \infty$ )} \end{cases} \tag{34}$$ Making use of the first boundary conditions (7)-(9) with Eqs.(10), we have the following system of singular integral equations for the determination of the unknown functions ( ) ( 1 2 3) Gt l <sup>l</sup> =,, : $$\begin{aligned} \left\| \int\_{0}^{c} t \Big\| \left< Z\_{11}^{\rm x} M\_{0}^{(1)}(t, r) + M\_{11}(t, r) \right> G\_{1}(t) + M\_{12}(t, r) G\_{2}(t) + \\ + \Big< Z\_{13}^{\rm x} M\_{0}^{(1)}(t, r) + M\_{13}(t, r) \Big> G\_{3}(t) \Big\| dt = \sigma\_{zz0}(r) \quad \text{ (} 0 \le r < c \text{)} \end{aligned} \tag{35}$$ $$\begin{aligned} \int\_0^c t \left[ M\_{21}(t, r) \mathcal{G}\_1(t) + \left( Z\_{22}^{\times} M\_1^{(1)}(t, r) + M\_{22}(t, r) \right) \mathcal{G}\_2(t) + \\ + M\_{23}(t, r) \mathcal{G}\_3(t) \right] dt &= \sigma\_{x0}(r) \quad \text{(} 0 \le r < c \text{)} \end{aligned} \tag{36}$$ $$\begin{aligned} \int\_0^c \mathbf{f} \Big\{ Z\_{31}^{\infty} M\_0^{(1)}(t, r) + M\_{31}(t, r) \Big\} \mathbf{G}\_1(t) + M\_{32}(t, r) \mathbf{G}\_2(t) + \\ \quad + \Big\{ Z\_{33}^{\infty} M\_0^{(1)}(t, r) + M\_{33}(t, r) \Big\} \mathbf{G}\_3(t)] dt &= D\_{z0}(r) \quad \text{(} 0 \le r < c \text{)} \end{aligned} \tag{37}$$ where the kernel functions (1) <sup>1</sup> M ( ) t r, , ( ) Mkl t r, and the constants ( 1 2 3) Z kl kl <sup>∞</sup> , =,, are given by { } ∞ ∞ ∫ kl kl 1 2 <sup>0</sup> $$M\_{1}^{(1)}(t,r) = \begin{cases} \frac{4}{\pi rt} \Big\{ K\left(\frac{r}{t}\right) - E\left(\frac{r}{t}\right) \right\} + \frac{2}{\pi rt} \Big[ \frac{t^{2}}{t^{2} - r^{2}} E\left(\frac{r}{t}\right) - K\left(\frac{r}{t}\right) \Big] & (r < t)\_{r} \\\\ \frac{4}{\pi t^{2}} \Big\{ K\left(\frac{t}{r}\right) - E\left(\frac{t}{r}\right) \Big\} + \frac{2}{\pi} \frac{1}{t^{2} - r^{2}} E\left(\frac{t}{r}\right) & (r > t) \end{cases} \tag{38}$$ $$M\_{kl}(t,r) = \begin{cases} \int\_{0}^{\infty} s \Big[ Z\_{kl}(s) - Z\_{kl}^{\infty} \right] I\_{0}(sr) I\_{1}(st) ds & (k = 1, 3, l = 1, 3), \\\\ \int\_{0}^{\infty} s \Big[ Z\_{kl}(s) - Z\_{kl}^{\infty} \right] I\_{0}(sr) I\_{2}(st) ds & (k = 1, 3, l = 2), \\\\ \int\_{0}^{\infty} s \Big[ Z\_{kl}(s) - Z\_{kl}^{\infty} \right] I\_{1}(sr) I\_{1}(st) ds & (k = 2, l = 1, 3), \\\\ \int\_{0}^{\infty} s \Big[ Z\_{kl}(s) - Z\_{kl}^{\infty} \right] I\_{1}(sr) I\_{2}(st) ds & (k = 2, l = 2) \end{cases} \tag{39}$$ ( ) ( ) ( ) ( 2 2) s Z s Z J sr J st ds k l Similar to the temperature analysis, the problem may be reduced to a system of singular integral equations by taking the second boundary conditions (7)-(9) into consideration and > { } (2) (2) 1 2 u r u r rc z z G r <sup>r</sup> ( 0) ( 0) (0 ) ( ) { } (2) (2) 1 2 <sup>1</sup> ( 0) ( 0) (0 ) ( ) { } (2) (2) 1 2 r r rc G r <sup>r</sup> φ { } (1) (1) (1) ∞ ∞ π ( ) kl M tr (1) π ∫ ∫ ∫ ∫ { } 23 3 0 { } { } ( 0) ( 0) (0 ) ( ) Making use of the first boundary conditions (7)-(9) with Eqs.(10), we have the following system of singular integral equations for the determination of the unknown functions <sup>11</sup> <sup>0</sup> 11 1 12 2 <sup>0</sup> [ ( ) ( ) () ( ) () 21 1 22 <sup>1</sup> 22 2 <sup>0</sup> zr [ ( ) ( ) () ( ) () t Z M tr M tr G t M trG t ( ) ( ) ( )] ( ) (0 ) + , + , = ≤< 2 2 2 π t t rr r r , = <sup>⎨</sup> <sup>⎬</sup> <sup>⎪</sup> ⎧ ⎫ ⎛⎞ ⎛⎞ ⎛ ⎞ <sup>⎪</sup> ⎨ ⎬ − + <sup>&</sup>gt; <sup>⎪</sup> ⎜⎟ ⎜⎟ ⎜ ⎟ <sup>⎪</sup> ⎩ ⎭ ⎩ ⎭ ⎝⎠ ⎝⎠ <sup>−</sup> ⎝ ⎠ s Z s Z J sr J st ds k l s Z s Z J sr J st ds k l <sup>⎧</sup> <sup>⎫</sup> − = , , <sup>=</sup> , <sup>⎪</sup> <sup>⎪</sup> <sup>⎪</sup> <sup>⎪</sup> <sup>⎪</sup> − = , , <sup>=</sup> <sup>⎪</sup> <sup>⎪</sup> <sup>⎪</sup> s Z s Z J sr J st ds k l , = <sup>⎨</sup> <sup>⎬</sup> <sup>⎪</sup> − = , <sup>=</sup> , <sup>⎪</sup> <sup>⎪</sup> <sup>⎪</sup> <sup>⎪</sup> <sup>⎪</sup> − = , <sup>=</sup> ⎩ ⎭ <sup>⎪</sup> <sup>⎪</sup> s Z s Z J sr J st ds k l Z M t r M t r G t dt D r r c t Z M tr M tr G t M trG t ( ) ( ) ( )] ( ) (0 ) [ ( ) () ( ) ( ) () tM trG t Z M tr M tr G t + , + , = ≤< Z M t r M t r G t dt r r c ,+ , + , + { } (1) <sup>∞</sup> , + ,+ , + ,+ , + , + <sup>1</sup> M ( ) t r, , ( ) Mkl t r, and the constants ( 1 2 3) Z kl kl 2 2 2 rr tr r K E E K rt <sup>4</sup> 2 1 ( ) ( ) ( ) ( ) ( 1 3 1 3), ( ) ( ) ( ) ( 1 3 2), ( ) ( ) ( ) ( 2 1 3), ( ) ( ) ( ) ( 2 2) tt t K E E rt 4 2 ( ), <sup>⎧</sup> ⎧ ⎫ ⎧ ⎫ <sup>⎫</sup> ⎛⎞ ⎛⎞ ⎪ ⎪ ⎛⎞ ⎛⎞ <sup>⎪</sup> ⎨ ⎬⎨ ⎜⎟ ⎜⎟ −+ − < ⎜⎟ ⎜⎟⎬ <sup>⎪</sup> <sup>⎪</sup> ⎩ ⎭ ⎝⎠ ⎝⎠ ⎪ ⎪ ⎩ ⎭ <sup>−</sup> ⎝⎠ ⎝⎠ <sup>⎪</sup> π 13 0 13 3 0 ( ) ( )] ( ) (0 ) + , = ≤< M t r G t dt r r c σ <sup>31</sup> <sup>0</sup> 31 1 32 2 <sup>0</sup> 33 0 33 3 0 rt t t rt t t t r M tr { } ∞ ∞ ∞ ∞ ∞ ∞ ∞ ∞ kl kl 0 1 <sup>0</sup> 0 2 <sup>0</sup> 1 1 <sup>0</sup> 1 2 <sup>0</sup> { } kl kl { } kl kl { } kl kl φ<sup>⎧</sup> <sup>∂</sup> <sup>⎫</sup> ⎪− , − , ≤< <sup>⎪</sup> <sup>=</sup> <sup>⎨</sup> <sup>∂</sup> <sup>⎬</sup> <sup>⎪</sup> <sup>≤</sup> < ∞ <sup>⎪</sup> ⎩ ⎭ <sup>⎧</sup> <sup>∂</sup> <sup>⎫</sup> <sup>⎪</sup> , − , ≤< <sup>⎪</sup> <sup>=</sup> ⎨∂ <sup>⎬</sup> <sup>⎪</sup> <sup>≤</sup> < ∞ <sup>⎪</sup> ⎩ ⎭ r r r u r u r rc <sup>⎧</sup> <sup>∂</sup> ⎡ ⎤ <sup>⎫</sup> <sup>⎪</sup> ⎢ ⎥ , − , ≤< <sup>⎪</sup> <sup>=</sup> <sup>⎨</sup> <sup>∂</sup> ⎣ ⎦ <sup>⎬</sup> <sup>⎪</sup> <sup>⎪</sup> ⎩ ⎭ <sup>≤</sup> < ∞ 0 () 0 () 0 () zz σ ∫ (36) z ∫ (37) ∫ (35) c r c r c r (32) (33) (34) <sup>∞</sup> , =,, are given by (38) (39) by defining the following new unknown functions ( ) ( 1 2 3) Gr l <sup>l</sup> = , , : 1 G r r r 2 3 ∞ ∞ c c where the kernel functions (1) (1) 1 ( ) c ( ) ( 1 2 3) Gt l <sup>l</sup> =,, : $$Z\_{kl}(\mathbf{s}) = \sum\_{j=1}^{6} p\_{k1j}^{(2)} d\_{1jl}(\mathbf{s}), \quad Z\_{kl}^{\Rightarrow} = \lim\_{s \to \infty} Z\_{kl}(\mathbf{s}) \quad \text{( $k,l = 1,2,3$ )}\tag{40}$$ In Eq.(40), the functions 1 ( ) ( 1 2 6 1 2 3) jl d sj l = , ,..., , = , , are given in Appendix C. The functions 0( ) zz σ r , 0( ) zr σ r and 0( ) D r <sup>z</sup> , which correspond to the stress and electric displacement components induced by the disturbed temperature field (2)( )( 1 2) T rz i <sup>i</sup> , = , on the r -axis in the plate without crack, are obtained as follows: $$\begin{aligned} \sigma\_{zx0}(r) &= \int\_0^\alpha R\_0(s) \left\{ \sum\_{j=1}^6 p\_{11j}^{(2)} d\_{1j}^T(s) + \sum\_{j=1}^2 p\_{11j}^{(1)} R\_{1j}(s) \right\} I\_0(sr) ds, \\ \sigma\_{zx0}(r) &= \int\_0^\alpha R\_0(s) \left\{ \sum\_{j=1}^6 p\_{21j}^{(2)} d\_{1j}^T(s) + \sum\_{j=1}^2 p\_{21j}^{(1)} R\_{1j}(s) \right\} I\_1(sr) ds, \\ D\_{z0}(r) &= \int\_0^\alpha R\_0(s) \left\{ \sum\_{j=1}^6 p\_{31j}^{(2)} d\_{1j}^T(s) - \sum\_{j=1}^2 p\_{31j}^{(1)} R\_{1j}(s) \right\} I\_0(sr) ds \end{aligned} \tag{41}$$ where the functions 1 ( ) <sup>T</sup> <sup>j</sup> d s ( 1 2 6) j = , ,..., are also given in Appendix C. These components are superficial quantities and have no physical meaning in this analysis. However, they are equivalent to the crack face tractions in solving the crack problem by a proper superposition. To solve the singular integral equations (21) and (35)- (37) by using the Gauss-Jacobi integration formula (Sih, 1972), we introduce the following functions ( ) ( 0 1 2 3) <sup>l</sup> Φt l = ,,, : $$\mathbf{G}\_{l}(t) = \left(\frac{c+t}{c-t}\right)^{1/2} \Phi\_{l}(t) \quad \text{( $l=0,1,2,3$ )}\tag{42}$$ Then the stress intensity factors K<sup>I</sup> , KII and the electric displacement intensity factor K<sup>D</sup> may be defined and evaluated as: $$\begin{aligned} K\_{\mathrm{I}} &= \lim\_{r \to c^{+}} \{ 2\pi (c - r) \}^{1/2} \sigma\_{zz1}(r, 0) = (\pi c)^{1/2} \left\langle Z\_{11}^{v} \Phi\_{1}(c) + Z\_{13}^{v} \Phi\_{3}(c) \right\rangle\_{\mathrm{I}} \\ K\_{\mathrm{II}} &= \lim\_{r \to c^{+}} \{ 2\pi (c - r) \}^{1/2} \sigma\_{zr1}(r, 0) = (\pi c)^{1/2} Z\_{22}^{v} \Phi\_{2}(c), \\ K\_{\mathrm{D}} &= \lim\_{r \to c^{+}} \{ 2\pi (c - r) \}^{1/2} D\_{z1}(r, 0) = (\pi c)^{1/2} \left\langle Z\_{31}^{v} \Phi\_{1}(c) + Z\_{33}^{v} \Phi\_{3}(c) \right\rangle\_{\mathrm{I}} \end{aligned} \tag{43}$$ #### 5. Numerical results and discussion For the numerical calculations, the thermo-electro-elastic properties of the plate are assumed to be ones of cadmium selenide with the following properties (Ashida & Tauchert, 1998). The values of the coefficients of heat conduction for cadmium selenide could not be found in the literature. Since the values of them for orthotropic Alumina (Al2O3) are 21.25[W/mK] κ <sup>r</sup> = and 29.82[W/mK] κ <sup>z</sup> = (Dag, 2006), the value <sup>2</sup> 1 15 κ κκ = r z / = /. is assumed. To examine the effects of the normalized crack size c h/ and the normalized crack Application of Hankel Transform for Solving a Fracture 0 0 1 2 c/h K / displacement intensity factor KD D Z 0 p T (πc) 1/2 0 0.01 0.02 σ zz<sup>0</sup> , σ r -axis without crack due to the temperature shown in Fig. 2 h1/h=0.25 (a) (b) h1/h=0.5 h1/h=0.75 (c) 0.02 0.04 (σzz0, σzr0)/λ33 Fig. 3. The stress components 0 K /λ T (πc) I 33 0 1/2 0.01 0.02 T0 0.06 h1/h=0.25 c/h=0.5 Problem of a Cracked Piezoelectric Strip Under Thermal Loading 217 0 1 2 σzz0 0.01 0.02 K /λ T (πc) II 33 0 0 1 2 h1/h=0.25 h1/h=0.5 h1/h=0.75 c/h Fig. 4. (a) The effect of the crack size on the stress intensity factor K<sup>I</sup> . (b) The effect of the crack size on the stress intensity factor KII . (c) The effect of the crack size on the electric 1/2 0.03 0.04 σzr0 Dz0 r/c zr<sup>0</sup> and the electric displacement component Dz<sup>0</sup> on the Dz0/pzT0 <sup>0</sup> <sup>1</sup> <sup>2</sup> <sup>0</sup> c/h h1/h=0.25, 0.75 h1/h=0.50 0 0.01 location 1 h h/ on the stress and electric displacement intensity factors, the solutions of the system of the singular integral equations have been computed numerically. $$\begin{aligned} c\_{11} &= 74.1 \times 10^9 \text{[N/ m}^2\text{]} \qquad c\_{12} = 45.2 \times 10^9 \text{[N/ m}^2\text{]} \\ c\_{13} &= 39.3 \times 10^9 \text{[N/ m}^2\text{]} \qquad c\_{33} = 83.6 \times 10^9 \text{[N/ m}^2\text{]} \\ c\_{44} &= 13.2 \times 10^9 \text{[N/ m}^2\text{]} \\ e\_{31} &= -0.16 \text{[C/ m}^2\text{]} \qquad e\_{33} = 0.347 \text{[C/ m}^2\text{]} \\ e\_{15} &= -0.138 \text{[C/ m}^2\text{]} \end{aligned} \qquad \begin{aligned} c\_{33} &= 83.6 \times 10^9 \text{[N/ m}^2\text{]} \\ e\_{33} &= 0.347 \text{[C/ m}^2\text{]} \qquad c\_{33} = 0.347 \text{[C/ Vm}^2\text{]} \\ e\_{16} &= 82.6 \times 10^{-12} \text{[C/ Vm}\text{]} \qquad c\_{33} = 90.3 \times 10^{-12} \text{[C/ Vm}\text{]} \qquad c\_{33} = 0.551 \times 10^6 \text{[N/ K m}^2\text{]} \qquad c\_{33} = 0.551 \times 10^6 \text{[N/ K m}^2\text{]} \qquad c\_{33} = 0.347 \text{[C/ K}^2\text{m}^{-2}\text{]} \qquad c\_{33} = 0.294 \times 10^{-6} \text{[C K}^{-1}\text{m}^{-2}\text{]} \} \end{aligned} \tag{44}$$ In the first set of calculations, we consider the temperature field and the electro-elastic fields without crack. Figure 2 shows the normalized temperature 20 0 ( ( ) ) ( 1 2) Tx T T i <sup>i</sup> − / =, on the crack faces (0 r cz 0 ) <sup>±</sup> ≤ <,→ and the crack extended line ( 2 0) c r cz <sup>≤</sup> ≤ ,= for 1 h h/ = . 0 25 and 0 5 c h/ = . , where TT T 0 10 20 = − . The maximum local temperature difference across the crack occurs at the center of the crack. Figure 3 exhibits the normalized stress components 0 0 33 0 ( ( ) ( )) zz zr σσ λ r rT , / and the electric displacement component 0 0 ( ) D r pT z z / on the r -axis in the strip without crack due to the temperature shown in Figure 2. The maximum absolute values of 0( ) zz σ r and 0( ) D r <sup>z</sup> occur at the center of the crack ( 0 0) r c/ = . , whereas the maximum value of 0( ) zr σ r occurs at the crack tip ( / 1 0) r c = . . Fig. 2. The temperature on the crack faces and the crack extended line for 0 5 c h/ = . and <sup>1</sup> h h/ = . 0 25 In the second set of calculations, we study the influence of the crack size on the stress and electric displacement intensity factors. Figures 4(a)-(c) show the plots of the normalized stress and electric displacement intensity factors 1 2 I II 33 0 ( ) () KK T c λ π / , / , 1 2 D 0( ) K pT c <sup>z</sup> π / / versus c h/ for 1 h h/ = . 0 25 , 0.5 and 0.75. Because of symmetry, the values of KI and KD location 1 h h/ on the stress and electric displacement intensity factors, the solutions of the = .× / / = .× = .× / / = .× =− . / / = . = .× / = .× / =. × 2 2 <sup>6</sup> 33 74 1 10 [ ], 45 2 10 [ ], N m N m 39 3 10 [ ], 83 6 10 [ ], N m N m 9 9 2 2 ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ (44) 9 9 2 2 12 12 82 6 10 [C Vm], 90 3 10 [C Vm], ε λ =− . × . ⎪⎭ In the first set of calculations, we consider the temperature field and the electro-elastic fields without crack. Figure 2 shows the normalized temperature 20 0 ( ( ) ) ( 1 2) Tx T T i <sup>i</sup> − / =, on the crack faces (0 r cz 0 ) <sup>±</sup> ≤ <,→ and the crack extended line ( 2 0) c r cz <sup>≤</sup> ≤ ,= for 1 h h/ = . 0 25 and 0 5 c h/ = . , where TT T 0 10 20 = − . The maximum local temperature difference across the displacement component 0 0 ( ) D r pT z z / on the r -axis in the strip without crack due to the 0 1 2 r/c Fig. 2. The temperature on the crack faces and the crack extended line for 0 5 c h/ = . and stress and electric displacement intensity factors 1 2 In the second set of calculations, we study the influence of the crack size on the stress and electric displacement intensity factors. Figures 4(a)-(c) show the plots of the normalized versus c h/ for 1 h h/ = . 0 25 , 0.5 and 0.75. Because of symmetry, the values of KI and KD − − 0 16[ ], C m 0 347[ ], C m 2 2 N Km ], 0 551 10 [ ], N Km =. × <sup>⎪</sup> / / <sup>⎪</sup> σσ : i=1 (z→0<sup>+</sup> ) : i=2 (z→0- ) I II 33 0 ( ) () KK T c λ π / , / , 1 2 D 0( ) K pT c <sup>z</sup> π/ / h1/h=0.25 c/h=0.5 λr rT , / and the electric σ r and 0( ) D r <sup>z</sup> occur r occurs at the σ system of the singular integral equations have been computed numerically. 11 12 c c c c 13 33 9 2 13 2 10 [ ], N m = .× / =− . / 11 33 6 0 621 10 [ 0.2 0.4 0.6 (T-Ti 20)/T0 0.8 1 pz 2 94 10 [ m ] CK 0 138[ ], C m 31 33 2 6 2 1 − − − Figure 3 exhibits the normalized stress components 0 0 33 0 ( ( ) ( )) zz zr temperature shown in Figure 2. The maximum absolute values of 0( ) zz at the center of the crack ( 0 0) r c/ = . , whereas the maximum value of 0( ) zr e e 44 c 11 ε λ crack occurs at the center of the crack. crack tip ( / 1 0) r c = . . <sup>1</sup> h h/ = . 0 25 15 e Fig. 3. The stress components σ zz<sup>0</sup> , σ zr<sup>0</sup> and the electric displacement component Dz<sup>0</sup> on the r -axis without crack due to the temperature shown in Fig. 2 Fig. 4. (a) The effect of the crack size on the stress intensity factor K<sup>I</sup> . (b) The effect of the crack size on the stress intensity factor KII . (c) The effect of the crack size on the electric displacement intensity factor KD Application of Hankel Transform for Solving a Fracture contact of the crack faces would occur. The constants (1) ( 1 2 1 2 6) kij p ij k , = , , = , ,..., are ( ) (1) 2 (1) 2 3 31 33 33 (1) (1) (1) 4 56 (1) ( ) τ ij C ij ij , τ k ( ) ij ij ij τ = − ′ ′ (1) 2 41 42 (2) 2 2 44 43 ij ij = − ′ ′ m a a m a a ( ) , , ε εε εε ε 1 2 2 τ 1122 rzrz (22) 2 ij z z ij n HH 13 33 31 33 13 11 15 31 334 33 11 15 33 15 33 33 11 c ee c ee HHH ee ee + + <sup>=</sup> <sup>=</sup> − − ε ε εε εε rzr ( ) τ ij ij ij ij ij ij τ p c ck C e N 2 44 15 p e ek C N p p k Cp Cp N 1 13 33 33 33 ij ij ij ij ij τ =− − − ⎫ ij ij ij ij ij ij z ij ij ij ij ij ij ij ij 1 , <sup>⎪</sup> =+ + <sup>⎪</sup> p c k C eN i j ετ 2 ij <sup>+</sup> ⎪⎪ <sup>=</sup> <sup>⎬</sup> ,=, + ⎪ <sup>+</sup> ⎪⎭ τ ′ ′ − ⎫ = ⎪ + ⎪ , ⎪ ⎪ , ( 1 2) τλ , , 21 22 (1) (2) (11) (12) (1) (2) 2 (11) (21) 2 (12) (22) m km n kn N i <sup>j</sup> m km b b ij ij ij ij ij ij ij ij ij ij ij ij n n <sup>+</sup> <sup>⎪</sup> = − <sup>⎪</sup> τ τ ij ij ij { } ( ) ij r r ij r ij ij ij z z ij z ij ij ( ) , τ τ 1 22 4 2 2 44 33 15 33 44 11 15 33 33 33 33 11 15 33 τ 33 11 15 33 15 33 33 11 33 11 15 33 15 33 33 11 ( ) , (11) 2 (1) 1 3 21 22 4 (21) 2 (1) 1 3 21 22 4 (12) 2 2 (2) 1 2 21 22 4 n HH b b H m =+ − + ′ ′ τ n HH b b H m =+ − + ′ ′ n HH b b H m =− − + ′ ′ , { } ( ) ij r r ij ij r ij ij { } ( )( ) ττ = − { } ( ) <sup>2</sup> (2) b b c ee c e c e c ee HHHH ee ee ee ee + + ++ <sup>=</sup> <sup>=</sup> <sup>=</sup> <sup>=</sup> −−−− n n =− = = ⎪ =− + + ⎪ large. Appendix A where with Problem of a Cracked Piezoelectric Strip Under Thermal Loading 219 3. For the case of 1 h h/ > 0.5 , mode I stress intensity factor becomes negative so that the 4. The intensity factors of crack near the free surfaces due to the thermal load are not so , ⎪ , ⎪ ⎭ ( 1 2) (A.1) (A.2) ⎬ , = , , εε , z z <sup>z</sup> p e p e <sup>H</sup> ee ee 33 33 33 15 33 11 4 33 11 15 33 15 33 33 11 <sup>⎬</sup> − − <sup>⎪</sup> <sup>=</sup> <sup>=</sup> − − <sup>⎪</sup> ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ = ⎪ ⎪ ⎪ ⎪ ( , 1,2) εε εε λ ε (A.3) ⎫ ⎪ ⎪ (A.4) ⎭ i j τ > τ τ > τ ij z ij ij <sup>⎪</sup> ′ ′ − + ⎪⎭ , , , , εε H m λ ε εε for 1 h h/ = . 0 5 are zero, and [ ] <sup>1</sup> <sup>I</sup> h h/ 0.25 <sup>K</sup> <sup>=</sup> [ ] <sup>1</sup> <sup>I</sup> h h/ 0.75 <sup>K</sup> <sup>=</sup> = − , [ ] <sup>1</sup> II h h/ 0.25 <sup>K</sup> <sup>=</sup> [ ] <sup>1</sup> II h h/ 0.75 <sup>K</sup> <sup>=</sup> <sup>=</sup> [ ] <sup>1</sup> <sup>D</sup> h h/ 0.25 <sup>K</sup> <sup>=</sup> [ ] <sup>1</sup> <sup>D</sup> h h/ 0.75 <sup>K</sup> <sup>=</sup> = − . The absolute value of 1 2 I 33 0 K Tc λ π( ) / / for 1 h h/ = . 0 25 and 0.75 monotonically increases with increasing c h/ , but the value of 1 2 II 33 0 K Tc λ π( ) / / and the absolute value of 1 2 D 0( ) K pT c <sup>z</sup> π / / increase at first, reach maximum values and then decrease with increasing c h/ . The value of KI for 1 h h/ = . 0 75 becomes negative so that the contact of the crack faces would occur. The results presented here without considering this effect may not be exact but would be more conservative. Since the contact of the crack faces will increase the friction between the faces and make thermo-electrical transfer across the crack faces easier, the stress and electric displacement intensity factors would be lowered by these two factors. In the final set of calculations, we investigate the influence of the crack location on the intensity factors. Figure 5 indicates the effect of the crack location on K<sup>I</sup> , KII and KD for c h/ = . 0 5 . As 1 h h/ increases, the values of KI and KD tend to decrease or increase monotonically. The value of 1 2 II 33 0 K Tc λ π( ) / / decreases if the crack approaches the free boundaries 1 ( 0.0 or 1.0) h h/ → , and the peak value of 1 2 II 33 0 K Tc λ π( ) / / =0.0277 occurs at <sup>1</sup> h h/ = 0.5 . Fig. 5 The effect of the crack location on the stress intensity factors K<sup>I</sup> , KII and the electric displacement intensity factor K<sup>D</sup> #### 6. Conclusion An example of the application of Hankel transform for solving a mixed-mode thermoelectro-elastic fracture problem of a piezoelectric material strip with a parallel penny-shaped crack is explained. The effects of the crack size ( ) c h/ and the crack location 1 ( ) h h/ on the fracture behavior are analyzed. The following facts can be found from the numerical results. ## Appendix A 218 Fourier Transform – Materials Analysis for 1 h h/ = . 0 5 are zero, and [ ] <sup>1</sup> <sup>I</sup> h h/ 0.25 <sup>K</sup> <sup>=</sup> [ ] <sup>1</sup> <sup>I</sup> h h/ 0.75 <sup>K</sup> <sup>=</sup> = − , [ ] <sup>1</sup> II h h/ 0.25 <sup>K</sup> <sup>=</sup> [ ] <sup>1</sup> II h h/ 0.75 <sup>K</sup> <sup>=</sup> <sup>=</sup> with increasing c h/ . The value of KI for 1 h h/ = . 0 75 becomes negative so that the contact of the crack faces would occur. The results presented here without considering this effect may not be exact but would be more conservative. Since the contact of the crack faces will increase the friction between the faces and make thermo-electrical transfer across the crack faces easier, the stress and electric displacement intensity factors would be lowered by these In the final set of calculations, we investigate the influence of the crack location on the intensity factors. Figure 5 indicates the effect of the crack location on K<sup>I</sup> , KII and KD for c h/ = . 0 5 . As 1 h h/ increases, the values of KI and KD tend to decrease or increase 0 0.2 0.4 0.6 0.8 1 c/h=0.5 KI KD KII h1/h Fig. 5 The effect of the crack location on the stress intensity factors K<sup>I</sup> , KII and the electric An example of the application of Hankel transform for solving a mixed-mode thermoelectro-elastic fracture problem of a piezoelectric material strip with a parallel penny-shaped crack is explained. The effects of the crack size ( ) c h/ and the crack location 1 ( ) h h/ on the fracture behavior are analyzed. The following facts can be found from the numerical results. 1. The large shear stress occurs in the strip without crack due to the disturbed temperature 2. The normalized intensity factors are under the great influence of the geometric 0.75 monotonically increases with increasing c h/ , but the value of 1 2 I 33 0 K Tc λ / / increase at first, reach maximum values and then decrease π ( ) / / decreases if the crack approaches the free II 33 0 K Tc λ π KD/pzT0(πc)1/2 0 0.01 0.02 ( ) / / for 1 h h/ = . 0 25 and II 33 0 K Tc λ π( ) / / and the ( ) / / =0.0277 occurs at [ ] <sup>1</sup> <sup>D</sup> h h/ 0.25 <sup>K</sup> <sup>=</sup> [ ] <sup>1</sup> <sup>D</sup> h h/ 0.75 <sup>K</sup> <sup>=</sup> = − . The absolute value of 1 2 II 33 0 K Tc λ boundaries 1 ( 0.0 or 1.0) h h/ → , and the peak value of 1 2 π absolute value of 1 2 two factors. <sup>1</sup> h h/ = 0.5 . D 0( ) K pT c <sup>z</sup> π monotonically. The value of 1 2 0 0.01 (KI, KII)/ displacement intensity factor K<sup>D</sup> parameters 1 h h/ and c h/ . 6. Conclusion field. λ33 T0(πc)1/2 0.02 0.03 The constants (1) ( 1 2 1 2 6) kij p ij k , = , , = , ,..., are $$\begin{aligned} p\_{1\dot{i}j}^{(1)} &= \left(c\_{13} - c\_{33}k\_{\dot{i}\dot{j}}\tau\_{\dot{i}\dot{j}}^{2}\right)\mathbf{C}\_{\dot{i}j} - e\_{33}\tau\_{\dot{i}\dot{j}}N\_{\dot{i}j} - \lambda\_{33}, \\ p\_{2\dot{i}j}^{(1)} &= c\_{44}\left(\mathbf{1} + k\_{\dot{i}j}\right)\tau\_{\dot{i}\dot{j}}\mathbf{C}\_{\dot{i}j} + e\_{15}N\_{\dot{i}j}, \\ p\_{3\dot{i}j}^{(1)} &= \left(e\_{31} - e\_{33}k\_{\dot{i}j}\tau\_{\dot{i}j}^{2}\right)\mathbf{C}\_{\dot{i}j} + e\_{33}\tau\_{\dot{i}j}N\_{\dot{i}j} + p\_{z^{\}} \\ p\_{4\dot{i}j}^{(1)} &= -k\_{\dot{i}j}\tau\_{\dot{i}\dot{j}}C\_{\dot{i}j} \ p\_{\dot{i}j}^{(1)} = \mathbf{C}\_{\dot{i}j} \ p\_{\dot{i}\dot{j}}^{(1)} = N\_{\dot{i}j} \end{aligned} \tag{A.1}$$ where $$\begin{aligned} C\_{ij} &= \frac{b\_{21}^{(1)} - b\_{22}^{\2} \tau\_{ij}^{2}}{m\_{ij}^{(1)} + k\_{ij} m\_{ij}^{(2)}}, \\ N\_{ij} &= \frac{n\_{ij}^{(11)} + k\_{ij} n\_{ij}^{(12)}}{m\_{ij}^{(1)} + k\_{ij} m\_{ij}^{(2)}}, \\ k\_{ij} &= -\frac{\tau\_{ij}^{2} n\_{ij}^{(11)} + n\_{ij}^{(21)}}{\tau\_{ij}^{2} n\_{ij}^{(12)} + n\_{ij}^{(22)}} \end{aligned} \tag{A.2}$$ with $$\begin{aligned} &m\_{ij}^{(1)} = a\_{41}^{\prime} - a\_{42}^{\prime}\tau\_{ij}^{2}, \\ &m\_{ij}^{(2)} = \left(a\_{41}^{\prime}\tau\_{ij}^{2} - a\_{43}^{\prime}\right)\tau\_{ij}^{2}, \\ &n\_{ij}^{(11)} = \left(\left(H\_{1r} + H\_{3r}\right)\left(b^{\prime}\underline{\boldsymbol{\boldsymbol{\tau}}}\_{21} - b^{\prime}\boldsymbol{\boldsymbol{\omega}}\_{22}^{\prime}\right) + H\_{4r}m\_{ij}^{(1)}\right)\tau\_{ij}, \\ &n\_{ij}^{(21)} = \left(\left(H\_{1z} + H\_{3z}\right)\left(b^{\prime}\underline{\boldsymbol{\boldsymbol{\tau}}}\_{21} - b^{\prime}\boldsymbol{\boldsymbol{\omega}}\_{22}\tau\_{ij}^{2}\right) + H\_{4z}m\_{ij}^{(1)}\right)\tau\_{ij}, \\ &n\_{ij}^{(21)} = \left(\left(H\_{1r} - H\_{2r}\tau\_{ij}^{2}\right)\left(b^{\prime}\underline{\boldsymbol{\boldsymbol{\tau}}}\_{21} - b^{\prime}\boldsymbol{\boldsymbol{\omega}}\_{22}\tau\_{ij}^{2}\right) + H\_{4r}m\_{ij}^{(1)}\right)\tau\_{ij}, \\ &n\_{ij}^{(22)} = \left(\left(H\_{1z} - H\_{2z}\tau\_{ij}^{2}\right)\left(b^{\prime}\underline{\boldsymbol{\boldsymbol{\tau}}}\_{21} - b^{\prime}\boldsymbol{\boldsymbol{\omega}}\_{22}\tau\_{ij}^{2}\right) + H\_{4z}m\_{ij}^{(2)}\right)\tau\_{ij} \end{aligned} \tag{A.3}$$ $$\begin{aligned} H\_{1r} &= \frac{c\_{44}\varepsilon\_{33} + c\_{15}\varepsilon\_{33}}{e\_{33}\varepsilon\_{11} - e\_{15}\varepsilon\_{33}}, \quad H\_{1z} = \frac{c\_{44}\varepsilon\_{11} + c\_{15}^2}{e\_{15}\varepsilon\_{33} - e\_{33}\varepsilon\_{11}}, \quad H\_{2r} = \frac{c\_{33}\varepsilon\_{33} + e\_{33}^2}{e\_{33}\varepsilon\_{11} - e\_{15}\varepsilon\_{33}}, \quad H\_{2z} = \frac{c\_{33}\varepsilon\_{11} + e\_{15}\varepsilon\_{33}}{e\_{15}\varepsilon\_{33} - e\_{33}\varepsilon\_{11}}\\ H\_{3r} &= \frac{c\_{13}\varepsilon\_{33} + e\_{31}\varepsilon\_{33}}{e\_{33}\varepsilon\_{11} - e\_{15}\varepsilon\_{33}}, \quad H\_{3z} = \frac{c\_{13}\varepsilon\_{11} + c\_{15}\varepsilon\_{31}}{e\_{15}\varepsilon\_{33} - e\_{33}\varepsilon\_{11}}, \quad H\_{4r} = \frac{p\_z\varepsilon\_{33} - \lambda\_{33}\varepsilon\_{33}}{e\_{33}\varepsilon\_{11} - e\_{15}\varepsilon\_{33}}, \quad H\_{4z} = \frac{p\_z\varepsilon\_{15} - \lambda\_{33}\varepsilon\_{11}}{e\_{15}\varepsilon\_{33} - e\_{33}\varepsilon\_{11}}\end{aligned} \quad \text{(A.4)}$$ Application of Hankel Transform for Solving a Fracture order 12. The elements ( ) ( 1 2 12) j k δ > , +,+ > > δ + , +,+ ij dsi j = , , = , ,..., are 1 T R s s s R s s T δ δ δ The functions ( ) ( 1 2 1 2 6) <sup>T</sup> + + The functions ( ) ( 1 2 1 2 6 1 2 3) ijk d si j k = , , = , ,..., , = , , are given by 1 9 dsq s jk j k ( ) ( ), , + = ⎫ ⎪ where the functions ( ) ( 1 2 12) j k q s jk , , = , ,..., are the elements of a square matrix <sup>1</sup> <sup>Q</sup> <sup>−</sup> <sup>=</sup> <sup>Δ</sup> of ( ) ( 1 2 6), ( ) ( 1 2 6) <sup>⎪</sup> = − = , ,..., <sup>⎪</sup> s p sh j <sup>k</sup> = − =,, ⎪ = =,, ⎫ () () 1 1 1 s p sh j γ (2) 6 1 12 ∑ 1 12 2 6 1 j j k k k d s q su s j jk k k d s q su s = 2 0 (1) ∑ 1 2 0 (1) 3 2 2 2 2 1 2 = = ∑ = ∑ k j k j j j k j k j j j k j k j k j j j Journal of Thermal Stresses, Vol. 19, pp. 237-265, 0149-5739 ∑ ( ) ( ) ( ), , = <sup>⎫</sup> <sup>=</sup> <sup>⎪</sup> () () () 0 (1) (1) 6 1 1 2 2 1 u s pRs pRs k R s u s pRs sh k + , u s pRs sh k <sup>⎪</sup> <sup>=</sup> <sup>⎪</sup> (2) sp j sp j γ ( ) exp( ) ( 1 2 3), +,+ <sup>⎪</sup> <sup>=</sup> <sup>⎭</sup> 2 69 dsq s jk j k (2) j k j k k j k j k k j k j k j k j k (2) 3 6 2 2 2 6 6 2 Appendix C where 7. References Problem of a Cracked Piezoelectric Strip Under Thermal Loading 221 ( 1 2 6 1 2 3) ⎪ ⎪ ⎭ ( 1 2 6) ⎬ = , ,..., j ⎪ ⎭ τ <sup>⎫</sup> = − =,, <sup>⎪</sup> <sup>⎪</sup> = − <sup>−</sup> =,, <sup>⎬</sup> <sup>⎪</sup> <sup>=</sup> − −= , ,..., <sup>⎪</sup> Embedded Crack in an Orthotropic FGM Coating, Proceedings of the International τ { } Ashida, F. & Tauchert, T.R. (1998). Transient Response of a Piezothermoelastic Circular Disk under Axisymmetric Heating. Acta Mechanica, Vol. 128, pp. 1-14, 0001-5970 Dag, S., Ilhan, K.A. & Erdogan, F. (2006), Mixed-Mode Stress Intensity Factors for an Conference FGM IX, 978-0-7354-0492-2, Oahu Island, Hawaii, October 2006 Erdogan, F. & Wu, B.H. (1996). Crack Problems in FGM Layers under Thermal Stresses. ( ) ( ) ( ) ( ) ( 1 2 6) 1 1 11 ( ) ( ) ( )exp( ) ( 1 2 3), ( ) ( ) ( )exp( ) ( 1 2 3), (C.1) (C.2) (C.4) (C.3) ⎪ ⎪ ⎪ ⎪ ⎭ j k s jk , , = , ,..., of the square matrix Δ are given by <sup>⎬</sup> <sup>=</sup> , ,..., <sup>=</sup> = , ,..., <sup>⎪</sup> ( ) exp( ) ( 1 2 3), ( 1 2 6) ⎬ = , ,..., , = , , $$\begin{aligned} a'\_{41} &= c\_{11'} & a'\_{42} &= c\_{44} + (e\_{15} + e\_{31})(H\_{1r} + H\_{3r}) \\ a'\_{43} &= c\_{13} + c\_{44} + (e\_{15} + e\_{31})H\_{1r'} & a'\_{44} &= (e\_{15} + e\_{31})H\_{2r'} \\ b'\_{21} &= \lambda\_{11'} & b'\_{22} &= -(e\_{15} + e\_{31})H\_{4r} \end{aligned} \tag{A.5}$$ ### Appendix B The constants ( 1 2 1 2 6) ij γi j = , , = , ,..., are the roots of the following characteristic equations: $$\begin{aligned} \left(f\_4 g\_2' + g\_4 f\_2'\right) \nu\_{\vec{\eta}}^6 + \left(f\_4 g\_0' + f\_2 g\_2' + g\_4 f\_0' + g\_2 f\_2'\right) \nu\_{\vec{\eta}}^4 + \\ + \left(f\_2 g\_0' + f\_0 g\_2' + g\_2 f\_0' + g\_0 f\_2'\right) \nu\_j^2 + \left(f\_0 g\_0' + g\_0 f\_0'\right) = 0 \end{aligned} \quad \text{(\$i = 1, 2, \$j = 1, 2, \dots, 6\$)}\tag{\text{B.1}}$$ where 1 11 [] [ ] j j γ γ ℜ <ℜ <sup>+</sup> , 2 21 [ ] [ ]( 1 2 5) j j γ γj ℜ > ℜ = , ,..., <sup>+</sup> and $$\begin{aligned} f\_4 &= c\_{44} e\_{33}, \\ f\_2 &= (c\_{13} + c\_{44})(e\_{15} + e\_{31}) - c\_{11} e\_{33} - c\_{44} e\_{15}, \\ f\_0 &= c\_{11} e\_{15}, \\ f\_2' &= c\_{33} (e\_{15} + e\_{31}) - e\_{33} (c\_{13} + c\_{44}), \\ f\_0' &= -c\_{44} (e\_{15} + e\_{31}) + e\_{15} (c\_{13} + c\_{44}) \end{aligned} \tag{B.2}$$ $$\begin{aligned} g\_4 &= c\_{44}\varepsilon\_{33\prime} \\ g\_2 &= -(e\_{15} + e\_{31})^2 - c\_{11}\varepsilon\_{33} - c\_{44}\varepsilon\_{11\prime} \\ g\_0 &= c\_{11}\varepsilon\_{11\prime} \\ g\_2' &= e\_{33}(e\_{15} + e\_{31}) + \varepsilon\_{33}(c\_{13} + c\_{44})\_{\prime} \\ g\_0' &= -e\_{15}(e\_{15} + e\_{31}) - \varepsilon\_{11}(c\_{13} + c\_{44}) \end{aligned} \tag{B.3}$$ The functions (2)( ) ( 1 2 1 2 6) kij p s i jk <sup>=</sup> , , , = , ,..., are $$\begin{aligned} p\_{1\stackrel{\uparrow}{i}}(^{2}\_{1}) &= c\_{13}a\_{\dot{i}} + \mathcal{V}\_{\dot{i}} \big( c\_{33} - c\_{33}b\_{\dot{i}\dot{j}} \big), \\ p\_{2\stackrel{\uparrow}{i}}^{(2)} &= c\_{44} \big( \mathcal{V}\_{\dot{i}\dot{j}} a\_{\dot{i}\dot{j}} - 1 \big) + c\_{15}b\_{\dot{i}\dot{j}}, \\ p\_{3\stackrel{\uparrow}{i}}^{(2)} &= c\_{31}a\_{\dot{i}\dot{j}} + \mathcal{V}\_{\dot{i}\dot{j}} \big( c\_{310} + c\_{330}b\_{\dot{i}\dot{j}} \big), \\ p\_{4\stackrel{\uparrow}{i}}^{(2)} &= 1, \\ p\_{5\stackrel{\uparrow}{i}}^{(2)} &= a\_{\dot{i}\dot{j}}, \\ p\_{8\stackrel{\uparrow}{i}}^{(2)} &= b\_{\dot{i}\dot{j}} \end{aligned} \quad \text{(\$i=1,2, \$j=1,2,...,6\$)} \tag{B.4}$$ where ij a and ( 1 2 1 2 6) ij bi j = , , = , ,..., are given by b $$\begin{aligned} a\_{ij} &= \frac{\mathbf{g}\_2' \mathbf{\hat{y}}\_{ij}^2 + \mathbf{g}\_0'}{\mathbf{g}\_4 \mathbf{\hat{y}}\_{ij}^4 + \mathbf{g}\_2 \mathbf{\hat{y}}\_{ij}^2 + \mathbf{g}\_0}, \\\ a\_{ij} &= -\frac{(\mathbf{c}\_{44} \mathbf{\hat{y}}\_{ij}^2 - \mathbf{c}\_{11}) a\_{ij} - c\_{13} - c\_{44}}{\mathbf{e}\_{15} + \mathbf{e}\_{31}} \end{aligned} \quad \text{(\$i = 1, 2, \$j = 1, 2, ..., 6\$)}\tag{B.5}$$ ## Appendix C 220 Fourier Transform – Materials Analysis ′ ′ = = + + + ⎫ <sup>⎪</sup> ′ ′ =++ + = + <sup>⎬</sup> <sup>⎪</sup> ′ ′ <sup>=</sup> <sup>=</sup> <sup>−</sup> <sup>+</sup> <sup>⎭</sup> , ( )( ), ( ), ( ), r r i j = , , = , ,..., are the roots of the following characteristic equations: γ + +++ + + = ′′ ′′ ′ ′ ( 1,2, 1 2 6) i j <sup>=</sup> = , ,..., (B.1) r r (A.5) (B.2) (B.3) (B.4) (B.5) r 41 11 42 44 15 31 1 3 a a c c e e H H 43 13 44 15 31 1 44 15 31 2 21 11 22 15 31 4 a a c c e eH e eH b b ( )( ) ( ) ( ) fg gf fg fg gf gf fg fg gf gf fg gf ℜ <ℜ <sup>+</sup> , 2 21 [ ] [ ]( 1 2 5) j j γ f ce 4 44 33 0 11 15 f ce 4 2 42 40 2 2 40 22 λ γ The constants ( 1 2 1 2 6) ij γ γ Appendix B where 1 11 [] [ ] j j γ , ( ) e eH 6 4 ij ij j j ℜ > ℜ = , ,..., <sup>+</sup> and 2 33 15 31 33 13 44 0 44 15 31 15 13 44 f ce e ec c f ce e ec c , , 2 13 44 15 31 11 33 44 15 f c c e e ce ce ( ) ( ), ( )( ) 2 2 15 31 11 33 44 11 g ee c c () , = ⎫ =− + − − ⎪ <sup>⎪</sup> <sup>=</sup> <sup>⎬</sup> <sup>⎪</sup> ′ = ++ + <sup>⎪</sup> ′ =− + − + ⎪ ε ε ⎪ ⎭ , ( 1 2 1 2 6) ( 1,2, 1 2 6) ⎬ = = , ,..., ⎬ = , , = , ,..., ( ) ( ), ( )( ) ε ε , ⎪ ⎪ ⎪ ⎭ 2 33 15 31 33 13 44 0 15 15 31 11 13 44 g ee e c c g ee e c c ( ) 1 , ( ) ε = ⎪ = ⎪ = ⎪ , ggg i j + ⎭ p ea e b i j <sup>⎪</sup> ′ = +− + <sup>⎪</sup> ′ =− + + + ⎪⎭ ( )( ) , = ⎫ <sup>⎪</sup> =+ +− − <sup>⎪</sup> <sup>⎪</sup> <sup>=</sup> <sup>⎬</sup> 2 20 0 2 20 02 00 00 0 γ ′ ′ ′′ ′′ + + +++ + γ , , 4 44 33 ε ε g c g c The functions (2)( ) ( 1 2 1 2 6) kij p s i jk <sup>=</sup> , , , = , ,..., are (2) (2) where ij a and ( 1 2 1 2 6) ij bi j = , , = , ,..., are given by ij a ij b (2) 0 11 11 ( ) ij ij ij ij 1, , =+ − ⎫ <sup>⎪</sup> = −+ <sup>⎪</sup> <sup>⎪</sup> =+ + <sup>⎪</sup> 1 13 33 33 p ca c eb p c a eb ij ij ij ij γ 2 44 15 γ 3 31 310 330 (2) 4 (2) 5 (2) 6 γ ij p a p b p 2 γ γ ( ) ij ij ij ij ij ij ij ij 2 2 0 4 2 420 g g ij γ ij ij ij ij 44 11 13 44 15 31 c ca c c − −− <sup>⎪</sup> = − <sup>⎪</sup> γ ′ ′ <sup>+</sup> <sup>⎫</sup> <sup>=</sup> <sup>⎪</sup> + + ⎪ e e The functions ( ) ( 1 2 1 2 6 1 2 3) ijk d si j k = , , = , ,..., , = , , are given by $$d\_{1jk}(\mathbf{s}) = q\_{j,k+9}(\mathbf{s})\_{\prime} \quad \text{(s)}\\d\_{2jk}(\mathbf{s}) = q\_{j+6,k+9}(\mathbf{s}) \quad \text{( $j=1,2,...,6, k=1,2,3$ )}\\ \tag{C.1}$$ where the functions ( ) ( 1 2 12) j k q s jk , , = , ,..., are the elements of a square matrix <sup>1</sup> <sup>Q</sup> <sup>−</sup> <sup>=</sup> <sup>Δ</sup> of order 12. The elements ( ) ( 1 2 12) j k δs jk , , = , ,..., of the square matrix Δ are given by $$\begin{aligned} \boldsymbol{\delta}\_{j,k}(\mathbf{s}) &= p\_{j1k}^{(2)} \exp(s\gamma\_{1k} h\_1) & (j = 1, 2, 3), \\ \boldsymbol{\delta}\_{j+3,k+6}(\mathbf{s}) &= p\_{j2k}^{(2)} \exp(-s\gamma\_{2k} h\_2) & (j = 1, 2, 3), \\ \boldsymbol{\delta}\_{j+6,k}(\mathbf{s}) &= p\_{j1k}^{(2)} & (j = 1, 2, ..., 6), \\ \boldsymbol{\delta}\_{j+6,k+6}(\mathbf{s}) &= -p\_{j2k}^{(2)} & (j = 1, 2, ..., 6) \end{aligned} \tag{C.2}$$ The functions ( ) ( 1 2 1 2 6) <sup>T</sup> ij dsi j = , , = , ,..., are $$\begin{aligned} d\_{1j}^T(\mathbf{s}) &= \sum\_{k=1}^{12} q\_{j,k}(\mathbf{s}) \mu\_k(\mathbf{s})\_\prime \\ d\_{2j}^T(\mathbf{s}) &= \sum\_{k=1}^{12} q\_{j+6,k}(\mathbf{s}) \mu\_k(\mathbf{s}) \end{aligned} \tag{C.3}$$ where $$\begin{aligned} u\_k(s) &= -\frac{R\_0(s)}{s} \sum\_{j=1}^2 p\_{k1j}^{(1)} R\_{1j}(s) \exp(s\tau\_1/h\_1) \qquad &(k=1,2,3), \\ u\_{k+3}(s) &= -\frac{R\_0(s)}{s} \sum\_{j=1}^2 p\_{k2j}^{(1)} R\_{2j}(s) \exp(-s\tau\_2/h\_2) \qquad &(k=1,2,3), \\ u\_{k+6}(s) &= -\frac{R\_0(s)}{s} \sum\_{j=1}^2 \left\{ p\_{k1j}^{(1)} R\_{1j}(s) - p\_{k2j}^{(1)} R\_{2j}(s) \right\} \quad &(k=1,2,...,6) \end{aligned} \tag{C.4}$$ ### 7. References 11 Eliminating the Undamaging S. Abdullah1, T. E. Putra2 and M. Z. Nuawi1 1Universiti Kebangsaan Malaysia 2Universitas Syiah Kuala 1Malaysia 2Indonesia Spectrum Filtering Techniques Fatigue Cycles Using the Frequency In 1982, the Battelle group has been found that between 80 – 90 % of all structural failures occur through a fatigue mechanism. Based on the finding, considerable effort has been applied in order to address this fact (Halfpenny, n.d.). The fatigue behaviour of mechanical components under service loading and its evaluation are usually affected by numerous uncertainties and characterized by several random variables such as material and structural properties, and load variation. The repeated fluctuating loads lead to microscopic physical Many vehicle components, like engine, steering, and suspension parts, are frequently subjected to a variety of driving conditions. When an automobile is driven on any road surface and hit a pothole, bump or curb, the lower suspension arm is effected by a significant shock amount compared to other components. The load is then transmitted through the control arm while it serves to maintain the contact between the wheel and the road. Furthermore, this component plays a vital role in the failure of automotive parts and it is submitted to multi axial fatigue loading under service conditions with generally With the advances in the digital signal processing research, there has been an increasingly strong interest in the application of the fatigue signal analysis for life prediction in automotive components. Several methods for analysis of signals have been proposed and performed for achieving this task. Among those is the Fast Fourier Transform (FFT) which has been widely applied to engineering problems. In the Fourier transform, the frequency components of an entire signal are analysed for producing the frequency information only. Since time information is lost, it is impossible to know when a particular event took place. For stationary signals, for which the frequency information is not changed by time, this nonproportional and variable amplitude loading (Nadot & Denier, 2004). 1. Introduction 1.1 Fatigue mechanism in structural failures damage of materials (Nizwan, et al., 2007). 1.2 Signal processing of fatigue ## Eliminating the Undamaging Fatigue Cycles Using the Frequency Spectrum Filtering Techniques S. Abdullah1, T. E. Putra2 and M. Z. Nuawi1 1Universiti Kebangsaan Malaysia 2Universitas Syiah Kuala 1Malaysia 2Indonesia ## 1. Introduction 222 Fourier Transform – Materials Analysis Rao, S.S. & Sunar, M. (1994). Piezoelectricity and Its Use in Disturbance Sensing and Control Sih, G.C. (Ed.). (1972). Methods of Analysis and Solution of Crack Problems, Noordhoff, Sneddon, I.N. & Lowengrub, M. (1969). Crack Problems in the Classical Theory of Elasticity, Tauchert, T.R. (1992). Piezothermoelastic Behavior of a Laminated Plate. Journal of Thermal Ueda, S. (2006a). The Crack Problem in Piezoelectric Strip under Thermoelectric Loading. Ueda, S. (2006b). Thermal Stress Intensity Factors for a Normal Crack in a Piezoelectric Strip. International Publishing, 978-9048182466, Leyden John Wiley & Sons, Inc., 978-0471808459, New York Journal of Thermal Stresses, Vol. 29, pp. 295-316, 0149-5739 Journal of Thermal Stresses, Vol. 29, pp. 1107-1126, 0149-5739 Stresses, Vol. 15, pp. 25-37, 0149-5739 0003-6900 of Flexible Structures: a Survey. Applied Mechanics Review, Vol. 47, pp. 113-123, ### 1.1 Fatigue mechanism in structural failures In 1982, the Battelle group has been found that between 80 – 90 % of all structural failures occur through a fatigue mechanism. Based on the finding, considerable effort has been applied in order to address this fact (Halfpenny, n.d.). The fatigue behaviour of mechanical components under service loading and its evaluation are usually affected by numerous uncertainties and characterized by several random variables such as material and structural properties, and load variation. The repeated fluctuating loads lead to microscopic physical damage of materials (Nizwan, et al., 2007). Many vehicle components, like engine, steering, and suspension parts, are frequently subjected to a variety of driving conditions. When an automobile is driven on any road surface and hit a pothole, bump or curb, the lower suspension arm is effected by a significant shock amount compared to other components. The load is then transmitted through the control arm while it serves to maintain the contact between the wheel and the road. Furthermore, this component plays a vital role in the failure of automotive parts and it is submitted to multi axial fatigue loading under service conditions with generally nonproportional and variable amplitude loading (Nadot & Denier, 2004). #### 1.2 Signal processing of fatigue With the advances in the digital signal processing research, there has been an increasingly strong interest in the application of the fatigue signal analysis for life prediction in automotive components. Several methods for analysis of signals have been proposed and performed for achieving this task. Among those is the Fast Fourier Transform (FFT) which has been widely applied to engineering problems. In the Fourier transform, the frequency components of an entire signal are analysed for producing the frequency information only. Since time information is lost, it is impossible to know when a particular event took place. For stationary signals, for which the frequency information is not changed by time, this Eliminating the Undamaging Fatigue Cycles Using the Frequency Spectrum Filtering Techniques 225 most recent solution to overcome the nonstationary signals. This time-frequency technique is applied by cutting time domain signal into various frequency components through the compromise between time and frequency-based views of the signal. It presents information in both time and frequency domain in a more useful form (Valens, 1999; Percival & Walden, This paper discussed on the study of fatigue data editing by using the frequency spectrum filtering techniques. Both the techniques were used to eliminate undamaging fatigue cycles in order to simplify raw signal for the simulation testing purposes The WT results were compared to the findings using the FFT extraction approach in order to see the suitability Signals are a form of information, as a function of time, coming from many sources obtained by measuring and using some recording method. Most data samples are normally measured using an analogue-to-digital converter, so as to produce an experimental signal at a series of regularly spaced times, known as a discrete time. The analysis of time series aims to determine the statistical characteristics of the original function by manipulating the series of Signals can be divided into two main categories, i.e. deterministic and nondeterministic. A deterministic signal can be described by a mathematical relationship between the value of the function and the value of time. Many signals in nature exhibit nondeterministic or random characteristics, which provide a challenge to analysis using signal processing techniques (Tacer & Loughlin, 1998). A signal representing a random physical phenomenon can not be described in a point by point manner by means of a deterministic mathematical A signal representing a random phenomenon can be characterised as either stationary or nonstationary. A stationary signal is characterised by values of the global signal statistical parameters, such as the mean, variance and root-mean-square (r.m.s.), which are unchanged across the signal length. In the case of nonstationary signals, the values are dependent on the time of measurement. Nonstationary signals can be divided into two categories: mildly nonstationary and heavily nonstationary. A mildly nonstationary signal is defined as a random process with a stable mean, variance and r.m.s. values for most of the recorded data, but with short periods of differing signal statistics due to the presence of transient behaviour. A heavily nonstationary signal is defined as being similar to a mildly nonstationary signal, but with the presence of transient events over a large interval of the time history. Since nonstationary loadings are common in the case of fatigue and vibrational analysis (Giancomin et al., 1999), signal modelling has often been used in the time domain In the case of fatigue researches, the signals consist of a measurement of cyclic loads, i.e. force, strain, and stress against time. A time series typically consists of a set of observations due to its simplicity and efficiency for the purpose of loading simulations. 2.2 Global signal statistical parameters 2000; Addison, 2002). approach in fatigue history editing. 2. Literature background 2.1 Fatigue signal discrete numbers. equation. drawback is not very important. However, most interesting signals contain numerous nonstationary or transitory characteristics which often occur in the most important part of an experimental measured signal with variable amplitude pattern. This means that this Fourier transform method was found to be an unsuitable method to investigate the behaviour of nonstationary patterns in a signal (Misiti et al., 2008; Valens, 1999). Therefore, the time-frequency localisation technique was identified to overcome this drawback of analysis of a nonstationary signal. During the last decade, a new mathematical technique, called the wavelet transform (WT), has been frequently used in the field of vibrational diagnostics and also in fault detection. In addition, the wavelet coefficient analysis has also have been applied to detect fatigue transverse cracks in rotors. Its peak absolute value is highly sensitive to the depth of a crack and even a very shallow crack can be detected. The rotor is not required to stop and the detection process can be applied to a rotating shaft making the methodology more versatile, convenient and unambiguous (Darpe, 2007). ## 1.3 Fatigue data editing For many automotive components, the primary mode of failure can be attributed to fatigue damage resulted from the application of variable amplitude loading (VAL). It contains large percentage of small amplitude cycles and the fatigue damage for these cycles can be small. For this reason, in many cases, the signal was edited by removing these cycles in order to produce representative and meaningful yet economical testing (Stephens et al., 1997). Therefore, it seems appropriate to see a method to summarise a fatigue strain signal. Without editing the service load, the testing time and cost become prohibitive (Abdullah, 2007). The effectiveness of the edited signals depends on the retention of the fatigue damage and signal statistical parameter values. Those values are measured in order to observe the signal behaviour. They are compared with the original signal to ensure the edited signal obtained can retain the original signal features. A 5 % difference of the fatigue damage and ± 10 % difference of the root-mean-square (r.m.s) and the kurtosis are required in order to produce an edited signal which has equivalent fatigue damage and signal statistics to the original signal. In the previous study, Nizwan et al., (2007) developed a fatigue data editing using the FFT. This algorithm was performed by eliminating unwanted cycles in a fatigue strain signal. Using Power Spectral Density (PSD), energy distribution in the frequency domain displayed strain characteristic of the input signal. In order to trace higher amplitude cycles, low pass filter was applied to filter out higher frequency containing small amplitude which not causes the fatigue damage. Thus, a smoothen signal was obtained. Despite using the frequency spectrum filtering technique could not shorten the signal, but it still could simplify the signal by reducing the numbers of cycle counting for the signal. The optimum Cut Off Frequency (COF) for lower pass filter application was determined to observe the effect of fatigue data editing technique in frequency domain. Since the technique could not shorten the original signal, it gave a motivation to the authors for developing a similar data extraction approach in the WT. Therefore, a new algorithm for fatigue feature extraction using the Morlet wavelet was developed. The WT is probably the most recent solution to overcome the nonstationary signals. This time-frequency technique is applied by cutting time domain signal into various frequency components through the compromise between time and frequency-based views of the signal. It presents information in both time and frequency domain in a more useful form (Valens, 1999; Percival & Walden, 2000; Addison, 2002). This paper discussed on the study of fatigue data editing by using the frequency spectrum filtering techniques. Both the techniques were used to eliminate undamaging fatigue cycles in order to simplify raw signal for the simulation testing purposes The WT results were compared to the findings using the FFT extraction approach in order to see the suitability approach in fatigue history editing. ## 2. Literature background ## 2.1 Fatigue signal 224 Fourier Transform – Materials Analysis drawback is not very important. However, most interesting signals contain numerous nonstationary or transitory characteristics which often occur in the most important part of an experimental measured signal with variable amplitude pattern. This means that this Fourier transform method was found to be an unsuitable method to investigate the Therefore, the time-frequency localisation technique was identified to overcome this drawback of analysis of a nonstationary signal. During the last decade, a new mathematical technique, called the wavelet transform (WT), has been frequently used in the field of vibrational diagnostics and also in fault detection. In addition, the wavelet coefficient analysis has also have been applied to detect fatigue transverse cracks in rotors. Its peak absolute value is highly sensitive to the depth of a crack and even a very shallow crack can be detected. The rotor is not required to stop and the detection process can be applied to a rotating shaft making the methodology more versatile, convenient and unambiguous For many automotive components, the primary mode of failure can be attributed to fatigue damage resulted from the application of variable amplitude loading (VAL). It contains large percentage of small amplitude cycles and the fatigue damage for these cycles can be small. For this reason, in many cases, the signal was edited by removing these cycles in order to produce representative and meaningful yet economical testing (Stephens et al., 1997). Therefore, it seems appropriate to see a method to summarise a fatigue strain signal. Without editing the service load, the testing time and cost become prohibitive (Abdullah, The effectiveness of the edited signals depends on the retention of the fatigue damage and signal statistical parameter values. Those values are measured in order to observe the signal behaviour. They are compared with the original signal to ensure the edited signal obtained can retain the original signal features. A 5 % difference of the fatigue damage and ± 10 % difference of the root-mean-square (r.m.s) and the kurtosis are required in order to produce an edited signal which has equivalent fatigue damage and signal statistics to the original In the previous study, Nizwan et al., (2007) developed a fatigue data editing using the FFT. This algorithm was performed by eliminating unwanted cycles in a fatigue strain signal. Using Power Spectral Density (PSD), energy distribution in the frequency domain displayed strain characteristic of the input signal. In order to trace higher amplitude cycles, low pass filter was applied to filter out higher frequency containing small amplitude which not causes the fatigue damage. Thus, a smoothen signal was obtained. Despite using the frequency spectrum filtering technique could not shorten the signal, but it still could simplify the signal by reducing the numbers of cycle counting for the signal. The optimum Cut Off Frequency (COF) for lower pass filter application was determined to observe the Since the technique could not shorten the original signal, it gave a motivation to the authors for developing a similar data extraction approach in the WT. Therefore, a new algorithm for fatigue feature extraction using the Morlet wavelet was developed. The WT is probably the effect of fatigue data editing technique in frequency domain. behaviour of nonstationary patterns in a signal (Misiti et al., 2008; Valens, 1999). (Darpe, 2007). 2007). signal. 1.3 Fatigue data editing Signals are a form of information, as a function of time, coming from many sources obtained by measuring and using some recording method. Most data samples are normally measured using an analogue-to-digital converter, so as to produce an experimental signal at a series of regularly spaced times, known as a discrete time. The analysis of time series aims to determine the statistical characteristics of the original function by manipulating the series of discrete numbers. Signals can be divided into two main categories, i.e. deterministic and nondeterministic. A deterministic signal can be described by a mathematical relationship between the value of the function and the value of time. Many signals in nature exhibit nondeterministic or random characteristics, which provide a challenge to analysis using signal processing techniques (Tacer & Loughlin, 1998). A signal representing a random physical phenomenon can not be described in a point by point manner by means of a deterministic mathematical equation. A signal representing a random phenomenon can be characterised as either stationary or nonstationary. A stationary signal is characterised by values of the global signal statistical parameters, such as the mean, variance and root-mean-square (r.m.s.), which are unchanged across the signal length. In the case of nonstationary signals, the values are dependent on the time of measurement. Nonstationary signals can be divided into two categories: mildly nonstationary and heavily nonstationary. A mildly nonstationary signal is defined as a random process with a stable mean, variance and r.m.s. values for most of the recorded data, but with short periods of differing signal statistics due to the presence of transient behaviour. A heavily nonstationary signal is defined as being similar to a mildly nonstationary signal, but with the presence of transient events over a large interval of the time history. Since nonstationary loadings are common in the case of fatigue and vibrational analysis (Giancomin et al., 1999), signal modelling has often been used in the time domain due to its simplicity and efficiency for the purpose of loading simulations. ## 2.2 Global signal statistical parameters In the case of fatigue researches, the signals consist of a measurement of cyclic loads, i.e. force, strain, and stress against time. A time series typically consists of a set of observations Eliminating the Undamaging Fatigue Cycles Using the Frequency Spectrum Filtering Techniques 227 localized region where fatigue cracks begin to the durability of a structure under the influence of a mean stress. It is often used for ductile materials at relatively short fatigue life. It also can be used where there is little plasticity at long fatigue life. Therefore, this is a comprehensive approach that can be used in place of the related stress - based approach The total strain amplitude εa produced by the combination of elastic and plastic amplitude is a ea pa <sup>b</sup> <sup>f</sup> <sup>a</sup> ea <sup>N</sup> <sup>f</sup> E E where σa is the stress amplitude, σ'f is the fatigue strength coefficient, E is the material modulus of elasticity, Nf is the numbers of cycle to failure for a particular stress range and mean, b is the fatigue strength exponent, ε'f is the fatigue ductility coefficient, and c is the Combining Eq. (5) and (6) gives the Coffin-Manson relationship, which is mathematically where εa is the true strain amplitude, σ'f is the fatigue strength coefficient, E is the material modulus of elasticity, Nf is the numbers of cycle to failure for a particular stress range and mean, b is the fatigue strength exponent, ε'f is the fatigue ductility coefficient, and c is the Fatigue damage is derived from the number of cycles to failure. The fatigue damage caused by each cycle of repeated loading is calculated by reference to material life curves, such as > 1 f > > i f <sup>N</sup> <sup>D</sup> N where Ni is the numbers of cycle within a particular stress range and mean. Therefore, fatigue damaging values have the range (0 - 1) where zero denotes no damage (extremely ⎛ ⎞ <sup>=</sup> ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ Σ D and the total fatigue damage ΣD caused by cycles is expressed as (Abdullah, 2005): Σ () () ' ' 2 2 b c f <sup>a</sup> N N f ff <sup>E</sup> ε ' 2( ) c σ ( ) ' <sup>2</sup> where the elastic strain amplitude εea and the plastic strain amplitude εpa are defined by: + (4) = = (5) = + (7) N= (8) (9) pa = f f N (6) ε = ε ε σ ε ε σ ε S-N or ε-N curves. The fatigue damage D for one cycle is calculated as: ε (Halfpenny, n.d.). fatigue ductility exponent. defined as (nCode, n.d.): fatigue ductility exponent. defined as: of a variable were taken at equally spaced intervals of time. Global signal statistical parameters are frequently used to classify random signals and monitor the pattern of analyzed signals. For a signal with a numbers of data point n in a sampled sequence, the mean x is given by: $$\overline{\hat{\mathbf{x}}} = \frac{1}{n} \sum\_{j=1}^{n} \mathbf{x}\_{j} \tag{1}$$ In the fatigue signals, the calculation of the r.m.s. and the kurtosis are very important in order to retain a certain amount of the signal amplitude range characteristics (Nuawi et al., 2009). The r.m.s. is the signal 2nd statistical moment used to quantify the overall energy content of the oscillatory signal. The r.m.s. relationship is defined as: $$r.m.s. = \left\{\frac{1}{n}\sum\_{j=1}^{n} {\boldsymbol{x}\_{j}}^{2}\right\}^{1/2} \tag{2}$$ The kurtosis is the signal 4th statistical moment. In an engineering field, it is used as a measure of nongaussianity for detection of fault symptoms since it is highly sensitive to spikiness or outlier signal among the instantaneous values. Mathematically, the kurtosis expression is defined as: $$K = \frac{1}{n\_{\{r,m,s\}}} \sum\_{j=1}^{n} \left(\mathbf{x}\_{j} - \overline{\mathbf{x}}\right)^{4} \tag{3}$$ where xj is the amplitude of signal. In some definitions of the kurtosis, a deduction of 3.0 is added to the definition in order to maintain the kurtosis of a Gaussian distribution to be equal to zero. For clarity and convenience, in this study the original definition of the kurtosis, where the Gaussian distribution has a kurtosis value of approximately 3.0 was used for the analysis. Therefore, a kurtosis value of higher than 3.0 indicates the presence of more extreme values than one that should be found in a Gaussian distribution (Abdullah et al., 2006). This situation indicates that the fatigue damage is higher than Gaussian stresses due to higher amplitude fatigue cycles (Braccesi et al., 2009). #### 2.3 Fatigue life assessment In material science, fatigue is defined as a process of progressive and localised structural damage duo to cyclic loads, which depend on the stresses and the strains at critical regions of a component (Prawoto, 2002). The repeated or otherwise varying loads never reach a level sufficient for causing failure in a single application. The loads cause plastic deformation (slip) locally which results in a crack beginning, crack propagation, and then a breakage. This plastic deformation might arise through the presence of a small crack or pre-existing defect on the surface of a component. In the case of the fatigue research, the signals consist of a measurement of cyclic loads, i.e. force, strain, and stress against time (Nuawi et al., 2009). Three major approaches to predicting fatigue life namely stress-life, strain-life, and fracture mechanics. The strain-life fatigue approach relates the plastic deformation that occurs in the of a variable were taken at equally spaced intervals of time. Global signal statistical parameters are frequently used to classify random signals and monitor the pattern of analyzed signals. For a signal with a numbers of data point n in a sampled sequence, the 1 <sup>n</sup> In the fatigue signals, the calculation of the r.m.s. and the kurtosis are very important in order to retain a certain amount of the signal amplitude range characteristics (Nuawi et al., 2009). The r.m.s. is the signal 2nd statistical moment used to quantify the overall energy > n j j rms x n <sup>=</sup> <sup>⎧</sup> <sup>⎫</sup> <sup>⎪</sup> <sup>⎪</sup> <sup>=</sup> <sup>⎨</sup> <sup>⎬</sup> The kurtosis is the signal 4th statistical moment. In an engineering field, it is used as a measure of nongaussianity for detection of fault symptoms since it is highly sensitive to spikiness or outlier signal among the instantaneous values. Mathematically, the kurtosis ( ) ( ) <sup>4</sup> . . 1 1 <sup>n</sup> <sup>j</sup> rms <sup>j</sup> K xx n <sup>=</sup> where xj is the amplitude of signal. In some definitions of the kurtosis, a deduction of 3.0 is added to the definition in order to maintain the kurtosis of a Gaussian distribution to be equal to zero. For clarity and convenience, in this study the original definition of the kurtosis, where the Gaussian distribution has a kurtosis value of approximately 3.0 was used for the analysis. Therefore, a kurtosis value of higher than 3.0 indicates the presence of more extreme values than one that should be found in a Gaussian distribution (Abdullah et al., 2006). This situation indicates that the fatigue damage is higher than Gaussian stresses In material science, fatigue is defined as a process of progressive and localised structural damage duo to cyclic loads, which depend on the stresses and the strains at critical regions of a component (Prawoto, 2002). The repeated or otherwise varying loads never reach a level sufficient for causing failure in a single application. The loads cause plastic deformation (slip) locally which results in a crack beginning, crack propagation, and then a breakage. This plastic deformation might arise through the presence of a small crack or pre-existing defect on the surface of a component. In the case of the fatigue research, the signals consist of a measurement of cyclic loads, i.e. force, strain, and stress against time (Nuawi et al., Three major approaches to predicting fatigue life namely stress-life, strain-life, and fracture mechanics. The strain-life fatigue approach relates the plastic deformation that occurs in the <sup>1</sup> . .. content of the oscillatory signal. The r.m.s. relationship is defined as: due to higher amplitude fatigue cycles (Braccesi et al., 2009). 1 j j x x n <sup>=</sup> > > 4 <sup>=</sup> ∑ (1) ⎩ ⎭ <sup>⎪</sup> <sup>⎪</sup> <sup>∑</sup> (2) <sup>=</sup> ∑ <sup>−</sup> (3) mean x is given by: expression is defined as: 2.3 Fatigue life assessment 2009). localized region where fatigue cracks begin to the durability of a structure under the influence of a mean stress. It is often used for ductile materials at relatively short fatigue life. It also can be used where there is little plasticity at long fatigue life. Therefore, this is a comprehensive approach that can be used in place of the related stress - based approach (Halfpenny, n.d.). The total strain amplitude εa produced by the combination of elastic and plastic amplitude is defined as: $$ \varepsilon\_a = \varepsilon\_{ea} + \varepsilon\_{pa} \tag{4} $$ where the elastic strain amplitude εea and the plastic strain amplitude εpa are defined by: $$ \varepsilon\_{ea} = \frac{\sigma\_a}{E} = \frac{\sigma'\_f}{E} \left(2N\_f\right)^b \tag{5} $$ $$ \varepsilon\_{\rm par} = \varepsilon'\_{f} \left( 2N\_{f} \right)^{c} \tag{6} $$ where σa is the stress amplitude, σ'f is the fatigue strength coefficient, E is the material modulus of elasticity, Nf is the numbers of cycle to failure for a particular stress range and mean, b is the fatigue strength exponent, ε'f is the fatigue ductility coefficient, and c is the fatigue ductility exponent. Combining Eq. (5) and (6) gives the Coffin-Manson relationship, which is mathematically defined as (nCode, n.d.): $$ \varepsilon\_a = \frac{\sigma \dot{\sigma}\_f}{E} \left( 2 \text{N}\_f \right)^b + \dot{\varepsilon}\_f \left( 2 \text{N}\_f \right)^c \tag{7} $$ where εa is the true strain amplitude, σ'f is the fatigue strength coefficient, E is the material modulus of elasticity, Nf is the numbers of cycle to failure for a particular stress range and mean, b is the fatigue strength exponent, ε'f is the fatigue ductility coefficient, and c is the fatigue ductility exponent. Fatigue damage is derived from the number of cycles to failure. The fatigue damage caused by each cycle of repeated loading is calculated by reference to material life curves, such as S-N or ε-N curves. The fatigue damage D for one cycle is calculated as: $$D = \frac{1}{N\_f} \tag{8}$$ and the total fatigue damage ΣD caused by cycles is expressed as (Abdullah, 2005): $$ \Sigma D = \Sigma \left(\frac{N\_i}{N\_f}\right) \tag{9} $$ where Ni is the numbers of cycle within a particular stress range and mean. Therefore, fatigue damaging values have the range (0 - 1) where zero denotes no damage (extremely Eliminating the Undamaging Fatigue Cycles Using the Frequency Spectrum Filtering Techniques 229 resolution problem because the window length is long for low frequency and short for high frequency. Therefore, the frequency resolution is good for low frequency (at high scales) and the time resolution is good at high frequency (at low scales). The major advantage is the ability to analyse a localised area of a larger signal, also known as the local analysis (Misiti et The Morlet wavelet is one of functions that are generally used in the Continuous Wavelet Transform (CWT) analyses (Gao et al., 2001). The wavelet decomposition calculates a resemblance index between signal being analyzed and the wavelet, called coefficient. It is a result of a regression of an original signal produced at different scales and different sections on the wavelet. It represents correlation between the wavelet and a section of the signal. If The WT of any time-varying signal f(t) is defined as the sum of all of the signals time multiplied by a scaled and shifted version of the wavelet function ψ(t) (Kim et al., 2007). The ( ) CWT a b, () () a b, f t t dt The parameter a represents the scale factor which is a reciprocal of frequency, the parameter b indicates the time shifting or translation factor, and t is the time. Ψ<sup>a</sup>,<sup>b</sup>(t) denotes the mother > <sup>1</sup> , ;0 a b t b t ab Ra a a t b CWT <sup>f</sup> t dt +∞ In addition, the wavelet coefficient indicates how energy in the signal is distributed in the time-frequency plane (Darpe, 2007). The energy spectrum (the energy density over frequency) is plotted in order to observe the signal behaviour and its content gives In a fatigue life assessment, fatigue signal extraction is described as a method for fatigue data editing which lead to summarise a fatigue signal. The method is performed by segment identification and extraction that contribute to the more fatigue damaging events to a metallic material. On the other hand, segments containing lower amplitude cycles are omitted, since these data type theoretically gave minimal or no fatigue damage. The goal of the removal of those parts from the original signal is to generate a new shortened mission signal, for which this signal type can be used to reduce the testing time and costs for fatigue The magnitude of time domain spectrum level is used as a parameter to set gate value for the eliminating process. The value is used to slice the original signal. The extracted segment identification is performed by searching two inversion points (one on either side of the peak ⎛−⎞ <sup>=</sup> ⎜ ⎟ ∈ ≠ ⎝ ⎠ 1 a a ψ ( ) ( ) , ψ ( ) ( ) , −∞ a b ψ+∞ −∞ <sup>=</sup> ∫ (10) ⎛ ⎞ <sup>−</sup> <sup>=</sup> ⎜ ⎟ ⎝ ⎠ <sup>∫</sup> (12) (11) the index is large, the resemblance is strong, otherwise it is slight (Misiti et al., 2008). CWT is expressed by the following integral: wavelet, i.e (Purushotham et al., 2005): 2.6 Fatigue data editing testing (Abdullah, 2005). ψ significant information about the random signal pattern. al., 2008). high or infinite numbers of cycle to failure) and 1 means total failure (one cycle to failure). For strain - based fatigue life prediction, current industrial practice uses the Palmgren-Miner linear cumulative damaging rule normally associated with the established strain-life fatigue damaging models, such as the Coffin-Manson. ## 2.4 The FFT Frequency domain is a term used to describe the analysis of mathematical functions or signals with respect to frequency. A frequency domain graph shows how much of the signal lies within each given frequency band over a range of frequency. A frequency domain representation can also include information on the phase shift that must be applied to each sinusoid in order to be able to recombine the frequency components to recover the original time signal. The frequency domain relates to the Fourier transform or Fourier series by decomposing a function into an infinite or finite number of frequency. This is based on the concept of Fourier series that any waveform can be expressed as a sum of sinusoids (Nizwan et al., 2007). The Fourier transform has been most commonly used to denoise signals for a frequency based editing method, which cannot provide any information regarding the time localization of the spectral components. This Fourier representation has been found inadequate in analyzing nonstationary signals (Oh, 2001). Frequency analysis data is typically presented in graphical form as PSD. Essentially, a PSD displays the amplitude of each sinusoidal wave of a particular frequency which is given on the x-axis. The mean squered amplitude of a sinusoidal wave at any frequency can be determined by finding the area under the PSD over that frequency range (nCode, 2005). A filter is used to remove undesirable frequency information from a dynamic signal. Filter can be broadly classified as being low pass, high pas, bandpass, and notch. Low pass filter permits frequency below a prescribed COF to pass while blocking the passage of frequency information above the COF. Similarly, a high-pass filter permits only frequency above the COF to pass. A bandpass filter combines features of both the low pass and high pass filter. It describes a lower and higher COF to define a band of frequency that is permitted to pass through the filter. A notch filter permits the passage of all frequency except those within narrow frequency band (Nizwan et al., 2007). ## 2.5 The Morlet wavelet The WT analysis is started with a basic function (called the mother wavelet) scaled and translated to represent the signal being analyzed (Berry, 1999). The transform shifts a window along the signal and calculates the spectrum for every position. The process is repeated many times with a slightly shorter (or longer) window for every new cycle. In the end, the result will be a collection of time-frequency representations of the signal with different resolutions. The WT provides information on when and at what frequency the change in signal behaviour occurs (Valens, 1999). The major advantage is the ability to analyze a localized area of larger signal (local analysis) (Misiti et al., 2008). Obviously, the WT represents a windowing technique with variable-sized regions. This technique allows the use of long time intervals (more precise low frequency information) and shorter regions (high frequency information). It means the wavelet method solves the resolution problem because the window length is long for low frequency and short for high frequency. Therefore, the frequency resolution is good for low frequency (at high scales) and the time resolution is good at high frequency (at low scales). The major advantage is the ability to analyse a localised area of a larger signal, also known as the local analysis (Misiti et al., 2008). The Morlet wavelet is one of functions that are generally used in the Continuous Wavelet Transform (CWT) analyses (Gao et al., 2001). The wavelet decomposition calculates a resemblance index between signal being analyzed and the wavelet, called coefficient. It is a result of a regression of an original signal produced at different scales and different sections on the wavelet. It represents correlation between the wavelet and a section of the signal. If the index is large, the resemblance is strong, otherwise it is slight (Misiti et al., 2008). The WT of any time-varying signal f(t) is defined as the sum of all of the signals time multiplied by a scaled and shifted version of the wavelet function ψ(t) (Kim et al., 2007). The CWT is expressed by the following integral: $$\text{CVWT}\_{\left(a,b\right)} = \int\_{-\infty}^{+\infty} f\left(t\right) \varphi\_{a,b}\left(t\right) dt\tag{10}$$ The parameter a represents the scale factor which is a reciprocal of frequency, the parameter b indicates the time shifting or translation factor, and t is the time. Ψ<sup>a</sup>,<sup>b</sup>(t) denotes the mother wavelet, i.e (Purushotham et al., 2005): $$ \psi\_{a,b}\left(t\right) = \frac{1}{\sqrt{\|a\|}} \psi\left(\frac{\left(t - b\right)}{a}\right) a, b \in \mathbb{R}; a \neq 0 \tag{11} $$ $$\text{CWT}\_{(a,b)} = \int\_{-\infty}^{+\infty} f\left(t\right) \frac{1}{\sqrt{a}} \nu\left(\frac{t-b}{a}\right) dt\tag{12}$$ In addition, the wavelet coefficient indicates how energy in the signal is distributed in the time-frequency plane (Darpe, 2007). The energy spectrum (the energy density over frequency) is plotted in order to observe the signal behaviour and its content gives significant information about the random signal pattern. ## 2.6 Fatigue data editing 228 Fourier Transform – Materials Analysis high or infinite numbers of cycle to failure) and 1 means total failure (one cycle to failure). For strain - based fatigue life prediction, current industrial practice uses the Palmgren-Miner linear cumulative damaging rule normally associated with the established strain-life fatigue Frequency domain is a term used to describe the analysis of mathematical functions or signals with respect to frequency. A frequency domain graph shows how much of the signal lies within each given frequency band over a range of frequency. A frequency domain representation can also include information on the phase shift that must be applied to each sinusoid in order to be able to recombine the frequency components to recover the original time signal. The frequency domain relates to the Fourier transform or Fourier series by decomposing a function into an infinite or finite number of frequency. This is based on the concept of Fourier series that any waveform can be expressed as a sum of sinusoids The Fourier transform has been most commonly used to denoise signals for a frequency based editing method, which cannot provide any information regarding the time localization of the spectral components. This Fourier representation has been found inadequate in analyzing nonstationary signals (Oh, 2001). Frequency analysis data is typically presented in graphical form as PSD. Essentially, a PSD displays the amplitude of each sinusoidal wave of a particular frequency which is given on the x-axis. The mean squered amplitude of a sinusoidal wave at any frequency can be determined by finding the area under the PSD over A filter is used to remove undesirable frequency information from a dynamic signal. Filter can be broadly classified as being low pass, high pas, bandpass, and notch. Low pass filter permits frequency below a prescribed COF to pass while blocking the passage of frequency information above the COF. Similarly, a high-pass filter permits only frequency above the COF to pass. A bandpass filter combines features of both the low pass and high pass filter. It describes a lower and higher COF to define a band of frequency that is permitted to pass through the filter. A notch filter permits the passage of all frequency except those within The WT analysis is started with a basic function (called the mother wavelet) scaled and translated to represent the signal being analyzed (Berry, 1999). The transform shifts a window along the signal and calculates the spectrum for every position. The process is repeated many times with a slightly shorter (or longer) window for every new cycle. In the end, the result will be a collection of time-frequency representations of the signal with different resolutions. The WT provides information on when and at what frequency the change in signal behaviour occurs (Valens, 1999). The major advantage is the ability to Obviously, the WT represents a windowing technique with variable-sized regions. This technique allows the use of long time intervals (more precise low frequency information) and shorter regions (high frequency information). It means the wavelet method solves the analyze a localized area of larger signal (local analysis) (Misiti et al., 2008). damaging models, such as the Coffin-Manson. 2.4 The FFT (Nizwan et al., 2007). that frequency range (nCode, 2005). narrow frequency band (Nizwan et al., 2007). 2.5 The Morlet wavelet In a fatigue life assessment, fatigue signal extraction is described as a method for fatigue data editing which lead to summarise a fatigue signal. The method is performed by segment identification and extraction that contribute to the more fatigue damaging events to a metallic material. On the other hand, segments containing lower amplitude cycles are omitted, since these data type theoretically gave minimal or no fatigue damage. The goal of the removal of those parts from the original signal is to generate a new shortened mission signal, for which this signal type can be used to reduce the testing time and costs for fatigue testing (Abdullah, 2005). The magnitude of time domain spectrum level is used as a parameter to set gate value for the eliminating process. The value is used to slice the original signal. The extracted segment identification is performed by searching two inversion points (one on either side of the peak Eliminating the Undamaging Fatigue Cycles Using the Frequency Spectrum Filtering Techniques 231 For the data acquisition purpose, strain gauge was placed at the highest stress location of the front lower suspension arm of a passenger car. The car was travelled on pavé (cobblestone) road surface with velocity was 20 - 30 km/h. Fig. 2 shows the strain gauge position, the test Fig. 2. The strain gauge position, a section of the pavé test track and the data acquisition The input signal was the variable amplitude loading sampled at 200 Hz since the fatigue damage occurs at lower frequency. From the data collection, it gave the total signal record length of 160 seconds. The collected signal recorded using a fatigue data acquisition system contained many small amplitude and high frequency in occurrence signal background. By Time (s) Fig. 3. The time history plot of the original test signal 0 50 100 150 3.2 Data acquisition set-up 60 40 20 0 -20 -40 -60 Strain (με) track and the data acquisition set-up. value) which define the temporal extent of the extracted segment. The identification is based on energy loss concept, i.e. selected segments are at the start and finish points. The example of the segment identification is described in Fig. 1. In the figure, the selected segment is at gate value of 400 με2/Hz. Start point is a valley point if the peak before is higher than the peak after the point. While the finish point is selected if peak after is higher than peak before the point. This concept is performed by Abdullah (2005) based on transient vibration where start and finish points are selected based on transient form. The points are determined based on the signal where the shortening in signal background occurs. Fig. 1. The extracted segment identification After all the segments are identified, the time history fatigue signal is then sliced to remove the lower amplitude (less than the gate value) contained in the original time history range with 100 % fatigue damaging retains. For this reason, the majority of the original fatigue damage is retained in the edited signal. All extracted segments (the complete section between the start and the end of the segments) selected based on time location of the wavelet coefficient amplitude are then combined together to produce a new mission time history. The mission signal replicates the signal statistical parameter and total fatigue damaging characteristics of the original time history. The optimum gate value is accordingly determined and it is based on the effectiveness of retaining the characteristics of the original signal in the mission signal. Ideally, the signal has shorter time length but is equivalent in the characteristic values. #### 3. Materials and methods #### 3.1 Finite element analysis Every structure will experiences a transformation if it accepts a load, and an internal force, called stress, will stops the transformation. The load causes the structure to experience direct stress, not only tensile stress but also compressive stress. Structure accepting the tensile stress will become smaller, while structure accepting the compressive stress will cause buckling or permanent damage (Balia & Putra, 2006). If A is the cross-sectional area and F is the applied load, the true stress σ at fracture can be defined mathematically as the following expression (Draper, 2007): $$ \sigma = \frac{P}{A} \tag{13} $$ ## 3.2 Data acquisition 230 Fourier Transform – Materials Analysis value) which define the temporal extent of the extracted segment. The identification is based The example of the segment identification is described in Fig. 1. In the figure, the selected segment is at gate value of 400 με2/Hz. Start point is a valley point if the peak before is higher than the peak after the point. While the finish point is selected if peak after is higher than peak before the point. This concept is performed by Abdullah (2005) based on transient vibration where start and finish points are selected based on transient form. The points are Time (s) After all the segments are identified, the time history fatigue signal is then sliced to remove the lower amplitude (less than the gate value) contained in the original time history range with 100 % fatigue damaging retains. For this reason, the majority of the original fatigue damage is retained in the edited signal. All extracted segments (the complete section between the start and the end of the segments) selected based on time location of the wavelet coefficient amplitude are then combined together to produce a new mission time history. The mission signal replicates the signal statistical parameter and total fatigue damaging characteristics of the original time history. The optimum gate value is accordingly determined and it is based on the effectiveness of retaining the characteristics of the original signal in the mission signal. Ideally, the signal has shorter time length but is equivalent in Every structure will experiences a transformation if it accepts a load, and an internal force, called stress, will stops the transformation. The load causes the structure to experience direct stress, not only tensile stress but also compressive stress. Structure accepting the tensile stress will become smaller, while structure accepting the compressive stress will cause buckling or permanent damage (Balia & Putra, 2006). If A is the cross-sectional area and F is the applied load, the true stress σ at fracture can be defined mathematically as the following > P A σ 0 1 2 3 4 5 Gate value End segment = (13) on energy loss concept, i.e. selected segments are at the start and finish points. determined based on the signal where the shortening in signal background occurs. Amplitude (με2/Hz) Start segment Fig. 1. The extracted segment identification the characteristic values. 3. Materials and methods 3.1 Finite element analysis expression (Draper, 2007): For the data acquisition purpose, strain gauge was placed at the highest stress location of the front lower suspension arm of a passenger car. The car was travelled on pavé (cobblestone) road surface with velocity was 20 - 30 km/h. Fig. 2 shows the strain gauge position, the test track and the data acquisition set-up. Fig. 2. The strain gauge position, a section of the pavé test track and the data acquisition set-up The input signal was the variable amplitude loading sampled at 200 Hz since the fatigue damage occurs at lower frequency. From the data collection, it gave the total signal record length of 160 seconds. The collected signal recorded using a fatigue data acquisition system contained many small amplitude and high frequency in occurrence signal background. By Fig. 3. The time history plot of the original test signal Eliminating the Undamaging Fatigue Cycles Using the Frequency Spectrum Filtering Techniques 233 fatigue damage to the original signal in order to produce a edited signal which retaining the Properties Value Ultimate tensile strength, Su (MPa) 621 Modulus of elasticity, E (GPa) 204 Fatigue strength coefficient, σ'f (MPa) 948 Fatigue strength exponent, b -0.092 Fatigue ductility exponent, c -0.445 Fatigue ductility coefficient, ε'f 0.26 The finite element modeling and analysis have been performed utilizing CATIA and MSC finite element analysis codes respectively. By using Eq. 13, this analysis determinates the maximum stress locations before doing the fatigue test. Based on the analysis performed by Al-Asady et al. (2008), it was obtained the stress distribution at the front lower suspension In this simulation, the load was assumed of 300 kg. This value came from the car weight and the passengers. The stress level is presented by a colour contour, where the white colour shows the highest stress content and followed by black, blue, and so on. Table 1. The mechanical properties of the SAE1045 carbon steel shaft Fig. 5. Stress distribution and strain gauge positions original fatigue damage. 4. Results and discussion 4.1 Stress distribution arm, as shown in Fig. 5. multiplying the frequency and the signal length, it gave 32,000 data points. Fig. 3 shows the 160 second time history plotted for the original signal. The flowchart of the study is shown in Fig. 4. Fig. 4. Simplified flowchart of the undamaging cycle eliminating process In this study, the selected material for the simulation purpose was the SAE1045 carbon steel shaft. This material was chosen because it was commonly used in automotive industries for fabricating a vehicle lower suspension arm structure (Khalil & Topper, 2003). The material properties and their definitions are given in Table 1 (nCode, 2005). The fatigue damage and signal statistical parameter values for the each edited signal which was produced from the extraction process with differences COF and gate values would then be determined in order to observe the effectiveness of the methods for the fatigue data editing technique. The fatigue damage was estimated by utilizing the Coffin-Manson strainlife model. The cumulative fatigue damage was then determined from the Palmgren-Miner linear cumulative damaging rule. The edited signal required lower than 5 % difference of fatigue damage to the original signal in order to produce a edited signal which retaining the original fatigue damage. Table 1. The mechanical properties of the SAE1045 carbon steel shaft ## 4. Results and discussion ## 4.1 Stress distribution 232 Fourier Transform – Materials Analysis multiplying the frequency and the signal length, it gave 32,000 data points. Fig. 3 shows the 160 second time history plotted for the original signal. The flowchart of the study is shown START Input the original signal Determinate the PSD distribution Filter the input signal using Butterwort filter Set COF Create filtered signal Analyze the Morlet wavelet coefficients Set gate value Extract significant segments Combine extracted segments and create new mission signal Fig. 4. Simplified flowchart of the undamaging cycle eliminating process Yes properties and their definitions are given in Table 1 (nCode, 2005). In this study, the selected material for the simulation purpose was the SAE1045 carbon steel shaft. This material was chosen because it was commonly used in automotive industries for fabricating a vehicle lower suspension arm structure (Khalil & Topper, 2003). The material Ensure adequate fatigue performance No No Choice more suitable approach for fatigue data editing STOP The fatigue damage and signal statistical parameter values for the each edited signal which was produced from the extraction process with differences COF and gate values would then be determined in order to observe the effectiveness of the methods for the fatigue data editing technique. The fatigue damage was estimated by utilizing the Coffin-Manson strainlife model. The cumulative fatigue damage was then determined from the Palmgren-Miner linear cumulative damaging rule. The edited signal required lower than 5 % difference of in Fig. 4. The finite element modeling and analysis have been performed utilizing CATIA and MSC finite element analysis codes respectively. By using Eq. 13, this analysis determinates the maximum stress locations before doing the fatigue test. Based on the analysis performed by Al-Asady et al. (2008), it was obtained the stress distribution at the front lower suspension arm, as shown in Fig. 5. Fig. 5. Stress distribution and strain gauge positions In this simulation, the load was assumed of 300 kg. This value came from the car weight and the passengers. The stress level is presented by a colour contour, where the white colour shows the highest stress content and followed by black, blue, and so on. Eliminating the Undamaging Fatigue Cycles Using the Frequency Spectrum Filtering Techniques 235 original signal. The filtered signal with COF 96 Hz was selected as the optimum values that giving lower than 5 % difference of fatigue damage. For the statistical parameter analysis, the values of the r.m.s. and the kurtosis for filtered signals were almost equivalent with the original signal, where those values were in the required range for any filtered signal with the differences were below than 10 %. Fig. 7 displays signal after the filtering process. For the Morlet wavelet based edited signal, it was started by analyzing the wavelet coefficients, as shown in Fig. 8. In the scalogram, the x-axis denoted the time parameter, the y-axis represented the scale that has an inversely related to the frequency value, and the colour intensity at each x-y point was proportional to the absolute value of the wavelet coefficients as a function of the dilation and translation parameters. It provided the signal energy distribution display with respect to the particular time and frequency information. Fig. 8. The Morlet wavelet coefficients in time-frequency representation distribution, as observed in time-frequency plane. Accordingly, a lower scale indicated higher frequency and had small amplitude that means these cycles had lower energy. They gave minimal or no fatigue damaging potential. A large scale was indicative of lower frequency and higher amplitude that indicates these cycles had higher energy causing the fatigue damage. Obviously, the lower frequency indicated higher magnitude distribution, and the lower magnitude distribution was presented at higher frequency event. Using the newly Morlet wavelet - based developed computational algorithm, the wavelet coefficient magnitude segments were transposed into time domain signal, as shown in Fig. 9. The representation showed a two dimensional view of the energy Data points This extraction process involved 150 με, 160 με, 170 με, 180 με, 190 με, and 200 με gate values. From the total fatigue damaging calculation results, it was found that 170 με was 4.3 The Morlet wavelet analysis Scale ## 4.2 The FFT analysis In this frequency spectrum filtering analysis, data in time domain was then transformed into the frequency domain using the FFT algorithm in order to obtain its PSD distribution. The FFT formed a complex vector of values which each value represents the amplitude and phase of the particular sinusoidal wave at a particular frequency. The energy distribution showed on the PSD was applied to see the frequency region with lower energy in order to find the COF value for filtering purpose. The PSD display for original data is shown in Fig. 6. Fig. 6. The PSD distribution of the original signal For fatigue data, lower amplitude region commonly located at higher frequency spectrum. Therefore, lower pass filter could be applied to remove higher frequency data which contain lower amplitude cycle in time history. Lower amplitude cycle could be removed because it gave minimal fatigue damage. The filtering analysis with 4th order of Butterworth low-pass filter has been used for this data set. The 30 Hz COF was selected as the initial COF value, where getting from the PSD plotted. With 30 Hz COF, filtering process would filter out the frequency information higher than that value and obtain the new signal which contains only the allowed frequency. The same procedure was then repeated by using other COF values i.e. 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, 65 Hz, 70 Hz, 75 Hz, 80 Hz, 85 Hz, 90 Hz, 92 Hz, 94 Hz, 96 Hz and 98 Hz. The difference in COF values were used in order to observe the effect of filtering behaviour related to fatigue life assessment for each filtered signal. From the analysis of each filtered signal, the optimum value of COF was accordingly determined and it was based on the effectiveness of retaining the value of the fatigue damage and the signal statistical parameters. Unfortunately, the filtered signal with lower COF gave an obvious deviation in retaining fatigue damage. Nevertheless, by increasing the COF value the fatigue damage was ascending and almost reaches the fatigue damage of the Fig. 7. Signal display after the filtering process original signal. The filtered signal with COF 96 Hz was selected as the optimum values that giving lower than 5 % difference of fatigue damage. For the statistical parameter analysis, the values of the r.m.s. and the kurtosis for filtered signals were almost equivalent with the original signal, where those values were in the required range for any filtered signal with the differences were below than 10 %. Fig. 7 displays signal after the filtering process. ## 4.3 The Morlet wavelet analysis 234 Fourier Transform – Materials Analysis In this frequency spectrum filtering analysis, data in time domain was then transformed into the frequency domain using the FFT algorithm in order to obtain its PSD distribution. The FFT formed a complex vector of values which each value represents the amplitude and phase of the particular sinusoidal wave at a particular frequency. The energy distribution showed on the PSD was applied to see the frequency region with lower energy in order to find the COF value for filtering purpose. The PSD display for original data is shown in Fig. 6. Frequency (Hz) For fatigue data, lower amplitude region commonly located at higher frequency spectrum. Therefore, lower pass filter could be applied to remove higher frequency data which contain lower amplitude cycle in time history. Lower amplitude cycle could be removed because it The filtering analysis with 4th order of Butterworth low-pass filter has been used for this data set. The 30 Hz COF was selected as the initial COF value, where getting from the PSD plotted. With 30 Hz COF, filtering process would filter out the frequency information higher than that value and obtain the new signal which contains only the allowed frequency. The same procedure was then repeated by using other COF values i.e. 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, 65 Hz, 70 Hz, 75 Hz, 80 Hz, 85 Hz, 90 Hz, 92 Hz, 94 Hz, 96 Hz and 98 Hz. The difference in COF values were used in order to observe the effect of filtering behaviour From the analysis of each filtered signal, the optimum value of COF was accordingly determined and it was based on the effectiveness of retaining the value of the fatigue damage and the signal statistical parameters. Unfortunately, the filtered signal with lower COF gave an obvious deviation in retaining fatigue damage. Nevertheless, by increasing the COF value the fatigue damage was ascending and almost reaches the fatigue damage of the Time (s) 0 50 100 150 <sup>0</sup> 0 20 40 60 80 100 4.2 The FFT analysis Amplitude (με2/Hz) Strain (με) 60 40 20 0 -20 -40 -60 Fig. 6. The PSD distribution of the original signal related to fatigue life assessment for each filtered signal. Fig. 7. Signal display after the filtering process gave minimal fatigue damage. For the Morlet wavelet based edited signal, it was started by analyzing the wavelet coefficients, as shown in Fig. 8. In the scalogram, the x-axis denoted the time parameter, the y-axis represented the scale that has an inversely related to the frequency value, and the colour intensity at each x-y point was proportional to the absolute value of the wavelet coefficients as a function of the dilation and translation parameters. It provided the signal energy distribution display with respect to the particular time and frequency information. Fig. 8. The Morlet wavelet coefficients in time-frequency representation Accordingly, a lower scale indicated higher frequency and had small amplitude that means these cycles had lower energy. They gave minimal or no fatigue damaging potential. A large scale was indicative of lower frequency and higher amplitude that indicates these cycles had higher energy causing the fatigue damage. Obviously, the lower frequency indicated higher magnitude distribution, and the lower magnitude distribution was presented at higher frequency event. Using the newly Morlet wavelet - based developed computational algorithm, the wavelet coefficient magnitude segments were transposed into time domain signal, as shown in Fig. 9. The representation showed a two dimensional view of the energy distribution, as observed in time-frequency plane. This extraction process involved 150 με, 160 με, 170 με, 180 με, 190 με, and 200 με gate values. From the total fatigue damaging calculation results, it was found that 170 με was Eliminating the Undamaging Fatigue Cycles Using the Frequency Spectrum Filtering Techniques 237 simulation testing purposes. Based on the simulation analysis, it was found that the Morlet wavelet was more suitable approach for eliminating undamaging cycles. The method removed 58 % of the original cycles in the 68 second edited signal with the retention of at least 95.5 % of the original fatigue damage. It removed 28 % more than the edited signal that was found using the FFT technique. The extraction of fatigue damaging events successfully removed the lower energy cycles in the time history. It created a new edited signal which retains higher fatigue damaging segments containing the majority of the fatigue damage. Hence, this fatigue data summarising computational algorithm can be used in laboratories in order to study the durability characteristics of automotive components. In overall, the analysis findings of this paper suggested that the Morlet wavelet was more suitable for the The authors would like to express their gratitude to Universiti Kebangsaan Malaysia and Abdullah, S. (2005). Wavelet Bump Extraction (WBE) for Editing Variable Amplitude Fatigue Abdullah, S.; Choi, J.C.; Giacomin, J.A. & Yates, J.R. (2006). Bump Extraction Algorithm for Abdullah, S. (2007). The Wavelet Transform for Fatigue History Editing: is It Applicable for Addison, P.S. (2002). The Illustrated Wavelet Transform Handbook, Institute of Physics Al-Asady, N.A.; Abdullah, S.; Arifin, A.K. & Rahman, M.M. (2008). Effects of Surface Finish Balia, F.N. & Putra, T.E. (2006). Strength Analysis Due To Static Load on Main Tribune Truss Berry, S. (1999). Practical Wavelet Signal Processing for Automated Testing, IEEE, pp. 653- Braccesi, C.; Cianetti, F.; Lori, G. & Pioli, D. (2009). The Frequency Domain Approach in Darpe, A.K. (2007). A Novel Way to Detect Transverse Surface Crack in a Rotating Shaft. Draper, J. (2007). Modern Metal Fatigue Analysis, EMAS Publishing Ltd., Warrington, UK Gao, J.H.; Wu, R.S. & Wang, B.J. (2001). A New Type of Analyzing Wavelet and Its Numerical Analysis (CMNA), pp. 297-300, Banda Aceh, Indonesia States of Stress. International Journal of Fatigue, Vol.31, pp. 766-775 Journal of Sound and Vibration, Vol.305, pp. 151-171 Variable Amplitude Fatigue Loading. International Journal of Fatigue, Vol.28, pp. Automotive Application? Journal of Engineering and Applied Sciences, Vol.2, No.2, pp. on the Fatigue Life Assessment Using Finite Element Analysis: a Case Study of a Lower Suspension Arm, Proceeding of 2nd Regional Conference on Vehicle Engineering of Harapan Bangsa Stadium, Proceedings of Conference on Computational Mechanics & Virtual Fatigue Estimation of Non-linear Systems: the Problem of Non-Gaussian Applications for Extraction of Instantaneous Spectrum Bandwidth, Proceedings of fatigue data editing. 7. References 6. Acknowledgement 675-691 342-349 and Technology 659, 0-7803-5432-X Universitas Syiah Kuala for supporting the research. Loadings, Ph.D. Thesis, The University of Sheffield Publishing, ISBN 0 7503 0692 0, Bristol, UK Fig. 9. The Morlet wavelet coefficients in time representation selected to be the optimum gate value giving lower than 5 % difference of the fatigue damage. In this gate value, the numbers of cycle counting were only 4,561 cycles, which was 58 % less than the original signal. Furthermore, the new edited signal was produced of 68 seconds, which was 92 seconds shorter than the original signal length. The values of the r.m.s. and the kurtosis for the signal were in the required range with the differences were below than 10 %. Fig. 10 shows the plot of the edited signal. Fig. 10. The 68 second edited signal Based on these two approaches, finally, the applicability of fatigue data editing with the adaptation of the Morlet wavelet method was proven for the situation to remove undamaging fatigue cycles with the retention of the majority of the original fatigue damage and shorten the signal length. The energy spectrum showed relatively adequate with damaging event in the fatigue signal and was a very useful tool for fatigue damaging detection in the fatigue signal. ## 5. Conclusions This paper discussed on the study of fatigue data editing by using the frequency spectrum filtering techniques. Both the methods have been used to analyze random signal that can be applied to extract fatigue damaging events in the fatigue strain loading. The techniques were used to eliminate undamaging fatigue cycles in order to simplify raw signal for the simulation testing purposes. Based on the simulation analysis, it was found that the Morlet wavelet was more suitable approach for eliminating undamaging cycles. The method removed 58 % of the original cycles in the 68 second edited signal with the retention of at least 95.5 % of the original fatigue damage. It removed 28 % more than the edited signal that was found using the FFT technique. The extraction of fatigue damaging events successfully removed the lower energy cycles in the time history. It created a new edited signal which retains higher fatigue damaging segments containing the majority of the fatigue damage. Hence, this fatigue data summarising computational algorithm can be used in laboratories in order to study the durability characteristics of automotive components. In overall, the analysis findings of this paper suggested that the Morlet wavelet was more suitable for the fatigue data editing. ## 6. Acknowledgement The authors would like to express their gratitude to Universiti Kebangsaan Malaysia and Universitas Syiah Kuala for supporting the research. ## 7. References 236 Fourier Transform – Materials Analysis Time (s) selected to be the optimum gate value giving lower than 5 % difference of the fatigue damage. In this gate value, the numbers of cycle counting were only 4,561 cycles, which was 58 % less than the original signal. Furthermore, the new edited signal was produced of 68 seconds, which was 92 seconds shorter than the original signal length. The values of the r.m.s. and the kurtosis for the signal were in the required range with the differences were Time (s) Based on these two approaches, finally, the applicability of fatigue data editing with the adaptation of the Morlet wavelet method was proven for the situation to remove undamaging fatigue cycles with the retention of the majority of the original fatigue damage and shorten the signal length. The energy spectrum showed relatively adequate with damaging event in the fatigue signal and was a very useful tool for fatigue damaging This paper discussed on the study of fatigue data editing by using the frequency spectrum filtering techniques. Both the methods have been used to analyze random signal that can be applied to extract fatigue damaging events in the fatigue strain loading. The techniques were used to eliminate undamaging fatigue cycles in order to simplify raw signal for the 0 50 100 150 Fig. 9. The Morlet wavelet coefficients in time representation below than 10 %. Fig. 10 shows the plot of the edited signal. 20 40 60 80 100 120 140 160 Amplitude (με2/Hz) Strain (με) 60 40 20 0 -20 -40 -60 Fig. 10. The 68 second edited signal detection in the fatigue signal. 5. Conclusions 12 China F. X. Xin and T. J. Lu Fourier Transform Sound Radiation School of Aerospace, Xi'an Jiaotong University, Xi'an, State Key Laboratory for Mechanical Structure Strength and Vibration, With the increasing use of periodically rib-stiffened composite sandwich structures as the cabin skin of aircrafts, marine ships and express trains etc. [1-5], great efforts have been made in the pursuit of efficient theoretical methods for predicting the vibration and acoustic behaviors of these lightweight structures, so as to design optimized configurations Active control algorithms with sensors and actuators have been developed to reduce structural vibration and sound radiation [6], which however inevitably brings the penalty of increasing system complexity and financial costs. Alternatively, passive measures such as inserting fibrous sound absorptive materials in the partitioned cavity of sandwich structures may be a preferable choice to achieve a compromise between noise-reduction efficiency and financial cost. For instance, the fuselages of commercial aircrafts are commonly made of periodically rib-stiffened composite structures filled with fiberglass to enhance thermal and sound insulation [6-9]. This provides strong impetus for the development of effective theoretical models to predict the sound radiation characteristics of periodically rib-stiffened There exist numerous theoretical models for the vibroacoustic behaviors of periodic ribstiffened structures, which may be grouped into two main categorizes: one is based on the Fourier transform method [2-4,10-13], which is able to handle both sound radiation and sound transmission problems; the other is built upon the space-harmonic approach [14-18], which are suited particularly for sound transmission problems. Mace [2] employed the Fourier transform method to solve the problem of sound radiation from a fluid-loaded infinite plate reinforced by two sets of parallel stiffeners when excited by a point force; for simplification, only the tensional force of the rib-stiffeners was considered. Subsequently, Mace [3] proposed a theoretical model for the radiation of sound from an infinite fluid loaded plate when the plate is reinforced with two sets of orthogonal line stiffeners; again, only the tensional force of the rib-stiffeners was accounted for. Similarly, by only taking account of the normal force interaction between panel and rib-stiffeners, Yin et al. [4] presented a simplified theoretical model for acoustic radiation from a point-driven, fluidloaded infinite laminated composite plate reinforced by periodic parallel rib-stiffeners. As an essentially equivalent method, the space-harmonic approach evolved from progressive wave propagation was initiated by Mead and Pujara [14] when they studied the acoustical 1. Introduction competent for practical low-noise requirements. sandwich structures filled with sound absorptive materials. SEG International Exposition and Annual Meeting, San Antonio, Texas, USA, September 9-14, 2001 ## Fourier Transform Sound Radiation F. X. Xin and T. J. Lu State Key Laboratory for Mechanical Structure Strength and Vibration, School of Aerospace, Xi'an Jiaotong University, Xi'an, China ## 1. Introduction 238 Fourier Transform – Materials Analysis Giancomin, J.; Steinwolf, A. & Staszewski, W.J. (1999). A Vibration Mission Synthesis Halfpenny, A. (n.d.). A Practical Introduction to Fatigue, nCode International Ltd., Sheffield, Kim, B.S.; Lee, S.H.; Lee, M.G.; Ni, J.; Song, J.Y. & Lee, C.W. (2007). A Comparative Study on Khalil, M. & Topper, T.H. (2003). Prediction of Crack-Opening Stress Levels for 1045 As- Misiti, M.; Misiti, Y.; Oppenheim, G. & Poggi, J.M. (2008). Matlab User's Guide: Wavelet Nadot, Y. & Denier, V. (2004). Fatigue Failure of Suspension Arm: Experimental Analysis and Multiaxial Criterion. Engineering Failure Analysis, Vol.11, pp. 485-499 nCode. (2005). ICE-flow: GlyphWorks 4.0 Tutorials, nCode International Ltd, Sheffield, UK nCode. (n.d.). The nCode Book of Fatigue Theory, nCode International Ltd, Sheffield, UK Nizwan, C.K.E.; Abdullah, S.; Nuawi, M.Z. & Lamin, F. (2007). A Study of Fatigue Data Nuawi, M.Z.; Abdullah, S.; Abdullah, S.; Haris, S.M. & Arifin, A. (2009). Matlab: A Oh, C.S. (2001). Application of Wavelet Transform in Fatigue History Editing. International Percival, D.B. & Walden, A.T. (2000). Wavelet Methods for Time Series Analysis, Cambridge Prawoto, Y. (2002). Linear Elastic Fracture Mechanics (LEFM) Analysis of the Effect of Purushotham, V.; Narayanan, S. & Prasad, S.A.N. (2005). Multi-fault Diagnosis of Rolling Stephens, R.I.; Dindinger, P.M. & Gunger, J.E. (1997). Fatigue Damage Editing for Tacer, B. & Loughlin, P.J. (1998). Non-stationary Signal Classification Using the Joint Recognition. NDT&E International, Vol.38, pp. 654-664 International Journal of Fatigue, Vol.19, No.8-9, pp. 599-606 Valens, C. (1999). A Really Friendly Guide to Wavelets, 07.05.2008, Available from http://pagesperso-orange.fr/polyvalens/clemens/wavelets/wavelets.html Development Process, Florence, Italy, November 17-19, 1999 ToolboxTM 4, The Math Works Inc, MA, USA September 9-14, 2001 UK pp. 30-36 Vol.25, pp. 149-157 August 5-9, 2007 University Press, UK 1635-1641 52-5, Kuala Lumpur, Malaysia Journal of Fatigue, Vol.23, pp. 241-250 SEG International Exposition and Annual Meeting, San Antonio, Texas, USA, Algorithm for Mildly Nonstationary Road Data, Proceedings of ATA 6th International Conference on the New Role of Experimentation in the Modern Automotive Product Damage Detection in Speed-Up and Coast-Down Process of Grinding Spindle-Typed Rotor-Bearing System. Journal of Materials Processing Technology, Vol.187-188, Received Steel Under Service Loading Spectra. International Journal of Fatigue, Editing Using Frequency Spectrum Filtering Technique, Proceedings of World Engineering Congress, pp. 372-378, ISBN 978–983–43571–1–5, Penang, Malaysia, Comprehensive Reference for Engineers, McGraw-Hill Sdn. Bhd, ISBN 978-983-3850- Residual Stress on Fatigue Crack Propagation Rate. PFANF8, Vol.2, No.5, pp. 75-83 Bearing Elements Using Wavelet Analysis and Hidden Markov Model Based Fault Accelerated Durability Testing Using Strain Range and SWT Parameter Criteria. Moments of Time-Frequency Distributions. Pattern Recognition, Vol.31, No. 11, pp. With the increasing use of periodically rib-stiffened composite sandwich structures as the cabin skin of aircrafts, marine ships and express trains etc. [1-5], great efforts have been made in the pursuit of efficient theoretical methods for predicting the vibration and acoustic behaviors of these lightweight structures, so as to design optimized configurations competent for practical low-noise requirements. Active control algorithms with sensors and actuators have been developed to reduce structural vibration and sound radiation [6], which however inevitably brings the penalty of increasing system complexity and financial costs. Alternatively, passive measures such as inserting fibrous sound absorptive materials in the partitioned cavity of sandwich structures may be a preferable choice to achieve a compromise between noise-reduction efficiency and financial cost. For instance, the fuselages of commercial aircrafts are commonly made of periodically rib-stiffened composite structures filled with fiberglass to enhance thermal and sound insulation [6-9]. This provides strong impetus for the development of effective theoretical models to predict the sound radiation characteristics of periodically rib-stiffened sandwich structures filled with sound absorptive materials. There exist numerous theoretical models for the vibroacoustic behaviors of periodic ribstiffened structures, which may be grouped into two main categorizes: one is based on the Fourier transform method [2-4,10-13], which is able to handle both sound radiation and sound transmission problems; the other is built upon the space-harmonic approach [14-18], which are suited particularly for sound transmission problems. Mace [2] employed the Fourier transform method to solve the problem of sound radiation from a fluid-loaded infinite plate reinforced by two sets of parallel stiffeners when excited by a point force; for simplification, only the tensional force of the rib-stiffeners was considered. Subsequently, Mace [3] proposed a theoretical model for the radiation of sound from an infinite fluid loaded plate when the plate is reinforced with two sets of orthogonal line stiffeners; again, only the tensional force of the rib-stiffeners was accounted for. Similarly, by only taking account of the normal force interaction between panel and rib-stiffeners, Yin et al. [4] presented a simplified theoretical model for acoustic radiation from a point-driven, fluidloaded infinite laminated composite plate reinforced by periodic parallel rib-stiffeners. As an essentially equivalent method, the space-harmonic approach evolved from progressive wave propagation was initiated by Mead and Pujara [14] when they studied the acoustical Fourier Transform Sound Radiation 241 coordinate system (x, y, z) is established, with its x-axis and y-axis positioned separately along one pair of the orthogonal rib-stiffeners, the positive direction of the z-axis pointing downward (Fig. 1). Three different kinds of sandwich structures will be considered in the proceeding sections, namely, the gap between the two parallel face panels and portioned by the orthogonal lattice cores is in vacuum, air filled, or filled with fibrous sound absorptive material (e.g., fiberglass), respectively. A theoretical model will be formulated for the complex structure (i.e., orthogonally rib-stiffened sandwich structure filled with fibrous sound absorptive material), which can be degraded to deal with the other two sandwiches. > i t q e ω location ( ) 0 0 x ,y ; see Fig. 1. Consequently, a radially outspreading bending wave propagates in the upper panel from the source ( <sup>0</sup> x , 0 y ). The vibration of the upper panel is transmitted to the bottom panel via the orthogonal rib-stiffeners and sound-absorbing material (or air cavity). Subsequently, the bottom panel vibrates and radiates sound pressure waves. Fig. 1. Schematic illustration of orthogonally rib-stiffened sandwich structure (three different kinds: vacuum cavity, air cavity, and fiberglass filled cavity) excited by time-harmonic point Fig. 2. Conventions for tensional forces, bending moments and torsional moments between upper panel and (a) x-wise and (b) y-wise stiffeners, which also hold at the interface between bottom panel and x- and y-wise stiffeners acts on the surface of the upper panel at Assume that a time-harmonic point force 0 force at ( <sup>0</sup> x , 0 y ) response of periodical stiffened beams subjected to a spatial and temporal harmonic pressure. It was demonstrated that as few as three terms of space harmonics could lead to solutions of acceptable accuracy. By combining the space-harmonic approach and virtual energy method, Lee and Kim [15] analyzed the sound transmission characteristics of a thin plate stiffened by equally spaced line stiffeners. By modeling the rib-stiffeners as a combination of translational springs and rotational springs, Wang et al. [16] proposed an analytical model for sound transmission loss across double-leaf partitions stiffened with periodically placed studs. Recently, Xin and Lu [18] developed a comprehensive analytical model for sound transmission through orthogonally rib-stiffened sandwich structures: all possible motions of the rib-stiffeners were accounted for by introducing the tensional forces, bending moments and torsional moments as well as the corresponding inertial terms into the governing equations of the two face panels. None of the above mentioned investigations dealt with sound radiation and/or sound transmission issues of composite sandwich structures filled with porous sound absorptive materials. As far as the sound radiation/transmission problems of double partitions with cavity absorption is of concern, a number of theoretical [12,13], numerical [19,20] and experimental [21] studies do exist. However, all of these studies did not consider the effects of structural rib-connections between two face panels, which may be far away from the factual engineering structures. To address this deficiency, a comprehensive theoretical model is developed here for the radiation of sound from an infinite orthogonally ribstiffened sandwich structure filled with fibrous sound absorptive material in the partitioned cavity when excited by a time-harmonic point force. The equivalent forces and moments (both bending and torsional) imposed on the two face panels by the rib-stiffeners are accounted for by considering all possible motions of the rib-stiffeners. By employing the well-known equivalent fluid model [12,22], wave propagation in the fibrous sound absorptive material can be accurately described. Both viscous drag forces and thermal exchanges between air and solid fibers are accounted for by introducing frequency dependent dynamic density and bulk modulus. Taking advantage of the periodical property of the composite sandwich structure, the Fourier transform technique is adopted to solve both the structural and acoustical governing equations. In limiting cases the developed model can be favorably degraded to deal with sound radiation issues of sandwich structures with vacuum or air cavities. Therefore, model validation is carried out by comparing the present predictions for simplified sandwich structures with those available in the open literature. To explore the influence of fibrous sound absorptive materials on sound radiation of orthogonally rib-stiffened composite structures, numerical results are presented, with relevant physical features interpreted in detail. Conclusions drawn from the present theoretical study may provide fundamental principles for factual engineering design of ribstiffened composite structures filled with fibrous sound absorptive materials. #### 2. Structural dynamic responses to time-harmonic point force #### 2.1 Analytical formulation of panel vibration Consider an infinite sandwich structure as shown schematically in Fig. 1, which is reinforced by two periodic sets of orthogonal rib-stiffeners having periodic uniform separations xl and yl in the x- and y-directions, respectively. A right-handed Cartesian response of periodical stiffened beams subjected to a spatial and temporal harmonic pressure. It was demonstrated that as few as three terms of space harmonics could lead to solutions of acceptable accuracy. By combining the space-harmonic approach and virtual energy method, Lee and Kim [15] analyzed the sound transmission characteristics of a thin plate stiffened by equally spaced line stiffeners. By modeling the rib-stiffeners as a combination of translational springs and rotational springs, Wang et al. [16] proposed an analytical model for sound transmission loss across double-leaf partitions stiffened with periodically placed studs. Recently, Xin and Lu [18] developed a comprehensive analytical model for sound transmission through orthogonally rib-stiffened sandwich structures: all possible motions of the rib-stiffeners were accounted for by introducing the tensional forces, bending moments and torsional moments as well as the corresponding inertial terms into None of the above mentioned investigations dealt with sound radiation and/or sound transmission issues of composite sandwich structures filled with porous sound absorptive materials. As far as the sound radiation/transmission problems of double partitions with cavity absorption is of concern, a number of theoretical [12,13], numerical [19,20] and experimental [21] studies do exist. However, all of these studies did not consider the effects of structural rib-connections between two face panels, which may be far away from the factual engineering structures. To address this deficiency, a comprehensive theoretical model is developed here for the radiation of sound from an infinite orthogonally ribstiffened sandwich structure filled with fibrous sound absorptive material in the partitioned cavity when excited by a time-harmonic point force. The equivalent forces and moments (both bending and torsional) imposed on the two face panels by the rib-stiffeners are accounted for by considering all possible motions of the rib-stiffeners. By employing the well-known equivalent fluid model [12,22], wave propagation in the fibrous sound absorptive material can be accurately described. Both viscous drag forces and thermal exchanges between air and solid fibers are accounted for by introducing frequency dependent dynamic density and bulk modulus. Taking advantage of the periodical property of the composite sandwich structure, the Fourier transform technique is adopted to solve both the structural and acoustical governing equations. In limiting cases the developed model can be favorably degraded to deal with sound radiation issues of sandwich structures with vacuum or air cavities. Therefore, model validation is carried out by comparing the present predictions for simplified sandwich structures with those available in the open literature. To explore the influence of fibrous sound absorptive materials on sound radiation of orthogonally rib-stiffened composite structures, numerical results are presented, with relevant physical features interpreted in detail. Conclusions drawn from the present theoretical study may provide fundamental principles for factual engineering design of rib- stiffened composite structures filled with fibrous sound absorptive materials. Consider an infinite sandwich structure as shown schematically in Fig. 1, which is reinforced by two periodic sets of orthogonal rib-stiffeners having periodic uniform separations xl and yl in the x- and y-directions, respectively. A right-handed Cartesian 2. Structural dynamic responses to time-harmonic point force 2.1 Analytical formulation of panel vibration the governing equations of the two face panels. coordinate system (x, y, z) is established, with its x-axis and y-axis positioned separately along one pair of the orthogonal rib-stiffeners, the positive direction of the z-axis pointing downward (Fig. 1). Three different kinds of sandwich structures will be considered in the proceeding sections, namely, the gap between the two parallel face panels and portioned by the orthogonal lattice cores is in vacuum, air filled, or filled with fibrous sound absorptive material (e.g., fiberglass), respectively. A theoretical model will be formulated for the complex structure (i.e., orthogonally rib-stiffened sandwich structure filled with fibrous sound absorptive material), which can be degraded to deal with the other two sandwiches. Assume that a time-harmonic point force 0 i t q e ω acts on the surface of the upper panel at location ( ) 0 0 x ,y ; see Fig. 1. Consequently, a radially outspreading bending wave propagates in the upper panel from the source ( <sup>0</sup> x , 0 y ). The vibration of the upper panel is transmitted to the bottom panel via the orthogonal rib-stiffeners and sound-absorbing material (or air cavity). Subsequently, the bottom panel vibrates and radiates sound pressure waves. Fig. 1. Schematic illustration of orthogonally rib-stiffened sandwich structure (three different kinds: vacuum cavity, air cavity, and fiberglass filled cavity) excited by time-harmonic point force at ( <sup>0</sup> x , 0 y ) Fig. 2. Conventions for tensional forces, bending moments and torsional moments between upper panel and (a) x-wise and (b) y-wise stiffeners, which also hold at the interface between bottom panel and x- and y-wise stiffeners Fourier Transform Sound Radiation 243 ( ) <sup>2</sup> <sup>2</sup> x x x x K KK m Qw w Km Km ( ) <sup>2</sup> <sup>2</sup> y y y y K KK m Qw w Km Km yy y y KK m K Q ww Km Km ω ω <sup>+</sup> <sup>−</sup> <sup>=</sup> − + − − ω xx xx xx x x <sup>x</sup> xx xx xx x x <sup>x</sup> xx xx xx xx yy yy yy y y Similarly, the bending moments of the rib-stiffeners can be expressed as [18]: ρ ω ρ ω ρ ω ρ ω ρ ω ρ ω obtains the torsional moments of the rib-stiffeners as [18]: where ( \* E Ix x , \* E I y y ) are the bending stiffness of half the rib-stiffeners, ( <sup>−</sup> <sup>−</sup> <sup>=</sup> − + − − xx x <sup>x</sup> <sup>x</sup> <sup>+</sup> <sup>−</sup> <sup>=</sup> − + − − ω xx x <sup>x</sup> <sup>x</sup> ω y y where ω stiffeners per unit length. M y y M indicating the direction of the stiffener. M M 2 2 1 2 2 2 ( ) <sup>2</sup> <sup>2</sup> 2 2 1 2 2 2 2 2 1 2 2 2 ( ) <sup>2</sup> <sup>2</sup> 2 2 1 2 2 2 y yy y is the circle frequency and ( Kx , Ky ) are the tensional stiffness of half the rib- ( ) \\ 2 2 2 2 \2 \ 2 2 \* 22 2 2 EI I x EI I x ( ) \\ 2 2 \2 2 2 E I w w EI EI I* \* 22 \* 2 2 2 2 EI I x EI I x ( ) \\ 2 2 \2 2 2 \ 2 2 \* 22 2 2 EI I y EI I y ( ) 2 \2 \\* 2 2 2 E I w w EI EI I \* 22 \* 2 2 2 2 y y yy yy yy EI I y EI I y yy yy yy yy are mass density and polar moment of inertia for the rib-stiffeners, with subscripts x and y Following similar procedures for deriving the tensional forces and bending moments, one ( ) \\ 2 2 2 2 \2 GJ J x y GJ J x y* ( ) \\ 2 2 \2 2 2 G J w w GJ GJ J* GJ J x y GJ J x y \2 \2 2 2 xx xx xx xx GJ GJ J w w G J <sup>M</sup> \2 \2 2 2 xx xx xx x x Tx xx xx xx xx xx xx xx x x Tx ρ ω ρ ω ρ ω yy yy yy yy EI EI I w w E I xx xx xx xx EI EI I w w E I <sup>M</sup> <sup>+</sup> <sup>−</sup> ∂ ∂ <sup>=</sup> <sup>−</sup> − ∂ −∂ <sup>−</sup> <sup>−</sup> ∂ ∂ <sup>=</sup> <sup>−</sup> −∂ − ∂ y y y y ω ω ω ω ω 1 2 ρ ω ρ ω 1 2 ρ ω 1 2 ρ ω <sup>+</sup> <sup>−</sup> ∂ ∂ <sup>=</sup> <sup>−</sup> − ∂ −∂ (9) 1 2 <sup>−</sup> <sup>−</sup> ∂ ∂ <sup>=</sup> <sup>−</sup> −∂ − ∂ (10) ρ ω ρ ω 1 2 <sup>+</sup> <sup>−</sup> ∂ ∂ <sup>=</sup> <sup>−</sup> − − <sup>∂</sup> <sup>∂</sup> ∂ ∂ (11) 1 2 <sup>−</sup> <sup>−</sup> ∂ ∂ <sup>=</sup> <sup>−</sup> − − <sup>∂</sup> <sup>∂</sup> ∂ ∂ (12) ρ ω ρ ω ρ ω ρx , ρ <sup>−</sup> <sup>−</sup> <sup>=</sup> − + − − (4) ω (3) (5) (6) (7) (8) <sup>y</sup> ) and ( xI , yI ) x x x x KK m <sup>K</sup> Q ww Km Km ω Upon point force excitation, the vibration of the upper and bottom panels can be described using two dynamic governing equations, where the influence of the rib-stiffeners exists in the form of tensional forces (general force plus inertial force), bending moments (general bending moment plus inertial bending moment), and torsional moments (general torsional moment plus inertial torsional moment). With the inertial effects of the rib-stiffeners accounted for, the resultant tensional forces, bending and torsional moments acting on the upper and bottom panels per rib-stiffener are not equal, denoted here by ( <sup>Q</sup><sup>+</sup> , M<sup>+</sup> , MT + ) and ( <sup>Q</sup><sup>−</sup> , M<sup>−</sup> , MT <sup>−</sup> ), respectively. Fig. 2 shows the convention employed for denoting the tensional forces as well as the bending and torsional moments between the upper panel and the x- and y-wise stiffeners. The same apply at the interface between the bottom panel and the x- and y-wise stiffeners. The dynamic responses of the sandwich structure are time-harmonic as the excitation is in the form of 0 i t q e ω . For simplicity, the harmonic time term i t e ω is suppressed henceforth. With the equivalent forces and moments of the lattice core and the pressure in the fibrous sound absorptive material (or air cavity) accounted for, the equations governing panel vibrations are given by: $$\begin{aligned} D\_1 \nabla^4 w\_1 + m\_1 \frac{\partial^2 w\_1}{\partial t^2} &= \sum\_{m = -\alpha}^{+\alpha} \left[ Q\_y^+ \delta\left(\mathbf{x} - m l\_x\right) + \frac{\partial}{\partial y} \left\{ M\_y^+ \delta\left(\mathbf{x} - m l\_x\right) \right\} + \frac{\partial}{\partial \mathbf{x}} \left\{ M\_{Ty}^+ \delta\left(\mathbf{x} - m l\_x\right) \right\} \right] \\ &+ \sum\_{n = -\alpha}^{+\alpha} \left[ Q\_x^+ \delta\left(y - n l\_y\right) + \frac{\partial}{\partial \mathbf{x}} \left\{ M\_x^+ \delta\left(y - n l\_y\right) \right\} + \frac{\partial}{\partial y} \left\{ M\_{Tx}^+ \delta\left(y - n l\_y\right) \right\} \right] \\ &+ q\_0 \delta\left(\mathbf{x} - \mathbf{x}\_0\right) \delta\left(y - y\_0\right) - p\_{\text{ave}}\left(\mathbf{x}, y\_1 h\_1\right) \end{aligned} \tag{1}$$ $$\begin{split} D\_{2} \nabla^{4} w\_{2} + m\_{2} \frac{\partial^{2} w\_{2}}{\partial t^{2}} &= -\sum\_{m=-\alpha}^{+\alpha} \Big[ Q\_{y}^{-} \delta \left( \mathbf{x} - m l\_{x} \right) + \frac{\partial}{\partial y} \Big\langle M\_{y}^{-} \delta \left( \mathbf{x} - m l\_{x} \right) \Big\rangle + \frac{\partial}{\partial \mathbf{x}} \Big\langle M\_{Ty}^{-} \delta \left( \mathbf{x} - m l\_{x} \right) \Big\rangle \Big] \\ &- \sum\_{n=-\alpha}^{+\alpha} \Big[ Q\_{x}^{-} \delta \left( y - n l\_{y} \right) + \frac{\partial}{\partial \mathbf{x}} \Big\langle M\_{x}^{-} \delta \left( y - n l\_{y} \right) \Big\rangle + \frac{\partial}{\partial y} \Big\langle M\_{Tx}^{-} \delta \left( y - n l\_{y} \right) \Big\rangle \Big] \\ &+ p\_{\text{core}} \left( \mathbf{x}, y, h\_{1} + d \right) \end{split} \tag{2}$$ where ( ) <sup>2</sup> 4 2222 ∇ ≡ ∂ ∂ +∂ ∂ x y ; δ (⋅) is the Dirac delta function; ( w<sup>1</sup> , w<sup>2</sup> ), ( m<sup>1</sup> , m<sup>2</sup> ) and ( D<sup>1</sup> , D<sup>2</sup> ) are the displacement, surface mass density and flexural rigidity of the upper panel and bottom panel, respectively. The material loss factor η <sup>j</sup> ( j = 1, 2 for upper panel and bottom panel, respectively) is introduced with the complex Young's modulus ( ) ( ) 3 2 1 12 1 D Eh i j jj j =+ − η ν<sup>j</sup> (where j =1, 2). As the factual forces and moments exerting on the upper and bottom panels are not the same due to the consideration of inertial forces and moments, the terms associated with the two panels are denoted separately by superscripts + (upper) and – (bottom). Subscripts x and y are introduced to represent those terms arising from the x- and y-wise stiffeners, respectively. Taking into account the inertial effects (due to stiffener mass) and applying both the Hooke's law and Newton's second law, one obtains the tensional forces arising from the ribstiffeners as [18]: Upon point force excitation, the vibration of the upper and bottom panels can be described using two dynamic governing equations, where the influence of the rib-stiffeners exists in the form of tensional forces (general force plus inertial force), bending moments (general bending moment plus inertial bending moment), and torsional moments (general torsional moment plus inertial torsional moment). With the inertial effects of the rib-stiffeners accounted for, the resultant tensional forces, bending and torsional moments acting on the upper and bottom panels per rib-stiffener are not equal, denoted here by ( <sup>Q</sup><sup>+</sup> , M<sup>+</sup> , MT tensional forces as well as the bending and torsional moments between the upper panel and the x- and y-wise stiffeners. The same apply at the interface between the bottom panel and The dynamic responses of the sandwich structure are time-harmonic as the excitation is in With the equivalent forces and moments of the lattice core and the pressure in the fibrous sound absorptive material (or air cavity) accounted for, the equations governing panel ( ) ()( ) <sup>w</sup> Dwm Q x ml M x ml M x ml t y x δδ δδ ∂ ∂∂ <sup>⎡</sup> <sup>⎤</sup> ∇ + =− − + − + − <sup>⎢</sup> <sup>⎥</sup> <sup>∂</sup> ∂ ∂ <sup>⎣</sup> <sup>⎦</sup> ( D<sup>1</sup> , D<sup>2</sup> ) are the displacement, surface mass density and flexural rigidity of the upper panel bottom panel, respectively) is introduced with the complex Young's modulus As the factual forces and moments exerting on the upper and bottom panels are not the same due to the consideration of inertial forces and moments, the terms associated with the two panels are denoted separately by superscripts + (upper) and – (bottom). Subscripts x and y are introduced to represent those terms arising from the x- and y-wise stiffeners, Taking into account the inertial effects (due to stiffener mass) and applying both the Hooke's law and Newton's second law, one obtains the tensional forces arising from the rib- <sup>w</sup> Dwm Q x ml M x ml M x ml t y x δδ δδ ∂ ∂∂ <sup>⎡</sup> <sup>⎤</sup> ∇+ = − + − + − <sup>⎢</sup> <sup>⎥</sup> <sup>∂</sup> ∂ ∂ <sup>⎣</sup> <sup>⎦</sup> cav +∞ −− − +∞ −− − +∞ ++ + +∞ ++ + 00 0 1 , , q x x y y p xyh δ + − −− ( ) <sup>j</sup> (where j =1, 2). <sup>1</sup> , , δ p xyh d + + . For simplicity, the harmonic time term i t <sup>−</sup> ), respectively. Fig. 2 shows the convention employed for denoting the e ω ( ) { } ( ) { } ( ) y x y x Ty x xy xy Tx y ⎡ ∂ ∂ ⎤ + −+ − + − <sup>⎢</sup> <sup>⎥</sup> ∂ ∂ <sup>⎣</sup> <sup>⎦</sup> Q y nl M y nl M y nl x y ( ) { } ( ) { } ( ) ∑ (1) ( ) { } ( ) { } ( ) (⋅) is the Dirac delta function; ( w<sup>1</sup> , w<sup>2</sup> ), ( m<sup>1</sup> , m<sup>2</sup> ) and ( ) { } ( ) { } ( ) η ∑ (2) xy xy Tx y ⎡ ∂ ∂ ⎤ − −+ − + − <sup>⎢</sup> <sup>⎥</sup> ∂ ∂ <sup>⎣</sup> <sup>⎦</sup> Q y nl M y nl M y nl x y y x y x Ty x and ( <sup>Q</sup><sup>−</sup> , M<sup>−</sup> , MT the form of 0 the x- and y-wise stiffeners. vibrations are given by: i t q e ω 2 4 1 1 11 2 2 4 2 2 22 2 <sup>2</sup> 4 2222 ∇ ≡ ∂ ∂ +∂ ∂ x y ; where ( ) ( ) ( ) 3 2 1 12 1 D Eh i j jj j =+ − η respectively. stiffeners as [18]: m n δ m =−∞ ∑ n cav ν =−∞ and bottom panel, respectively. The material loss factor =−∞ =−∞ ∑ + ) is suppressed henceforth. δ δ > δ <sup>j</sup> ( j = 1, 2 for upper panel and δ $$Q\_{\mathbf{x}}^{+} = -\frac{K\_{\mathbf{x}}\left(K\_{\mathbf{x}} - m\_{\mathbf{x}}o^{2}\right)}{2K\_{\mathbf{x}} - m\_{\mathbf{x}}o^{2}}w\_{1} + \frac{K\_{\mathbf{x}}^{2}}{2K\_{\mathbf{x}} - m\_{\mathbf{x}}o^{2}}w\_{2} \tag{3}$$ $$Q\_x^- = -\frac{K\_\times^2}{2K\_\times - m\_\times o^2} w\_1 + \frac{K\_\times \left(K\_\times - m\_\times o^2\right)}{2K\_\times - m\_\times o^2} w\_2 \tag{4}$$ $$Q\_y^+ = -\frac{K\_y \left(K\_y - m\_y o^2\right)}{2K\_y - m\_y o^2} w\_1 + \frac{K\_y^2}{2K\_y - m\_y o^2} w\_2 \tag{5}$$ $$Q\_y^- = -\frac{K\_y^2}{2K\_y - m\_y o^2} w\_1 + \frac{K\_y \left(K\_y - m\_y o^2\right)}{2K\_y - m\_y o^2} w\_2 \tag{6}$$ where ω is the circle frequency and ( Kx , Ky ) are the tensional stiffness of half the ribstiffeners per unit length. Similarly, the bending moments of the rib-stiffeners can be expressed as [18]: $$\mathcal{M}\_{\mathbf{x}}^{+} = \frac{E\_{\mathbf{x}}I\_{\mathbf{x}}^{"\prime}(E\_{\mathbf{x}}I\_{\mathbf{x}}^{"\prime} - \rho\_{\mathbf{x}}I\_{\mathbf{x}}oo^{2})}{2E\_{\mathbf{x}}I\_{\mathbf{x}}^{"\prime} - \rho\_{\mathbf{x}}I\_{\mathbf{x}}oo^{2}} \frac{\partial^{2}w\_{1}}{\partial\mathbf{x}^{2}} - \frac{E\_{\mathbf{x}}^{2}I\_{\mathbf{x}}^{"\prime 2}}{2E\_{\mathbf{x}}I\_{\mathbf{x}}^{"\prime} - \rho\_{\mathbf{x}}I\_{\mathbf{x}}oo^{2}} \frac{\partial^{2}w\_{2}}{\partial\mathbf{x}^{2}}\tag{7}$$ $$M\_{\mathbf{x}} = \frac{E\_{\mathbf{x}}^{2}I\_{\mathbf{x}}^{\2}}{2E\_{\mathbf{x}}I\_{\mathbf{x}}^{\} - \rho\_{\mathbf{x}}I\_{\mathbf{x}}o\rho^{2}}\frac{\hat{\sigma}^{2}w\_{\mathbf{1}}}{\hat{\alpha}^{2}} - \frac{E\_{\mathbf{x}}I\_{\mathbf{x}}^{\}\left(E\_{\mathbf{x}}I\_{\mathbf{x}}^{\} - \rho\_{\mathbf{x}}I\_{\mathbf{x}}o\rho^{2}\right)}{2E\_{\mathbf{x}}I\_{\mathbf{x}}^{\} - \rho\_{\mathbf{x}}I\_{\mathbf{x}}o\rho^{2}}\frac{\hat{\sigma}^{2}w\_{\mathbf{2}}}{\hat{\alpha}^{2}}\tag{8}$$ $$\mathcal{M}\_y^+ = \frac{E\_y I\_y^"\left(E\_y I\_y^"-\rho\_y I\_y \rho^2\right)}{2E\_y I\_y^"-\rho\_y I\_y \rho^2} \frac{\partial^2 w\_1}{\partial y^2} - \frac{E\_y^2 I\_y^{"\2}}{2E\_y I\_y^"-\rho\_y I\_y \rho^2} \frac{\partial^2 w\_2}{\partial y^2} \tag{9}$$ $$\mathbf{M}\_y^- = \frac{E\_y^2 I\_y^{\2}}{2E\_y I\_y^\ - \rho\_y I\_y \alpha^2} \frac{\partial^2 w\_1}{\partial y^2} - \frac{E\_y I\_y^\* \left(E\_y I\_y^\* - \rho\_y I\_y \alpha^2\right)}{2E\_y I\_y^\* - \rho\_y I\_y \alpha^2} \frac{\partial^2 w\_2}{\partial y^2} \tag{10}$$ where ( \* E Ix x , \* E I y y ) are the bending stiffness of half the rib-stiffeners, ( ρx , ρ <sup>y</sup> ) and ( xI , yI ) are mass density and polar moment of inertia for the rib-stiffeners, with subscripts x and y indicating the direction of the stiffener. Following similar procedures for deriving the tensional forces and bending moments, one obtains the torsional moments of the rib-stiffeners as [18]: $$\mathbf{M}\_{\rm Tx}^{+} = \frac{\mathbf{G}\_{\rm x} \mathbf{J}\_{\rm x}^{\} \left(\mathbf{G}\_{\rm x} \mathbf{J}\_{\rm x}^{\} - \rho\_{\rm x} \mathbf{J}\_{\rm x} \boldsymbol{\alpha}^{2}\right)}{2 \mathbf{G}\_{\rm x} \mathbf{J}\_{\rm x}^{\} - \rho\_{\rm x} \mathbf{J}\_{\rm x} \boldsymbol{\alpha}^{2}} \frac{\hat{\boldsymbol{\alpha}}^{2} \mathbf{w}\_{1}}{\hat{\boldsymbol{\alpha}} \mathbf{x} \boldsymbol{\alpha}} - \frac{\mathbf{G}\_{\rm x}^{2} \mathbf{J}\_{\rm x}^{\2}}{2 \mathbf{G}\_{\rm x} \mathbf{J}\_{\rm x}^{\} - \rho\_{\rm x} \mathbf{J}\_{\rm x} \boldsymbol{\alpha}^{2}} \frac{\hat{\boldsymbol{\alpha}}^{2} \mathbf{w}\_{2}}{\hat{\boldsymbol{\alpha}} \mathbf{x} \boldsymbol{\alpha}}\tag{11}$$ $$\mathbf{M}\_{\rm Tx}^{-} = \frac{\mathbf{G}\_{\rm x}^{2} \mathbf{J}\_{\rm x}^{\2}}{2\mathbf{G}\_{\rm x} \mathbf{J}\_{\rm x}^{\} - \rho\_{\rm x} \mathbf{J}\_{\rm x} \boldsymbol{\phi}^{2}} \frac{\hat{\boldsymbol{\phi}}^{2} \mathbf{w}\_{1}}{\hat{\boldsymbol{\alpha}} \mathbf{x} \boldsymbol{\phi}} - \frac{\mathbf{G}\_{\rm x} \mathbf{J}\_{\rm x}^{\} \left(\mathbf{G}\_{\rm x} \mathbf{J}\_{\rm x}^{\} - \rho\_{\rm x} \mathbf{J}\_{\rm x} \boldsymbol{\alpha}^{2}\right)}{2\mathbf{G}\_{\rm x} \mathbf{J}\_{\rm x}^{\} - \rho\_{\rm x} \mathbf{J}\_{\rm x} \boldsymbol{\alpha}^{2}} \frac{\hat{\boldsymbol{\alpha}}^{2} \mathbf{w}\_{2}}{\hat{\boldsymbol{\alpha}} \mathbf{x} \boldsymbol{\alpha}} \tag{12}$$ Fourier Transform Sound Radiation 245 ( ) \\ 2 2 \2 1 2 \2 \2 , 2 2 xx xx xx x x M M* EI EI I E I R R ω GJ GJ J G J R R GJ GJ J G J R R ω <sup>−</sup> <sup>=</sup> <sup>=</sup> − − ρ ω <sup>−</sup> <sup>=</sup> <sup>=</sup> − − ρ ω ρ ω T T Q Rw Rw xQ Q 11 22 Q Rw Rw yQ Q 31 42 structure can be expressed by using space harmonic series, as: ρ <sup>−</sup> <sup>=</sup> <sup>=</sup> − − ρ ω ρ ω ρ ω M M ρ R R xx xx xx xx yy yy yy yy EI I EI I ( ) \\ 2 2 \2 1 2 \2 \2 , 2 2 xx xx xx x x T T* xx xx xx xx yy yy yy yy GJ J GJ J Using Eqs. (21)-(26), one can simplify the expressions of the tensional forces, bending <sup>+</sup> =− + , Q Rw Rw xQ Q 21 12 <sup>+</sup> =− + , Q Rw Rw yQ Q 41 32 12 21 22 22 , xM M xM M ww ww MR R MR R 34 43 22 22 , yM M yM M ww ww MR R MR R + − ∂∂ ∂∂ =− =− ∂∂ ∂∂ 12 21 , Tx T T Tx T T ww ww MR R MR R 34 43 , Ty T <sup>T</sup> Ty T <sup>T</sup> ww ww MR R MR R Given the 2D (two-dimensional) periodic nature of the sandwich structure as shown in Fig. 1, applying the Poisson summation formula [3,23], the wave components in the + − ∂∂ ∂∂ =− =− <sup>∂</sup> ∂ ∂∂ 22 22 12 12 xx xx 22 22 12 12 yy yy 22 22 12 12 22 22 12 12 xy xy xy xy y x yx yx yx + − ∂∂ ∂∂ =− =− <sup>∂</sup> ∂ ∂∂ ∂∂ ∂∂ , (31) + − ∂∂ ∂∂ =− =− <sup>∂</sup> ∂ ∂∂ ∂∂ ∂∂ , (32) GJ J GJ J ( ) \\ 2 2 \2 3 4 \2 \2 , 2 2 yy yy yy y y* ρ ω ρ ω ρ ω ρ ω <sup>−</sup> =− + (27) <sup>−</sup> =− + (28) (23) , (24) , (25) , (26) , (29) , (30) EI I EI I ( ) \\ 2 2 \2 3 4 \2 \2 , 2 2 yy yy yy y y* EI EI I E I <sup>−</sup> <sup>=</sup> <sup>=</sup> − − 2. Replacement of bending moment coefficients: 3. Replacement of torsional moment coefficients: moments and torsional moments, as: 1. Tensional forces 2. Bending moments 3. Torsional moments 2.2 Solutions $$\mathbf{M}\_{Ty}^{+} = \frac{\mathbf{G}\_{y}\mathbf{J}\_{y}^{\}\left(\mathbf{G}\_{y}\mathbf{J}\_{y}^{\} - \rho\_{y}\mathbf{J}\_{y}\rho\sigma^{2}\right)}{2\mathbf{G}\_{y}\mathbf{J}\_{y}^{\} - \rho\_{y}\mathbf{J}\_{y}\rho\sigma^{2}} \frac{\hat{\sigma}^{2}w\_{1}}{\hat{\sigma}y\hat{\sigma}\mathbf{x}} - \frac{\mathbf{G}\_{y}^{2}\mathbf{J}\_{y}^{\2}}{2\mathbf{G}\_{y}\mathbf{J}\_{y}^{\} - \rho\_{y}\mathbf{J}\_{y}\rho\sigma^{2}} \frac{\hat{\sigma}^{2}w\_{2}}{\hat{\sigma}y\hat{\sigma}\mathbf{x}}\tag{13}$$ $$\boldsymbol{M}\_{\rmTy}^{-} = \frac{\mathbf{G}\_{\rm y}^{2} \mathbf{J}\_{\rm y}^{\2}}{2 \mathbf{G}\_{\rm y} \mathbf{J}\_{\rm y}^{\} - \rho\_{\rm y} \mathbf{J}\_{\rm y} \boldsymbol{o} \boldsymbol{o}^{2}} \frac{\hat{\boldsymbol{\sigma}}^{2} \mathbf{w}\_{1}}{\hat{\boldsymbol{\sigma}} \boldsymbol{y} \boldsymbol{\hat{\sigma}}} - \frac{\mathbf{G}\_{\rm y} \mathbf{J}\_{\rm y}^{\} \left( \mathbf{G}\_{\rm y} \mathbf{J}\_{\rm y}^{\} - \rho\_{\rm y} \mathbf{J}\_{\rm y} \boldsymbol{o} \boldsymbol{o}^{2} \right)}{2 \mathbf{G}\_{\rm y} \mathbf{J}\_{\rm y}^{\} - \rho\_{\rm y} \mathbf{J}\_{\rm y} \boldsymbol{o}^{2}} \frac{\hat{\boldsymbol{\sigma}}^{2} \mathbf{w}\_{2}}{\hat{\boldsymbol{\sigma}} \boldsymbol{y} \boldsymbol{\hat{\sigma}} \mathbf{x}} \tag{14}$$ where ( \* G J x x , \* G J y y ) are the torsional stiffness of half the rib-stiffeners and ( xJ , yJ ) are the torsional moment of inertia for the rib-stiffeners. In the above expressions for the tensional forces, bending moments and torsional moments of a rib-stiffener, the geometrical properties of its cross-section are given by: $$K\_x = \frac{E\_x t\_x}{d \,/\, 2},\,\, K\_y = \frac{E\_y t\_y}{d \,/\, 2} \tag{15}$$ $$I\_x^\* = \frac{t\_x \left(d \;/\; \mathcal{D}\right)^3}{12}, \; I\_y^\* = \frac{t\_y \left(d \;/\; \mathcal{D}\right)^3}{12}, \; I\_x = \frac{t\_x d^3}{12}, \; I\_y = \frac{t\_y d^3}{12} \tag{16}$$ $$f\_x^\* = \frac{t\_x^3 d}{2} \left[ \frac{1}{3} - \frac{64}{\pi^5} \frac{2t\_x}{d} \sum\_{n=1,3,5,\dots}^{\infty} \frac{\tanh(n\pi d / 4t\_x)}{n^5} \right] \tag{17}$$ $$J\_y^\* = \frac{t\_y^3 d}{2} \left[ \frac{1}{3} - \frac{64}{\pi^5} \frac{2t\_y}{d} \sum\_{n=1,3,5,\dots}^{\infty} \frac{\tanh(n\pi d / 4t\_y)}{n^5} \right] \tag{18}$$ $$J\_x = t\_x^3 d \left[ \frac{1}{3} - \frac{64}{\pi^5} \frac{t\_x}{d} \sum\_{n=1,3,5,\dots}^{\infty} \frac{\tanh(n\pi d / 2t\_x)}{n^5} \right] \tag{19}$$ $$J\_y = t\_y^3 d \left[ \frac{1}{3} - \frac{64}{\pi^5} \frac{t\_y}{d} \sum\_{n=1,3,5,\dots}^{\infty} \frac{\tanh(n\pi d / 2t\_y)}{n^5} \right] \tag{20}$$ where Ex and Ey are separately the Young's modulus of the x- and y-wise stiffener materials. To simplify Eqs. (3)-(14), the following set of specified characteristics is introduced to replace the coefficients of general displacements. 1. Replacement of tensional force coefficients: $$R\_{Q1} = \frac{K\_{\text{x}} \left(K\_{\text{x}} - m\_{\text{x}}oo^2\right)}{2K\_{\text{x}} - m\_{\text{x}}oo^2}, \ R\_{Q2} = \frac{K\_{\text{x}}^2}{2K\_{\text{x}} - m\_{\text{x}}oo^2} \tag{21}$$ $$R\_{Q3} = \frac{K\_y \left(K\_y - m\_y ao^2\right)}{2K\_y - m\_y ao^2}, \ R\_{Q4} = \frac{K\_y^2}{2K\_y - m\_y ao^2} \tag{22}$$ ρ ω of a rib-stiffener, the geometrical properties of its cross-section are given by: K K \* / 2 12 y y t d I = , ρ ω ρ ω ( )<sup>3</sup> \* / 2 12 x x 2 3 2 3 3 3 y y replace the coefficients of general displacements. 1. Replacement of tensional force coefficients: Q Q R R materials. 3 3 3 <sup>I</sup> <sup>=</sup> , ( )<sup>3</sup> π π π π t d <sup>3</sup> \* \* y ( ) \\ 2 2 \2 2 2 GJ J y x GJ J y x* ( ) 2 \2 \\* 2 2 2 G J w w GJ GJ J GJ J y x GJ J y x \2 \2 2 2 yy yy yy y y yy yy yy yy GJ GJ J w w G J <sup>M</sup> > \2 \2 2 2 y y yy yy yy yy yy yy yy where ( \* G J x x , \* G J y y ) are the torsional stiffness of half the rib-stiffeners and ( xJ , yJ ) are the In the above expressions for the tensional forces, bending moments and torsional moments , /2 /2 y y x x x y E t E t 5 5 1,3,5,... 1 64 2 tanh( / 4 ) 5 5 1,3,5,... 1 64 2 tanh( / 4 ) 5 5 1,3,5,... 1 64 tanh( / 2 ) > 5 5 1,3,5,... 1 64 tanh( / 2 ) y y t nd t y y y ∞ td t nd t x x <sup>x</sup> <sup>x</sup> n td t nd t <sup>J</sup> <sup>d</sup> <sup>n</sup> = n = x x x x n <sup>t</sup> nd t J td <sup>d</sup> <sup>n</sup> = n = J td <sup>d</sup> <sup>n</sup> ( ) <sup>2</sup> ( ) <sup>2</sup> ω ω ω ω x x y y <sup>−</sup> <sup>=</sup> <sup>−</sup> , K m <sup>−</sup> <sup>=</sup> <sup>−</sup> , K m <sup>1</sup> <sup>2</sup> 2 xx x <sup>3</sup> <sup>2</sup> 2 yy y KK m KK m ∞ ∞ where Ex and Ey are separately the Young's modulus of the x- and y-wise stiffener To simplify Eqs. (3)-(14), the following set of specified characteristics is introduced to <sup>J</sup> <sup>d</sup> <sup>n</sup> ∞ Ty Ty torsional moment of inertia for the rib-stiffeners. M 1 2 <sup>+</sup> <sup>−</sup> ∂ ∂ <sup>=</sup> <sup>−</sup> − − <sup>∂</sup> <sup>∂</sup> ∂ ∂ (13) 1 2 <sup>−</sup> <sup>−</sup> ∂ ∂ <sup>=</sup> <sup>−</sup> − − <sup>∂</sup> <sup>∂</sup> ∂ ∂ (14) ρ ω ρ ω ρ ω d d = = (15) 3 I = (16) 12 y y t d 3 12 <sup>x</sup> <sup>x</sup> t d <sup>I</sup> <sup>=</sup> , π π π π <sup>⎡</sup> <sup>⎤</sup> = − <sup>⎢</sup> <sup>⎥</sup> ⎢⎣ ⎥⎦ <sup>∑</sup> (18) <sup>⎡</sup> <sup>⎤</sup> = − <sup>⎢</sup> <sup>⎥</sup> ⎢⎣ ⎥⎦ <sup>∑</sup> (19) <sup>⎡</sup> <sup>⎤</sup> = − <sup>⎢</sup> <sup>⎥</sup> ⎢⎣ ⎥⎦ <sup>∑</sup> (20) 2 <sup>2</sup> <sup>2</sup> 2 <sup>x</sup> <sup>Q</sup> x x 2 y y K ω ω <sup>=</sup> <sup>−</sup> (21) <sup>=</sup> <sup>−</sup> (22) K m <sup>4</sup> <sup>2</sup> 2 y K m <sup>K</sup> <sup>R</sup> Q R <sup>⎡</sup> <sup>⎤</sup> = − <sup>⎢</sup> <sup>⎥</sup> ⎢⎣ ⎥⎦ <sup>∑</sup> (17) 2. Replacement of bending moment coefficients: $$R\_{M1} = \frac{E\_x I\_x^"\left(E\_x I\_x^" - \rho\_x I\_x \alpha^2\right)}{2E\_x I\_x^" - \rho\_x I\_x \alpha^2}, \ R\_{M2} = \frac{E\_x^2 I\_x^{"2}}{2E\_x I\_x^" - \rho\_x I\_x \alpha^2} \tag{23}$$ $$R\_{M3} = \frac{E\_y I\_y^\* \left( E\_y I\_y^\* - \rho\_y I\_y a \sigma^2 \right)}{2E\_y I\_y^\* - \rho\_y I\_y a \sigma^2}, \ R\_{M4} = \frac{E\_y^2 I\_y^{\2}}{2E\_y I\_y^\ - \rho\_y I\_y a \sigma^2},\tag{24}$$ 3. Replacement of torsional moment coefficients: $$R\_{T1} = \frac{G\_{\rm x} \int\_{\rm x}^{\rm x} \left( G\_{\rm x} \int\_{\rm x}^{\rm x} - \rho\_{\rm x} \mathbb{I}\_{\rm x} \rho \sigma^2 \right)}{2 \text{G}\_{\rm x} \int\_{\rm x}^{\rm x} - \rho\_{\rm x} \mathbb{I}\_{\rm x} \rho \sigma^2}, \ R\_{T2} = \frac{\text{G}\_{\rm x}^2 \text{J}\_{\rm x}^{\2}}{2 \text{G}\_{\rm x} \text{J}\_{\rm x}^{\} - \rho\_{\rm x} \mathbb{I}\_{\rm x} \rho \sigma^2}, \tag{25}$$ $$R\_{T3} = \frac{\mathcal{G}\_y \mathcal{I}\_y^\* \left(\mathcal{G}\_y \mathcal{I}\_y^\* - \rho\_y \mathcal{I}\_y a \sigma^2\right)}{2 \mathcal{G}\_y \mathcal{I}\_y^\* - \rho\_y \mathcal{I}\_y a^2}, \\ R\_{T4} = \frac{\mathcal{G}\_y^2 \mathcal{I}\_y^{\2}}{2 \mathcal{G}\_y \mathcal{I}\_y^\ - \rho\_y \mathcal{I}\_y a^2}, \tag{26}$$ Using Eqs. (21)-(26), one can simplify the expressions of the tensional forces, bending moments and torsional moments, as: 1. Tensional forces $$\mathbf{Q}\_x^+ = -R\_{Q1}w\_1 + R\_{Q2}w\_2 \ \ \ \ \mathbf{Q}\_x^- = -R\_{Q2}w\_1 + R\_{Q1}w\_2 \tag{27}$$ $$\mathbf{Q}\_y^+ = -R\_{Q3}w\_1 + R\_{Q4}w\_2 \ \ \ \mathbf{Q}\_y^- = -R\_{Q4}w\_1 + R\_{Q3}w\_2 \tag{28}$$ 2. Bending moments $$M\_{\mathbf{x}}^{+} = R\_{M1} \frac{\partial^2 w\_1}{\partial \mathbf{x}^2} - R\_{M2} \frac{\partial^2 w\_2}{\partial \mathbf{x}^2},\ \ M\_{\mathbf{x}}^{-} = R\_{M2} \frac{\partial^2 w\_1}{\partial \mathbf{x}^2} - R\_{M1} \frac{\partial^2 w\_2}{\partial \mathbf{x}^2} \tag{29}$$ $$\hat{\sigma}\_{\text{M}}\!M\_{\text{y}}^{+} = \text{R}\_{\text{M}3}\frac{\hat{\sigma}^{2}w\_{1}}{\hat{\sigma}y^{2}} - \text{R}\_{\text{M4}}\frac{\hat{\sigma}^{2}w\_{2}}{\hat{\sigma}y^{2}}\,,\;M\_{\text{y}}^{-} = \text{R}\_{\text{M4}}\frac{\hat{\sigma}^{2}w\_{1}}{\hat{\sigma}y^{2}} - \text{R}\_{\text{M3}}\frac{\hat{\sigma}^{2}w\_{2}}{\hat{\sigma}y^{2}}\,,\tag{30}$$ 3. Torsional moments $$M\_{\rm Tx}^{+} = R\_{\rm T1} \frac{\partial^2 w\_1}{\partial \mathbf{x} \partial y} - R\_{\rm T2} \frac{\partial^2 w\_2}{\partial \mathbf{x} \partial y},\ \ M\_{\rm Tx}^{-} = R\_{\rm T2} \frac{\partial^2 w\_1}{\partial \mathbf{x} \partial y} - R\_{\rm T1} \frac{\partial^2 w\_2}{\partial \mathbf{x} \partial y} \tag{31}$$ $$M\_{Ty}^{+} = R\_{T3} \frac{\partial^2 w\_1}{\partial y \partial \mathbf{x}} - R\_{T4} \frac{\partial^2 w\_2}{\partial y \partial \mathbf{x}} \text{ , }\\M\_{Ty}^{-} = R\_{T4} \frac{\partial^2 w\_1}{\partial y \partial \mathbf{x}} - R\_{T3} \frac{\partial^2 w\_2}{\partial y \partial \mathbf{x}} \text{ , }\tag{32}$$ #### 2.2 Solutions Given the 2D (two-dimensional) periodic nature of the sandwich structure as shown in Fig. 1, applying the Poisson summation formula [3,23], the wave components in the structure can be expressed by using space harmonic series, as: Fourier Transform Sound Radiation 247 ( ) ( ) ( ) <sup>2</sup> 3 1 4 2 , ,, M a R wa R wa y m ( ) ( ) ( ) <sup>2</sup> 4 1 3 2 , ,, M a R wa R wa y m Mm Mm <sup>+</sup> <sup>=</sup> − − <sup>⎡</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> (44) Mm Mm <sup>−</sup> <sup>=</sup> − − <sup>⎡</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> (45) n T ) 2 2 ( ) 1 2 ( ) <sup>+</sup> <sup>=</sup> − − <sup>⎡</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> (48) ) <sup>−</sup> <sup>=</sup> − − <sup>⎡</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> (49) ) <sup>−</sup> <sup>=</sup> − − <sup>⎡</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> (47) <sup>n</sup> ) <sup>+</sup> <sup>=</sup> − − <sup>⎡</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> (46) β β βmT m T m Rwa Rwa 3 1 ( ) 4 2 ( βmT m T m Rwa Rwa 4 1 ( ) 3 2 ( β β β β β β ββ ββ M a Rwa Rwa Tx n n T ( , ,, M a Rwa Rwa Tx n nT n T n ( , ,, M a Ty m ( , ,, M a Ty m ( , ,, The absorption of sound absorption by porous materials mainly arises from viscous drag forces and thermal exchange loss when sound penetrates through the material [19,24-26]. There exist numerous theoretical models to address these issues, while different models may be specialized to deal with different types of porous materials. For instance, Lu et al. proposed a model for high porosity cellular metallic foams with open cells [24,25,27,28] and another model for semi-open metal foams [26]. As for fibrous materials considered here, there are two main classes of models [19]. The first one models the fibrous material as an equivalent fluid with effective density and bulk modulus [22,29,30]: under the assumption of the solid fibers being a rigid skeleton, only one compression wave propagates in the airsaturated medium, which thereby is governed by the Helmholtz equation. The other one employs the more rigorous theory of Biot [31,32] with the elasticity of the skeleton taken into account, the solution of which often seeks help from the finite element method (FEM) and In view of the complexity of the proposed structural vibration model and the primary focus of the present study on sound radiation of the sandwich structure as a whole, the welldeveloped empirical expressions (i.e., equivalent fluid model) of Allard and Champoux [22] is adopted to model the acoustic pressure in fibrous absorption materials such as glass/rock wools widely used in noise absorption engineering. In terms of scholar description, these may be defined as Newtonian fluid saturated rigid frame fibrous materials, with the frame fibers randomly distributed. Although Allard and Champoux [22] called their empirical equations as the equivalent fluid model, this model is in fact based on Johnson et al.'s two phases theory [33]. It accurately accounted for the viscous forces between fluid and solid and the physical transposition in the process of sound propagation, by adopting two the fibrous material is isotropic. The equivalent fluid model has been demonstrated to be capable of providing accurate predictions of sound wave propagation across fibrous sound absorptive materials, over a wide frequency range, and hence has been widely acknowledged by the acoustic community [19,24-26]. To be more precise, the equivalent ) and the dynamic bulk modulus K( ω ) - assuming that β β β β β) β) 2.3 The acoustic pressure and fluid-structure coupling suffers from huge computational expenses. variables - the dynamic density ρ (ω αβ) 1 1 ( αβ) 2 1 ( αβ αβ 3. Fourier transforms of torsional moments $$\sum\_{m=-\infty}^{+\infty} \mathcal{S}\left(\mathbf{x} - ml\_x\right) = \frac{1}{l\_x} \sum\_{m=-\infty}^{+\infty} e^{-i\left(2m\pi/l\_x\right)\mathbf{x}}, \quad \sum\_{n=-\infty}^{+\infty} \mathcal{S}\left(y - nl\_y\right) = \frac{1}{l\_y} \sum\_{n=-\infty}^{+\infty} e^{-i\left(2n\pi/l\_y\right)\mathbf{y}}\tag{33}$$ The Fourier transform pair of a function with respect to (x, y) and (α , β) can be defined as: $$w(x,y) = \int\_{-\infty}^{+\infty} \int\_{-\alpha}^{+\alpha} \tilde{w}(\alpha, \beta) \qquad \qquad e^{i(\alpha x + \beta y)} da d\beta \tag{34}$$ $$ \tilde{w}\left(\alpha,\beta\right) = \left(\frac{1}{2\pi}\right)^2 \int\_{-\infty}^{+\infty} \int\_{-\infty}^{+\infty} w\left(x,y\right) \quad e^{-i(\alpha x + \beta y)} dx dy \tag{35} $$ Applying the Poisson summation formula and then taking the Fourier transform of Eqs. (1) and (2), one gets: $$\begin{aligned} \left[D\_1\left(\alpha^2 + \beta^2\right)^2 - m\_1\alpha^2\right] \tilde{w}\_1\left(\alpha, \beta\right) &= \frac{1}{l\_x} \sum\_m \left[\tilde{Q}\_y^+\left(\alpha\_m, \beta\right) + i\beta \tilde{M}\_y^+\left(\alpha\_m, \beta\right) + i\alpha \tilde{M}\_{Ty}^+\left(\alpha\_m, \beta\right)\right] \\ + \frac{1}{l\_y} \sum\_n \left[\tilde{Q}\_x^+\left(\alpha, \beta\_n\right) + i\alpha \tilde{M}\_x^+\left(\alpha, \beta\_n\right) + i\beta \tilde{M}\_{Tx}^+\left(\alpha, \beta\_n\right)\right] &+ \frac{q\_0}{\left(2\pi\right)^2} e^{-i\left(\alpha x\_0 + \beta y\_0\right)} - \tilde{p}\_{\rm cav}\left(\alpha, \beta, h\_1\right) \\ \left[D\_2\left(\alpha^2 + \beta^2\right)^2 - m\_2\alpha^2\right] \tilde{w}\_2\left(\alpha, \beta\right) &= -\frac{1}{l\_x} \sum\_m \left[\tilde{Q}\_y^-\left(\alpha\_m, \beta\right) + i\beta \tilde{M}\_y^-\left(\alpha\_m, \beta\right) + i\alpha \tilde{M}\_{Ty}^-\left(\alpha\_m, \beta\right)\right] \\ - \frac{1}{l\_y} \sum\_n \left[\tilde{Q}\_x^-\left(\alpha, \beta\_n\right) + i\alpha \tilde{M}\_x^-\left(\alpha, \beta\_n\right) + i\beta \tilde{M}\_{Tx}^-\left(\alpha, \beta\_n\right)\right] + \tilde{p}\_{\rm cav}\left(\alpha, \beta, h\_1 + d\right) \end{aligned} \tag{37}$$ where α m x = + α π 2 / m l , 2 / β <sup>n</sup> = β π + n ly , and the dependence of a term on the wavenumbers (α , β ) is indicated using the hat sign ∼, meaning the corresponding Fourier transform of this term. Expressions for the Fourier transform of the tensional forces, bending and torsional moments are presented below. 1. Fourier transforms of tensional forces y n $$ \tilde{Q}\_x^+\left(a,\beta\_n\right) = -R\_{Q1}\tilde{w}\_1\left(a,\beta\_n\right) + R\_{Q2}\tilde{w}\_2\left(a,\beta\_n\right) \tag{38} $$ $$ \tilde{Q}\_x^- \left( a\_\prime \beta\_n \right) = -R\_{Q2} \tilde{w}\_1 \left( a\_\prime \beta\_n \right) + R\_{Q1} \tilde{w}\_2 \left( a\_\prime \beta\_n \right) \tag{39} $$ $$ \tilde{Q}\_y^+ \left( a\_{m'} \beta \right) = -R\_{Q3} \tilde{w}\_1 \left( a\_{m'} \beta \right) + R\_{Q4} \tilde{w}\_2 \left( a\_{m'} \beta \right) \tag{40} $$ $$ \tilde{Q}\_y^- \left( a\_{m'} \beta \right) = -R\_{Q4} \tilde{w}\_1 \left( a\_{m'} \beta \right) + R\_{Q3} \tilde{w}\_2 \left( a\_{m'} \beta \right) \tag{41} $$ 2. Fourier transforms of bending moments $$\tilde{M}\_{\mathbf{x}}^{+}\left(a,\beta\_{n}\right) = -\alpha^{2}\left[R\_{M1}\tilde{w}\_{1}\left(a,\beta\_{n}\right) - R\_{M2}\tilde{w}\_{2}\left(a,\beta\_{n}\right)\right] \tag{42}$$ $$ \tilde{M}\_{\mathbf{x}}^{-}\left(\boldsymbol{a}\_{\prime}\boldsymbol{\beta}\_{n}\right) = -\alpha^{2} \left[ \boldsymbol{R}\_{M2} \tilde{\boldsymbol{w}}\_{1}\left(\boldsymbol{a}\_{\prime}\boldsymbol{\beta}\_{n}\right) - \boldsymbol{R}\_{M1} \tilde{\boldsymbol{w}}\_{2}\left(\boldsymbol{a}\_{\prime}\boldsymbol{\beta}\_{n}\right) \right] \tag{43} $$ ∑ ∑ − = , ( ) <sup>1</sup> ( ) 2 / <sup>y</sup> in l <sup>y</sup> ( ) ( ) ( ) , , ix y w x y w e dd ( ) ( ) ( ) −∞ −∞ ix y w w x y e dxdy Applying the Poisson summation formula and then taking the Fourier transform of Eqs. (1) ( ) ( ) () () () α β β αβ <sup>1</sup> , , , , , <sup>2</sup> x n x n Tx n cav αβ ( ) ( ) () () () ⎡ ⎤ + − =− + + <sup>⎡</sup> <sup>⎤</sup> ⎢ ⎥ <sup>⎣</sup> <sup>⎦</sup> ⎣ ⎦ <sup>q</sup> Q iM iM e ph <sup>l</sup> ⎡ ⎤ +− = <sup>⎡</sup> + + <sup>⎤</sup> ⎢ ⎥ <sup>⎣</sup> <sup>⎦</sup> ⎣ ⎦ x m +++ − + () () () ( ) αβ transform of this term. Expressions for the Fourier transform of the tensional forces, bending Qa Rwa Rwa xn Q nQ n ( , ,, Qa Rwa Rwa xn Q nQ n ( , ,, Qa Rwa Rwa ym Q m Q m ( , ,, Qa Rwa Rwa ym Q m Q m ( , ,, ( ) ( ) ( ) <sup>2</sup> 1 1 2 2 , ,, M a R wa R wa xn M nM n ( ) ( ) ( ) <sup>2</sup> 2 1 1 2 , ,, M a R wa R wa xn M nM n β β β β β β x m ∑ <sup>D</sup> m w Q iM iM <sup>l</sup> β + ++ + − ⎡ ⎤ <sup>D</sup> m w Q iM iM <sup>l</sup> ∑ <sup>2</sup> <sup>1</sup> , , <sup>2</sup> π ⎛ ⎞ <sup>=</sup> ⎜ ⎟ () () () <sup>1</sup> , , , ,, x n x n Tx n cav − + + ++ ⎡ ⎤ ⎣ ⎦ Q iM iM p h d <sup>l</sup> β > π ) 1 1 ( ) 2 1 ( ) 3 1 ( ) 4 1 ( αβ αβ αβ αβ 2 / m l , 2 / β <sup>n</sup> = β β β β β βα βα −−− ⎣ ⎦ α β δ +∞ +∞ <sup>+</sup> +∞ +∞ − + y n n y > α β > > α β ⎝ ⎠ ∫ ∫ (35) ( ) π <sup>1</sup> , ,, , β > αβ ) is indicated using the hat sign ∼, meaning the corresponding Fourier 0 <sup>1</sup> , ,, , y m y m Ty m α β α +++ ix y α β 1 + n ly , and the dependence of a term on the β) 2 2 ( ) <sup>+</sup> <sup>=</sup> − + (38) β) 1 2 ( ) <sup>−</sup> <sup>=</sup> − + (39) ) 4 2 ( ) <sup>+</sup> <sup>=</sup> − + (40) ) 3 2 ( ) <sup>−</sup> <sup>=</sup> − + (41) <sup>+</sup> <sup>=</sup> − − <sup>⎡</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> (42) <sup>−</sup> <sup>=</sup> − − <sup>⎡</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> (43) β β > β > β y m y m Ty m αβ −−− −∞ −∞ <sup>=</sup> ∫ ∫ (34) +∞ +∞ <sup>−</sup> =−∞ =−∞ y nl e <sup>l</sup> π ( ) ( ) 0 0 α β α β > αβ (36) (37) 2 1 α ) can be defined as: ∑ ∑ − = (33) α , β α β π ( ) <sup>1</sup> ( ) 2 / <sup>x</sup> im lx The Fourier transform pair of a function with respect to (x, y) and ( x m mx +∞ +∞ <sup>−</sup> =−∞ =−∞ δ <sup>2</sup> 22 2 1 1 1 αβ <sup>2</sup> 22 2 2 2 2 αβ m x = + α > α , β π and torsional moments are presented below. 1. Fourier transforms of tensional forces 2. Fourier transforms of bending moments β ω > α ω > α β and (2), one gets: y n α y n α wavenumbers ( where ∑ ∑ α x ml e l > α β $$ \tilde{M}\_y^+ \left( a\_{m'} \beta \right) = -\beta^2 \left[ R\_{M3} \tilde{w}\_1 \left( a\_{m'} \beta \right) - R\_{M4} \tilde{w}\_2 \left( a\_{m'} \beta \right) \right] \tag{44} $$ $$ \tilde{M}\_y^- \left( a\_{m'} \beta \right) = -\beta^2 \left[ R\_{M4} \tilde{w}\_1 \left( a\_{m'} \beta \right) - R\_{M3} \tilde{w}\_2 \left( a\_{m'} \beta \right) \right] \tag{45} $$ 3. Fourier transforms of torsional moments $$\tilde{M}\_{\text{Tx}}^{+}\left(\boldsymbol{a},\boldsymbol{\beta}\_{n}\right) = -\alpha \boldsymbol{\beta}\_{n} \left[\boldsymbol{R}\_{\text{T}1} \tilde{\boldsymbol{w}}\_{1}\left(\boldsymbol{a},\boldsymbol{\beta}\_{n}\right) - \boldsymbol{R}\_{\text{T}2} \tilde{\boldsymbol{w}}\_{2}\left(\boldsymbol{a},\boldsymbol{\beta}\_{n}\right)\right] \tag{46}$$ $$ \tilde{M}\_{\rm Tx}^{-}\left(a\_{\prime}\beta\_{n}\right) = -a\beta\_{n}\left[R\_{\rm T2}\tilde{w}\_{1}\left(a\_{\prime}\beta\_{n}\right) - R\_{\rm T1}\tilde{w}\_{2}\left(a\_{\prime}\beta\_{n}\right)\right] \tag{47} $$ $$ \tilde{M}\_{\Gamma y}^{+}\left(a\_{m'}\beta\right) = -\alpha\_m \beta \left[R\_{\Gamma 3}\tilde{w}\_1\left(a\_{m'}\beta\right) - R\_{\Gamma 4}\tilde{w}\_2\left(a\_{m'}\beta\right)\right] \tag{48} $$ $$ \tilde{M}\_{\rm Ty}^{-}\left(a\_{m},\beta\right) = -a\_{m}\beta \left[R\_{\rm T4}\tilde{w}\_{1}\left(a\_{m},\beta\right) - R\_{\rm T3}\tilde{w}\_{2}\left(a\_{m},\beta\right)\right] \tag{49} $$ #### 2.3 The acoustic pressure and fluid-structure coupling The absorption of sound absorption by porous materials mainly arises from viscous drag forces and thermal exchange loss when sound penetrates through the material [19,24-26]. There exist numerous theoretical models to address these issues, while different models may be specialized to deal with different types of porous materials. For instance, Lu et al. proposed a model for high porosity cellular metallic foams with open cells [24,25,27,28] and another model for semi-open metal foams [26]. As for fibrous materials considered here, there are two main classes of models [19]. The first one models the fibrous material as an equivalent fluid with effective density and bulk modulus [22,29,30]: under the assumption of the solid fibers being a rigid skeleton, only one compression wave propagates in the airsaturated medium, which thereby is governed by the Helmholtz equation. The other one employs the more rigorous theory of Biot [31,32] with the elasticity of the skeleton taken into account, the solution of which often seeks help from the finite element method (FEM) and suffers from huge computational expenses. In view of the complexity of the proposed structural vibration model and the primary focus of the present study on sound radiation of the sandwich structure as a whole, the welldeveloped empirical expressions (i.e., equivalent fluid model) of Allard and Champoux [22] is adopted to model the acoustic pressure in fibrous absorption materials such as glass/rock wools widely used in noise absorption engineering. In terms of scholar description, these may be defined as Newtonian fluid saturated rigid frame fibrous materials, with the frame fibers randomly distributed. Although Allard and Champoux [22] called their empirical equations as the equivalent fluid model, this model is in fact based on Johnson et al.'s two phases theory [33]. It accurately accounted for the viscous forces between fluid and solid and the physical transposition in the process of sound propagation, by adopting two variables - the dynamic density ρ (ω) and the dynamic bulk modulus K(ω) - assuming that the fibrous material is isotropic. The equivalent fluid model has been demonstrated to be capable of providing accurate predictions of sound wave propagation across fibrous sound absorptive materials, over a wide frequency range, and hence has been widely acknowledged by the acoustic community [19,24-26]. To be more precise, the equivalent Fourier Transform Sound Radiation 249 the facesheet to fibrous-material interaction, note that the stiffener separations xl and yl are generally much larger than the stiffener height d , implying that the contact surface area between the facesheet and fibrous material is much larger than that between the stiffener and fibrous material. Therefore, whilst the vibration of the facesheet is affected significantly by the fibrous material in contact, it has negligible influence on the motion of the short stiffeners. As a result, the fluid-structure coupling here only needs to consider the facesheet to fibrous-material interaction. To ensure the equality of panel velocity and fluid velocity on the panel surface, the momentum equation (i.e., continuity condition of fluid-structure 2 2 2 0 0 cav s cav k γ σρ ρ 2 1 2 α β =+− k . More specifically, the pressures acting on the upper and bottom ⎧⎡ ⎤ <sup>⎫</sup> <sup>−</sup> ⎪⎣ ⎦ <sup>⎪</sup> = − <sup>⎨</sup> <sup>⎬</sup> <sup>−</sup> ⎪+ − ⎡ ⎤ <sup>⎪</sup> ⎩ ⎭ ⎣ ⎦ α β k , , , , , 2 2 p h <sup>d</sup> ρ ω cav <sup>2</sup> <sup>2</sup> <sup>2</sup> 22 2 1 1 1 2 γ > γ d Dm w <sup>w</sup> d d p hd <sup>d</sup> ρ ω ( ) 3 3 4 4 4 2 2 2 R iR i R w R iR i α β Q M mT m Q M + ++ ⎡ ⎤ + ++ ⎣ ⎦ 3 3 = −− − ⎡ ⎤⎡ ⎤ + −− − ⎣ ⎦⎣ ⎦ α β cav γ cav cav d d h d <sup>h</sup> <sup>z</sup> ( ) ( ) ( ) ( ) αβ , cosh , , , , sinh ( ) ( ) ( ) ( ) αβ , , cosh , , , sinh Substituting Eqs. (57) and (58) into Eqs. (36) and (37), respectively, one can rewrite the 3 3 3 1 1 1 11 Q M mT m Q M nT n n 1 1 , , R iR i R w R iR i R w α β cav cav γαβ as [12]: 2 <sup>=</sup> (55) 1 1 α β h d h z + − α β > αβ > > γ > > > α β αββ ββ α γ γ γ α β (59) , α β 2 2 R w n T n (56) γ 1 1 cav cav <sup>2</sup> zh d p z w ρ ω= + ∂∂ = (54) ( ) ( ) ( ) , , 1 2 γ − − + − e e w e w ee γ ( ) () 1 2 <sup>⎡</sup> − ⎤ <sup>⎣</sup> <sup>⎦</sup> = − (57) ( ) () ρ ω <sup>⎡</sup> − ⎤ <sup>⎣</sup> <sup>⎦</sup> + =− (58) γ > γ ww d αβ > γ ( ) ( ) ⎡ ⎤ ⎣ ⎦ ∑ α α ( ) γ w dw 1 2 γαβ ( ) () ( ) ( ) () ( ) γαβ cosh , , , sinh , sinh ( ) ( ) ( ) w e w ee cav of the fibrous material is related to the complex coupling [7,10,35]) is applied: where the complex density p z where 2 2 22 governing equations as: αβ ∑ 0 2 <sup>q</sup> <sup>e</sup> ( ) π 2 + panels are given by: γαβ α β wavenumber cav k and porosity [ ] <sup>1</sup> ( ) ( )( ) γαβ cav α β cav cav α β ( ) ( ) ⎣ ⎦ ω β β ( ) 0 0 ix y α β − + 1 1 , l l x y m l l αα β x y m n ∑ ∑ αα β ρω ⎡ ⎤ ⎢ ⎥ +− − <sup>+</sup> γαβ cav cav <sup>1</sup> z h p z w ρ ω<sup>=</sup> ∂∂ = , [ ] <sup>1</sup> ρ σ Applying the Fourier transform to Eqs. (50) and (54), one obtains: γ ρ ω 1 1 fluid model is valid for most glass/rock wools for f R smaller than 1.0 kg-1m3, where f is the frequency and R is the flow resistivity of the fibrous material [22]. Generally, the flow resistivity R of typical glass/rock wools is approximately 20000 Nm/s4, and hence the equivalent fluid model works well for frequencies below 20 kHz. According to the equivalent fluid model, wave propagation in fibrous sound absorptive material (e.g., fiberglass or mineral wool) is governed by [12,13,22,34]: $$\left(\hat{\boldsymbol{\phi}}^{2}\left\langle\hat{\boldsymbol{\phi}}\mathbf{x}^{2}+\hat{\boldsymbol{\phi}}^{2}\left\langle\hat{\boldsymbol{\phi}}\mathbf{y}^{2}+\hat{\boldsymbol{\phi}}^{2}\left\langle\hat{\boldsymbol{\phi}}\mathbf{z}^{2}\right\rangle\right\rangle\mathbf{p}\_{\text{cav}}+\boldsymbol{k}\_{\text{cav}}^{2}\boldsymbol{p}\_{\text{cav}}=\mathbf{0}\tag{50}$$ where cav p is the sound pressure in the fibrous material and cav k is the corresponding complex wavenumber, which is related to the dynamic density ρ (ω) and dynamic bulk modulus K(ω) of the fibrous material by: $$k\_{\rm cav} = 2\pi f \sqrt{\rho(o)/\mathcal{K}(o)}\tag{51}$$ In accordance with the complex physical phenomena taking place in the fibrous material, such as thermal exchanges between air and fibers showing a significant transition with increasing frequency (i.e., isothermal process at low frequency turning to adiabatic process at high frequency) [22], the equivalent density and bulk modulus are both dynamic. In other words, the dynamic density and dynamic bulk modulus are frequency dependent, given by [22]: $$\rho\left(\alpha\right) = \rho\_0 \left[1 + \left(R/\rho\_0 f\right)G\_1\left(\rho\_0 f/R\right)/i2\pi\right] \tag{52}$$ $$K(\alpha) = \gamma\_s P\_0 \left( \gamma\_s - \frac{\gamma\_s - 1}{1 + \left( 1/i 8 \pi N\_{\rm pr} \right) \left( \rho\_0 f/R \right)^{-1} G\_2 \left( \rho\_0 f/R \right)} \right)^{-1} \tag{53}$$ where 1 0 ( ) ( ) <sup>0</sup> G fR i fR ρ = +1 π ρ , 20 1 0 ( ) ( ) pr G fR G fR N ρ ρ <sup>=</sup> <sup>⎡</sup> <sup>4</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> , R is the static flow resistivity of the fibrous material, <sup>s</sup> γ and ρ0 are separately the specific heat ratio (i.e., s p v γ = c c , <sup>p</sup> c and <sup>v</sup> c being the specific heat per unit mass of the air at constant pressure and constant volume) and density of air, P0 is the air equilibrium pressure and Npr is the Prandtl number. As a further understanding of physical meanings, the dynamic density ρ (ω) contains the inertial and viscous forces per unit volume of air in fibrous material, while the dynamic bulk modulus K(ω) gives the relationship between the averaged molecular displacement of air and the averaged variation of pressure. As a conclusion of Lu et al.'s model, it is found that the viscous drag forces operating at the fiber surface govern the complex density ρ (ω) and the thermal forces control the complex bulk modulus K(ω) . As seen in Eqs. (52) and (53), these two quantities is strongly dependent on the term 0 ρ f R , reflecting the inherent dynamic property of sound absorbing process and flow resistance being the fundamental origin of sound absorption. Generally, in contrast with the facesheet to stiffener interaction and the facesheet to fibrousmaterial (or air) interaction, the stiffener to fibrous-material (or air) interaction is negligible. It is easy to understand that the direct structural connection between the facesheets and the rib-stiffeners is far stronger than the stiffener to fibrous-material (or air) interaction. As for fluid model is valid for most glass/rock wools for f R smaller than 1.0 kg-1m3, where f is the frequency and R is the flow resistivity of the fibrous material [22]. Generally, the flow resistivity R of typical glass/rock wools is approximately 20000 Nm/s4, and hence the According to the equivalent fluid model, wave propagation in fibrous sound absorptive where cav p is the sound pressure in the fibrous material and cav k is the corresponding kf K cav = 2π > ρ 0 1 ρ ω As seen in Eqs. (52) and (53), these two quantities is strongly dependent on the term 0 πρ , 20 1 0 ( ) ( ) pr G fR G fR N ) contains the inertial and viscous forces per unit volume of air in fibrous material, = c c , <sup>p</sup> c and <sup>v</sup> c being the specific heat per unit mass of the air at constant pressure and constant volume) and density of air, P0 is the air equilibrium pressure and Npr is the Prandtl number. As a further understanding of physical meanings, the dynamic density molecular displacement of air and the averaged variation of pressure. As a conclusion of Lu et al.'s model, it is found that the viscous drag forces operating at the fiber surface govern reflecting the inherent dynamic property of sound absorbing process and flow resistance Generally, in contrast with the facesheet to stiffener interaction and the facesheet to fibrousmaterial (or air) interaction, the stiffener to fibrous-material (or air) interaction is negligible. It is easy to understand that the direct structural connection between the facesheets and the rib-stiffeners is far stronger than the stiffener to fibrous-material (or air) interaction. As for ⎛ ⎞ <sup>−</sup> ⎜ ⎟ = − 1 18 <sup>s</sup> K Ps s > γ and ρ ρω In accordance with the complex physical phenomena taking place in the fibrous material, such as thermal exchanges between air and fibers showing a significant transition with increasing frequency (i.e., isothermal process at low frequency turning to adiabatic process at high frequency) [22], the equivalent density and bulk modulus are both dynamic. In other words, the dynamic density and dynamic bulk modulus are frequency dependent, given by ω ρ γ <sup>+</sup> ⎝ ⎠ ( ) pr ( ) ( ) i N fR G fR ρ ) and the thermal forces control the complex bulk modulus K( 1 0 20 − π) = + 0 0 10 ⎡1 2 (R fG fR i ) ( ) ⎤ ⎣ ⎦ (52) > ρ ( ) 222 222 2 0 cav cav cav ∂ ∂ +∂ ∂ +∂ ∂ + = x y zp kp (50) ρ (ω () () (51) 1 − <sup>=</sup> <sup>⎡</sup> <sup>4</sup> <sup>⎤</sup> <sup>⎣</sup> <sup>⎦</sup> , R is the static flow 0 are separately the specific heat ratio (i.e., ) gives the relationship between the averaged ) and dynamic bulk (53) ω) . ρf R , equivalent fluid model works well for frequencies below 20 kHz. complex wavenumber, which is related to the dynamic density ) of the fibrous material by: ρ (ωρ > γ ( ) where 1 0 ( ) ( ) <sup>0</sup> G fR i fR = +1 resistivity of the fibrous material, <sup>s</sup> while the dynamic bulk modulus K( ρ (ω being the fundamental origin of sound absorption. ρ the complex density s p v γ ρ (ω ωγ π ρ modulus K( [22]: ω material (e.g., fiberglass or mineral wool) is governed by [12,13,22,34]: the facesheet to fibrous-material interaction, note that the stiffener separations xl and yl are generally much larger than the stiffener height d , implying that the contact surface area between the facesheet and fibrous material is much larger than that between the stiffener and fibrous material. Therefore, whilst the vibration of the facesheet is affected significantly by the fibrous material in contact, it has negligible influence on the motion of the short stiffeners. As a result, the fluid-structure coupling here only needs to consider the facesheet to fibrous-material interaction. To ensure the equality of panel velocity and fluid velocity on the panel surface, the momentum equation (i.e., continuity condition of fluid-structure coupling [7,10,35]) is applied: $$\left[\left\\|\mathcal{p}\_{\rm cav}/\partial\boldsymbol{z}\right\\|\_{\boldsymbol{z}=h\_{1}}=\rho\_{\rm cav}\alpha^{2}w\_{1}\,,\left[\left\\|\mathcal{p}\_{\rm cav}/\partial\boldsymbol{z}\right\\|\_{\boldsymbol{z}=h\_{1}+d}=\rho\_{\rm cav}\alpha^{2}w\_{2}\right] \tag{54}$$ where the complex density ρcav of the fibrous material is related to the complex wavenumber cav k and porosity σas [12]: $$\frac{k\_{\rm cav}^2}{k\_0^2} = \frac{\chi\_s \sigma \rho\_{\rm cav}}{\rho\_0} \tag{55}$$ Applying the Fourier transform to Eqs. (50) and (54), one obtains: $$\tilde{p}\_{\rm cav}(\alpha,\beta,z) = -\frac{\rho\_{\rm cav}\alpha^2}{\gamma\left(\alpha,\beta\right)\left(e^{\gamma d} - e^{-\gamma d}\right)} \begin{vmatrix} \left[\tilde{w}\_1(\alpha,\beta)e^{\gamma\left(h\_1+d\right)} - \tilde{w}\_2\left(\alpha,\beta\right)e^{\gamma h\_1}\right]e^{-\gamma z} \\ + \left[\tilde{w}\_1\left(\alpha,\beta\right)e^{-\gamma\left(h\_1+d\right)} - \tilde{w}\_2\left(\alpha,\beta\right)e^{-\gamma h\_1}\right]e^{\gamma z} \end{vmatrix} \tag{56}$$ where 2 2 22 cav γαβ =+− k . More specifically, the pressures acting on the upper and bottom panels are given by: $$\tilde{p}\_{cav}(a,\beta,h\_1) = -\frac{\rho\_{cav}a^2\left[\tilde{w}\_1(a,\beta)\cosh(\gamma d) - \tilde{w}\_2(a,\beta)\right]}{\gamma(a,\beta)\sinh(\gamma d)}\tag{57}$$ $$\tilde{p}\_{cav}\left(a,\beta,h\_1+d\right) = -\frac{\rho\_{cav}a^2\left[\tilde{w}\_1\left(a,\beta\right) - \tilde{w}\_2\left(a,\beta\right)\cosh\left(\gamma d\right)\right]}{\gamma\left(a,\beta\right)\sinh\left(\gamma d\right)}\tag{58}$$ Substituting Eqs. (57) and (58) into Eqs. (36) and (37), respectively, one can rewrite the governing equations as: $$\begin{aligned} & \left[ D\_1 \left( \alpha^2 + \beta^2 \right)^2 - m\_1 \alpha^2 - \frac{\rho\_{\text{un}} \rho^2 \cosh(\gamma d)}{\gamma(\alpha, \beta) \sinh(\gamma d)} \right] \tilde{w}\_1(\alpha, \beta) + \frac{\rho\_{\text{un}} \alpha^2}{\gamma(\alpha, \beta) \sinh(\gamma d)} \tilde{w}\_2(\alpha, \beta) \\ & = \frac{1}{l\_x} \sum\_m \left[ -R\_{Q3} - i \beta^3 R\_{M3} - i \alpha a\_m \beta R\_{T3} \right] \tilde{w}\_1(a\_m, \beta) + \frac{1}{l\_y} \sum\_n \left[ -R\_{Q1} - i \alpha^3 R\_{M1} - i \alpha \beta \theta\_n R\_{T1} \right] \tilde{w}\_1(a, \beta\_n) \\ & + \frac{1}{l\_x} \sum\_m \left[ R\_{Q4} + i \beta^3 R\_{M4} + i \alpha a\_m \beta R\_{T4} \right] \tilde{w}\_2(a\_m, \beta) + \frac{1}{l\_y} \sum\_n \left[ R\_{Q2} + i \alpha^3 R\_{M2} + i \alpha \beta \theta\_n R\_{T2} \right] \tilde{w}\_2(a, \beta\_n) \\ & + \frac{q\_{\text{ff}}}{\left( 2\pi \right)^2} e^{-i \left( \alpha a\_b + \beta \theta\_0 \right)} \end{aligned} \tag{59}$$ Fourier Transform Sound Radiation 251 () () ( ) ( )( ) α β , ,, , sinh , ′′ ′′ ′′ <sup>+</sup> ′′ ′′ ⎡ ⎤ <sup>⋅</sup> ⎣ ⎦ cav m n mn mn αβ αβ αβ 1 2 2 2 ( ) ( ) iR w αβ > α > β 3 2 β α n mnT m m n α m n ′ ,β α m n ′ ,β αβ , ′ ) and w <sup>2</sup> ( ′ ) and w <sup>2</sup> ( ) located at the excitation point ( <sup>0</sup> x , 0 y ), (66) ) radiated from a vibrating surface with displacement α β 0 are separately the sound speed and air density, and the α β<sup>+</sup> = − (67) α m n ′ ′ ,β α m n ′ ′ ,β (64) ) , with ) , with α,βis α β α β > α β α β w Df w <sup>d</sup> ( ) ( ) ( ) ⎡ ⎤ − + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> Q nM m n mnT m m n ∑ ∑ 4 41 4 1 <sup>1</sup> , , R iR w i R w α β 2 21 2 1 β <sup>1</sup> , , R iR w i R w α β αβ α x m m y n n y n n ( ) ( ) ( ) ⎡ ⎤ − + ′ ′ ′′ <sup>+</sup> ′ ⎢ ⎥ ⎣ ⎦ Q mM m n mnT n m n ( ) ( ) ( ) m = −∞ + ∞ to and n = −∞ ∞ to + . Insofar as the solution converges, these equations can be mm m = − ˆ ˆ to and nn n = − ˆ ˆ to (both mˆ and nˆ are positive integers), and hence can be Owing to the fluid-structure interaction of the vibrating panel (bottom panel in the present case) and its surrounding fluid, sound pressure will be radiated from the fluid-structure interface into the far field. Therefore, once the response of the bottom panel w <sup>2</sup> ( ) numerically solved, the radiated sound pressure at the far field can be obtained by ( ) ( ) ( ) ( ) <sup>0</sup> <sup>2</sup> 0 0 ik r <sup>e</sup> ix y ix y <sup>p</sup> <sup>r</sup> <sup>e</sup> w x y e dxdy ( ) ( ) ( ) ( ) <sup>2</sup> <sup>0</sup> 0 0 <sup>0</sup> ,, 2 , ik r ix y p r e re w For reference, the high frequency asymptote of far field sound pressure radiated by an + +∞ +∞ − + −∞ −∞ = − ∫ ∫ (65) > = k sin sin ϕ θ > > α β <sup>0</sup> , , , <sup>2</sup> r α β π 0 α By adopting the Fourier transform of Eq. (35), Eq. (65) becomes: θ ϕ = k cos sin ϕ θ , 0 β > πρ ω θ ϕ ⎡ ⎤ + + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> <sup>⎡</sup> <sup>⎤</sup> ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> Q m M m n mnT n m n ∑ ∑ m m ∑ ∑ 1 12 1 2 <sup>1</sup> , , R iR w i R w α β ∑ ∑ ( ) β α x 0 = numerically solved. l + + ′ 2.4 Far field sound radiated pressure the far field sound pressure p r( , , wxy ( ) , is given by [30]: where k c 0 0 = ω wavenumbers employing the established sound radiation theory. With the origin of the spherical coordinates (r, , θϕ / , 0c and unstiffened plate [3] is introduced, as: α and βare: ρω ρ θ ϕ l l γα β l 2 3 γα β m n m n β α ρ ω 3 3 3 32 R iR w 3 which contain two sets of infinite unknowns: w <sup>1</sup> ( truncated to retain one set of finite unknowns w <sup>1</sup> ( <sup>1</sup> , Q nM m $$\begin{aligned} & \left[ D\_2(\alpha^2 + \beta^2) \right]^2 - m\_2 \alpha^2 - \frac{\rho\_{\rm av} \rho^2 \cosh(\gamma d)}{\gamma(a, \beta) \sinh(\gamma d)} \Big] \tilde{w}\_2(a, \beta) + \frac{\rho\_{\rm av} \rho^2}{\gamma(a, \beta) \sinh(\gamma d)} \tilde{w}\_1(a, \beta) \\ & = -\frac{1}{l\_x} \sum\_m \left[ -R\_{Q4} - i\beta^3 R\_{M4} - i\alpha a\_m \beta R\_{T4} \right] \tilde{w}\_1(a\_m, \beta) \\ & - \frac{1}{l\_y} \sum\_n \left[ -R\_{Q2} - i\alpha^3 R\_{M2} - i\alpha \beta \theta\_n R\_{T2} \right] \tilde{w}\_1(a, \beta\_n) \\ & - \frac{1}{l\_x} \sum\_m \left[ R\_{Q3} + i\beta^3 R\_{M3} + i\alpha a\_m \beta R\_{T3} \right] \tilde{w}\_2(a\_m, \beta) \\ & - \frac{1}{l\_y} \sum\_n \left[ R\_{Q1} + i\alpha^3 R\_{M1} + i\alpha \beta \theta\_n R\_{T1} \right] \tilde{w}\_2(a, \beta\_n) \end{aligned} \tag{60}$$ To simplify the derivation procedures, the following definitions are introduced: $$f\_1(\alpha, \beta) = \left(\alpha^2 + \beta^2\right)^2 - \frac{m\_1 \alpha^2}{D\_1} - \frac{\rho\_{\alpha v} \alpha^2 \cosh(\gamma d)}{D\_1 \gamma(\alpha, \beta) \sinh(\gamma d)}\tag{61}$$ $$f\_2(\boldsymbol{a}, \boldsymbol{\beta}) = \left(\boldsymbol{\alpha}^2 + \boldsymbol{\beta}^2\right)^2 - \frac{m\_2 \boldsymbol{o}^2}{D\_2} - \frac{\rho\_{\rm cav} \boldsymbol{o}^2 \cosh(\boldsymbol{\gamma} \boldsymbol{d})}{D\_2 \boldsymbol{\gamma}(\boldsymbol{a}, \boldsymbol{\beta}) \sinh(\boldsymbol{\gamma} \boldsymbol{d})} \tag{62}$$ As seen in Eqs. (59) and (60), the panel displacements w <sup>1</sup> (α,β ) and w <sup>2</sup> (α,β ) to be solved are not independent but have coupling terms w <sup>1</sup> (α <sup>m</sup> ,β ) , w <sup>1</sup> (α,β <sup>n</sup> ) , w <sup>2</sup> ( ) α <sup>m</sup> ,β and w <sup>2</sup> (α,β <sup>n</sup> ) in the corresponding sum formula. To solve these unknowns, one needs to replace (α , β ) by (α m ′ , β n ′ ) , leading to two sets of simultaneous algebraic equations, as: ( )( ) () () ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 1 1 1 2 3 3 31 3 1 3 1 11 1 1 3 4 42 , , , , sinh , <sup>1</sup> , , <sup>1</sup> , , <sup>1</sup> , cav mn mn m n m n m n Q n M m n mnT m m n x m m Q mM m n mnT n m n y n n Q nM m x Df w w d R iR w i R w l R iR w i R w l R iR w l ρ ω α β αβ α β γα β γα β β α β αβ α α β α α β αβ β α β β α ′ ′ ′′ <sup>+</sup> ′ ′ ′′ ′′ ⎡ ⎤ <sup>⋅</sup> ⎣ ⎦ ⎡ ⎤ + + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> ⎡ ⎤ + + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> − + ′ ∑ ∑ ∑ ∑ ( ) ( ) ( ) ( ) ( ) ( ) ( ) 0 0 4 2 3 2 22 2 2 0 2 , <sup>1</sup> , , 2 m n n mnT m m n m m Q mM m n mnT n m n y n n ix y iR w R iR w i R w l <sup>q</sup> <sup>e</sup> α β β αβ α α β α α β αβ β α β π − + ′ ′ <sup>⎡</sup> <sup>⎤</sup> ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> ⎡ ⎤ − + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> = ∑ ∑ ∑ ∑ (63) d Dm w w <sup>2</sup> <sup>2</sup> <sup>2</sup> 22 2 2 2 2 1 γ > γ > > ( ) α β α β α β α β ( ) ( ) ( ) ω , inh , <sup>s</sup> ( ) ( ) ( ) ω , inh , <sup>s</sup> <sup>D</sup> <sup>f</sup> <sup>d</sup> m D <sup>D</sup> <sup>f</sup> <sup>d</sup> m D 1 1 2 2 <sup>n</sup> ) in the corresponding sum formula. To solve these unknowns, one needs to ( )( ) () () ( ) , , , , sinh , cav mn mn m n m n m n ′ ′ ′′ <sup>+</sup> ′ ′ ′′ ′′ ⎡ ⎤ <sup>⋅</sup> ⎣ ⎦ ρ ω αβ αβ αβ αβ ( ) ( ) ( ) ⎡ ⎤ + + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> Q n M m n mnT m m n ∑ ∑ 3 31 3 1 <sup>1</sup> , , R iR w i R w x m m y n n y n n 1 11 1 1 β <sup>1</sup> , , R iR w i R w α β α 1 1 1 2 Df w w γα β > α β ( ) ( ) ( ) ( ) ( ) ( ) ⎡ ⎤ − + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> <sup>⎡</sup> <sup>⎤</sup> ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> Q mM m n mnT n m n ∑ ∑ m m ∑ ∑ 2 22 2 2 <sup>1</sup> , , R iR w i R w α β ⎡ ⎤ + + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> Q mM m n mnT n m n ∑ ∑ cav cav α β ( ) ( ) ( ) () ( ) ( ) () ( ) d d γαβ ( ) () ( ) () cosh cav d =+ − − (62) γ γ γ > γ ) and w <sup>2</sup> ( d α β α β α β α β , α > β 4 2 β α n mnT m m n α,β α,β α β <sup>n</sup> ) , w <sup>2</sup> ( ) α <sup>m</sup> ,β ) to be solved (63) and cosh cav d =+ − − (61) 2 2 α,β ρω ρω α <sup>m</sup> ,β) , w <sup>1</sup> ( 2 ( ) ( ) iR w γα β ′ ) , leading to two sets of simultaneous algebraic equations, as: α β γ α β γ ρ ω > γ α β (60) cosh , , , sinh , sinh ( ) ( ) ⎡ ⎤ ⎣ ⎦ 4 4 4 1 αα β ρω ⎢ ⎥ +− − + γαβ Q M mT m Q M nT n ( ) <sup>3</sup> 1 12 M nT , <sup>n</sup> To simplify the derivation procedures, the following definitions are introduced: <sup>2</sup> <sup>2</sup> 2 2 <sup>1</sup> <sup>2</sup> <sup>2</sup> 2 2 <sup>2</sup> <sup>1</sup> , R iR i R w R iR i R w 2 2 21 αββ 3 3 3 2 α β α β αα β R iR i R w ⎡ ⎤ R i Rw + ∑⎣ ⎦ Q M mT m αββ 3 β =− − − − ⎡ ⎤ ⎣ ⎦ ω 3 − −− − ⎡ ⎤ ⎣ ⎦ 1 , 1 , 3 − ++ ⎡ ⎤ ⎣ ⎦ β α 1 2 αβ αβ As seen in Eqs. (59) and (60), the panel displacements w <sup>1</sup> ( are not independent but have coupling terms w <sup>1</sup> ( 3 β α αβ 3 3 4 42 ( ) 0 0 m n ix y − + ′ ′ α β R iR w 3 <sup>1</sup> , Q nM m ( ) β α x ( ) π 2 0 2 <sup>q</sup> <sup>e</sup> − + ′ l l = l α β l α 1 R i Q 1 w <sup>2</sup> (α,β replace ( α , β ) by (α m ′ , βn x m ∑ ∑ ∑ α β l l l y n x m − + n y l () () ( ) ( )( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 1 2 2 2 3 4 41 4 1 3 2 21 2 1 3 3 32 , ,, , sinh , <sup>1</sup> , , <sup>1</sup> , , <sup>1</sup> , cav m n mn mn m n m n Q nM m n mnT m m n x m m Q mM m n mnT n m n y n n Q nM m x w Df w <sup>d</sup> R iR w i R w l R iR w i R w l R iR w l ρ ω α β αβ αβ γα β γα β β α β αβ α α β α α β αβ β α β β α ′′ ′′ ′′ <sup>+</sup> ′′ ′′ ⎡ ⎤ <sup>⋅</sup> ⎣ ⎦ ⎡ ⎤ − + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> ⎡ ⎤ − + ′ ′ ′′ <sup>+</sup> ′ ⎢ ⎥ ⎣ ⎦ + + ′ ∑ ∑ ∑ ∑ ( ) ( ) ( ) ( ) ( ) 3 2 3 1 12 1 2 , <sup>1</sup> , , 0 n mnT m m n m m Q m M m n mnT n m n y n n iR w R iR w i R w l β αβ α α β α α β αβ β α β <sup>⎡</sup> <sup>⎤</sup> ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> ⎡ ⎤ + + ′ ′ ′′ <sup>+</sup> ′ <sup>⎢</sup> <sup>⎥</sup> <sup>⎣</sup> <sup>⎦</sup> = ∑ ∑ ∑ ∑ (64) which contain two sets of infinite unknowns: w <sup>1</sup> (α m n ′ ,β′ ) and w <sup>2</sup> (α m n ′ ′ ,β ) , with m = −∞ + ∞ to and n = −∞ ∞ to + . Insofar as the solution converges, these equations can be truncated to retain one set of finite unknowns w <sup>1</sup> (α m n ′ ,β′ ) and w <sup>2</sup> (α m n ′ ′ ,β ) , with mm m = − ˆ ˆ to and nn n = − ˆ ˆ to (both mˆ and nˆ are positive integers), and hence can be numerically solved. #### 2.4 Far field sound radiated pressure Owing to the fluid-structure interaction of the vibrating panel (bottom panel in the present case) and its surrounding fluid, sound pressure will be radiated from the fluid-structure interface into the far field. Therefore, once the response of the bottom panel w <sup>2</sup> ( ) α,β is numerically solved, the radiated sound pressure at the far field can be obtained by employing the established sound radiation theory. With the origin of the spherical coordinates (r, , θ ϕ ) located at the excitation point ( <sup>0</sup> x , 0 y ), the far field sound pressure p r( , , θ ϕ ) radiated from a vibrating surface with displacement wxy ( ) , is given by [30]: $$p\left(r,\theta,\varphi\right) = -\rho\_0 \alpha^2 \frac{e^{jk\_0r}}{2\pi r} e^{i(\alpha x\_0 + \beta y\_0)} \int\_{-\alpha}^{+\alpha} \int\_{-\alpha}^{+\alpha} w\left(x,y\right) e^{-i(\alpha x + \beta y)} dx dy\tag{65}$$ where k c 0 0 = ω / , 0c and ρ0 are separately the sound speed and air density, and the wavenumbers α and βare: $$ \alpha = k\_0 \cos \varphi \sin \theta \; \; \; \; \; \beta = k\_0 \sin \varphi \sin \theta \; \tag{66} $$ By adopting the Fourier transform of Eq. (35), Eq. (65) becomes: $$p\left(r,\theta,\phi\right) = -2\pi\rho\_0 a^2 \left(e^{ik\_0r}/r\right) e^{i(ax\_0+\beta y\_0)} \tilde{w}\left(a,\beta\right) \tag{67}$$ For reference, the high frequency asymptote of far field sound pressure radiated by an unstiffened plate [3] is introduced, as: Fourier Transform Sound Radiation 253 above mentioned criterion, the values of mˆ and nˆ are both taken as 10 (i.e., retaining 441 unknowns in the finite system) for all frequencies below 10kHz, which is sufficient to ensure Fig. 3. Convergence check of numerical solution for sound radiation of an infinite orthogonally rib-stiffened sandwich structure with stiffener separations ( xl , yl ) = (0.20m, 0.20m) when To check the validity of the proposed model, the model (simplified version) is used to calculate the sound pressure level radiated from an orthogonally rib-stiffened single panel and the predictions are compared in Fig. 4 with those obtained by Mace [3]. To degrade the present model for sandwich structures to cover rib-stiffened single panels, negligibly small thickness h ) of one face panel of the sandwich whilst the remaining system parameters are It can be seen from Fig. 4 that overall the present predictions agree excellently well with those of Mace: only slight deviations exist beyond 5000 Hz. These discrepancies in the high frequency range are expected, which can be attributed to the difference in vibration modeling of the rib-stiffeners between the present model and Mace's theory. The ribstiffeners were modeled as Euler beams in Mace's theory [3], meaning that only the bending moments and the inertial effect of the tensional forces of the rib-stiffeners are considered. In contrast, the present model accounts for all possible motions of the rib-stiffeners, including tensional forces, bending and torsional moments as well as their inertial effects. Therefore, insofar as the dynamic responses and sound radiation of rib-stiffened plates are of concern, ρand values are assigned to the prime parameters (i.e., Yong's modulus E , density the convergence and accuracy of the solution. excited by a harmonic point force at 10 kHz 3.2 Validation of theoretical modeling identical to those used by Mace [3]. $$p\_{\rm asy} = \rho\_0 q\_0 e^{ik\_0 r} \int \mathbf{2} \pi m r \tag{68}$$ The far field sound pressure radiated by the present orthogonally rib-stiffened sandwich structure with cavity absorption is then given in the form of sound pressure level (SPL) in decibel scales (dB) with respect to asy p , as: $$SPL = 10 \cdot \log\_{10} \left( p/p\_{asy} \right)^2 \tag{69}$$ #### 3. Numerical results and discussions In this section, representative examples for the on-axis (i.e., on the axis θ = ϕ = 0) far field pressure is numerically calculated to explore the sound radiation characteristics of infinite orthogonally rib-stiffened sandwich structures with fibrous material filled cavity. Note that, on the selected axis (i.e., θ = ϕ = 0), the stationary phase wavenumbers α and β are both equal to zero. The material properties and structural dimensions of the sandwich structure are taken as follows. Both the face panels and rib-stiffeners are made of aluminum, with Young's modulus E = 70 GPa, density ρ = 2700 kg/m3, Poisson ratio ν = 0.33, and loss factor η1 = η2 = 0.01. The thickness of the two face panel are 1 h = 2 h = 2 mm, and that of ribstiffeners are xt = yt = 1 mm. The depth of air cavity (i.e., height of rib connections; see Fig. 1) is d = 0.025 m. For air at normal temperature and atmospheric pressure, it is assumed that ρ0 = 1.21 kg/m3, Npr = 0.702, <sup>s</sup> γ = 1.4, P0 = 101320 N/m2 and 0c = 343 m/s. Fiberglass is selected as the cavity filling material, with porosity σ = 0.95 and flow resistivity R = 24000 Nm/s4. The time-harmonic point force with unit amplitude acts on the upper panel at location ( xl /2, yl /2). With these system parameters, the present theoretical model is used to examine the influence of fiberglass material in the cavity (partitioned by the lattice core) on the sound radiation characteristics of the sandwich structure. For comparison, the sound radiation behaviors of sandwich structures with pure air cavity (i.e., air-structure coupling effect included) and vacuum cavity (i.e., fluid-structure coupling effect ignored) are also considered. #### 3.1 Convergence check for numerical solution As previously mentioned, the infinite simultaneous algebraic equations are truncated so that one only needs to solve a finite system of equations containing a finite number of unknowns. More specifically, only M m = 2 1 ˆ + and N n = 2 1 ˆ + unknowns are retained, associated separately with subscripts m and n , leading to the same number of harmonic wave components in the x - and y -directions. Insofar as a sufficiently large number of terms are retained, the finite system is capable of ensuring the convergence and accuracy of the solution. The well acknowledged criterion [1,15] is employed, which assumes that once the solution converges at a given frequency, it converges for all lower frequencies. Therefore, the required number of unknowns is determined by the highest frequency of interest (10 kHz in the present study). To check the convergence of the solution, a numerical test is carried out by calculating the SPL at 10 kHz, with increasingly more terms used in Eqs. (63) and (64), as shown in Fig. 3. It can be seen from Fig. 3 that, when mˆ and nˆ both have a value of 10, solution convergence is ensured at 10 kHz. Consequently, following the The far field sound pressure radiated by the present orthogonally rib-stiffened sandwich structure with cavity absorption is then given in the form of sound pressure level (SPL) in pressure is numerically calculated to explore the sound radiation characteristics of infinite orthogonally rib-stiffened sandwich structures with fibrous material filled cavity. Note that, The material properties and structural dimensions of the sandwich structure are taken as follows. Both the face panels and rib-stiffeners are made of aluminum, with Young's resistivity R = 24000 Nm/s4. The time-harmonic point force with unit amplitude acts on the upper panel at location ( xl /2, yl /2). With these system parameters, the present theoretical model is used to examine the influence of fiberglass material in the cavity (partitioned by the lattice core) on the sound radiation characteristics of the sandwich structure. For comparison, the sound radiation behaviors of sandwich structures with pure air cavity (i.e., air-structure coupling effect included) and vacuum cavity (i.e., fluid-structure coupling As previously mentioned, the infinite simultaneous algebraic equations are truncated so that one only needs to solve a finite system of equations containing a finite number of unknowns. More specifically, only M m = 2 1 ˆ + and N n = 2 1 ˆ + unknowns are retained, associated separately with subscripts m and n , leading to the same number of harmonic wave components in the x - and y -directions. Insofar as a sufficiently large number of terms are retained, the finite system is capable of ensuring the convergence and accuracy of the solution. The well acknowledged criterion [1,15] is employed, which assumes that once the solution converges at a given frequency, it converges for all lower frequencies. Therefore, the required number of unknowns is determined by the highest frequency of interest (10 kHz in the present study). To check the convergence of the solution, a numerical test is carried out by calculating the SPL at 10 kHz, with increasingly more terms used in Eqs. (63) and (64), as shown in Fig. 3. It can be seen from Fig. 3 that, when mˆ and nˆ both have a value of 10, solution convergence is ensured at 10 kHz. Consequently, following the γ = 2700 kg/m3, Poisson ratio 2 = 0.01. The thickness of the two face panel are 1 h = 2 h = 2 mm, and that of ribstiffeners are xt = yt = 1 mm. The depth of air cavity (i.e., height of rib connections; see Fig. 1) is d = 0.025 m. For air at normal temperature and atmospheric pressure, it is assumed asy p = ρ In this section, representative examples for the on-axis (i.e., on the axis ρ Fiberglass is selected as the cavity filling material, with porosity decibel scales (dB) with respect to asy p , as: 3. Numerical results and discussions θ = ϕ 0 = 1.21 kg/m3, Npr = 0.702, <sup>s</sup> 3.1 Convergence check for numerical solution on the selected axis (i.e., modulus E = 70 GPa, density effect ignored) are also considered. equal to zero. ρ η1 = η that 0 0 0 <sup>2</sup> ik r π ( ) 2 = 0), the stationary phase wavenumbers q e mr (68) θ = ϕ > α and β = 0.33, and loss factor = 0.95 and flow = 0) far field are both <sup>10</sup> SPL 10 log asy = ⋅ p p (69) ν = 1.4, P0 = 101320 N/m2 and 0c = 343 m/s. σ above mentioned criterion, the values of mˆ and nˆ are both taken as 10 (i.e., retaining 441 unknowns in the finite system) for all frequencies below 10kHz, which is sufficient to ensure the convergence and accuracy of the solution. Fig. 3. Convergence check of numerical solution for sound radiation of an infinite orthogonally rib-stiffened sandwich structure with stiffener separations ( xl , yl ) = (0.20m, 0.20m) when excited by a harmonic point force at 10 kHz #### 3.2 Validation of theoretical modeling To check the validity of the proposed model, the model (simplified version) is used to calculate the sound pressure level radiated from an orthogonally rib-stiffened single panel and the predictions are compared in Fig. 4 with those obtained by Mace [3]. To degrade the present model for sandwich structures to cover rib-stiffened single panels, negligibly small values are assigned to the prime parameters (i.e., Yong's modulus E , density ρ and thickness h ) of one face panel of the sandwich whilst the remaining system parameters are identical to those used by Mace [3]. It can be seen from Fig. 4 that overall the present predictions agree excellently well with those of Mace: only slight deviations exist beyond 5000 Hz. These discrepancies in the high frequency range are expected, which can be attributed to the difference in vibration modeling of the rib-stiffeners between the present model and Mace's theory. The ribstiffeners were modeled as Euler beams in Mace's theory [3], meaning that only the bending moments and the inertial effect of the tensional forces of the rib-stiffeners are considered. In contrast, the present model accounts for all possible motions of the rib-stiffeners, including tensional forces, bending and torsional moments as well as their inertial effects. Therefore, insofar as the dynamic responses and sound radiation of rib-stiffened plates are of concern, Fourier Transform Sound Radiation 255 To a large extent, the comparisons made above may be regarded as acceptable validations for the proposed theoretical model, because all the theoretical formulations have been involved in the numerical calculation. In particular, if a theoretical model can be degraded to obtain the same results for simplified cases, its accuracy and feasibility would be better than the case when it can only give results similar to those obtained with its counterpart models. Together with the equivalent fluid model for fibrous sound absorptive materials, the present model is able to characterize the sound radiation characteristics of lightweight lattice-cored sandwich structures filled with fibrous materials, such as fiberglass considered here. Note also that the model can be degraded to describe sandwich structures with either air cavity (i.e., air-structure coupling effect included) or vacuum cavity (i.e., fluid-structure coupling effect ignored). Therefore, comparisons amongst the three different kinds of sandwiches under time-harmonic point force excitation can be performed to assess the influences of air- To better evaluate the influences of air-structure coupling effect and fibrous material, the location of point force acting on the face panel is selected at the center of one lattice cell, i.e., ( ) /2, /2 x y l l , away from the conjunction between the face panel and rib-stiffeners. The predicted sound pressure level (SPL) radiated by the three different sandwich structures is plotted in Figs. 6, 7, and 8 as a function of frequency for ( xl , yl ) = (0.20m, 0.20m), (0.35m, 0.35m) and (0.50m, 0.50m), respectively. For each pair of stiffener spacing selected, three kinds of sandwich configurations are compared: (i) vacuum cavity; (ii) air cavity; (iii) cavity Fig. 6. Sound pressure levels radiated by different orthogonally rib-stiffened sandwich structures plotted as functions of frequency for stiffener separations ( xl , yl ) = (0.20m, 0.20m) structure coupling effect and fibrous filling material on sound radiation. 3.3 Influence of air-structure coupling effect filled with fiberglass. the present model provides a more precise theoretical tool than the beam-based theory of Mace. The discrepancies between the two theories in the high frequency range of Fig. 4 just demonstrate the necessity of accurately modeling the motion of the rib-stiffeners. To further check the accuracy of the present model for the double-panel case, the model is degraded to reproduce Takahashi's results [11] for rib-stiffened double-panel structures, as shown in Fig. 5. The relevant geometrical dimensions and material property parameters are identical as those of Takahashi. Again, the model predictions fit well with Takahashi's theoretical results, with only slight divergences appearing at relatively high frequencies. These divergences are attributed to the additional consideration of inertial effects corresponding to the bending moments and torsional moments in the present model, which Takahashi did not take into account. Fig. 4. Comparison between present model predictions and those by Mace [3] for orthogonally rib-stiffened single panel excited by time-harmonic point force at location (0, 0) Fig. 5. Comparison between model predictions and theoretical results of Takahashi [11] for ribstiffened double-panel structure excited by time-harmonic point force at location (l l x y 2, 2) the present model provides a more precise theoretical tool than the beam-based theory of Mace. The discrepancies between the two theories in the high frequency range of Fig. 4 just To further check the accuracy of the present model for the double-panel case, the model is degraded to reproduce Takahashi's results [11] for rib-stiffened double-panel structures, as shown in Fig. 5. The relevant geometrical dimensions and material property parameters are identical as those of Takahashi. Again, the model predictions fit well with Takahashi's theoretical results, with only slight divergences appearing at relatively high frequencies. These divergences are attributed to the additional consideration of inertial effects corresponding to the bending moments and torsional moments in the present model, which demonstrate the necessity of accurately modeling the motion of the rib-stiffeners. Fig. 4. Comparison between present model predictions and those by Mace [3] for orthogonally rib-stiffened single panel excited by time-harmonic point force at location (0, 0) Fig. 5. Comparison between model predictions and theoretical results of Takahashi [11] for ribstiffened double-panel structure excited by time-harmonic point force at location (l l x y 2, 2) Takahashi did not take into account. To a large extent, the comparisons made above may be regarded as acceptable validations for the proposed theoretical model, because all the theoretical formulations have been involved in the numerical calculation. In particular, if a theoretical model can be degraded to obtain the same results for simplified cases, its accuracy and feasibility would be better than the case when it can only give results similar to those obtained with its counterpart models. ### 3.3 Influence of air-structure coupling effect Together with the equivalent fluid model for fibrous sound absorptive materials, the present model is able to characterize the sound radiation characteristics of lightweight lattice-cored sandwich structures filled with fibrous materials, such as fiberglass considered here. Note also that the model can be degraded to describe sandwich structures with either air cavity (i.e., air-structure coupling effect included) or vacuum cavity (i.e., fluid-structure coupling effect ignored). Therefore, comparisons amongst the three different kinds of sandwiches under time-harmonic point force excitation can be performed to assess the influences of airstructure coupling effect and fibrous filling material on sound radiation. To better evaluate the influences of air-structure coupling effect and fibrous material, the location of point force acting on the face panel is selected at the center of one lattice cell, i.e., ( ) /2, /2 x y l l , away from the conjunction between the face panel and rib-stiffeners. The predicted sound pressure level (SPL) radiated by the three different sandwich structures is plotted in Figs. 6, 7, and 8 as a function of frequency for ( xl , yl ) = (0.20m, 0.20m), (0.35m, 0.35m) and (0.50m, 0.50m), respectively. For each pair of stiffener spacing selected, three kinds of sandwich configurations are compared: (i) vacuum cavity; (ii) air cavity; (iii) cavity filled with fiberglass. Fig. 6. Sound pressure levels radiated by different orthogonally rib-stiffened sandwich structures plotted as functions of frequency for stiffener separations ( xl , yl ) = (0.20m, 0.20m) Fourier Transform Sound Radiation 257 At first glance, it can be seen from Figs. 6-8 that the air cavity case shows several additional peaks and dips on the SPL versus frequency curve. This is caused by air cavity interacting with the face panels through air-structure coupling. Besides these additional peaks and dips, it is also observed that the air-structure coupling effect plays an increasingly significant role in structure sound radiation with increasing rib-stiffener separation. This is reflected by the enlarged deviations between the two curves associated separately with the vacuum case and the air cavity case as the rib-stiffener separation is increased. In particular, when the separation is relatively large, air-structure coupling exerts a visible effect on the location of maximum sound radiation especially in low frequency range. The air-structure coupling is in effect by means of pumping effect, that is, the air cavity partitioned by the face panels and rib-stiffeners has timely changing pressure as its volume alters with the dynamic displacements of these two face panels, often imposing a converse force on the panels. In the case of rib-stiffener separation being relatively large, a considerable area of the panels is exposed to the impinging of air cavity pressure. It is thence understandable that the airstructure coupling effect may not be ignored when the rib-stiffeners are sparsely distributed. In contrast to the air cavity case, the fiberglass case exhibits almost the same trends as the vacuum one, especially when the stiffener separation is relatively small, although the discrepancies between the two cases increase as the stiffener separation is increased. Note that the air-structure coupling effect is not present in the vacuum case whilst it is eliminated in the fiberglass case (the presence of fiberglass in the cavity significantly changes the behavior of the cavity). This is also the reason why the fiberglass case exhibits almost the same trend as the vacuum one: the discrepancies between the two cases enlarging with increasing stiffener separation actually reflect the combined effect of fiberglass stiffness and It is understandable that the stiffness of the cavity-filling fiberglass reinforces the structural connection between the two face panels, enabling more vibration energies transmitted from the upper panel to the bottom one and thus causing larger sound radiation pressure levels. Conversely, fiberglass can dissipate acoustic energy via viscous drag forces and thermal exchange between the air and fibers, and hence decreases sound radiation. In addition, both the stiffness and damping of the fiberglass material are frequency dependent [17,19,22]. Consequently, the fact that the discrepancies between the vacuum and fiberglass cases The periodically distributed rib-stiffeners with relatively narrow separations restrict the deformation of fiberglass in-between, offering therefore the fiberglass a larger stiffness than that inserted between those stiffeners having wider separations. That the fiberglass case exhibits the same trend as the vacuum one when the separation is small (e.g., xl = 0.20 m and yl = 0.20 m, as shown in Fig. 6) may be attributed to the balance of the converse effects of fiberglass stiffness and damping on sound radiation. More specifically, whilst damping is dominant at low frequencies, causing decreased sound radiation in the first peak, stiffness dominates at high frequencies, resulting in increased sound radiation in the following peaks (Figs. 7 and 8). As mentioned above, the stiffness of fiberglass decreases with increasing stiffener separation. Therefore, as the separation is increased, the frequency range dominated by stiffness (i.e., stiffness-controlled region) is shifted to higher frequencies and 3.4 Influence of fibrous sound absorptive filling material increase with increasing stiffener separation can be well explained. damping on structure responses. Fig. 7. Sound pressure levels radiated by different orthogonally rib-stiffened sandwich structures plotted as functions of frequency for stiffener separations ( xl =0.35m, yl =0.35m) Fig. 8. Sound pressure levels radiated by different orthogonally rib-stiffened sandwich structures plotted as functions of frequency for stiffener separations ( xl =0.50m, yl =0.50m) Fig. 7. Sound pressure levels radiated by different orthogonally rib-stiffened sandwich structures plotted as functions of frequency for stiffener separations ( xl =0.35m, yl =0.35m) Fig. 8. Sound pressure levels radiated by different orthogonally rib-stiffened sandwich structures plotted as functions of frequency for stiffener separations ( xl =0.50m, yl =0.50m) At first glance, it can be seen from Figs. 6-8 that the air cavity case shows several additional peaks and dips on the SPL versus frequency curve. This is caused by air cavity interacting with the face panels through air-structure coupling. Besides these additional peaks and dips, it is also observed that the air-structure coupling effect plays an increasingly significant role in structure sound radiation with increasing rib-stiffener separation. This is reflected by the enlarged deviations between the two curves associated separately with the vacuum case and the air cavity case as the rib-stiffener separation is increased. In particular, when the separation is relatively large, air-structure coupling exerts a visible effect on the location of maximum sound radiation especially in low frequency range. The air-structure coupling is in effect by means of pumping effect, that is, the air cavity partitioned by the face panels and rib-stiffeners has timely changing pressure as its volume alters with the dynamic displacements of these two face panels, often imposing a converse force on the panels. In the case of rib-stiffener separation being relatively large, a considerable area of the panels is exposed to the impinging of air cavity pressure. It is thence understandable that the airstructure coupling effect may not be ignored when the rib-stiffeners are sparsely distributed. ## 3.4 Influence of fibrous sound absorptive filling material In contrast to the air cavity case, the fiberglass case exhibits almost the same trends as the vacuum one, especially when the stiffener separation is relatively small, although the discrepancies between the two cases increase as the stiffener separation is increased. Note that the air-structure coupling effect is not present in the vacuum case whilst it is eliminated in the fiberglass case (the presence of fiberglass in the cavity significantly changes the behavior of the cavity). This is also the reason why the fiberglass case exhibits almost the same trend as the vacuum one: the discrepancies between the two cases enlarging with increasing stiffener separation actually reflect the combined effect of fiberglass stiffness and damping on structure responses. It is understandable that the stiffness of the cavity-filling fiberglass reinforces the structural connection between the two face panels, enabling more vibration energies transmitted from the upper panel to the bottom one and thus causing larger sound radiation pressure levels. Conversely, fiberglass can dissipate acoustic energy via viscous drag forces and thermal exchange between the air and fibers, and hence decreases sound radiation. In addition, both the stiffness and damping of the fiberglass material are frequency dependent [17,19,22]. Consequently, the fact that the discrepancies between the vacuum and fiberglass cases increase with increasing stiffener separation can be well explained. The periodically distributed rib-stiffeners with relatively narrow separations restrict the deformation of fiberglass in-between, offering therefore the fiberglass a larger stiffness than that inserted between those stiffeners having wider separations. That the fiberglass case exhibits the same trend as the vacuum one when the separation is small (e.g., xl = 0.20 m and yl = 0.20 m, as shown in Fig. 6) may be attributed to the balance of the converse effects of fiberglass stiffness and damping on sound radiation. More specifically, whilst damping is dominant at low frequencies, causing decreased sound radiation in the first peak, stiffness dominates at high frequencies, resulting in increased sound radiation in the following peaks (Figs. 7 and 8). As mentioned above, the stiffness of fiberglass decreases with increasing stiffener separation. Therefore, as the separation is increased, the frequency range dominated by stiffness (i.e., stiffness-controlled region) is shifted to higher frequencies and Fourier Transform Sound Radiation 259 the porosity, cell size and other topological parameters of fiberglass (indirectly altering its stiffness and damping) in conjunction with stiffener separation to reduce the structure As a future research forecast, the theoretical model for sandwich composite structures considered here (i.e., square lattice-cored sandwich structures filled with fibrous materials) can be further extended to study the acoustical performance of sandwich composite structures having laminated composites as skins, since these structures have been This work is supported by the National Basic Research Program of China (2011CB6103005), the National Natural Science Foundation of China (11072188, 11102148, 10825210 and [1] Xin FX, Lu TJ, Chen CQ. Sound transmission through simply supported finite double- [2] Mace BR. Sound radiation from a plate reinforced by two sets of parallel stiffeners. [3] Mace BR. Sound radiation from fluid loaded orthogonally stiffened plates. J Sound Vib [4] Yin XW, Gu XJ, Cui HF, Shen RY. Acoustic radiation from a laminated composite plate [5] Xin FX, Lu TJ, Chen CQ. Dynamic response and acoustic radiation of double-leaf metallic panel partition under sound excitation. Comput Mater Sci 2009;46(3):728–732. [6] Maury C, Gardonio P, Elliott SJ. Active control of the flow-induced noise transmitted [7] Xin FX, Lu TJ, Chen CQ. External mean flow influence on noise transmission through [8] Xin FX, Lu TJ. Analytical and experimental investigation on transmission loss of clamped [9] Xin FX, Lu TJ, Chen CQ. Vibroacoustic behavior of clamp mounted double-panel partition with enclosure air cavity. J Acoust Soc Am 2008;124(6):3604-3612. [10] Lin G-F, Garrelick JM. Sound transmission through periodically framed parallel plates. [11] Takahashi D. Sound radiation from periodically connected double-plate structures. [12] Trochidis A, Kalaroutis A. Sound transmission through double partitions with cavity [13] Alba J, Ramis J, Sanchez-Morcillo VJ. Improvement of the prediction of transmission loss of double partitions with cavity absorption by minimization techniques. panel partitions with enclosed air cavity. ASME J Vib Acoust 2010;132(1):011008: reinforced by doubly periodic parallel stiffeners. J Sound Vib 2007;306(3-5):877-889. double panels: Implication of boundary effects. J Acoust Soc Am 2009;125(3):1506- 11021202) and the Fundamental Research Funds for the Central Universities. vibration and sound radiation to an acceptable level required in specific situations. increasingly applied in aerospace and astronautic fields. J Sound Vib 1980;71(3):435-441. through a panel. AIAA J. 2001;39(10):1860-1867. J Acoust Soc Am 1977;61(4):1014-1018. absorption. J Sound Vib 1986;107(2):321-327. J Sound Vib 1983;90(4):541-557. J Sound Vib 2004;273(4-5):793-804. double-leaf aeroelastic plates. AIAA J. 2009;47(8):1939-1951. 1981;79(3):439-452. 5. Acknowledgements 6. References 1-11. 1517. that dominated by damping (i.e., damping-controlled region) is widened. Correspondingly, in Fig. 7 the first three sound radiation peaks of the fiberglass case are lower than those of the vacuum one, and all the sound radiation peaks of the fiberglass case are significantly lower than those of the vacuum one in Fig. 8. It may thence be deduced that the fiberglass-filled cavity affects structural radiation through the combined effects of fiberglass stiffness and damping (both being frequency dependent), the balance of which is significantly influenced by stiffener separation. It is therefore possible to optimize the stiffener separation and fiberglass porosity (both indirectly related to the stiffness and damping of fiberglass) to reduce structure sound radiation to an acceptable level required in specific cases. ## 4. Conclusions The sound radiation characteristics of an infinite orthogonally rib-stiffened sandwich structure having cavity-filling fibrous sound absorptive material have been formulated by a comprehensive theoretical model when the structure is excited by a time-harmonic point force. The novelty of this work is to provide a general theoretical framework to address sound radiation issues of sandwich structures filled with fibrous sound absorptive materials, which can be degraded to deal with relatively simple structures. In the theoretical model, the vibration behaviors of the rib-stiffeners are accounted for by including all possible forces and moments exerted on the face panels by the rib-stiffeners in the governing equations. The propagation of sound in the fibrous material is modeled by adopting an equivalent fluid model with frequency dependent dynamic density and bulk modulus, with viscous drag force and thermal exchanges between air and fibers taken into account. The technique of Fourier transform is applied to solve the governing equations, resulting in an infinite set of simultaneous algebraic equations, which can be truncated and numerically solved. Numerical calculations are subsequently carried out to explore the influences of airstructure coupling effect and fibrous sound absorptive materials on structure sound radiation. The model is validated by comparing the present model predictions with previously published data, with excellent agreement achieved especially at low frequencies. Nevertheless, slight deviations emerge at high frequencies, which just demonstrate the superiority of the present model. Special attention is then focused on the effects of air-structure coupling and fibrous sound absorptive materials on sound penetration. This is explored by comparing three different sandwich structures: partitioned cavity in vacuum, filled with air, and filled with fiberglass. Interesting physical features emerging from the comparison are well interpreted by considering the combined effects of fiberglass stiffness and damping as well as the influence of rib-stiffener separation. It is found that the air-structure coupling effect induces additional peaks and dips in the SPL versus frequency curves, which plays an increasingly significant effect on structure sound radiation as the stiffener separation is increased. In particular, it is concluded that the fiberglass-filled cavity exerts its impact on wave penetration (finally on structural radiation) through the combined effects of fiberglass stiffness and damping (both frequency dependent), the balance of which is significantly affected by stiffener separation. This may provide a convenient and efficient tool to optimize that dominated by damping (i.e., damping-controlled region) is widened. Correspondingly, in Fig. 7 the first three sound radiation peaks of the fiberglass case are lower than those of the vacuum one, and all the sound radiation peaks of the fiberglass case are significantly It may thence be deduced that the fiberglass-filled cavity affects structural radiation through the combined effects of fiberglass stiffness and damping (both being frequency dependent), the balance of which is significantly influenced by stiffener separation. It is therefore possible to optimize the stiffener separation and fiberglass porosity (both indirectly related to the stiffness and damping of fiberglass) to reduce structure sound radiation to an The sound radiation characteristics of an infinite orthogonally rib-stiffened sandwich structure having cavity-filling fibrous sound absorptive material have been formulated by a comprehensive theoretical model when the structure is excited by a time-harmonic point force. The novelty of this work is to provide a general theoretical framework to address sound radiation issues of sandwich structures filled with fibrous sound absorptive materials, which can be degraded to deal with relatively simple structures. In the theoretical model, the vibration behaviors of the rib-stiffeners are accounted for by including all possible forces and moments exerted on the face panels by the rib-stiffeners in the governing equations. The propagation of sound in the fibrous material is modeled by adopting an equivalent fluid model with frequency dependent dynamic density and bulk modulus, with viscous drag force and thermal exchanges between air and fibers taken into account. The technique of Fourier transform is applied to solve the governing equations, resulting in an infinite set of simultaneous algebraic equations, which can be truncated and numerically Numerical calculations are subsequently carried out to explore the influences of airstructure coupling effect and fibrous sound absorptive materials on structure sound radiation. The model is validated by comparing the present model predictions with previously published data, with excellent agreement achieved especially at low frequencies. Nevertheless, slight deviations emerge at high frequencies, which just demonstrate the Special attention is then focused on the effects of air-structure coupling and fibrous sound absorptive materials on sound penetration. This is explored by comparing three different sandwich structures: partitioned cavity in vacuum, filled with air, and filled with fiberglass. Interesting physical features emerging from the comparison are well interpreted by considering the combined effects of fiberglass stiffness and damping as well as the influence of rib-stiffener separation. It is found that the air-structure coupling effect induces additional peaks and dips in the SPL versus frequency curves, which plays an increasingly significant effect on structure sound radiation as the stiffener separation is increased. In particular, it is concluded that the fiberglass-filled cavity exerts its impact on wave penetration (finally on structural radiation) through the combined effects of fiberglass stiffness and damping (both frequency dependent), the balance of which is significantly affected by stiffener separation. This may provide a convenient and efficient tool to optimize lower than those of the vacuum one in Fig. 8. acceptable level required in specific cases. 4. Conclusions solved. superiority of the present model. the porosity, cell size and other topological parameters of fiberglass (indirectly altering its stiffness and damping) in conjunction with stiffener separation to reduce the structure vibration and sound radiation to an acceptable level required in specific situations. As a future research forecast, the theoretical model for sandwich composite structures considered here (i.e., square lattice-cored sandwich structures filled with fibrous materials) can be further extended to study the acoustical performance of sandwich composite structures having laminated composites as skins, since these structures have been increasingly applied in aerospace and astronautic fields. ## 5. Acknowledgements This work is supported by the National Basic Research Program of China (2011CB6103005), the National Natural Science Foundation of China (11072188, 11102148, 10825210 and 11021202) and the Fundamental Research Funds for the Central Universities. ## 6. References [14] Mead DJ, Pujara KK. Space-harmonic analysis of periodically supported beams: response to convected random loading. J Sound Vib 1971;14(4):525-532. [15] Lee JH, Kim J. Analysis of sound transmission through periodically stiffened panels by space-harmonic expansion method. J Sound Vib 2002;251(2):349-366. [16] Wang J, Lu TJ, Woodhouse J, Langley RS et al. Sound transmission through lightweight double-leaf partitions: Theoretical modelling. J Sound Vib 2005;286(4-5):817-847. [17] Legault J, Atalla N. Numerical and experimental investigation of the effect of structural [18] Xin FX, Lu TJ. Analytical modeling of fluid loaded orthogonally rib-stiffened sandwich structures: Sound transmission. J Mech Phys Solids 2010;58(9):1374-1396. [19] Panneton R, Atalla N. Numerical prediction of sound transmission through finite [20] Sgard FC, Atalla N, Nicolas J. A numerical model for the low frequency diffuse field [21] Brown SM, Niedzielski J, Spalding GR. Effect of sound-absorptive facings on partition airborne-sound transmission loss. J Acoust Soc Am 1978;63(6):1851-1856. [22] Allard J-F, Champoux Y. New empirical equations for sound propagation in rigid frame [23] Rumerman ML. Vibration and wave propagation in ribbed plates. J Acoust Soc Am [24] Lu TJ, Hess A, Ashby MF. Sound absorption in metallic foams. J Appl Phys [25] Wang XL, Lu TJ. Optimized acoustic properties of cellular solids. J Acoust Soc Am [26] Lu TJ, Chen F, He D. Sound absorption of cellular metals with semiopen cells. J Acoust [27] Lu TJ, Kepets M, Dowling A. Acoustic properties of sintered FeCrAlY foams with open cells (I): Static flow resistance. Sci China Ser E: Tech Sci 2008;51(11):1803-1811. [28] Lu TJ, Kepets M, Dowling A. Acoustic properties of sintered FeCrAlY foams with open cells (II): Sound attenuation. Sci China Ser E: Tech Sci 2008;51(11):1812-1837. [31] Biot MA. Theory of propagation of elastic waves in a fluid-saturated porous solid. [32] Biot MA. Theory of propagation of elastic waves in a fluid-saturated porous solid. [33] Johnson DL, Koplik J, Dashen R. Theory of Dynamic Permeability and Tortuosity in [34] Xin FX, Lu TJ. Sound radiation of orthogonally rib-stiffened sandwich structures with [35] Xin FX, Lu TJ. Analytical modeling of sound transmission across finite aeroelastic [29] Zwikker C, Kosten CW, 1949, Sound absorbing materials, Elsevier, New York. [30] Morse PM, Ingard KU, 1968, Theoretical acoustics, McGraw-Hill, New York. I. Low-frequency range. J Acoust Soc Am 1956;28(2):168-178. II. Higher frequency range. J Acoust Soc Am 1956;28(2):179-191. Fluid-Saturated Porous-Media. J. Fluid Mech. 1987;176(379-402. cavity absorption. Compos Sci Technol 2010;70(15):2198-2206. panels in convected fluids. J Acoust Soc Am 2010;128(3):1097-1107. Vib 2009;324(3-5):712-732. 1975;57(2):370-373. 1999;85(11):7528-7539. 1999;106(2):756-765. Soc Am 2000;108(4):1697-1709. J Acoust Soc Am 2000;108(6):2865-2872. fibrous materials. J Acoust Soc Am 1992;91(6):3346-3353. links on the sound transmission of a lightweight double panel structure. J Sound multilayer systems with poroelastic materials. J Acoust Soc Am 1996;100(1):346-354. sound transmission loss of double-wall sound barriers with elastic porous linings. ## Edited by Salih Mohammed Salih The field of material analysis has seen explosive growth during the past decades. Almost all the textbooks on materials analysis have a section devoted to the Fourier transform theory. For this reason, the book focuses on the material analysis based on Fourier transform theory. The book chapters are related to FTIR and the other methods used for analyzing different types of materials. It is hoped that this book will provide the background, reference and incentive to encourage further research and results in this area as well as provide tools for practical applications. It provides an applicationsoriented approach to materials analysis written primarily for physicist, Chemists, Agriculturalists, Electrical Engineers, Mechanical Engineers, Signal Processing Engineers, and the Academic Researchers and for the Graduate Students who will also find it useful as a reference for their research activities. Photo by JaysonPhotography / iStock Fourier Transform - Materials Analysis Fourier Transform Materials Analysis	doab	2025-04-07T03:56:57.756120	1-12-2023 15:59	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "00064f14-4c6c-4123-bed8-b5ae765fbfc8", "url": "https://mts.intechopen.com/storage/books/2270/authors_book/authors_book.pdf", "author": "", "title": "Fourier Transform", "publisher": "IntechOpen", "isbn": "9789535105947", "section_idx": 3 }
0008a8ec-01ff-4139-b26e-22862621b722.0	Edited by Miroslav Radenković Gestational diabetes mellitus is defined as hyperglycemia with onset or first recognition during pregnancy. The incidence of gestational diabetes is still increasing and this pathological condition has strong association with adverse pregnancy outcomes. Since gestational diabetes can have long-term pathological consequences for both mother and the child, it is important that it is promptly recognized and adequately managed. Treatment of gestational diabetes is aimed to maintain euglycemia and it should involve regular glucose monitoring, dietary modifications, life style changes, appropriate physical activity, and when necessary, pharmacotherapy. Adequate glycemic control throughout the pregnancy can notably reduce the occurrence of specific adverse perinatal and maternal outcomes. In a longterm prospect, in order to prevent development of diabetes later in life, as well to avoid associated complications, an adequate education on lifestyle modifications should start in pregnancy and continue postpartum. ISBN 978-953-307-581-5 Gestational Diabetes Photo by 10174593\_258 / iStock	doab	2025-04-07T03:56:57.842936	20-4-2021 17:12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0008a8ec-01ff-4139-b26e-22862621b722", "url": "https://mts.intechopen.com/storage/books/337/authors_book/authors_book.pdf", "author": "", "title": "Gestational Diabetes", "publisher": "IntechOpen", "isbn": "9789533075815", "section_idx": 0 }
0008a8ec-01ff-4139-b26e-22862621b722.1	Gestational Diabetes Edited by Miroslav Radenković GESTATIONAL DIABETES Edited by Miroslav Radenković INTECHOPEN.COM	doab	2025-04-07T03:56:57.843264	20-4-2021 17:12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0008a8ec-01ff-4139-b26e-22862621b722", "url": "https://mts.intechopen.com/storage/books/337/authors_book/authors_book.pdf", "author": "", "title": "Gestational Diabetes", "publisher": "IntechOpen", "isbn": "9789533075815", "section_idx": 1 }
0008a8ec-01ff-4139-b26e-22862621b722.2	GESTATIONAL DIABETES Edited by Miroslav Radenković INTECHOPEN.COM http://dx.doi.org/10.5772/863 Edited by Miroslav Radenkovic #### Contributors Valentina Petkova, Irina Nikolova, Michelle Mottola, Stephanie-May Ruchat, Veerasamy Seshiah, Vijayam Balaji, Balaji Madhuri, Qingcheng Mao, Diana Shuster, Mary Hebert, Mohammed Shalayel, Mohammed Al-Noaemi, Salah Ahmed, William WK To, Sedigheh Soheilikhah, Zilma Silveira Nogueria Reis, Gabriel Costa Osanan, Theresa Scholl, Xinhua Chen, T. Peter Stein, Robert Steer, Miroslav Radenkovic, Harry Michael Georgiou, Gregory Edward Rice, Sebastián Illanes, Ravi Retnakaran, Leanne R De Souza, Joel G Ray, Bernard Portha, Audrey Chavey, Jamileh Movassat, Aleida M. Rivas, Melania Amorim, Leila Katz, Mostafa A. Abolfotouh, Mohammed A. Al-Rowaily, Johannes Ott, Stefanie Aust, Sara J Meltzer, Rima Alsayari, Rebeca Monroy-Torres, Jaime Naves-Sánchez, Alexander Emeakpor Omu #### © The Editor(s) and the Author(s) 2011 The moral rights of the and the author(s) have been asserted. All rights to the book as a whole are reserved by INTECH. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECH's written permission. Enquiries concerning the use of the book should be directed to INTECH rights and permissions department ([email protected]). Violations are liable to prosecution under the governing Copyright Law. Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be foundat http://www.intechopen.com/copyright-policy.html. #### Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. First published in Croatia, 2011 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia Legal deposit, Croatia: National and University Library in Zagreb Additional hard and PDF copies can be obtained from [email protected] Gestational Diabetes Edited by Miroslav Radenkovic p. cm. ISBN 978-953-307-581-5 eBook (PDF) ISBN 978-953-51-6548-4	doab	2025-04-07T03:56:57.843413	20-4-2021 17:12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0008a8ec-01ff-4139-b26e-22862621b722", "url": "https://mts.intechopen.com/storage/books/337/authors_book/authors_book.pdf", "author": "", "title": "Gestational Diabetes", "publisher": "IntechOpen", "isbn": "9789533075815", "section_idx": 2 }
0008a8ec-01ff-4139-b26e-22862621b722.3	We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists 4,000+ Open access books available 116,000+ International authors and editors 120M+ Downloads Our authors are among the Top 1% most cited scientists 12.2% Contributors from top 500 universities Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) ## Interested in publishing with us? Contact [email protected] Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com	doab	2025-04-07T03:56:57.843658	20-4-2021 17:12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0008a8ec-01ff-4139-b26e-22862621b722", "url": "https://mts.intechopen.com/storage/books/337/authors_book/authors_book.pdf", "author": "", "title": "Gestational Diabetes", "publisher": "IntechOpen", "isbn": "9789533075815", "section_idx": 3 }
0008a8ec-01ff-4139-b26e-22862621b722.4	Meet the editor Dr. Miroslav Radenković was born in 1968. He graduated at the School of Medicine – University of Belgrade in 1995, and since 1996 he is working at the Department of Pharmacology, Clinical Pharmacology and Toxicology (School of Medicine – University of Belgrade), currently in the position of the associate professor. He received an MSc, board certified in clinical pharmacology and PhD in 1999, 2000 and 2004, respectively, from the School of Medicine – University of Belgrade. Since 2002 Dr. Radenković officially participates in research activities of several scientific projects supported by the Ministry of Science – Republic of Serbia. In 2007 he was a principal investigator in the scientific project financed by the Austrian Science Fund (Lise Meitner Program) and senior postdoc at the Medical University of Vienna. In the capacity of guest researcher he worked at the University Medical Center Hamburg – Eppendorf and the University of Graz. Dr. Radenković is a member of the Austrian Pharmacological Society, Austrian Atherosclerosis Society, German Atherosclerosis Society, European Atherosclerosis Society, Bioethical Society of Serbia, and Serbian Medical Society. Contents Preface IX and Gregory E. Rice Chapter 4 Validity of Fasting Blood Glucose Chapter 1 Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus 1 Harry M. Georgiou, Sebastián Illanes López Chapter 2 Gestational Diabetes Mellitus - A Perspective 21 Chapter 3 Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 41 Melania Maria Ramos Amorim and Leila Katz Test in Screening for the Pre-Diabetes State Among Pregnant Females 61 Chapter 5 Prevalence and Risk Factors for the Development Chapter 6 Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future 91 for the Choice of Treatment 69 Valentina Petkova and Irina Nikolova Mohammed Chyad Al-Noaemi and Mohammed Helmy Faris Shalayel Sedigheh Soheilykhah Chapter 7 The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes 115 Chapter 8 Gestational Diabetes and the Metabolic Syndrome 141 Leanne R. De Souza, Joel G. Ray and Ravi Retnakaran Veerasamy Seshiah, Vijayam Balaji and Balaji Madhuri Mostafa A Abolfotouh and Mohammed A Al-Rowaily of GD in Some Eastern European Countries – Tendencies and Pharmacoeconomical Assessment ### Contents #### Preface XIII X Contents Contents VII Chapter 20 Exercise Guidelines for Women with Gestational Diabetes 339 Aleida M. Rivas Michelle F. Mottola and Stephanie-May Ruchat Chapter 21 Gestational Diabetes Mellitus After Delivery 363 #### Chapter 20 Exercise Guidelines for Women with Gestational Diabetes 339 Michelle F. Mottola and Stephanie-May Ruchat VI Contents Chapter 9 Insulin Resistance in the Third Trimester of Mohammed Helmy Faris Shalayel, Chapter 10 The Impact of Polycystic Ovarian Syndrome on Gestational Diabetes 183 Stefanie Aust and Johannes Ott Chapter 11 Impact and Mechanisms of Pancreatic Beta-Cell Chapter 12 Unravelling the Connection Between Gestational Chapter 13 Gestational Hyperglycemia, Excessive Pregnancy Chapter 14 Congenital Cardiopathies Screening Associated with Diabetes Mellitus Using Maternal Fructosamine Plasma Concentration 255 Zilma Silveira Nogueira Reis and Gabriel Costa Osanan Surveillance in Diabetic Pregnancies 263 Pragmatic Protocols for Self-Management Chapter 16 The Influence of Diet to Control the Metabolism in Gestational Diabetes Mellitus 277 Rebeca Monroy-Torres and Jaime Naves-Sanchez > and for Labour and Delivery 293 Sara J Meltzer and Rima Alsayari Chapter 19 Glyburide Disposition During Pregnancy 325 Chapter 18 Treatment Considerations for Gestational Diabetes Mellitus and Long-Term Postpartum Options 315 Diana L. Shuster, Mary F. Hebert and Qingcheng Mao Xinhua Chen, Theresa O. Scholl, Robert A. Steer and T. Peter Stein Chapter 15 Applications of Doppler Studies for Fetal Chapter 17 Insulin Use in Gestational Diabetes – Miroslav Radenković William WK To Alexander E. Omu Frcog Mass Programming by Maternal Diabetes - Insight from Animal Model Studies 201 Audrey Chavey, Jamileh Movassat and Bernard Portha Diabetes Mellitus and Butyrylcholinesterase 227 Weight Gain and Risk of Fetal Overgrowth 243 Pregnancy Suffering from Gestational Diabetes Mellitus or Impaired Glucose Tolerance 169 Mohammed Chyad Al-Noaemi and Salah A.M. Ahmed #### Chapter 21 Gestational Diabetes Mellitus After Delivery 363 Aleida M. Rivas Preface in pregnancy and continue postpartum. specialists, teachers and students. and expertise. Gestational diabetes mellitus is generally defined as hyperglycemia with onset or first recognition during pregnancy. The incidence of gestational diabetes is still increasing and this pathological condition has strong association with adverse pregnancy outcomes. Given that gestational diabetes may have long-term pathological consequences for both mother and the child, it is important that it is promptly recognized and adequately managed. Preconceptional screening and medical informing of women with pre-existing diabetes, older age, family history of gestational diabetes, metabolic syndrome, obesity, polycystic ovary syndrome or hypertension would be significant in order to reduce the risk to the fetus and mother connected to gestational diabetes. Treatment of gestational diabetes is aimed to maintain euglycemia and it should involve regular glucose monitoring, dietary modifications, life style changes, exercise program, and when necessary, pharmacotherapy. In a longterm view, in order to prevent development of diabetes later in life, as well to avoid associated complications, an adequate education on lifestyle modifications should start This book brings together 21 chapters of up-to-date information on gestational diabetes. The idea of this book is to direct the reader to comprehensively explore etiology and different aspects of prevention, screening, diagnosis, treatment, and postpartum follow-up in consideration to gestational diabetes. Furthermore, the presented facts provide a useful framework for both clinicians and basic investigators to further explore and update existing knowledge on diabetes related to pregnancy. We hope that this book will be used as a reference textbook for researchers, medical We express sincere appreciation to all the authors of the chapters for their enthusiasm Department of Pharmacology, Clinical Pharmacology and Toxicology Miroslav Radenković, MD, PhD School of Medicine, University of Belgrade Associate Professor Serbia ### Preface Gestational diabetes mellitus is generally defined as hyperglycemia with onset or first recognition during pregnancy. The incidence of gestational diabetes is still increasing and this pathological condition has strong association with adverse pregnancy outcomes. Given that gestational diabetes may have long-term pathological consequences for both mother and the child, it is important that it is promptly recognized and adequately managed. Preconceptional screening and medical informing of women with pre-existing diabetes, older age, family history of gestational diabetes, metabolic syndrome, obesity, polycystic ovary syndrome or hypertension would be significant in order to reduce the risk to the fetus and mother connected to gestational diabetes. Treatment of gestational diabetes is aimed to maintain euglycemia and it should involve regular glucose monitoring, dietary modifications, life style changes, exercise program, and when necessary, pharmacotherapy. In a longterm view, in order to prevent development of diabetes later in life, as well to avoid associated complications, an adequate education on lifestyle modifications should start in pregnancy and continue postpartum. This book brings together 21 chapters of up-to-date information on gestational diabetes. The idea of this book is to direct the reader to comprehensively explore etiology and different aspects of prevention, screening, diagnosis, treatment, and postpartum follow-up in consideration to gestational diabetes. Furthermore, the presented facts provide a useful framework for both clinicians and basic investigators to further explore and update existing knowledge on diabetes related to pregnancy. We hope that this book will be used as a reference textbook for researchers, medical specialists, teachers and students. We express sincere appreciation to all the authors of the chapters for their enthusiasm and expertise. > Miroslav Radenković, MD, PhD Associate Professor Department of Pharmacology, Clinical Pharmacology and Toxicology School of Medicine, University of Belgrade Serbia 1 1,3Australia 2Chile Novel Screening Approaches for the 3University of Queensland Centre for Clinical Research, Queensland Reproducción, Universidad de los Andes, Santiago Early Detection of Gestational Diabetes Mellitus Within the discipline of clinical obstetrics, our understanding of the aetiology of complications of pregnancy is lacking. This lack of understanding limits our ability to identify and implement efficacious management and intervention strategies to ameliorate any adverse effects on both the mother and her baby. This situation is further confounded by the lack of reliable screening test(s) to identify pre-symptomatic women who subsequently develop complications of pregnancy. Gestational diabetes mellitus (GDM), extremes of birth weight (intrauterine growth restriction (IUGR) and fetal macrosomia (FM)), preeclampsia toxaemia (PET) and preterm labour ((PTL), including preterm rupture of membranes) are the most important complications of pregnancy that have no effective With an incidence each of about 5-10% of all pregnancies, these complications are common, responsible for the majority of obstetric and paediatric morbidity and mortality, and can permanently impact on lifelong health. For example, extreme preterm birth, whether spontaneous, or iatrogenic to protect the mother or fetus from progressive disease, can result in perinatal death or serious permanent disability such as blindness, deafness or neurological injury (Chandiramani, et al., 2007). Very low birth weight is not only an immediate threat to the fetus, but can programme adult onset hypertension, stroke and diabetes (Barker, 2006). Gestational diabetes is associated with a range of perinatal morbidities including fetal macrosomia, hyperinsulinaemia and hypoglycaemia, but may also programme childhood obesity and adult onset cardiovascular disease and diabetes (Moore, 2010, Nolan, 2011); diseases with the greatest impact on health economics. Moreover, women who develop gestational diabetes are at greatly increased risk of developing type 2 diabetes in later life (Henry and Beischer, 1991, Lee, et al., 2007). Early detection of disease risk and onset is the first step in implementing efficacious treatment. If such early detection tests were available they would represent a major advance and contribution to the discipline and afford the opportunity to evaluate alternate treatment 1. Introduction 1.1 Complications of pregnancy antenatal preventative treatment. Harry M. Georgiou1, Sebastián Illanes López2 and Gregory E. Rice3 1Department of Obstetrics & Gynaecology, University of Melbourne, Victoria 2Departamento de Obstetricia & Ginecología y laboratorio de Biología de la ## Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus Harry M. Georgiou1, Sebastián Illanes López2 and Gregory E. Rice3 1Department of Obstetrics & Gynaecology, University of Melbourne, Victoria 2Departamento de Obstetricia & Ginecología y laboratorio de Biología de la Reproducción, Universidad de los Andes, Santiago 3University of Queensland Centre for Clinical Research, Queensland 1,3Australia 2Chile #### 1. Introduction #### 1.1 Complications of pregnancy Within the discipline of clinical obstetrics, our understanding of the aetiology of complications of pregnancy is lacking. This lack of understanding limits our ability to identify and implement efficacious management and intervention strategies to ameliorate any adverse effects on both the mother and her baby. This situation is further confounded by the lack of reliable screening test(s) to identify pre-symptomatic women who subsequently develop complications of pregnancy. Gestational diabetes mellitus (GDM), extremes of birth weight (intrauterine growth restriction (IUGR) and fetal macrosomia (FM)), preeclampsia toxaemia (PET) and preterm labour ((PTL), including preterm rupture of membranes) are the most important complications of pregnancy that have no effective antenatal preventative treatment. With an incidence each of about 5-10% of all pregnancies, these complications are common, responsible for the majority of obstetric and paediatric morbidity and mortality, and can permanently impact on lifelong health. For example, extreme preterm birth, whether spontaneous, or iatrogenic to protect the mother or fetus from progressive disease, can result in perinatal death or serious permanent disability such as blindness, deafness or neurological injury (Chandiramani, et al., 2007). Very low birth weight is not only an immediate threat to the fetus, but can programme adult onset hypertension, stroke and diabetes (Barker, 2006). Gestational diabetes is associated with a range of perinatal morbidities including fetal macrosomia, hyperinsulinaemia and hypoglycaemia, but may also programme childhood obesity and adult onset cardiovascular disease and diabetes (Moore, 2010, Nolan, 2011); diseases with the greatest impact on health economics. Moreover, women who develop gestational diabetes are at greatly increased risk of developing type 2 diabetes in later life (Henry and Beischer, 1991, Lee, et al., 2007). Early detection of disease risk and onset is the first step in implementing efficacious treatment. If such early detection tests were available they would represent a major advance and contribution to the discipline and afford the opportunity to evaluate alternate treatment Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus 3 collective efforts is the generation of multivariate, classification models that, at the very least, will allow the triage of early pregnant, asymptomatic women into low- and high-risk Fig. 1. The evolving healthcare system. The role of proteomics in the evolution of the healthcare system may be in the provision of multiple analyte protein and peptide profiles that facilitate risk assessment, earlier diagnosis and more effective treatment response monitoring. (Modified from "Personalised Healthcare 2010", IBM Business Consulting Services) Fig. 2. Rationale for antenatal screening. The objective of implementing an antenatal screening test is to identify pre-symptomatic women who will subsequently develop complications of pregnancy and implement efficacious treatment to reduce morbidity and mortality. Currently, complications of pregnancy are not diagnosed until mid-late gestation. cohorts (Figure 2). and clinical management strategies to improve health outcomes for both mother and baby. Based upon recent technological developments and studies, it is now realistic that clinically useful antenatal screening test(s) can be developed. Unlike diseases such as cancer where biomarkers need to be exquisitely specific, a useful antenatal screening test would ideally be highly sensitive, but not necessarily highly specific. The consequence of a false positive would be no worse than an erroneous triage to high-risk care. #### 1.2 The future of diagnostics In the context of antenatal screening, the objective of proteomic approaches is to identify proteins or peptides that are informative of the risk of pre-symptomatic early pregnant women who subsequently develop complications of pregnancy. That is, how the antecedents of complications of pregnancy alter the expression of the genome and how this is manifested as altered protein and peptide expression. Informative proteins and peptides identified may be used to develop classification models (e.g. multiple biomarker diagnostic or prognostic tests) that assign the likelihood that an individual test sample came from a normal or "at-risk" group. Such tests (as with all in vitro diagnostic medical devices) inform clinical decision-making and provide an opportunity for timely and appropriate intervention. The performance of the test (i.e. its diagnostic efficiency) determines the quality of the information provided and ultimately patient management. The application of proteomics, thus, extends beyond mapping and comparing the protein complement of healthy and at-risk individuals and needs to be considered in the context of its contribution to the healthcare system. Global health care is rapidly evolving, being driven by two processes - technological development and information management systems. Technological developments now provide opportunity to acquire complex information about patients. For example, rather than relying on a single measurement (e.g. a single biomarker diagnostic blood test) to detect disease, multiple disease markers may be simultaneously measured and combined to provide earlier and more accurate diagnosis. Information management systems are allowing such complex data to be ascribed to the individual over the course of their lifetime. The anticipated outcome of these forces is a move from the episodic, reactionary medicine of today to personalised medicine where pharmacogenetics and molecular medicine will afford the opportunity to identify predisposition to disease, risk assessment, and assign individuals to personalised, efficacious treatment/intervention groups. Both genomics and proteomics will be useful contributors to the evolving healthcare system by providing a better understanding of physiology, by defining disease risk, by enabling earlier diagnosis and by monitoring treatment responses (Figure 1). It is now widely acknowledged that single biomarkers are unlikely to deliver the significant incremental gain in sensitivity and specificity required for the development of effective screening and classification tests requisite for the implementation of personalised medicine. New approaches based upon the measurement of multiple biomarkers of disease risk afford opportunity to increase diagnostic test sensitivity and specificity. Over the past decade, the advent and optimisation of new proteomic technologies has paved the way for new strategies for the development of such multiple biomarker diagnostic tests. Our research team has applied both candidate-based and discovery-based proteomic approaches to identify biomarkers that are informative of disease risk. The ultimate objective of these and clinical management strategies to improve health outcomes for both mother and baby. Based upon recent technological developments and studies, it is now realistic that clinically useful antenatal screening test(s) can be developed. Unlike diseases such as cancer where biomarkers need to be exquisitely specific, a useful antenatal screening test would ideally be highly sensitive, but not necessarily highly specific. The consequence of a false positive In the context of antenatal screening, the objective of proteomic approaches is to identify proteins or peptides that are informative of the risk of pre-symptomatic early pregnant women who subsequently develop complications of pregnancy. That is, how the antecedents of complications of pregnancy alter the expression of the genome and how this is manifested as altered protein and peptide expression. Informative proteins and peptides identified may be used to develop classification models (e.g. multiple biomarker diagnostic or prognostic tests) that assign the likelihood that an individual test sample came from a normal or "at-risk" group. Such tests (as with all in vitro diagnostic medical devices) inform clinical decision-making and provide an opportunity for timely and appropriate intervention. The performance of the test (i.e. its diagnostic efficiency) determines the quality of the information provided and ultimately patient management. The application of proteomics, thus, extends beyond mapping and comparing the protein complement of healthy and at-risk individuals and needs to be considered in the context of its contribution Global health care is rapidly evolving, being driven by two processes - technological development and information management systems. Technological developments now provide opportunity to acquire complex information about patients. For example, rather than relying on a single measurement (e.g. a single biomarker diagnostic blood test) to detect disease, multiple disease markers may be simultaneously measured and combined to provide earlier and more accurate diagnosis. Information management systems are allowing such complex data to be ascribed to the individual over the course of their lifetime. The anticipated outcome of these forces is a move from the episodic, reactionary medicine of today to personalised medicine where pharmacogenetics and molecular medicine will afford the opportunity to identify predisposition to disease, risk assessment, and assign individuals to personalised, efficacious treatment/intervention groups. Both genomics and proteomics will be useful contributors to the evolving healthcare system by providing a better understanding of physiology, by defining disease risk, by enabling earlier diagnosis It is now widely acknowledged that single biomarkers are unlikely to deliver the significant incremental gain in sensitivity and specificity required for the development of effective screening and classification tests requisite for the implementation of personalised medicine. New approaches based upon the measurement of multiple biomarkers of disease risk afford opportunity to increase diagnostic test sensitivity and specificity. Over the past decade, the advent and optimisation of new proteomic technologies has paved the way for new strategies for the development of such multiple biomarker diagnostic tests. Our research team has applied both candidate-based and discovery-based proteomic approaches to identify biomarkers that are informative of disease risk. The ultimate objective of these would be no worse than an erroneous triage to high-risk care. 1.2 The future of diagnostics to the healthcare system. and by monitoring treatment responses (Figure 1). collective efforts is the generation of multivariate, classification models that, at the very least, will allow the triage of early pregnant, asymptomatic women into low- and high-risk cohorts (Figure 2). Fig. 1. The evolving healthcare system. The role of proteomics in the evolution of the healthcare system may be in the provision of multiple analyte protein and peptide profiles that facilitate risk assessment, earlier diagnosis and more effective treatment response monitoring. (Modified from "Personalised Healthcare 2010", IBM Business Consulting Services) Fig. 2. Rationale for antenatal screening. The objective of implementing an antenatal screening test is to identify pre-symptomatic women who will subsequently develop complications of pregnancy and implement efficacious treatment to reduce morbidity and mortality. Currently, complications of pregnancy are not diagnosed until mid-late gestation. Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus 5 Numerous GDM risk-factor assessments have been attempted during first trimester pregnancy and include, but not limited to, family history of GDM and/or diabetes (Savvidou, et al., 2010), maternal demographics (Alanis, et al., 2010, Phaloprakarn, et al., 2009, Shirazian, et al., 2009, Wein, et al., 1995), maternal pregnancy weight gain (Morisset, et al., 2011), fasting plasma glucose (Riskin-Mashiah, et al., 2009, Riskin-Mashiah, et al., 2010), one-hour glucose challenge test (Maegawa, et al., 2003, Nahum, et al., 2002, Punthumapol and Tekasakul, 2008), oral glucose tolerance test (Bhattacharya, 2004, Phaloprakarn and Tangjitgamol, 2008, Sacks, et al., 2003) and haemoglobin A1c levels (Maegawa, et al., 2003). Although some tests have provided a good negative predictive measure for subsequent GDM, most tests suffer from poor positive predictive values and therefore are of limited efficacy. It is evident that other metabolic markers that precede hyperglycaemia would need Recently, a number of first trimester studies have identified various biomarkers associated with subsequent development of GDM. In some cases these can be regarded as surrogate markers of inflammation such as C-reactive protein (Wolf, et al., 2003), of oxidative stress such as 8-isoPGF2α (Rogers, et al., 2006) or of obesity such as serum triglycerides (Nolan, et al., 1995, Son, et al., 2010) and may not necessarily be specific for impending GDM. Perhaps the more exciting studies are those that have investigated serum or plasma protein biomarkers associated with early pregnancy placental function and carbohydrate/lipid metabolism. For example, it has been shown that lower sex hormone-binding globulin (Thadhani, et al., 2003), increased placental growth factor (Ong, et al., 2004), elevated leptin concentrations (Qiu, et al., 2004), reduced plasma adiponectin concentrations (Retnakaran, et al., 2004, Williams, et al., 2004) and lower follistatin-like-3 levels (Thadhani, et al., 2010) are all risk factors for subsequent development GDM. Although the associations are compelling further investigations are warranted as it appears that none of these markers alone provide More recent studies have focused on multiple candidate-based profiling of blood-borne biomarkers to identify lead candidates for developing early pregnancy screening tests for gestational diabetes. For example, we measured multiple plasma biomarkers at 11 weeks' gestation in women who subsequently experienced a normal pregnancy outcome (n=14) and women who subsequently developed gestational diabetes (n=14) (Georgiou, et al., 2008). Of the biomarkers considered (insulin, adiponectin, leptin, resistin and glucose), receiver operator characteristic (ROC) curves for three biomarkers (adiponectin, insulin and random blood glucose) are presented together with a ROC curve based on the predicted posterior probability values (ppv) generated by a classification model that combined information from all three biomarkers (Figure 3). The combined model out performed individual biomarkers based upon the area under the ROC curve (combined model = 0.94; adiponectin = 0.867; insulin = 0.872 and glucose = 0.827). This simple example demonstrates the putative benefit of a multimarker approach for improving diagnostic efficiency. Similar multiple biomarker investigations in association with GDM early (Nanda, et al., 2011) or late (Bomba- to be identified if GDM were to be predicted from a test in early pregnancy. 3. Early screening of impending GDM adequate positive predictive values for subsequent GDM. Opon, et al., 2010, Lowe, et al., 2010) in pregnancy have been described. 3.1 Single biomarker investigations 3.2 Multiple biomarker investigations #### 2. Gestational diabetes mellitus #### 2.1 Current screening Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance of varying severity with onset or first recognition during pregnancy (Metzger and Coustan, 1998). GDM usually manifests in the latter half of pregnancy and is typically diagnosed by an oral glucose tolerance test. If GDM is diagnosed, women will usually be counselled and advised to adopt a healthy lifestyle for the duration of their pregnancy and where it is deemed appropriate, women may also be required to undergo pharmacologic or insulin therapy. During this time, increased surveillance of the pregnancy is undertaken to help ameliorate the consequent maternal and fetal morbidity associated with GDM. The clinical importance of this surveillance is highlighted by recent published findings that demonstrate that even mild hyperglycaemia over a prolonged period is associated with numerous adverse perinatal outcomes (Schafer-Graf, 2009) and that intensive intervention therapy can significantly reduce maternal and fetal morbidity (Crowther, et al., 2005, Landon, et al., 2009). The effects of hyperglycaemia on pregnancy outcome are underpinned by experimental studies that identify putative effector pathways by which exposure to glucose concentrations may alter placental and maternal adipose tissue phenotype and responsiveness (Coughlan, et al., 2001, Coughlan, et al., 2004, Coughlan, et al., 2004, Lappas, et al., 2004). The current 'gold standard' for the diagnosis of GDM is the oral glucose tolerance test (OGTT) and although there is no international consensus on the diagnostic methods or the blood glucose thresholds, it has become more widely accepted that the OGTT should be performed between 24-28 weeks' gestation using a 75 g glucose load with fasting, one-hour and two-hour venous glucose determinations (Coustan, et al., 2010, Metzger, et al., 2010). Universal screening and blood glucose thresholds remain contentious and may sometimes be based on resource availability and economic factors rather than clinical factors. Often, the more 'restrictive' blood glucose limits, leading to the diagnosis of the more severe hyperglycaemic patients, is all that can be provided in some instances. By contrast, professional associations advocate more 'inclusive' diagnostic criteria, and consequently more patients being diagnosed with less severe hyperglycaemia (Agarwal, et al., 2005, Lindsay, 2011, Metzger, et al., 2010, Ryan, 2011). With the obesity epidemic well entrenched in the Western world and with more women delaying pregnancy and the associated increase in pre-pregnancy body mass index (BMI), the incidence of GDM is increasing irrespective of the diagnostic criteria used (Kerrigan and Kingdon, 2010, Wein and Beischer, 2000). #### 2.2 The problem with current tests It is important to recognise that by the time GDM is diagnosed in the late second or early third trimester of pregnancy, the 'pathology' is probably established and that reversal of the potential adverse perinatal outcomes may be limited. Many health professionals advocate the need for an earlier diagnostic/predictive test for GDM while at the same time acknowledging that avenues for preventative treatment may be limited (Guedj, 2010). In fact, it is the lack of a reliable early test for GDM that has hampered the development of useful intervention therapies. Although a direct clinical benefit of the early diagnosis of GDM remains to be established conclusively, identification of women at greatest risk would allow triage of patients to an appropriate model of care and identify those who are at greatest need of glucose tolerance assessment (Caliskan, et al., 2004, Shirazian, et al., 2009). Numerous GDM risk-factor assessments have been attempted during first trimester pregnancy and include, but not limited to, family history of GDM and/or diabetes (Savvidou, et al., 2010), maternal demographics (Alanis, et al., 2010, Phaloprakarn, et al., 2009, Shirazian, et al., 2009, Wein, et al., 1995), maternal pregnancy weight gain (Morisset, et al., 2011), fasting plasma glucose (Riskin-Mashiah, et al., 2009, Riskin-Mashiah, et al., 2010), one-hour glucose challenge test (Maegawa, et al., 2003, Nahum, et al., 2002, Punthumapol and Tekasakul, 2008), oral glucose tolerance test (Bhattacharya, 2004, Phaloprakarn and Tangjitgamol, 2008, Sacks, et al., 2003) and haemoglobin A1c levels (Maegawa, et al., 2003). Although some tests have provided a good negative predictive measure for subsequent GDM, most tests suffer from poor positive predictive values and therefore are of limited efficacy. It is evident that other metabolic markers that precede hyperglycaemia would need to be identified if GDM were to be predicted from a test in early pregnancy. #### 3. Early screening of impending GDM #### 3.1 Single biomarker investigations 4 Gestational Diabetes Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance of varying severity with onset or first recognition during pregnancy (Metzger and Coustan, 1998). GDM usually manifests in the latter half of pregnancy and is typically diagnosed by an oral glucose tolerance test. If GDM is diagnosed, women will usually be counselled and advised to adopt a healthy lifestyle for the duration of their pregnancy and where it is deemed appropriate, women may also be required to undergo pharmacologic or insulin therapy. During this time, increased surveillance of the pregnancy is undertaken to help ameliorate the consequent maternal and fetal morbidity associated with GDM. The clinical importance of this surveillance is highlighted by recent published findings that demonstrate that even mild hyperglycaemia over a prolonged period is associated with numerous adverse perinatal outcomes (Schafer-Graf, 2009) and that intensive intervention therapy can significantly reduce maternal and fetal morbidity (Crowther, et al., 2005, Landon, et al., 2009). The effects of hyperglycaemia on pregnancy outcome are underpinned by experimental studies that identify putative effector pathways by which exposure to glucose concentrations may alter placental and maternal adipose tissue phenotype and responsiveness (Coughlan, et al., 2001, Coughlan, et al., 2004, Coughlan, et al., 2004, Lappas, The current 'gold standard' for the diagnosis of GDM is the oral glucose tolerance test (OGTT) and although there is no international consensus on the diagnostic methods or the blood glucose thresholds, it has become more widely accepted that the OGTT should be performed between 24-28 weeks' gestation using a 75 g glucose load with fasting, one-hour and two-hour venous glucose determinations (Coustan, et al., 2010, Metzger, et al., 2010). Universal screening and blood glucose thresholds remain contentious and may sometimes be based on resource availability and economic factors rather than clinical factors. Often, the more 'restrictive' blood glucose limits, leading to the diagnosis of the more severe hyperglycaemic patients, is all that can be provided in some instances. By contrast, professional associations advocate more 'inclusive' diagnostic criteria, and consequently more patients being diagnosed with less severe hyperglycaemia (Agarwal, et al., 2005, Lindsay, 2011, Metzger, et al., 2010, Ryan, 2011). With the obesity epidemic well entrenched in the Western world and with more women delaying pregnancy and the associated increase in pre-pregnancy body mass index (BMI), the incidence of GDM is increasing irrespective of the diagnostic criteria used (Kerrigan and It is important to recognise that by the time GDM is diagnosed in the late second or early third trimester of pregnancy, the 'pathology' is probably established and that reversal of the potential adverse perinatal outcomes may be limited. Many health professionals advocate the need for an earlier diagnostic/predictive test for GDM while at the same time acknowledging that avenues for preventative treatment may be limited (Guedj, 2010). In fact, it is the lack of a reliable early test for GDM that has hampered the development of useful intervention therapies. Although a direct clinical benefit of the early diagnosis of GDM remains to be established conclusively, identification of women at greatest risk would allow triage of patients to an appropriate model of care and identify those who are at greatest need of glucose tolerance assessment (Caliskan, et al., 2004, Shirazian, et al., 2009). 2. Gestational diabetes mellitus Kingdon, 2010, Wein and Beischer, 2000). 2.2 The problem with current tests 2.1 Current screening et al., 2004). Recently, a number of first trimester studies have identified various biomarkers associated with subsequent development of GDM. In some cases these can be regarded as surrogate markers of inflammation such as C-reactive protein (Wolf, et al., 2003), of oxidative stress such as 8-isoPGF2α (Rogers, et al., 2006) or of obesity such as serum triglycerides (Nolan, et al., 1995, Son, et al., 2010) and may not necessarily be specific for impending GDM. Perhaps the more exciting studies are those that have investigated serum or plasma protein biomarkers associated with early pregnancy placental function and carbohydrate/lipid metabolism. For example, it has been shown that lower sex hormone-binding globulin (Thadhani, et al., 2003), increased placental growth factor (Ong, et al., 2004), elevated leptin concentrations (Qiu, et al., 2004), reduced plasma adiponectin concentrations (Retnakaran, et al., 2004, Williams, et al., 2004) and lower follistatin-like-3 levels (Thadhani, et al., 2010) are all risk factors for subsequent development GDM. Although the associations are compelling further investigations are warranted as it appears that none of these markers alone provide adequate positive predictive values for subsequent GDM. #### 3.2 Multiple biomarker investigations More recent studies have focused on multiple candidate-based profiling of blood-borne biomarkers to identify lead candidates for developing early pregnancy screening tests for gestational diabetes. For example, we measured multiple plasma biomarkers at 11 weeks' gestation in women who subsequently experienced a normal pregnancy outcome (n=14) and women who subsequently developed gestational diabetes (n=14) (Georgiou, et al., 2008). Of the biomarkers considered (insulin, adiponectin, leptin, resistin and glucose), receiver operator characteristic (ROC) curves for three biomarkers (adiponectin, insulin and random blood glucose) are presented together with a ROC curve based on the predicted posterior probability values (ppv) generated by a classification model that combined information from all three biomarkers (Figure 3). The combined model out performed individual biomarkers based upon the area under the ROC curve (combined model = 0.94; adiponectin = 0.867; insulin = 0.872 and glucose = 0.827). This simple example demonstrates the putative benefit of a multimarker approach for improving diagnostic efficiency. Similar multiple biomarker investigations in association with GDM early (Nanda, et al., 2011) or late (Bomba-Opon, et al., 2010, Lowe, et al., 2010) in pregnancy have been described. Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus 7 centrifugation, the resultant plasma or serum was stored at -80o C for mass spectrometry peptide profiling. Both plasma and serum samples were subjected to protein dye binding depletion (Affi-gel Blue™) as previously described (Ahmed, et al., 2003) followed by solid phase peptide enrichment using hydrophilic-lipophilic balanced solid phase extraction sorbent in 96-well micro-elution plates, eluted and analysed using matrix assisted laser desorption ionisation – time-of-flight (MALDI-ToF) mass spectrometry (AutoFlex II, Bruker Daltonics). The resultant peptide profiles of plasma and serum while showing some concordance at m/z >4000, exhibited dramatically different peptide ion profiles at m/z < 2000 (Figure 4). It is likely that these ions represent peptides generated during the coagulation process and/or by the action of peptidases during the 60 min incubation at room temperature. Thus, for the purpose of primary peptidomic profiling of blood peptides, serum presents significant methodological challenges. Indeed, even different anticoagulant methods (e.g. EDTA, heparin, citrate) have been found to alter peptide ion profiles (Banks, et Fig. 4. Method of blood collection. Mass spectrometric peptidomic profiling of paired plasma and serum showing marked spectral differences. al., 2005). Fig. 3. The advantage of multiple biomarker screening. A comparison of ROC curves of the performance of individual biomarkers (adiponectin, insulin and glucose) and a combined model (ppv) to correctly classify women who subsequently developed gestational diabetes. #### 4. Proteomic approaches for early detection of GDM #### 4.1 Sample selection and processing As with all analytical techniques, sample heterogeneity (e.g. variation from individual patients, sample collection and processing) needs to be minimised for proteomic analysis. This is particularly relevant to the collection of blood, where both the method of collection and processing may dramatically alter the peptide profile (e.g. clotting, temperature and time taken to process samples). For example, while the collection of serum may be suitable for some candidate-based approaches (e.g. protein solution array and immunoassay), the peptides generated during coagulation confound peptidomic analysis. The impact of the method used to collect blood is demonstrated in Figure 4, in which paired plasma and serum samples were collected from pregnant women. Blood was either collected into EDTA or Serum Clot Activator tubes. The former was immediately centrifuged for 15 min at room temperature, while the latter was allowed to clot at room temperature for 60 min and then centrifuged for 15 min at room temperature. Following Fig. 3. The advantage of multiple biomarker screening. A comparison of ROC curves of the performance of individual biomarkers (adiponectin, insulin and glucose) and a combined model (ppv) to correctly classify women who subsequently developed gestational diabetes. As with all analytical techniques, sample heterogeneity (e.g. variation from individual patients, sample collection and processing) needs to be minimised for proteomic analysis. This is particularly relevant to the collection of blood, where both the method of collection and processing may dramatically alter the peptide profile (e.g. clotting, temperature and time taken to process samples). For example, while the collection of serum may be suitable for some candidate-based approaches (e.g. protein solution array and immunoassay), the The impact of the method used to collect blood is demonstrated in Figure 4, in which paired plasma and serum samples were collected from pregnant women. Blood was either collected into EDTA or Serum Clot Activator tubes. The former was immediately centrifuged for 15 min at room temperature, while the latter was allowed to clot at room temperature for 60 min and then centrifuged for 15 min at room temperature. Following 4. Proteomic approaches for early detection of GDM peptides generated during coagulation confound peptidomic analysis. 4.1 Sample selection and processing centrifugation, the resultant plasma or serum was stored at -80o C for mass spectrometry peptide profiling. Both plasma and serum samples were subjected to protein dye binding depletion (Affi-gel Blue™) as previously described (Ahmed, et al., 2003) followed by solid phase peptide enrichment using hydrophilic-lipophilic balanced solid phase extraction sorbent in 96-well micro-elution plates, eluted and analysed using matrix assisted laser desorption ionisation – time-of-flight (MALDI-ToF) mass spectrometry (AutoFlex II, Bruker Daltonics). The resultant peptide profiles of plasma and serum while showing some concordance at m/z >4000, exhibited dramatically different peptide ion profiles at m/z < 2000 (Figure 4). It is likely that these ions represent peptides generated during the coagulation process and/or by the action of peptidases during the 60 min incubation at room temperature. Thus, for the purpose of primary peptidomic profiling of blood peptides, serum presents significant methodological challenges. Indeed, even different anticoagulant methods (e.g. EDTA, heparin, citrate) have been found to alter peptide ion profiles (Banks, et al., 2005). Fig. 4. Method of blood collection. Mass spectrometric peptidomic profiling of paired plasma and serum showing marked spectral differences. Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus 9 Fig. 5. Variation in plasma proteins displayed during early pregnancy (6-12 weeks of gestation). Serial peripheral blood samples were collected from women and displayed using 2D-DIGE. The data presented represent normalised spot volumes for 89 protein spot that Fig. 6. Non-parametric, significance of microarray (SAM) analysis of the variation in normalised protein spot volumes during early pregnancy. Five protein spot (shown in colour) are identified as varying significantly. were common to all gels. #### 4.2 Consideration of gestational change Of particular relevance to any discussion of biomarkers for the development of antenatal screening tests is gestational variation. To be of clinical utility, any early pregnancy screening test would need to be independent of or well outside the normal early gestational changes in the subset of the proteome being interrogated. For example, in seeking to identify plasma protein biomarkers that may be of utility in identifying women at risk of developing a complication of pregnancy, it is critical to first establish the variation (both gestational and inter-patient) that occurs within the temporal window in which the test is to be applied. That is, if the objective is to identify plasma protein biomarkers using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for a test that will be applied between 6-12 weeks of gestation then the variation that occurs in the subset of proteins being screened must be established. To assess the early-pregnancy variation that plasma proteins display by 2D electrophoresis, we completed an initial study that begins to define the gestational variation in a subset of plasma proteins. Weekly peripheral blood samples were collected from women from 6-12 weeks of pregnancy. Plasma samples were immuno-depleted of high-abundance proteins (IgY14 column, Sigma) and then labelled with fluorescent CyDyes (Cy3, Cy5) for pair-wise comparison (GE Healthcare). A pooled plasma sample was labelled with Cy2 for normalisation across gels. Labelled proteins were pooled and then separated in the first dimension (24 cm Immobiline™ Dry- Strips, pH 3-11NL) and then in the second dimension (12.5% 24 cm hand-cast acrylamide gels with low fluorescent glass). Gel were imaged using a Typhoon Trio 9100 (GE Healthcare) and then analysed using Progenesis SameSpots software (v3.2.3107.24565, Nonlinear Dynamics). The analysis focussed on spots with a greater than 1.5 fold difference. Protein spots that were common to all gels (n=89) were further investigated. Figure 5 presents a box-plot summary of the gestational variation in these proteins for one patient. To identify protein spots that varied significantly across gestation and accounting for 'false discovery rates', the combined data set was subjected to nonparametric analysis methodology using Significance of Microarray (SAM) analysis. Using a false discovery rate of 1%, 5 protein spots were identified that varied significantly during 6-12 weeks of pregnancy (Figure 6). #### 4.3 Candidate-based profiling approaches (solution array workflow) Protein and antibody arrays and multiple immunoassay methodologies represent examples of candidate or targeted proteomic approaches. The advantages of these approaches include: rapid, high throughput screening of known targets and quantitative endpoints. Multiplex protein solution array is one application that represents a generation of antibody-based detection technology that allows the simultaneous quantification of multiple analytes in a single, small volume sample. Multiplex protein solution array has a number of advantages over current analyte quantification technologies, including: measurement of many biomarkers (up to 100 different analytes) in a single sample; wider operational dynamic range; and increased sensitivity and specificity derived from multivariate modelling of combinations of biomarker analytes. This system utilises a sandwich ELISA-like protocol, in which capture antibodies are coupled to spectrally distinct polystyrene or metal beads (5-6 μm diameter). Biotinylated sandwich antibody and streptavidin-phycoerytherin (PE) fluorophore are used as a reporter complex. Assays are conducted in 96-well filter-bottom Of particular relevance to any discussion of biomarkers for the development of antenatal screening tests is gestational variation. To be of clinical utility, any early pregnancy screening test would need to be independent of or well outside the normal early gestational changes in the subset of the proteome being interrogated. For example, in seeking to identify plasma protein biomarkers that may be of utility in identifying women at risk of developing a complication of pregnancy, it is critical to first establish the variation (both gestational and inter-patient) that occurs within the temporal window in which the test is to be applied. That is, if the objective is to identify plasma protein biomarkers using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for a test that will be applied between 6-12 weeks of gestation then the variation that occurs in the subset of proteins being screened To assess the early-pregnancy variation that plasma proteins display by 2D electrophoresis, we completed an initial study that begins to define the gestational variation in a subset of plasma proteins. Weekly peripheral blood samples were collected from women from 6-12 weeks of pregnancy. Plasma samples were immuno-depleted of high-abundance proteins (IgY14 column, Sigma) and then labelled with fluorescent CyDyes (Cy3, Cy5) for pair-wise comparison (GE Healthcare). A pooled plasma sample was labelled with Cy2 for normalisation across gels. Labelled proteins were pooled and then separated in the first dimension (24 cm Immobiline™ Dry- Strips, pH 3-11NL) and then in the second dimension (12.5% 24 cm hand-cast acrylamide gels with low fluorescent glass). Gel were imaged using a Typhoon Trio 9100 (GE Healthcare) and then analysed using Progenesis SameSpots software (v3.2.3107.24565, Nonlinear Dynamics). The analysis focussed on spots with a greater than 1.5 fold difference. Protein spots that were common to all gels (n=89) were further investigated. Figure 5 presents a box-plot summary of the gestational variation in these proteins for one patient. To identify protein spots that varied significantly across gestation and accounting for 'false discovery rates', the combined data set was subjected to nonparametric analysis methodology using Significance of Microarray (SAM) analysis. Using a false discovery rate of 1%, 5 protein spots were identified that varied significantly 4.2 Consideration of gestational change during 6-12 weeks of pregnancy (Figure 6). 4.3 Candidate-based profiling approaches (solution array workflow) Protein and antibody arrays and multiple immunoassay methodologies represent examples of candidate or targeted proteomic approaches. The advantages of these approaches include: rapid, high throughput screening of known targets and quantitative endpoints. Multiplex protein solution array is one application that represents a generation of antibody-based detection technology that allows the simultaneous quantification of multiple analytes in a single, small volume sample. Multiplex protein solution array has a number of advantages over current analyte quantification technologies, including: measurement of many biomarkers (up to 100 different analytes) in a single sample; wider operational dynamic range; and increased sensitivity and specificity derived from multivariate modelling of combinations of biomarker analytes. This system utilises a sandwich ELISA-like protocol, in which capture antibodies are coupled to spectrally distinct polystyrene or metal beads (5-6 μm diameter). Biotinylated sandwich antibody and streptavidin-phycoerytherin (PE) fluorophore are used as a reporter complex. Assays are conducted in 96-well filter-bottom must be established. Fig. 5. Variation in plasma proteins displayed during early pregnancy (6-12 weeks of gestation). Serial peripheral blood samples were collected from women and displayed using 2D-DIGE. The data presented represent normalised spot volumes for 89 protein spot that were common to all gels. Fig. 6. Non-parametric, significance of microarray (SAM) analysis of the variation in normalised protein spot volumes during early pregnancy. Five protein spot (shown in colour) are identified as varying significantly. Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus 11 Bio-Rad Laboratories). Using this approach more than 600 protein spots were visualised. Of these up to 20 proteins were significantly differentially expressed in pre-symptomatic women. Some of these protein spots are unique to pre-GDM (12 weeks' gestation) while others are also differentially expressed during overt disease (28 weeks' gestation). In some cases only specific isomers of a particular protein were differentially expressed (Figure 7). The limitations of this methodology include (i) time consuming and sometimes unreliable matching of hundreds of spots in multiple gels, (ii) problems associated with spot normalisation, (iii) limited in-built statistical capacity of software to compare protein abundance, (iv) difficulty with excision of spots especially in small gel formats, and (v) the failure to reliably characterise proteins by MALDI-ToF mass spectrometry due to low protein abundance. This necessitates the need to scale-up methods for protein Fig. 7. 2D-PAGE Gaussian image of human plasma taken at approximately 12 weeks' gestation. Boxes indicate protein spots that were significantly differentially expressed in women who subsequently developed GDM compared to gestation-matched women who Some of the limitations of gel-based approaches have been overcome with the development of difference gel electrophoresis. This minimal labelling approach using fluorescent cyanine dyes increases throughput by reducing sample processing and both gel-to-gel and analytical variation by combining case and control samples into a single processing step, and by the characterisation (orthogonal identification). had a normal pregnancy. 4.4.2 2-Difference Gel Electrophoresis (2D-DIGE) plates and beads are washed by vacuum filtration. Bead identity and analyte-specific fluorescence are assessed using a flow cytometre (Luminex) fitted with dual lasers. Solution array offers excellent reproducibility (CV <10%) and analyte quantification and has the capacity to multiplex up to 100 different analytes in small sample volumes (e.g. 50-100 μl plasma). Various manufacturers have now produced a myriad of multiplex assay kits consisting of premixed panels of biologically related biomarkers (e.g. cytokines/chemokines, endocrine hormones, matrix metalloproteinases, phosphoproteins etc) or disease-related panels (e.g. cancer markers, autoimmunity biomarkers and more recently, diabetes biomarkers). All suppliers also offer single-plex biomarkers that can be custom mixed to produce any panel of choice. We have previously used cytokine/chemokine multiplex panels to investigate pregnancy related complications such as GDM (Georgiou, et al., 2008), intrauterine growth restriction (Georgiou, et al., 2011) and preterm prelabour rupture of membranes (Hodges, et al., 2010) as well as ovarian cancer (Edgell, et al., 2010). Although the differentially expressed biomarkers may only be associative rather than causative of disease, these and other similar studies highlight the advantages of multiple biomarker screening for improved diagnostic/predictive modelling. An important consideration for any method that utilises multiple analyte determination is the appropriate control for the false discovery rate when multiple comparisons (hypotheses) are being tested. #### 4.4 Gel-based profiling approaches Gel-based platforms such as 1-dimensional and 2-dimensional polyacrylamide gel electrophoresis (1D or 2D-PAGE) and fluorescence 2D difference gel electrophoresis (2D-DIGE) have been used in both expression and comparative studies to define plasma protein abundance and disease-associated or treatment-induced changes. The advantage of these approaches resides in their ability to identify post-translational modified protein isoforms. The limitation of gel-based systems is their relatively low throughput, the necessity for sample processing and fractionation prior to display and limited mass range (~10-200 kDa). In addition, procedural protein losses and the overall experimental variation in estimating endpoints by 2D-PAGE may be considerable. Procedural losses of proteins during 2D-PAGE display have been reported to be as high as 80% but this can vary depending on the starting protein load (Zhou, et al., 2005). As with any other technique, variation is apportioned between technical replication, both within assay and between assay, and biologic variation (i.e. sample-to-sample). Estimates of the variation attributable to technical replication average 25- 40%. Biological variation has been estimated to be between 24 and 70% (Molloy, et al., 2003). #### 4.4.1 2-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) Using a traditional 2D-PAGE approach, we analysed the maternal plasma proteome from women with a normal pregnancy and compared this with women who subsequently developed GDM. Plasma samples were obtained at approximately 12 weeks' (pre-GDM) and 28 weeks' gestation (overt GDM) and gestation-matched with an equal number of normal controls. Individual plasma samples were depleted of high abundance proteins (albumin and immunoglobulins) by matrix binding centrifugation (Affi-gel Blue and Affigel Protein A respectively), solubilised in a multiple chaotrope buffer and focused on 11 cm, pH4-7 immobilised pH gradient strips. Second dimension electrophoresis was performed on 10% polyacrylamide gels and proteins visualised with Sypro Ruby staining. Protein spots were matched and relative abundance was determined using PD-Quest software (v7.3.1, plates and beads are washed by vacuum filtration. Bead identity and analyte-specific fluorescence are assessed using a flow cytometre (Luminex) fitted with dual lasers. Solution array offers excellent reproducibility (CV <10%) and analyte quantification and has the capacity to multiplex up to 100 different analytes in small sample volumes (e.g. 50-100 μl Various manufacturers have now produced a myriad of multiplex assay kits consisting of premixed panels of biologically related biomarkers (e.g. cytokines/chemokines, endocrine hormones, matrix metalloproteinases, phosphoproteins etc) or disease-related panels (e.g. cancer markers, autoimmunity biomarkers and more recently, diabetes biomarkers). All suppliers also offer single-plex biomarkers that can be custom mixed to produce any panel of choice. We have previously used cytokine/chemokine multiplex panels to investigate pregnancy related complications such as GDM (Georgiou, et al., 2008), intrauterine growth restriction (Georgiou, et al., 2011) and preterm prelabour rupture of membranes (Hodges, et al., 2010) as well as ovarian cancer (Edgell, et al., 2010). Although the differentially expressed biomarkers may only be associative rather than causative of disease, these and other similar studies highlight the advantages of multiple biomarker screening for improved diagnostic/predictive modelling. An important consideration for any method that utilises multiple analyte determination is the appropriate control for the false discovery rate when Gel-based platforms such as 1-dimensional and 2-dimensional polyacrylamide gel electrophoresis (1D or 2D-PAGE) and fluorescence 2D difference gel electrophoresis (2D-DIGE) have been used in both expression and comparative studies to define plasma protein abundance and disease-associated or treatment-induced changes. The advantage of these approaches resides in their ability to identify post-translational modified protein isoforms. The limitation of gel-based systems is their relatively low throughput, the necessity for sample processing and fractionation prior to display and limited mass range (~10-200 kDa). In addition, procedural protein losses and the overall experimental variation in estimating endpoints by 2D-PAGE may be considerable. Procedural losses of proteins during 2D-PAGE display have been reported to be as high as 80% but this can vary depending on the starting protein load (Zhou, et al., 2005). As with any other technique, variation is apportioned between technical replication, both within assay and between assay, and biologic variation (i.e. sample-to-sample). Estimates of the variation attributable to technical replication average 25- 40%. Biological variation has been estimated to be between 24 and 70% (Molloy, et al., 2003). Using a traditional 2D-PAGE approach, we analysed the maternal plasma proteome from women with a normal pregnancy and compared this with women who subsequently developed GDM. Plasma samples were obtained at approximately 12 weeks' (pre-GDM) and 28 weeks' gestation (overt GDM) and gestation-matched with an equal number of normal controls. Individual plasma samples were depleted of high abundance proteins (albumin and immunoglobulins) by matrix binding centrifugation (Affi-gel Blue and Affigel Protein A respectively), solubilised in a multiple chaotrope buffer and focused on 11 cm, pH4-7 immobilised pH gradient strips. Second dimension electrophoresis was performed on 10% polyacrylamide gels and proteins visualised with Sypro Ruby staining. Protein spots were matched and relative abundance was determined using PD-Quest software (v7.3.1, 4.4.1 2-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) multiple comparisons (hypotheses) are being tested. 4.4 Gel-based profiling approaches plasma). Bio-Rad Laboratories). Using this approach more than 600 protein spots were visualised. Of these up to 20 proteins were significantly differentially expressed in pre-symptomatic women. Some of these protein spots are unique to pre-GDM (12 weeks' gestation) while others are also differentially expressed during overt disease (28 weeks' gestation). In some cases only specific isomers of a particular protein were differentially expressed (Figure 7). The limitations of this methodology include (i) time consuming and sometimes unreliable matching of hundreds of spots in multiple gels, (ii) problems associated with spot normalisation, (iii) limited in-built statistical capacity of software to compare protein abundance, (iv) difficulty with excision of spots especially in small gel formats, and (v) the failure to reliably characterise proteins by MALDI-ToF mass spectrometry due to low protein abundance. This necessitates the need to scale-up methods for protein characterisation (orthogonal identification). Fig. 7. 2D-PAGE Gaussian image of human plasma taken at approximately 12 weeks' gestation. Boxes indicate protein spots that were significantly differentially expressed in women who subsequently developed GDM compared to gestation-matched women who had a normal pregnancy. #### 4.4.2 2-Difference Gel Electrophoresis (2D-DIGE) Some of the limitations of gel-based approaches have been overcome with the development of difference gel electrophoresis. This minimal labelling approach using fluorescent cyanine dyes increases throughput by reducing sample processing and both gel-to-gel and analytical variation by combining case and control samples into a single processing step, and by the Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus 13 normal and disease). Based upon the analysis of a training sample set (e.g. disease-free patients), pattern recognition software and multivariate modelling are employed to build peptide profiles or motifs that characterise a disease-free condition. Once established, such reference profiles may be used as a template to detect variance and thus deliver a diagnosis or predictive capacity. Two of the mass spectrometry-based profiling approaches we have Matrix-affinity peptide capture coupled with mass spectrometry is a discovery-based tool for comparing peptide mass fingerprints between individual or groups of samples. Numerous 'magnetic bead capture' chemistries are available including metal affinity (Cu, Fe), cationic exchange and hydrophobic reverse phase. In a prospective study of GDM, plasma samples were collected from pregnant women at 10-14 weeks' and 26-30 weeks' gestation and retrospectively allocated to gestation-matched GDM and normal groups. Samples were analysed after removal of high abundance proteins (Affi-gel Blue/Affi-gel Protein A) following a single fractionation process. Processing of samples with magnetic beads was performed in quadruplicate using a robotic workstation. Samples were mixed with Copper Immobilised Metal Affinity Chromatography beads (IMAC-Cu, Bruker Daltonics) in 96-well plates. Unbound peptides in the supernatant were aspirated and discarded while magnetic beads were washed and bound peptides eluded. Extracted samples were then processed by traditional MALD-ToF methods and raw spectral files were analysed with ClinProTools software (v2.2, Bruker Daltonics). Based upon the analysis of peptide profiles, we were able to identify disease-specific differentially-detected peptide ion peaks (Figure 8) and to develop multivariate classification models (Support Vector Machine and Genetic Mutation Models) using ClinProtTools software that discriminated between women who subsequently experienced a normal or GDM pregnancy. For example, using a genetic mutation classification model, 5 peptides were selected that had the ability to correctly classify 100% of women to a low risk group (i.e. those women who subsequently experienced a normal pregnancy). Furthermore, the model correctly classified greater than 93% of those women who subsequently experienced a GDM pregnancy. An independent The other mass-spectrometry based approach we have used to identify disease-specific proteins is iTRAQ. This labelling method is arguably the benchmark for relative protein quantification. One significant benefit is that it allows sample multiplexing and hence the ability to perform comparative analyses of up to eight different samples. For example, seven disease conditions or treatment groups and a pooled internal control could be processed in The same plasma samples described in section 4.4.2 above were subjected to iTRAQ analysis. Each pooled sample group was initially depleted of high abundance plasma proteins using the IgY14/Supermix system (Sigma). Depleted samples were digested with trypsin and each was labelled with one of four different iTRAQ reagents (ABSciex, normal - 114, IUGR - 116, GDM - 118 and FM - 121). After labelling, all 4 labelled reaction mixtures were combined and applied to a strong cation exchange (SCX) cartridge. A single fraction utilised to identify peptides that may be informative of disease risk are described. 4.5.1 MALDI-ToF peptide profiling (ClinProt™ workflow) and larger cohort is now required to validate these observations. tandem, allowing identification and quantification relative to control. 4.5.2 Stable isotope labelling (iTRAQ workflow) use of an internal standard for normalisation of data across gels (as described in 4.2 above). 2D-DIGE also delivers useful relative quantification of protein expression profiles where the dyes are purported to have sub-nanogram sensitivity and a linear response to protein concentrations of over five orders of magnitude. The dyes are also compatible with mass spectrometric analysis. With respect to analysing the plasma proteome, 2D-DIGE is still limited by the compositional complexity of plasma and similarly benefits from sample fractionation and the removal of high-abundance proteins. In these experiments, we collected plasma from asymptomatic pregnant women at 12-18 weeks' gestation. Pregnancies were retrospectively classified as normal (n = 10), GDM (n=5), small-for-gestational age (n=5) and large-for-gestational age (n=5). Plasma was pooled for each group and depleted of high abundance proteins (top 14) using the IgY14/Supermix system (Sigma). Samples were then concentrated and labelled with Cy2, Cy3 and Cy5 CyDye DIGE fluors (GE Healthcare) and subjected to 2D-PAGE using 13 cm, pH4-11 immobilised pH gradient strips (1st dimension) followed by 12.5% gel electrophoresis (2nd dimension) as described in section 4.2 above. The analysis focused on spots with a greater than 2-fold difference in expression. Comparison between GDM and normal yielded 10 proteins that were down-regulated and 4 proteins that were up-regulated (some being different isoforms of the same protein). Proteins associated with small or large for gestation fetal complications were also identified long before disease onset. #### 4.5 Mass-spectrometry based quantitative profiling approaches There are now a number of mass spectrometry (MS)-based, relative quantification approaches currently available including: (i) Multidimensional Chromatography (e.g. MudPIT); (ii) Stable Isotope Labelling (e.g. metabolic-SILAC, enzymatic-18O labelling, chemical ICPL and iTRAQ labelling); (iii) MALDI-ToF Profiling (e.g. SELDI™ and ClinProt™) and (iv) Label Free Quantification (e.g. spectral counting). Of these, stable isotope labelling is becoming the method of choice for quantitative proteomics. Stable isotope labelling has the advantages of being more sensitive and reproducible than gelbased methods. These approaches utilise either a mass tag coding strategy (e.g. ICPL - Isotope Coded Protein Labelling, ICAT - Isotope Coded Affinity Tag or iTRAQ - isobaric Tags for Relative and Absolute Quantification) that allow pooling of samples to reduce technical variation. Label-free quantification is an approach that holds the promise of true MudPIT-type 'shotgun' quantification but has some disadvantages in sample preparation, cost and the challenge of normalizing the data so that accurate quantification can be done across multiple samples and multiple analyses. Comparison of protein expression profiles between samples is based upon two metrics: ion peak intensities of extracted peptide signals from LC/MS profiles or spectral counting (number of times peptide precursor is selected for fragmentation) of identified proteins after MS/MS analysis (Zhu, et al., 2010). In addition to its analytical applications, mass spectrometry affords opportunities to identify signature profiles contained within biological samples for the purpose of classification. The application of mass spectrometry is a burgeoning area within the domain of diagnostic and predictive medicine. This approach now affords the opportunity to develop disease-specific patterns or profiles based upon the presence of specific peptides in a patient sample. MSbased protein profiling relies on the presence and spatial relationships between peptide peaks to facilitate the classification of biological samples into different categories (e.g. normal and disease). Based upon the analysis of a training sample set (e.g. disease-free patients), pattern recognition software and multivariate modelling are employed to build peptide profiles or motifs that characterise a disease-free condition. Once established, such reference profiles may be used as a template to detect variance and thus deliver a diagnosis or predictive capacity. Two of the mass spectrometry-based profiling approaches we have utilised to identify peptides that may be informative of disease risk are described. #### 4.5.1 MALDI-ToF peptide profiling (ClinProt™ workflow) 12 Gestational Diabetes use of an internal standard for normalisation of data across gels (as described in 4.2 above). 2D-DIGE also delivers useful relative quantification of protein expression profiles where the dyes are purported to have sub-nanogram sensitivity and a linear response to protein concentrations of over five orders of magnitude. The dyes are also compatible with mass spectrometric analysis. With respect to analysing the plasma proteome, 2D-DIGE is still limited by the compositional complexity of plasma and similarly benefits from sample In these experiments, we collected plasma from asymptomatic pregnant women at 12-18 weeks' gestation. Pregnancies were retrospectively classified as normal (n = 10), GDM (n=5), small-for-gestational age (n=5) and large-for-gestational age (n=5). Plasma was pooled for each group and depleted of high abundance proteins (top 14) using the IgY14/Supermix system (Sigma). Samples were then concentrated and labelled with Cy2, Cy3 and Cy5 CyDye DIGE fluors (GE Healthcare) and subjected to 2D-PAGE using 13 cm, pH4-11 immobilised pH gradient strips (1st dimension) followed by 12.5% gel electrophoresis (2nd dimension) as described in section 4.2 above. The analysis focused on spots with a greater than 2-fold difference in expression. Comparison between GDM and normal yielded 10 proteins that were down-regulated and 4 proteins that were up-regulated (some being different isoforms of the same protein). Proteins associated with small or large for gestation There are now a number of mass spectrometry (MS)-based, relative quantification approaches currently available including: (i) Multidimensional Chromatography (e.g. MudPIT); (ii) Stable Isotope Labelling (e.g. metabolic-SILAC, enzymatic-18O labelling, chemical ICPL and iTRAQ labelling); (iii) MALDI-ToF Profiling (e.g. SELDI™ and ClinProt™) and (iv) Label Free Quantification (e.g. spectral counting). Of these, stable isotope labelling is becoming the method of choice for quantitative proteomics. Stable isotope labelling has the advantages of being more sensitive and reproducible than gelbased methods. These approaches utilise either a mass tag coding strategy (e.g. ICPL - Isotope Coded Protein Labelling, ICAT - Isotope Coded Affinity Tag or iTRAQ - isobaric Tags for Relative and Absolute Quantification) that allow pooling of samples to reduce technical variation. Label-free quantification is an approach that holds the promise of true MudPIT-type 'shotgun' quantification but has some disadvantages in sample preparation, cost and the challenge of normalizing the data so that accurate quantification can be done across multiple samples and multiple analyses. Comparison of protein expression profiles between samples is based upon two metrics: ion peak intensities of extracted peptide signals from LC/MS profiles or spectral counting (number of times peptide precursor is selected for fractionation and the removal of high-abundance proteins. fetal complications were also identified long before disease onset. 4.5 Mass-spectrometry based quantitative profiling approaches fragmentation) of identified proteins after MS/MS analysis (Zhu, et al., 2010). In addition to its analytical applications, mass spectrometry affords opportunities to identify signature profiles contained within biological samples for the purpose of classification. The application of mass spectrometry is a burgeoning area within the domain of diagnostic and predictive medicine. This approach now affords the opportunity to develop disease-specific patterns or profiles based upon the presence of specific peptides in a patient sample. MSbased protein profiling relies on the presence and spatial relationships between peptide peaks to facilitate the classification of biological samples into different categories (e.g. Matrix-affinity peptide capture coupled with mass spectrometry is a discovery-based tool for comparing peptide mass fingerprints between individual or groups of samples. Numerous 'magnetic bead capture' chemistries are available including metal affinity (Cu, Fe), cationic exchange and hydrophobic reverse phase. In a prospective study of GDM, plasma samples were collected from pregnant women at 10-14 weeks' and 26-30 weeks' gestation and retrospectively allocated to gestation-matched GDM and normal groups. Samples were analysed after removal of high abundance proteins (Affi-gel Blue/Affi-gel Protein A) following a single fractionation process. Processing of samples with magnetic beads was performed in quadruplicate using a robotic workstation. Samples were mixed with Copper Immobilised Metal Affinity Chromatography beads (IMAC-Cu, Bruker Daltonics) in 96-well plates. Unbound peptides in the supernatant were aspirated and discarded while magnetic beads were washed and bound peptides eluded. Extracted samples were then processed by traditional MALD-ToF methods and raw spectral files were analysed with ClinProTools software (v2.2, Bruker Daltonics). Based upon the analysis of peptide profiles, we were able to identify disease-specific differentially-detected peptide ion peaks (Figure 8) and to develop multivariate classification models (Support Vector Machine and Genetic Mutation Models) using ClinProtTools software that discriminated between women who subsequently experienced a normal or GDM pregnancy. For example, using a genetic mutation classification model, 5 peptides were selected that had the ability to correctly classify 100% of women to a low risk group (i.e. those women who subsequently experienced a normal pregnancy). Furthermore, the model correctly classified greater than 93% of those women who subsequently experienced a GDM pregnancy. An independent and larger cohort is now required to validate these observations. #### 4.5.2 Stable isotope labelling (iTRAQ workflow) The other mass-spectrometry based approach we have used to identify disease-specific proteins is iTRAQ. This labelling method is arguably the benchmark for relative protein quantification. One significant benefit is that it allows sample multiplexing and hence the ability to perform comparative analyses of up to eight different samples. For example, seven disease conditions or treatment groups and a pooled internal control could be processed in tandem, allowing identification and quantification relative to control. The same plasma samples described in section 4.4.2 above were subjected to iTRAQ analysis. Each pooled sample group was initially depleted of high abundance plasma proteins using the IgY14/Supermix system (Sigma). Depleted samples were digested with trypsin and each was labelled with one of four different iTRAQ reagents (ABSciex, normal - 114, IUGR - 116, GDM - 118 and FM - 121). After labelling, all 4 labelled reaction mixtures were combined and applied to a strong cation exchange (SCX) cartridge. A single fraction Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus 15 was eluted, collected, acidified and analysed by LC-MS/MS (QSTAR Elite, ABSciex) for simultaneous protein identification and peptide quantification (ProteinPilot ABSciex). Relative abundance of proteins in depleted plasma was determined by comparing the peak heights of reporter ions for each sample (m/z at 116, 118, 121) with those from the normal pregnancy (m/z at 114) pool. Using iTRAQ reagents and high resolution mass spectrometry, eight proteins that were differentially expressed (greater than 2-fold) in maternal plasma in association with GDM were unambiguously identified. Three of these proteins were upregulated while five proteins were down-regulated. It is important to note that there was partial concordance between the identified proteins using iTRAQ and 2D-DIGE methods. The methods we have described in this brief chapter provide proof-of-principle both technically and conceptually that biomarkers associated with disease can be reliably identified before the onset of overt disease. The challenge now remains to validate these findings in large independent cohorts to determine the predictive efficacy of these biomarkers. These emerging technologies and sophisticated modelling approaches now afford a realistic opportunity to develop and robustly evaluate the risk of asymptomatic early pregnant women developing complications of pregnancy such as GDM, IUGR, FM, PET and PTL. The development of such test(s) will provide data that better informs clinical decision-making and patient management that will not only directly benefit the immediate pregnancy, but will also help mitigate the longer-term ramifications of these conditions for All studies described above received prior approval from a relevant institutional human research ethics committee. The authors wish to acknowledge the invaluable help provided by research midwives from the Mercy Hospital for Women who have consented participants and processed blood samples. Technical assistance was provided by Gill Barker and Karen Oliva while mass spectrometry expertise was provided by Drs Mark Bailey, Alun Jones and Mustafa Ayhan. We acknowledge the expertise provided by representatives from Bruker Daltonics (Freemont, CA) and Bio-Rad Laboratories (Hercules, CA) for their platform technologies. This work was funded by the Medical Research Foundation for Women and Babies and grants from the National Health and Medical Research Council (NHMRC, Australia) 454451, 509127, 526686 and 586651. GER is in receipt of an NHMRC Agarwal, M. M., Dhatt, G. S., Punnose, J. & Koster, G. (2005). Gestational diabetes: dilemma Ahmed, N., Barker, G., Oliva, K., Garfin, D., Talmadge, K., Georgiou, H., Quinn, M. & Rice, caused by multiple international diagnostic criteria. Diabet Med, Vol.22, No.12, pp. G. (2003). An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum. Proteomics, Vol.3, No.10, pp. 1980-1987, 5. Conclusion both mother and baby. 6. Acknowledgement Principal Research Fellowship. issn 1615-9853 1731-1736, issn 0742-3071 7. References Fig. 8. MALDI-ToF peptide profile comparisons. Top. Example of the average peptide profiles over a limited spectral range (2300-2800 m/z) is presented to illustrate identified differences in peptide profiles between women with a normal pregnancy (red, n=19, 12 weeks) and women who subsequently developed GDM (green, n=16, 12 weeks). Bottom. A peptide peak cluster plot highlighting the potential for using differentially-expressed peptides to classify women into low- and high-risk groups for subsequent GDM. The plot presents the data (integrated area) of two peptide peaks (1669 vs 2021 m/z) observed in plasma obtained from women (12 weeks' gestation) who subsequently experienced a normal (red) or GDM pregnancy (green). Standard deviation envelopes are presented. was eluted, collected, acidified and analysed by LC-MS/MS (QSTAR Elite, ABSciex) for simultaneous protein identification and peptide quantification (ProteinPilot ABSciex). Relative abundance of proteins in depleted plasma was determined by comparing the peak heights of reporter ions for each sample (m/z at 116, 118, 121) with those from the normal pregnancy (m/z at 114) pool. Using iTRAQ reagents and high resolution mass spectrometry, eight proteins that were differentially expressed (greater than 2-fold) in maternal plasma in association with GDM were unambiguously identified. Three of these proteins were upregulated while five proteins were down-regulated. It is important to note that there was partial concordance between the identified proteins using iTRAQ and 2D-DIGE methods. #### 5. Conclusion 14 Gestational Diabetes Fig. 8. MALDI-ToF peptide profile comparisons. Top. Example of the average peptide profiles over a limited spectral range (2300-2800 m/z) is presented to illustrate identified differences in peptide profiles between women with a normal pregnancy (red, n=19, 12 weeks) and women who subsequently developed GDM (green, n=16, 12 weeks). Bottom. A peptide peak cluster plot highlighting the potential for using differentially-expressed peptides to classify women into low- and high-risk groups for subsequent GDM. The plot presents the data (integrated area) of two peptide peaks (1669 vs 2021 m/z) observed in plasma obtained from women (12 weeks' gestation) who subsequently experienced a normal (red) or GDM pregnancy (green). Standard deviation envelopes are presented. The methods we have described in this brief chapter provide proof-of-principle both technically and conceptually that biomarkers associated with disease can be reliably identified before the onset of overt disease. The challenge now remains to validate these findings in large independent cohorts to determine the predictive efficacy of these biomarkers. These emerging technologies and sophisticated modelling approaches now afford a realistic opportunity to develop and robustly evaluate the risk of asymptomatic early pregnant women developing complications of pregnancy such as GDM, IUGR, FM, PET and PTL. The development of such test(s) will provide data that better informs clinical decision-making and patient management that will not only directly benefit the immediate pregnancy, but will also help mitigate the longer-term ramifications of these conditions for both mother and baby. #### 6. Acknowledgement All studies described above received prior approval from a relevant institutional human research ethics committee. The authors wish to acknowledge the invaluable help provided by research midwives from the Mercy Hospital for Women who have consented participants and processed blood samples. Technical assistance was provided by Gill Barker and Karen Oliva while mass spectrometry expertise was provided by Drs Mark Bailey, Alun Jones and Mustafa Ayhan. We acknowledge the expertise provided by representatives from Bruker Daltonics (Freemont, CA) and Bio-Rad Laboratories (Hercules, CA) for their platform technologies. This work was funded by the Medical Research Foundation for Women and Babies and grants from the National Health and Medical Research Council (NHMRC, Australia) 454451, 509127, 526686 and 586651. GER is in receipt of an NHMRC Principal Research Fellowship. #### 7. References Novel Screening Approaches for the Early Detection of Gestational Diabetes Mellitus 17 Georgiou, H. M., Thio, Y. S., Russell, C., Permezel, M., Heng, Y. J., Lee, S. & Tong, S. (2011). Guedj, A. M. (2010). When should screening be performed for gestational diabetes? Henry, O. A. & Beischer, N. A. (1991). Long-term implications of gestational diabetes for the mother. 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Diabetes Care, Vol.27, No.3, pp. 799-800, issn hyperglycemia and adverse pregnancy outcomes. Diabetes Care, Vol.32, No.9, pp. 2 India 1Diabetes Research Institute, 2Diabetes Care Centre Gestational Diabetes Mellitus - A Perspective The prevalence of diabetes is increasing globally and the total number of people with this condition is projected to rise from 171 million in 2000 to 366 million in 2030 (Wild et al, 2004). India is no exception, with projected rates of 79.4 million in 2030—a 151% increase from 31.7 million in 2000 (Wild et al, 2004). The increased prevalence is attributed to the aging population structure, urbanization, the obesity epidemic, and physical inactivity (Hunt & Schuller, 2007). While all these factors contribute to the epidemic of diabetes, intrauterine exposures are emerging as potential risk factors (Barker, 1995). The "fetal origin of adult disease" hypothesis proposes that gestational programming may critically influence adult health and disease (Barker, 1995). Gestational programming is a process whereby stimuli or stresses occurring at critical or sensitive periods of fetal development, permanently change structure, physiology, and metabolism, which predisposes individuals to disease in adult life (Lucas, 1991). If the stimulus happens to be glucose intolerance in pregnancy, it predisposes the offspring to an increased risk of developing glucose intolerance in the future. This vicious cycle is likely to influence and perpetuate the incidence and prevalence of glucose intolerance in any population (Seshiah et al., 2004). Therefore, preventive measures against type 2 diabetes should start during the intrauterine period and continue from early childhood throughout life (Tuomilehto, 2005). In this respect, detection of gestational diabetes mellitus (GDM), defined as carbohydrate intolerance of variable severity with onset or first recognition during the present pregnancy (Metzger, 1991), becomes an important public health issue. The etiopathogenesis of glucose intolerance that develops in women with GDM could be the result of their inability to increase insulin secretion enough to overcome insulin resistance that occurs even in non diabetic pregnancy (Kuhl et al., 1985). The present concept is that GDM represents, detection of chronic β cell dysfunction, rather than development of relative insulin deficiency as insulin resistance increases during pregnancy (Buchanan et al., 2007). The usual recommendation of lifestyle modifications or drug intervention for prevention of diabetes is likely to delay or postpone the development of overt diabetes in persons diagnosed with abnormal glucose tolerance. These measures essentially target only the post primary prevention of diabetes whereas the aim should be primary prevention of diabetes by keeping the genetically or otherwise susceptible individuals normoglycemic, apart from preventing them from developing type 2 DM (Tuomilehto, 2005). In this context, women 1. Introduction 2. Implications Veerasamy Seshiah1, Vijayam Balaji2 and Balaji Madhuri2 ## Gestational Diabetes Mellitus - A Perspective Veerasamy Seshiah1, Vijayam Balaji2 and Balaji Madhuri2 1Diabetes Research Institute, 2Diabetes Care Centre India #### 1. Introduction 20 Gestational Diabetes Zhou, S., Bailey, M. J., Dunn, M. J., Preedy, V. R. & Emery, P. W. (2005). A quantitative issn 1110-7243 investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure. Proteomics, Vol.5, No.11, pp. 2739-2747, issn 1615-9853 Zhu, W., Smith, J. W. & Haung, C.-M. (2010). Mass Spectrometry-Based Label Free Quantitative Proteomics. J Biomed BioTechnol, Vol.2010, Article ID 840518, 6 pages, The prevalence of diabetes is increasing globally and the total number of people with this condition is projected to rise from 171 million in 2000 to 366 million in 2030 (Wild et al, 2004). India is no exception, with projected rates of 79.4 million in 2030—a 151% increase from 31.7 million in 2000 (Wild et al, 2004). The increased prevalence is attributed to the aging population structure, urbanization, the obesity epidemic, and physical inactivity (Hunt & Schuller, 2007). While all these factors contribute to the epidemic of diabetes, intrauterine exposures are emerging as potential risk factors (Barker, 1995). The "fetal origin of adult disease" hypothesis proposes that gestational programming may critically influence adult health and disease (Barker, 1995). Gestational programming is a process whereby stimuli or stresses occurring at critical or sensitive periods of fetal development, permanently change structure, physiology, and metabolism, which predisposes individuals to disease in adult life (Lucas, 1991). If the stimulus happens to be glucose intolerance in pregnancy, it predisposes the offspring to an increased risk of developing glucose intolerance in the future. This vicious cycle is likely to influence and perpetuate the incidence and prevalence of glucose intolerance in any population (Seshiah et al., 2004). Therefore, preventive measures against type 2 diabetes should start during the intrauterine period and continue from early childhood throughout life (Tuomilehto, 2005). In this respect, detection of gestational diabetes mellitus (GDM), defined as carbohydrate intolerance of variable severity with onset or first recognition during the present pregnancy (Metzger, 1991), becomes an important public health issue. The etiopathogenesis of glucose intolerance that develops in women with GDM could be the result of their inability to increase insulin secretion enough to overcome insulin resistance that occurs even in non diabetic pregnancy (Kuhl et al., 1985). The present concept is that GDM represents, detection of chronic β cell dysfunction, rather than development of relative insulin deficiency as insulin resistance increases during pregnancy (Buchanan et al., 2007). #### 2. Implications The usual recommendation of lifestyle modifications or drug intervention for prevention of diabetes is likely to delay or postpone the development of overt diabetes in persons diagnosed with abnormal glucose tolerance. These measures essentially target only the post primary prevention of diabetes whereas the aim should be primary prevention of diabetes by keeping the genetically or otherwise susceptible individuals normoglycemic, apart from preventing them from developing type 2 DM (Tuomilehto, 2005). In this context, women Gestational Diabetes Mellitus - A Perspective 23 Dr. K. P. Paulose Trivandrum, Kerala 750 15% Dr. Mary John Ludhiana, Punjab 220 17.5% Dr. Prasanna Kumar Bangalore, Karnataka 49 12% Dr. Shyam Mukundan Alwaye, Kerala 200 21% TOTAL 3674 16.55% Crowther et al found that treatment of GDM diagnosed by WHO criterion reduces serious perinatal morbidity and may also improve the women's health-related quality of life (Crowther at al., 2005). Similarly a long term outcome study conducted by Franks et al documented that when maternal 2-h PG was ≥ 7.8 mmol/L, the cumulative risk of offspring developing type 2 DM was 30% at the age 24 yrs (Franks et al., 2006). Both these short term and long term outcome studies validate the WHO criterion and hence the authors chose this criterion for the DIPAP project. In this project GDM was detected in 739 (17.8%) women in urban, 548 (13.8%) in semi urban and 392(9.9%) in rural areas. In this community based study, the overall prevalence of GDM was 13.9% (Seshiah et al., 2008a). The prevalence of GDM had increased from 16.55% to 17.8% in the urban areas in two years (Seshiah et al., 2004, 2008a). There is a definite divide between the rural and urban areas in the prevalence of GDM. The possible cause for the low prevalence in the rural settings may be due to the less mechanized, agriculture based lifestyle. In this population the risk factors for the development of GDM Number of pregnant women screened Tamil Nadu 891 16.2% Tamil Nadu 1002 15% Tamil Nadu 562 18.8% Prevalence Rate Centre Dr. Balaji et al North Chennai, Dr. Anjalakshi et al South Chennai, Dr. Aruyerchelvan Erode, Fig. 1. Risk factors for the development of GDM Table 1. Prevalence of GDM in different parts of India – 2002 were: age ≥ 25 years, BMI ≥ 25 and family history of diabetes (Figure 1). with GDM become the ideal group for primary prevention of diabetes (Girling & Dornhorst, 2003), as women with GDM are at increased risk of developing diabetes predominantly type 2 DM as are their children (Dornhorst & Rossi, 1998). The diagnosis of GDM offers a unique opportunity in identifying individuals who will be benefited by early therapeutic intervention with diet and exercise, thus normalizing the weight to delay or even possibly prevent the onset of diabetes. #### 3. Prevalence The epidemiology of GDM is subject to various factors such as the population to be screened, the screening methods, the gestational weeks for screening and the glycemic criteria for diagnosis. Screening recommendations range from inclusion of all pregnant women (universal) to the exclusion of all other women except those with very specific risk factors (selective): (e.g., age > 25 years, obesity: BMI > 30, ethnicity: Hispanic, Native American, Asian-American, African-American, family history: first degree relative, and previous GDM or large for gestational age infant) (Mazze, 2006). Different ethnic groups when exposed to the same environmental setting, experienced a widely variable risk. Among ethnic groups in South Asian countries, Indian women have the highest frequency of GDM (15%), followed by Chinese (13.9%), Vietnam-born (7.8%) and Australian-born (4.3%) (King, 1998). For a given population and ethnicity, the risk of diabetes in pregnancy, mirrors that of the underlying frequency of type 2 DM in that population (King, 1998). Impaired Glucose Tolerance (IGT) is generally much more prevalent than diabetes in women of child bearing age (King, 1998). Among Indians, the prevalence of IGT in the age group of 20 to 29 years and 30 to 39 years was found to be 12.2% and 15.3% respectively. No gender difference was seen in the prevalence of IGT (Ramachandran et al., 2001). It was observed in a national survey performed in 2002, the frequency of the occurrence of GDM was 16.55% by the World Health Organization (WHO) criteria (Seshiah et al., 2004) which was closer to the prevalence of IGT in the child-bearing age group of women in India (Ramachandran et al., 2001). Parallel to the increased prevalence of IGT in the general population, the frequency of GDM had also increased. The prevalence of GDM was 2% in 1982 (Agarwal & Gupta, 1982) [IGT - 2% (Ramachandran et al., 1988)] which increased to 7.62% in 1991 (Narendra et al., 1991) [IGT – 8.2% (Ramachandran et al., 1992)], and doubled to 16.55% in 2002 (Seshiah et al., 2004) [IGT – 14.5% (Ramachandran et al., 2001)]. The prevalence data published (Seshiah et al., 2004) included pregnant women attending different health care providing centres spread in different parts of the country (Table – 1). This phenomenal increase in the prevalence of GDM prompted the authors to initiate a project on 'Diabetes In Pregnancy Awareness and Prevention (DIPAP)', funded by the World Diabetes Foundation and supported by the government of Tamil Nadu, India. To have a community based prevalence data under the DIPAP project, the author's group screened a total of 4151, 3960 and 3945 pregnant women in the urban, semi urban and rural areas of Tamil Nadu, respectively (Seshiah et al., 2008a). This was the largest prospective study (N=12,056) other than Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study. GDM diagnosis was based on the WHO criterion of 2-h plasma glucose (PG) ≥ 7.8 mmol/L with 75g oral glucose. WHO recommendation serves both as one step screening and diagnostic procedure, and is easy to perform besides being economical (Seshiah et al., 2004, 2005). WHO criterion of 2-h PG ≥ 7.8 mmol/L identifying a large number of cases may have a greater potential for prevention (Schmidt et al., 2001). In addition, a study performed by with GDM become the ideal group for primary prevention of diabetes (Girling & Dornhorst, 2003), as women with GDM are at increased risk of developing diabetes predominantly type 2 DM as are their children (Dornhorst & Rossi, 1998). The diagnosis of GDM offers a unique opportunity in identifying individuals who will be benefited by early therapeutic intervention with diet and exercise, thus normalizing the weight to delay or even possibly The epidemiology of GDM is subject to various factors such as the population to be screened, the screening methods, the gestational weeks for screening and the glycemic criteria for diagnosis. Screening recommendations range from inclusion of all pregnant women (universal) to the exclusion of all other women except those with very specific risk factors (selective): (e.g., age > 25 years, obesity: BMI > 30, ethnicity: Hispanic, Native American, Asian-American, African-American, family history: first degree relative, and previous GDM or large for gestational age infant) (Mazze, 2006). Different ethnic groups when exposed to the same environmental setting, experienced a widely variable risk. Among ethnic groups in South Asian countries, Indian women have the highest frequency of GDM (15%), followed by For a given population and ethnicity, the risk of diabetes in pregnancy, mirrors that of the underlying frequency of type 2 DM in that population (King, 1998). Impaired Glucose Tolerance (IGT) is generally much more prevalent than diabetes in women of child bearing age (King, 1998). Among Indians, the prevalence of IGT in the age group of 20 to 29 years and 30 to 39 years was found to be 12.2% and 15.3% respectively. No gender difference was seen in the prevalence of IGT (Ramachandran et al., 2001). It was observed in a national survey performed in 2002, the frequency of the occurrence of GDM was 16.55% by the World Health Organization (WHO) criteria (Seshiah et al., 2004) which was closer to the prevalence of IGT in the child-bearing age group of women in India (Ramachandran et al., 2001). Parallel to the increased prevalence of IGT in the general population, the frequency of GDM had also increased. The prevalence of GDM was 2% in 1982 (Agarwal & Gupta, 1982) [IGT - 2% (Ramachandran et al., 1988)] which increased to 7.62% in 1991 (Narendra et al., 1991) [IGT – 8.2% (Ramachandran et al., 1992)], and doubled to 16.55% in 2002 (Seshiah et al., 2004) [IGT – 14.5% (Ramachandran et al., 2001)]. The prevalence data published (Seshiah et al., 2004) included pregnant women attending different health care providing centres This phenomenal increase in the prevalence of GDM prompted the authors to initiate a project on 'Diabetes In Pregnancy Awareness and Prevention (DIPAP)', funded by the World Diabetes Foundation and supported by the government of Tamil Nadu, India. To have a community based prevalence data under the DIPAP project, the author's group screened a total of 4151, 3960 and 3945 pregnant women in the urban, semi urban and rural areas of Tamil Nadu, respectively (Seshiah et al., 2008a). This was the largest prospective study (N=12,056) other than Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study. GDM diagnosis was based on the WHO criterion of 2-h plasma glucose (PG) ≥ 7.8 mmol/L with 75g oral glucose. WHO recommendation serves both as one step screening and diagnostic procedure, and is easy to perform besides being economical (Seshiah et al., 2004, 2005). WHO criterion of 2-h PG ≥ 7.8 mmol/L identifying a large number of cases may have a greater potential for prevention (Schmidt et al., 2001). In addition, a study performed by Chinese (13.9%), Vietnam-born (7.8%) and Australian-born (4.3%) (King, 1998). spread in different parts of the country (Table – 1). prevent the onset of diabetes. 3. Prevalence Table 1. Prevalence of GDM in different parts of India – 2002 Crowther et al found that treatment of GDM diagnosed by WHO criterion reduces serious perinatal morbidity and may also improve the women's health-related quality of life (Crowther at al., 2005). Similarly a long term outcome study conducted by Franks et al documented that when maternal 2-h PG was ≥ 7.8 mmol/L, the cumulative risk of offspring developing type 2 DM was 30% at the age 24 yrs (Franks et al., 2006). Both these short term and long term outcome studies validate the WHO criterion and hence the authors chose this criterion for the DIPAP project. In this project GDM was detected in 739 (17.8%) women in urban, 548 (13.8%) in semi urban and 392(9.9%) in rural areas. In this community based study, the overall prevalence of GDM was 13.9% (Seshiah et al., 2008a). The prevalence of GDM had increased from 16.55% to 17.8% in the urban areas in two years (Seshiah et al., 2004, 2008a). There is a definite divide between the rural and urban areas in the prevalence of GDM. The possible cause for the low prevalence in the rural settings may be due to the less mechanized, agriculture based lifestyle. In this population the risk factors for the development of GDM were: age ≥ 25 years, BMI ≥ 25 and family history of diabetes (Figure 1). Fig. 1. Risk factors for the development of GDM Gestational Diabetes Mellitus - A Perspective 25 For the successful implementation of universal screening, a test has to be casual and reliable. A procedure that does not impose any restriction would be ideal for universal screening. The test performed should be able to diagnose GDM, as they walk into the prenatal clinic or clinical laboratory irrespective of their last meal timings. Hence the authors undertook a study to evaluate, whether a 2-h 75g oral glucose test performed in a non-fasting state, irrespective of last meal timing, is as efficacious as 2-h 75g oral glucose test done in the fasting state recommended by WHO in detecting GDM (Anjalakshi et al., 2009). A total of 862 consecutive pregnant women were subjected to 75g oral glucose test irrespective of time of the last meal. Venous samples were collected at 2-h after oral glucose administration. They were advised to follow a diet containing atleast 150g carbohydrate daily and usual activity for atleast 3 days and come to the prenatal clinic after an overnight fasting of 10-12 h. At the second visit 800 of them responded and underwent 2-h 75g oral glucose test in the fasting state recommended by WHO. The observation in this study was, all women diagnosed as GDM (N=87) by 75g glucose test irrespective of the last meal timings also satisfied the diagnostic criteria of 75-g oral glucose test performed in the fasting state recommended by WHO. It was also found that there was no statistically significant difference (P > 0.05) between the PG levels of the 75g glucose test in fasting and non fasting state, irrespective of last meal timing, performed in the GDM and in NGT pregnant women. The rationale behind this study outcome is that, a normal glucose tolerant woman would be able to maintain euglycemia despite glucose challenge due to adequate insulin response, whereas in a woman with GDM who has impaired insulin secretion (Kuhl, 1991), her glycemic level increases with a meal and with glucose challenge, the glycemic excursion is expected to exaggerate. This cascading effect is advantageous as this would not result in false positive diagnosis of GDM. Performing this test procedure in the non-fasting state, irrespective of last meal timing, is prudent as glucose concentrations during the glucose tolerance are affected little by the time since the last meal (Gough et al., 1970). Pregnancy Study Group India (DIPSI) (Seshiah et al., 2009) 5.2 Comparison of WHO and IADPSG criteria Pettitt et al. observed that WHO criteria based on the glucose concentration 2-h after 75g oral glucose administered to non-fasting women correctly identified subjects with GDM (Pettitt et al., 1994). The non-fasting 2-h post 75g glucose concentration strongly predicts adverse outcome for the mother and her offspring (Pettitt et al., 1991). Philips et al also observed that plasma glucose value with a glucose challenge test was unaffected by the time after a meal or time of the day in Normal Glucose Tolerant non pregnant subjects (Philips et al., 2009). Thus, this single test procedure performed irrespective of the last meal timing is rational and a patient friendly approach, which causes least disturbance in her routine activities. This procedure is a modified version of WHO criteria in that, only 2-h PG is taken into consideration for the diagnosis of GDM and is being followed by the Diabetes In All the diagnostic criteria, except the existing diagnostic criterion of WHO 2-h plasma glucose (PG) ≥ 7.8 mmol/L with 75g oral glucose load (King, 1998), are country specific or recommended by various associations. Recently, based on the HAPO study, the International Association of Diabetes and Pregnancy Study Groups (IADPSG) consensus panel recommended that GDM can be diagnosed, if any one value of fasting plasma glucose (FPG), 1-h and 2-h PG concentrations meet or exceed 5.1 mmol/L, 10.0 mmol/L and 8.5 mmol/L respectively, with 75g oral glucose tolerance test (OGTT) (Metzger et al., 2010). India one of the most populous countries in the world was not part of the HAPO study. Hence the authors group undertook a prospective, collaborative study to ascertain whether #### 3.1 Geographical variations in the prevalence of GDM Prevalence varies between 1% to 16% depending on the geographical variation and ethnicity and from one region to another in the same country (Yogev et al., 2003). The prevalence of GDM corresponds to the prevalence of IGT within a given population (King, 1998). The prevalence of GDM in India was 16.55% in the urban area and the frequency varied from 12% to 21% in different parts of the country (Seshiah et al., 2004) (Table 1). A low prevalence of GDM was observed in Kashmir (Zargar et al., 2004) (northern tip of India) 4.4% and a high prevalence of 16.55% in the southern part of India (Seshiah et al., 2004). The prevalence of GDM in other developing countries also showed regional variations. In Mexico, the prevalence of GDM varied from 4.3% to 11% when screening was done in different parts of the country (Forsbach et al., 1998). The rate of abnormal screening test results ranged from 8.0% to 20.7% for different regions of Poland (Wojcikowski et al, 2002). Among Pan Arab countries, Saudi Arabia (12.5%) and Bahrain (13.5%) had the highest prevalence of GDM (Al Mahroos et al, 2005; Ardawi et al, 2000). The frequency of GDM in Argentina was between 2% and 12% depending upon the population studied and geographical variations (Liliana et al, 2003). #### 4. Rationale for universal screening Selective screening based on risk factors scored poorly in predicting GDM (Shamsuddin et al, 2001). If selective screening is employed, it is likely that 27% of GDM women will go undetected (Shamsuddin et al, 2001). GDM diagnosis is overlooked in about 1/3rd of the women, where selective rather than Universal screening is performed (Cosson et al., 2004a). Further selective screening recommended by American Diabetes Association (ADA) may be applicable for women belonging to the ethnic group with low prevalence of GDM. Risk factor screening does not take into account the inevitable difficulties in implementation, including the potential for substantial under-diagnosis of GDM (Simmons et al., 2009). Among ethnic groups in South Asian countries, Indian women have the highest frequency of GDM necessitating Universal Screening (Beischer et al., 1991). The recognition of glucose intolerance during pregnancy is more relevant as Indian women have 11 fold increased risk of developing GDM compared to Caucasians (Dornhorst et al., 1992). Compared to selective screening, Universal screening for GDM detects more cases and improves maternal and offspring prognosis (Cosson, 2004b). Thus Universal screening appears to be the most reliable and desired method for the detection of GDM (Shamsuddin et al., 2001). For universal screening the test should be simple and cost effective. The two step procedure of screening with 50g Glucose challenge test (GCT) and then diagnosing GDM based on 75g OGTT is not feasible in a country like India, because the pregnant women may have to visit the antenatal clinic twice and at least 3 to 5 blood samples have to be drawn, which they resent. The scenario is likely to be the same in most of the developing countries. #### 5. Diagnosis of GDM #### 5.1 A single step procedure to diagnose GDM All the diagnostic criteria require women to be in fasting, but most of the time pregnant women do not come in the fasting state because of commutation and belief not to fast for long hours. Attending the first prenatal visit in the fasting state is impractical in many settings (Metzger et al., 2010). The dropout rate is very high when a pregnant woman is asked to come again for the glucose tolerance test (Seshiah et al., 2004; Magee et al., 2001). Prevalence varies between 1% to 16% depending on the geographical variation and ethnicity and from one region to another in the same country (Yogev et al., 2003). The prevalence of GDM corresponds to the prevalence of IGT within a given population (King, 1998). The prevalence of GDM in India was 16.55% in the urban area and the frequency varied from 12% to 21% in different parts of the country (Seshiah et al., 2004) (Table 1). A low prevalence of GDM was observed in Kashmir (Zargar et al., 2004) (northern tip of India) 4.4% and a high prevalence of 16.55% in the southern part of India (Seshiah et al., 2004). The prevalence of GDM in other developing countries also showed regional variations. In Mexico, the prevalence of GDM varied from 4.3% to 11% when screening was done in different parts of the country (Forsbach et al., 1998). The rate of abnormal screening test results ranged from 8.0% to 20.7% for different regions of Poland (Wojcikowski et al, 2002). Among Pan Arab countries, Saudi Arabia (12.5%) and Bahrain (13.5%) had the highest prevalence of GDM (Al Mahroos et al, 2005; Ardawi et al, 2000). The frequency of GDM in Argentina was between 2% and 12% depending upon the population studied and geographical variations (Liliana et al, 2003). Selective screening based on risk factors scored poorly in predicting GDM (Shamsuddin et al, 2001). If selective screening is employed, it is likely that 27% of GDM women will go undetected (Shamsuddin et al, 2001). GDM diagnosis is overlooked in about 1/3rd of the women, where selective rather than Universal screening is performed (Cosson et al., 2004a). Further selective screening recommended by American Diabetes Association (ADA) may be applicable for women belonging to the ethnic group with low prevalence of GDM. Risk factor screening does not take into account the inevitable difficulties in implementation, including the potential for substantial under-diagnosis of GDM (Simmons et al., 2009). Among ethnic groups in South Asian countries, Indian women have the highest frequency of GDM necessitating Universal Screening (Beischer et al., 1991). The recognition of glucose intolerance during pregnancy is more relevant as Indian women have 11 fold increased risk Compared to selective screening, Universal screening for GDM detects more cases and improves maternal and offspring prognosis (Cosson, 2004b). Thus Universal screening appears to be the most reliable and desired method for the detection of GDM (Shamsuddin et al., 2001). For universal screening the test should be simple and cost effective. The two step procedure of screening with 50g Glucose challenge test (GCT) and then diagnosing GDM based on 75g OGTT is not feasible in a country like India, because the pregnant women may have to visit the antenatal clinic twice and at least 3 to 5 blood samples have to be drawn, which they resent. The scenario is likely to be the same in most of the developing countries. All the diagnostic criteria require women to be in fasting, but most of the time pregnant women do not come in the fasting state because of commutation and belief not to fast for long hours. Attending the first prenatal visit in the fasting state is impractical in many settings (Metzger et al., 2010). The dropout rate is very high when a pregnant woman is asked to come again for the glucose tolerance test (Seshiah et al., 2004; Magee et al., 2001). of developing GDM compared to Caucasians (Dornhorst et al., 1992). 3.1 Geographical variations in the prevalence of GDM 4. Rationale for universal screening 5. Diagnosis of GDM 5.1 A single step procedure to diagnose GDM For the successful implementation of universal screening, a test has to be casual and reliable. A procedure that does not impose any restriction would be ideal for universal screening. The test performed should be able to diagnose GDM, as they walk into the prenatal clinic or clinical laboratory irrespective of their last meal timings. Hence the authors undertook a study to evaluate, whether a 2-h 75g oral glucose test performed in a non-fasting state, irrespective of last meal timing, is as efficacious as 2-h 75g oral glucose test done in the fasting state recommended by WHO in detecting GDM (Anjalakshi et al., 2009). A total of 862 consecutive pregnant women were subjected to 75g oral glucose test irrespective of time of the last meal. Venous samples were collected at 2-h after oral glucose administration. They were advised to follow a diet containing atleast 150g carbohydrate daily and usual activity for atleast 3 days and come to the prenatal clinic after an overnight fasting of 10-12 h. At the second visit 800 of them responded and underwent 2-h 75g oral glucose test in the fasting state recommended by WHO. The observation in this study was, all women diagnosed as GDM (N=87) by 75g glucose test irrespective of the last meal timings also satisfied the diagnostic criteria of 75-g oral glucose test performed in the fasting state recommended by WHO. It was also found that there was no statistically significant difference (P > 0.05) between the PG levels of the 75g glucose test in fasting and non fasting state, irrespective of last meal timing, performed in the GDM and in NGT pregnant women. The rationale behind this study outcome is that, a normal glucose tolerant woman would be able to maintain euglycemia despite glucose challenge due to adequate insulin response, whereas in a woman with GDM who has impaired insulin secretion (Kuhl, 1991), her glycemic level increases with a meal and with glucose challenge, the glycemic excursion is expected to exaggerate. This cascading effect is advantageous as this would not result in false positive diagnosis of GDM. Performing this test procedure in the non-fasting state, irrespective of last meal timing, is prudent as glucose concentrations during the glucose tolerance are affected little by the time since the last meal (Gough et al., 1970). Pettitt et al. observed that WHO criteria based on the glucose concentration 2-h after 75g oral glucose administered to non-fasting women correctly identified subjects with GDM (Pettitt et al., 1994). The non-fasting 2-h post 75g glucose concentration strongly predicts adverse outcome for the mother and her offspring (Pettitt et al., 1991). Philips et al also observed that plasma glucose value with a glucose challenge test was unaffected by the time after a meal or time of the day in Normal Glucose Tolerant non pregnant subjects (Philips et al., 2009). Thus, this single test procedure performed irrespective of the last meal timing is rational and a patient friendly approach, which causes least disturbance in her routine activities. This procedure is a modified version of WHO criteria in that, only 2-h PG is taken into consideration for the diagnosis of GDM and is being followed by the Diabetes In Pregnancy Study Group India (DIPSI) (Seshiah et al., 2009) #### 5.2 Comparison of WHO and IADPSG criteria All the diagnostic criteria, except the existing diagnostic criterion of WHO 2-h plasma glucose (PG) ≥ 7.8 mmol/L with 75g oral glucose load (King, 1998), are country specific or recommended by various associations. Recently, based on the HAPO study, the International Association of Diabetes and Pregnancy Study Groups (IADPSG) consensus panel recommended that GDM can be diagnosed, if any one value of fasting plasma glucose (FPG), 1-h and 2-h PG concentrations meet or exceed 5.1 mmol/L, 10.0 mmol/L and 8.5 mmol/L respectively, with 75g oral glucose tolerance test (OGTT) (Metzger et al., 2010). India one of the most populous countries in the world was not part of the HAPO study. Hence the authors group undertook a prospective, collaborative study to ascertain whether Gestational Diabetes Mellitus - A Perspective 27 Specificity (95% CI) 89.4 (87.6-91.0) 93.0 (91.4-94.3) 97.4 (96.3-98.2) 99.2 (98.5-99.6) 99.8 (99.4-100.0) Table 2. Performance of FPG test for the predictor of gestational diabetes and macrosomia 5.4 The validation of WHO criterion (DIPSI criterion) based on the fetal outcome The authors investigated whether the diagnosis of GDM by WHO criterion is rational based on the fetal outcome (N = 1463). Macrosomia was the end point of this study as this is the most common morbidity of GDM (Jovanovic, 2001). They observed that there was no statistically significant difference in the mean birth weight of neonates born to women in the normal glucose tolerance (NGT) and with intervention in GDM groups (P=0.705) (Balaji et al., 2011b). This was due to the medical nutrition therapy (MNT) and/or insulin in maintaining FPG ~ 5.0 mmol/L and 2-h post meal ~ 6.7 mmol/L in GDM women. Intervention helped in maintaining the pregnancy outcome in GDM women equivalent to that of NGT women. Gayle et al also observed that diagnosis of GDM with OGTT 2-h PG ≥ 7.8 mmol/L and treatment in a combined diabetes antenatal clinic is worthwhile with a decreased macrosomia rate and fewer emergency cesarean sections (Gayle et al., 2010). The distribution of birth weight of neonates born to GDM and NGT women were similar (Figure 2) in the study conducted by the authors, indicating that the intervention given to pregnant women with 2-h PG ≥ 7.8 mmol/L had a significant effect in obtaining neonatal birth weight appropriate for gestational age. The level of association between macrosomia and GDM status after controlling the factors: maternal age, gestational age, family history of diabetes and BMI was elucidated. It was found that, the GDM status (2-h PG ≥ 7.8 mmol/L) of the pregnant women after intervention was not associated with macrosomia (adjusted OR = 0.752; 95% CI (0.406-1.390); P=0.363). There are publications confirming that treatment of GDM women as defined by WHO criterion was associated with a reduced risk of pregnancy outcome (Crowther et al., 2005; Gayle et al., 2010). In pregnancy, the decision to perform a placebo controlled trial requires clinical equipoise (Gifford et al., 2001). Hence, in this study, the authors did not have a control group of untreated pregnant women with 2-h PG ≥ 7.8 mmol/L, as there are evidences confirming that the treatment of GDM women as defined by WHO criterion was associated with a reduced risk of pregnancy outcome (Crowther et al., 2005; Gayle et al., 2010). The policy of not treating women with 2-h PG ≥ 7.8 mmol/L amounts to deliberately exposing the pregnant mothers to unphysiological glycemic level despite our extensive knowledge of the benefits of treatment of mild hyperglycemia during pregnancy (Seshiah et al., 2008a; Landon et al., 2009; Bevier et al., Sensitivity (95% CI) (22.9-36.1) (18.3-30.7) (9.4-19.6) (4.1-11.9) (2.3-8.8) 2- h PG 7.8 13.4 13.6 2- h PG value Macrosomia Sensitivity (95% CI) 21.2 (12.5-33.3) 15.2 (7.9-26.6) 6.1 (2.0-15.6) 1.5 (0.1-9.3) 0.0 (0.0-6.9) (6.8-24.8) Specificity (95% CI) 87.2 (85.0-89.2) 90.2 (88.2-91.9) 95.6 (94.1-96.7) 98.2 (97.1-98.9) 99.3 (98.6-99.7) 86.3 (84.0-88.3) FPG (mmol/L) Test positive 5.0 3.9 29.1 5.1 3.2 24.0 5.5 1.8 13.8 6.1 0.9 7.1 6.6 0.6 4.6 the present practice of diagnosing GDM by the guidelines recommended by Diabetes In Pregnancy Study Group India (DIPSI) (Seshiah et al., 2009) based on WHO criterion of 2-h PG ≥ 7.8 mmol/L can still be followed in India or adopt IADPSG recommendations. A total of 1,463 consecutive pregnant women with no previous history of GDM/pre GDM underwent a 75g OGTT and fasting, 1-h and 2-h PG were measured. Using the DIPSI criterion, 196 (13.4%) women were diagnosed as GDM. By applying IADPSG recommendation the cumulative prevalence of GDM was 14.6% (n=214). There was no significant difference (P > 0.05) in the discordant pair of diagnosing GDM by the two criteria which in turn implies, that the disagreement in diagnosing GDM by both criteria was not significant (P = 0.21, by Mc Nemar test). The difference in the diagnostic capability between IADPSG and DIPSI was 1.2% which was not significant (P>0.02) (Seshiah et al., 2011). IADPSG recommendation necessarily requires estimation of PG in three blood samples after administrating 75g oral glucose load. Pregnant women despise this procedure, as venous blood is drawn three times and they feel too much of blood is drained. Whereas, DIPSI criterion requires one blood sample drawn at 2-h following a 75g glucose load for estimating the PG. The cost involved in performing IADPSG recommended procedure is high, as this procedure requires three blood tests compared to one blood test of DIPSI. The cost will escalate further, if IADPSG diagnostic procedure is performed in each trimester in high risk population in whom GDM manifests in all trimesters of pregnancy (Seshiah et al., 2007). Among women with normal OGTT results in the first visit when tested in the subsequent visits, 28% of them were detected to have GDM (Seshiah et al., 2007). Hence, DIPSI procedure based on WHO criterion is feasible, sustainable, cost-effective and best buy to diagnose GDM in any country and particularly in less resource nations. IADPSG recommendations are suitable in clinical settings where financial and technical supports are available. The performance of both IADPSG and WHO criteria are similar as per GRADE ratings. #### 5.3 Inadequacy of fasting plasma glucose to diagnose GDM The IADPSG criteria suggests FPG ≥ 5.1 mmol/L but ≤ 7.0 mmol/L to diagnose GDM in the first prenatal visit (Metzger et al., 2010) whereas the authors observed in their study that by applying this criterion of FPG ≥ 5. 1 mmol/L, only 24% (3.2% of the total population) of those diagnosed as GDM using WHO criterion 2-h PG ≥ 7.8 mmol/L would have been classified as GDM (Balaji et al., 2011a). Further, FPG of 5.1 mmol/L was not able to diagnose GDM in comparison to 2-h PG ≥ 7.8 mmol/L (Table 2). This is due to the ethnicity of Asian Indians who have high insulin resistance (IR) and as a consequence, their postprandial plasma glucose is higher compared to Caucasians (Mohan et al., 2007; Snehalatha et al., 2009). Asian and South Asian ethnicity are both independently associated with increased IR in late pregnancy (Retnakaran et al., 2006). Siddhartha Das et al documented an increased IR during pregnancy in Asian Indian Women and IR escalates further in GDM (Das et al., 2010). These studies provide evidence that FPG may not be an appropriate option to diagnose GDM in Asian Indian women. Further, in all GDM, the FPG values do not reflect the postprandial hyperglycemia (Valensi et al., 2009), which is the hallmark of GDM (Weiss et al., 2000). In addition, there is a paucity of data regarding the reproducibility of the FPG test (Sacks et al., 2010). Hence, administering 75g oral glucose load and measuring 2-h PG serves as a one-step definitive procedure to diagnose GDM in less serviced regions. Perucchini et al also suggest one-step diagnostic procedure, though their observation was based on different ethnic population (Perucchini et al., 1999). the present practice of diagnosing GDM by the guidelines recommended by Diabetes In Pregnancy Study Group India (DIPSI) (Seshiah et al., 2009) based on WHO criterion of 2-h PG ≥ 7.8 mmol/L can still be followed in India or adopt IADPSG recommendations. A total of 1,463 consecutive pregnant women with no previous history of GDM/pre GDM underwent a 75g OGTT and fasting, 1-h and 2-h PG were measured. Using the DIPSI criterion, 196 (13.4%) women were diagnosed as GDM. By applying IADPSG recommendation the cumulative prevalence of GDM was 14.6% (n=214). There was no significant difference (P > 0.05) in the discordant pair of diagnosing GDM by the two criteria which in turn implies, that the disagreement in diagnosing GDM by both criteria was not significant (P = 0.21, by Mc Nemar test). The difference in the diagnostic capability between IADPSG and DIPSI was 1.2% which was not significant (P>0.02) (Seshiah et al., 2011). IADPSG recommendation necessarily requires estimation of PG in three blood samples after administrating 75g oral glucose load. Pregnant women despise this procedure, as venous blood is drawn three times and they feel too much of blood is drained. Whereas, DIPSI criterion requires one blood sample drawn at 2-h following a 75g glucose load for estimating the PG. The cost involved in performing IADPSG recommended procedure is high, as this procedure requires three blood tests compared to one blood test of DIPSI. The cost will escalate further, if IADPSG diagnostic procedure is performed in each trimester in high risk population in whom GDM manifests in all trimesters of pregnancy (Seshiah et al., 2007). Among women with normal OGTT results in the first visit when tested in the subsequent visits, 28% of them were detected to have GDM (Seshiah et al., 2007). Hence, DIPSI procedure based on WHO criterion is feasible, sustainable, cost-effective and best buy to diagnose GDM in any country and particularly in less resource nations. IADPSG recommendations are suitable in clinical settings where financial and technical supports are available. The performance of both IADPSG and WHO criteria are similar as per GRADE ratings. The IADPSG criteria suggests FPG ≥ 5.1 mmol/L but ≤ 7.0 mmol/L to diagnose GDM in the first prenatal visit (Metzger et al., 2010) whereas the authors observed in their study that by applying this criterion of FPG ≥ 5. 1 mmol/L, only 24% (3.2% of the total population) of those diagnosed as GDM using WHO criterion 2-h PG ≥ 7.8 mmol/L would have been classified as GDM (Balaji et al., 2011a). Further, FPG of 5.1 mmol/L was not able to diagnose GDM in comparison to 2-h PG ≥ 7.8 mmol/L (Table 2). This is due to the ethnicity of Asian Indians who have high insulin resistance (IR) and as a consequence, their postprandial plasma glucose is higher compared to Caucasians (Mohan et al., 2007; Snehalatha et al., 2009). Asian and South Asian ethnicity are both independently associated with increased IR in late pregnancy (Retnakaran et al., 2006). Siddhartha Das et al documented an increased IR during pregnancy in Asian Indian Women and IR escalates further in GDM (Das et al., 2010). These studies provide evidence that FPG may not be an appropriate option to diagnose GDM in Asian Indian women. Further, in all GDM, the FPG values do not reflect the postprandial hyperglycemia (Valensi et al., 2009), which is the hallmark of GDM (Weiss et al., 2000). In addition, there is a paucity of data regarding the reproducibility of the FPG test (Sacks et al., 2010). Hence, administering 75g oral glucose load and measuring 2-h PG serves as a one-step definitive procedure to diagnose GDM in less serviced regions. Perucchini et al also suggest one-step diagnostic procedure, though their observation was 5.3 Inadequacy of fasting plasma glucose to diagnose GDM based on different ethnic population (Perucchini et al., 1999). #### 5.4 The validation of WHO criterion (DIPSI criterion) based on the fetal outcome The authors investigated whether the diagnosis of GDM by WHO criterion is rational based on the fetal outcome (N = 1463). Macrosomia was the end point of this study as this is the most common morbidity of GDM (Jovanovic, 2001). They observed that there was no statistically significant difference in the mean birth weight of neonates born to women in the normal glucose tolerance (NGT) and with intervention in GDM groups (P=0.705) (Balaji et al., 2011b). This was due to the medical nutrition therapy (MNT) and/or insulin in maintaining FPG ~ 5.0 mmol/L and 2-h post meal ~ 6.7 mmol/L in GDM women. Intervention helped in maintaining the pregnancy outcome in GDM women equivalent to that of NGT women. Gayle et al also observed that diagnosis of GDM with OGTT 2-h PG ≥ 7.8 mmol/L and treatment in a combined diabetes antenatal clinic is worthwhile with a decreased macrosomia rate and fewer emergency cesarean sections (Gayle et al., 2010). The distribution of birth weight of neonates born to GDM and NGT women were similar (Figure 2) in the study conducted by the authors, indicating that the intervention given to pregnant women with 2-h PG ≥ 7.8 mmol/L had a significant effect in obtaining neonatal birth weight appropriate for gestational age. The level of association between macrosomia and GDM status after controlling the factors: maternal age, gestational age, family history of diabetes and BMI was elucidated. It was found that, the GDM status (2-h PG ≥ 7.8 mmol/L) of the pregnant women after intervention was not associated with macrosomia (adjusted OR = 0.752; 95% CI (0.406-1.390); P=0.363). There are publications confirming that treatment of GDM women as defined by WHO criterion was associated with a reduced risk of pregnancy outcome (Crowther et al., 2005; Gayle et al., 2010). In pregnancy, the decision to perform a placebo controlled trial requires clinical equipoise (Gifford et al., 2001). Hence, in this study, the authors did not have a control group of untreated pregnant women with 2-h PG ≥ 7.8 mmol/L, as there are evidences confirming that the treatment of GDM women as defined by WHO criterion was associated with a reduced risk of pregnancy outcome (Crowther et al., 2005; Gayle et al., 2010). The policy of not treating women with 2-h PG ≥ 7.8 mmol/L amounts to deliberately exposing the pregnant mothers to unphysiological glycemic level despite our extensive knowledge of the benefits of treatment of mild hyperglycemia during pregnancy (Seshiah et al., 2008a; Landon et al., 2009; Bevier et al., Gestational Diabetes Mellitus - A Perspective 29 pregnancy are likely to have delayed diagnosis and may not receive appropriate medical care. Further, early screening for glucose intolerance and care could avoid some diabetes related complications in women with gestational diabetes (Bartha et al., 2003). To substantiate the above observation the present author's group screened 207 pregnant women attending their referral centre for diabetes and pregnancy with a 75g OGTT (Seshiah et al., 2006). Among them, 87 (42.03%) were diagnosed with GDM. Women in whom GDM was detected between 0 - 23 weeks of gestation were classified as Group 1 [54 (62.7%)] and beyond 24 weeks of gestation as Group 2 [33 (37.93%)]. All of them were treated and followed till confinement. There was no statistically significant difference (P < 0.05) between the birth weight of the neonates born to Normal Glucose Tolerance (NGT) women (3.28 0.50 kg) and GDM women in group 1 (3.13 ± 0.55 kg). In group 2, the neonatal birth weight was 3.42 ± 0.58 kg which is the upper limit of the normal range in Indian new born babies. In India, the normal birth weight varies between 2.5 to 3.5 kg (Paul et al., 2002). The observation of this study was that, by early detection of glucose intolerance during pregnancy and by giving adequate care to the antenatal women, a good fetal outcome can be achieved similar to that of The goal in the management is to avoid both low birth weight and macrosomic babies, as they are prone to develop diabetes in their adolescent and adult life (Jovanovic, 1998). In India, both under nutrition and over nutrition exists during pregnancy. There are two reported studies in India that relates size at birth to future risk of type 2 DM. In Mysore, low birth weight did not increase the risk of diabetes but babies who were short and fat at birth (higher BMI) were at increased risk (Fall et al., 1998). Fall et al speculate that the rise in type 2 DM in Indian urban populations would have been triggered by mild obesity in mothers, leading to glucose intolerance during pregnancy, macrosomic changes in the fetus and insulin deficiency in adult life (Fall et al., 1998). Yet another study attributes high prevalence of type 2 DM and IGT in Indian people linked to poor fetal growth (Yajnik et al., 1995) The meal pattern should provide adequate calories and nutrients to meet the needs of pregnancy metabolism. The meal plan advised has to be simple and easy to practice. The MNT recommended is based on their routine diet habit and glycemic excursions that occur with the meal. In a normal person, the peaking of the plasma glucose is high after breakfast (due to 'Dawn phenomenon') than after lunch and dinner, and the insulin secretion also matches the glycemic excursions that occur with these three meals (Polonsky et al., 1988). Since GDM mothers have deficiency in first phase insulin secretion, the quantity of food at one time should also be less, to overcome this insulin deficiency, particularly after breakfast. To avoid the post prandial plasma glucose peaking with breakfast, the authors guide their women with GDM to distribute calorie consumption especially the breakfast into two portions 'Split Breakfast'. This implies splitting the usual breakfast into two halves and consuming these portions with a two-hour gap in between. By this, the undue peak in plasma glucose levels after ingestion of the total quantity of breakfast at one time is avoided. NGT pregnant women (Seshiah et al., 2006, 2008b). which is at variant to Fall et al observation (Fall et al., 1998). 7.1 Medical Nutrition Therapy (MNT) 7. Management 1999; Negrato et al., 2008). Wahi et al observed in their prospective study, the advantage of adhering to a cut-off level of 2-h PG ≥ 7.8 mmol/L in diagnosis and management of GDM for a significantly positive effect on pregnancy outcomes (Wahi et al., 2011). Fetal exposure to high maternal glucose (1-h PG > 7.2 mmol/L with 50g GCT) in the absence of preexisting diabetes/GDM may contribute to the development of overweight/obesity in the offspring, independent of maternal pre-pregnancy BMI (Deierlein et al., 2011). All these studies validate WHO/DIPSI criterion for the diagnosis of GDM Fig. 2. Neonate Birth weight distribution of women with NGT and GDM #### 6. Gestational weeks for screening The current recommendation is to perform screening test between 24 - 28 weeks of gestation, though there are reports that claim, about 40% to 66% of women with GDM can be detected early during pregnancy (Super et al., 1991; Nahum et al., 2002). Nahum et al also suggest that the ideal period to screen for GDM is around 16 weeks of gestation and even earlier in high-risk groups with a history of fetal wastage (Nahum et al., 2002). This is due to the embryological development of fetal β cells. Each islet cell functions as an endocrine organ and differentiates between 10th and 12th weeks of gestation. They recognize and respond to maternal glycemia before 15 weeks of gestation, suggesting that metabolic perturbations are underway before diagnosis and that earlier screening and intervention may be warranted (Tisi et al., 2011). The study performed by the present authors group in the DIPAP project revealed that, 16.3% had glucose intolerance within 16 weeks, 22.4% between 17 - 23 weeks and remaining 61.3% more than 24 weeks of gestation (Seshiah et., 2007). If a pregnant woman has an A1c level > 6%, she is more likely to be an overt diabetic (Balaji et al., 2007). These studies stress the need for screening for GDM during the early weeks of gestation. If the test is normal in the first visit, the test has to be repeated in the subsequent visits. GDM diagnosis may not be missed by screening around 24 -28 weeks of gestation, but a substantial number of pregnant women who develop GDM in the earlier weeks of 1999; Negrato et al., 2008). Wahi et al observed in their prospective study, the advantage of adhering to a cut-off level of 2-h PG ≥ 7.8 mmol/L in diagnosis and management of GDM for a significantly positive effect on pregnancy outcomes (Wahi et al., 2011). Fetal exposure to high maternal glucose (1-h PG > 7.2 mmol/L with 50g GCT) in the absence of preexisting diabetes/GDM may contribute to the development of overweight/obesity in the offspring, independent of maternal pre-pregnancy BMI (Deierlein et al., 2011). All these studies validate WHO/DIPSI criterion for the diagnosis of GDM 6. Gestational weeks for screening Fig. 2. Neonate Birth weight distribution of women with NGT and GDM The current recommendation is to perform screening test between 24 - 28 weeks of gestation, though there are reports that claim, about 40% to 66% of women with GDM can be detected early during pregnancy (Super et al., 1991; Nahum et al., 2002). Nahum et al also suggest that the ideal period to screen for GDM is around 16 weeks of gestation and even earlier in high-risk groups with a history of fetal wastage (Nahum et al., 2002). This is due to the embryological development of fetal β cells. Each islet cell functions as an endocrine organ and differentiates between 10th and 12th weeks of gestation. They recognize and respond to maternal glycemia before 15 weeks of gestation, suggesting that metabolic perturbations are underway before diagnosis and that earlier screening and intervention may be warranted (Tisi et al., 2011). The study performed by the present authors group in the DIPAP project revealed that, 16.3% had glucose intolerance within 16 weeks, 22.4% between 17 - 23 weeks and remaining 61.3% more than 24 weeks of gestation (Seshiah et., 2007). If a pregnant woman has an A1c level > 6%, she is more likely to be an overt diabetic (Balaji et al., 2007). These studies stress the need for screening for GDM during the early weeks of gestation. If the test is normal in the first visit, the test has to be repeated in the subsequent visits. GDM diagnosis may not be missed by screening around 24 -28 weeks of gestation, but a substantial number of pregnant women who develop GDM in the earlier weeks of pregnancy are likely to have delayed diagnosis and may not receive appropriate medical care. Further, early screening for glucose intolerance and care could avoid some diabetes related complications in women with gestational diabetes (Bartha et al., 2003). To substantiate the above observation the present author's group screened 207 pregnant women attending their referral centre for diabetes and pregnancy with a 75g OGTT (Seshiah et al., 2006). Among them, 87 (42.03%) were diagnosed with GDM. Women in whom GDM was detected between 0 - 23 weeks of gestation were classified as Group 1 [54 (62.7%)] and beyond 24 weeks of gestation as Group 2 [33 (37.93%)]. All of them were treated and followed till confinement. There was no statistically significant difference (P < 0.05) between the birth weight of the neonates born to Normal Glucose Tolerance (NGT) women (3.28 0.50 kg) and GDM women in group 1 (3.13 ± 0.55 kg). In group 2, the neonatal birth weight was 3.42 ± 0.58 kg which is the upper limit of the normal range in Indian new born babies. In India, the normal birth weight varies between 2.5 to 3.5 kg (Paul et al., 2002). The observation of this study was that, by early detection of glucose intolerance during pregnancy and by giving adequate care to the antenatal women, a good fetal outcome can be achieved similar to that of NGT pregnant women (Seshiah et al., 2006, 2008b). #### 7. Management The goal in the management is to avoid both low birth weight and macrosomic babies, as they are prone to develop diabetes in their adolescent and adult life (Jovanovic, 1998). In India, both under nutrition and over nutrition exists during pregnancy. There are two reported studies in India that relates size at birth to future risk of type 2 DM. In Mysore, low birth weight did not increase the risk of diabetes but babies who were short and fat at birth (higher BMI) were at increased risk (Fall et al., 1998). Fall et al speculate that the rise in type 2 DM in Indian urban populations would have been triggered by mild obesity in mothers, leading to glucose intolerance during pregnancy, macrosomic changes in the fetus and insulin deficiency in adult life (Fall et al., 1998). Yet another study attributes high prevalence of type 2 DM and IGT in Indian people linked to poor fetal growth (Yajnik et al., 1995) which is at variant to Fall et al observation (Fall et al., 1998). #### 7.1 Medical Nutrition Therapy (MNT) The meal pattern should provide adequate calories and nutrients to meet the needs of pregnancy metabolism. The meal plan advised has to be simple and easy to practice. The MNT recommended is based on their routine diet habit and glycemic excursions that occur with the meal. In a normal person, the peaking of the plasma glucose is high after breakfast (due to 'Dawn phenomenon') than after lunch and dinner, and the insulin secretion also matches the glycemic excursions that occur with these three meals (Polonsky et al., 1988). Since GDM mothers have deficiency in first phase insulin secretion, the quantity of food at one time should also be less, to overcome this insulin deficiency, particularly after breakfast. To avoid the post prandial plasma glucose peaking with breakfast, the authors guide their women with GDM to distribute calorie consumption especially the breakfast into two portions 'Split Breakfast'. This implies splitting the usual breakfast into two halves and consuming these portions with a two-hour gap in between. By this, the undue peak in plasma glucose levels after ingestion of the total quantity of breakfast at one time is avoided. Gestational Diabetes Mellitus - A Perspective 31 1991; Rossetti et al., 1990). Another advantage is that, the human placental transfer of glibenclamide is negligible. Maternally administered glibenclamide in pharmacologic doses, and even doses greatly exceeding therapeutic levels, may not reach the fetus (Elliott et al., 1991). The landmark study of Langer et al concluded that glyburide was as effective as insulin in maintaining the desired glycemic levels and resulted in a comparable outcome (Langer et al., 2000). The author's group undertook a prospective study comparing insulin and glibenclamide in GDM. In this study, both Glibenclamide and insulin treatment achieved equally good glycemic control and the perinatal outcome was not different (Anjalakshi et al., 2007). The observation of this study was that the mean dose of glibenclamide required at term was 1.45 ± 0.57 mg/day and mean insulin requirement at term was 21.7 ± 13.55 units/day to achieve the same glycemic level (Anjalakshi et al., 2007). It is noteworthy that Glibenclamide is very much economical and cost effective compared to insulin, which is not only expensive but also inconvenient as it has to be taken parenterally. Yet an another observation was that in Indian population, the dose of glibenclamide required is very much less compared to the other published studies (Langer et al., 2005). Women with polycystic ovary syndrome (PCOS) are advised metformin to induce ovulation. The drug is not withdrawn if a woman conceives while on metformin therapy and the maximum dose prescribed in the author's clinical practice is 1500 mg. If the plasma glucose is not under control with metformin, insulin is always added. No adverse pregnancy outcome with metformin therapy was observed. A preliminary study showed that metformin was safe in pregnant, glucose intolerant women either as an adjunct to insulin treatment or even as a monotherapy (Ramachandran et al., 2005). A prospective study found no adverse influence on the pregnancy outcome in PCOS women treated Metformin in gestational diabetes (MiG) trial found that in women with gestational diabetes mellitus, metformin (alone or with supplemental insulin) was not associated with increased The success of the treatment for a woman with GDM depends on the glycemic control maintained with meal plan or pharmacological intervention. To know the effectiveness of Once diagnosis is made, medical nutritional therapy (MNT) is advised initially for two weeks. If MNT fails to achieve control i.e., FPG ≥ 5.0 mmol/L and/or 2-h PG ≥ 6.7 Once target blood glucose is achieved, woman with GDM till the 28th week of gestation require laboratory monitoring of both fasting and 2-h post breakfast once a month and After the 28th week of gestation, the laboratory monitoring should be more frequent After 32 weeks of gestation, laboratory monitoring should be done once a week till In high risk pregnancies, frequency of monitoring may be intensified with self throughout pregnancy with Metformin (Glueck et al., 2004). treatment, monitoring of glycemic control is essential. at other time of the day as the clinician decides. atleast once in 2 weeks, if need be more frequently. 8. Monitoring glycemic control mmol/L, insulin may be initiated. monitoring of blood glucose (SMBG). perinatal complications as compared with insulin (Rowan et al., 2008). 7.3.2 Metformin delivery #### 7.2 Insulin therapy 7.2.1 Human insulin The policy followed in India is to advise human insulin in women with GDM who failed to achieve FPG of ≤ 5.0 mmol/L and 2-h post meal plasma glucose level of ≤ 6.7 mmol/L with MNT. The aim is to maintain post meal peak plasma glucose level of ≤ 6.7 mmol/L. This time point is suggested as the diagnosis of GDM is made with 2-h PG and it is easy to remember the same timing. A number of studies have established the benefits of maintaining the plasma glucose at this level (Franks et al., 2006; de Sereday et al., 2003; Ben-Haroush et al., 2004). However whichever time is targeted for monitoring glycemic control and adjusting the insulin dose, the blood tests have to be done at the same time at each visit. GDM women usually have high post breakfast plasma glucose level compared to post lunch and post dinner. The period between breakfast and lunch are often problematical because of the physiological tendency to hyperglycemia at this time and may necessitate substantial increases in the morning dose of short acting insulin, together with careful adjustment of meal timing and snacks to avoid pre-lunch hypoglycemia (Langer et al., 2000). #### 7.2.2 Insulin analogues Due to the pharmacokinetic action of human regular insulin, a considerable segment of pregnant women with GDM, fail to achieve optimum glycemic control, mostly the post prandial plasma glucose. In them, the best option is to administer ultra short acting analogues, insulin lispro (Humalog) or insulin aspart (Novo rapid). These analogues improve the post prandial glucose control in pregnant women with type 1, type 2 DM and GDM, and are also safe and effective (Hermansen et al., 2002; Jovanovic et al., 1999). The authors group conducted an open label trial using a large independent cohort of GDM patients to evaluate the efficacy, safety and foetal outcome for Biphasic Insulin aspart (BIAsp 30) compared with biphasic human insulin (BHI 30) in the management of GDM (Balaji et al., 2010). GDM women (N = 323) who remained unable to maintain a FPG ≤ 5.0 mmol/L and 2-h PG ≤ 6.7 mmol/L with MNT were randomly allocated in a 1:1 ratio to receive either BIAsp 30 (Group A) or BHI 30 (Group B). There was no statistical significance in the levels of glycaemic control achieved by the groups by labour onset. However, the mean total insulin dose administered by the last visit was significantly lower for Group A [19.83±15.75 U compared with 26.34±23.15 U for Group B (p=0.006)], implying that those receiving BHI 30 required a higher dose to achieve a similar degree of glycaemic control. The frequency of macrosomia was 6.3% in Group A and 6.9% in Group B. Although the proportion of macrosomia was numerically higher for Group B than Group A, the difference was not statistically significant (p=0.819). It was found that BIAsp 30 was non-inferior to BHI 30 and was well tolerated during pregnancy. Yet in another study, the authors observed that pregnant women found BIAsp convenient as this preparation allows flexibility in the meal time insulin dosing and did not disturb their routine life pattern. Most importantly, BIAsp was found to be safe during pregnancy (Balaji et al., 2010). #### 7.3 Oral hypoglycemic agents 7.3.1 Glibenclamide Glibenclamide (Glyburide) may be an alternative safe therapy for many GDM women who are hesitant to take insulin. This drug decreases the insulin resistance and improves insulin secretion, the pathogenic factors in the causation of hyperglycemia in GDM (Groop et al., 1991; Rossetti et al., 1990). Another advantage is that, the human placental transfer of glibenclamide is negligible. Maternally administered glibenclamide in pharmacologic doses, and even doses greatly exceeding therapeutic levels, may not reach the fetus (Elliott et al., 1991). The landmark study of Langer et al concluded that glyburide was as effective as insulin in maintaining the desired glycemic levels and resulted in a comparable outcome (Langer et al., 2000). The author's group undertook a prospective study comparing insulin and glibenclamide in GDM. In this study, both Glibenclamide and insulin treatment achieved equally good glycemic control and the perinatal outcome was not different (Anjalakshi et al., 2007). The observation of this study was that the mean dose of glibenclamide required at term was 1.45 ± 0.57 mg/day and mean insulin requirement at term was 21.7 ± 13.55 units/day to achieve the same glycemic level (Anjalakshi et al., 2007). It is noteworthy that Glibenclamide is very much economical and cost effective compared to insulin, which is not only expensive but also inconvenient as it has to be taken parenterally. Yet an another observation was that in Indian population, the dose of glibenclamide required is very much less compared to the other published studies (Langer et al., 2005). #### 7.3.2 Metformin 30 Gestational Diabetes The policy followed in India is to advise human insulin in women with GDM who failed to achieve FPG of ≤ 5.0 mmol/L and 2-h post meal plasma glucose level of ≤ 6.7 mmol/L with MNT. The aim is to maintain post meal peak plasma glucose level of ≤ 6.7 mmol/L. This time point is suggested as the diagnosis of GDM is made with 2-h PG and it is easy to remember the same timing. A number of studies have established the benefits of maintaining the plasma glucose at this level (Franks et al., 2006; de Sereday et al., 2003; Ben-Haroush et al., 2004). However whichever time is targeted for monitoring glycemic control and adjusting the insulin dose, the blood tests have to be done at the same time at each visit. GDM women usually have high post breakfast plasma glucose level compared to post lunch and post dinner. The period between breakfast and lunch are often problematical because of the physiological tendency to hyperglycemia at this time and may necessitate substantial increases in the morning dose of short acting insulin, together with careful adjustment of Due to the pharmacokinetic action of human regular insulin, a considerable segment of pregnant women with GDM, fail to achieve optimum glycemic control, mostly the post prandial plasma glucose. In them, the best option is to administer ultra short acting analogues, insulin lispro (Humalog) or insulin aspart (Novo rapid). These analogues improve the post prandial glucose control in pregnant women with type 1, type 2 DM and The authors group conducted an open label trial using a large independent cohort of GDM patients to evaluate the efficacy, safety and foetal outcome for Biphasic Insulin aspart (BIAsp 30) compared with biphasic human insulin (BHI 30) in the management of GDM (Balaji et al., 2010). GDM women (N = 323) who remained unable to maintain a FPG ≤ 5.0 mmol/L and 2-h PG ≤ 6.7 mmol/L with MNT were randomly allocated in a 1:1 ratio to receive either BIAsp 30 (Group A) or BHI 30 (Group B). There was no statistical significance in the levels of glycaemic control achieved by the groups by labour onset. However, the mean total insulin dose administered by the last visit was significantly lower for Group A [19.83±15.75 U compared with 26.34±23.15 U for Group B (p=0.006)], implying that those receiving BHI 30 required a higher dose to achieve a similar degree of glycaemic control. The frequency of macrosomia was 6.3% in Group A and 6.9% in Group B. Although the proportion of macrosomia was numerically higher for Group B than Group A, the difference was not statistically significant (p=0.819). It was found that BIAsp 30 was non-inferior to BHI 30 and was well tolerated during pregnancy. Yet in another study, the authors observed that pregnant women found BIAsp convenient as this preparation allows flexibility in the meal time insulin dosing and did not disturb their routine life pattern. Most importantly, BIAsp was found to be safe during Glibenclamide (Glyburide) may be an alternative safe therapy for many GDM women who are hesitant to take insulin. This drug decreases the insulin resistance and improves insulin secretion, the pathogenic factors in the causation of hyperglycemia in GDM (Groop et al., GDM, and are also safe and effective (Hermansen et al., 2002; Jovanovic et al., 1999). meal timing and snacks to avoid pre-lunch hypoglycemia (Langer et al., 2000). 7.2 Insulin therapy 7.2.1 Human insulin 7.2.2 Insulin analogues pregnancy (Balaji et al., 2010). 7.3.1 Glibenclamide 7.3 Oral hypoglycemic agents Women with polycystic ovary syndrome (PCOS) are advised metformin to induce ovulation. The drug is not withdrawn if a woman conceives while on metformin therapy and the maximum dose prescribed in the author's clinical practice is 1500 mg. If the plasma glucose is not under control with metformin, insulin is always added. No adverse pregnancy outcome with metformin therapy was observed. A preliminary study showed that metformin was safe in pregnant, glucose intolerant women either as an adjunct to insulin treatment or even as a monotherapy (Ramachandran et al., 2005). A prospective study found no adverse influence on the pregnancy outcome in PCOS women treated throughout pregnancy with Metformin (Glueck et al., 2004). Metformin in gestational diabetes (MiG) trial found that in women with gestational diabetes mellitus, metformin (alone or with supplemental insulin) was not associated with increased perinatal complications as compared with insulin (Rowan et al., 2008). #### 8. Monitoring glycemic control The success of the treatment for a woman with GDM depends on the glycemic control maintained with meal plan or pharmacological intervention. To know the effectiveness of treatment, monitoring of glycemic control is essential. Gestational Diabetes Mellitus - A Perspective 33 increased prevalence of diabetes in India. This is likely to be true as GDM has a far reaching consequence in predisposing their offsprings to glucose intolerance. This observation was substantiated and documented in Pima Indians (Dabelea et al., 2000). The children born in 1965 to women with GDM were followed up till 2000. By the time they reached 35 years, more than half of the group had diabetes (Dabelea et al., 2000). Hence as a policy to identify GDM and its consequences on the infant, a 75 g OGTT has been recommended to all women in the population during the third trimester of pregnancy (Dabelea et al., 2000). Now it is obvious that taking care of women with GDM is the first step in the primary prevention of The important aspect of diabetes and pregnancy is that, the intrauterine millieu interieur, whether one of nutritional deprivation or one of nutritional plenty, results in changes in fetal pancreatic development and peripheral response to insulin that may lead to adultonset GDM and type 2 DM (Savona-Ventura & Chircop, 2003). Thus, the timely action taken now in screening all pregnant women for glucose intolerance, achieving euglycemia in them and ensuring adequate nutrition may prevent in all probability, the vicious cycle of transmitting glucose intolerance from one generation to another (Aerts, 2004). GDM offers an important opportunity for the development, testing and implementation of clinical 'No single period in human development provides a greater potential than pregnancy for a long range pay off via relatively short range period of enlightened metabolic manipulation' - GDM women are at increased risk of future diabetes as are their children and following Asian women are ethnically more prone to develop glucose intolerance compared to GDM based on 2-h 75g OGTT defined by WHO predicts adverse pregnancy outcome A 2-h 75g post plasma glucose ≥ 7.8 mmol/L serves both as screening and diagnostic criteria which is a technically simple economical and evidence based one step IADPSG recommendations are suitable in clinical settings where technical and financial Early screening for glucose intolerance and care could avoid some diabetes related Women with NGT in the first visit are advised to undergo glucose tolerance test in the Occurrence of macrosomia was continuum as the FPG increased from 5.0 mmol/L and The meal pattern advised has to be simple, and easy to understand and follow. The goal is to maintain mean plasma glucose of 5.8 to 6.1 mmol/L Prevalence of GDM varies from one region to another region in the same country. Compared with selective screening, Universal screening for GDM detects more cases strategies for diabetes prevention (Buchanan et al., 2007). and improves maternal and offspring prognosis. complications in women with gestational diabetes diabetes. Norbert Frienkel. 11. Summary generations. procedure. other ethnic groups. and warrants treatment. supports are available. subsequent trimesters. 2-h PG increased from 6.7 mmol/L. Continuous glucose monitoring devices are available but these equipments need special training and are expensive. These devices may be useful in high risk pregnancies to know the glycemic fluctuations and to plan proper insulin dosage. #### 8.1 Glycosylated haemoglobin – A1c levels If the glucose intolerance is detected in the early pregnancy, A1c level will be helpful to differentiate between a pre gestational diabetic and GDM. If A1c level is more than 6% (Balaji et al., 2007), the chances are that she may be a pre GDM or GDM, in whom the glucose intolerance was detected in the early weeks of pregnancy; all the more validating that the screening needs to be performed in the early weeks of gestation. The estimation of A1c may help in distinguishing a pre GDM from an early onset GDM, but not essential, as this differentiation is of no consequence in clinical practice, as the treatment approach is going to be the same (Seshiah et al., 2007). Further, A1c is not estimated in the community health centres, barring a few tertiary care hospitals due to the difficulty in standardization, inadequate technical support and the cost. #### 8.2 Measuring other parameters The blood pressure has to be monitored during every visit. Examination of the fundus and estimation of microalbuminuria, every trimester is recommended. #### 8.3 Ultrasound fetal measurement The management of gestational diabetes, based on the foetal growth by ultrasonogram demands that the fetus at risk must first manifest overgrowth before treatment decisions are made. Further, the cost of performing a number of ultrasonograms to monitor the foetal growth and recommending therapy has to be kept in mind. Until there is evidence to absolutely prove that ignoring maternal hyperglycemia when the fetal growth patterns appear normal on the ultrasonogram, it is prudent to achieve and maintain normoglycemia in every pregnancy complicated by gestational diabetes. #### 9. Target glycemic level Increased birth weight of neonates occurred even when the mother's glucose tolerance was less than the glycemic criteria recommended by WHO (2-h PG > 7.8 mmol/L) for diagnosis of GDM. Increasing carbohydrate intolerance in women without overt GDM was associated with graded increase in the incidence of macrosomia (Sermer et al., 1998). The author's group documented that the occurrence of macrosomia was continuum as the FPG increased > 5.0 mmol/L (Seshiah et al., 2008c) and the 2-h PG > 6.7 mmol/L (Balaji et al., 2006). Thus maintenance of mean plasma glucose level ~ 5.8 to 6.1 mmol/L is desirable for a good fetal outcome (Langer et al., 1989). This is possible if FPG and peak postprandial glucose levels are maintained ~ 5.0 mmol/L (4.4-5.0 mmol/L) and ~ 6.7 mmol/L (6.1 – 6.7 mmol/L, respectively) #### 10. Prevention of type 2 dm The screening for glucose intolerance during pregnancy is not done routinely and probably the undiagnosed glucose intolerance that has been occurring in the past has resulted in the Continuous glucose monitoring devices are available but these equipments need special training and are expensive. These devices may be useful in high risk pregnancies to If the glucose intolerance is detected in the early pregnancy, A1c level will be helpful to differentiate between a pre gestational diabetic and GDM. If A1c level is more than 6% (Balaji et al., 2007), the chances are that she may be a pre GDM or GDM, in whom the glucose intolerance was detected in the early weeks of pregnancy; all the more validating that the screening needs to be performed in the early weeks of gestation. The estimation of A1c may help in distinguishing a pre GDM from an early onset GDM, but not essential, as this differentiation is of no consequence in clinical practice, as the treatment approach is going to be the same (Seshiah et al., 2007). Further, A1c is not estimated in the community health centres, barring a few tertiary care hospitals due to the difficulty in standardization, The blood pressure has to be monitored during every visit. Examination of the fundus and The management of gestational diabetes, based on the foetal growth by ultrasonogram demands that the fetus at risk must first manifest overgrowth before treatment decisions are made. Further, the cost of performing a number of ultrasonograms to monitor the foetal growth and recommending therapy has to be kept in mind. Until there is evidence to absolutely prove that ignoring maternal hyperglycemia when the fetal growth patterns appear normal on the ultrasonogram, it is prudent to achieve and maintain normoglycemia Increased birth weight of neonates occurred even when the mother's glucose tolerance was less than the glycemic criteria recommended by WHO (2-h PG > 7.8 mmol/L) for diagnosis of GDM. Increasing carbohydrate intolerance in women without overt GDM was associated with graded increase in the incidence of macrosomia (Sermer et al., 1998). The author's group documented that the occurrence of macrosomia was continuum as the FPG increased > 5.0 mmol/L (Seshiah et al., 2008c) and the 2-h PG > 6.7 mmol/L (Balaji et al., 2006). Thus maintenance of mean plasma glucose level ~ 5.8 to 6.1 mmol/L is desirable for a good fetal outcome (Langer et al., 1989). This is possible if FPG and peak postprandial glucose levels are maintained ~ 5.0 mmol/L (4.4-5.0 mmol/L) and ~ 6.7 mmol/L (6.1 – 6.7 mmol/L, The screening for glucose intolerance during pregnancy is not done routinely and probably the undiagnosed glucose intolerance that has been occurring in the past has resulted in the estimation of microalbuminuria, every trimester is recommended. in every pregnancy complicated by gestational diabetes. know the glycemic fluctuations and to plan proper insulin dosage. 8.1 Glycosylated haemoglobin – A1c levels inadequate technical support and the cost. 8.2 Measuring other parameters 8.3 Ultrasound fetal measurement 9. Target glycemic level 10. Prevention of type 2 dm respectively) increased prevalence of diabetes in India. This is likely to be true as GDM has a far reaching consequence in predisposing their offsprings to glucose intolerance. This observation was substantiated and documented in Pima Indians (Dabelea et al., 2000). The children born in 1965 to women with GDM were followed up till 2000. By the time they reached 35 years, more than half of the group had diabetes (Dabelea et al., 2000). Hence as a policy to identify GDM and its consequences on the infant, a 75 g OGTT has been recommended to all women in the population during the third trimester of pregnancy (Dabelea et al., 2000). Now it is obvious that taking care of women with GDM is the first step in the primary prevention of diabetes. The important aspect of diabetes and pregnancy is that, the intrauterine millieu interieur, whether one of nutritional deprivation or one of nutritional plenty, results in changes in fetal pancreatic development and peripheral response to insulin that may lead to adultonset GDM and type 2 DM (Savona-Ventura & Chircop, 2003). Thus, the timely action taken now in screening all pregnant women for glucose intolerance, achieving euglycemia in them and ensuring adequate nutrition may prevent in all probability, the vicious cycle of transmitting glucose intolerance from one generation to another (Aerts, 2004). GDM offers an important opportunity for the development, testing and implementation of clinical strategies for diabetes prevention (Buchanan et al., 2007). 'No single period in human development provides a greater potential than pregnancy for a long range pay off via relatively short range period of enlightened metabolic manipulation' - Norbert Frienkel. #### 11. Summary Gestational Diabetes Mellitus - A Perspective 35 Ben-Haroush, A.; Yogev, Y.; Chen R.; et al (2004). The post prandial glucose profile in the Bevier, WC.; Fischer, R.; Jovanovic, L. (1999). Treatment of women with an abnormal Buchanan, TA.; Xiang, A.; Kjos, SL.; et al. (2007). What is Gestational Diabetes.Diabetes Care. Cosson, E.; Benthimol, M.; Carbilon, L.; et al. (2004). Universal screening for gestational Cosson, E. (2004). Screening and insulin sensitivity in gestational diabetes. Abstract volume Crowther, CA.; Hiller, JE.; Moss, JR.; et al. (2005). Effect of treatment of gestational diabetes Dabelea, D.; Knowler, WC.; Pettitt, DJ. (2000). Effect of diabetes in pregnancy and offspring: Das, S.; Behera, MK.; Misra, S.; et al. (2010). Β-cell function and insulin resistance in de Sereday, MS.; Damiano, MM.; Gonzalez, CD.; et al. (2003). Diagnostic criteria for Deierlein, AL.; Siega-Riz, AM.; Chantala, K.; et al. (2011). The association between maternal Dornhorst, A & Rossi, M. (1998). Risk and Prevention of Type 2 Diabetes in women with Dornhost, A.; Paterson, CM. ; Nicholls, JS. ; et al. (1992) High prevalence of GDM in women Elliott, BD.; Langer, O.; Schenker, S.; et al. (1991). Insignificant transfer of glyburide occurs Fall, CH.; Stein, CE.; Kumaran, K.; et al. (1998). 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Mc Cance DR, Maresh M, Sacks DA,. pp. 45-55, Blackwell 3 Brazil Gestational Diabetes: Evidence-Based Instituto de Medicina Integral Prof. Fernando Figueira (IMIP) – Recife, Pernambuco, Gestational diabetes (GD) is defined as carbohydrate intolerance that begins or is first diagnosed during pregnancy. Hyperglycemia is found only during pregnancy and diagnosis is confirmed when glucose tolerance test results return to normal levels in the postpartum (Metzger et al., 2007). GD occurs in around 4-10% of pregnancies; however, its incidence varies as a function of nutritional habits and differences in genetic patterns The importance of GD was first described around forty years ago when it was noticed that women with this disorder were more likely to develop diabetes mellitus later on in their lives. The original diagnostic criteria proposed by O'Sullivan and Mahan were in fact never validated for the development of gestational complications or adverse perinatal outcomes (O'Sullivan and Mahan, 1964). Throughout all this time, the importance of this diagnosis for the prognosis of the pregnancy has been the subject of debate (Holt et al., 2011). Whereas some specialists feared that even mild levels of hyperglycemia would negatively affect pregnancy outcome, others have questioned the very existence of GD as a disease Recently, however, the harmful effects of hyperglycemia during pregnancy have been demonstrated (HAPO, 2008) and evidence is mounting on the risks of hyperglycemia during pregnancy, not only in terms of adverse perinatal outcome, but also for the future of the infant In this chapter, the rationale and current recommendations for the diagnosis and treatment of gestational diabetes will be evaluated based on the best scientific evidence currently available. The MEDLINE, EMBASE, SCOPUS and SciELO databases and the systematic reviews of the Cochrane Library were reassessed using the key words: gestational diabetes, screening, diagnosis and therapy. Preference was given to randomized clinical trials and meta-analyses, with observational studies and review articles being included only when studies with a better level of evidence were unavailable. Guidelines and recommendations established by medical societies for the screening, diagnosis and treatment of gestational diabetes were also consulted. As any carbohydrate metabolism disorder, GD is characterized by insufficient insulin levels for insulin demand (Metzger et al., 2007). The cause of this insulin insufficiency in in adult life (Catalano, 2010; Chandler-Laney et al., 2011; Fall, 2010; Lawlor et al., 2011). 1. Introduction between populations (Metzger et al., 2007). (Buchanan and Kjos, 1999). 2. Physiopathology Screening, Diagnosis and Treatment Melania Maria Ramos Amorim and Leila Katz ## Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment Melania Maria Ramos Amorim and Leila Katz Instituto de Medicina Integral Prof. Fernando Figueira (IMIP) – Recife, Pernambuco, Brazil #### 1. Introduction 40 Gestational Diabetes Super, DM.; Edelberg, SC.; Philipson, EH.; et al. (1991). Diagnosis of gestational diabetes in Tisi, DK.; Burns, DH.; Luskey, GW.; et al. (2011). Fetal exposure to altered amniotic fluid Tuomilehto, J.; (2005). A paradigm shift is needed in the primary prevention of type 2 Valensi, P.; Benroubi, M.; Borzi, V.; et al. (2009). Initiating insulin therapy with, or switching Voto, LS.; Uranga Imaz, M.; Margulies, M. (2003). Gestational diabetes in developing Mellitus (GDM) and its Outcomes in Jammu Region. J Assoc Physicians India. Vol. 59, No. 4, Weiss, PA.; Haeusler, M.; Tamussino, K.; et al. (2000). Can glucose tolerance test predict fetal Wild, S.; Roglic, G.; Green, A.; et al. (2004). Global prevalence of diabetes: estimates for the Wojcikowski, C.; Krolikowska, B.; Konarzewska, J.; et al. (2002). The prevalence of Yajnik, CS.; Fall, CH.; Vaidya, U.; et al. (1995). Fetal growth and glucose and insulin Yogev, Y.; Avi Ben-Haroush, Moshe Hod: Pathogenesis of gestational diabetes mellitus; In: Zargar, AH.; Sheikh, MI.; Bashir, MI.; et al. (2004). Prevalence of gestational diabetes 66, No. 2, (November 2004), pp. 139-45. ISSN 0168-8227. Wahi, P.; Dogra, V.; Jandial, K.; et al. (2011). Prevalence of Gestational Diabetes 5992. 1368-5031. 0328. 2011), pp. 139-44. ISSN 0149-5992. Ltd, England, ISBN: 978-0-470-85734-2. Group plc, ISBN 1 84184 110 2, London. (April 2011), pp. 227-30. ISSN 0004-5772. (October 2002), pp. 811-6. ISSN 0017-0011. 1995), pp. 330-6. ISSN 0742-3071. 1047-53. ISSN 0149-5992. 84184 110 2, London. early pregnancy. Diabetes Care. Vol. 14, No. 4, (April 1991), pp. 288-94. ISSN 0149- glucose, insulin, and insulin-like growth factor-binding protein 1 occurs before screening for gestational diabetes mellitus. Diabetes Care. Vol. 34, No. 1, (January diabetes. In: Prevention of type 2 diabetes. Ganz M, pp. 153-65, John Willey & Sons existing insulin therapy to, biphasic insulin aspart 30/70 (NovoMix 30) in routine care: safety and effectiveness in patients with type 2 diabetes in the IMPROVE observational study. Int J Clin Pract. Vol. 63, No. 3, (March 2009), pp. 522–31. ISSN countries. In: Textbook of Diabetes and Pregnancy. Hod, M.; Jovanovic, L.; Di Renzo, GC.; de Leiva, A.; Langer, O.; 1st ed. pp. 183- 90. Martin Dunitz, Taylor & Francis hyperinsulinism?. BJOG. Vol. 107, No. 12, (December 2000), pp. 1480-5. ISSN 1470- year 2000 and projections for 2030. Diabetes Care. Vol. 27, No. 5, (May 2004), pp. gestational diabetes mellitus in Polish population. Ginekol Pol. Vol. 73, No. 10, metabolism in four year old Indian children. Diabetic Medicine. Vol. 12, No. 4, (April Textbook of Diabetes and Pregnancy. Hod, M.; Jovanovic, L.; Di Renzo, GC.; de Leiva, A.; Langer, O.; 1st ed. pp. 46. Martin Dunitz, Taylor & Francis Group plc, ISBN 1 mellitus in Kashmiri women from the Indian subcontinent. Dia Res Clin Pract. Vol. Gestational diabetes (GD) is defined as carbohydrate intolerance that begins or is first diagnosed during pregnancy. Hyperglycemia is found only during pregnancy and diagnosis is confirmed when glucose tolerance test results return to normal levels in the postpartum (Metzger et al., 2007). GD occurs in around 4-10% of pregnancies; however, its incidence varies as a function of nutritional habits and differences in genetic patterns between populations (Metzger et al., 2007). The importance of GD was first described around forty years ago when it was noticed that women with this disorder were more likely to develop diabetes mellitus later on in their lives. The original diagnostic criteria proposed by O'Sullivan and Mahan were in fact never validated for the development of gestational complications or adverse perinatal outcomes (O'Sullivan and Mahan, 1964). Throughout all this time, the importance of this diagnosis for the prognosis of the pregnancy has been the subject of debate (Holt et al., 2011). Whereas some specialists feared that even mild levels of hyperglycemia would negatively affect pregnancy outcome, others have questioned the very existence of GD as a disease (Buchanan and Kjos, 1999). Recently, however, the harmful effects of hyperglycemia during pregnancy have been demonstrated (HAPO, 2008) and evidence is mounting on the risks of hyperglycemia during pregnancy, not only in terms of adverse perinatal outcome, but also for the future of the infant in adult life (Catalano, 2010; Chandler-Laney et al., 2011; Fall, 2010; Lawlor et al., 2011). In this chapter, the rationale and current recommendations for the diagnosis and treatment of gestational diabetes will be evaluated based on the best scientific evidence currently available. The MEDLINE, EMBASE, SCOPUS and SciELO databases and the systematic reviews of the Cochrane Library were reassessed using the key words: gestational diabetes, screening, diagnosis and therapy. Preference was given to randomized clinical trials and meta-analyses, with observational studies and review articles being included only when studies with a better level of evidence were unavailable. Guidelines and recommendations established by medical societies for the screening, diagnosis and treatment of gestational diabetes were also consulted. #### 2. Physiopathology As any carbohydrate metabolism disorder, GD is characterized by insufficient insulin levels for insulin demand (Metzger et al., 2007). The cause of this insulin insufficiency in Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 43 The screening and diagnosis of GD is the subject of intense debate and controversy worldwide (Holt et al., 2011; Leary et al., 2010). All aspects of diagnosis (who should be investigated, using which tests and what values are considered diagnostic) have been widely discussed over the past two decades (Holt et al., 2011). Consequently, the guidelines published by the major societies differ with respect to these aspects with the result that the The World Health Organization (WHO) recommends screening high-risk women with a 75 gram oral glucose tolerance test in the first trimester of pregnancy and all other women at 24-28 weeks, with fasting glucose measurements of 126 mg/dl and two-hour glucose levels of 140 mg/dl being considered abnormal (WHO, 1999). However, until recently (up to autumn 2010), the American Diabetes Association (ADA) recommended screening only women with risk factors and advocated an oral load of 100 grams of anhydrous glucose Recently, the International Association of Diabetes and Pregnancy Study Groups (IADPSG) assembled the evidence accumulated over recent years and published new criteria for the Screening is performed to select individuals to be investigated. Since 1999, the World Health Organization (WHO) has recommended the screening of all women or all women except In 2003, a US taskforce was formed to evaluate screening for gestational diabetes. The authors' conclusion was that better-quality evidence was required to determine whether the benefits of screening are greater than the risks. They recommended that until such evidence was available, to screen or not to screen should be left to the discretion of each individual physician according to his/her own clinical judgment and that both options are reasonable The same taskforce evaluated the risk factors for gestational diabetes and found a strong association with: maternal obesity (body mass index > 25), age > 25 years, personal or family history of a carbohydrate metabolism disorder or a history of gestational diabetes in a previous pregnancy. Some ethnic groups such as Hispanics, Blacks, native American Indians and Asians are also at an increased risk of developing gestational diabetes. If all these criteria are taken into consideration, 90% of all women at risk of developing A systematic review was conducted of observational studies published in the past thirty years to evaluate the presence and strength of the association between pre-gestational body mass index (BMI) and the presence of gestational diabetes. Seventy studies were included involving 671,945 women (59 cohort studies and 11 case-control studies). Compared to women with normal pregestational BMI, in accordance with the odds ratio (OR) the estimated risk of developing gestational diabetes was 1.97 [95% confidence interval (95%CI) 1.77 – 2.19], 3.01 [95%CI: 2.34 – 3.87] and 5.55 [95%CI: 4.27 – 7.21] for overweight, moderate In addition to the principal risk factors, Table 2 provides a detailed list of risk factors from previous pregnancies as well as risk factors that may appear during the course of pregnancy practices of physicians worldwide differ to the same extent (Leary et al., 2010). (ADA, 2004). The values adopted in each one of the guidelines also differed greatly. 4. Screening and diagnosis of gestational diabetes screening and diagnosis of GD (IADPSG, Metzger et al., 2010). (The US Preventive Services Task-Force – USPSTF, Brody et al., 2003-A). gestational diabetes will be identified (Brody et al., 2003-A). obesity and severe obesity, respectively (Torloni et al., 2009). and may merit investigation. those considered low-risk (WHO, 1999). 4.1 Screening pregnancy remains to be fully established; however, it is believed that the occurrence of this event during pregnancy reveals underlying maternal pancreatic disorders that would otherwise only become apparent later on in the woman's life (Metzger et al., 2007). In a normal pregnancy, fetal and placental growth increases cortisol, growth hormone, human placental lactogen, progesterone, estrogen and prolactin levels. The presence of these stimuli triggers hyperinsulinemia, insulin resistance, fasting hypoglycemia and postprandial hyperglycemia. Consequently, there is a reduction in peripheral sensitivity to insulin and an increase in demand. As a compensatory mechanism, an increase occurs in pancreatic function at the cost of both hypertrophy and hyperplasia. Furthermore, in response to the high insulin levels, peripheral utilization of glucose by the muscles and peripheral glycogen storage increase in an attempt to maintain balance (Metzger et al., 2007; Pridjian and Benjamin, 2010). As pregnancy advances, these compensatory mechanisms may be insufficient in susceptible women, resulting in an imbalance between insulin production and insulin requirements in pregnancy. Compared to a normal pregnant woman, a woman with GD has pancreatic βcell dysfunction and a reduction in adaptive β-cell capacity. This results in insufficient insulin secretion and consequent hyperglycemia (Metzger et al., 2007; Pridjian and Benjamin, 2010). #### 3. Consequences for the mother and child The consequences of gestational diabetes for the mother and child are summarized in Table 1. Modified from Pridjian and Benjamin, 2010 Table 1. Risks of Gestational Diabetes #### 4. Screening and diagnosis of gestational diabetes The screening and diagnosis of GD is the subject of intense debate and controversy worldwide (Holt et al., 2011; Leary et al., 2010). All aspects of diagnosis (who should be investigated, using which tests and what values are considered diagnostic) have been widely discussed over the past two decades (Holt et al., 2011). Consequently, the guidelines published by the major societies differ with respect to these aspects with the result that the practices of physicians worldwide differ to the same extent (Leary et al., 2010). The World Health Organization (WHO) recommends screening high-risk women with a 75 gram oral glucose tolerance test in the first trimester of pregnancy and all other women at 24-28 weeks, with fasting glucose measurements of 126 mg/dl and two-hour glucose levels of 140 mg/dl being considered abnormal (WHO, 1999). However, until recently (up to autumn 2010), the American Diabetes Association (ADA) recommended screening only women with risk factors and advocated an oral load of 100 grams of anhydrous glucose (ADA, 2004). The values adopted in each one of the guidelines also differed greatly. Recently, the International Association of Diabetes and Pregnancy Study Groups (IADPSG) assembled the evidence accumulated over recent years and published new criteria for the screening and diagnosis of GD (IADPSG, Metzger et al., 2010). #### 4.1 Screening 42 Gestational Diabetes pregnancy remains to be fully established; however, it is believed that the occurrence of this event during pregnancy reveals underlying maternal pancreatic disorders that would In a normal pregnancy, fetal and placental growth increases cortisol, growth hormone, human placental lactogen, progesterone, estrogen and prolactin levels. The presence of these stimuli triggers hyperinsulinemia, insulin resistance, fasting hypoglycemia and postprandial hyperglycemia. Consequently, there is a reduction in peripheral sensitivity to insulin and an increase in demand. As a compensatory mechanism, an increase occurs in pancreatic function at the cost of both hypertrophy and hyperplasia. Furthermore, in response to the high insulin levels, peripheral utilization of glucose by the muscles and peripheral glycogen storage increase in an attempt to maintain balance (Metzger et al., 2007; Pridjian and As pregnancy advances, these compensatory mechanisms may be insufficient in susceptible women, resulting in an imbalance between insulin production and insulin requirements in pregnancy. Compared to a normal pregnant woman, a woman with GD has pancreatic βcell dysfunction and a reduction in adaptive β-cell capacity. This results in insufficient insulin secretion and consequent hyperglycemia (Metzger et al., 2007; Pridjian and The consequences of gestational diabetes for the mother and child are summarized in Table 1. Risks of Gestational Diabetes Mother Fetus Newborn infant Child/Adult Respiratory distress Hypocalcemia Metabolic Obesity Mellitus syndrome syndrome Cardiomyopathy Hypoglycemia Type 2 Diabetes Polycythemia: • Hyperviscosity • Hyperbilirubinemia Cardiomyopathy otherwise only become apparent later on in the woman's life (Metzger et al., 2007). Benjamin, 2010). Benjamin, 2010). Obstetric trauma Higher rate of Cesarean sections Preeclampsia/ gestational hypertension Type 2 Diabetes mellitus Metabolic syndrome 3. Consequences for the mother and child Hyperinsulinemia: • Macrosomia Obstetric trauma: • Shoulder dystocia • Brachial plexus lesion Stillbirth Hypomagnesemia • Fractures Modified from Pridjian and Benjamin, 2010 Table 1. Risks of Gestational Diabetes • Large for gestational age Screening is performed to select individuals to be investigated. Since 1999, the World Health Organization (WHO) has recommended the screening of all women or all women except those considered low-risk (WHO, 1999). In 2003, a US taskforce was formed to evaluate screening for gestational diabetes. The authors' conclusion was that better-quality evidence was required to determine whether the benefits of screening are greater than the risks. They recommended that until such evidence was available, to screen or not to screen should be left to the discretion of each individual physician according to his/her own clinical judgment and that both options are reasonable (The US Preventive Services Task-Force – USPSTF, Brody et al., 2003-A). The same taskforce evaluated the risk factors for gestational diabetes and found a strong association with: maternal obesity (body mass index > 25), age > 25 years, personal or family history of a carbohydrate metabolism disorder or a history of gestational diabetes in a previous pregnancy. Some ethnic groups such as Hispanics, Blacks, native American Indians and Asians are also at an increased risk of developing gestational diabetes. If all these criteria are taken into consideration, 90% of all women at risk of developing gestational diabetes will be identified (Brody et al., 2003-A). A systematic review was conducted of observational studies published in the past thirty years to evaluate the presence and strength of the association between pre-gestational body mass index (BMI) and the presence of gestational diabetes. Seventy studies were included involving 671,945 women (59 cohort studies and 11 case-control studies). Compared to women with normal pregestational BMI, in accordance with the odds ratio (OR) the estimated risk of developing gestational diabetes was 1.97 [95% confidence interval (95%CI) 1.77 – 2.19], 3.01 [95%CI: 2.34 – 3.87] and 5.55 [95%CI: 4.27 – 7.21] for overweight, moderate obesity and severe obesity, respectively (Torloni et al., 2009). In addition to the principal risk factors, Table 2 provides a detailed list of risk factors from previous pregnancies as well as risk factors that may appear during the course of pregnancy and may merit investigation. Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 45 2010) and in the risk factors for the occurrence of GD such as, for example, obesity (Catalano, 2010). In addition, it ensures that cases of early-onset GD will be identified (Leary In addition, IADPSG recommends universal investigation at 24-28 weeks of all women not The argument used by those who support universal screening is based on the randomized clinical trial entitled the Australian Carbohydrate Intolerance Study in Pregnant Women (ACHOIS) trial group (Crowther et al., 2005), which showed a reduction in healthcare costs with universal screening. Nevertheless, criticism to the use of these results for recommending IADPSG's proposal is based on the fact that the ACHOIS study used an oral glucose tolerance test of 75 grams, measuring fasting glucose levels and two-hour glucose levels alone, whereas the IADPSG recommendations also include measurement of glucose levels one hour after overload (Leary et al., 2010). Further studies should be conducted to The origin of all this controversy surrounding the diagnosis of GD lies in the form in which testing was initially developed. The first authors to develop a diagnostic test for GD were John O'Sullivan and Clare Mahan in 1964 (O'Sullivan and Mahan, 1964). The test was developed to predict the risk of developing diabetes mellitus years after the pregnancy Although it constituted a watershed in the diagnosis of GD, faults were found in the study conducted by O'Sullivan and Mahan when analyzed from a methodological point of view, particularly with respect to the conclusions drawn and the validation of the test as a diagnostic technique, which was clearly demonstrated by Naylor in a study published in 1989 (Naylor, 1989). One of the questions raised was that gestational diabetes is more important as a predictor of a pregnancy with a higher maternal-fetal risk, whereas the endpoint initially evaluated was the presence of carbohydrate intolerance after the end of pregnancy. Since the investigators' objective was to predict the development of diabetes mellitus over the long-term, this characteristic was taken into consideration to select the cutoff points. It was later shown that when pregnant women considered to be diabetic in accordance with the values selected were treated with insulin, the rate of macrosomia decreased when glucose levels returned to normal (O'Sullivan, 1996 and 1973). This is an indirect way of reaching conclusions that is, nonetheless, far from ideal and does not allow the diagnostic technique to be adequately validated. According to Naylor, it would have been more appropriate to try to record the immediate and long-term neonatal complications In the following years, changes were made to the diagnostic techniques used and glucose levels were no longer measured in full blood but rather in venous plasma. Furthermore, enzymatic methods began to be used to measure plasma glucose levels instead of the Somogyi-Nelson technique. These technical modifications led to the mathematical correction of the values initially proposed by O'Sullivan and Mahan with the appearance of different sets of values adopted by different organizations involved in the study of GD (National Later, an intense debate ensued among investigators regarding the best form of diagnosing GD. Many investigators suggested the adoption of more rigid diagnostic criteria, including a previously diagnosed as having clinical or gestational diabetes (IADPSG et al., 2010). et al., 2010). Early screening reflects the preference for universal investigation. evaluate the costs and benefits associated with this form of management. rather than the risk of an adverse perinatal outcome. and test their association using an oral glucose tolerance test. Diabetes Data Group [NDDG], 1979; Carpenter and Coustan, 1982). 4.2 Diagnosis #### Personal characteristics: Age > 35 years Obesity (BMI > 25) Arterial hypertension Family history of diabetes Obstetric history: Diabetes in previous pregnancy Multiparity Recurrent miscarriage Prematurity Recurrent preeclampsia Fetal death, principally in the final weeks of pregnancy Neonatal morbidity and mortality: - Hypoglycemia - Respiratory distress syndrome - Hyperbilirubinemia - Hypocalcemia - Malformations Complications in current pregnancy: Excessive weight gain Excessive growth of uterine fundal height Polyhydramnios Fetal macrosomia Glycosuria Use of hyperglycemic drugs (betamimetics, corticoids) Amorim and Katz, 2011 Table 2. Risk factors for gestational diabetes More recently, another systematic review evaluated the available literature, searching for further evidence on screening for gestational diabetes (Tieu et al.,2011). The authors searched for articles which evaluated any individual screening tool or screening program, protocol or guideline for gestational diabetes compared with the absence of screening; or any individual screening tool or screening program, protocol or guideline for gestational diabetes with another. Thirty-one trials were considered for inclusion into the review but after application of eligibility criteria, only four of these trials were included. After analysis the authors found that there was insufficient evidence to determine the effects of screening for gestational diabetes and its subsequent management, or the comparative effects of different protocols for screening. Although women who were routinely screened by 50 g glucose challenge testing were more likely to be diagnosed with gestational diabetes than those screened by their risk factors, effects of subsequent management on health outcome are unclear. IADPSG recommends investigating "all women or all high-risk women" at their first prenatal consultation (IADPSG, Metzger et al., 2010). Universal investigation is justified by the increase in the prevalence of undiagnosed type 2 diabetes in young women (Leary et al., 2010) and in the risk factors for the occurrence of GD such as, for example, obesity (Catalano, 2010). In addition, it ensures that cases of early-onset GD will be identified (Leary et al., 2010). Early screening reflects the preference for universal investigation. In addition, IADPSG recommends universal investigation at 24-28 weeks of all women not previously diagnosed as having clinical or gestational diabetes (IADPSG et al., 2010). The argument used by those who support universal screening is based on the randomized clinical trial entitled the Australian Carbohydrate Intolerance Study in Pregnant Women (ACHOIS) trial group (Crowther et al., 2005), which showed a reduction in healthcare costs with universal screening. Nevertheless, criticism to the use of these results for recommending IADPSG's proposal is based on the fact that the ACHOIS study used an oral glucose tolerance test of 75 grams, measuring fasting glucose levels and two-hour glucose levels alone, whereas the IADPSG recommendations also include measurement of glucose levels one hour after overload (Leary et al., 2010). Further studies should be conducted to evaluate the costs and benefits associated with this form of management. #### 4.2 Diagnosis 44 Gestational Diabetes Personal characteristics: Diabetes in previous pregnancy Neonatal morbidity and mortality: Complications in current pregnancy: Excessive growth of uterine fundal height Fetal death, principally in the final weeks of pregnancy Use of hyperglycemic drugs (betamimetics, corticoids) factors, effects of subsequent management on health outcome are unclear. More recently, another systematic review evaluated the available literature, searching for further evidence on screening for gestational diabetes (Tieu et al.,2011). The authors searched for articles which evaluated any individual screening tool or screening program, protocol or guideline for gestational diabetes compared with the absence of screening; or any individual screening tool or screening program, protocol or guideline for gestational diabetes with another. Thirty-one trials were considered for inclusion into the review but after application of eligibility criteria, only four of these trials were included. After analysis the authors found that there was insufficient evidence to determine the effects of screening for gestational diabetes and its subsequent management, or the comparative effects of different protocols for screening. Although women who were routinely screened by 50 g glucose challenge testing were more likely to be diagnosed with gestational diabetes than those screened by their risk IADPSG recommends investigating "all women or all high-risk women" at their first prenatal consultation (IADPSG, Metzger et al., 2010). Universal investigation is justified by the increase in the prevalence of undiagnosed type 2 diabetes in young women (Leary et al., Age > 35 years Obesity (BMI > 25) Arterial hypertension Family history of diabetes Obstetric history: Recurrent miscarriage Recurrent preeclampsia Excessive weight gain Polyhydramnios Fetal macrosomia Table 2. Risk factors for gestational diabetes Glycosuria Amorim and Katz, 2011 Multiparity Prematurity The origin of all this controversy surrounding the diagnosis of GD lies in the form in which testing was initially developed. The first authors to develop a diagnostic test for GD were John O'Sullivan and Clare Mahan in 1964 (O'Sullivan and Mahan, 1964). The test was developed to predict the risk of developing diabetes mellitus years after the pregnancy rather than the risk of an adverse perinatal outcome. Although it constituted a watershed in the diagnosis of GD, faults were found in the study conducted by O'Sullivan and Mahan when analyzed from a methodological point of view, particularly with respect to the conclusions drawn and the validation of the test as a diagnostic technique, which was clearly demonstrated by Naylor in a study published in 1989 (Naylor, 1989). One of the questions raised was that gestational diabetes is more important as a predictor of a pregnancy with a higher maternal-fetal risk, whereas the endpoint initially evaluated was the presence of carbohydrate intolerance after the end of pregnancy. Since the investigators' objective was to predict the development of diabetes mellitus over the long-term, this characteristic was taken into consideration to select the cutoff points. It was later shown that when pregnant women considered to be diabetic in accordance with the values selected were treated with insulin, the rate of macrosomia decreased when glucose levels returned to normal (O'Sullivan, 1996 and 1973). This is an indirect way of reaching conclusions that is, nonetheless, far from ideal and does not allow the diagnostic technique to be adequately validated. According to Naylor, it would have been more appropriate to try to record the immediate and long-term neonatal complications and test their association using an oral glucose tolerance test. In the following years, changes were made to the diagnostic techniques used and glucose levels were no longer measured in full blood but rather in venous plasma. Furthermore, enzymatic methods began to be used to measure plasma glucose levels instead of the Somogyi-Nelson technique. These technical modifications led to the mathematical correction of the values initially proposed by O'Sullivan and Mahan with the appearance of different sets of values adopted by different organizations involved in the study of GD (National Diabetes Data Group [NDDG], 1979; Carpenter and Coustan, 1982). Later, an intense debate ensued among investigators regarding the best form of diagnosing GD. Many investigators suggested the adoption of more rigid diagnostic criteria, including a Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 47 100 grams of anhydrous glucose (O'Sullivan and Mahan, 1964), a procedure that was also recommended by the American Diabetes Association in 2004 (ADA, 2004). Nonetheless, since the WHO proposal in 1980 to use a 75-gram overload, as used in non-pregnant women, this practice became more and more common and even the HAPO study used the 75-gram glucose overload. With the power obtained in the HAPO study showing the adverse perinatal effects of hyperglycemia associated with the levels obtained following a 75-gram overload of anhydrous glucose, this test will probably increase in popularity Another interesting outcome of the HAPO study was the recommendation that a finding of only one abnormal value on the glucose curve should also be considered abnormal and diagnostic. This recommendation was made with the aim of increasing the likelihood of identifying milder degrees of hyperglycemia that had been associated with poorer maternal The cut-off points recommended by IADPSG are arbitrarily defined based on an odds ratio of 1.75 relative to the mean glucose levels at each time-point, i.e. those measurements that result in a 75% greater likelihood of adverse perinatal outcomes. The selection had to be made in this way, since, as previously mentioned, the association between glucose levels and adverse outcome is continuous. These values, however, are subject to criticism and the occurrence of adverse outcomes with glucose levels below the proposed values is to be expected. Nevertheless, use of a lower cut-off point would certainly result in a greater percentage of diagnosed cases and the impact of this excess number of diagnoses on The absence of any IADPSG recommendations regarding a category referred to as "carbohydrate intolerance" is noteworthy. According to current criteria, either a woman has normal glucose levels or she has gestational diabetes. However, cases need to be taken into consideration in which women have glucose levels outside the limits considered normal yet without reaching levels that would be considered diagnostic. The adoption of the term The findings of the HAPO study appear to indicate that only normal glucose levels would eliminate the risk of adverse perinatal outcome; therefore, it could be argued that any deviation above normal levels should be considered abnormal (Leary et al., 2010). However, the economic and even the psychological impact of so many diagnoses of an "abnormal To determine the ideal cut-off point, a cost-benefit analysis has to be performed of different cut-off points. In addition to the cost of the diagnostic test itself, the financial burden caused by the additional cases diagnosed in terms of follow-up and treatment has to be taken into consideration. In addition, it has to be confirmed that treating these diagnosed cases will indeed lead to a reduction in the number of adverse outcomes (Leary et al., 2010) and, furthermore, that this reduction will cause a positive impact that will compensate for the The change in the diagnostic criteria defined by IADPSG will certainly have significant clinical implications for women and for the healthcare system. The number of women diagnosed as having gestational diabetes will rise. This increase in the prevalence of GD may cause a significant impact on all the additional women who will be diagnosed (Holt et al., 2011). In addition to the greater volume of resources required to follow-up and treat these women, the compared to the 100-gram overload (Leary et al., 2010). perinatal outcomes has yet to be established (Leary et al., 2010). "carbohydrate intolerance" is suggested for such cases (Leary et al., 2010). pregnancy" needs to be taken into consideration and may be immense. effect on patients of the very existence of a diagnosis should be kept in mind. and perinatal prognosis in previous studies. costs of diagnosis and follow-up. reduction in blood glucose levels or the adoption of fewer points on the curve as being sufficient for diagnosis. It was even proposed that the presence of hyperglycemia below the levels established for diagnosis could be sufficient to lead to adverse maternal and perinatal outcomes, or that the hyperglycemia occurring irrespective of an overload should be taken into consideration (Aberg et al., 2001; Jensen et al., 2001; Langer et al., 1987; Rudge et al., 1990; Sermer et al., 1995). If on the one hand evidence was accumulating to the effect that milder degrees of hyperglycemia, albeit below the levels established for a diagnosis of GD, could lead to unfavorable perinatal outcomes, on the other hand some authors questioned the very existence of this diagnosis as a valid entity and called attention to the possible negative effects of this diagnosis (Buchanan, 1999; Lucas et al., 1993). A diagnosis of GD may result in excessive medicalization of pregnancy, which in itself would be negative. Furthermore, this diagnosis may result in an increase in the number of interventions performed, including even Cesarean sections, in situations in which the need for this type of delivery is questionable, due to the mere presence of a diagnosis of diabetes. In addition, it is important to remember the psychological burden caused by a label of diabetes. In 2005, an Australian group (ACHOIS) published the findings of a randomized clinical trial in which mild hyperglycemia was treated in women who did not fulfill the diagnostic criteria for GD, but who had measurements of 140 to 199 mg/dl in a 75-gram oral glucose tolerance test (OGTT) and were consequently considered to be carbohydrate intolerant (Crowther et al., 2005). These investigators found a reduction in composite final outcome (perinatal death, shoulder dystocia, fractures and brachial plexus palsy) compared with women managed in the usual manner. In 2009, a clinical trial was conducted to treat women who were found not to fulfill the diagnostic criteria for GD after being submitted to an oral glucose tolerance test with 100 grams of carbohydrate, but whose glucose levels were not completely normal. Likewise, when this group was treated, a reduction was found in macrosomia, shoulder dystocia, Cesarean section and hypertensive diseases (Landon et al., 2009). Finally, the HAPO (Hyperglycemia and Adverse Pregnancy Outcome) study was conducted to evaluate the risks of hyperglycemia during pregnancy for the mother, the fetus and the newborn infant (HAPO, 2008). A total of 25,505 women were included and followed up prospectively to evaluate a possible association between glucose levels and maternal and perinatal outcome. The women were submitted to an OGTT using 75 grams of anhydrous glucose. The HAPO results were conclusive with respect to the existence of a linear association between elevated glucose levels in the pregnant woman and the rates of large-forgestational-age infants, preeclampsia and Cesarean sections. Moreover, an association was found between maternal hyperglycemia and neonatal hypoglycemia, C-peptide in umbilical cord blood, premature delivery, admission to the neonatal intensive care unit (ICU) and hyperbilirubinemia. No association was found between maternal hyperglycemia and neonatal death; however, the sample size may have been insufficient to evaluate this particular outcome. One of the most important findings of HAPO was the demonstration of an association that is continuous; hence no clear cut-off point can be defined above which adverse events occur (Leary et al., 2010). The choice of the 75-gram curve to investigate these women has never been validated scientifically. The first glucose overload to be described in pregnancy was performed using reduction in blood glucose levels or the adoption of fewer points on the curve as being sufficient for diagnosis. It was even proposed that the presence of hyperglycemia below the levels established for diagnosis could be sufficient to lead to adverse maternal and perinatal outcomes, or that the hyperglycemia occurring irrespective of an overload should be taken into consideration (Aberg et al., 2001; Jensen et al., 2001; Langer et al., 1987; Rudge et al., If on the one hand evidence was accumulating to the effect that milder degrees of hyperglycemia, albeit below the levels established for a diagnosis of GD, could lead to unfavorable perinatal outcomes, on the other hand some authors questioned the very existence of this diagnosis as a valid entity and called attention to the possible negative effects of this diagnosis (Buchanan, 1999; Lucas et al., 1993). A diagnosis of GD may result in excessive medicalization of pregnancy, which in itself would be negative. Furthermore, this diagnosis may result in an increase in the number of interventions performed, including even Cesarean sections, in situations in which the need for this type of delivery is questionable, due to the mere presence of a diagnosis of diabetes. In addition, it is important In 2005, an Australian group (ACHOIS) published the findings of a randomized clinical trial in which mild hyperglycemia was treated in women who did not fulfill the diagnostic criteria for GD, but who had measurements of 140 to 199 mg/dl in a 75-gram oral glucose tolerance test (OGTT) and were consequently considered to be carbohydrate intolerant (Crowther et al., 2005). These investigators found a reduction in composite final outcome (perinatal death, shoulder dystocia, fractures and brachial plexus palsy) compared with In 2009, a clinical trial was conducted to treat women who were found not to fulfill the diagnostic criteria for GD after being submitted to an oral glucose tolerance test with 100 grams of carbohydrate, but whose glucose levels were not completely normal. Likewise, when this group was treated, a reduction was found in macrosomia, shoulder dystocia, Finally, the HAPO (Hyperglycemia and Adverse Pregnancy Outcome) study was conducted to evaluate the risks of hyperglycemia during pregnancy for the mother, the fetus and the newborn infant (HAPO, 2008). A total of 25,505 women were included and followed up prospectively to evaluate a possible association between glucose levels and maternal and perinatal outcome. The women were submitted to an OGTT using 75 grams of anhydrous The HAPO results were conclusive with respect to the existence of a linear association between elevated glucose levels in the pregnant woman and the rates of large-forgestational-age infants, preeclampsia and Cesarean sections. Moreover, an association was found between maternal hyperglycemia and neonatal hypoglycemia, C-peptide in umbilical cord blood, premature delivery, admission to the neonatal intensive care unit (ICU) and hyperbilirubinemia. No association was found between maternal hyperglycemia and neonatal death; however, the sample size may have been insufficient to evaluate this One of the most important findings of HAPO was the demonstration of an association that is continuous; hence no clear cut-off point can be defined above which adverse events occur The choice of the 75-gram curve to investigate these women has never been validated scientifically. The first glucose overload to be described in pregnancy was performed using to remember the psychological burden caused by a label of diabetes. Cesarean section and hypertensive diseases (Landon et al., 2009). 1990; Sermer et al., 1995). women managed in the usual manner. glucose. particular outcome. (Leary et al., 2010). 100 grams of anhydrous glucose (O'Sullivan and Mahan, 1964), a procedure that was also recommended by the American Diabetes Association in 2004 (ADA, 2004). Nonetheless, since the WHO proposal in 1980 to use a 75-gram overload, as used in non-pregnant women, this practice became more and more common and even the HAPO study used the 75-gram glucose overload. With the power obtained in the HAPO study showing the adverse perinatal effects of hyperglycemia associated with the levels obtained following a 75-gram overload of anhydrous glucose, this test will probably increase in popularity compared to the 100-gram overload (Leary et al., 2010). Another interesting outcome of the HAPO study was the recommendation that a finding of only one abnormal value on the glucose curve should also be considered abnormal and diagnostic. This recommendation was made with the aim of increasing the likelihood of identifying milder degrees of hyperglycemia that had been associated with poorer maternal and perinatal prognosis in previous studies. The cut-off points recommended by IADPSG are arbitrarily defined based on an odds ratio of 1.75 relative to the mean glucose levels at each time-point, i.e. those measurements that result in a 75% greater likelihood of adverse perinatal outcomes. The selection had to be made in this way, since, as previously mentioned, the association between glucose levels and adverse outcome is continuous. These values, however, are subject to criticism and the occurrence of adverse outcomes with glucose levels below the proposed values is to be expected. Nevertheless, use of a lower cut-off point would certainly result in a greater percentage of diagnosed cases and the impact of this excess number of diagnoses on perinatal outcomes has yet to be established (Leary et al., 2010). The absence of any IADPSG recommendations regarding a category referred to as "carbohydrate intolerance" is noteworthy. According to current criteria, either a woman has normal glucose levels or she has gestational diabetes. However, cases need to be taken into consideration in which women have glucose levels outside the limits considered normal yet without reaching levels that would be considered diagnostic. The adoption of the term "carbohydrate intolerance" is suggested for such cases (Leary et al., 2010). The findings of the HAPO study appear to indicate that only normal glucose levels would eliminate the risk of adverse perinatal outcome; therefore, it could be argued that any deviation above normal levels should be considered abnormal (Leary et al., 2010). However, the economic and even the psychological impact of so many diagnoses of an "abnormal pregnancy" needs to be taken into consideration and may be immense. To determine the ideal cut-off point, a cost-benefit analysis has to be performed of different cut-off points. In addition to the cost of the diagnostic test itself, the financial burden caused by the additional cases diagnosed in terms of follow-up and treatment has to be taken into consideration. In addition, it has to be confirmed that treating these diagnosed cases will indeed lead to a reduction in the number of adverse outcomes (Leary et al., 2010) and, furthermore, that this reduction will cause a positive impact that will compensate for the costs of diagnosis and follow-up. The change in the diagnostic criteria defined by IADPSG will certainly have significant clinical implications for women and for the healthcare system. The number of women diagnosed as having gestational diabetes will rise. This increase in the prevalence of GD may cause a significant impact on all the additional women who will be diagnosed (Holt et al., 2011). In addition to the greater volume of resources required to follow-up and treat these women, the effect on patients of the very existence of a diagnosis should be kept in mind. Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 49 the different organizations, it is clear that there is no consensus with respect to the quantity of glucose that should be used in the oral glucose tolerance test (OGTT), to the glucose levels that should be considered abnormal, or to the number of abnormal measurements on the Table 4 shows the different criteria currently adopted for a diagnosis of gestational diabetes. Fasting glucose levels 92mg/dl 126mg/dl 95mg/dl 100mg/dl 95mg/dl 108mg/dl 100mg/dl ADA: American Diabetes Association (until autumn 2010); ADIPS: Australasian Diabetes in Pregnancy Society; CDA: Canadian Diabetes Association; EASD: European Association for the Study of Diabetes; IADPSG: International Association of Diabetes and Pregnancy Study Groups; NZSSD: New Zealand Society for the Study of Diabetes; WHO: World Health Organization. \The ADA adopted the IAPDSG criteria in the autumn of 2010. Until randomized clinical trials are conducted to compare the different strategies for screening and diagnosis and to define possible differences in maternal and perinatal outcome, the ideal test and the ideal criteria remain to be defined. The characteristics of each population should be evaluated, principally with respect to the frequency of gestational diabetes and macrosomia. In populations with a high risk for diabetes, we suggest that the IADPSG criteria be used; however, in low-risk populations in which there is no significant association between macrosomia and gestational diabetes, these criteria may not be Normally, the proposal of any strategy for screening and diagnosis of gestational diabetes is aimed at establishing a therapeutic plan for diagnosed cases, since available evidence Glucose levels after 1 hour 10.0mmol/l 180mg/dl 10.0mmol/l 180mg/dl 10.6mmol/l 190mg/dl Glucose levels after 2 hours 8.5mmol/l 153mg/dl 7.8mmol/l 140mg/dl 8.6mmol/l 155mg/dl 8.0mmol/l 144mg/dl 8.9mmol/l 160mg/dl 9.0mmol/l 162mg/dl 9.0mmol/l 162mg/dl curve that would permit a diagnosis of GD to be made (Holt et al., 2011). Number of abnormal values required Organization Glucose Holt et al, 2011 applicable (Leary et al., 2010). 5.1 Rationale for treatment* 5. Treatment of gestational diabetes overload IADPSG 75g > 1 5.1mmol/l WHO 75g > 1 7.0mmol/l ADA 100g > 2 5.3mmol/l ADIPS 75g > 1 5.5mmol/l CDA 75g > 2 5.3mmol/l EASD 75g > 1 6.0mmol/l NZSSD 75g > 1 5.5mmol/l Table 4. Comparison of diagnostic criteria for gestational diabetes Therefore, this change needs to be supported with convincing data showing beyond doubt that its adoption will improve pregnancy outcome. Since the HAPO study was merely observational, it is limited to associating adverse perinatal outcomes with higher glucose levels; however, it does not prove that normalizing glucose levels will necessarily result in any improvement in prognosis (Holt et al., 2011). Two studies evaluated the benefits of treating milder degrees of hyperglycemia in pregnancy: the ACHOIS study (Crowther, 2005) and the US Multicenter Randomized Trial for Treatment of Mild GMD (Landon, 2009). Despite promising results, it should be remembered that these studies differed in relation to the glucose levels considered treatable and in the number of glucose measurements performed for diagnosis. This hampers extrapolation of these results to the findings of the IADPSG study (Holt et al., 2011). #### 4.3 Investigation The IADPSG proposal for the screening and diagnosis of GD is shown in Table 3: First prenatal consultation Fasting glucose level or hemoglobin A1 (HgA1) or random measurement in women - and fasting glucose level is > 92 and < 126 => diagnosis of GD - and fasting glucose level is < 92 => test at 24-28 weeks with OGTT, 75 grams. 24 - 28 weeks of pregnancy OGTT, 75 grams: fasting glucose measurement/1 hour/2 hours #### For a diagnosis of GD (OGTT, 75g) Table 3. Screening and diagnosis of GD according to the IADPSG It is important, however, to call attention to the fact that controversies persist, despite the enormous number of studies conducted in this field. Analyzing the guidelines drawn up by the different organizations, it is clear that there is no consensus with respect to the quantity of glucose that should be used in the oral glucose tolerance test (OGTT), to the glucose levels that should be considered abnormal, or to the number of abnormal measurements on the curve that would permit a diagnosis of GD to be made (Holt et al., 2011). Table 4 shows the different criteria currently adopted for a diagnosis of gestational diabetes. ADA: American Diabetes Association (until autumn 2010); ADIPS: Australasian Diabetes in Pregnancy Society; CDA: Canadian Diabetes Association; EASD: European Association for the Study of Diabetes; IADPSG: International Association of Diabetes and Pregnancy Study Groups; NZSSD: New Zealand Society for the Study of Diabetes; WHO: World Health Organization. \The ADA adopted the IAPDSG criteria in the autumn of 2010. Holt et al, 2011 48 Gestational Diabetes Therefore, this change needs to be supported with convincing data showing beyond doubt that its adoption will improve pregnancy outcome. Since the HAPO study was merely observational, it is limited to associating adverse perinatal outcomes with higher glucose levels; however, it does not prove that normalizing glucose levels will necessarily result in Two studies evaluated the benefits of treating milder degrees of hyperglycemia in pregnancy: the ACHOIS study (Crowther, 2005) and the US Multicenter Randomized Trial for Treatment of Mild GMD (Landon, 2009). Despite promising results, it should be remembered that these studies differed in relation to the glucose levels considered treatable and in the number of glucose measurements performed for diagnosis. This hampers extrapolation of these results to the findings of the IADPSG study (Holt et al., 2011). The IADPSG proposal for the screening and diagnosis of GD is shown in Table 3: • If clinical diabetes => treatment and follow-up for preexisting diabetes. • and fasting glucose level is > 92 and < 126 => diagnosis of GD • Consider GD if ONE or more measurements are above the cut-off points. For a diagnosis of clinical diabetes during pregnancy (any one of these tests)* It is important, however, to call attention to the fact that controversies persist, despite the enormous number of studies conducted in this field. Analyzing the guidelines drawn up by OGTT, 75 grams: fasting glucose measurement/1 hour/2 hours • Consider normal if all the values are below the cut-off points. Fasting glucose > 92 mg/dl Fasting glucose > 126 mg/dl Hemoglobin A1 > 6.5% Table 3. Screening and diagnosis of GD according to the IADPSG Fasting glucose level or hemoglobin A1 (HgA1) or random measurement in women • and fasting glucose level is < 92 => test at 24-28 weeks with OGTT, 75 grams. >180 mg/dl >153 mg/dl > 200 mg/dl any improvement in prognosis (Holt et al., 2011). • If results are non-diagnostic for clinical diabetes: • Consider clinical diabetes if fasting glucose > 126 4.3 Investigation First prenatal consultation 24 - 28 weeks of pregnancy For a diagnosis of GD (OGTT, 75g) Glucose level at 1 hour after Glucose level at 2 hours after Random plasma glucose overload overload measurement Table 4. Comparison of diagnostic criteria for gestational diabetes Until randomized clinical trials are conducted to compare the different strategies for screening and diagnosis and to define possible differences in maternal and perinatal outcome, the ideal test and the ideal criteria remain to be defined. The characteristics of each population should be evaluated, principally with respect to the frequency of gestational diabetes and macrosomia. In populations with a high risk for diabetes, we suggest that the IADPSG criteria be used; however, in low-risk populations in which there is no significant association between macrosomia and gestational diabetes, these criteria may not be applicable (Leary et al., 2010). #### 5. Treatment of gestational diabetes #### 5.1 Rationale for treatment Normally, the proposal of any strategy for screening and diagnosis of gestational diabetes is aimed at establishing a therapeutic plan for diagnosed cases, since available evidence Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 51 versus 4.0%) and Cesarean sections (26.9% versus 33.8%). The rates of preeclampsia and A more recent systematic review on the treatment of gestational diabetes included 18 studies, five of which compared the specific treatment of diabetes with routine treatment (including the 2009 US trial). Modest effects of treatment were found, including a reduction in the risk of fetal macrosomia and shoulder dystocia and a trend, albeit non-significant, towards a reduction in the rate of Cesarean sections. Different levels and intensity of treatment were compared in 13 trials, with findings showing a significant reduction in risk only with respect to shoulder dystocia in the group receiving intensive treatment (Horvath Based on the results of these more recent studies, we believe that it is important to diagnose and treat gestational diabetes in order to reduce maternal and neonatal morbidity. Data from the Hyperglycemia and Adverse Pregnancy Outcomes study (HAPO, 2008) reinforce this recommendation, since findings showed a significant association between increasing glucose levels and maternal complications such as preeclampsia, and neonatal complications such as macrosomia and metabolic alterations (Leary et al., 2010), leading, as previously discussed, to changes in the diagnostic criteria for gestational diabetes, as defined by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) (Metzger et al., 2010). Nonetheless, some criticism remains with respect to the comparability of the HAPO study with the more recent clinical trials, since different The universal recommendation has been that all women with a confirmed diagnosis of gestational diabetes should receive dietary counseling and initiate an appropriate diet with the aim of normalizing glucose levels, preventing ketosis, ensuring adequate weight gain and contributing towards fetal well-being. The number of calories will depend on the woman's current weight, with an allowance of 30 kcal/kg of current weight for women with a normal body mass index (BMI), 24 kcal/kg for overweight women and 12-15 kcal/kg for obese women. Carbohydrates (preferably complex carbohydrates) should correspond to 33- 40% of the total number of calories, with protein corresponding to 20% and fat to 40%, provided in the form of three main meals and three snacks. Moderate use of sweeteners such as aspartame is permitted. Following these dietary guidelines, glucose levels will Nevertheless, the most adequate strategy for the control of gestational diabetes still remains to be defined, since diet alone may fail to prevent macrosomia. A systematic review available in the Cochrane Library included four studies with 612 women with gestational diabetes and failed to find any differences in the rates of macrosomia (OR = 0.78; 95%CI: 0.45 – 1.35) or Cesarean section (Walkinshaw, 2011). However, the clinical trials included in this review were all small and old, with variations in quality and very wide confidence intervals that did not permit evaluation of the validity of dietetic therapy. The reviewers concluded that there is insufficient evidence to evaluate the use of primary dietetic therapy for women with impaired glucose metabolism in pregnancy. They recommended that larger studies should be conducted to evaluate the effects of diet on maternal outcome normalize in around 75-80% of women with gestational diabetes (ADA, 2004). screening strategies were used (Horvath et al., 2010). (particularly Cesarean sections) and perinatal outcome. 5.2 Treatment strategies 5.2.1 Diet gestational hypertension were also lower in the treated group (8.6% versus 13.6%). et al., 2010). suggests that adequate treatment successfully reduces maternal and fetal morbidity, particularly macrosomia (Crowther et al., 2005; Langer et al., 2005; Landon et al., 2009). Various therapeutic options are available such as diet, physical exercise, blood glucose monitoring and insulin therapy in cases in which diet alone fails to maintain adequate glucose levels (Pridjian and Benjamin, 2010). This chapter does not explore the details of each individual treatment, but simply reviews the available evidence regarding the need for treatment and its effectiveness. A systematic review in the Cochrane Library specifically deals with the various alternative therapeutic options for gestational diabetes (Alwan et al., 2011). Eight randomized clinical trials involving a total of 1,418 women were included in which any form of treatment was compared with routine prenatal care or different treatments were compared with each other. Except for one large Australian study published in 2005 that included 1,000 women (ACHOIS trial) (Crowther et al., 2005), the sample sizes were small in all the other studies. When the treatment of mild hyperglycemia was compared with routine prenatal care, a significant reduction was found in the risk of preeclampsia and an increase in the risk of induced labor in the group that received treatment. There were no differences in Cesarean section rates, rates of hospital admission, instrumental delivery or postpartum hemorrhage or in the duration of hospital stay. With respect to perinatal outcome, the treatment of diabetes resulted in a significant reduction in composite perinatal morbidity (death, shoulder dystocia, bone fracture and nerve palsy), as well as in the frequency of macrosomia (birthweight > 4000 grams) and shoulder dystocia. Although in the ACHOIS study the rate of admittance to a neonatal intensive care unit was higher for the infants of mothers who received treatment for hyperglycemia, in the meta-analysis this difference was not found to be statistically significant. There were no significant differences between the two groups with respect to gestational age at delivery, incidence of bone fracture in newborn infants, incidence of nerve palsy in the newborn, perinatal death, neonatal hypoglycemia, incidence of respiratory distress syndrome in the newborn infant or in the need for mechanical ventilation. The conclusion reached by these reviewers was that specific treatment for mild gestational diabetes, including diet and insulin, reduces the risk of maternal and perinatal morbidity; however, the risk of labor induction increases. Further studies need to be conducted to evaluate the effect of the different therapeutic modalities, including oral hypoglycemic drugs and insulin, on infant short and long-term outcomes. Specific analysis of the findings of the ACHOIS trial reveals a frequency of severe neonatal morbidity of 1% in the treated group compared to 4% in the group that received routine care. The incidence of neonatal admission to hospital was 71% versus 61%, respectively, whereas rates of labor induction were 39% in the treatment group versus 29% in the group receiving routine care. The rate of Cesarean sections was similar in both groups, 31% versus 32%. In addition, lower rates of depression and better quality of life scores were found in the treated women in the ACHOIS trial at three months postpartum (Crowther et al., 2005). Another large randomized clinical trial conducted in the United States was published in 2009 and has yet to be included in the Cochrane systematic review (Landon et al., 2009). The study included 958 women. There was no statistically significant difference in composite outcome (32.4% in the treated group and 37% in the control group) and no perinatal deaths occurred in either group. Nevertheless, birthweight was significantly lower in the treated group (3302 grams versus 3408 grams), as was the frequency of large-for-gestational-age infants (7.1% versus 14.5%), fetal macrosomia (5.9% versus 4.0%), shoulder dystocia (1.5% versus 4.0%) and Cesarean sections (26.9% versus 33.8%). The rates of preeclampsia and gestational hypertension were also lower in the treated group (8.6% versus 13.6%). A more recent systematic review on the treatment of gestational diabetes included 18 studies, five of which compared the specific treatment of diabetes with routine treatment (including the 2009 US trial). Modest effects of treatment were found, including a reduction in the risk of fetal macrosomia and shoulder dystocia and a trend, albeit non-significant, towards a reduction in the rate of Cesarean sections. Different levels and intensity of treatment were compared in 13 trials, with findings showing a significant reduction in risk only with respect to shoulder dystocia in the group receiving intensive treatment (Horvath et al., 2010). Based on the results of these more recent studies, we believe that it is important to diagnose and treat gestational diabetes in order to reduce maternal and neonatal morbidity. Data from the Hyperglycemia and Adverse Pregnancy Outcomes study (HAPO, 2008) reinforce this recommendation, since findings showed a significant association between increasing glucose levels and maternal complications such as preeclampsia, and neonatal complications such as macrosomia and metabolic alterations (Leary et al., 2010), leading, as previously discussed, to changes in the diagnostic criteria for gestational diabetes, as defined by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) (Metzger et al., 2010). Nonetheless, some criticism remains with respect to the comparability of the HAPO study with the more recent clinical trials, since different screening strategies were used (Horvath et al., 2010). #### 5.2 Treatment strategies #### 5.2.1 Diet 50 Gestational Diabetes suggests that adequate treatment successfully reduces maternal and fetal morbidity, particularly macrosomia (Crowther et al., 2005; Langer et al., 2005; Landon et al., 2009). Various therapeutic options are available such as diet, physical exercise, blood glucose monitoring and insulin therapy in cases in which diet alone fails to maintain adequate glucose levels (Pridjian and Benjamin, 2010). This chapter does not explore the details of each individual treatment, but simply reviews the available evidence regarding the need for A systematic review in the Cochrane Library specifically deals with the various alternative therapeutic options for gestational diabetes (Alwan et al., 2011). Eight randomized clinical trials involving a total of 1,418 women were included in which any form of treatment was compared with routine prenatal care or different treatments were compared with each other. Except for one large Australian study published in 2005 that included 1,000 women (ACHOIS trial) (Crowther et al., 2005), the sample sizes were small in all the other studies. When the treatment of mild hyperglycemia was compared with routine prenatal care, a significant reduction was found in the risk of preeclampsia and an increase in the risk of induced labor in the group that received treatment. There were no differences in Cesarean section rates, rates of hospital admission, instrumental delivery or postpartum hemorrhage or in the duration of hospital stay. With respect to perinatal outcome, the treatment of diabetes resulted in a significant reduction in composite perinatal morbidity (death, shoulder dystocia, bone fracture and nerve palsy), as well as in the frequency of macrosomia (birthweight > 4000 grams) and shoulder dystocia. Although in the ACHOIS study the rate of admittance to a neonatal intensive care unit was higher for the infants of mothers who received treatment for hyperglycemia, in the meta-analysis this difference was not found to be statistically significant. There were no significant differences between the two groups with respect to gestational age at delivery, incidence of bone fracture in newborn infants, incidence of nerve palsy in the newborn, perinatal death, neonatal hypoglycemia, incidence of respiratory distress syndrome in the newborn infant or in the need for mechanical ventilation. The conclusion reached by these reviewers was that specific treatment for mild gestational diabetes, including diet and insulin, reduces the risk of maternal and perinatal morbidity; however, the risk of labor induction increases. Further studies need to be conducted to evaluate the effect of the different therapeutic modalities, including oral hypoglycemic drugs and insulin, on infant short and long-term outcomes. Specific analysis of the findings of the ACHOIS trial reveals a frequency of severe neonatal morbidity of 1% in the treated group compared to 4% in the group that received routine care. The incidence of neonatal admission to hospital was 71% versus 61%, respectively, whereas rates of labor induction were 39% in the treatment group versus 29% in the group receiving routine care. The rate of Cesarean sections was similar in both groups, 31% versus 32%. In addition, lower rates of depression and better quality of life scores were found in the treated women in the ACHOIS trial at three months postpartum (Crowther et al., 2005). Another large randomized clinical trial conducted in the United States was published in 2009 and has yet to be included in the Cochrane systematic review (Landon et al., 2009). The study included 958 women. There was no statistically significant difference in composite outcome (32.4% in the treated group and 37% in the control group) and no perinatal deaths occurred in either group. Nevertheless, birthweight was significantly lower in the treated group (3302 grams versus 3408 grams), as was the frequency of large-for-gestational-age infants (7.1% versus 14.5%), fetal macrosomia (5.9% versus 4.0%), shoulder dystocia (1.5% treatment and its effectiveness. The universal recommendation has been that all women with a confirmed diagnosis of gestational diabetes should receive dietary counseling and initiate an appropriate diet with the aim of normalizing glucose levels, preventing ketosis, ensuring adequate weight gain and contributing towards fetal well-being. The number of calories will depend on the woman's current weight, with an allowance of 30 kcal/kg of current weight for women with a normal body mass index (BMI), 24 kcal/kg for overweight women and 12-15 kcal/kg for obese women. Carbohydrates (preferably complex carbohydrates) should correspond to 33- 40% of the total number of calories, with protein corresponding to 20% and fat to 40%, provided in the form of three main meals and three snacks. Moderate use of sweeteners such as aspartame is permitted. Following these dietary guidelines, glucose levels will normalize in around 75-80% of women with gestational diabetes (ADA, 2004). Nevertheless, the most adequate strategy for the control of gestational diabetes still remains to be defined, since diet alone may fail to prevent macrosomia. A systematic review available in the Cochrane Library included four studies with 612 women with gestational diabetes and failed to find any differences in the rates of macrosomia (OR = 0.78; 95%CI: 0.45 – 1.35) or Cesarean section (Walkinshaw, 2011). However, the clinical trials included in this review were all small and old, with variations in quality and very wide confidence intervals that did not permit evaluation of the validity of dietetic therapy. The reviewers concluded that there is insufficient evidence to evaluate the use of primary dietetic therapy for women with impaired glucose metabolism in pregnancy. They recommended that larger studies should be conducted to evaluate the effects of diet on maternal outcome (particularly Cesarean sections) and perinatal outcome. Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 53 glucose levels, a lower rate of large-for-gestational-age infants and a lower rate of Cesarean sections with the latter protocol (deVeciana et al., 1995). Comparing monitoring one hour postprandial with two hours postprandial, a prospective, observational study found less need for insulin therapy and a trend towards lower rates of fetal macrosomia and Cesarean sections with one-hour postprandial glucose measurements (Weisz et al., 2005). There is insufficient evidence to determine the role of continuous glucose monitoring in patients with gestational diabetes, although this may be useful in women who require insulin and who have difficulty in achieving adequate control of glucose levels (Hawkins, 2010). With respect to insulin therapy, there is no consensus on the glucose levels that would indicate that insulin should be initiated after the implementation of dietetic therapy. The American College of Obstetricians and Gynecologists (ACOG) suggests that insulin should be administered to reduce the risk of macrosomia with fasting glucose levels ≥ 95 mg% OR one-hour postprandial glucose levels > 130-140 mg% OR two-hour postprandial glucose levels ≥ 120 mg% (ACOG, 2001). There are no randomized clinical trials available in which different glucose levels were compared with the objective of determining the cut-off point for the implementation of insulin therapy. Three randomized clinical trials suggest initiating insulin therapy irrespective of glucose levels if ultrasonographic measurement of fetal abdominal circumference exceeds the 75th percentile (Bonomo et al., 2004; Kjos et al., 2001; Rossi et al., 2000). The doses and types of insulin will not be discussed in this chapter. Oral hypoglycemic drugs are classically contraindicated in pregnancy. First generation drugs such as chlorpropamide and tolbutamide cross the placental barrier and may potentially cause prolonged and profound states of hypoglycemia, leading to fetal malformation. Nevertheless, the newer hypoglycemic drugs such as glibenclamide do not Furthermore, considering that in patients with gestational diabetes, the need for treatment initiates after embryogenesis (Langer, 2007), the newer oral hypoglycemic drugs were seen as a practical therapeutic option for this group of patients. Patient satisfaction with this route of administration may result in better compliance with treatment. Interest in evaluating these drugs as an option for the control of gestational diabetes has been intense and various randomized clinical trials using these agents have been published over the past In 2008, a systematic review was published that included a meta-analysis of all the clinical trials in which the use of insulin was compared with glibenclamide in women with gestational diabetes. Nine clinical trials were included involving 1,382 women with gestational diabetes. The use of glibenclamide was not found to be associated with any increased risk of macrosomia nor with differences in relation to fetal weight or the frequency of large-forgestational-age infants, admission to the neonatal ICU or an increased risk of neonatal hypoglycemia. These findings suggest that there is no increased perinatal risk with the use of this drug; however, the effectiveness and safety of its use still need to be confirmed, since the ten years (Langer et al., 2000; Moore et al., 2010; Rowan et al., 2008). majority of the studies included were not randomized (Moretti et al., 2008). 5.2.4 Pharmacological treatment 5.2.4.2 Antihyperglycemic drugs enter fetal circulation (Langer, 2007). 5.2.4.1 Insulin therapy Compliance with treatment and weight gain constitute factors capable of modifying response to dietetic treatment. One large retrospective study including more than 30,000 women with gestational diabetes showed that in the women in whom weight gain was adequate maternal and perinatal outcomes were favorable, whereas those in whom weight gain was excessive had a higher risk of large-for-gestational-age infants, premature delivery and Cesarean sections (Cheng et al., 2008). #### 5.2.2 Physical exercise Physical exercise has been proposed as part of the treatment for gestational diabetes based on the fact that, in adults, an improvement in physical fitness increases insulin sensitivity, improves glucose control and reduces the need for insulin (Colberg et al., 2010). A systematic review available in the Cochrane Library evaluated the effects of exercise programs alone or in association with other therapies on maternal and perinatal morbidity in pregnant women with diabetes. The review included four small, randomized clinical trials involving 114 women with gestational diabetes recruited during the third trimester of pregnancy. The intervention (exercise) was performed over six weeks. There were no statistically significant differences between the group that performed exercise and the controls for any one of the endpoints evaluated. The authors' conclusion was that the evidence was insufficient to either recommend or contraindicate exercise for pregnant women with diabetes and that larger randomized clinical trials should be conducted to further evaluate this form of intervention (Ceysens et al., 2011). Despite the consistent lack of evidence on the effects of exercise on maternal and perinatal prognosis in women with gestational diabetes, the American Association of Diabetes (ADA) suggests a program of moderate exercise as part of the therapeutic management of women with gestational diabetes as long as there are no medical or obstetrical contraindications to this level of physical activity (ADA, 2004). #### 5.2.3 Monitoring glucose levels Monitoring glucose levels may also alter the progression of the condition in women with gestational diabetes. One study showed that daily monitoring of pregnant women treated with diet allows identification of those who could benefit from treatment with an antihyperglycemic agent, which may lead to a reduction in the rates of macrosomia (Hawkins et al., 2009; Hawkins, 2010). Nevertheless, the ideal frequency of self-monitoring in women with diet controlled gestational diabetes remains to be established and there is insufficient evidence regarding the ideal glucose levels and the duration of control that would allow longer intervals between capillary glucose measurements (Metzger, 2007). With respect to the timing and frequency of capillary glucose monitoring, although there are still some controversies between investigators, most of them currently recommend measuring fasting levels immediately after waking and one hour after meals. The proposal for self-monitoring made by some specialists is to test capillary glucose levels four times a day in cases of diet controlled gestational diabetes (fasting and one hour after each meal) and six times a day in gestational diabetes requiring the use of insulin (fasting, one hour prior to and one hour after each meal) (Jovanovic, 2008). A clinical trial comparing monitoring with schedules that involve either the measurement of pre-prandial glucose levels or fasting glucose and postprandial levels (one hour after meals) in patients with gestational diabetes using insulin therapy showed a better control of glucose levels, a lower rate of large-for-gestational-age infants and a lower rate of Cesarean sections with the latter protocol (deVeciana et al., 1995). Comparing monitoring one hour postprandial with two hours postprandial, a prospective, observational study found less need for insulin therapy and a trend towards lower rates of fetal macrosomia and Cesarean sections with one-hour postprandial glucose measurements (Weisz et al., 2005). There is insufficient evidence to determine the role of continuous glucose monitoring in patients with gestational diabetes, although this may be useful in women who require insulin and who have difficulty in achieving adequate control of glucose levels (Hawkins, 2010). #### 5.2.4 Pharmacological treatment #### 5.2.4.1 Insulin therapy 52 Gestational Diabetes Compliance with treatment and weight gain constitute factors capable of modifying response to dietetic treatment. One large retrospective study including more than 30,000 women with gestational diabetes showed that in the women in whom weight gain was adequate maternal and perinatal outcomes were favorable, whereas those in whom weight gain was excessive had a higher risk of large-for-gestational-age infants, premature delivery Physical exercise has been proposed as part of the treatment for gestational diabetes based on the fact that, in adults, an improvement in physical fitness increases insulin sensitivity, A systematic review available in the Cochrane Library evaluated the effects of exercise programs alone or in association with other therapies on maternal and perinatal morbidity in pregnant women with diabetes. The review included four small, randomized clinical trials involving 114 women with gestational diabetes recruited during the third trimester of pregnancy. The intervention (exercise) was performed over six weeks. There were no statistically significant differences between the group that performed exercise and the controls for any one of the endpoints evaluated. The authors' conclusion was that the evidence was insufficient to either recommend or contraindicate exercise for pregnant women with diabetes and that larger randomized clinical trials should be conducted to Despite the consistent lack of evidence on the effects of exercise on maternal and perinatal prognosis in women with gestational diabetes, the American Association of Diabetes (ADA) suggests a program of moderate exercise as part of the therapeutic management of women with gestational diabetes as long as there are no medical or obstetrical contraindications to Monitoring glucose levels may also alter the progression of the condition in women with gestational diabetes. One study showed that daily monitoring of pregnant women treated with diet allows identification of those who could benefit from treatment with an antihyperglycemic agent, which may lead to a reduction in the rates of macrosomia (Hawkins et al., 2009; Hawkins, 2010). Nevertheless, the ideal frequency of self-monitoring in women with diet controlled gestational diabetes remains to be established and there is insufficient evidence regarding the ideal glucose levels and the duration of control that would allow With respect to the timing and frequency of capillary glucose monitoring, although there are still some controversies between investigators, most of them currently recommend measuring fasting levels immediately after waking and one hour after meals. The proposal for self-monitoring made by some specialists is to test capillary glucose levels four times a day in cases of diet controlled gestational diabetes (fasting and one hour after each meal) and six times a day in gestational diabetes requiring the use of insulin (fasting, one hour A clinical trial comparing monitoring with schedules that involve either the measurement of pre-prandial glucose levels or fasting glucose and postprandial levels (one hour after meals) in patients with gestational diabetes using insulin therapy showed a better control of longer intervals between capillary glucose measurements (Metzger, 2007). prior to and one hour after each meal) (Jovanovic, 2008). improves glucose control and reduces the need for insulin (Colberg et al., 2010). further evaluate this form of intervention (Ceysens et al., 2011). and Cesarean sections (Cheng et al., 2008). this level of physical activity (ADA, 2004). 5.2.3 Monitoring glucose levels 5.2.2 Physical exercise With respect to insulin therapy, there is no consensus on the glucose levels that would indicate that insulin should be initiated after the implementation of dietetic therapy. The American College of Obstetricians and Gynecologists (ACOG) suggests that insulin should be administered to reduce the risk of macrosomia with fasting glucose levels ≥ 95 mg% OR one-hour postprandial glucose levels > 130-140 mg% OR two-hour postprandial glucose levels ≥ 120 mg% (ACOG, 2001). There are no randomized clinical trials available in which different glucose levels were compared with the objective of determining the cut-off point for the implementation of insulin therapy. Three randomized clinical trials suggest initiating insulin therapy irrespective of glucose levels if ultrasonographic measurement of fetal abdominal circumference exceeds the 75th percentile (Bonomo et al., 2004; Kjos et al., 2001; Rossi et al., 2000). The doses and types of insulin will not be discussed in this chapter. #### 5.2.4.2 Antihyperglycemic drugs Oral hypoglycemic drugs are classically contraindicated in pregnancy. First generation drugs such as chlorpropamide and tolbutamide cross the placental barrier and may potentially cause prolonged and profound states of hypoglycemia, leading to fetal malformation. Nevertheless, the newer hypoglycemic drugs such as glibenclamide do not enter fetal circulation (Langer, 2007). Furthermore, considering that in patients with gestational diabetes, the need for treatment initiates after embryogenesis (Langer, 2007), the newer oral hypoglycemic drugs were seen as a practical therapeutic option for this group of patients. Patient satisfaction with this route of administration may result in better compliance with treatment. Interest in evaluating these drugs as an option for the control of gestational diabetes has been intense and various randomized clinical trials using these agents have been published over the past ten years (Langer et al., 2000; Moore et al., 2010; Rowan et al., 2008). In 2008, a systematic review was published that included a meta-analysis of all the clinical trials in which the use of insulin was compared with glibenclamide in women with gestational diabetes. Nine clinical trials were included involving 1,382 women with gestational diabetes. The use of glibenclamide was not found to be associated with any increased risk of macrosomia nor with differences in relation to fetal weight or the frequency of large-forgestational-age infants, admission to the neonatal ICU or an increased risk of neonatal hypoglycemia. These findings suggest that there is no increased perinatal risk with the use of this drug; however, the effectiveness and safety of its use still need to be confirmed, since the majority of the studies included were not randomized (Moretti et al., 2008). Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 55 drugs, it has been suggested that monitoring should be performed twice weekly beginning at 32 weeks (ACOG, 2001). The method of evaluating vitality and the periodicity of this evaluation, however, remains to be determined and varies in accordance with the protocol Macrosomia may be investigated by performing a single ultrasonography scan in the 36th week of pregnancy or by serial scans from 28 weeks onwards (Ben-Haroush et al., 2007). Nevertheless, the poor accuracy of ultrasonography for the prediction of fetal weight limits its use for this purpose (Wong et al., 2001). Based on specialist opinion, it has been suggested that fetal growth monitoring and the investigation of macrosomia is unnecessary in cases of gestational diabetes controlled by diet, principally because false-positive results may lead to unnecessary Cesarean sections (Melamed et al., 2010). Fetal weight estimated by ultrasonography would have to be ≥ 4,800 grams to have at least a 50% chance of predicting an Treatment of gestational diabetes may include anticipating delivery through induction or by elective Cesarean section. In a systematic review of the Cochrane Library, the policy of electively interrupting pregnancy by inducing labor in full-term diabetic women was evaluated (Boulvain et al., 2011). Only one study involving 200 women was included. Results showed that induction at 38 weeks reduced the frequency of newborn infants weighing > 4000 grams and above the 90th percentile, which is not surprising, since gestational age at delivery was lower in the induction group. This intervention, however, failed to reduce the risk of Cesarean section or of neonatal morbidities. Therefore, the authors concluded that further studies involving larger sample sizes are required in order to confirm the advantages of this intervention. Up to the present moment, there is insufficient More recently, a systematic review including five studies (the same clinical trial included in the Cochrane review plus four observational studies) compared active management at term (induction or Cesarean section) with expectant management. The results of the randomized clinical trial were similar to the findings of the previous systematic review. When the four observational studies were analyzed, however, a potential reduction was found in the rate of macrosomia, of shoulder dystocia in induced deliveries and in Cesarean sections indicated because of fetal macrosomia. The authors concluded that active management appears to reduce the rates of macrosomia and its complications; however, further clinical trials are clearly necessary to strengthen the evidence and support clinical practice (Witkop et al., 2009). The ACOG suggests performing elective Cesarean sections as a means of reducing the risk of shoulder dystocia in cases of gestational diabetes when estimated fetal weight is ≥ 4,500 grams (ACOG, 2001). In diabetic pregnant women in whom estimated fetal weight is below 4,000 grams, Cesarean section is unjustified on the basis of fetal weight alone (Hawkins and Casey, 2007). On the other hand, the management of cases in which estimated fetal weight is between 4,000 and 4,500 grams remains controversial. In addition to estimated fetal weight, the size of the mother's pelvis and the progression of labor should also be taken into consideration when deciding on the type of delivery (Hawkins and Casey, 2007). It should also be noted that the limited accuracy of ultrasonography for adequately estimating fetal weight leads to unnecessary Cesarean sections because of the suspicion of macrosomia (Chauhan et al., 2005). implemented in the service and the clinical situation (Conway, 2007). infant being born with a birthweight of 4,500 kg or more (McLaren et al., 1995). 5.2.5.2 Screening for fetal macrosomia 5.2.5.3 Anticipating delivery evidence to enable this practice to be recommended. Another systematic review published by the Johns Hopkins University Evidence-Based Practice Center for the Agency for Healthcare Research and Quality evaluated oral hypoglycemic drugs in women with gestational diabetes. Nine studies were selected, four of which consisted of randomized clinical trials involving 1,229 participants, while five were observational studies involving 831 participants. Two clinical trials compared insulin with glibenclamide, while one compared glibenclamide with acarbose and another compared insulin with metformin. No statistically significant differences were found with respect to glycemic control, the weight of the newborn infant or in the rate of Cesarean sections when insulin was compared with glibenclamide. There was a greater proportion of newborn infants with hypoglycemia in the group that used insulin (8.1% versus 3.3%; p = 0.008). No statistically significant difference was found in the rate of congenital malformations when the pregnancies treated with insulin were compared with those treated with oral hypoglycemic drugs. The authors concluded that there are no substantial differences in maternal and neonatal outcomes between women with gestational diabetes using insulin and those using oral hypoglycemic drugs (glibenclamide and metformin) (Nicholson et al., 2009). The most recent systematic review on oral hypoglycemic drugs for the treatment of gestational diabetes showed no difference either in glycemic control or in the outcome of pregnancy when insulin was compared with hypoglycemic drugs in six randomized clinical trials involving a total of 1,388 pregnant women. There was no increased risk of neonatal hypoglycemia, macrosomia or Cesarean section, and maternal glucose levels were similar (Dhulkotia et al., 2010). Results with the use of glibenclamide for the treatment of gestational diabetes are encouraging and although the ADA and ACOG consensuses recommend not prescribing glibenclamide for women with gestational diabetes (ACOG, 2001; ADA 2004), it would appear that there is already sufficient and consistent evidence confirming its safety and effectiveness in this condition. Another issue to be evaluated with respect to glibenclamide is its cost, which is significantly lower compared to treatment with insulin (Melamed and Yogev, 2009). Nevertheless, the United States Food and Drug Administration (FDA) has not approved these drugs for this purpose. #### 5.2.5 Obstetric treatment #### 5.2.5.1 Evaluation of fetal vitality Randomized clinical trials have yet to be conducted to evaluate the need for antenatal testing or the type of antenatal tests for the assessment of fetal well-being. Nonetheless, the fetuses of women with gestational diabetes, depending on glycemic control, may be at an increased risk of macrosomia (Durnwald et al., 2011) and intrauterine death (Yogev and Visser, 2009), and some observational studies have reported a reduction in the risk of fetal loss with various protocols for evaluating vitality (Graves, 2007; Kjos et al., 2005). In 2001, the ACOG concluded that there is insufficient evidence to determine the ideal scheme for monitoring antepartum fetal vitality in women with gestational diabetes controlled by diet and in whom there are no additional perinatal risks (ACOG, 2001). The evaluation of fetal vitality in cases of gestational diabetes may include fetal biophysical profile and antepartum cardiotocography. Doppler blood flow measurement is not useful for evaluating fetal vitality in this context (Graves, 2007). The frequency with which these tests should be performed depends on the classification of diabetes and is not routinely recommended in cases controlled with diet (ACOG, 2001; Conway, 2007). In women who require insulin or antihyperglycemic drugs, it has been suggested that monitoring should be performed twice weekly beginning at 32 weeks (ACOG, 2001). The method of evaluating vitality and the periodicity of this evaluation, however, remains to be determined and varies in accordance with the protocol implemented in the service and the clinical situation (Conway, 2007). #### 5.2.5.2 Screening for fetal macrosomia 54 Gestational Diabetes Another systematic review published by the Johns Hopkins University Evidence-Based Practice Center for the Agency for Healthcare Research and Quality evaluated oral hypoglycemic drugs in women with gestational diabetes. Nine studies were selected, four of which consisted of randomized clinical trials involving 1,229 participants, while five were observational studies involving 831 participants. Two clinical trials compared insulin with glibenclamide, while one compared glibenclamide with acarbose and another compared insulin with metformin. No statistically significant differences were found with respect to glycemic control, the weight of the newborn infant or in the rate of Cesarean sections when insulin was compared with glibenclamide. There was a greater proportion of newborn infants with hypoglycemia in the group that used insulin (8.1% versus 3.3%; p = 0.008). No statistically significant difference was found in the rate of congenital malformations when the pregnancies treated with insulin were compared with those treated with oral hypoglycemic drugs. The authors concluded that there are no substantial differences in maternal and neonatal outcomes between women with gestational diabetes using insulin and those using oral hypoglycemic The most recent systematic review on oral hypoglycemic drugs for the treatment of gestational diabetes showed no difference either in glycemic control or in the outcome of pregnancy when insulin was compared with hypoglycemic drugs in six randomized clinical trials involving a total of 1,388 pregnant women. There was no increased risk of neonatal hypoglycemia, macrosomia or Cesarean section, and maternal glucose levels were similar Results with the use of glibenclamide for the treatment of gestational diabetes are encouraging and although the ADA and ACOG consensuses recommend not prescribing glibenclamide for women with gestational diabetes (ACOG, 2001; ADA 2004), it would appear that there is already sufficient and consistent evidence confirming its safety and effectiveness in this condition. Another issue to be evaluated with respect to glibenclamide is its cost, which is significantly lower compared to treatment with insulin (Melamed and Yogev, 2009). Nevertheless, the United States Food and Drug Administration (FDA) has not Randomized clinical trials have yet to be conducted to evaluate the need for antenatal testing or the type of antenatal tests for the assessment of fetal well-being. Nonetheless, the fetuses of women with gestational diabetes, depending on glycemic control, may be at an increased risk of macrosomia (Durnwald et al., 2011) and intrauterine death (Yogev and Visser, 2009), and some observational studies have reported a reduction in the risk of fetal In 2001, the ACOG concluded that there is insufficient evidence to determine the ideal scheme for monitoring antepartum fetal vitality in women with gestational diabetes controlled by diet and in whom there are no additional perinatal risks (ACOG, 2001). The evaluation of fetal vitality in cases of gestational diabetes may include fetal biophysical profile and antepartum cardiotocography. Doppler blood flow measurement is not useful for evaluating fetal vitality in this context (Graves, 2007). The frequency with which these tests should be performed depends on the classification of diabetes and is not routinely recommended in cases controlled with diet (ACOG, 2001; Conway, 2007). In women who require insulin or antihyperglycemic loss with various protocols for evaluating vitality (Graves, 2007; Kjos et al., 2005). drugs (glibenclamide and metformin) (Nicholson et al., 2009). (Dhulkotia et al., 2010). approved these drugs for this purpose. 5.2.5 Obstetric treatment 5.2.5.1 Evaluation of fetal vitality Macrosomia may be investigated by performing a single ultrasonography scan in the 36th week of pregnancy or by serial scans from 28 weeks onwards (Ben-Haroush et al., 2007). Nevertheless, the poor accuracy of ultrasonography for the prediction of fetal weight limits its use for this purpose (Wong et al., 2001). Based on specialist opinion, it has been suggested that fetal growth monitoring and the investigation of macrosomia is unnecessary in cases of gestational diabetes controlled by diet, principally because false-positive results may lead to unnecessary Cesarean sections (Melamed et al., 2010). Fetal weight estimated by ultrasonography would have to be ≥ 4,800 grams to have at least a 50% chance of predicting an infant being born with a birthweight of 4,500 kg or more (McLaren et al., 1995). #### 5.2.5.3 Anticipating delivery Treatment of gestational diabetes may include anticipating delivery through induction or by elective Cesarean section. In a systematic review of the Cochrane Library, the policy of electively interrupting pregnancy by inducing labor in full-term diabetic women was evaluated (Boulvain et al., 2011). Only one study involving 200 women was included. Results showed that induction at 38 weeks reduced the frequency of newborn infants weighing > 4000 grams and above the 90th percentile, which is not surprising, since gestational age at delivery was lower in the induction group. This intervention, however, failed to reduce the risk of Cesarean section or of neonatal morbidities. Therefore, the authors concluded that further studies involving larger sample sizes are required in order to confirm the advantages of this intervention. Up to the present moment, there is insufficient evidence to enable this practice to be recommended. More recently, a systematic review including five studies (the same clinical trial included in the Cochrane review plus four observational studies) compared active management at term (induction or Cesarean section) with expectant management. The results of the randomized clinical trial were similar to the findings of the previous systematic review. When the four observational studies were analyzed, however, a potential reduction was found in the rate of macrosomia, of shoulder dystocia in induced deliveries and in Cesarean sections indicated because of fetal macrosomia. The authors concluded that active management appears to reduce the rates of macrosomia and its complications; however, further clinical trials are clearly necessary to strengthen the evidence and support clinical practice (Witkop et al., 2009). The ACOG suggests performing elective Cesarean sections as a means of reducing the risk of shoulder dystocia in cases of gestational diabetes when estimated fetal weight is ≥ 4,500 grams (ACOG, 2001). In diabetic pregnant women in whom estimated fetal weight is below 4,000 grams, Cesarean section is unjustified on the basis of fetal weight alone (Hawkins and Casey, 2007). On the other hand, the management of cases in which estimated fetal weight is between 4,000 and 4,500 grams remains controversial. In addition to estimated fetal weight, the size of the mother's pelvis and the progression of labor should also be taken into consideration when deciding on the type of delivery (Hawkins and Casey, 2007). It should also be noted that the limited accuracy of ultrasonography for adequately estimating fetal weight leads to unnecessary Cesarean sections because of the suspicion of macrosomia (Chauhan et al., 2005). Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 57 American College of Obstetricians and Gynecologists Committee on Practice Bulletins— American Diabetes Association. Gestational diabetes mellitus. Diabetes Care 2004 Jan; 27 Ben-Haroush, A., Chen, R., Hadar, E., Hod, M., Yogev, Y. Accuracy of a single fetal weight gestational-age infants at term? Am J Obstet Gynecol 2007 Nov; 197:497.e1-6. Bonomo, M., Cetin, I., Pisoni, M.P., Faden, D., Mion, E., Taricco, E., et al. Flexible treatment a controlled randomized clinical trial. Diabetes Metab 2004 Jun; 30: 237-44. Boulvain, M., Stan, C.M., Irion, O. Elective delivery in diabetic pregnant women. Cochrane Brody, S.C., Harris, R., Lohr, K. Screening for gestational diabetes: a summary of the Buchanan, A.T., Kjos, L.S.: Gestational diabetes: Risk or myth? J Clin Endocrinol Metab 1999 Carpenter, M.W., Coustan, D.R.B. Criteria for screening tests for gestational diabetes. Am J Catalano, P.M. Obesity, insulin resistance, and pregnancy outcome. Reproduction. 2010 May; Ceysens, G., Rouiller, D., Boulvain, M. Exercise for diabetic pregnant women. Cochrane Chandler-Laney, P.C., Bush, N.C., Rouse, D.J., Mancuso, M.S., Gower, B.A. Maternal glucose Chauhan, S.P., Grobman, W.A., Gherman, R.A., Chauhan, V.B., Chang, G., Magann, E.F., Cheng, Y.W., Chung, J.H., Kurbisch-Block, I., Inturrisi, M., Shafer, S., Caughey, A.B. Colberg, S.R., Sigal, R.J., Fernhall, B., Regensteiner, J.G., Blissmer, B.J., Rubin, R.R., Chasan- Conway, D.L. Obstetric management in gestational diabetes. Diabetes Care 2007 Jul; 30 Suppl Crowther, C.A., Hiller, J.E., Moss , J.R., McPhee, A.J., Jeffries, W.S., Robinson, J.S.; Australian Database of Systematic Reviews. In: The Cochrane Library, Issue 04, 2011, Art. No. concentration during pregnancy predicts fat and lean mass of prepubertal Hendrix, N.W. Suspicion and treatment of the macrosomic fetus: a review. Am J Gestational weight gain and gestational diabetes mellitus: perinatal outcomes. Taber, L., Albright, A.L., Braun, B.; American College of Sports Medicine; American Diabetes Association. Exercise and type 2 diabetes: the American College of Sports Medicine and the American Diabetes Association: Joint Position Statement. Diabetes Carbohydrate Intolerance Study in Pregnant Women (ACHOIS) Trial Group. Effect of treatment of gestational diabetes mellitus on pregnancy outcomes. N Engl J Med CD001997. DOI: 10.1002/14651858.CD001997.pub1 CD004225. DOI: 10.1002/14651858.CD004225.pub3 offspring. Diabetes Care. 2011 Mar; 34:741-5. Obstet Gynecol 2005 Aug; 193: 332-46. Obstet Gynecol 2008 Nov; 112: 1015-22. Care 2010 Dec; 33: e147-67. 2005 Jun; 352: 2477-86. 2: S175-9. Obstet Gynecol 2001; 98: 525-38. Suppl 1: S88-90. 101:380-92 Jun; 84: 854-7. 140: 365-71. Obstet Gynecol 1982 Jun; 144:768-73 Obstetrics. ACOG Practice Bulletin. Clinical management guidelines for obstetrician-gynecologists. Number 30, September 2001. Gestational diabetes. estimation at 29-34 weeks in diabetic pregnancies: can it predict large-for- of gestational diabetes modulated on ultrasound evaluation of intrauterine growth: Database of Systematic Reviews. In: The Cochrane Library, Issue 04, 2011, Art. No. evidence for the U.S. Preventive Services Task Force. Obstet Gynecol. 2003 Feb; In the absence of macrosomia, specialists suggest that patients with gestational diabetes controlled by diet may be able to reach 40/41 weeks and recommend induction at this gestational age. In patients in use of insulin or oral antihyperglycemic drugs, labor should be induced at 39 weeks. In diabetic patients in use of insulin or those in whom glycemic control is poor, labor should be induced at 38 weeks and even prior to this gestational age if there are associated conditions such as severe preeclampsia, for example, or if fetal wellbeing is compromised. There is no need for amniocentesis to evaluate fetal lung maturity in patients after 38 weeks of pregnancy when gestational age is well documented (Conway et al., 2007; Nicholson et al., 2008). #### 6. Conclusions The most recent evidence suggests that screening for gestational diabetes is beneficial; however, the best screening strategy remains to be defined. Clinical trials also need to be conducted to compare various diagnostic tests and glucose levels; however, until these studies are performed, clinicians and societies have to define their own protocols for screening and diagnosis taking the characteristics of the population to be screened into consideration. In populations with a high risk for diabetes and consequently for macrosomia, a universal screening policy leads to a significant reduction in perinatal morbidity. With respect to treatment, although the Cochrane systematic review found only modest benefits with treatment, more recent randomized clinical trials suggest an improvement in perinatal outcome. Based on specialist opinion, initial dietetic therapy is recommended, with pharmacological treatment indicated when diet alone fails to control glucose levels. Despite recent evidence that treatment with antihyperglycemic drugs may represent a safe, reliable alternative for the pharmacological treatment of diabetes in pregnancy, the ADA and other guidelines continue recommending insulin therapy as standard treatment. There is insufficient evidence either to indicate or contraindicate exercise in women with gestational diabetes. The types of tests and the ideal frequency at which fetal well-being should be monitored are factors that are yet to be determined; however, they are unnecessary in cases in which glucose levels are controlled by diet. In addition, there is insufficient evidence to recommend ultrasonography for the prediction of macrosomia and scans should not be performed routinely for this purpose in pregnant women on dietetic therapy in whom glucose levels are under control. With respect to delivery, elective Cesarean sections are recommended by ACOG in the case of fetuses over 4,500 grams. In cases of gestational diabetes controlled by diet, it is possible to wait for spontaneous labor to occur up to a limit of 40/41 weeks. In patients in use of insulin or oral hypoglycemic drugs, labor should be induced at 39 weeks. In cases in which glucose control is poor, delivery should be anticipated at 38 weeks or earlier if fetal wellbeing is compromised or there are other associated morbid conditions. #### 7. References In the absence of macrosomia, specialists suggest that patients with gestational diabetes controlled by diet may be able to reach 40/41 weeks and recommend induction at this gestational age. In patients in use of insulin or oral antihyperglycemic drugs, labor should be induced at 39 weeks. In diabetic patients in use of insulin or those in whom glycemic control is poor, labor should be induced at 38 weeks and even prior to this gestational age if there are associated conditions such as severe preeclampsia, for example, or if fetal wellbeing is compromised. There is no need for amniocentesis to evaluate fetal lung maturity in patients after 38 weeks of pregnancy when gestational age is well documented (Conway et The most recent evidence suggests that screening for gestational diabetes is beneficial; however, the best screening strategy remains to be defined. Clinical trials also need to be conducted to compare various diagnostic tests and glucose levels; however, until these studies are performed, clinicians and societies have to define their own protocols for screening and diagnosis taking the characteristics of the population to be screened into consideration. In populations with a high risk for diabetes and consequently for macrosomia, a universal With respect to treatment, although the Cochrane systematic review found only modest benefits with treatment, more recent randomized clinical trials suggest an improvement in perinatal outcome. Based on specialist opinion, initial dietetic therapy is recommended, with pharmacological treatment indicated when diet alone fails to control glucose levels. Despite recent evidence that treatment with antihyperglycemic drugs may represent a safe, reliable alternative for the pharmacological treatment of diabetes in pregnancy, the ADA and other guidelines continue recommending insulin therapy as standard treatment. There is insufficient evidence either to indicate or contraindicate exercise in women with The types of tests and the ideal frequency at which fetal well-being should be monitored are factors that are yet to be determined; however, they are unnecessary in cases in which glucose levels are controlled by diet. In addition, there is insufficient evidence to recommend ultrasonography for the prediction of macrosomia and scans should not be performed routinely for this purpose in pregnant women on dietetic therapy in whom With respect to delivery, elective Cesarean sections are recommended by ACOG in the case of fetuses over 4,500 grams. In cases of gestational diabetes controlled by diet, it is possible to wait for spontaneous labor to occur up to a limit of 40/41 weeks. In patients in use of insulin or oral hypoglycemic drugs, labor should be induced at 39 weeks. In cases in which glucose control is poor, delivery should be anticipated at 38 weeks or earlier if fetal well- Aberg, A., Rydhstroem, H., Frid, A. Impaired glucose tolerance associated with adverse Alwan, N., Tuffnell, D.J., West, J. Treatments for gestational diabetes. Cochrane Database of pregnancy outcome: a population-based study in southern Sweden. Am J Obstet Systematic Reviews. In: The Cochrane Library, Issue 04, 2011, Art. No. CD003395. screening policy leads to a significant reduction in perinatal morbidity. being is compromised or there are other associated morbid conditions. al., 2007; Nicholson et al., 2008). 6. Conclusions gestational diabetes. 7. References glucose levels are under control. Gynecol. 2001 Jan; 184:77-83. DOI: 10.1002/14651858.CD003395.pub3 Gestational Diabetes: Evidence-Based Screening, Diagnosis and Treatment 59 Langer, O., Brustman, L., Anyaegbunam, A., Mazze, R. The significance of one abnormal Langer, O., Conway, D.L., Berkus, M.D., Xenakis, E.M., Gonzales, O. A comparison of Langer, O., Yogev, Y., Most, O., Xenakis, E.M. Gestational diabetes: the consequences of not Langer O. From educated guess to accepted practice: the use of oral antidiabetic agents in Landon, M.B., Spong, C.Y., Thom, E., Carpenter, M.W., Ramin, S.M., Casey, B. et al. A Lawlor, D.A., Lichtenstein, P., Långström, N. Association of maternal diabetes mellitus in Lucas, M.J., Lowe, T.W., Bowe, L., McIntire, D.D. Class A1 gestational diabetes: a Melamed, N., Yogev, Y. Can pregnant diabetics be treated with glyburide? Womens Health Melamed, N., Yogev, Y., Meizner, I., Mashiach, R., Ben-Haroush, A. Sonographic prediction Metzger, B.E., Buchanan, T.A., Coustan, D.R., de Leiva, A., Dunger, D.B., Hadden, D.R., on Gestational Diabetes Mellitus. Diabetes Care. 2007 Jul;30 Suppl 2:S251-60. Metzger, B.E., Gabbe, S.G., Persson, B., Buchanan, T.A., Catalano, P.A., Damm, P. et al. Moore, L.E., Clokey, D., Rappaport, V.J., Curet, L.B. Metformin compared with glyburide in gestational diabetes: a randomized controlled trial. Obstet Gynecol 2010 Jan; 115: 55-9. Moretti, M.E., Rezvani, M., Koren, G. Safety of glyburide for gestational diabetes: a metaanalysis of pregnancy outcomes. Ann Pharmacother 2008 Apr; 42: 483-90. National Diabetes Data Group: Classification and diagnosis of diabetes mellitus and other Naylor, C.D. Diagnosing Gestational Diabetes mellitus. Is the Gold Standard Valid? Diabetes Nicholson, W.K., Wilson, L.M., Witkop, C.T., Baptiste-Roberts, K., Bennett, W.L., Bolen, S. et al. gestational diabetes. Evid Rep Technol Assess (Full Rep) 2008 Mar; 162:1-96. Nicholson, W., Bolen, S., Witkop, C.T., Neale, D., Wilson, L., Bass, E. Benefits and risks of Therapeutic management, delivery, and postpartum risk assessment and screening in oral diabetes agents compared with insulin in women with gestational diabetes: a categories of glucose intolerance. Diabetes 1979; 28: 1039-1057, systematic review. Obstet Gynecol 2009 Jan; 113: 193-205. of fetal macrosomia: the consequences of false diagnosis. J Ultrasound Med 2010 Feb; Hod, M., Kitzmiller, J.L., Kjos, S.L., Oats, J.N., Pettitt, D.J., Sacks, D.A., Zoupas, C. Summary and recommendations of the Fifth International Workshop-Conference International association of diabetes and pregnancy study groups recommendations on the diagnosis and classification of hyperglycemia in cohort of 280,866 men from 248,293 families. Circulation. 2011 Jan;123:258-65. Leary, J., Pettitt, D.J., Jovanovic, L. Gestational diabetes guidelines in a HAPO world. Best treating. Am J Obstet Gynecol 2005 Apr; 192: 989-97. pregnancy. Clin Obstet Gynecol 2007 Dec; 50: 959-71. Pract Res Clin Endocrinol Metab 2010 Aug; 24: 673-85. pregnancy. Diabetes Care 2010 Mar; 33: 676–82. meaningful diagnosis? Obstet Gynecol 1993 Aug; 82:260-5. 1987 Sep; 157:758-63. 2000 Oct; 343: 1134-8. Med 2009 Oct; 361: 1339-48. (Lond Engl) 2009 Nov; 5: 649-58. Care 1989 Sep; 12: 565-72. 29: 225-30. glucose tolerance test value on adverse outcome in pregnancy. Am J Obstet Gynecol glyburide and insulin in women with gestational diabetes mellitus. N Engl J Med multicenter, randomized trial of treatment for mild gestational diabetes. N Engl J pregnancy with offspring adiposity into early adulthood: sibling study in a prospective de Veciana, M., Major, C.A., Morgan, M.A., Asrat, T., Toohey, J.S., Lien, J.M., Evans, A.T. diabetes mellitus requiring insulin therapy. N Engl J Med 1995 Nov; 333:1237-41. Dhulkotia, J.S., Ola, B., Fraser, R., Farrell, T. Oral hypoglycemic agents vs insulin in Durnwald, C.P., Mele, L., Spong, C.Y., Ramin, S.M., Varner, M.W., Rouse, D.J. et al. for the Fall, C. Maternal nutrition: effects on health in the next generation. Indian J Med Res. 2009 Graves, C.R. Antepartum fetal surveillance and timing of delivery in the pregnancy complicated by diabetes mellitus. Clin Obstet Gynecol 2007 Dec; 50: 1007-13. HAPO Study Cooperative Research Group. Hyperglycemia and adverse pregnancy Hawkins, J.S., Casey, B.M. Labor and delivery management for women with diabetes. Obstet Hawkins, J.S., Casey, B.M., Lo, J.Y., Moss, K., McIntire, D.D., Leveno, K.J. Weekly compared Horvath, K., Koch, K., Jeitler, K., Matyas, E., Bender, R., Bastian, H., Lange, S., Siebenhofer, International Association of Diabetes and Pregnancy Study Groups. International Jensen, D.M., Damm, P., Sørensen, B., Mølsted-Pedersen, L., Westergaard, J.G., Klebe, J., Jovanovic, L.G. Using meal-based self-monitoring of blood glucose as a tool to improve outcomes in pregnancy complicated by diabetes. Endocr Pract 2008 Mar; 14: 239-47. Kjos, S.L., Leung, A., Henry, O.A., Victor, M.R., Paul, R.H., Medearis, A.L. Antepartum Kjos, S.L., Schaefer-Graf, U., Sardesi, S., Peters, R.K., Buley, A., Xiang, A.H., et al. A Hawkins, J.S. Glucose Monitoring During Pregnancy. Curr Diab Rep 2010 Jun; 10: 229–34. Holt, R.I., Coleman, M.A., McCance, D.R. The implications of the new International with daily blood glucose monitoring in women with diet-treated gestational Association of Diabetes and Pregnancy Study Groups (IADPSG) diagnostic criteria A. Effects of treatment in women with gestational diabetes mellitus: systematic Association of Diabetes and Pregnancy Study Groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care 2010 Mar; Beck-Nielsen, H. Clinical impact of mild carbohydrate intolerance in pregnancy: a study of 2904 nondiabetic Danish women with risk factors for gestational diabetes surveillance in diabetic pregnancies: predictors of fetal distress in labor. Am J Obstet randomized controlled trial using glycemic plus fetal ultrasound parameters versus glycemic parameters to determine insulin therapy in gestational diabetes with Gestational Diabetes. Obstet Gynecol 2011 Apr; 117: 819-27. outcomes. N Engl J Med 2008 May; 358: 1991-2002. Gynecol Clin North Am 2007 Jun; 34:323-34. diabetes. Obstet Gynecol 2009 Jun; 113:1307-12. for gestational diabetes. Diabet Med. 2011 Apr; 28: 382-5. review and meta-analysis. BMJ 2010 Apr; 340: c1395. mellitus. Am J Obstet Gynecol. 2001 Aug; 185:413-9. fasting hyperglycemia. Diabetes Care 2001 Nov; 24: 1904-10. Gynecol 1995 Nov; 173: 1532-9. Obstet Gynecol 2010 Nov; 203: 457.e1-9. Nov;130: 593-9. 33: 676–82. Postprandial versus preprandial blood glucose monitoring in women with gestational management of gestational diabetes: a systematic review and metaanalysis. Am J Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Maternal-Fetal Medicine Units Network (MFMU). Glycemic Characteristics and Neonatal Outcomes of Women Treated for Mild 4 Saudi Arabia Validity of Fasting Blood Glucose State Among Pregnant Females Mostafa A Abolfotouh and Mohammed A Al-Rowaily King Saud Bin-Abdulaziz University for Health Sciences, Riyadh, Test in Screening for the Pre-Diabetes Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance of varying degrees of severity with onset or first recognition during pregnancy (Metzer & Coustan, 1998). Glucose tolerance deteriorates in human pregnancy, but about 97% to 98% of all pregnant women retain a normal glucose tolerance and only 2% to 3% develops GDM (Kuhl, 1991). However, failure to diagnose and treat GDM will result in increased morbidity in some pregnancies, while an aggressive approach to diagnosis and treatment may result in The prevalence of GDM ranges from 1% to 14% of all pregnancies, depending on the population studied and the diagnostic tests and criteria employed (World Health Organization [WHO], 1985). In a recent study by Al-Rowaily and Abolfotouh in Riyadh, Saudi Arabia, the prevalence of GDM was 12.5% and 3.8% by the WHO and American Diabetes Association criteria respectively (Al-Rowaily & Abolfotouh, 2010). The appropriateness of these different diagnostic criteria has been debated (Gabir et al., 2000); nevertheless women meeting the definition for GDM by either set of criteria are at greater The 75-g glucose load has been the international standard for the diagnosis of diabetes in nonpregnant adults for several decades. This oral glucose tolerance test (OGTT) identifies pregnant women who are at risk of pre-eclampsia and whose babies are at risk of macrosomia and perinatal mortality (Schmidt et al., 2001). Although the American Diabetes Association (ADA) still recommends a 3-h 100-g OGTT for the diagnosis of GDM, it has recently included in its recommendations the use of a 2-h 75-g OGTT (American Diabetes A recent WHO panel, although in general maintaining previous diagnostic recommendations, now characterizes GDM as the joint category of diabetes and impaired glucose tolerance (fasting glucose ≥ 7.0 mmol/L or 2-h glucose ≥ 7.8mmol/L (WHO, 1999). At present, screening for gestational diabetes appears to be hampered by the lack of a clear definition, agreed diagnostic criteria and evidence to show that intervention and treatment for this condition leads to improved outcomes for the mother and fetus. Although fasting plasma glucose and Glucose Challenge Test (GCT) have the highest reported sensitivities unnecessary intervention in others (Kjos & Buchanan, 1999). risk of complications than women without the diagnosis. Association [ADA], 2000; Metzger & Coustan, 1998). 1. Introduction ## Validity of Fasting Blood Glucose Test in Screening for the Pre-Diabetes State Among Pregnant Females Mostafa A Abolfotouh and Mohammed A Al-Rowaily King Saud Bin-Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia #### 1. Introduction 60 Gestational Diabetes O'Sullivan, J.B., Mahan, C.M.: Criteria for the oral glucose tolerance test in pregnancy. O'Sullivan, J.B., Gellis, S.S., Dandrow, R.V., Tenney, B.O. The potential diabetic and her O'Sullivan, J.B., Charles, D., Mahan, C.M., Dandrow, R.V. Gestational diabetes and perinatal Pridjian, G., Benjamin, T.D. Update on gestational diabetes. Obstet Gynecol Clin North Am Rossi, G., Somigliana, E., Moschetta, M., Bottani, B., Barbieri, M., Vignali, M. Adequate timing Rudge, M.V.C., Peraçoli, J.C., Berezowski, A.T., Calderon, I.M., Brasil, M.A.M. The oral Sermer, M., Naylor, D.C., Gare, D.J., Kenshole, A.B., Ritchie, J.W.K., Farine, D., Cohen, H.R., Tieu, J., Middleton, P., McPhee, A.J., Crowther, C.A. Screening and subsequent management Torloni, M.R., Betrán, A.P., Horta, B.L., Nakamura, M.U., Atallah, A.N., Moron, A.F., review of the literature with meta-analysis. Obes Rev. 2009 Mar; 10: 194-203. Walkinshaw, S.A. Dietary regulation for 'gestational diabetes'. Cochrane Database of Weisz, B., Shrim, A., Homko, C.J., Schiff, E., Epstein, G.S., Sivan, E. One hour versus two WHO Expert Committee on Diabetes Mellitus. second report. WHO Technical Report Series, World Health Organization. Definition, diagnosis, and classification of diabetes mellitus and Witkop, C.T., Neale, D., Wilson, L.M., Bass, E.B., Nicholson, W.K. Active compared with Wong, S.F., Chan, F.Y., Cincotta, R.B., Oats, J.J., McIntyre, H.D. Sonographic estimation of Yogev, Y., Visser, G.H. Obesity, gestational diabetes and pregnancy outcome. Semin Fetal gestational diabetes. Am J Obstet Gynecol 1995 Jul; 173: 146-56. CD007222. DOI: 10.1002/14651858.CD007222.pub7 DOI: 10.1002/14651858.CD000070.pub3 study. J Perinatol 2005 Apr; 25: 241-4. Production Services, 1999. pp 19-20. review. Obstet Gynecol 2009 Jan; 113: 206-17. N Z J Obstet Gynaecol 2001 Nov; 41: 429-32. Neonatal Med 2009 Apr; 14:77-84. 1980; 646: 1-80. of fetal ultrasound to guide metabolic therapy in mild gestational diabetes mellitus. Results from a randomized study. Acta Obstet Gynecol Scand 2000 Aug; 79: 649-54. Rowan, J.A., Hague, W.M., Gao, W., Battin, M.R., Moore, M.P; MiG Trial Investigators. Metformin versus insulin for the treatment of gestational diabetes. N Engl J Med glucose tolerance test is a poor predictor of hyperglycemia during pregnancy. Braz J McArthur, K., Holzapfel, S., Biringer, A., Chen, E. Impact of increasing carbohydrate intolerance on maternal-fetal outcomes in 3637 women without for gestational diabetes for improving maternal and infant health. Cochrane Database of Systematic Reviews. In: The Cochrane Library, Issue 04, 2011, Art. No. Valente, O. Prepregnancy BMI and the risk of gestational diabetes: a systematic Systematic Reviews. In: The Cochrane Library, Issue 04, 2011, Art. No. CD000070. hours postprandial glucose measurement in gestational diabetes: a prospective its complications: report of a WHO consultation. Geneva: WHO document expectant delivery management in women with gestational diabetes: a systematic fetal weight in macrosomic fetuses: diabetic versus non-diabetic pregnancies. Aust treatment during pregnancy. Obstet Gynecol 1966 May; 27:683-9 mortality rates. Am J Obstet Gynecol 1973 Aug; 116:901-4 Diabetes 1964 May-Jun; 13:278-85. 2010 Jun; 37: 255-67. 2008 May; 358: 2003-15. Med Biol Res 1990; 23:1079-89. Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance of varying degrees of severity with onset or first recognition during pregnancy (Metzer & Coustan, 1998). Glucose tolerance deteriorates in human pregnancy, but about 97% to 98% of all pregnant women retain a normal glucose tolerance and only 2% to 3% develops GDM (Kuhl, 1991). However, failure to diagnose and treat GDM will result in increased morbidity in some pregnancies, while an aggressive approach to diagnosis and treatment may result in unnecessary intervention in others (Kjos & Buchanan, 1999). The prevalence of GDM ranges from 1% to 14% of all pregnancies, depending on the population studied and the diagnostic tests and criteria employed (World Health Organization [WHO], 1985). In a recent study by Al-Rowaily and Abolfotouh in Riyadh, Saudi Arabia, the prevalence of GDM was 12.5% and 3.8% by the WHO and American Diabetes Association criteria respectively (Al-Rowaily & Abolfotouh, 2010). The appropriateness of these different diagnostic criteria has been debated (Gabir et al., 2000); nevertheless women meeting the definition for GDM by either set of criteria are at greater risk of complications than women without the diagnosis. The 75-g glucose load has been the international standard for the diagnosis of diabetes in nonpregnant adults for several decades. This oral glucose tolerance test (OGTT) identifies pregnant women who are at risk of pre-eclampsia and whose babies are at risk of macrosomia and perinatal mortality (Schmidt et al., 2001). Although the American Diabetes Association (ADA) still recommends a 3-h 100-g OGTT for the diagnosis of GDM, it has recently included in its recommendations the use of a 2-h 75-g OGTT (American Diabetes Association [ADA], 2000; Metzger & Coustan, 1998). A recent WHO panel, although in general maintaining previous diagnostic recommendations, now characterizes GDM as the joint category of diabetes and impaired glucose tolerance (fasting glucose ≥ 7.0 mmol/L or 2-h glucose ≥ 7.8mmol/L (WHO, 1999). At present, screening for gestational diabetes appears to be hampered by the lack of a clear definition, agreed diagnostic criteria and evidence to show that intervention and treatment for this condition leads to improved outcomes for the mother and fetus. Although fasting plasma glucose and Glucose Challenge Test (GCT) have the highest reported sensitivities Validity of Fasting Blood Glucose Test in Screening Fasting Blood Glucose [N=769] Normal [<140mg/dl(<7.8mmol/L)] Normal [<100mg/dl(<5.6mmol/L)] Impaired fasting tolerance [100-125mg/dl(5.6-6.9mmol/L)] Glucose Challenge Test (2 hr after 75-g glucose drink) [N=408] Impaired glucose tolerance [140-199mg/dl (7.8-11mmol/L)] Provisional diagnosis of GDM [≥ 200mg/dl ( 11.1mmol/L)] and glucose challenge tests based on the ADA classification (ADA, 2010). 279 84.8 59 78.7 3 75.0 among pregnant females at the National Guard Hospital. challenge test, and x is the result of fasting blood sugar testing. Glucose Challenge Test Fasting Blood Glucose Provisional Table 2. Association between the results of Fasting blood sugar and Glucose challenge test Figure 1 shows the scatter plot for the values and the correlation between the values obtained by the fasting blood glucose and those obtained by the 2hr-glucose challenge test. The correlation coefficient (r) was 0.25 (t-value = 5.24, p<0.0001) indicating a highly significant direct correlation between the values of blood sugar by the two methods. The linear regression equation was: y= 0.66 x + 2.98, where y is the result of the glucose GDM Normal IGT No. % No. % No. % 47 14.3 14 18.7 1 25.0 Provisional diagnosis of GDM [≥126mg/dl(7mmol/L)] 3. Results (Table 2) Normal IFT Provisional GDM K = 0.054, p=0.25 for the Pre-Diabetes State Among Pregnant Females 63 Based on the cut-off values recommended by the American Diabetic Association (ADA, 2010) for diagnosis of GDM, the results of fasting blood sugar for 769 pregnant females showed that 17.2% had Impaired Fasting Tolerance (IFT) and 1.4% had provisional diagnosis of GDM. The corresponding results for those who responded to the GCT, are 15.2% IGT and 1.2% provisional diagnosis of GDM (Table 1), although the results of these two tests were seen to be comparable, yet the agreement level, as tested by kappa, was low. Category No % Table 1. Distribution of pregnant females according to their results of fasting blood glucose 81.4 17.2 1.4 83.6 15.2 1.2 626 133 10 341 62 5 3 0.9 2 2.7 - - and specificities in the literature, there also exists considerable debate about which screening test should be used if there is to be screening. A continuum of risk for GDM should be researched and risk of adverse pregnancy outcomes clarified on such a continuum. This would help to form the basis for diagnosis. The most appropriate strategies for screening, diagnosing and managing asymptomatic GDM remain controversial (Moody, 2003). The pregnant females who attend the antenatal clinic of King Abdulaziz Medical City at the National Guard Health Affairs (usually between 24-28 weeks of gestation) are prepared for the test by fasting for a minimum of 8 hrs. After that, a fasting sample is analyzed for glucose. Then, every pregnant female is subjected to the Glucose Challenge Test (GCT) by being given 75-g Glucose solution [named Glucola which is a chilled glucose syrup with 75-g of glucose with orange flavor], and another blood sample is collected 2 hours after the drink (Berger et al., 2002). The diagnosis of GDM is based upon the results of both the fasting sample and the 2hr-glucose challenge test. Although GCT showed good sensitivity and specificity in a previous study (79% and 87% respectively), yet it has been observed that with Glucola drink, there is always a tendency for the pregnant female to vomit (O'Sullivan et al., 1973). This usually leads to non-compliance with the glucose challenge test by most of the pregnant females. Thus, the aim of the present study was to determine the threshold value of fasting blood sugar that suggests the prediabetes status that needs further investigations including the GCT. #### 2. Methods All pregnant females who attended the antenatal clinic of King Fahd Hospital at Riyadh National Guard Health Affairs during the period from July, 2005 to July, 2006 for the first time (n=769) constituted the target of the present study. For all respondents to the GCT (n=408,53.1%), all values of the fasting blood sugar were cross classified according to their status by the GCT, and by various cut-off points along the range of FBS values above which subjects may be considered having Impaired Glucose Tolerance (IGT) by the GCT(≥ 7.8mmol/L) result. From these tabulations, the sensitivity, specificity and positive predictive value were computed for Fasting Blood Sugar (FBS) at each cut of point. The sensitivity of FBS diagnosis for the GCT diagnosis "gold standard" was determined by calculating how frequent the correct FBS diagnosis was made in each GCT diagnosis. The specificity of FBS diagnosis was determined by calculating how frequently the FBS diagnosis was not made when the corresponding GCT diagnosis was not present. Positive predictability indicated how frequently the FBS diagnosis was not made when the corresponding GCT diagnosis was not present. Positive predictability indicated how frequently the FBS diagnosis correctly reflected the GCT diagnosis. Also, the level of agreement between these two methods was determined at each cut-off point by the calculation of kappa coefficient (k). The Receiver Operating Characteristic (ROC) curve of a diagnostic test is a graph of the pairs of sensitivity and 1 minus specificity that correspond to each possible cut-off for the diagnostic test result (Richardson et al., 1993). This curve was used to determine the threshold value of FBS that correspond to the value of 7.8mmol/L by the GCT. The Statistical Package for the Social Sciences (SPSS) software program version 17 was used for all statistical analyses. ### 3. Results 62 Gestational Diabetes and specificities in the literature, there also exists considerable debate about which screening test should be used if there is to be screening. A continuum of risk for GDM should be researched and risk of adverse pregnancy outcomes clarified on such a continuum. This would help to form the basis for diagnosis. The most appropriate strategies for screening, The pregnant females who attend the antenatal clinic of King Abdulaziz Medical City at the National Guard Health Affairs (usually between 24-28 weeks of gestation) are prepared for the test by fasting for a minimum of 8 hrs. After that, a fasting sample is analyzed for glucose. Then, every pregnant female is subjected to the Glucose Challenge Test (GCT) by being given 75-g Glucose solution [named Glucola which is a chilled glucose syrup with 75-g of glucose with orange flavor], and another blood sample is collected 2 hours after the drink (Berger et al., 2002). The diagnosis of GDM is based upon the results of both the fasting Although GCT showed good sensitivity and specificity in a previous study (79% and 87% respectively), yet it has been observed that with Glucola drink, there is always a tendency for the pregnant female to vomit (O'Sullivan et al., 1973). This usually leads to non-compliance with the glucose challenge test by most of the pregnant females. Thus, the aim of the present study was to determine the threshold value of fasting blood sugar that suggests the pre- All pregnant females who attended the antenatal clinic of King Fahd Hospital at Riyadh National Guard Health Affairs during the period from July, 2005 to July, 2006 for the first time (n=769) constituted the target of the present study. For all respondents to the GCT (n=408,53.1%), all values of the fasting blood sugar were cross classified according to their status by the GCT, and by various cut-off points along the range of FBS values above which subjects may be considered having Impaired Glucose Tolerance (IGT) by the GCT(≥ 7.8mmol/L) result. From these tabulations, the sensitivity, specificity and positive predictive The sensitivity of FBS diagnosis for the GCT diagnosis "gold standard" was determined by calculating how frequent the correct FBS diagnosis was made in each GCT diagnosis. The specificity of FBS diagnosis was determined by calculating how frequently the FBS diagnosis was not made when the corresponding GCT diagnosis was not present. Positive predictability indicated how frequently the FBS diagnosis was not made when the corresponding GCT diagnosis was not present. Positive predictability indicated how frequently the FBS diagnosis correctly reflected the GCT diagnosis. Also, the level of agreement between these two methods was determined at each cut-off point by the The Receiver Operating Characteristic (ROC) curve of a diagnostic test is a graph of the pairs of sensitivity and 1 minus specificity that correspond to each possible cut-off for the diagnostic test result (Richardson et al., 1993). This curve was used to determine the threshold value of FBS that correspond to the value of 7.8mmol/L by the GCT. The Statistical Package for the Social Sciences (SPSS) software program version 17 was used for diabetes status that needs further investigations including the GCT. value were computed for Fasting Blood Sugar (FBS) at each cut of point. diagnosing and managing asymptomatic GDM remain controversial (Moody, 2003). sample and the 2hr-glucose challenge test. calculation of kappa coefficient (k). all statistical analyses. 2. Methods Based on the cut-off values recommended by the American Diabetic Association (ADA, 2010) for diagnosis of GDM, the results of fasting blood sugar for 769 pregnant females showed that 17.2% had Impaired Fasting Tolerance (IFT) and 1.4% had provisional diagnosis of GDM. The corresponding results for those who responded to the GCT, are 15.2% IGT and 1.2% provisional diagnosis of GDM (Table 1), although the results of these two tests were seen to be comparable, yet the agreement level, as tested by kappa, was low. (Table 2) Table 1. Distribution of pregnant females according to their results of fasting blood glucose and glucose challenge tests based on the ADA classification (ADA, 2010). K = 0.054, p=0.25 Table 2. Association between the results of Fasting blood sugar and Glucose challenge test among pregnant females at the National Guard Hospital. Figure 1 shows the scatter plot for the values and the correlation between the values obtained by the fasting blood glucose and those obtained by the 2hr-glucose challenge test. The correlation coefficient (r) was 0.25 (t-value = 5.24, p<0.0001) indicating a highly significant direct correlation between the values of blood sugar by the two methods. The linear regression equation was: y= 0.66 x + 2.98, where y is the result of the glucose challenge test, and x is the result of fasting blood sugar testing. Validity of Fasting Blood Glucose Test in Screening characteristic curve as shown in. (Figure 2) 1.00 .75 .50 .25 0.00 Sensitivity 4. Discussion for the Pre-Diabetes State Among Pregnant Females 65 Computations for sensitivity and specificity were made for all cut-off levels along the range of values of the fasting blood glucose test. The resulting values for sensitivity were plotted against the corresponding values of (1 – specificity) to obtain the receiver operating 1 - Specificity Fig. 2. Receiver operating characteristic curve of fasting blood glucose measurements at the As mentioned earlier, the point on the curve that was closest to the upper left hand corner would be the optimum trade-off level for the test. The figure shows that point corresponding to ≥5.1 mmol/L. At this cut-off level, the test had a sensitivity = 0.64 and a specificity = 0.53. The computed positive predictive value was 0.21. Thus, at this cut-off level, the test correctly diagnosed 64% of the true pre-diabetics, missed 36% of these prediabetics, but misclassified 47% of normal ( of low risk for GDM) pregnant females as prediabetics (false positives). The Area Under the Curve was reasonable (AUC=0.60). At this cut-off level, the level of agreement between the fasting blood glucose levels and those of glucose challenge test, as calculated by kappa coefficient is significant (k=0.093, p=0.011). If the sensitivity is to be increased, the cut-off level should be lowered, but it will be at the expense of specificity. For example, at a cut of 4.7 mmol/L ( 85mg/dl) only 16% of the true pre-diabetics would be misclassified as normal, but on the other hand, 80% of normal GDM is an asymptomatic condition most of the time, and effectiveness of its detection has not been adequately tested. Based on the American Diabetic Association criteria for value of 7.8mmol/L for the glucose challenge test. The optimal threshold value of 5.1mmol/L for fasting blood glucose test is marked with the arrow. 5.1mmol/L females would be misclassified as pre-diabetics( false positives). 0.0 .3 .5 .8 1.0 fasting blood glucose (mmol/L) Fig. 1. Correlation between fasting blood glucose and 75g-2hr glucose challenge test. Solid line represents the linear regression line (y= 0.66 x + 2.98) Table 3 shows an example for the measurements of all terms used in evaluating the fasting blood glucose test based on the studied data. In this example, a measurement of ≥5.1mmol/L by the fasting blood glucose test was considered a cut-off point at which subjects having that measurement or above was labeled to have pre-diabetes status by the FBS test result. At this particular cut-off level, it is noted that the sensitivity of the test was 0.64, specificity was 0.53 and the false positive rate (1– specificity) was 0.21. Sensitivity = 43/67 = 0.64 Specificity = 180/341 = 0.53 Positive predictive value = 43/204 = 0.21 False positive rate= 1 – specificity = 1 – 0.53 = 0.47 Table 3. 2x2 classification of 408 pregnant females by "true pre-diabetes" status and by a cutoff point ≥5.1 mmol/L for the fasting blood glucose test as one diagnostic criterion. 75 g glucose challenge test (mmol/L) 14 12 10 8 6 4 2 True Pre-diabetic by Pre-diabetic–yes Pre-diabetic - no GCT line represents the linear regression line (y= 0.66 x + 2.98) fasting blood glucose (mmol/L) Row total ≥5.1 mmol/L < 5.1 mmol/L (non-prediabetic) Fig. 1. Correlation between fasting blood glucose and 75g-2hr glucose challenge test. Solid Table 3 shows an example for the measurements of all terms used in evaluating the fasting blood glucose test based on the studied data. In this example, a measurement of ≥5.1mmol/L by the fasting blood glucose test was considered a cut-off point at which subjects having that measurement or above was labeled to have pre-diabetes status by the FBS test result. At this particular cut-off level, it is noted that the sensitivity of the test was 0.64, specificity was 0.53 and the false positive rate (1– specificity) was 0.21. (≥7.8mmol/L) 43 24 67 (<7.8mmol/L) 161 180 341 Column total 204 204 408 off point ≥5.1 mmol/L for the fasting blood glucose test as one diagnostic criterion. Positive predictive value = 43/204 = 0.21 False positive rate= 1 – specificity = 1 – 0.53 = 0.47 Table 3. 2x2 classification of 408 pregnant females by "true pre-diabetes" status and by a cut- FBS test result (Pre-diabetic) Sensitivity = 43/67 = 0.64 Specificity = 180/341 = 0.53 3 4 5 6 7 8 9 10 Computations for sensitivity and specificity were made for all cut-off levels along the range of values of the fasting blood glucose test. The resulting values for sensitivity were plotted against the corresponding values of (1 – specificity) to obtain the receiver operating characteristic curve as shown in. (Figure 2) 1 - Specificity Fig. 2. Receiver operating characteristic curve of fasting blood glucose measurements at the value of 7.8mmol/L for the glucose challenge test. The optimal threshold value of 5.1mmol/L for fasting blood glucose test is marked with the arrow. As mentioned earlier, the point on the curve that was closest to the upper left hand corner would be the optimum trade-off level for the test. The figure shows that point corresponding to ≥5.1 mmol/L. At this cut-off level, the test had a sensitivity = 0.64 and a specificity = 0.53. The computed positive predictive value was 0.21. Thus, at this cut-off level, the test correctly diagnosed 64% of the true pre-diabetics, missed 36% of these prediabetics, but misclassified 47% of normal ( of low risk for GDM) pregnant females as prediabetics (false positives). The Area Under the Curve was reasonable (AUC=0.60). At this cut-off level, the level of agreement between the fasting blood glucose levels and those of glucose challenge test, as calculated by kappa coefficient is significant (k=0.093, p=0.011). If the sensitivity is to be increased, the cut-off level should be lowered, but it will be at the expense of specificity. For example, at a cut of 4.7 mmol/L ( 85mg/dl) only 16% of the true pre-diabetics would be misclassified as normal, but on the other hand, 80% of normal females would be misclassified as pre-diabetics( false positives). #### 4. Discussion GDM is an asymptomatic condition most of the time, and effectiveness of its detection has not been adequately tested. Based on the American Diabetic Association criteria for Validity of Fasting Blood Glucose Test in Screening this threshold, and thus, the unnecessary GCT will be avoided. Vol 25, pp. 29-33, ISSN 0256-4947 636-641, PMID 20799591 pp. S77–S79, PMID 12017686 threshold value of 5.1mmol/L. 6. Acknowledgement 7. References 5. Conclusion for the Pre-Diabetes State Among Pregnant Females 67 Dornhorst and Rossi, 1998), this perhaps will increase the validity of the fasting blood glucose From the collective findings of this study, and considering its limitations in terms of testing the validity of fasting blood glucose in defining the pre-diabetics and not the diabetics, it is concluded that the results may be considered preliminary and suggestive for potential validity of advantage of fasting blood glucose test at the specified cut-off point. This strategy allowed 50% of the study population to avoid the glucose challenge test altogether without compromising detection rates. Women with a cut-off value of fasting blood glucose below the threshold of 5.1mmol/L may not need to be subjected to further testing by the GCT. Also, a special consideration to women with high risk for GDM will improve the validity of This project was approved by the institutional review board of King Abdulaziz Medical City, National Guard Health Affairs, Riyadh, Saudi Arabia. The authors would like to express their gratitude and thanks to the staff members of King Abdullah International Medical Research Center (KAIMRC) for their constructive support throughout the study. Special thanks to Mr. Mahmoud Salam, the research coordinator in the center, for his efforts in formatting the text in accordance with the requirements of the InTech –MS instructions. Al-Mahroos, S., Nagalla, DS., Yousif, W., & Sanad, H. (2005). A population-based screening Al-Rowaily, MA. & Abolfotouh MA (2010). Predictors of gestational diabetes in a high- American Diabetes Association (2000). Gestational diabetes mellitus. Diabetes Care 23 (Suppl.1) American Diabetes Association (2010). Diagnosis and classification of Diabetes Mellitus. Retrieved from: http://care.diabetesjournals.org/content/29/suppl\_1/s43.full Berger, H., Crane, J. & Farine, D. (2002). Screening for gestational diabetes mellitus. American Journal of Obstetrics and Gynecology, Vol. 24(11), pp. 894-912, PMID 12417905 Dornhorst, A. & Rossi, M. (1998). Risk and prevention of type 2 diabetes in women with gestational diabetes. Diabetes Care, Vol. 21(2), pp. 43-49, ISSN 0149-5992 World Health Organization (1985). Diabetes mellitus report of a study group. WHO Gabir, MM. et al. (2000). The 1997 American Diabetes Association and 1999 World Health IDF (2006). A digest of the global guideline for type 2 diabetes. Diabetes voice (Supplement), Organization criteria for hyperglycemia in the diagnosis and prediction of diabetes. Technical Report Series, Geneva (No. 727), PMID 3934850 Diabetes care. Vol. 23(8), pp. 1108-1112, ISSN 0149-5992 Vol. 51, pp. 32-35, ISBN 2-930229-43-8 for gestational diabetes mellitus in non-diabetic women in Bahrain. Ann Saudi Med, parity community in Saudi Arabia. Eastern Mediterranean Health Journal. Vol 16, pp. diagnosis of GDM, 17.2% of the studied pregnant females had impaired fasting tolerance (IFT) and 1.4% had provisional diagnosis of GDM. The corresponding results for those who responded to the GCT are 15.2% IGT and 1.2% provisional diagnosis of GDM. These figures are suggestive of the necessity for screening for GDM in our community. They are comparable with the figures of other nearby countries (Al-Mahroos et al., 2005), but still higher than those of the western countries (Naylor et al., 1996; Tsutomu et al., 2002). Diagnostic levels for GDM remain uncertain. The guidelines present a confusing picture as regards screening tests for GDM. The use of a 4.7 mmol/L (85mg/dl) cut-off for fasting plasma glucose is suggested by some researchers, but others have suggested higher cut-offs (IDF, 2006). Fasting glucose may not be the most appropriate measure however, and the 75g glucose challenge test advocated by the WHO is increasingly used internationally (IDF, 2006). The aim of the present study was to determine the threshold value of fasting blood glucose for which further testing by the 2hr-glucose challenge test is needed to confirm the prediabetic status among pregnant females attending the antenatal clinics of the King Fahd hospital at the National Guard in Riyadh city, Saudi Arabia.. Of all pregnant females subjected to the fasting blood glucose testing ( n=769), only 408 subjects (53%) complied with the 2hr-glucose challenge test, a finding that may reflect the need for specifying the feasible indication for such GCT, so as not to subject all females for an unnecessary as well as unacceptable test. The figures of the pre-diabetes by both the fasting blood glucose test (17.2%) and the GCT (15.2%) are nearly comparable, based on the cut-off points recommended by the American Diabetic Association (ADA, 2010). Correlation between the individual values of fasting blood glucose testing and those of 2hr-glucose challenge testing was highly significant (r=0.25,p<0.0001).However, the agreement between the categorical results of both tests ( in terms of pre-diabetes, provisional GDM, and normal) was low as calculated by the kappa coefficient (k=0.054,p=0.25). This finding reflect the fact that the cut-off value of 5.6mmol/L for fasting blood glucose test is not the threshold value for the pre-diabetes status, especially when we see that out of those with impaired fasting test, only 18.7 were found with IGT by the GCT. (Table 2) Thus, this suboptimal accuracy of the cut-off level of 5.6mmol/L will result in the misclassification of subjects. To overcome this problem to a great extent, the receiver operating characteristic curve analysis was used to determine the threshold value of fasting blood glucose. Based on the previously reported high sensitivity and specificity of the GCT (O'Sullivan et al., 1973), it was considered as a gold standard (in detection of the pre-diabetic status) against which to test the validity of different values of fasting blood glucose test. The levels of sensitivity and specificity found in this study for the cut-off value of 5.1mmol/L for fasting blood glucose (64% and 53%) might not suggest the use of this test, especially that in Brazil, examining a range of thresholds, maximum sensitivity (88%) and specificity (78%) was found at 4.9mmol/L (O'Sullivan et al., 1973). However, in the present study, at this cut-off level of 4.9mmol/L, in spite of the high sensitivity (82%), very low and unacceptable specificity (24%) was attained. Thus, the cut-off level of 5.1mmol/L could be potentially useful. At this level, of all pregnant females subjected to the fasting blood glucose test, only 50% would be tested by the GCT. This may result in better compliance to the GCT. Moreover, if women with clinical characteristics consistent with a high risk of GDM (marked obesity, personal history of GDM, glycosuria, or a strong family history of diabetes) would be considered as the target group for screening (ADA, 2006 & Dornhorst and Rossi, 1998), this perhaps will increase the validity of the fasting blood glucose threshold value of 5.1mmol/L. #### 5. Conclusion 66 Gestational Diabetes diagnosis of GDM, 17.2% of the studied pregnant females had impaired fasting tolerance (IFT) and 1.4% had provisional diagnosis of GDM. The corresponding results for those who responded to the GCT are 15.2% IGT and 1.2% provisional diagnosis of GDM. These figures are suggestive of the necessity for screening for GDM in our community. They are comparable with the figures of other nearby countries (Al-Mahroos et al., 2005), but still Diagnostic levels for GDM remain uncertain. The guidelines present a confusing picture as regards screening tests for GDM. The use of a 4.7 mmol/L (85mg/dl) cut-off for fasting plasma glucose is suggested by some researchers, but others have suggested higher cut-offs (IDF, 2006). Fasting glucose may not be the most appropriate measure however, and the 75g glucose The aim of the present study was to determine the threshold value of fasting blood glucose for which further testing by the 2hr-glucose challenge test is needed to confirm the prediabetic status among pregnant females attending the antenatal clinics of the King Fahd hospital at the National Guard in Riyadh city, Saudi Arabia.. Of all pregnant females subjected to the fasting blood glucose testing ( n=769), only 408 subjects (53%) complied with the 2hr-glucose challenge test, a finding that may reflect the need for specifying the feasible indication for such GCT, so as not to subject all females for an unnecessary as well The figures of the pre-diabetes by both the fasting blood glucose test (17.2%) and the GCT (15.2%) are nearly comparable, based on the cut-off points recommended by the American Diabetic Association (ADA, 2010). Correlation between the individual values of fasting blood glucose testing and those of 2hr-glucose challenge testing was highly significant (r=0.25,p<0.0001).However, the agreement between the categorical results of both tests ( in terms of pre-diabetes, provisional GDM, and normal) was low as calculated by the kappa coefficient (k=0.054,p=0.25). This finding reflect the fact that the cut-off value of 5.6mmol/L for fasting blood glucose test is not the threshold value for the pre-diabetes status, especially when we see that out of those with impaired fasting test, only 18.7 were found with IGT by Thus, this suboptimal accuracy of the cut-off level of 5.6mmol/L will result in the misclassification of subjects. To overcome this problem to a great extent, the receiver operating characteristic curve analysis was used to determine the threshold value of fasting blood glucose. Based on the previously reported high sensitivity and specificity of the GCT (O'Sullivan et al., 1973), it was considered as a gold standard (in detection of the pre-diabetic status) against which to test the validity of different values of fasting blood glucose test. The levels of sensitivity and specificity found in this study for the cut-off value of 5.1mmol/L for fasting blood glucose (64% and 53%) might not suggest the use of this test, especially that in Brazil, examining a range of thresholds, maximum sensitivity (88%) and specificity (78%) However, in the present study, at this cut-off level of 4.9mmol/L, in spite of the high sensitivity (82%), very low and unacceptable specificity (24%) was attained. Thus, the cut-off level of 5.1mmol/L could be potentially useful. At this level, of all pregnant females subjected to the fasting blood glucose test, only 50% would be tested by the GCT. This may result in better compliance to the GCT. Moreover, if women with clinical characteristics consistent with a high risk of GDM (marked obesity, personal history of GDM, glycosuria, or a strong family history of diabetes) would be considered as the target group for screening (ADA, 2006 & higher than those of the western countries (Naylor et al., 1996; Tsutomu et al., 2002). challenge test advocated by the WHO is increasingly used internationally (IDF, 2006). as unacceptable test. the GCT. (Table 2) was found at 4.9mmol/L (O'Sullivan et al., 1973). From the collective findings of this study, and considering its limitations in terms of testing the validity of fasting blood glucose in defining the pre-diabetics and not the diabetics, it is concluded that the results may be considered preliminary and suggestive for potential validity of advantage of fasting blood glucose test at the specified cut-off point. This strategy allowed 50% of the study population to avoid the glucose challenge test altogether without compromising detection rates. Women with a cut-off value of fasting blood glucose below the threshold of 5.1mmol/L may not need to be subjected to further testing by the GCT. Also, a special consideration to women with high risk for GDM will improve the validity of this threshold, and thus, the unnecessary GCT will be avoided. #### 6. Acknowledgement This project was approved by the institutional review board of King Abdulaziz Medical City, National Guard Health Affairs, Riyadh, Saudi Arabia. The authors would like to express their gratitude and thanks to the staff members of King Abdullah International Medical Research Center (KAIMRC) for their constructive support throughout the study. Special thanks to Mr. Mahmoud Salam, the research coordinator in the center, for his efforts in formatting the text in accordance with the requirements of the InTech –MS instructions. #### 7. References 5 Bulgaria Prevalence and Risk Factors for the Development of GD in Some Eastern Pharmacoeconomical Assessment for the Choice of Treatment Valentina Petkova and Irina Nikolova Medical University – Sofia, Faculty of Pharmacy European Countries – Tendencies and Diabetes is a metabolic disorder of fat, carbohydrate, and protein metabolism, characterized by resistance to the action of insulin, insufficient insulin secretion, or both. The two major classifications of diabetes mellitus (DM) are type 1 (insulin deficient) and type 2 (combined insulin resistance and relative deficiency in insulin secretion). They differ in clinical presentation, onset, etiology, and progression of disease. Two to five percent of pregnancies are complicated by diabetes, of which 90% are classified as gestational diabetes mellitus (GD) (Satman et al, 2002). GD is defined as glucose intolerance of variable severity which is first recognized during pregnancy, including individuals with previously undiagnosed diabetes as well as those in whom high glucose levels are provoked by pregnancy. This term should not be used for gravid women with previously diagnosed diabetes. GD has much in common with type 2 diabetes with similar genetic susceptibility, corresponding prevalence within a given population or ethnic group and similar risk factors. Both conditions can be considered as a mixture of insulin resistance (IR) and impaired insulin secretion. Indeed, GD is a predictor of future type 2 diabetes with a cumulative incidence of about 50% at 5 years. GD is also a predictor of the metabolic (resistance) syndrome and should probably be considered a cardiovascular risk factor for later life. Since its first description in the early 1950s, GD has been one of the most controversial syndromes in the field of diabetes. Pregnancy is a state of insulin resistance, characterized by raised circulating insulin concentrations as the maternal pancreas compensates for increased peripheral demands. If adequate compensation does not occur, GD develops. GD is a well-established risk factor for adverse infant health outcomes, including fetal macrosomia, birth trauma, neonatal hypoglycemia, and fetal death (Cetin et al., 1997). GD can predict that the children of women who have GD are at an increased risk for obesity, glucose intolerance, and diabetes during adulthood. There is still confusion about the type of diagnostic tests and diagnostic criteria for GD and a screening protocol (e.g. universal versus selective screening). Gestational diabetes complicates about 1-14% of all pregnancies, depending on the 1. Introduction ## Prevalence and Risk Factors for the Development of GD in Some Eastern European Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment Valentina Petkova and Irina Nikolova Medical University – Sofia, Faculty of Pharmacy Bulgaria #### 1. Introduction 68 Gestational Diabetes Kjos, SL. & Buchanan, TA. (1999). Gestational diabetes. The New England Journal of Medicine. Kuhl, C. (1991). Aetiology of gestational diabetes. Baillière's Clinical Obstetrics and Metzger, BE. & Coustan, DR. (1998). The Organizing Committee: Summary and Moody, J. (Edi). (October 2003). Antenatal care- routine care for the healthy pregnant woman : Clinical guideline, RCOG press publisher, pp. 96-99, London, ISBN 1900364 913 Naylor, CD., Serner, M., Chen, E. & Sykora, K. (1996). Caesarian delivery in relation to birth weight and gestational glucose tolerance: pathophysiology or practice style. The Journal of the American Medical Association. Vol. 275, pp. 1165-1170, ISSN 0098-7484 O'Sullivan, JB., Mahan, CM., Charles, D. & Dandrow, RV. (1973). Screening criteria for high- Richardson, DK., Schwartz, JS., Weinbaum, PJ., & Gabbe, SG. (1993). Diagnostic tests in Schmidt, MI. et al. (2001). Gestational Diabetes Mellitus Diagnosed with a 2-h 75-g Oral Tsutomu, K. et al. (2002). Young men with high normal blood pressure have lower serum WHO (1999). Consultation: Definition, Diagnosis and Classification of Diabetes Mellitus and blood pressure. Diabetes Care. Vol. 25, pp. 971, PMID 12032101 recommendations of the Fourth International Workshop-Conference on Gestational Diabetes Mellitus. Diabetes Care. Vol. 21, suppl. 1: pp. 161–167, PMID 9704245 Metzer, BE. & Coustan, DM. (1998). Organizing committee. Summary and recommendations of Fourth international Workshop. Conference on Gestational diabetes mellitus. risk gestational diabetic patients. American Journal of Obstetrics and Gynecology. Vol. Obstetrics: a method for improved evaluation. American Journal of Obstetrics and Glucose Tolerance Test and Adverse Pregnancy Outcomes. Diabetes Care. Vol. 24, adiponectin, smaller LDL, size and higher elevated heart rate than those optimal Its Complications: Report of a WHO Consultation. Part 1. Diagnosis and Classification of Diabetes Mellitus., WHO/NCD/NCS. Vol. 99.2, Geneva, ISSN Vol. 341, pp. 1749-1756, PMID 10580075 116, pp. 895–900, PMID 4718216 pp. 1151–1155, ISSN 1935-5548 07423071 Gynecology. Vol. 152, pp. 613-618, PMID 4025419 Gynaecology. Vo. 5(2), pp. 279-292, PMID 1954714 Diabetes Care. Vol. 21, suppl 2: pp. 161-167, PMID 9704245 Diabetes is a metabolic disorder of fat, carbohydrate, and protein metabolism, characterized by resistance to the action of insulin, insufficient insulin secretion, or both. The two major classifications of diabetes mellitus (DM) are type 1 (insulin deficient) and type 2 (combined insulin resistance and relative deficiency in insulin secretion). They differ in clinical presentation, onset, etiology, and progression of disease. Two to five percent of pregnancies are complicated by diabetes, of which 90% are classified as gestational diabetes mellitus (GD) (Satman et al, 2002). GD is defined as glucose intolerance of variable severity which is first recognized during pregnancy, including individuals with previously undiagnosed diabetes as well as those in whom high glucose levels are provoked by pregnancy. This term should not be used for gravid women with previously diagnosed diabetes. GD has much in common with type 2 diabetes with similar genetic susceptibility, corresponding prevalence within a given population or ethnic group and similar risk factors. Both conditions can be considered as a mixture of insulin resistance (IR) and impaired insulin secretion. Indeed, GD is a predictor of future type 2 diabetes with a cumulative incidence of about 50% at 5 years. GD is also a predictor of the metabolic (resistance) syndrome and should probably be considered a cardiovascular risk factor for later life. Since its first description in the early 1950s, GD has been one of the most controversial syndromes in the field of diabetes. Pregnancy is a state of insulin resistance, characterized by raised circulating insulin concentrations as the maternal pancreas compensates for increased peripheral demands. If adequate compensation does not occur, GD develops. GD is a well-established risk factor for adverse infant health outcomes, including fetal macrosomia, birth trauma, neonatal hypoglycemia, and fetal death (Cetin et al., 1997). GD can predict that the children of women who have GD are at an increased risk for obesity, glucose intolerance, and diabetes during adulthood. There is still confusion about the type of diagnostic tests and diagnostic criteria for GD and a screening protocol (e.g. universal versus selective screening). Gestational diabetes complicates about 1-14% of all pregnancies, depending on the Prevalence and Risk Factors for the Development of GD in Some Eastern European contribute to vascular and metabolic complications in obese pregnancy. times more likely to develop GD than lean women. Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment 71 upper thigh. A large population-based Swedish study highlighted that an increase in BMI of 3 kg/m2 between two consecutive pregnancies resulted in an increased risk of preeclampsia, GD, gestational hypertension, caesarean delivery, stillbirth and large for gestation age births even if a woman has a healthy BMI during pregnancy. (Villamor & Cnattingius, 2006; Hossein-Nezhad et al, 2007) It reported 35% prevalence of obesity (BMI of more than 27) in women with GD, compared with 11.3% in normal women (p < 0.0001). Not only weight gained during pregnancy, but variations in body habitus or constitution, like high waist-hip ratios, short stature and higher body fat percentages at given BMI levels could contribute as a risk factors (Davis, 2008; Mensing et al., 2002; Scholl et al., 2002) Not surprisingly, obese women are 4 times more likely and severely obese women almost 9 Pregnancy is also characterised by marked increases in plasma lipid concentrations as gestation advances. Plasma cholesterol and triglyceride concentrations rise by 25–50% and 200–400% respectively. The hyperlipidaemia of pregnancy is exaggerated further in obesity with higher serum triglyceride and very low density lipoprotein (LDL) cholesterol concentrations than those observed in lean women. This is seen together with lower high density lipoprotein (HDL) cholesterol, although LDL cholesterol and total cholesterol concentrations appear similar. This pattern of dyslipidaemia is similar to that of the metabolic syndrome in the non-pregnant population. Some of these pathways may well It is well established that people with high levels of visceral or intra-abdominal body fat are associated with increased risk for type 2 diabetes (Huxley et al., 2008) and impaired glucose tolerance even after adjustment for BMI. Recent evidence suggests that even modest fasting hyperglycaemia (between 4.2 and 5.6 mmol/L), currently thought to be within normal limits, is linearly associated with adverse pregnancy outcomes including increased birth weight, caesarean delivery and neonatal hypoglycaemia (Huda et al, 2010). Women with a history of GD are metabolically vulnerable with insufficient ß-cell reserve, and many are IR. Approximately 17-63% of women who are diagnosed with GD during pregnancy will develop it in future pregnancies, and are at a much greater risk of developing type 2 diabetes within 5-16 years. The risk varies with the magnitude of insulin resistance, for example, if the patient needed insulin in pregnancy or was noted to be obese, or developed GD before 24 weeks gestation, the risk is greater. There is also some evidence that further pregnancies accelerate the rate of decline of beta cell function in women with GD. Several studies have reported links between GD and the subsequent risk of type 2 diabetes. Recent meta-analysis reports that GD corresponds to a 7.4 fold increased risk for developing type 2 diabetes mellitus (Bellamy et al., 2009). In addition, numerous studies have reported an increased risk of GD in women who are overweight or obese compared with lean or normalweight women (Chatzi et al., 2009). GD identifies pregnancies at increased risk for adverse perinatal outcome. It also identifies mothers who are at increased risk of developing type 2 DM in the future and offers the possibility of interventions and early detection. Severity of glucose intolerance during pregnancy, insulin requirement during pregnancy, earlier diagnosis during pregnancy, family history of diabetes, recurrence of GD, increasing parity, maternal age, prepregnancy obesity, weight gain during and after pregnancy, presence of islet cell antibodies, and delivery of a macrosomic infant were reportedly the key risk factors for type 2 DM in women with history of GD (Cho et al., 2006). One of the major determinants of the risk for the development of subsequent type 2 diabetes is ethnic origin. Ethnic group with high risk of developing diabetes are Hispanic, African, Native American, population and diagnostic tests that are used and ranges from mild degrees of hyperglycemia to insulin-dependent diabetes (Kim et al., 2007). Gestational diabetes usually manifests in the latter half of pregnancy and is characterized by insulin levels that are insufficient to meet insulin demands. #### 2. Risk factors for GD Risk factors for GD include obesity, family history of diabetes, age greater than 35 years of age, pre-diabetes detected before pregnancy, previous delivery of babies with birth weights greater than 4 kg, sedentary lifestyles and some ethnical groups. The frequency of GD significantly increases with increasing number of risk factors. However, even a combination of risk factors does not reliably predict the likelihood of developing GD, missing up to 50% of cases in population based studies (Fraser & Heller, 2007; Jang et al., 2003) estimates that if age > 30 years, obesity (BMI > 27.3), family history of diabetes mellitus and glycosuria in the present pregnancy were included as risk factors, 56.5% of the total population had risk factors for GD. However, in the stated report, the prevalence of GD in Korea was 2.0%, Thailand - 2.0% and U.S. Caucasians - 2.3%. One large study of women with GD showed that an increased incidence of GD was associated with age over 35, thin or obese prepregnancy weight, previous stillbirth, previous spontaneous or induced abortion, previous low- or high-birth-weight infant and chronic hypertension (Johnstone, 1999; Hossein-Nezhad et al., 2007) found the age distribution and GD prevalence in 15–24 age group was 0.39% and 2.68% in the 35–45 age group. The authors reported 33.33% of women with GD who had a positive family history of diabetes, compared with 10.3% of women in the normal group (p < 0.0001). It is now well established that women with a family history of diabetes had three times higher odds for GD than women without a family history of diabetes. Perhaps other maternal health and lifestyle behaviors, such as levels of physical activity, or selective immigration, could play an important role (Scholl et al., 2002). Obesity in pregnancy is increasing worldwide and is associated with increased risk of adverse outcomes for both mother and child. Maternal obesity is also accompanied by alterations in glucose metabolism and by perturbations in inflammatory markers, adipokines and vascular dysfunction. The link between obesity and IR has been recognised for many years and much of this association has been attributed to disturbances in adipocyte function and metabolism (Huda et al. 2010). Obesity has considerable effects on glucose metabolism in pregnancy with a loss of the reduction in fasting glucose in early pregnancy and significant enhancement of peripheral and hepatic IR. All women increase maternal fat stores in early pregnancy irrespective of prepregnancy adiposity to meet the feto-placental and maternal demands of late gestation and lactation. Normal pregnancy is associated with marked changes in glucose metabolism and IR to facilitate provision of fuel substrate for the fetus. In early pregnancy insulin secretion increases, while insulin sensitivity is unchanged, or even slightly improved. However, as pregnancy progresses, insulin-mediated glucose utilization worsens by 40–60% and insulin secretion increases several fold in order to maintain euglycaemia in the mother. The mechanisms for this increased risk are multifactorial and include effects on insulin signalling similar to those seen in obese non-pregnant women. Women of normal weight gain around 3.8 kg of fat during pregnancy although there is substantial variation. In women of normal weight the majority of fat is accumulated centrally in the subcutaneous compartment of the trunk and population and diagnostic tests that are used and ranges from mild degrees of Gestational diabetes usually manifests in the latter half of pregnancy and is characterized by Risk factors for GD include obesity, family history of diabetes, age greater than 35 years of age, pre-diabetes detected before pregnancy, previous delivery of babies with birth weights greater than 4 kg, sedentary lifestyles and some ethnical groups. The frequency of GD significantly increases with increasing number of risk factors. However, even a combination of risk factors does not reliably predict the likelihood of developing GD, missing up to 50% of cases in population based studies (Fraser & Heller, 2007; Jang et al., 2003) estimates that if age > 30 years, obesity (BMI > 27.3), family history of diabetes mellitus and glycosuria in the present pregnancy were included as risk factors, 56.5% of the total population had risk factors for GD. However, in the stated report, the prevalence of GD in Korea was 2.0%, Thailand - 2.0% and U.S. Caucasians - 2.3%. One large study of women with GD showed that an increased incidence of GD was associated with age over 35, thin or obese prepregnancy weight, previous stillbirth, previous spontaneous or induced abortion, previous low- or high-birth-weight infant and chronic hypertension (Johnstone, 1999; Hossein-Nezhad et al., 2007) found the age distribution and GD prevalence in 15–24 age group was 0.39% and 2.68% in the 35–45 age group. The authors reported 33.33% of women with GD who had a positive family history of diabetes, compared with 10.3% of women in the normal group (p < 0.0001). It is now well established that women with a family history of diabetes had three times higher odds for GD than women without a family history of diabetes. Perhaps other maternal health and lifestyle behaviors, such as levels of physical activity, or selective immigration, could play an important role (Scholl et al., 2002). Obesity in pregnancy is increasing worldwide and is associated with increased risk of adverse outcomes for both mother and child. Maternal obesity is also accompanied by alterations in glucose metabolism and by perturbations in inflammatory markers, adipokines and vascular dysfunction. The link between obesity and IR has been recognised for many years and much of this association has been attributed to disturbances in adipocyte function and metabolism (Huda et al. 2010). Obesity has considerable effects on glucose metabolism in pregnancy with a loss of the reduction in fasting glucose in early pregnancy and significant enhancement of peripheral and hepatic IR. All women increase maternal fat stores in early pregnancy irrespective of prepregnancy adiposity to meet the feto-placental and maternal demands of late gestation and lactation. Normal pregnancy is associated with marked changes in glucose metabolism and IR to facilitate provision of fuel substrate for the fetus. In early pregnancy insulin secretion increases, while insulin sensitivity is unchanged, or even slightly improved. However, as pregnancy progresses, insulin-mediated glucose utilization worsens by 40–60% and insulin secretion increases several fold in order to maintain euglycaemia in the mother. The mechanisms for this increased risk are multifactorial and include effects on insulin signalling similar to those seen in obese non-pregnant women. Women of normal weight gain around 3.8 kg of fat during pregnancy although there is substantial variation. In women of normal weight the majority of fat is accumulated centrally in the subcutaneous compartment of the trunk and hyperglycemia to insulin-dependent diabetes (Kim et al., 2007). insulin levels that are insufficient to meet insulin demands. 2. Risk factors for GD upper thigh. A large population-based Swedish study highlighted that an increase in BMI of 3 kg/m2 between two consecutive pregnancies resulted in an increased risk of preeclampsia, GD, gestational hypertension, caesarean delivery, stillbirth and large for gestation age births even if a woman has a healthy BMI during pregnancy. (Villamor & Cnattingius, 2006; Hossein-Nezhad et al, 2007) It reported 35% prevalence of obesity (BMI of more than 27) in women with GD, compared with 11.3% in normal women (p < 0.0001). Not only weight gained during pregnancy, but variations in body habitus or constitution, like high waist-hip ratios, short stature and higher body fat percentages at given BMI levels could contribute as a risk factors (Davis, 2008; Mensing et al., 2002; Scholl et al., 2002) Not surprisingly, obese women are 4 times more likely and severely obese women almost 9 times more likely to develop GD than lean women. Pregnancy is also characterised by marked increases in plasma lipid concentrations as gestation advances. Plasma cholesterol and triglyceride concentrations rise by 25–50% and 200–400% respectively. The hyperlipidaemia of pregnancy is exaggerated further in obesity with higher serum triglyceride and very low density lipoprotein (LDL) cholesterol concentrations than those observed in lean women. This is seen together with lower high density lipoprotein (HDL) cholesterol, although LDL cholesterol and total cholesterol concentrations appear similar. This pattern of dyslipidaemia is similar to that of the metabolic syndrome in the non-pregnant population. Some of these pathways may well contribute to vascular and metabolic complications in obese pregnancy. It is well established that people with high levels of visceral or intra-abdominal body fat are associated with increased risk for type 2 diabetes (Huxley et al., 2008) and impaired glucose tolerance even after adjustment for BMI. Recent evidence suggests that even modest fasting hyperglycaemia (between 4.2 and 5.6 mmol/L), currently thought to be within normal limits, is linearly associated with adverse pregnancy outcomes including increased birth weight, caesarean delivery and neonatal hypoglycaemia (Huda et al, 2010). Women with a history of GD are metabolically vulnerable with insufficient ß-cell reserve, and many are IR. Approximately 17-63% of women who are diagnosed with GD during pregnancy will develop it in future pregnancies, and are at a much greater risk of developing type 2 diabetes within 5-16 years. The risk varies with the magnitude of insulin resistance, for example, if the patient needed insulin in pregnancy or was noted to be obese, or developed GD before 24 weeks gestation, the risk is greater. There is also some evidence that further pregnancies accelerate the rate of decline of beta cell function in women with GD. Several studies have reported links between GD and the subsequent risk of type 2 diabetes. Recent meta-analysis reports that GD corresponds to a 7.4 fold increased risk for developing type 2 diabetes mellitus (Bellamy et al., 2009). In addition, numerous studies have reported an increased risk of GD in women who are overweight or obese compared with lean or normalweight women (Chatzi et al., 2009). GD identifies pregnancies at increased risk for adverse perinatal outcome. It also identifies mothers who are at increased risk of developing type 2 DM in the future and offers the possibility of interventions and early detection. Severity of glucose intolerance during pregnancy, insulin requirement during pregnancy, earlier diagnosis during pregnancy, family history of diabetes, recurrence of GD, increasing parity, maternal age, prepregnancy obesity, weight gain during and after pregnancy, presence of islet cell antibodies, and delivery of a macrosomic infant were reportedly the key risk factors for type 2 DM in women with history of GD (Cho et al., 2006). One of the major determinants of the risk for the development of subsequent type 2 diabetes is ethnic origin. Ethnic group with high risk of developing diabetes are Hispanic, African, Native American, Prevalence and Risk Factors for the Development of GD in Some Eastern European Fasting ≥ 92 mg/dL (5.1 mmol/L) 1 hour ≥ 180 mg/dL (10.0 mmol/L) 2 hours ≥ 153 mg/dL (8.5 mmol/L) not a member of an ethnic group with a high prevalence of GD, no history of abnormal glucose tolerance, no history of abnormal obstetric outcome. Table 1. Diagnosis of GD with a 75 g Glucose Load diagnosis of diabetes to be made. retesting at 24 to 28 weeks' gestation. Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment 73 Screening for GD utilizes the oral glucose challenge test. New ADA guidelines (ADA, 2011) and WHO guidelines stated to perform a 75 g oral glucose tolerance test (OGTT), with plasma glucose measurement fasting and at 1 and 2 h at 24-28 weeks. OGTT should be performed in the morning after an overnight fast of at least 10 hours and after at least 3 days of unrestricted diet and unlimited physical activity. The patient should remain seated and should not smoke during the test (Johnstone, 1999). Criteria for diagnosis of GD based on the OGTT are summarized in Table 1. Two or more values must be met or exceeded for a Results of the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study (Metzger et al., 2008), a large-scale (~25,000 pregnant women) multinational epidemiologic study, demonstrated that risk of adverse maternal, fetal, and neonatal outcomes continuously increased as a function of maternal glycemia at 24–28 weeks, even within ranges previously considered normal for pregnancy. For most complications there was no threshold for risk. These results have led to careful reconsideration of the diagnostic criteria for GD. The International Association of Diabetes and Pregnancy Study Groups (IADPSG) recommended that all women not known to have prior diabetes undergo a 75 g OGTT at 24– 28 weeks of gestation. This probably will significantly increase the prevalence of GD, however there is mounting evidence that treating even mild GD reduces morbidity for both mother and baby (International Diabetes Federation [IDF], 2004). Women at high risk should be screened as soon as feasible. If the initial screening is negative they should undergo There are two principles (selective and universal) in testing which need to be distinguished. First, there is selective (or at-risk) clinical need for high risk patient (obesity, a family history of diabetes, a history of macrosomic stillbirth, etc.). Second, there is universal screening, for women not clinically identified as being at particular risk. Universal screening is more sensitive, while selective screening is less costly. Unfortunately, the use of risk factors for individuals is not very effective, can result in great inconsistencies, and is a source of uncertainty for obstetricians and midwives. Universal screening should, at the moment, depend on the local prevalence of type 2 DM in the reproductive age group. Low-risk status requires no glucose testing, but this category is limited to those women meeting all of the following characteristics: age <25 years, normal weight before pregnancy, member of a population with a low prevalence of GD, no known diabetes in first-degree relatives, no history of abnormal glucose tolerance, no history of poor obstetric outcome. Where type 2 DM is common (for example many units in the USA, Saudi Arabia and the Gulf, and certain ethnic minorities), then a total population screen is justified (Forsbach et al., 1997). Universal screening is more sensitive and more practical when a family history of diabetes cannot be obtained reliably (Karcaaltincaba et al., 2009). The diagnosis of GD is associated with a risk of type 2 diabetes later in life, so it is unsurprising that the prevalence of GD in any South or East Asian, Pacific Islands or Indigenous Australian. Women with previous GD have a higher prevalence of polycystic ovary syndrome (PCOS), which is a condition associated with IR. Similarly in a study of women with PCOS, GD developed in 20% of these women compared with only 9% of controls with an odds ratio of 1.9. Women with PCOS often attend assisted fertility clinics and should be warned of the likelihood of developing GD if they become pregnant (Hyer & Shehata, 2005; Metzger et al., 2010). Maternal diabetes during pregnancy exposes the fetus to hyperglycemia resulting in increased fetal insulin levels, which are associated with increased birth weight, increased childhood and adult obesity, and increased risk of GD and type-2 diabetes during childbearing age (Kim et al., 2007). Perinatal and maternal morbidity can be reduced by maintaining normoglycaemia in GD. To achieve good glycaemic control, women diagnosed with GD should monitor their blood glucose levels, exercise, and undergo nutrition counselling. Besides, medical treatment options should be incorporated in care plans. However, despite intensive treatment aiming at near normoglycemia, a surprisingly high risk of macrosomia and birth trauma has been reported among the neonates of mothers with GD. It would seem that the prevention of macrosomia should be the primary goal of GD management. Several studies suggest an increased rate of preeclampsia among women with GD, and a combination of maternal diabetes and pre-eclampsia is associated with poor perinatal outcome (Fan et al., 2006). Untreated or poorly controlled gestational diabetes can hurt the baby. It can raise the risk of certain pregnancy complications, like high blood pressure in the mother and having a larger-than-normal baby (macrosomia), which may require a C-section. Studies have shown that in GD the frequency of having an overweight baby is almost double, the frequency of having preeclampsia is almost double, and the frequency of early delivery is 40 percent greater. It was proven that the proper identification and management of GD are associated with a decrease in mortality and morbidity in infants. #### 3. Testing programs GD is associated with maternal (pre-eclampsia, hypertension, caesarean section) and foetal morbidity (macrosomia, birth trauma, hypoglycaemia, hyperbilirubinemia, hypocalcemia, respiratory distress syndrome). Moreover, GD uncovers a pre-existing metabolic abnormality that may precede the development of overt diabetes mellitus. Therefore, prompt diagnosis of GD is essential to reduce maternal and foetal morbidity and to allow subsequent attempt at preventing or delaying the onset of Type 2 diabetes. Whether or not to screen for gestational diabetes is an issue of significant controversy. The ADA favors screening a woman who has risk factors for developing GD (e.g., severe obesity, personal history of GD or previous delivery of large-for-gestational age infant, glycosuria, PCOS or a strong family history of diabetes) at her first prenatal visit (American Diabetes Association [ADA], 2011). If test is abnormal, these individuals should be considered to have "overt" (not gestational) diabetes (ADA, 2010). If this screen is normal, testing should be repeated between weeks 24 and 28 of gestation. Pregnant women without these risk factors should undergo screening for GD between weeks 24 and 28 of gestation unless they are considered low risk. To be low risk, a woman must fulfill all the following criteria: South or East Asian, Pacific Islands or Indigenous Australian. Women with previous GD have a higher prevalence of polycystic ovary syndrome (PCOS), which is a condition associated with IR. Similarly in a study of women with PCOS, GD developed in 20% of these women compared with only 9% of controls with an odds ratio of 1.9. Women with PCOS often attend assisted fertility clinics and should be warned of the likelihood of developing Maternal diabetes during pregnancy exposes the fetus to hyperglycemia resulting in increased fetal insulin levels, which are associated with increased birth weight, increased childhood and adult obesity, and increased risk of GD and type-2 diabetes during Perinatal and maternal morbidity can be reduced by maintaining normoglycaemia in GD. To achieve good glycaemic control, women diagnosed with GD should monitor their blood glucose levels, exercise, and undergo nutrition counselling. Besides, medical treatment options should be incorporated in care plans. However, despite intensive treatment aiming at near normoglycemia, a surprisingly high risk of macrosomia and birth trauma has been reported among the neonates of mothers with GD. It would seem that the prevention of macrosomia should be the primary goal of GD management. Several studies suggest an increased rate of preeclampsia among women with GD, and a combination of maternal diabetes and pre-eclampsia is associated with poor perinatal outcome (Fan et al., 2006). Untreated or poorly controlled gestational diabetes can hurt the baby. It can raise the risk of certain pregnancy complications, like high blood pressure in the mother and having a larger-than-normal baby (macrosomia), which may require a C-section. Studies have shown that in GD the frequency of having an overweight baby is almost double, the frequency of having preeclampsia is almost double, and the frequency of early delivery is 40 percent greater. It was proven that the proper identification and management of GD are associated GD is associated with maternal (pre-eclampsia, hypertension, caesarean section) and foetal morbidity (macrosomia, birth trauma, hypoglycaemia, hyperbilirubinemia, hypocalcemia, respiratory distress syndrome). Moreover, GD uncovers a pre-existing metabolic abnormality that may precede the development of overt diabetes mellitus. Therefore, prompt diagnosis of GD is essential to reduce maternal and foetal morbidity and to allow Whether or not to screen for gestational diabetes is an issue of significant controversy. The ADA favors screening a woman who has risk factors for developing GD (e.g., severe obesity, personal history of GD or previous delivery of large-for-gestational age infant, glycosuria, PCOS or a strong family history of diabetes) at her first prenatal visit (American Diabetes Association [ADA], 2011). If test is abnormal, these individuals should be considered to have "overt" (not gestational) diabetes (ADA, 2010). If this screen is normal, testing should be repeated between weeks 24 and 28 of gestation. Pregnant women without these risk factors should undergo screening for GD between weeks 24 and 28 of gestation unless they are considered low risk. To be low risk, a woman must fulfill all the following criteria: subsequent attempt at preventing or delaying the onset of Type 2 diabetes. GD if they become pregnant (Hyer & Shehata, 2005; Metzger et al., 2010). childbearing age (Kim et al., 2007). 3. Testing programs age younger than 25 years, normal prepregnancy weight, no known diabetes in first-degree relatives, with a decrease in mortality and morbidity in infants. no history of abnormal obstetric outcome. Screening for GD utilizes the oral glucose challenge test. New ADA guidelines (ADA, 2011) and WHO guidelines stated to perform a 75 g oral glucose tolerance test (OGTT), with plasma glucose measurement fasting and at 1 and 2 h at 24-28 weeks. OGTT should be performed in the morning after an overnight fast of at least 10 hours and after at least 3 days of unrestricted diet and unlimited physical activity. The patient should remain seated and should not smoke during the test (Johnstone, 1999). Criteria for diagnosis of GD based on the OGTT are summarized in Table 1. Two or more values must be met or exceeded for a diagnosis of diabetes to be made. Results of the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study (Metzger et al., 2008), a large-scale (~25,000 pregnant women) multinational epidemiologic study, demonstrated that risk of adverse maternal, fetal, and neonatal outcomes continuously increased as a function of maternal glycemia at 24–28 weeks, even within ranges previously considered normal for pregnancy. For most complications there was no threshold for risk. These results have led to careful reconsideration of the diagnostic criteria for GD. The International Association of Diabetes and Pregnancy Study Groups (IADPSG) recommended that all women not known to have prior diabetes undergo a 75 g OGTT at 24– 28 weeks of gestation. This probably will significantly increase the prevalence of GD, however there is mounting evidence that treating even mild GD reduces morbidity for both mother and baby (International Diabetes Federation [IDF], 2004). Women at high risk should be screened as soon as feasible. If the initial screening is negative they should undergo retesting at 24 to 28 weeks' gestation. There are two principles (selective and universal) in testing which need to be distinguished. First, there is selective (or at-risk) clinical need for high risk patient (obesity, a family history of diabetes, a history of macrosomic stillbirth, etc.). Second, there is universal screening, for women not clinically identified as being at particular risk. Universal screening is more sensitive, while selective screening is less costly. Unfortunately, the use of risk factors for individuals is not very effective, can result in great inconsistencies, and is a source of uncertainty for obstetricians and midwives. Universal screening should, at the moment, depend on the local prevalence of type 2 DM in the reproductive age group. Low-risk status requires no glucose testing, but this category is limited to those women meeting all of the following characteristics: age <25 years, normal weight before pregnancy, member of a population with a low prevalence of GD, no known diabetes in first-degree relatives, no history of abnormal glucose tolerance, no history of poor obstetric outcome. Where type 2 DM is common (for example many units in the USA, Saudi Arabia and the Gulf, and certain ethnic minorities), then a total population screen is justified (Forsbach et al., 1997). Universal screening is more sensitive and more practical when a family history of diabetes cannot be obtained reliably (Karcaaltincaba et al., 2009). The diagnosis of GD is associated with a risk of type 2 diabetes later in life, so it is unsurprising that the prevalence of GD in any Prevalence and Risk Factors for the Development of GD in Some Eastern European GD (6.3%) than whites (3.8%), blacks (3.5%), or Hispanics (3.6%). 2007; Ricart et al., 2005; Carolan et al., 2010) 33.3% in patients with 4 risk factors. USA Asia Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment 75 2001, Rosenberg et al., 2003; Ben-Haroush et al., 2004). Moreover, women of non-Caucasian ethnicity represent a considerable portion of childbearing populations in developed countries, and as rates of obesity and GD grow, there is a concordant increase in the risk of poorer pregnancy outcomes among these groups. GD is also associated with an increased maternal likelihood of developing diabetes type 2 in later life, and recent research postulates a link between GD, childhood obesity and later onset of diabetes in the offspring (Ferrara, In the USA more than 200,000 cases annually are complicated by GD (Bottalico, 2007). Only 1.5–2% of Midwestern white women develop GD, while up to 15% of Native American women from southwestern USA have been reported to develop GD. In the Hispanic, African-American and Asian populations of the USA, the incidence of GD is 5–8%. Most women will return to normoglycemia postpartum, but 30% to 50% will develop type 2 DM or glucose intolerance later in life. State-, city-, hospital-level GD prevalence estimates have consistently shown GD rates to be higher among Asian and Pacific Islander (API) mothers than among white, black, or Hispanic mothers (Lawrence et al., 2008; Chu et al., 2009) found that among 3,108,877 births, US APIs had a substantially higher age-adjusted prevalence of Oriental populations have different demographic characteristics (maternal age, height, BMI and frequency of obesity) to other ethnic populations and it is not clear to what extent the effects of age and obesity are interrelated. In Korea the life styles have changed rapidly to a westernized pattern characterized by sedentary occupations and consumption of refined food and a high-fat diet. Therefore it is reasonable to predict that the prevalence of GD in Korean women will increase as the prevalence of type 2 DM increases in the developing countries (Jang et al., 1995; 2003). The overall prevalence of GD in Korean women was estimated as 2.2%. The patients with GD were older, shorter and had a higher prepregnancy weight, BMI and parity than normal controls. The prevalence of GD in women with a single risk factor was 1.3% higher than that in women without risk factors (0.6%, p < 0.05), but was lower than that in the population at large (2.2%). However the prevalence increased to The DECODA study showed that India has a higher prevalence of diabetes than China or Japan (Ferrara, 2007) reported 3.8% prevalence of GD in China (20512 women screened), however they could not determine if there is an increase in the incidence of GD, but state that it is possible that absolute rate of GD has increased in China, probably due to changes in the lifestyle, decreased physical activity and increased incidence of obesity. Shai et al. (Shai et al., 2006) found that Asian women had a higher risk of diabetes than White, Hispanic, or Black women. In addition, they found that weight gain increased risk of diabetes more for Asian than for other women. Hunsberger et al. (Hunsberger et al., 2010) found that, compared with women of other race/ethnicities, both high BMI and low BMI Asian women were at the greatest risk of having GD. Additionally, diabetes risk among Asians seems to increase with prolonged exposure to Western lifestyle. The Turkish Diabetes Epidemiology Study reported the highest prevalence of diabetes in southern Turkey compared with northern and central parts of Turkey (Satman et al., 2002). The approximately rate of GD in Turkey is 4% (Karcaaltincaba et al., 2009). population will be largely determined by those factors which predict such a risk (Fraser & Heller, 2007) Any woman diagnosed with GD should be screened for diabetes 6–12 weeks postpartum and should be followed up with subsequent screening for the development of diabetes or pre-diabetes. If the fasting plasma glucose (FPG) level is normal, then reassessment for DM should occur every 3 years. Family planning for subsequent pregnancies should be discussed, and monitoring for the development of symptoms of DM should be undertaken. The goal of treatment is to reduce the risks of GD for mother and child by keeping blood glucose levels equal to those of pregnant women who don't have such a disease. The highest risk of complications is established when there is an increase of the initial level of preprandial blood sugar and when there is an increase of the postprandial glucose. Scientific evidences show that controlling glucose levels can result in less serious fetal complications and increased maternal quality of life and insulin administered twice daily during the third trimester to mothers who have even a mild degree of hyperglycemia will reduce fetal size, and in particular fetal adiposity. The proper management GD includes special meal plans and scheduled physical activity and of course - daily blood glucose testing. The further steps may require insulin injections. #### 4. Prevalence of GD GD is the most common metabolic disease of pregnancy. The prevalence of GD varies across the globe, as well as between racial and ethnic groups within the same country. In recent years there has been a reported increased trend in the prevalence of GD throughout the world, with huge health-care and economic costs (Bellamy et al., 2009). This is particularly the case in developed countries, such as the USA, the UK, Australia, China and New Zealand (Sinha et al., 2003; Ferrara et al., 2004; Joshy & Simmons, 2006; Metzger & Counstan, 1998; Fan et al., 2006; Carolan et al., 2010). The increased prevalence has been recently disputed, suggesting that more cases were identified through more aggressive universal screening. For example, Lopez-de-Andres et al. (Lopez-de-Andres et al., 2011) did not found increase in the prevalence of GD is Spain from 2001 to 2008. This partly coud be explained with the fact that with the selective approach, women at low risk for GD do not need to be screened and some cases could be missed. Several teams have tried to improve the sensitivity of selective screening by making the relevant protocols more inclusive. Through this 'tinkering', age has emerged as the most important risk factor for type 2 DM and, by extension, gestational diabetes. Accordingly, the lower threshold for screening has decreased from 30 years in earlier studies to 25 years in more recent ones. Approximately 8% of all pregnancies with wide-ranging differences between countries (ranging from 1 to 16% depending on the population studied, screening protocols and diagnostic criteria used) are complicated by GD (Hossein-Nezhad et al., 2007). Furthermore, within the same country, the prevalence of GD is strongly influenced by race and culture. High prevalence rates have been reported in studies from Australia (Indian-born 15%, Chinese 13.9%) and the United States (Zuni Indians 14.3%). Increasing prevalence relates to a range of factors including advanced maternal age, obesity and migratory patterns (Ferrara et al., 2004; Joshy & Simmons, 2006). Of particular interest here is the increase seen among specific ethnic groups (Joshy & Simmons, 2006, Kim et al., 2007), which may relate to ethnic differences in maternal glucose concentrations (Scholl et al., 2002; Esakoff et al., 2005) and recent trends of obesity (Harris et al., 1997, Xiong et al., 2001, Rosenberg et al., 2003; Ben-Haroush et al., 2004). Moreover, women of non-Caucasian ethnicity represent a considerable portion of childbearing populations in developed countries, and as rates of obesity and GD grow, there is a concordant increase in the risk of poorer pregnancy outcomes among these groups. GD is also associated with an increased maternal likelihood of developing diabetes type 2 in later life, and recent research postulates a link between GD, childhood obesity and later onset of diabetes in the offspring (Ferrara, 2007; Ricart et al., 2005; Carolan et al., 2010) #### USA 74 Gestational Diabetes population will be largely determined by those factors which predict such a risk (Fraser & Any woman diagnosed with GD should be screened for diabetes 6–12 weeks postpartum and should be followed up with subsequent screening for the development of diabetes or pre-diabetes. If the fasting plasma glucose (FPG) level is normal, then reassessment for DM should occur every 3 years. Family planning for subsequent pregnancies should be discussed, and monitoring for the development of symptoms of DM should be undertaken. The goal of treatment is to reduce the risks of GD for mother and child by keeping blood glucose levels equal to those of pregnant women who don't have such a disease. The highest risk of complications is established when there is an increase of the initial level of pre- Scientific evidences show that controlling glucose levels can result in less serious fetal complications and increased maternal quality of life and insulin administered twice daily during the third trimester to mothers who have even a mild degree of hyperglycemia will reduce fetal size, and in particular fetal adiposity. The proper management GD includes special meal plans and scheduled physical activity and of course - daily blood glucose GD is the most common metabolic disease of pregnancy. The prevalence of GD varies across the globe, as well as between racial and ethnic groups within the same country. In recent years there has been a reported increased trend in the prevalence of GD throughout the world, with huge health-care and economic costs (Bellamy et al., 2009). This is particularly the case in developed countries, such as the USA, the UK, Australia, China and New Zealand (Sinha et al., 2003; Ferrara et al., 2004; Joshy & Simmons, 2006; Metzger & Counstan, 1998; Fan et al., 2006; Carolan et al., 2010). The increased prevalence has been recently disputed, suggesting that more cases were identified through more aggressive universal screening. For example, Lopez-de-Andres et al. (Lopez-de-Andres et al., 2011) did not found increase in the prevalence of GD is Spain from 2001 to 2008. This partly coud be explained with the fact that with the selective approach, women at low risk for GD do not need to be screened and some cases could be missed. Several teams have tried to improve the sensitivity of selective screening by making the relevant protocols more inclusive. Through this 'tinkering', age has emerged as the most important risk factor for type 2 DM and, by extension, gestational diabetes. Accordingly, the lower threshold for screening has Approximately 8% of all pregnancies with wide-ranging differences between countries (ranging from 1 to 16% depending on the population studied, screening protocols and diagnostic criteria used) are complicated by GD (Hossein-Nezhad et al., 2007). Furthermore, within the same country, the prevalence of GD is strongly influenced by race and culture. High prevalence rates have been reported in studies from Australia (Indian-born 15%, Increasing prevalence relates to a range of factors including advanced maternal age, obesity and migratory patterns (Ferrara et al., 2004; Joshy & Simmons, 2006). Of particular interest here is the increase seen among specific ethnic groups (Joshy & Simmons, 2006, Kim et al., 2007), which may relate to ethnic differences in maternal glucose concentrations (Scholl et al., 2002; Esakoff et al., 2005) and recent trends of obesity (Harris et al., 1997, Xiong et al., prandial blood sugar and when there is an increase of the postprandial glucose. decreased from 30 years in earlier studies to 25 years in more recent ones. Chinese 13.9%) and the United States (Zuni Indians 14.3%). testing. The further steps may require insulin injections. Heller, 2007) 4. Prevalence of GD In the USA more than 200,000 cases annually are complicated by GD (Bottalico, 2007). Only 1.5–2% of Midwestern white women develop GD, while up to 15% of Native American women from southwestern USA have been reported to develop GD. In the Hispanic, African-American and Asian populations of the USA, the incidence of GD is 5–8%. Most women will return to normoglycemia postpartum, but 30% to 50% will develop type 2 DM or glucose intolerance later in life. State-, city-, hospital-level GD prevalence estimates have consistently shown GD rates to be higher among Asian and Pacific Islander (API) mothers than among white, black, or Hispanic mothers (Lawrence et al., 2008; Chu et al., 2009) found that among 3,108,877 births, US APIs had a substantially higher age-adjusted prevalence of GD (6.3%) than whites (3.8%), blacks (3.5%), or Hispanics (3.6%). #### Asia Oriental populations have different demographic characteristics (maternal age, height, BMI and frequency of obesity) to other ethnic populations and it is not clear to what extent the effects of age and obesity are interrelated. In Korea the life styles have changed rapidly to a westernized pattern characterized by sedentary occupations and consumption of refined food and a high-fat diet. Therefore it is reasonable to predict that the prevalence of GD in Korean women will increase as the prevalence of type 2 DM increases in the developing countries (Jang et al., 1995; 2003). The overall prevalence of GD in Korean women was estimated as 2.2%. The patients with GD were older, shorter and had a higher prepregnancy weight, BMI and parity than normal controls. The prevalence of GD in women with a single risk factor was 1.3% higher than that in women without risk factors (0.6%, p < 0.05), but was lower than that in the population at large (2.2%). However the prevalence increased to 33.3% in patients with 4 risk factors. The DECODA study showed that India has a higher prevalence of diabetes than China or Japan (Ferrara, 2007) reported 3.8% prevalence of GD in China (20512 women screened), however they could not determine if there is an increase in the incidence of GD, but state that it is possible that absolute rate of GD has increased in China, probably due to changes in the lifestyle, decreased physical activity and increased incidence of obesity. Shai et al. (Shai et al., 2006) found that Asian women had a higher risk of diabetes than White, Hispanic, or Black women. In addition, they found that weight gain increased risk of diabetes more for Asian than for other women. Hunsberger et al. (Hunsberger et al., 2010) found that, compared with women of other race/ethnicities, both high BMI and low BMI Asian women were at the greatest risk of having GD. Additionally, diabetes risk among Asians seems to increase with prolonged exposure to Western lifestyle. The Turkish Diabetes Epidemiology Study reported the highest prevalence of diabetes in southern Turkey compared with northern and central parts of Turkey (Satman et al., 2002). The approximately rate of GD in Turkey is 4% (Karcaaltincaba et al., 2009). Prevalence and Risk Factors for the Development of GD in Some Eastern European acts: priorities of all institutions in Bulgaria. treatment of a patient with diabetes. (IDF, 2011) National program. diabetes like Russia. Assembly during the autumn session of 2008. Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment 77 According to the data, provided by the Diabetes Atlas, Estonia, Latvia and Ukraine are those eastern European countries with highest percentage of diabetes for 2010. Albania is the country with lowest percentage. Totally 18 438 400 is the number of the patients with diabetes in these sixteen countries. The mean percentage with diabetes for 2010 is 8.76. And the decrease for the next 20 years will be insignificant – 8.24. Of course all the eastern European countries are extremely diverse as the population and gross domestic product (GDP) are taken in mind. Russia is the richest, while Moldova, Macedonia and Albania are the poorest. The number of deaths attributable to diabetes (20-79 years) is 317 955. And there is no significant differentiation by sex 143 810 – males and 174 145 – females. A very interesting fact is that only five countries - Greece, Russia, Belarus, Ukraine and Romania have National Diabetes program according to a survey of International Diabetes Federation (IDF) member associations. Bulgaria for example is working in order to develop and implement a national diabetes program. The Bulgarian Diabetes Association (BDA) that is the only one national representative association for patients and diabetics in Bulgaria is working towards achieving this goal. It was created in 1990. On the initiative of the Group for parliamentary consensus to combat socially significant diseases in the 40th National Assembly of Bulgaria and under the auspices of the National Assembly on 2-3 February 2008 in Plovdiv, Bulgaria there was a consensus around three significant 1. Restriction of diabetes in Bulgaria and particularly the preparation and implementation of diabetic register and national program to combat diabetes should be one of the 2. It is the creation of a working group of representatives of all stakeholders to draw up an action plan for prevention, early diagnosis and proper treatment of diabetes in Bulgaria. These documents must be submitted for consideration and adoption by the National 3. After the program and action plan, responsible institutions is imperative to take all necessary actions to ensure adequate funding for their practical application in the country. So we can conclude that Bulgaria is working towards the development of a Of course not only the disease itself has to be assessed but also the complication that accelerates the mortality. Close to four million deaths in the 20-79 age group may be attributable to diabetes in 2010, accounting for 6.8% of global all-cause mortality in this age group. According to the literay sources - this number resembles the deaths in this age group from several infectious diseases. The highest number of deaths due to diabetes is expected to occur in countries with large populations as they have the largest numbers of people with Another very important aspect is the health expenditures per person with diabetes in 2010. As it can be seen there are very big fluctuations in the Eastern European region. For example Greece spent 2742 USD, while Moldova – only 76 USD. In order to be precise in the conclusions it has to be analyzed the GDP in order to compare these expenditures. For Greece it is 325 088 billion USD , while for Moldova – 5 403 billlion. For Greece these mean health expenditures per person with diabetes are 0.0084% from the GDP of the country, while for Moldova – 0.014%. Bulgaria spent 0.007% from the GDP of the country for #### Europe Di Cianni et al. (Di Cianni et al., 2003) performed a retrospective study to evaluate the prevalence of GD by using both the selective and the universal screening approach and the presence of risk factors for GD in a cohort of Italian women. From June 1st, 1995 to December 31st, 2001, universal screening for GD was performed in 3950 women. In this analysis GD was significantly and independently associated to age, pre-pregnancy BMI, weight gain, height and family history of diabetes. The generated figures indicate that GCT was positive in 35.2% of cases, while the true prevalence of GD was 8.7%. When the OGTT was performed in a random sample of women with negative GCT, about 6.5% were found to have GD. By extrapolation to the whole cohort, it was then calculated that the approximate true prevalence of GD in the general population can be as high as 12.3%.Lopez-de-Andres et al. (Lopez-de-Andres et al., 2011) reported the prevalence of GD is Spain from 2001 to 2008 to be 3.6%. In Sweden, 7817 infants (of total number 892084) were born of mothers with GD between 1992 and 2004. This represents an incidence of GD of 0.9% in the studied population (Ahlsson et al., 2010). The number of adults with diabetes in the European region is expected to reach 55.2 million. And 33.40% are from the eastern European region. #### Eastern Europe The prevalence of GD in Eastern Europe and especially in Bulgaria shows a very diverse prevalence. (IDF, 2011) Table 2. The DM morbidity in Eastern European region. Di Cianni et al. (Di Cianni et al., 2003) performed a retrospective study to evaluate the prevalence of GD by using both the selective and the universal screening approach and the presence of risk factors for GD in a cohort of Italian women. From June 1st, 1995 to December 31st, 2001, universal screening for GD was performed in 3950 women. In this analysis GD was significantly and independently associated to age, pre-pregnancy BMI, weight gain, height and family history of diabetes. The generated figures indicate that GCT was positive in 35.2% of cases, while the true prevalence of GD was 8.7%. When the OGTT was performed in a random sample of women with negative GCT, about 6.5% were found to have GD. By extrapolation to the whole cohort, it was then calculated that the approximate true prevalence of GD in the general population can be as high as 12.3%.Lopez-de-Andres et al. (Lopez-de-Andres et al., 2011) reported the prevalence of GD is Spain from 2001 to 2008 to be 3.6%. In Sweden, 7817 infants (of total number 892084) were born of mothers with GD between 1992 and 2004. This represents an incidence of GD of 0.9% in the studied population (Ahlsson et al., 2010). The number of adults with diabetes in the European region is expected to reach 55.2 The prevalence of GD in Eastern Europe and especially in Bulgaria shows a very diverse 1. Albania 4.8 102 800 5.1 2 986 952 12.224 billion 2. Belarus 9.1 661 100 9.0 9 648 533 52.887 billion 4. Bulgaria 9.0 519 500 7.8 7 351 234 44.843 billion 5. Croatia 9.2 315 900 8.0 4 486 881 59 917 billion 6. Estonia 9.9 97 900 9.0 1 340 021 19.123 billion 7. Greece 8.8 754 000 7.4 11 305 118 325 088 billion 8. Latvia 9.9 169 700 9.0 2 217 969 23 955 billion 9. Lithuania 9.7 239 800 9.0 3 244 000 35.152 billion 10. Macedonia 8.0 119 300 8.0 2 052 722 9470 billion 11. Moldova 8.7 233 500 9.0 3 567 500 5 403 billlion 13. Romania 8.4 1 351 400 8.0 21 959 278 158 393 billion 14. Russia 9.0 9 624 900 9.0 142 905 200 1 477 trillion 15. Serbia 8.6 613 400 8.0 9 981 929 43.6 billion 16. Ukraine 9.6 3 328 400 9.0 49 100 000 37.6 billion percent with diabetes (20-79 years) 2030 9.1 271 100 8.6 3 842 566 16 631 billion population GDP nominal total (USD) number of people with diabetes 2010 12. Montenegro 8.4 35 700 8.0 620 000 Table 2. The DM morbidity in Eastern European region. million. And 33.40% are from the eastern European region. Europe Eastern Europe 3. Bosnia and Herzegovina prevalence. (IDF, 2011) Country percent with diabetes (20-79 years) 2010 According to the data, provided by the Diabetes Atlas, Estonia, Latvia and Ukraine are those eastern European countries with highest percentage of diabetes for 2010. Albania is the country with lowest percentage. Totally 18 438 400 is the number of the patients with diabetes in these sixteen countries. The mean percentage with diabetes for 2010 is 8.76. And the decrease for the next 20 years will be insignificant – 8.24. Of course all the eastern European countries are extremely diverse as the population and gross domestic product (GDP) are taken in mind. Russia is the richest, while Moldova, Macedonia and Albania are the poorest. The number of deaths attributable to diabetes (20-79 years) is 317 955. And there is no significant differentiation by sex 143 810 – males and 174 145 – females. A very interesting fact is that only five countries - Greece, Russia, Belarus, Ukraine and Romania have National Diabetes program according to a survey of International Diabetes Federation (IDF) member associations. Bulgaria for example is working in order to develop and implement a national diabetes program. The Bulgarian Diabetes Association (BDA) that is the only one national representative association for patients and diabetics in Bulgaria is working towards achieving this goal. It was created in 1990. On the initiative of the Group for parliamentary consensus to combat socially significant diseases in the 40th National Assembly of Bulgaria and under the auspices of the National Assembly on 2-3 February 2008 in Plovdiv, Bulgaria there was a consensus around three significant acts: Of course not only the disease itself has to be assessed but also the complication that accelerates the mortality. Close to four million deaths in the 20-79 age group may be attributable to diabetes in 2010, accounting for 6.8% of global all-cause mortality in this age group. According to the literay sources - this number resembles the deaths in this age group from several infectious diseases. The highest number of deaths due to diabetes is expected to occur in countries with large populations as they have the largest numbers of people with diabetes like Russia. Another very important aspect is the health expenditures per person with diabetes in 2010. As it can be seen there are very big fluctuations in the Eastern European region. For example Greece spent 2742 USD, while Moldova – only 76 USD. In order to be precise in the conclusions it has to be analyzed the GDP in order to compare these expenditures. For Greece it is 325 088 billion USD , while for Moldova – 5 403 billlion. For Greece these mean health expenditures per person with diabetes are 0.0084% from the GDP of the country, while for Moldova – 0.014%. Bulgaria spent 0.007% from the GDP of the country for treatment of a patient with diabetes. (IDF, 2011) Prevalence and Risk Factors for the Development of GD in Some Eastern European survival rate of girls born with weight above 4 kg. workmates and organisations in: health maintenance. major components. with diabetes: 5. St. Vincent declaration – Review and principles The management of their own diabetes and education for it. The planning, provision and quality audit of health care. Promoting and applying research. (IDF, 2004) The five-year targets that are granted in the declaration are: expectation in quality and quantity. Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment 79 These statistics are calculated extrapolations of various prevalence or incidence rates against the populations of a particular country or region. The statistics used for prevalence/incidence of GD are typically based on US, UK, Canadian or Australian prevalence or incidence statistics, which are then extrapolated using only the population of a given country. The base is that 0.05% from the population (women) will get GD annually. The literary data shows that the frequency of development of GD varies from 2 to 4-5 % from the pregnant women. Some authors even state that 9% from the pregnant women develop GD. In Bulgaria a pilot study shows that the frequency is even greater – 14%, but till today there was not perform a systematic screening. Unfortunatelly in Bulgaria there is a tendency for doubling the GD morbidity, because of the obesity and because of the increase In 1989, in St. Vincent, Italy was signed the St. Vincent declaration, a joint initiative of the International Diabetes Federation European region and the WHO European regional office. It is a program for strategic action to reduce the human and economic burden of diabetes in Europe and has been adopted by most of the European governments. (IDF, 2004) The St. Vincent initiative has few target areas, which seek to improve the quality of life of people with diabetes and to promote education of patients so to prevent diabetes complications. Patient education is very important and a team approach, including physicians, pharmacists and nurses, is beneficial. Some of the main conclusions during the meeting were that "diabetes mellitus is a major and growing European health problem, a problem at all ages and in all countries. It currently threatens at least ten million European citizens. It is within the power of national governments and health departments to create conditions in which a major reduction in this heavy burden of disease and death can be achieved. Countries should give formal recognition to the diabetes problem and deploy resources for its solution. Plans for the prevention, identification and treatment of diabetes and particularly its complications should be formulated at local, national and European regional levels. General goals and five-year targets can be achieved by the organised activities of the medical services in active partnership with diabetic patients, their families, friends and National, regional and international organisations for disseminating information about The St. Vincent's declaration outlined the following general goals for children and adults sustained improvement in health experience and a life experience approaching normal elaboration, initiation and evaluation comprehensive programmes for the detection and control of diabetes and its complications with self-care and community support as prevention and cure of diabetes and its complications by intensifying research effort. Table 3. Mean health expenditures per person with DM in 2010 for the Eastern European region. The statistics by country for gestational diabetes shows that apporximatelly 1 in 2 014 or 0.05% or 135 000 women get GD every year in the USA. The analysis of the data for the eastern European region are shown in Table 4. Table 4. Extrapolated incidence of GD (wrongdiagnosis.com, 2011) These statistics are calculated extrapolations of various prevalence or incidence rates against the populations of a particular country or region. The statistics used for prevalence/incidence of GD are typically based on US, UK, Canadian or Australian prevalence or incidence statistics, which are then extrapolated using only the population of a given country. The base is that 0.05% from the population (women) will get GD annually. The literary data shows that the frequency of development of GD varies from 2 to 4-5 % from the pregnant women. Some authors even state that 9% from the pregnant women develop GD. In Bulgaria a pilot study shows that the frequency is even greater – 14%, but till today there was not perform a systematic screening. Unfortunatelly in Bulgaria there is a tendency for doubling the GD morbidity, because of the obesity and because of the increase survival rate of girls born with weight above 4 kg. ### 5. St. Vincent declaration – Review and principles 78 Gestational Diabetes Country Mean health expenditure per person with diabetes in 2010 (USD) Table 3. Mean health expenditures per person with DM in 2010 for the Eastern European The statistics by country for gestational diabetes shows that apporximatelly 1 in 2 014 or 0.05% or 135 000 women get GD every year in the USA. The analysis of the data for the Population estimated used base 2004) (US Census Bureau, International data Albania 261 Belarus 238 Bosnia and Herzegovina 307 Bulgaria 301 Croatia 736 Estonia 584 Greece 2 742 Latvia 493 Lithuania 521 Macedonia 287 Moldova 76 Montenegro 14 Romania 145 Russia 261 Serbia 238 Ukraine 307 eastern European region are shown in Table 4. Incidence Albania 1 759 3 544 808 Belarus 5 117 10 310 520 Herzegovina 202 407 608 Bulgaria 3 731 7 517 973 Croatia 2 231 4 496 869 Estonia 665 1 341 664 Greece 5 284 10 647 529 Latvia 1 144 2 306 306 Lithuania 1 790 3 607 899 Macedonia 1 012 2 040 085 Romania 11 095 22 355 551 Russia 71 457 143 974 059 Montenegro 5 373 10 825 900 Ukraine 23 690 47 732 079 Table 4. Extrapolated incidence of GD (wrongdiagnosis.com, 2011) Country Extrapolated region. Bosnia and Serbia and In 1989, in St. Vincent, Italy was signed the St. Vincent declaration, a joint initiative of the International Diabetes Federation European region and the WHO European regional office. It is a program for strategic action to reduce the human and economic burden of diabetes in Europe and has been adopted by most of the European governments. (IDF, 2004) The St. Vincent initiative has few target areas, which seek to improve the quality of life of people with diabetes and to promote education of patients so to prevent diabetes complications. Patient education is very important and a team approach, including physicians, pharmacists and nurses, is beneficial. Some of the main conclusions during the meeting were that "diabetes mellitus is a major and growing European health problem, a problem at all ages and in all countries. It currently threatens at least ten million European citizens. It is within the power of national governments and health departments to create conditions in which a major reduction in this heavy burden of disease and death can be achieved. Countries should give formal recognition to the diabetes problem and deploy resources for its solution. Plans for the prevention, identification and treatment of diabetes and particularly its complications should be formulated at local, national and European regional levels. General goals and five-year targets can be achieved by the organised activities of the medical services in active partnership with diabetic patients, their families, friends and workmates and organisations in: The St. Vincent's declaration outlined the following general goals for children and adults with diabetes: Prevalence and Risk Factors for the Development of GD in Some Eastern European insulin and with complications that is 398.90 USD (p=0,03). (Todorova et al., 2007) women is CCE is 5141.17 USD effective treatment for each of them. (334.43 x 0,4) + (363.2x 0,6) = 351.69 USD (346.84x0,95) + (398.2x0,05) = 349.35 USD USD (351.64/0,4). (Todorova et al., 2007) The total costs of the treatment with diet is 351.69 USD The total cost of the treatment with insulin is 349.35 USD. 51.4117x 100 = 5141.17 USD CCE= 398.90 – 363.94/6,1 – 5,42 = 51.4117USD The cost-effectiveness coefficient is calculated based on the total direct medical costs, used for the reduction of the average 24-houred glucose under 5,8 mmol/l as for higher precision the calculations are proceeded with the received difference in the level of the glycated hemoglobin after the treatment. (Todorova et al., 2007) The over calculated CCE for 100 The so calculated coefficient reflects the costs for complications that is saved by the insulin treatment and that should occur after an ineffective diet treatment. The interpretation of this coefficient shows that the spent 12.40 USD for insulin treatment for every woman in fact saves 51.41 USD that should be spent for the treatment of unfavorable peripartal maternal complications. Through the application of a model of mathematical modeling the total final cost for one beneficially treated woman with GD is calculated. The calculations for the two therapeutical alternatives are performed by the use of the probabilities for occurrence of The applied analytical model shows that on the base of the higher per cent successfully treated women, the spent final costs for successfully treated woman after the treatment with insulin are 388,13 USD (349.32/0,9), and the final costs after the treatment with diet are 879.1 Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment 81 importance for the neonatal end of the pregnancy. That is why it is very important to determine the cost and effectiveness of the drug treatment for women with GD. It can be achieved by studying the clinical effectiveness of the treatment with diet and diet + insulin for pregnant women with GD and determination of the ratio treatment cost/effectiveness. A prospective study of 50 women with gestational diabetes from Sofia, Bulgaria were studied from pharmacoeconomical point of view. They were divided into 2 groups: Group I (n = 30) pregnant women only on a diet and Group II (n = 20) - pregnant women treated by a diet and insulin. The following including criteria were applied: age above 18 years, one fetus pregnancy, insignificant additional disease without organ damages, without infectious diseases, without obesity and HbA1-c under 7%. The key excluding criteria are the availability of diabetes before pregnancy, or prior insulin therapy, treatment with oral hypoglycemic drugs before pregnancy, income of drugs that have influence on the carbohydrate tolerance, existing obstetrics complications till the demonstration of the gestation diabetes. The diet treatment was with diet No 9 according to M.I. Pevzner. The diet satisfies the recommendations for feeding. Human insulin, in intensified insulin regimen type- bazalprandial, including treatment with three doses rapid acting insulin and one or two doses intermediate acting insulin was applied. The total costs of the treatment of a patient, on a diet without complications in the peripartal period is 335.02 USD and is lower in comparison with the total cost of the treatment of a woman treated only with insulin that is 347.42 USD. (p=0.04). The difference in the treatment cost is 12.40 USD. The total treatment cost of a pregnant woman, treated with diet with complications in the peripartal period is 363.94 USD and is also lower in comparison with the total cost of the treatment of a woman treated with Since 1989 further implementation and evaluation meetings have been held in Budapest (1992), Athens (1995), Lisbon (1997) and Istanbul (1999), where representatives of ISPAD have helped to formulate recommendations on behalf of children and adolescents. #### 6. Pharmacoeconomical assessment of GD drug treatment. The role of the pharmacist for GD management The standard treatment of GD is insulin treatment. The metabolic control target in the GD treatment is blood sugar before meal under 5.8 mmo/l, postprandial blood sugar under 7.5 mmo/l and hemoglobin HbA1-c under 6.5%. The postprandial blood glucose is of critical importance for the neonatal end of the pregnancy. That is why it is very important to determine the cost and effectiveness of the drug treatment for women with GD. It can be achieved by studying the clinical effectiveness of the treatment with diet and diet + insulin for pregnant women with GD and determination of the ratio treatment cost/effectiveness. A prospective study of 50 women with gestational diabetes from Sofia, Bulgaria were studied from pharmacoeconomical point of view. They were divided into 2 groups: Group I (n = 30) pregnant women only on a diet and Group II (n = 20) - pregnant women treated by a diet and insulin. The following including criteria were applied: age above 18 years, one fetus pregnancy, insignificant additional disease without organ damages, without infectious diseases, without obesity and HbA1-c under 7%. The key excluding criteria are the availability of diabetes before pregnancy, or prior insulin therapy, treatment with oral hypoglycemic drugs before pregnancy, income of drugs that have influence on the carbohydrate tolerance, existing obstetrics complications till the demonstration of the gestation diabetes. The diet treatment was with diet No 9 according to M.I. Pevzner. The diet satisfies the recommendations for feeding. Human insulin, in intensified insulin regimen type- bazalprandial, including treatment with three doses rapid acting insulin and one or two doses intermediate acting insulin was applied. The total costs of the treatment of a patient, on a diet without complications in the peripartal period is 335.02 USD and is lower in comparison with the total cost of the treatment of a woman treated only with insulin that is 347.42 USD. (p=0.04). The difference in the treatment cost is 12.40 USD. The total treatment cost of a pregnant woman, treated with diet with complications in the peripartal period is 363.94 USD and is also lower in comparison with the total cost of the treatment of a woman treated with insulin and with complications that is 398.90 USD (p=0,03). (Todorova et al., 2007) The cost-effectiveness coefficient is calculated based on the total direct medical costs, used for the reduction of the average 24-houred glucose under 5,8 mmol/l as for higher precision the calculations are proceeded with the received difference in the level of the glycated hemoglobin after the treatment. (Todorova et al., 2007) The over calculated CCE for 100 women is CCE is 5141.17 USD CCE= 398.90 – 363.94/6,1 – 5,42 = 51.4117USD 51.4117x 100 = 5141.17 USD 80 Gestational Diabetes steps for raising the awareness in the population and amongst health care professionals of the present opportunities and the future needs for the prevention of diabetes and its organisation training and teaching programs in diabetes management and care for people of all ages with diabetes, for their families, friends and working associates and ensuring that care for children with diabetes is provided by individuals and teams specialised in the management of both diabetes and children, and that families with a reinforcement of the existing centres of excellence in diabetes care, education and promotion of independence, equity and self-sufficiency for all people with diabetes, i.e. attempts for reducing the hindrances to the fullest possible integration of the diabetic b reduce the number of people entering end-stage diabetic renal failure by at least d cut morbidity and mortality from coronary heart disease in the diabetic by e achieve a pregnancy outcome in the diabetic woman that approximates that of establishment of monitoring and control systems, using new information technology for quality assurance of diabetes health care revision and for laboratory and technical promotion and granting of collaboration of European and international programmes of diabetes research and development through national, regional and World Health Organisation agencies and in active partnership with diabetes patients' organisations. taking urgent action in the spirit of the WHO programme 'Health for All' to establish joint initiative between the WHO and the International Diabetes Federation (European region) to initiate, accelerate and facilitate the implementation of these Since 1989 further implementation and evaluation meetings have been held in Budapest (1992), Athens (1995), Lisbon (1997) and Istanbul (1999), where representatives of ISPAD 6. Pharmacoeconomical assessment of GD drug treatment. The role of the The standard treatment of GD is insulin treatment. The metabolic control target in the GD treatment is blood sugar before meal under 5.8 mmo/l, postprandial blood sugar under 7.5 mmo/l and hemoglobin HbA1-c under 6.5%. The postprandial blood glucose is of critical have helped to formulate recommendations on behalf of children and adolescents. diabetic child get the necessary social, economic and emotional support. children, adolescents, those in the working years of life and the elderly. c reduce by one half the rate of limb amputations for diabetic gangrene. procedures in diabetes diagnosis, treatment and self-management. a reduce new blindness due to diabetes by one third or more. vigorous programmes of risk factor reduction. implementation of effective measures for the prevention of costly complications: complications. research. citizen into society. one third. the non-diabetic woman. recommendations." (IDF, 2004) pharmacist for GD management for the health care team. The so calculated coefficient reflects the costs for complications that is saved by the insulin treatment and that should occur after an ineffective diet treatment. The interpretation of this coefficient shows that the spent 12.40 USD for insulin treatment for every woman in fact saves 51.41 USD that should be spent for the treatment of unfavorable peripartal maternal complications. Through the application of a model of mathematical modeling the total final cost for one beneficially treated woman with GD is calculated. The calculations for the two therapeutical alternatives are performed by the use of the probabilities for occurrence of effective treatment for each of them. The total costs of the treatment with diet is 351.69 USD (334.43 x 0,4) + (363.2x 0,6) = 351.69 USD The total cost of the treatment with insulin is 349.35 USD. (346.84x0,95) + (398.2x0,05) = 349.35 USD The applied analytical model shows that on the base of the higher per cent successfully treated women, the spent final costs for successfully treated woman after the treatment with insulin are 388,13 USD (349.32/0,9), and the final costs after the treatment with diet are 879.1 USD (351.64/0,4). (Todorova et al., 2007) Prevalence and Risk Factors for the Development of GD in Some Eastern European The educational course included the following teaching units: The educational materials used during the program included: carbs, comments and activities, insulin treatment); educational lecture, provided to the patient after every session; physical activity for both of the groups. selected pregnant women with GD. was to learn the seriousness of GD. every woman the effects on the fetus. units; Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment 83 study design is a pilot case-control study that includes 45 women with GD from city of Sofia, Bulgaria. The pregnant women were divided into two groups – Group I (n=22), that were educated on the proper management of their disease and were on a diet and treated with insulin, and Group II (n=23), that were not educated and on a diet, but treated with insulin on a regimen, prescribed by the physician. The assignment was based on the principle of random numbers through custom random number generator. The educational program continued 3 months. It was designed and adapted to the patients comfort, provided at the pharmacy they are attending. The course was presented to the 22 previously The first unit acquainted each of the women with the aim of the educational program, provided general concept about GD and about self-monitoring and emphasized on the active patient participation in the treatment. The personal information of each of the patients was collected, concerning the duration of the disease, the prescribed drug treatment if any, the frequency of the hypoglycaemic and hyperglycaemic incidents. At the end of the first unit, each of the patients was supplied with written materials on the essence of GD. The goal The main topic discussed during the second teaching unit was complication of improper management. The educator explained the complications of GD. The educator discussed with The main topic discussed during the third teaching unit was proper diet regimen (based on the GD diet menu of the Endocrinology clinic of Minneapolis, USA). At the end of the session the patients were supplied with the Sample GD diet menu. Each of the patients was supplied In the beginning and at the end of the educational process a patient satisfaction questionnaire was applied (Diabetes Questionnaire (IMG)). During the 3-months education and its end, the observed behavioural parameters performed changes. The results from the twice-applied questionnaire assessing the quality of life of the patients in the beginning and in the end of the educational programme show that the five main indices have been improved with on the average of 5% for the both groups, but greater for those included in Group I (Table 7). The greater increase was observed in the positive changes in the mood – 7,9% for Group I and 8,2% for Group II , followed by number of days "being easy" with 6.9 % for Group I and with 6,2% for Group II (6.7%) and possibility to perform physical activities 5.8 % for Group I and 5% for Group II. It could be considered that the educational with written materials on proper nourishing for diabetic patients and physical activity. Table 5. "Three of decisions" of the alternatives for the treatment of GD. There are four clinical paths, concerning diabetes that are included in the list of the Bulgarian Health Insurance fund. Unfortunatelly there is no clinical path concerning DM or the complications of this disease that decreases the accessibility towards proper management of this disease. And on this base it can be explained the prospective data about doubling the GD morbidity. Table 6. Clinical paths in Bulgaria, concerning diabetes. Another aspect that is innovative in the Bulgarian pharmaceutical practice, concerning the management of GD is the role of the pharmacist as a health-care educator in order to be accomplished the St. Vincent declaration aims is demonstrated. Patient education is very important and a team approach, including physicians, pharmacists and nurses, is beneficial.(38). A study that included pregnant women attending antenatal clinic with GD January 2009 - December 2009 demonstrated the beneficial role of patients education. The following including criteria are used: age above 18 years, one-fetus pregnancy, GD, insignificant additional disease without organ damage, without infectious diseases. The study design is a pilot case-control study that includes 45 women with GD from city of Sofia, Bulgaria. The pregnant women were divided into two groups – Group I (n=22), that were educated on the proper management of their disease and were on a diet and treated with insulin, and Group II (n=23), that were not educated and on a diet, but treated with insulin on a regimen, prescribed by the physician. The assignment was based on the principle of random numbers through custom random number generator. The educational program continued 3 months. It was designed and adapted to the patients comfort, provided at the pharmacy they are attending. The course was presented to the 22 previously selected pregnant women with GD. The educational course included the following teaching units: 82 Gestational Diabetes There are four clinical paths, concerning diabetes that are included in the list of the Bulgarian Health Insurance fund. Unfortunatelly there is no clinical path concerning DM or the complications of this disease that decreases the accessibility towards proper management of this disease. And on this base it can be explained the prospective data about stay Drug treatment Prize carbamazepine - 400- 800 mg/daily. gabapentin – from 3 mg daily; pregabalin – up to 2 х 150 mg; times x 300 mg till 1500 Electrolities 9.57 - once Electrolities 9.57 - once (USD)/daily 0.23-0.46 2.34 – 3.90 3.92 Table 5. "Three of decisions" of the alternatives for the treatment of GD. patients above 18 years 3 days Insulin patients bellow 18 years 3 days Insulin 3 days Another aspect that is innovative in the Bulgarian pharmaceutical practice, concerning the management of GD is the role of the pharmacist as a health-care educator in order to be accomplished the St. Vincent declaration aims is demonstrated. Patient education is very important and a team approach, including physicians, pharmacists and nurses, is beneficial.(38). A study that included pregnant women attending antenatal clinic with GD January 2009 - December 2009 demonstrated the beneficial role of patients education. The following including criteria are used: age above 18 years, one-fetus pregnancy, GD, insignificant additional disease without organ damage, without infectious diseases. The doubling the GD morbidity. path No disease Hospital 8 Diabetes polineuropathia 3 days Surgical Intervention Of Diabetic Foot without vessel reconstruction Table 6. Clinical paths in Bulgaria, concerning diabetes. <sup>104</sup>Decompensated DM for <sup>105</sup>Decompensated DM for Clinical 178 The first unit acquainted each of the women with the aim of the educational program, provided general concept about GD and about self-monitoring and emphasized on the active patient participation in the treatment. The personal information of each of the patients was collected, concerning the duration of the disease, the prescribed drug treatment if any, the frequency of the hypoglycaemic and hyperglycaemic incidents. At the end of the first unit, each of the patients was supplied with written materials on the essence of GD. The goal was to learn the seriousness of GD. The main topic discussed during the second teaching unit was complication of improper management. The educator explained the complications of GD. The educator discussed with every woman the effects on the fetus. The main topic discussed during the third teaching unit was proper diet regimen (based on the GD diet menu of the Endocrinology clinic of Minneapolis, USA). At the end of the session the patients were supplied with the Sample GD diet menu. Each of the patients was supplied with written materials on proper nourishing for diabetic patients and physical activity. The educational materials used during the program included: In the beginning and at the end of the educational process a patient satisfaction questionnaire was applied (Diabetes Questionnaire (IMG)). During the 3-months education and its end, the observed behavioural parameters performed changes. The results from the twice-applied questionnaire assessing the quality of life of the patients in the beginning and in the end of the educational programme show that the five main indices have been improved with on the average of 5% for the both groups, but greater for those included in Group I (Table 7). The greater increase was observed in the positive changes in the mood – 7,9% for Group I and 8,2% for Group II , followed by number of days "being easy" with 6.9 % for Group I and with 6,2% for Group II (6.7%) and possibility to perform physical activities 5.8 % for Group I and 5% for Group II. It could be considered that the educational Prevalence and Risk Factors for the Development of GD in Some Eastern European changes to try and prevent them developing type 2 diabetes in later life. Diabetes Care, No 33, (Suppl 1):S11-61 Diabetes Care, No 34(Suppl 1):S11-61 =2906&TEMPLATE=/CM/HTMLDisplay.cfm) carrying any specific benefit for pregnancy outcome. 7. Conclusion 8. References 538 Available from: Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment 85 Gestational diabetes is a subject of endless debate, uncertainty and confusion. Although it is a alteration during pregnancy, the true prevalence of GD remains a matter of discussion. The prevalence of GD in the general population is varied from 1% to 16% depending on both the country of origin and the nature of the indigenous population. The rate can vary due to differences in data collection methods, low response rates, non-random selection of the women, and lack of uniformity in the diagnostic criteria. More recently, the high rate of GD has been claimed to be an unrealistic estimation caused by universal screening, not GD is considered to be a state of prediabetes. The diagnosis of GD identifies women at high risk of diabetes after the pregnancy. Therefore, it is important for these women who may develop type 2 diabetes during their life to take preventive measures as well as to prevent pregnancy-related complications. It is well known that raised glucose levels in women with GD increase both morbidity and mortality among their offspring due mainly to an increased incidence of congenital abnormalities and excessive fetal growth in the third trimester. Women with a family history of diabetes had three times higher odds for GD than women without a family history of diabetes. With the prevalence of type 2 diabetes increasing across the world, and given that the prevalence of GD is thought to shadow that of type 2 diabetes, most populations will expect to see a rise in GD figures during the coming years. It is very important to establish clear policies to ensure that those at risk are reliably identified, appropriately treated during pregnancy and then equipped to make the necessary lifestyle ADA: American Diabetes Association. (2010). Standards of medical care in diabetes-2010. ADA: American Diabetes Association. (2011). Standards of medical care in diabetes-2011. Ahlsson F, Lundgren M, Tuvemo T, Gustafsson J, Haglund B. (2010). Gestational diabetes American College of Obstetricians and Gynaecologists Committee on Practice Bulletins American Diabetes Association.(2008) Clinical Practice Recommendations - Diagnosis and Classification of Diabetes Mellitus. Diabetes Care; No 31(Suppl. 1), S55-S60 American Pharmaceutical Association. (2011). Principles of practice for pharmaceutical care. – Obstetrics (2001) ACOG practice bulletin. Clinical management guidelines for obstetrician-gynecologists. No 30, September 2001 (replaces Technical Bulletin Number 200, December 1994). Gestational diabetes. Obstet Gynecol 98, pp.525- http://www.pharmacist.com/AM/Template.cfm?Section=Home2&CONTENTID and offspring body disproportion. Acta Pediatrica, No 99, pp.89-93 process affects both the physical and the psychological well-being and thus it is beneficial for the global patient's quality of life. Data are %. QL – "quality of life". Table 7. Changes in the patients sample after the educational process The advanced pharmacy practitioner in diabetes management is a relatively new approach. The role of the pharmacist in it, integrates drug management, patients' compliance assessment, blood glucose monitoring, skills training, prospective and retrospective drug utilization review, adverse drug reaction and toxicity screening and education of the patients.(Valentine et al, 2003). These skills in fact are not new for the pharmacist but their introduction, as systematized approach in everyday practice should correspond to the local circumstances. To match the context of the pharmaceutical care, defined by the APA as "Patient-centered, outcomes-oriented pharmacy practice that requires the pharmacist to work in concert with the patient to promote health, to prevent disease and to assess, monitor, initiate and modify medication use", is a real challenge for the management of diabetes, especially for Bulgaria.(American Pharmaceutical Association [APA], 2011). Despite the relatively small sample size, this study shows the role of education program for improvement of patient's outcomes. The results confirm the necessity of individual approach in the selection of therapeutic strategy for the women with GD. As the St. Vincent declaration assumed, the quality of life of people with diabetes has to be improved and to be promoted education of patients so to prevent diabetes complications. According to the St. Vincent declaration the aim of the treatment of GD is the achievement of child birth similar to the child birth by women without diabetes.(IDF, 2004) In this project are involved all healthcare givers, including the pharmacists in order to be achieved the goals. (Douglas et al., 2000; Dixon, 2002; Campbell et al., 1990; Mensing et al., 2002) The educational approach is a necessary step for better management of the disease in order to minimize the risk of maternal and fetal complications and the pharmacists are capable to perform it. The results obtained confirm the need for consistent patients' education, using variety of educational models, as an essential part of the diabetes care that will result in improvement of patient's quality of life. ### 7. Conclusion 84 Gestational Diabetes process affects both the physical and the psychological well-being and thus it is beneficial Group I Group II Group I Group II Variable 0 3 month Table 7. Changes in the patients sample after the educational process QL-positive changes in the mood 10,8% 9,9% 18,7% 18,1% QL-increase in days "being easy" 18,4% 17,9% 25,3% 24,1% QL- increase in social activity 11,1% 12,2% 14% 15,1% QL- increase in days being "rested" 15% 14,5% 17,4% 16,8% QL- increase in physical activity 13,4% 14,0% 19,2% 19,0% total 68,70% 68,50% 94,60% 93,10% mean 13,74% 13,70% 18,92% 18,62% The advanced pharmacy practitioner in diabetes management is a relatively new approach. The role of the pharmacist in it, integrates drug management, patients' compliance assessment, blood glucose monitoring, skills training, prospective and retrospective drug utilization review, adverse drug reaction and toxicity screening and education of the patients.(Valentine et al, 2003). These skills in fact are not new for the pharmacist but their introduction, as systematized approach in everyday practice should correspond to the local circumstances. To match the context of the pharmaceutical care, defined by the APA as "Patient-centered, outcomes-oriented pharmacy practice that requires the pharmacist to work in concert with the patient to promote health, to prevent disease and to assess, monitor, initiate and modify medication use", is a real challenge for the management of diabetes, especially for Bulgaria.(American Pharmaceutical Association [APA], 2011). Despite the relatively small sample size, this study shows the role of education program for improvement of patient's outcomes. The results confirm the necessity of individual approach in the selection of therapeutic strategy for the women with GD. As the St. Vincent declaration assumed, the quality of life of people with diabetes has to be improved and to be promoted education of patients so to prevent diabetes complications. According to the St. Vincent declaration the aim of the treatment of GD is the achievement of child birth similar to the child birth by women without diabetes.(IDF, 2004) In this project are involved all healthcare givers, including the pharmacists in order to be achieved the goals. (Douglas et al., 2000; Dixon, 2002; Campbell et al., 1990; Mensing et al., 2002) The educational approach is a necessary step for better management of the disease in order to minimize the risk of maternal and fetal complications and the pharmacists are capable to perform it. The results obtained confirm the need for consistent patients' education, using variety of educational models, as an essential part of the diabetes care that will result in improvement of patient's for the global patient's quality of life. Data are %. QL – "quality of life". quality of life. Time period Gestational diabetes is a subject of endless debate, uncertainty and confusion. Although it is a alteration during pregnancy, the true prevalence of GD remains a matter of discussion. The prevalence of GD in the general population is varied from 1% to 16% depending on both the country of origin and the nature of the indigenous population. The rate can vary due to differences in data collection methods, low response rates, non-random selection of the women, and lack of uniformity in the diagnostic criteria. More recently, the high rate of GD has been claimed to be an unrealistic estimation caused by universal screening, not carrying any specific benefit for pregnancy outcome. GD is considered to be a state of prediabetes. The diagnosis of GD identifies women at high risk of diabetes after the pregnancy. Therefore, it is important for these women who may develop type 2 diabetes during their life to take preventive measures as well as to prevent pregnancy-related complications. It is well known that raised glucose levels in women with GD increase both morbidity and mortality among their offspring due mainly to an increased incidence of congenital abnormalities and excessive fetal growth in the third trimester. Women with a family history of diabetes had three times higher odds for GD than women without a family history of diabetes. With the prevalence of type 2 diabetes increasing across the world, and given that the prevalence of GD is thought to shadow that of type 2 diabetes, most populations will expect to see a rise in GD figures during the coming years. It is very important to establish clear policies to ensure that those at risk are reliably identified, appropriately treated during pregnancy and then equipped to make the necessary lifestyle changes to try and prevent them developing type 2 diabetes in later life. #### 8. References http://www.pharmacist.com/AM/Template.cfm?Section=Home2&CONTENTID =2906&TEMPLATE=/CM/HTMLDisplay.cfm) Prevalence and Risk Factors for the Development of GD in Some Eastern European perspective. Diabetes Care. Jul, Vol. 30 Suppl 2, pp.:S141-6. pp.229-32. pp.1991–2002 Vol.20, No. 9, pp.1422-5. Med, Vol.17, No.12, pp.:345-348. Acta Diabetologica, No. 45, pp.157-165 Countries – Tendencies and Pharmacoeconomical Assessment for the Choice of Treatment 87 Ferrara A. (2007). Increasing prevalence of gestational diabetes mellitus: a public health Forsbach G, Cantú-Diaz C, Vázquez-Lara J, Villanueva-Cuellar MA, Alvarez y García C, Fraser R, Heller SR. (2007). Gestational diabetes: aetiology and management. Obst Gin Rep Georgiou H, Lappas M, Georgiou G, Marita A, Bryant V, Hiscock R, Permezel M, Khail Z, Metzger BE, Lowe LP, Dyer AR, Trimble ER, Chaovarindr U, Coustan DR, Hadden DR, Harris SB, Caulfield LE, Sugamori ME, Whalen EA, Henning B. (1997). The epidemiology of Hillier TA, Pedula KL, Schmidt MM, Mullen JA, Charles MA, Pettitt DJ. (2007). Childhood Hillier TA, Vesco KK, Pedula K,. Beil TL, Whitlock EP, Pettitt DJ. (2008). Screening for Hossein-Nezhad A, Maghbooli Z, Vassigh AR, Larijani B. (2007). Prevalence of gestational Huda SS, Brodie LE, Sattar N. (2010). Obesity in pregnancy: prevalence and metabolic Hunsberger M, Rosenberg KD, Donatelle RJ. (2010). Racial/ethnic disparities in gestational Huxley R, James WP, Barzi F, Patel JV, Lear SA, Suriyawongpaisal P, Janus E, Caterson I, Hyer SL, Shehata HA. (2005). Gestational diabetes mellitus. Curr Obstet Gynecol, No. 15, Metzger BE, Gabbe SG, Persson B, Buchanan TA, Catalano PA, Damm P, Dyer AR, Leiva A, consequences. Semin Fetal Neonatal Med, Apr, Vol.15, No. 2, pp.70-6. Diabetes Care, September, Vol. 30, No. 9, pp.2287-92. Task Force. Ann Intern Med, No 148, pp. 766-775. Gynecol, September, Vol. 46, No. 3, pp.:236-41. pregnancy. 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Spanish Group for the Study of the Impact of Carpenter and Coustan GD thresholds. Potential impact of American Diabetes Association (2000) criteria for diagnosis of gestational diabetes mellitus in Spain. Diabetologia, Vol. 48, No.6, pp.1135–41 Wrongdiagnosis.com, Statistics by Country for Gestational diabetes http://www.wrongdiagnosis.com/g/gestdiab/stats-country.htm?ktrack=kcplink (2011) 6 Sudan Pathophysiology of Gestational Diabetes 2National College for Medical and Technical Studies, Khartoum, Mohammed Chyad Al-Noaemi1 and Mohammed Helmy Faris Shalayel2 1Al-Yarmouk College, Khartoum, Mellitus: The Past, the Present and the Future It is just to remember that "Pathophysiology" refers to the study of alterations in normal body function (physiology and biochemistry) which result in disease. E.g. changes in the normal thyroid hormone level causes either hyper or hypothyroidism. Changes in insulin level as a decrease in its blood level or a decrease in its action will cause hyperglycemia and Scientists agreed that gestational diabetes mellitus (GDM) is a condition in which women without previously diagnosed diabetes exhibit high blood glucose levels during pregnancy. From our experience most women with GDM in the developing countries are not aware of the symptoms (i.e., the disease will be symptomless). While some of the women will have few symptoms and their GDM is most commonly diagnosed by routine blood examinations during pregnancy which detect inappropriate high level of glucose in their blood samples. GDM should be confirmed by doing fasting blood glucose and oral glucose tolerance test A decrease in insulin sensitivity (i.e. an increase in insulin resistance) is normally seen during pregnancy to spare the glucose for the fetus. This is attributed to the effects of placental hormones. In a few women the physiological changes during pregnancy result in impaired glucose tolerance which might develop diabetes mellitus (GDM). The prevalence of GDM ranges from 1% to 14% of all pregnancies depending on the population studied and the diagnostic tests used. Although the majority of women with GDM return to normal glucose tolerance immediately after delivery, a significant number will remain diabetic or To understand how gestational diabetes occurs, it is necessary to understand the normal physiological metabolism of glucose during pregnancy and the physiological changes mainly the endocrine changes during pregnancy in the feto-placental unit, which might Only about 1-2% of the pancreatic structure is endocrine tissues which are represented by the presence of 1-2 million islets of langerhans. These islets contain four main types of cells (A, B, D, and F cells). Insulin is secreted by B (beta) cells which constitute about 60-70% of the islets (OGTT), according to the WHO diagnostic criteria for diabetes. continue to have impaired glucose tolerance (IGT). explain the development of insulin resistance and GDM. 1. Introduction 1.1 Insulin finally diabetes mellitus. Xiong X, Saunders LD, Wang FL, Demianczuk NN. (2001).Gestational diabetes mellitus: prevalence, risk factors, maternal and infant outcomes. Int J Gynaecol Obstet, Dec;Vol.75, No.3, pp.221-8. ### Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future Mohammed Chyad Al-Noaemi1 and Mohammed Helmy Faris Shalayel2 1Al-Yarmouk College, Khartoum, 2National College for Medical and Technical Studies, Khartoum, Sudan #### 1. Introduction 90 Gestational Diabetes Xiong X, Saunders LD, Wang FL, Demianczuk NN. (2001).Gestational diabetes mellitus: Dec;Vol.75, No.3, pp.221-8. prevalence, risk factors, maternal and infant outcomes. Int J Gynaecol Obstet, It is just to remember that "Pathophysiology" refers to the study of alterations in normal body function (physiology and biochemistry) which result in disease. E.g. changes in the normal thyroid hormone level causes either hyper or hypothyroidism. Changes in insulin level as a decrease in its blood level or a decrease in its action will cause hyperglycemia and finally diabetes mellitus. Scientists agreed that gestational diabetes mellitus (GDM) is a condition in which women without previously diagnosed diabetes exhibit high blood glucose levels during pregnancy. From our experience most women with GDM in the developing countries are not aware of the symptoms (i.e., the disease will be symptomless). While some of the women will have few symptoms and their GDM is most commonly diagnosed by routine blood examinations during pregnancy which detect inappropriate high level of glucose in their blood samples. GDM should be confirmed by doing fasting blood glucose and oral glucose tolerance test (OGTT), according to the WHO diagnostic criteria for diabetes. A decrease in insulin sensitivity (i.e. an increase in insulin resistance) is normally seen during pregnancy to spare the glucose for the fetus. This is attributed to the effects of placental hormones. In a few women the physiological changes during pregnancy result in impaired glucose tolerance which might develop diabetes mellitus (GDM). The prevalence of GDM ranges from 1% to 14% of all pregnancies depending on the population studied and the diagnostic tests used. Although the majority of women with GDM return to normal glucose tolerance immediately after delivery, a significant number will remain diabetic or continue to have impaired glucose tolerance (IGT). To understand how gestational diabetes occurs, it is necessary to understand the normal physiological metabolism of glucose during pregnancy and the physiological changes mainly the endocrine changes during pregnancy in the feto-placental unit, which might explain the development of insulin resistance and GDM. #### 1.1 Insulin Only about 1-2% of the pancreatic structure is endocrine tissues which are represented by the presence of 1-2 million islets of langerhans. These islets contain four main types of cells (A, B, D, and F cells). Insulin is secreted by B (beta) cells which constitute about 60-70% of the islets Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future 93 which explain the diversity of insulin action, activating or inactivating certain enzymes to produce the desired effect on the cellular carbohydrate, fat, and protein metabolism (Zwick Within seconds after insulin binds with its membrane receptors, glucose transporters are moved to the cell membrane to facilitate glucose entry into the cell especially to the muscle The endocrinology of human pregnancy involves endocrine and metabolic changes that result from physiological alterations at the boundary between mother and fetus, known as the feto-placental unit (FPU), this interface is a major site of protein and steroid hormone production and secretion. Many of the endocrine and metabolic changes that occur during pregnancy can be directly attributed to hormonal signals originating from the FPU (Ganong, During early pregnancy, glucose tolerance is normal or slightly improved and peripheral (muscle) sensitivity to insulin and hepatic basal glucose production is normal (Catalano et al., 1991; Catalano et al., 1992; Catalano et al., 1993). These could be caused by the increased maternal estrogen and progesterone in early pregnancy which increase and promote pancreatic ß-cell hyperplasia (Expansion of beta-cell mass in response to pregnancy) causing an increased insulin release (Carr & Gabbe, 1998; Rieck & Kaestner, 2010). This explains the rapid increase in insulin level in early pregnancy, in response to insulin resistance. In the second and third trimester, the continuous increase in the feto-placental factors will decrease maternal insulin sensitivity, and this will stimulate mother cells to use sources of fuels (energy) other than glucose as free fatty acids, and this will increase supply of glucose to the fetus (Catalano et al., 1991; Catalano et al., 1992; Ryan & Enns, 1988). In the normal physiological conditions, the fetal blood glucose is 10-20% less than maternal blood glucose allowing the transport of glucose in the placenta to the fetal blood by the process of simple diffusion and facilitated transport. Therefore, glucose is the main fuel required by the developing fetus, whether as a source of energy for cellular metabolism or to provide energy During pregnancy, the insulin resistance of the whole body is increased to about three times In general, the resistance to insulin can be characterized as pre-receptor (insulin antibodies) as in autoimmune diseases, receptor (decreased number of receptors on the cell surface) as in obesity, or post-receptor (defects in the intracellular insulin signaling pathway). In pregnancy, the decreased insulin sensitivity is best characterized by a post-receptor defect resulting in the decreased ability of insulin to bring about SLC2A4 (GLUT4) mobilization from the interior of the cell to the cell surface (Catalano, 2010). This could be due to increase in the plasma levels of one or more of the pregnancy-associated hormones (Kühl, 1991; Although, pregnancy is associated with increase in the beta-cell mass and increase in insulin level throughout pregnancy but certain pregnant women are unable to up-regulate insulin production relative to the degree of insulin resistance, and consequently become et al., 2001; Pawson, 1995; Hans-Georg,1995; Perz & Torlińska, 2001). and adipose tissues (Guyton & Hall, 2006; Sherwood, 2010). 2003; Guyton & Hall, 2006; Monga & Baker, 2006). for the synthesis of protein, lipids, and glycogen. hyperglycemic, developing gestational diabetes (Kühl, 1991). the resistance in the non-pregnant state. Hornns, 1985). 2. Physiology of pregnancy cells. Insulin is a 51-amino acid polypeptide (small protein) hormone consist of A and B-chains connected together by disulphide bridges (Ganong, 2003; Guyton & Hall, 2006). #### 1.2 Insulin Receptor (IR) The IR is a large heterotetrameric, transmembrane glycoprotein, having a molecular weight of about 300,000. Each receptor consists of two alpha (α) subunit that lie outside the cell membrane and two beta (β) subunits that penetrate the cell membrane protruding into the cytoplasm connected together by disulphide bridges in a β-α- α-β configuration. IR is assembled from a single polypeptide pro-receptor, by dimerization, proteolytic cleavage, and glycosylation within the cytoplasm and Golgi apparatus, before trafficking of the mature receptor to the plasma membrane. These insulin receptors have also been designated recently as CD220 (cluster of differentiation 220) (Ganong, 2003; Guyton & Hall, 2006; Ward & Lawrence, 2009). #### 1.3 Insulin action Insulin has many metabolic functions such as enhancing cellular uptake of glucose, fatty acids, amino acids, and potassium ions. It also has an anabolic action by increasing cellular formation of glycogen, lipids, and protein. These physiological functions will be reversed if insulin action is decreased as seen with the increase in insulin resistance during pregnancy. The main function of insulin concerning gestational diabetes mellitus (GDM) is its action on glucose and lipid metabolism. #### 1.3.1 Insulin effect on lipid metabolism Normally insulin stimulates the synthesis and release of lipoprotein lipase from the endothelial cells of blood vessels causing lipolysis of triglycerides in the blood and release of free fatty acids (FFA). Insulin enhances the transport of FFA to the fatty cells (adipocytes) to be stored as lipids. Furthermore, insulin inhibits lipoprotein lipase in adipose cells preventing lipolysis. #### 1.3.2 Insulin effect on glucose metabolism Insulin enhances entrance of glucose to the cells through its action on the insulin receptors. Insulin receptor complex will stimulates mobilization of glucose carrier protein (GLUT- 4 transporter) from the interior of the cell to the plasma membrane which will transport glucose inside the cell by the process of facilitated diffusion. Furthermore, insulin-receptor complex will activates the storage of some glucose as glycogen while others will be metabolized into pyruvate and then fatty acids which are stored as triglycerides (fat) (Ganong, 2003; Guyton & Hall, 2006). #### 1.4 Insulin-receptor interaction To initiate insulin effects on target cells, it first binds with and activates a membrane receptor protein. [4] It is the activated receptor, not the insulin that causes the subsequent effects. The combination of insulin with the alpha subunits will induce autophosphorylation of the beta subunits which will activates a local tyrosine kinase [(phosphatidylinositol 3 kinase (PI3-K)] , which in turn begins a cascade of cell phosphorylation that increase or decrease the activity of enzymes, including insulin receptor substrates (IRSs). There are different types of IRSs (IRS-1, IRS-2, and IRS-3) which are expressed in different tissues which explain the diversity of insulin action, activating or inactivating certain enzymes to produce the desired effect on the cellular carbohydrate, fat, and protein metabolism (Zwick et al., 2001; Pawson, 1995; Hans-Georg,1995; Perz & Torlińska, 2001). Within seconds after insulin binds with its membrane receptors, glucose transporters are moved to the cell membrane to facilitate glucose entry into the cell especially to the muscle and adipose tissues (Guyton & Hall, 2006; Sherwood, 2010). #### 2. Physiology of pregnancy 92 Gestational Diabetes cells. Insulin is a 51-amino acid polypeptide (small protein) hormone consist of A and B-chains The IR is a large heterotetrameric, transmembrane glycoprotein, having a molecular weight of about 300,000. Each receptor consists of two alpha (α) subunit that lie outside the cell membrane and two beta (β) subunits that penetrate the cell membrane protruding into the cytoplasm connected together by disulphide bridges in a β-α- α-β configuration. IR is assembled from a single polypeptide pro-receptor, by dimerization, proteolytic cleavage, and glycosylation within the cytoplasm and Golgi apparatus, before trafficking of the mature receptor to the plasma membrane. These insulin receptors have also been designated recently as CD220 (cluster of differentiation 220) (Ganong, 2003; Guyton & Hall, 2006; Ward Insulin has many metabolic functions such as enhancing cellular uptake of glucose, fatty acids, amino acids, and potassium ions. It also has an anabolic action by increasing cellular formation of glycogen, lipids, and protein. These physiological functions will be reversed if insulin action is decreased as seen with the increase in insulin resistance during pregnancy. The main function of insulin concerning gestational diabetes mellitus (GDM) is its action on Normally insulin stimulates the synthesis and release of lipoprotein lipase from the endothelial cells of blood vessels causing lipolysis of triglycerides in the blood and release of free fatty acids (FFA). Insulin enhances the transport of FFA to the fatty cells (adipocytes) to be stored as lipids. Furthermore, insulin inhibits lipoprotein lipase in adipose cells Insulin enhances entrance of glucose to the cells through its action on the insulin receptors. Insulin receptor complex will stimulates mobilization of glucose carrier protein (GLUT- 4 transporter) from the interior of the cell to the plasma membrane which will transport glucose inside the cell by the process of facilitated diffusion. Furthermore, insulin-receptor complex will activates the storage of some glucose as glycogen while others will be metabolized into pyruvate and then fatty acids which are stored as triglycerides (fat) To initiate insulin effects on target cells, it first binds with and activates a membrane receptor protein. [4] It is the activated receptor, not the insulin that causes the subsequent effects. The combination of insulin with the alpha subunits will induce autophosphorylation of the beta subunits which will activates a local tyrosine kinase [(phosphatidylinositol 3 kinase (PI3-K)] , which in turn begins a cascade of cell phosphorylation that increase or decrease the activity of enzymes, including insulin receptor substrates (IRSs). There are different types of IRSs (IRS-1, IRS-2, and IRS-3) which are expressed in different tissues connected together by disulphide bridges (Ganong, 2003; Guyton & Hall, 2006). 1.2 Insulin Receptor (IR) & Lawrence, 2009). 1.3 Insulin action preventing lipolysis. glucose and lipid metabolism. 1.3.1 Insulin effect on lipid metabolism 1.3.2 Insulin effect on glucose metabolism (Ganong, 2003; Guyton & Hall, 2006). 1.4 Insulin-receptor interaction The endocrinology of human pregnancy involves endocrine and metabolic changes that result from physiological alterations at the boundary between mother and fetus, known as the feto-placental unit (FPU), this interface is a major site of protein and steroid hormone production and secretion. Many of the endocrine and metabolic changes that occur during pregnancy can be directly attributed to hormonal signals originating from the FPU (Ganong, 2003; Guyton & Hall, 2006; Monga & Baker, 2006). During early pregnancy, glucose tolerance is normal or slightly improved and peripheral (muscle) sensitivity to insulin and hepatic basal glucose production is normal (Catalano et al., 1991; Catalano et al., 1992; Catalano et al., 1993). These could be caused by the increased maternal estrogen and progesterone in early pregnancy which increase and promote pancreatic ß-cell hyperplasia (Expansion of beta-cell mass in response to pregnancy) causing an increased insulin release (Carr & Gabbe, 1998; Rieck & Kaestner, 2010). This explains the rapid increase in insulin level in early pregnancy, in response to insulin resistance. In the second and third trimester, the continuous increase in the feto-placental factors will decrease maternal insulin sensitivity, and this will stimulate mother cells to use sources of fuels (energy) other than glucose as free fatty acids, and this will increase supply of glucose to the fetus (Catalano et al., 1991; Catalano et al., 1992; Ryan & Enns, 1988). In the normal physiological conditions, the fetal blood glucose is 10-20% less than maternal blood glucose allowing the transport of glucose in the placenta to the fetal blood by the process of simple diffusion and facilitated transport. Therefore, glucose is the main fuel required by the developing fetus, whether as a source of energy for cellular metabolism or to provide energy for the synthesis of protein, lipids, and glycogen. During pregnancy, the insulin resistance of the whole body is increased to about three times the resistance in the non-pregnant state. In general, the resistance to insulin can be characterized as pre-receptor (insulin antibodies) as in autoimmune diseases, receptor (decreased number of receptors on the cell surface) as in obesity, or post-receptor (defects in the intracellular insulin signaling pathway). In pregnancy, the decreased insulin sensitivity is best characterized by a post-receptor defect resulting in the decreased ability of insulin to bring about SLC2A4 (GLUT4) mobilization from the interior of the cell to the cell surface (Catalano, 2010). This could be due to increase in the plasma levels of one or more of the pregnancy-associated hormones (Kühl, 1991; Hornns, 1985). Although, pregnancy is associated with increase in the beta-cell mass and increase in insulin level throughout pregnancy but certain pregnant women are unable to up-regulate insulin production relative to the degree of insulin resistance, and consequently become hyperglycemic, developing gestational diabetes (Kühl, 1991). Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future 95 Since 1984, professor Alwan AAS and collaborators have adopted the measurement of HbA1c levels as another index for follow-up of pregnant diabetic patients, and reported a significant relationship between elevated levels of HbA1c late in the third trimester and fetomaternal complications (Al-Dahwi et al., 1986; Al- Dahwi et al., 1987; Al-Dahwi et al., 1988; Al-Dahwi et al., 1989). Recently, the American Diabetic Association (2009) added that HbA1c ≥ 6.5% is another criterion for the diagnosis of diabetes (Nathan, 2009). Therefore we highly recommend the measurement of HbA1c during pregnancy, as an additional diagnostic criteria and to anticipate the maternal and fetal complications if it is abnormally The past; In the last century insulin resistance and the decrease in insulin sensitivity during pregnancy is mainly attributed to the increase in the levels of pregnancy-associated hormones as estrogen, progesterone, cortisol, and placental lactogen in the maternal circulation (Ryan, 1988; Hornns, 1985; Ahmed & Shalayel, 1999; Polderman et al., 1994; Barbour et al., 2002). Normally the insulin resistance of the whole body is increased to about three times that seen in the non-pregnant state (Kuhl, 1998; Catalano et al., 1999). The increased resistance is caused by post-insulin receptor events and is probably brought about by the cellular effects of the increased levels of one or all of the above hormones (Davis, 1990). As pregnancy progresses and the placenta grow larger, hormone production also increases and so does the level of insulin resistance. This process usually starts between 20 and 24 weeks of pregnancy. At birth, when the placenta is delivered, the hormone production stops and so does the condition, strongly suggesting that these hormones cause The placenta synthesizes pregnenolone and progesterone from cholesterol. Some of the progesterone enters the fetal circulation and provides the substrate for the formation of cortisol and corticosterone in the fetal adrenal glands. Some of the pregnenolone enters the fetus and, along with pregnenolone synthesized in the fetal liver, is the substrate for the formation of dehydroepiandrosterone sulfate (DHEAS) and 16-hydroxydehydroepiandrosterone sulfate (16-OHDHEAS) in the fetal adrenal. Some 16-hydroxylation also occurs in the fetal liver. DHEAS and 16-OHDHEAS are transported back to the placenta, where DHEAS forms estradiol and 16-OHDHEAS forms estriol. The principal estrogen formed is estriol, and since fetal 16-OHDHEAS is the principal substrate for the estrogens, the urinary estriol excretion of the mother can be monitored as an index of 4.1.2 Diabetic action of steroid hormones (cortisol, estrogen, and progesterone) These hormones are increased steadily with the advance of pregnancy. The anti-insulin action of these hormones is a well known fact since the last century (Ryan & Enns, 1988; 3.2 Glycosylated hemoglobin (HbA1c) as a diagnostic test for GDM In the pathophysiology of GDM we have to consider two main points. 4.1 The role of feto-placental unit in the development of GDM GDM (Ryan & Enns, 1988; Kuhl, 1975; Buchanan & Xiang, 2005). elevated. 4. Pathophysiology of GDM 4.1.1 Feto-placental unit the state of the fetus (Ganong, 2003). 4.1 Role of feto-placental unit in GDM. 4.2 Role of the adipose tissue in GDM. #### 3. Diagnosis of gestational diabetes mellitus Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance resulting in hyperglycemia of variable severity, with onset or first recognition during pregnancy. It does not exclude the possibility that unrecognized glucose intolerance may have antedated but has been previously unrecognized (Metzger, 1991; Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycemia World Health Organization [WHO], 2006). Women who become pregnant and who are known to have diabetes mellitus which antedates pregnancy do not have gestational diabetes but have "diabetes mellitus and pregnancy" and should be treated accordingly before, during, and after the pregnancy (WHO, 2006). Gestational diabetes generally has few symptoms and it is most commonly diagnosed by screening during pregnancy. Diagnostic tests detect inappropriately high levels of glucose in blood samples. #### 3.1 WHO diagnostic criteria for hyperglycemia and GDM (2006) In the early part of pregnancy (e.g. first trimester and first half of second trimester) fasting and postprandial glucose concentrations are normally lower than in normal, non-pregnant women. Elevated fasting or postprandial plasma glucose levels at this time in pregnancy may well reflect the presence of diabetes which has antedated pregnancy. The occurrence of higher than usual plasma glucose levels at this time in pregnancy mandates careful management and may be an indication for carrying out an oral glucose tolerance test (OGTT). Nevertheless, normal glucose tolerance in the early part of pregnancy does not by itself establish that gestational diabetes will not develop later. It may be appropriate to screen pregnant women belonging to high-risk populations during the first trimester of pregnancy in order to detect previously undiagnosed diabetes mellitus. Formal systematic testing for gestational diabetes is usually done between 24 and 28 weeks of gestation. To determine if gestational diabetes is present in pregnant women, a standard OGTT should be performed after overnight fasting (8-14 hours) by giving 75 g anhydrous glucose in 250- 300 ml water. Plasma glucose is measured fasting and after 2 hours. Pregnant women who meet WHO criteria for diabetes mellitus or impaired glucose tolerance (IGT) are classified as having GDM. After the pregnancy ends, the woman should be re-classified as having either diabetes mellitus, or IGT, or normal glucose tolerance based on the results of a 75 g OGTT six weeks or more after delivery. The following table (table 1) summarizes the 2006 WHO recommendations for the diagnostic criteria for diabetes and intermediate hyperglycemia (WHO, 2006). \* Venous plasma 2-h after ingestion of 75gm oral glucose load (OGTT) Table 1. Diagnostic criteria for diabetes and intermediate hyperglycemia #### 3.2 Glycosylated hemoglobin (HbA1c) as a diagnostic test for GDM Since 1984, professor Alwan AAS and collaborators have adopted the measurement of HbA1c levels as another index for follow-up of pregnant diabetic patients, and reported a significant relationship between elevated levels of HbA1c late in the third trimester and fetomaternal complications (Al-Dahwi et al., 1986; Al- Dahwi et al., 1987; Al-Dahwi et al., 1988; Al-Dahwi et al., 1989). Recently, the American Diabetic Association (2009) added that HbA1c ≥ 6.5% is another criterion for the diagnosis of diabetes (Nathan, 2009). Therefore we highly recommend the measurement of HbA1c during pregnancy, as an additional diagnostic criteria and to anticipate the maternal and fetal complications if it is abnormally elevated. ### 4. Pathophysiology of GDM 94 Gestational Diabetes Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance resulting in hyperglycemia of variable severity, with onset or first recognition during pregnancy. It does not exclude the possibility that unrecognized glucose intolerance may have antedated but has been previously unrecognized (Metzger, 1991; Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycemia World Health Organization [WHO], 2006). Women who become pregnant and who are known to have diabetes mellitus which antedates pregnancy do not have gestational diabetes but have "diabetes mellitus and pregnancy" and should be treated accordingly before, during, and after the pregnancy Gestational diabetes generally has few symptoms and it is most commonly diagnosed by screening during pregnancy. Diagnostic tests detect inappropriately high levels of glucose in In the early part of pregnancy (e.g. first trimester and first half of second trimester) fasting and postprandial glucose concentrations are normally lower than in normal, non-pregnant women. Elevated fasting or postprandial plasma glucose levels at this time in pregnancy may well reflect the presence of diabetes which has antedated pregnancy. The occurrence of higher than usual plasma glucose levels at this time in pregnancy mandates careful management and may be an indication for carrying out an oral glucose tolerance test (OGTT). Nevertheless, normal glucose tolerance in the early part of pregnancy does not by It may be appropriate to screen pregnant women belonging to high-risk populations during the first trimester of pregnancy in order to detect previously undiagnosed diabetes mellitus. Formal systematic testing for gestational diabetes is usually done between 24 and 28 weeks of gestation. To determine if gestational diabetes is present in pregnant women, a standard OGTT should be performed after overnight fasting (8-14 hours) by giving 75 g anhydrous glucose in 250- 300 ml water. Plasma glucose is measured fasting and after 2 hours. Pregnant women who meet WHO criteria for diabetes mellitus or impaired glucose tolerance (IGT) are classified as having GDM. After the pregnancy ends, the woman should be re-classified as having either diabetes mellitus, or IGT, or normal glucose tolerance based on the results of a 75 g OGTT The following table (table 1) summarizes the 2006 WHO recommendations for the diagnostic criteria for diabetes and intermediate hyperglycemia (WHO, 2006). 2–h plasma glucose\* ≥7.8 and <11.1mmol/l (140mg/dl and 00mg/dl) Fasting plasma glucose 6.1 to 6.9 mmol/L (110mg/dl to 125 mg/dl) Table 1. Diagnostic criteria for diabetes and intermediate hyperglycemia Fasting plasma glucose ≥7.0mmol/l (126mg/dl), or 2–h plasma glucose \* ≥11.1mmol/l (200mg/dl) Fasting plasma glucose <7.0mmol/l (126mg/dl) 2-h Plasma glucose\* < 7.8 mmol/dl (140mg/dl) \* Venous plasma 2-h after ingestion of 75gm oral glucose load (OGTT) 3.1 WHO diagnostic criteria for hyperglycemia and GDM (2006) itself establish that gestational diabetes will not develop later. six weeks or more after delivery. Impaired Glucose Tolerance (IGT) Impaired Fasting Glucose (IFG) Diabetes 3. Diagnosis of gestational diabetes mellitus (WHO, 2006). blood samples. In the pathophysiology of GDM we have to consider two main points. 4.1 Role of feto-placental unit in GDM. 4.2 Role of the adipose tissue in GDM. #### 4.1 The role of feto-placental unit in the development of GDM The past; In the last century insulin resistance and the decrease in insulin sensitivity during pregnancy is mainly attributed to the increase in the levels of pregnancy-associated hormones as estrogen, progesterone, cortisol, and placental lactogen in the maternal circulation (Ryan, 1988; Hornns, 1985; Ahmed & Shalayel, 1999; Polderman et al., 1994; Barbour et al., 2002). Normally the insulin resistance of the whole body is increased to about three times that seen in the non-pregnant state (Kuhl, 1998; Catalano et al., 1999). The increased resistance is caused by post-insulin receptor events and is probably brought about by the cellular effects of the increased levels of one or all of the above hormones (Davis, 1990). As pregnancy progresses and the placenta grow larger, hormone production also increases and so does the level of insulin resistance. This process usually starts between 20 and 24 weeks of pregnancy. At birth, when the placenta is delivered, the hormone production stops and so does the condition, strongly suggesting that these hormones cause GDM (Ryan & Enns, 1988; Kuhl, 1975; Buchanan & Xiang, 2005). #### 4.1.1 Feto-placental unit The placenta synthesizes pregnenolone and progesterone from cholesterol. Some of the progesterone enters the fetal circulation and provides the substrate for the formation of cortisol and corticosterone in the fetal adrenal glands. Some of the pregnenolone enters the fetus and, along with pregnenolone synthesized in the fetal liver, is the substrate for the formation of dehydroepiandrosterone sulfate (DHEAS) and 16-hydroxydehydroepiandrosterone sulfate (16-OHDHEAS) in the fetal adrenal. Some 16-hydroxylation also occurs in the fetal liver. DHEAS and 16-OHDHEAS are transported back to the placenta, where DHEAS forms estradiol and 16-OHDHEAS forms estriol. The principal estrogen formed is estriol, and since fetal 16-OHDHEAS is the principal substrate for the estrogens, the urinary estriol excretion of the mother can be monitored as an index of the state of the fetus (Ganong, 2003). #### 4.1.2 Diabetic action of steroid hormones (cortisol, estrogen, and progesterone) These hormones are increased steadily with the advance of pregnancy. The anti-insulin action of these hormones is a well known fact since the last century (Ryan & Enns, 1988; Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future 97 PGH is the product of the GH-V gene specifically expressed in the syncytiotrophoblast layer of the human placenta. PGH (20-kDa HGH-V) differs from pituitary growth hormone by 13 amino acids. It has high somatogenic and low lactogenic activities (Lacroix et al., 2002). PGH is produced by the placenta and found predominantly in the maternal circulation. It progressively replaces pituitary growth hormone (hGH) in the human maternal circulation from mid-gestation onwards, peaking towards term (Chellakooty et al., 2004). PGH appears to be an important potential regulator of maternal insulin resistance in human pregnancy and may influence fetal growth both by modifying substrate availability and through Barbour et al (2004) demonstrated a unique mechanism of insulin resistance in non-pregnant transgenic mice and suggested that human placental growth hormone (hPGH) may contribute to the insulin resistance of normal pregnancy secondary to its effect on p85 Nevertheless, in a recent experimental study by Vickers and Gilmour (2009), it was demonstrated that rats treated with HGH enhanced insulin sensitivity and suggested that It seems that there is a controversy about the involvement of HGH with insulin resistance In conclusion, considering the previously discussed hormones, HPL is considered as the main diabetogenic hormone synthesized and released from the feto-placental unit. But during pregnancy, there is another maternal hormone which is involved in insulin Prolactin level begins to rise at 5-8 weeks of gestation, followed by a progressively increase in its level as pregnancy advances (Shalayel et al., 2010; Glass & Kase, 1984). The increase in prolactin secretion is due the increase in the size and number of maternal pituitary lactotrophs (Kuhl et al., 1985) and its secretion from the uterine decidual cells seems to be Shalayel et al (2010) revealed that prolactin increases progressively as pregnancy advances, reaching a peak in the third trimester when many pregnant ladies may develop gestational diabetes due to the state of insulin resistance which may occur although there is no evidence that prolactin may be directly incorporated with the pathogenesis of glucose intolerance in pregnancy. A decline in insulin secretion may lead to a decline in prolactin since insulin There were no significant differences in the level of plasma prolactin in normal or diabetic pregnancies; in fact its level might be lower in the pregnancies with GDM (Guyton & Hall, 2006). Therefore, prolactin might have no effect on glucose intolerance during pregnancy Historically, placental hormones have been considered as the primary mediators of insulin resistance during gestation. Over the past decade, adipose tissue has been shown to produce numerous factors (adipocytokines), most of them act as hormones. These adipocyte-derived hormones have been implicated in the regulation of maternal metabolism and gestational insulin resistance. Adipocytokines, including leptin, adiponectin, tumor necrosis factor stimulated by progesterone and insulin (Ahmed & Shalayel, 1999; Davis, 1990). stimulates both acute secretion and de novo synthesis of decidual prolactin. 4.2 The role of adipose tissue in the development of GDM 4.1.4 Placental growth hormone (PGH) HGH have an antidiabetic action. resistance which is prolactin. (Milasinovic et al., 1997). 4.2.1 Adipocytokines and GDM. 4.1.5 Prolactin paracrine actions in the placental bed (McIntyre et al., 2009). expression and its interference with PI 3-kinase activity in skeletal muscle. Barbour et al., 2002; Barbieri, 1999; Kirwan et al., 2002; Shalayel et al., 2010). The fetus and the placenta interact in the formation of these steroid hormones. It has been shown that the increase in cortisol level during pregnancy is considered as the main hormone which cause decrease in glucose tolerance in normal pregnancy (Hornns, 1985; Ahmed & Shalayel, 1999). While others considered that estrogen and progesterone which are elevated steadily during pregnancy are the main hormones which influence beta cell function in early pregnancy and insulin resistance especially in late pregnancy (Ryan & Enns, 1988; Polderman et al., 1994; Glass & Kase, 1984). Although some scientists have considered that human chorionic gonadotropin (HCG) may participates in the development of insulin resistance during pregnancy as it shows higher level in women with GDM in comparison with normal pregnancies (Merviel et al., 2001). But, as we know from the normal changes during pregnancy, the main increase of HCG occurs during the first trimester, and this period is associated with an increase in insulin sensitivity and improvement of glucose tolerance. Therefore, we consider that HCG has no direct role as a cause of GDM. #### 4.1.3 Human placental lactogen (hPL), [human chorionic somatomammotropin (hCS)] It is a single polypeptide chain held together by disulphide bonds. It is about 96% similar to human growth hormone (HGH), but has only 3% of HGH activity. Its half life is short (15minutes); hence its appeal as an index of placental problems (Glass & Kase, 1984). HPL, which is the product of the HPL-A and HPL-B genes, is secreted into both the maternal and fetal circulations after the sixth week of pregnancy (Handwerger & Freemark, 2000). The level of HPL in the maternal circulation is correlated with fetal and placental weight, plateauing in the last 4 weeks of pregnancy. Therefore, measurement of HPL levels is used as a screening test for fetal distress and neonatal asphyxia (Glass & Kase, 1984; Letchworth & Chard, 1972). #### 4.1.3.1 Physiologicalfunction of HPL During pregnancy the maternal level of HPL can be altered by changing the circulating level of glucose. HPL is elevated with hypoglycemia and depressed with hyperglycemia (Barbour et al., 2002; Kuhl, 1998). The metabolic role of HPL is to mobilize lipids and free fatty acids. In the fed state, there is abundant glucose available, leading to increased insulin level, lipogenesis, and glucose utilization. This is associated with decreased gluconeogenesis, and a decrease in the circulating free fatty acid levels, as the free fatty acids are utilized in the process of lipogenesis to deposit storage packets of triglycerides (Glass & Kase, 1984; Kim & Feling, 1971). #### 4.1.3.2 Diabetogenic action of HPL In the second half of pregnancy, HPL level rises approximately 10 folds. HPL stimulates lipolysis leading to an increase in circulating free fatty acids in order to provide a different fuel for the mother so that glucose and amino acids can be conserved for the fetus. The increase in free fatty acid levels, in turn directly interferes with insulin-directed entry of glucose into cells. Therefore, HPL is considered as a potent antagonist to insulin action during pregnancy (Glass & Kase, 1984; Mills et al., 1985). Furthermore, HPL and placental growth hormone act in concert in the mother to stimulate insulin-like growth factor (IGF) production and modulate intermediary metabolism, resulting in an increase in the availability of glucose and amino acids to the fetus (Handwerger & Freemark, 2000). #### 4.1.4 Placental growth hormone (PGH) 96 Gestational Diabetes Barbour et al., 2002; Barbieri, 1999; Kirwan et al., 2002; Shalayel et al., 2010). The fetus and the placenta interact in the formation of these steroid hormones. It has been shown that the increase in cortisol level during pregnancy is considered as the main hormone which cause decrease in glucose tolerance in normal pregnancy (Hornns, 1985; Ahmed & Shalayel, 1999). While others considered that estrogen and progesterone which are elevated steadily during pregnancy are the main hormones which influence beta cell function in early pregnancy and insulin resistance especially in late pregnancy (Ryan & Enns, 1988; Polderman et al., Although some scientists have considered that human chorionic gonadotropin (HCG) may participates in the development of insulin resistance during pregnancy as it shows higher level in women with GDM in comparison with normal pregnancies (Merviel et al., 2001). But, as we know from the normal changes during pregnancy, the main increase of HCG occurs during the first trimester, and this period is associated with an increase in insulin sensitivity and improvement of glucose tolerance. Therefore, we consider that HCG has no 4.1.3 Human placental lactogen (hPL), [human chorionic somatomammotropin (hCS)] It is a single polypeptide chain held together by disulphide bonds. It is about 96% similar to human growth hormone (HGH), but has only 3% of HGH activity. Its half life is short (15minutes); hence its appeal as an index of placental problems (Glass & Kase, 1984). HPL, which is the product of the HPL-A and HPL-B genes, is secreted into both the maternal and fetal circulations after the sixth week of pregnancy (Handwerger & Freemark, 2000). The level of HPL in the maternal circulation is correlated with fetal and placental weight, plateauing in the last 4 weeks of pregnancy. Therefore, measurement of HPL levels is used as a screening test for fetal distress and neonatal asphyxia (Glass & Kase, 1984; Letchworth During pregnancy the maternal level of HPL can be altered by changing the circulating level of glucose. HPL is elevated with hypoglycemia and depressed with hyperglycemia (Barbour et al., 2002; Kuhl, 1998). The metabolic role of HPL is to mobilize lipids and free fatty acids. In the fed state, there is abundant glucose available, leading to increased insulin level, lipogenesis, and glucose utilization. This is associated with decreased gluconeogenesis, and a decrease in the circulating free fatty acid levels, as the free fatty acids are utilized in the process of lipogenesis to deposit storage packets of triglycerides (Glass & Kase, 1984; Kim & In the second half of pregnancy, HPL level rises approximately 10 folds. HPL stimulates lipolysis leading to an increase in circulating free fatty acids in order to provide a different fuel for the mother so that glucose and amino acids can be conserved for the fetus. The increase in free fatty acid levels, in turn directly interferes with insulin-directed entry of glucose into cells. Therefore, HPL is considered as a potent antagonist to insulin action during pregnancy (Glass & Kase, 1984; Mills et al., 1985). Furthermore, HPL and placental growth hormone act in concert in the mother to stimulate insulin-like growth factor (IGF) production and modulate intermediary metabolism, resulting in an increase in the availability of glucose and amino acids to the fetus (Handwerger & Freemark, 2000). 1994; Glass & Kase, 1984). direct role as a cause of GDM. 4.1.3.1 Physiologicalfunction of HPL 4.1.3.2 Diabetogenic action of HPL & Chard, 1972). Feling, 1971). PGH is the product of the GH-V gene specifically expressed in the syncytiotrophoblast layer of the human placenta. PGH (20-kDa HGH-V) differs from pituitary growth hormone by 13 amino acids. It has high somatogenic and low lactogenic activities (Lacroix et al., 2002). PGH is produced by the placenta and found predominantly in the maternal circulation. It progressively replaces pituitary growth hormone (hGH) in the human maternal circulation from mid-gestation onwards, peaking towards term (Chellakooty et al., 2004). PGH appears to be an important potential regulator of maternal insulin resistance in human pregnancy and may influence fetal growth both by modifying substrate availability and through paracrine actions in the placental bed (McIntyre et al., 2009). Barbour et al (2004) demonstrated a unique mechanism of insulin resistance in non-pregnant transgenic mice and suggested that human placental growth hormone (hPGH) may contribute to the insulin resistance of normal pregnancy secondary to its effect on p85 expression and its interference with PI 3-kinase activity in skeletal muscle. Nevertheless, in a recent experimental study by Vickers and Gilmour (2009), it was demonstrated that rats treated with HGH enhanced insulin sensitivity and suggested that HGH have an antidiabetic action. It seems that there is a controversy about the involvement of HGH with insulin resistance and GDM. In conclusion, considering the previously discussed hormones, HPL is considered as the main diabetogenic hormone synthesized and released from the feto-placental unit. But during pregnancy, there is another maternal hormone which is involved in insulin resistance which is prolactin. #### 4.1.5 Prolactin Prolactin level begins to rise at 5-8 weeks of gestation, followed by a progressively increase in its level as pregnancy advances (Shalayel et al., 2010; Glass & Kase, 1984). The increase in prolactin secretion is due the increase in the size and number of maternal pituitary lactotrophs (Kuhl et al., 1985) and its secretion from the uterine decidual cells seems to be stimulated by progesterone and insulin (Ahmed & Shalayel, 1999; Davis, 1990). Shalayel et al (2010) revealed that prolactin increases progressively as pregnancy advances, reaching a peak in the third trimester when many pregnant ladies may develop gestational diabetes due to the state of insulin resistance which may occur although there is no evidence that prolactin may be directly incorporated with the pathogenesis of glucose intolerance in pregnancy. A decline in insulin secretion may lead to a decline in prolactin since insulin stimulates both acute secretion and de novo synthesis of decidual prolactin. There were no significant differences in the level of plasma prolactin in normal or diabetic pregnancies; in fact its level might be lower in the pregnancies with GDM (Guyton & Hall, 2006). Therefore, prolactin might have no effect on glucose intolerance during pregnancy (Milasinovic et al., 1997). #### 4.2 The role of adipose tissue in the development of GDM 4.2.1 Adipocytokines Historically, placental hormones have been considered as the primary mediators of insulin resistance during gestation. Over the past decade, adipose tissue has been shown to produce numerous factors (adipocytokines), most of them act as hormones. These adipocyte-derived hormones have been implicated in the regulation of maternal metabolism and gestational insulin resistance. Adipocytokines, including leptin, adiponectin, tumor necrosis factor Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future 99 ii. Effect of adiponectin on glucose metabolism: It has been reported that an acute increase in circulating adiponectin levels triggers a transient decrease in basal glucose levels by inhibiting both the expression of hepatic gluconeogenic enzymes and the rate of endogenous glucose production in both wild-type mice and a type 2 diabetic mouse model (Berg et al., 2001; Combs et al., 2001). Furthermore, Kubota et al (2002) provided the first direct evidence that adiponectin plays a protective role against insulin resistance by generating adiponectin-deficient mice. Adiponectin improves insulin resistance and glucose tolerance in both heterozygous (+/-) and homozygous (-/-) iii. Effects of adiponectin on lipid metabolism: Adiponectin activates AMP-Kinase (AMPK) and peroxisome proliferator-actvated receptor (PPAR α) in the liver and muscle, thereby stimulating fatty-acid oxidation and decreasing tissue TG content in the liver and muscle (Thamer et al., 2002; Fruebis et al., 2001; Yamauchi et al., 2003a). Furthermore, adiponectin decreases lipid synthesis and glucose production in the liver and causes a decrease in glucose and fatty acid concentration in the blood (Meier & Many studies have shown that plasma adiponectin concentration is negatively correlated with body mass index (BMI) and accordingly, lower in obese than in lean subjects (Ouchi et al., 1999; Hotta et al., 2000; Pena et al., 2009). Furthermore, scientists extended these finding by demonstrating that plasma adiponectin concentrations are inversely related to percentage of body fat, a direct measure of adiposity. And that is consistent across different ethnic groups. These results thus confirm that adiponectin is the main adipose-specific protein known to date that despite its excusive production in white adipose tissue, is negatively regulated in obesity (Hu et al., 1996; Weyer et al., 2001; Statnick et al., 2000). These scientific data suggest that adiponectin may have a role in the pathogenesis of obesity. As obesity is a predisposing factor for the development of diabetes mellitus in general and GDM in specific, this might explain the indirect involvement of a decreased adiponectin in the pathogenesis of diabetes mellitus. It has also been shown that in pregnant women there is a decrease in adiponectin which is associated with an increase in insulin resistance in the third trimester and a further decrease in women with IGT or GDM compared to pregnant women with normal glucose tolerance test, even after adjustment for varying degree of adiposity. Hypoadiponectinemia was also found in women with GDM independently of 2002). adiponectin-deficient mice. Gressner, 2004; Yool et al., 2006). 4.2.1.1.2 Adiponectin and the pathophysiology of obesity and diabetes addition to that, adiponectin has indirect insulin-sensitizing effect by decreasing tissue triglyceride (TG) content (Shulman, 2000). It is well known that tissue triglycerides interfere with insulin-stimulated phosphatidylinositol (PI) 3-kinase activation and subsequent glucose transporter 4 (GLUT-4) translocations and glucose uptake, leading to insulin resistance (Shulman, 2000; Godoy-Matos et al., 2010). Thus, decreased tissue TG content in muscle may contribute to the improved insulin signal transduction. Interestingly, in skeletal muscle, adiponectin increases expression of molecules involved in fatty acid transport such as CD36, in combustion of fatty-acid such as acylcoenzyme-A oxidase, and in energy dissipation such as uncoupling protein 2. These changes led to decreased tissue TG content in skeletal muscle whether in experimental animals or in human (Godoy-Matos et al., 2010; Yamauchi et al., 2001; Thamer et al., alpha, interleukin-6, as well as the newly discovered resistin, visfatin, and apelin, are also known to be produced within the intrauterine environment (Catalano, 2010; Briana & Malamitsi-Puchner, 2009; Henry & Clarke, 2008). Although human placental lactogen has often been cited as the cause of the decreased insulin sensitivity in pregnancy, because of its production from the placenta and increasing concentrations with advancing gestation as described previously (Ryan & Enns, 1988), more recently the role of adipocytokines and elevated lipid concentrations in pregnancy have been correlated with the longitudinal changes in insulin sensitivity in non-pregnant women (Hotamisligil et al., 1994) as well as in pregnant women (Kirwan et al., 2002; Hotamisligil et al., 1996). Evidence suggests that one or more of these adipokines (as TNF-α and leptin) could impair insulin signaling and cause insulin resistance (Briana & Malamitsi-Puchner, 2009; Xiang et al., 1999). TNF-α in specific has a potential effect in decreasing insulin sensitivity (Catalano, 2010). While other adipocytokines might increase insulin sensitivity as adiponectin which has been shown to be decreased especially in late pregnancy (Al-Noaemi & Shalayel, 2009). #### 4.2.1.1 Adiponectin Adiponectin is a novel adipocyte secreting protein hormone discovered in 1995/1996 (Scherer et al., 1995; Nakano et al., 1996; Maeda et al., 1996; Hu et al., 1996; Tsao et al., 2002). Adiponectin is abundant in the circulation of humans, with plasma levels in the microgram per ml range, thus accounting for approximately 0.01% of total plasma protein. Chen et al. (2006) reported that the human placenta produces and secretes adiponectin and that adiponectin and its receptors are differentially regulated by cytokines and their expression altered in women with gestational diabetes mellitus, suggesting that adiponectin may play a role in adapting energy metabolism at the materno-fetal interface. #### 4.2.1.1.1 Functions of Adiponectin Although the physiological role of adiponectin is not yet fully determined, but it has been shown that there are a variety of physiological functions induced by adiponectin such as: 4.2.1.1.1.A General functions 4.2.1.1.1.B Specific anti-diabetic functions such as its actions on glucose and lipid metabolism i. Effects of adiponectin on insulin and glucose metabolism: Adiponectin has insulinsensitizing effects. Replenishment of a physiological dose of recombinant adiponectin to lipoatrophic mice significantly ameliorated insulin resistance. Moreover, insulin resistance in lipoatrophic mice was completely reversed by the combination of physiological doses of adiponectin and leptin (Hotta et al., 2000; Berg et al., 2001). In alpha, interleukin-6, as well as the newly discovered resistin, visfatin, and apelin, are also known to be produced within the intrauterine environment (Catalano, 2010; Briana & Although human placental lactogen has often been cited as the cause of the decreased insulin sensitivity in pregnancy, because of its production from the placenta and increasing concentrations with advancing gestation as described previously (Ryan & Enns, 1988), more recently the role of adipocytokines and elevated lipid concentrations in pregnancy have been correlated with the longitudinal changes in insulin sensitivity in non-pregnant women (Hotamisligil et al., 1994) as well as in pregnant women (Kirwan et al., 2002; Hotamisligil et al., 1996). Evidence suggests that one or more of these adipokines (as TNF-α and leptin) could impair insulin signaling and cause insulin resistance (Briana & Malamitsi-Puchner, 2009; Xiang et al., 1999). TNF-α in specific has a potential effect in decreasing insulin sensitivity (Catalano, 2010). While other adipocytokines might increase insulin sensitivity as adiponectin which has been shown to be decreased especially in late pregnancy (Al-Noaemi & Shalayel, 2009). Adiponectin is a novel adipocyte secreting protein hormone discovered in 1995/1996 (Scherer et al., 1995; Nakano et al., 1996; Maeda et al., 1996; Hu et al., 1996; Tsao et al., 2002). Adiponectin is abundant in the circulation of humans, with plasma levels in the microgram Chen et al. (2006) reported that the human placenta produces and secretes adiponectin and that adiponectin and its receptors are differentially regulated by cytokines and their expression altered in women with gestational diabetes mellitus, suggesting that adiponectin Although the physiological role of adiponectin is not yet fully determined, but it has been shown that there are a variety of physiological functions induced by adiponectin such as: i. Anti-atherosclerotic action: By inhibiting lipid-laden foam cell formation (Ouchi et al., 2001), and inhibiting the inflammatory adipokine, tumor necrosis factor-α (TNF-α) ii. Anti-inflammatory action: By inhibiting the phagocytic activity of macrophages and inhibiting the production of TNF-α by these macrophages (Yokota et al., 2000). iii. Anti-oxidant action: By stimulating the endothelial cells to produce nitric oxide (NO) iv. Anti-tumor action: There is a significant inverse association of adiponectin with postmenopausal endometrial and breast cancer (Mantzoros et al., 2004; Petridou et al., 4.2.1.1.1.B Specific anti-diabetic functions such as its actions on glucose and lipid i. Effects of adiponectin on insulin and glucose metabolism: Adiponectin has insulinsensitizing effects. Replenishment of a physiological dose of recombinant adiponectin to lipoatrophic mice significantly ameliorated insulin resistance. Moreover, insulin resistance in lipoatrophic mice was completely reversed by the combination of physiological doses of adiponectin and leptin (Hotta et al., 2000; Berg et al., 2001). In per ml range, thus accounting for approximately 0.01% of total plasma protein. may play a role in adapting energy metabolism at the materno-fetal interface. Malamitsi-Puchner, 2009; Henry & Clarke, 2008). 4.2.1.1 Adiponectin 4.2.1.1.1 Functions of Adiponectin 4.2.1.1.1.A General functions (Ouchi et al., 2000). (Ouchi et al., 1999). 2003). metabolism addition to that, adiponectin has indirect insulin-sensitizing effect by decreasing tissue triglyceride (TG) content (Shulman, 2000). It is well known that tissue triglycerides interfere with insulin-stimulated phosphatidylinositol (PI) 3-kinase activation and subsequent glucose transporter 4 (GLUT-4) translocations and glucose uptake, leading to insulin resistance (Shulman, 2000; Godoy-Matos et al., 2010). Thus, decreased tissue TG content in muscle may contribute to the improved insulin signal transduction. Interestingly, in skeletal muscle, adiponectin increases expression of molecules involved in fatty acid transport such as CD36, in combustion of fatty-acid such as acylcoenzyme-A oxidase, and in energy dissipation such as uncoupling protein 2. These changes led to decreased tissue TG content in skeletal muscle whether in experimental animals or in human (Godoy-Matos et al., 2010; Yamauchi et al., 2001; Thamer et al., 2002). #### 4.2.1.1.2 Adiponectin and the pathophysiology of obesity and diabetes Many studies have shown that plasma adiponectin concentration is negatively correlated with body mass index (BMI) and accordingly, lower in obese than in lean subjects (Ouchi et al., 1999; Hotta et al., 2000; Pena et al., 2009). Furthermore, scientists extended these finding by demonstrating that plasma adiponectin concentrations are inversely related to percentage of body fat, a direct measure of adiposity. And that is consistent across different ethnic groups. These results thus confirm that adiponectin is the main adipose-specific protein known to date that despite its excusive production in white adipose tissue, is negatively regulated in obesity (Hu et al., 1996; Weyer et al., 2001; Statnick et al., 2000). These scientific data suggest that adiponectin may have a role in the pathogenesis of obesity. As obesity is a predisposing factor for the development of diabetes mellitus in general and GDM in specific, this might explain the indirect involvement of a decreased adiponectin in the pathogenesis of diabetes mellitus. It has also been shown that in pregnant women there is a decrease in adiponectin which is associated with an increase in insulin resistance in the third trimester and a further decrease in women with IGT or GDM compared to pregnant women with normal glucose tolerance test, even after adjustment for varying degree of adiposity. Hypoadiponectinemia was also found in women with GDM independently of Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future 101 circulating TNF-α during late pregnancy is mainly due to placental secretion. These findings may also help to explain the rapid reversal of insulin resistance after delivery, since maternal levels of TNF-α decrease substantially after delivery of the placenta (Kirwan et al., In-vitro studies have described a direct role for TNF-α in the pathophysiology of insulin resistance. TNF-α downregulates insulin receptor signaling in cultured adipocytes (Catalano, 2010), hepatocytes (Feinstein et al., 1993), and skeletal muscle (del Aguila et al., 1999). TNF-α activates a pathway that increases sphingomyelinase and ceramides and appears to interfere with insulin receptor autophosphorylation (Catalano, 2010). Also it has been shown that TNFα promotes serine phosphorylation of insulin receptor substrate (IRS)-1, thus impairing its association with the insulin receptor (Rui et al., 2001). In pregnancy, there is an evidence that insulin receptor and IRS-1 tyrosine phosphorylation are impaired, and serine phosphorylation is increased in late gestation in skeletal muscle (Friedman et al., 1999; Shao et al., 2000). Therefore, it seems that elevated levels of TNF-α in late gestation could attenuate insulin Barbour et al (2007) demonstrated that in skeletal muscle there is 40% decrease in glucose entrance in normal pregnant women and 65% decrease in GDM compared with obese pregnant women. Although there is no decrease in GLUT4 protein transporter in skeletal muscle (Garvey et al., 1992), the GLUT4 transporters are decreased in adipose tissue (Garvey & Birnbaum, 1993). The increase in circulating TNF-α in women with GDM is also associated with an increased TNF-α in the skeletal muscle and the impaired insulin signaling persist in obese women with gestational diabetes mellitus up to one year TNF- α is considered as one of the factors which suppress PPAR-γ (Kirwan et al., 2002). Furthermore, it has been shown that TNF-α downregulates PPAR-γ expression in 3T3-L1 Catalano et al (2002) observed a decrease in steady-state PPARγ mRNA and protein concentration in normal and GDM subjects during late gestation. Furthermore, it has been demonstrated that TNF-α decreases adiponectin gene expression in human adipocytes (Kappes & Loffler, 2000), and 3t3-L1 adipocytes (Fasshauer et al., 2002). Whereas thiazolidinediones (synthetic PPAR-gamma ligand) significantly increases the plasma adiponectin concentrations in insulin resistant humans and rodents without affecting their body weight, suggesting that the anti TNF-α will restore the adiponectin and improve the Thus the increase in TNF-α whether in subjects with normal pregnancy or with GDM might explain the lower level of adiponectin (insulin-sensitizing hormone). The above data highly Steppan et al. (2001) showed that adipocytes secrete a unique signalling molecule, which is considered as a hormone and named 'resistin' (for resistance to insulin). Resistin is a 114- There is a great argument about the involvement of resistin in the pathogenesis of diabetes mellitus. Some scientists reported the involvement of resistin in the pathogenesis of diabetes mellitus relying on their studies that revealed strong correlations between resistin and obesity as serum resistin levels increased with increased adiposity (Steppan et al., 2001; Vendrell et al., signaling, thus causing the decreased insulin sensitivity observed in pregnancy. cells and can inhibit adipose differentiation (Zhang et al., 1996). suggest the involvement of TNF-α in the development of GDM. amino acid polypeptide hormone (Doshani & Konje, 2009). 2002; Coughlan et al., 2001). 4.2.1.2.1 The Diabetogenic action of TNF-α postpartum (Kirwan et al., 2004). insulin sensitivity (Maeda et al., 2005). 4.2.1.3 Resistin their body fat mass compared to women with normal glucose tolerance during and after pregnancy (Yamauchi et al., 2003; Weyer et al., 2001; Kadowaki & Yamaushi, 2005). On experimental animal studies, it was shown that adiponectin causes glucose-lowering effects and ameliorates insulin resistance in mice (Yamauchi et al., 2003b). Thus, decreased plasma adiponectin concentrations (hypoadiponectinemia) could be involved in the pathophysiology of pregnancy-driven insulin resistance and in the pathogenesis of GDM and Diabetes mellitus type 2 (DM2). It is known that peroxisome proliferator-activated receptors (PPARs) are transcriptional factors involved in the regulation of insulin resistance, fat cell differentiation, and adipogenesis (Joosen et al., 2006; Schoonjans et al., 1996; Zeghari et al., 2000). It has been shown that adiponectin activates AMP-kinase and PPARγ and α which improves insulin resistance and reduces fasting glucose level (Tsuchida et al., 2005; Kadowaki & Yamauchi, 2005). Low plasma adiponectin correlates highly with insulin resistance in obesity, type 2 DM and GDM (Weyer et al., 2001; Worda et al., 2004; Cseh et al., 2004). Low adiponectin level in normal pregnancy and GDM could be due to the suppression effect of TNF-α and other inflammatory factors on adiponectin transcription in adipocytes (Bruun et al., 2003; Fasshauer et al., 2003). These data highly support the antidiabetic effect of adiponectin. #### 4.2.1.2 Tumor necrosis factor-α (TNF-α) In 1975 Carswell et al discovered the so-called tumor necrosis factor (TNF) which is released from macrophages and induces tumor necrosis. Increased circulating TNF-α levels have been associated with insulin resistance in obesity, aging, sepsis, muscle damage, and burn patients (Hotamisligil et al., 1996; del Aguila et al., 2000; Kirwan et al., 2001; Ling et al., 1994; Conrad et al., 1998). Obese animals and humans show a positive correlation between TNF-α levels and BMI and hyperinsulinemia (Ling et al., 1994; Clapp & Kiess, 2000; Laham et al., 1994). Kirwan et al (2001) reported that TNF-α is a significant predictor of insulin resistance during pregnancy. Together with a small additive contribution from leptin and cortisol, TNF-α exerted a significant influence on insulin-mediated glucose disposal. Circulating TNF-α showed a downward trend during early pregnancy and increased during the third trimester, thus mirroring insulin sensitivity changes during those periods. This observation is consistent with studies showing an increase in plasma TNF-α in late pregnancy (Kirwan et al., 2002; Clapp & Kiess, 2000; Boyd et al., 2007). TNF-α correlates inversely with insulin secretion in normal pregnancy and was significantly higher in GDM group (McLachlan et al., 2006). TNF-alpha mRNA and protein are present in human placenta and uterine cells at both early and late stages of gestation (Chen et al., 1991). In maternal obesity, the level of TNF-α is increased in the placenta compared with the non-obese pregnant women (Denison et al., 2010). Furthermore, it has been shown that placenta and subcutaneous adipose tissues obtained from women with GDM release greater amount of TNF-α in response to high glucose compared with normal glucose. On the other hand, there was no stimulatory effect of high glucose on TNF-α release by tissues obtained from normal pregnant women which suggests that TNF-α might be involved in the pathogenesis and /or progression of GDM (Coughlan et al., 2001). These results could highly explain the increase in the level of TNF-α throughout pregnancy. The increased TNF-α levels in pregnancy fall rapidly after delivery (Kirwan et al., 2002; Uvena et al., 1999), which is consistent with the idea that the increase in circulating TNF-α during late pregnancy is mainly due to placental secretion. These findings may also help to explain the rapid reversal of insulin resistance after delivery, since maternal levels of TNF-α decrease substantially after delivery of the placenta (Kirwan et al., 2002; Coughlan et al., 2001). #### 4.2.1.2.1 The Diabetogenic action of TNF-α 100 Gestational Diabetes their body fat mass compared to women with normal glucose tolerance during and after On experimental animal studies, it was shown that adiponectin causes glucose-lowering effects and ameliorates insulin resistance in mice (Yamauchi et al., 2003b). Thus, decreased plasma adiponectin concentrations (hypoadiponectinemia) could be involved in the pathophysiology of pregnancy-driven insulin resistance and in the pathogenesis of GDM It is known that peroxisome proliferator-activated receptors (PPARs) are transcriptional factors involved in the regulation of insulin resistance, fat cell differentiation, and adipogenesis (Joosen et al., 2006; Schoonjans et al., 1996; Zeghari et al., 2000). It has been shown that adiponectin activates AMP-kinase and PPARγ and α which improves insulin resistance and reduces fasting glucose level (Tsuchida et al., 2005; Kadowaki & Yamauchi, 2005). Low plasma adiponectin correlates highly with insulin resistance in obesity, type 2 DM and GDM Low adiponectin level in normal pregnancy and GDM could be due to the suppression effect of TNF-α and other inflammatory factors on adiponectin transcription in adipocytes (Bruun et al., 2003; Fasshauer et al., 2003). These data highly support the antidiabetic effect of In 1975 Carswell et al discovered the so-called tumor necrosis factor (TNF) which is released from macrophages and induces tumor necrosis. Increased circulating TNF-α levels have been associated with insulin resistance in obesity, aging, sepsis, muscle damage, and burn patients (Hotamisligil et al., 1996; del Aguila et al., 2000; Kirwan et al., 2001; Ling et al., 1994; Conrad et al., 1998). Obese animals and humans show a positive correlation between TNF-α levels and Kirwan et al (2001) reported that TNF-α is a significant predictor of insulin resistance during pregnancy. Together with a small additive contribution from leptin and cortisol, TNF-α exerted a significant influence on insulin-mediated glucose disposal. Circulating TNF-α showed a downward trend during early pregnancy and increased during the third trimester, thus mirroring insulin sensitivity changes during those periods. This observation is consistent with studies showing an increase in plasma TNF-α in late pregnancy (Kirwan et al., 2002; Clapp & Kiess, 2000; Boyd et al., 2007). TNF-α correlates inversely with insulin secretion in BMI and hyperinsulinemia (Ling et al., 1994; Clapp & Kiess, 2000; Laham et al., 1994). normal pregnancy and was significantly higher in GDM group (McLachlan et al., 2006). TNF-alpha mRNA and protein are present in human placenta and uterine cells at both early and late stages of gestation (Chen et al., 1991). In maternal obesity, the level of TNF-α is increased in the placenta compared with the non-obese pregnant women (Denison et al., 2010). Furthermore, it has been shown that placenta and subcutaneous adipose tissues obtained from women with GDM release greater amount of TNF-α in response to high glucose compared with normal glucose. On the other hand, there was no stimulatory effect of high glucose on TNF-α release by tissues obtained from normal pregnant women which suggests that TNF-α might be involved in the pathogenesis and /or progression of GDM (Coughlan et al., 2001). These results could highly explain the increase in the level of TNF-α throughout pregnancy. The increased TNF-α levels in pregnancy fall rapidly after delivery (Kirwan et al., 2002; Uvena et al., 1999), which is consistent with the idea that the increase in pregnancy (Yamauchi et al., 2003; Weyer et al., 2001; Kadowaki & Yamaushi, 2005). and Diabetes mellitus type 2 (DM2). 4.2.1.2 Tumor necrosis factor-α (TNF-α) adiponectin. (Weyer et al., 2001; Worda et al., 2004; Cseh et al., 2004). In-vitro studies have described a direct role for TNF-α in the pathophysiology of insulin resistance. TNF-α downregulates insulin receptor signaling in cultured adipocytes (Catalano, 2010), hepatocytes (Feinstein et al., 1993), and skeletal muscle (del Aguila et al., 1999). TNF-α activates a pathway that increases sphingomyelinase and ceramides and appears to interfere with insulin receptor autophosphorylation (Catalano, 2010). Also it has been shown that TNFα promotes serine phosphorylation of insulin receptor substrate (IRS)-1, thus impairing its association with the insulin receptor (Rui et al., 2001). In pregnancy, there is an evidence that insulin receptor and IRS-1 tyrosine phosphorylation are impaired, and serine phosphorylation is increased in late gestation in skeletal muscle (Friedman et al., 1999; Shao et al., 2000). Therefore, it seems that elevated levels of TNF-α in late gestation could attenuate insulin signaling, thus causing the decreased insulin sensitivity observed in pregnancy. Barbour et al (2007) demonstrated that in skeletal muscle there is 40% decrease in glucose entrance in normal pregnant women and 65% decrease in GDM compared with obese pregnant women. Although there is no decrease in GLUT4 protein transporter in skeletal muscle (Garvey et al., 1992), the GLUT4 transporters are decreased in adipose tissue (Garvey & Birnbaum, 1993). The increase in circulating TNF-α in women with GDM is also associated with an increased TNF-α in the skeletal muscle and the impaired insulin signaling persist in obese women with gestational diabetes mellitus up to one year postpartum (Kirwan et al., 2004). TNF- α is considered as one of the factors which suppress PPAR-γ (Kirwan et al., 2002). Furthermore, it has been shown that TNF-α downregulates PPAR-γ expression in 3T3-L1 cells and can inhibit adipose differentiation (Zhang et al., 1996). Catalano et al (2002) observed a decrease in steady-state PPARγ mRNA and protein concentration in normal and GDM subjects during late gestation. Furthermore, it has been demonstrated that TNF-α decreases adiponectin gene expression in human adipocytes (Kappes & Loffler, 2000), and 3t3-L1 adipocytes (Fasshauer et al., 2002). Whereas thiazolidinediones (synthetic PPAR-gamma ligand) significantly increases the plasma adiponectin concentrations in insulin resistant humans and rodents without affecting their body weight, suggesting that the anti TNF-α will restore the adiponectin and improve the insulin sensitivity (Maeda et al., 2005). Thus the increase in TNF-α whether in subjects with normal pregnancy or with GDM might explain the lower level of adiponectin (insulin-sensitizing hormone). The above data highly suggest the involvement of TNF-α in the development of GDM. #### 4.2.1.3 Resistin Steppan et al. (2001) showed that adipocytes secrete a unique signalling molecule, which is considered as a hormone and named 'resistin' (for resistance to insulin). Resistin is a 114 amino acid polypeptide hormone (Doshani & Konje, 2009). There is a great argument about the involvement of resistin in the pathogenesis of diabetes mellitus. Some scientists reported the involvement of resistin in the pathogenesis of diabetes mellitus relying on their studies that revealed strong correlations between resistin and obesity as serum resistin levels increased with increased adiposity (Steppan et al., 2001; Vendrell et al., Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future 103 Chen et al (2010) carried out a study in which twenty women with normal pregnancy and 20 with GDM were recruited and blood samples were taken on the day of delivery and Days 1, 3 and 5 after delivery. Serum leptin levels were significantly higher in women with GDM than in the controls before delivery and decreased significantly after delivery (p < 0.001). After delivery there were no significant differences in serum leptin concentrations between women with GDM and the controls. Serum soluble leptin receptor concentrations did not differ neither between the two groups, nor before or after delivery. Thus, they concluded that Leptin may play a role in GDM through a positive correlation with insulin resistance. Fukuhara et al (2005) isolated a newly adipocytokine, named as 'visfatin', which is highly enriched in the visceral fat of both humans and mice and whose expression level in plasma increases during the development of obesity. Visfatin exerted insulin-mimetic effects in cultured cells and lowered plasma glucose levels in mice by binding and activating the insulin receptors. Suggesting that visfatin's physiological role may lead to new insights into According to some authors, plasma concentrations of visfatin are elevated in obesity (Berndt et al., 2005), type 2 diabetes (Chen et al., 2006) and the increase is typically observed in GDM (Krzyzanowska et al., 2006; Lewandowski et al., 2007), all of which are states characterized by insulin resistance. There are also, however, data pointing to possible lower visfatin levels in obese subjects (Pagano et al., 2006), similarly, Chan et al. (2006) have reported lower visfatin levels in women of Chinese origin with GDM. The precise reason for these Shali et al (2009) reported that maternal GDM, as well as delivery of a large-for-gestationalage (LGA) neonate were independently associated with higher maternal plasma visfatin concentrations. The linkage between increased maternal circulating visfatin and the presence of GDM or delivery of an LGA neonate supports the hypothesis that perturbation of adipokines homeostasis may play a role in the pathophysiology of GDM or excess fetal The current data regarding the relationship between visfatin and insulin sensitivity in humans are conflicting. Some authors report a lack of correlation (Berndt et al., 2005; Pagano et al., 2006; Zhang et al., 2010), while others observed a significant correlation (Chen et al., The role of visfatin in human physiology and pathophysiology remains to be elucidated and further work is needed to establish the exact function of visfatin and its mode of action on Tatemoto et al (1998) isolated an APJ receptor ligand, designated apelin, from bovine stomach extracts. The preproproteins consisted of 77 amino acid residues, and the apelin sequence was encoded in the C-terminal regions indicating that apelin is an endogenous Apelin has been described as an adipocyte-secreted factor (adipokine), that is up-regulated in obesity and the expression of apelin gene in adipose tissue is reported to increase by insulin and TNF-α (Carpéné et al., 2007). Apelin synthesis in adipocytes is stimulated by insulin, and plasma apelin level markedly increases in obesity associated with insulin glucose homeostasis and/or new therapies for metabolic disorders such as diabetes. 4.2.1.5 Visfatin differences is unclear. 2006; Lewandowski et al., 2007). ligand for the APJ receptor. insulin resistance during normal pregnancy and GDM. resistance and hyperinsulinemia (Bełtowski, 2006). growth. 4.2.1.6 Apelin 2004; Lee et al., 2005). Conversely, serum resistin levels have been found to decline with decreased adiposity following medical treatment (Valsamakis et al., 2004). This discovery is further authenticated by studies which confirmed a direct correlation between resistin levels and subjects with type 2DM (Steppan et al., 2001; Fujinami et al., 2004; McTernan et al., 2003). Nevertheless, this theory lacks support from the entire scientific community at large as an increasingly greater number of studies presenting contradictory evidences continue to emerge (Lee et al., 2003; Nagaev & Smith, 2001). Some studies found significant decreased serum concentrations of resistin with increased adiposity (Heilbronn et al., 2004; Way et al., 2001) suggesting that not only resistin is downregulated in obese subjects but that it also presents itself as an unlikely candidate for linking obesity to Type 2DM. Milan et al (2002) mentioned that a decrease of resistin mRNA after weight loss does not support the hypothesis that resistin may play a causative role in insulin resistance in obese rats. Many studies reported that in patients with type 2 diabetes or obesity, both resistin levels and resistin expression in fat cells are increased, correlating with hepatic, but not muscle, insulin resistance. In humans, the major source of resistin is the immune cells rather than the adipocytes, resistin being a potent inflammatory agent. Insulin inhibits resistin expression in adipocytes. Therefore, the elevated basal plasma resistin levels found in patients with type 2 diabetes, despite increased insulin concentrations, may be the result of adipocyte insulin resistance. Resistin inhibits the phosphorylation of hepatic AMPK, decreasing β oxidation and increasing fatty acid esterification in triglycerides, and eventually leading to lipid accumulation (Maiorana et al., 2007). #### 4.2.1.4 Leptin It was discovered as an antiobesity hormone in ob/ob mice (Zhang et al., 1994). In human adult, the white adipose tissue is the main source of leptin, and its circulating concentration is positively correlated with body mass index and fat mass (Maffei et al., 1995; Hellstrom et al., 2000). Leptin has been detected in the placenta (Masuzaki et al., 1997), and shown to be increased in early pregnancy, remained elevated in late pregnancy (Kirwan et al., 2002; Highman et al., 1998), and was highest in the more obese GDM group (Kirwan et al., 2002). The increased leptin during pregnancy is not proportional with the change in adipose tissue mass, and it return to the normal level after delivery suggesting that leptin production by the placenta contributes to maternal leptinemia during pregnancy (Lepercq et al., 2001). In vitro study on muscle, Muoio et al (1997) demonstrated that leptin attenuated both the antioxidative and the lipogenic effects of insulin by 50%. Cseh K et al (2002) suggested that the increased TNF-alpha and leptin levels may contribute to insulin resistance in GDM and in the third trimester of normal pregnancy. Furthermore, Qiu et al (2004) demonstrated that Hyperleptinemia, independent of maternal adiposity, in early pregnancy appears to be predictive of an increased risk of GDM later in pregnancy. Kirwan et al (2002) reported that leptin was increased in all women in early pregnancy, remained elevated in late pregnancy, and was highest in the more obese GDM group. But to adjust for the possible confounding effect of obesity and increased fat mass on the relationship between leptin and insulin sensitivity, they covaried for body fat and found that the correlation was no longer significant, because the increased leptin per se was not predictive of insulin sensitivity. They interpreted that, in addition to insulin resistance, leptin resistance may also develop in late pregnancy. Chen et al (2010) carried out a study in which twenty women with normal pregnancy and 20 with GDM were recruited and blood samples were taken on the day of delivery and Days 1, 3 and 5 after delivery. Serum leptin levels were significantly higher in women with GDM than in the controls before delivery and decreased significantly after delivery (p < 0.001). After delivery there were no significant differences in serum leptin concentrations between women with GDM and the controls. Serum soluble leptin receptor concentrations did not differ neither between the two groups, nor before or after delivery. Thus, they concluded that Leptin may play a role in GDM through a positive correlation with insulin resistance. #### 4.2.1.5 Visfatin 102 Gestational Diabetes 2004; Lee et al., 2005). Conversely, serum resistin levels have been found to decline with decreased adiposity following medical treatment (Valsamakis et al., 2004). This discovery is further authenticated by studies which confirmed a direct correlation between resistin levels and subjects with type 2DM (Steppan et al., 2001; Fujinami et al., 2004; McTernan et al., 2003). Nevertheless, this theory lacks support from the entire scientific community at large as an increasingly greater number of studies presenting contradictory evidences continue to emerge (Lee et al., 2003; Nagaev & Smith, 2001). Some studies found significant decreased serum concentrations of resistin with increased adiposity (Heilbronn et al., 2004; Way et al., 2001) suggesting that not only resistin is downregulated in obese subjects but that it also presents itself as an unlikely candidate for linking obesity to Type 2DM. Milan et al (2002) mentioned that a decrease of resistin mRNA after weight loss does not support the hypothesis that resistin Many studies reported that in patients with type 2 diabetes or obesity, both resistin levels and resistin expression in fat cells are increased, correlating with hepatic, but not muscle, insulin resistance. In humans, the major source of resistin is the immune cells rather than the adipocytes, resistin being a potent inflammatory agent. Insulin inhibits resistin expression in adipocytes. Therefore, the elevated basal plasma resistin levels found in patients with type 2 diabetes, despite increased insulin concentrations, may be the result of adipocyte insulin resistance. Resistin inhibits the phosphorylation of hepatic AMPK, decreasing β oxidation and increasing fatty acid esterification in triglycerides, and eventually leading to lipid It was discovered as an antiobesity hormone in ob/ob mice (Zhang et al., 1994). In human adult, the white adipose tissue is the main source of leptin, and its circulating concentration is positively correlated with body mass index and fat mass (Maffei et al., 1995; Hellstrom et Leptin has been detected in the placenta (Masuzaki et al., 1997), and shown to be increased in early pregnancy, remained elevated in late pregnancy (Kirwan et al., 2002; Highman et al., 1998), and was highest in the more obese GDM group (Kirwan et al., 2002). The increased leptin during pregnancy is not proportional with the change in adipose tissue mass, and it return to the normal level after delivery suggesting that leptin production by the placenta contributes to maternal leptinemia during pregnancy (Lepercq et al., 2001). In vitro study on muscle, Muoio et al (1997) demonstrated that leptin attenuated both the antioxidative and the lipogenic effects of insulin by 50%. Cseh K et al (2002) suggested that the increased TNF-alpha and leptin levels may contribute to insulin resistance in GDM and in the third trimester of normal pregnancy. Furthermore, Qiu et al (2004) demonstrated that Hyperleptinemia, independent of maternal adiposity, in early pregnancy appears to be predictive of an increased risk of GDM later in pregnancy. Kirwan et al (2002) reported that leptin was increased in all women in early pregnancy, remained elevated in late pregnancy, and was highest in the more obese GDM group. But to adjust for the possible confounding effect of obesity and increased fat mass on the relationship between leptin and insulin sensitivity, they covaried for body fat and found that the correlation was no longer significant, because the increased leptin per se was not predictive of insulin sensitivity. They interpreted that, in addition to insulin resistance, leptin resistance may also develop in late may play a causative role in insulin resistance in obese rats. accumulation (Maiorana et al., 2007). 4.2.1.4 Leptin al., 2000). pregnancy. Fukuhara et al (2005) isolated a newly adipocytokine, named as 'visfatin', which is highly enriched in the visceral fat of both humans and mice and whose expression level in plasma increases during the development of obesity. Visfatin exerted insulin-mimetic effects in cultured cells and lowered plasma glucose levels in mice by binding and activating the insulin receptors. Suggesting that visfatin's physiological role may lead to new insights into glucose homeostasis and/or new therapies for metabolic disorders such as diabetes. According to some authors, plasma concentrations of visfatin are elevated in obesity (Berndt et al., 2005), type 2 diabetes (Chen et al., 2006) and the increase is typically observed in GDM (Krzyzanowska et al., 2006; Lewandowski et al., 2007), all of which are states characterized by insulin resistance. There are also, however, data pointing to possible lower visfatin levels in obese subjects (Pagano et al., 2006), similarly, Chan et al. (2006) have reported lower visfatin levels in women of Chinese origin with GDM. The precise reason for these differences is unclear. Shali et al (2009) reported that maternal GDM, as well as delivery of a large-for-gestationalage (LGA) neonate were independently associated with higher maternal plasma visfatin concentrations. The linkage between increased maternal circulating visfatin and the presence of GDM or delivery of an LGA neonate supports the hypothesis that perturbation of adipokines homeostasis may play a role in the pathophysiology of GDM or excess fetal growth. The current data regarding the relationship between visfatin and insulin sensitivity in humans are conflicting. Some authors report a lack of correlation (Berndt et al., 2005; Pagano et al., 2006; Zhang et al., 2010), while others observed a significant correlation (Chen et al., 2006; Lewandowski et al., 2007). The role of visfatin in human physiology and pathophysiology remains to be elucidated and further work is needed to establish the exact function of visfatin and its mode of action on insulin resistance during normal pregnancy and GDM. #### 4.2.1.6 Apelin Tatemoto et al (1998) isolated an APJ receptor ligand, designated apelin, from bovine stomach extracts. The preproproteins consisted of 77 amino acid residues, and the apelin sequence was encoded in the C-terminal regions indicating that apelin is an endogenous ligand for the APJ receptor. Apelin has been described as an adipocyte-secreted factor (adipokine), that is up-regulated in obesity and the expression of apelin gene in adipose tissue is reported to increase by insulin and TNF-α (Carpéné et al., 2007). Apelin synthesis in adipocytes is stimulated by insulin, and plasma apelin level markedly increases in obesity associated with insulin resistance and hyperinsulinemia (Bełtowski, 2006). Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future 105 challenge OGTT had demonstrated that up to 17% of pregnant women develop GDM. That means there is a significant increase in the development of GDM in these days. Could there be more in future? Environmental factors, changes in life style, and change of diet, such as decrease in using fresh diet while increase in using canned food, in addition to the use of high caloric diet and food with high glycemic index. All these factors, could highly participate in the development of GDM whether directly or indirectly by increasing the The precise mechanisms causing GDM remain unknown. All the previously described maternal and feto-placental factors interact in an integrated manner in the development of insulin resistance and GDM. The most prominent factors, which are involved in the pathogenesis of GDM, are the increase in HPL and TNF-α and the decrease in adiponectin Most of the women reverting to normal after delivery, will suggest that the placenta is the The main cause of insulin resistance during GDM is post-cellular defect manifested by a decreased phosphorylation of tyrosine residues in insulin receptors and insulin receptor substrate-1, while serine phosphorylation is increased which inhibit insulin signaling from Finally, GDM is probably produced by a complex and variable interaction of all the previously mentioned factors - pregnancy-induced factors, genetic, diet, environmental, Ahmed SA, Shalayel MH. (1999). Role of cortisol in the deterioration of glucose tolerance in Al- Dahwi AN, Al-Noaemi M, Alwan AAS, Al-Dahwi RN and Al-Taie ID. (1987) Al-Dahwi AN, Al-Noaemi M, Alwan AAS, et al. (1988). Glycosylated haemoglobin in normal and diabetic pregnancies. Iraqi Medical Journal, 37 (2): 68-371. Al-Dahwi AN, Al-Noaemi M, Alwan AAS, et al. (1986). Glycosylated hemoglobin in diabetic Al-Dahwi RN, Alwan AAS, Al-Noaemi M and Al-Dahwi AN. (1989). Clinical experience Baker P N (2006) Physiology of pregnancy In: Obstetrics by ten teachers (18th Edition), pp 48- Barbieri RL (1999) Endocrine disorders in pregnancy In: Reproductive Endocrinology (4th edition). Yen SSC, Jaffe RB, Barbieri RL, Eds. Philadelphia, W.B. Saunder. Glycosylated hemoglobin as an index for maternal and fetal complications. Abstract submitted in the first scientific congress of the Iraqi Society for obstetrics Pergnancy. Abstract submitted in the first scientific congress of Al- Mustansiryah with fifty pregnancies in diabetic patients Abstract submitted to the 4th congress of the Mediterranean Medical Association. Tunis 10-13th September 1989. Tunisia. Al-Noaemi M and Shalayel MHF. (2009). Adiponectin (review article). Sudan Journal of Sudanese pregnant women. East Afr Med J, 76(8):465-7. 62. A Hodder Arnold Publication. ISBN: 0340816651. incidence of obesity. 6. Conclusions during pregnancy. autoimmunity, etc. 7. References activating GLUT4 translocation. major contributing organ in the development of GDM. and gynecology. Baghdad, Iraq. Medical College. Baghdad, Iraq. Medical Sciences, 4 (30): 297-305. Dray et al (2010) reported that apelin is increased in adipose tissue in different mice models of obesity and in the type2 diabetic patients. They reported that apelin plasma levels were significantly increased in type 2 diabetic patients. They suggested that apelin and APJ expression in mice and humans are regulated in a tissue dependent manner and according to the severity of insulin resistance. But, Meral et al (2010) reported that no significant relation was found between apelin and BMI, glucose, lipids levels, and also insulin sensitivity. In addition, Telejko et al (2010) reported that there is no associations between circulating apelin or apelin/APJ mRNA expression and GDM and no indices of insulin resistance were noted in their study. Furthermore, Tapan et al (2010) recently reported a significant decrease in plasma apelin and adiponectin levels in pubertal obese children. More work is needed to establish the involvement/or not of apelin and insulin resistance in normal pregnancy and GDM. #### 5. The future Every now and then, there will be a new factor, hormone, adipocytokine, etc. which is involved in the development of insulin resistance and GDM. But the precise pathophysiological mechanisms which make the women unable to balance insulin needs and develop GDM, remain unknown. However, a number of future studies could explain some of these mechanisms. #### 5.1 Genotyping Genetic variants might be involved in the defect in B-cell function and/or subcellular insulin signaling which contribute to the development of GDM. Therefore, we suggest a screening genotyping test for a significant number of pregnant women, to identify any genetic variants in those who develop GDM. Extending this genotyping to involve familial studies, to demonstrate any involvement of these genetic variants and whether they run in families. Any positive result will help to give a special care to those women anticipated to develop GDM, and thus reducing the associated maternal or fetal complications. #### 5.2 Subcellular studies Certain studies should be done to identify the exact subcellular reactions in the normal insulin resistance that develop during normal pregnancy compared with those subcellular changes that lead to glucose intolerance and GDM. The use of radioactive substances might help to identify the change in phosphorylation of serine instead of tyrosine residues, or in any other subcellular reaction change which occurs in GDM. #### 5.3 Autoimmunity To study certain aspects of the immune system, which could demonstrate any B-cell dysfunction that develops GDM and is related to autoimmunity. Furthermore, if there is any interference of the immune system with insulin-receptor interaction, this might contribute to the development of GDM. #### 5.4 Environmental and diet factors 30-40 years ago or more, it has been reported that the percent of GDM range from 1-2%, 2- 3% then the percent increased to 1-4%, 2-7%, 5-10%. Until recently using 1-hour glucose challenge OGTT had demonstrated that up to 17% of pregnant women develop GDM. That means there is a significant increase in the development of GDM in these days. Could there be more in future? Environmental factors, changes in life style, and change of diet, such as decrease in using fresh diet while increase in using canned food, in addition to the use of high caloric diet and food with high glycemic index. All these factors, could highly participate in the development of GDM whether directly or indirectly by increasing the incidence of obesity. ### 6. Conclusions 104 Gestational Diabetes Dray et al (2010) reported that apelin is increased in adipose tissue in different mice models of obesity and in the type2 diabetic patients. They reported that apelin plasma levels were significantly increased in type 2 diabetic patients. They suggested that apelin and APJ expression in mice and humans are regulated in a tissue dependent manner and according to the severity of insulin resistance. But, Meral et al (2010) reported that no significant relation was found between apelin and BMI, glucose, lipids levels, and also insulin sensitivity. In addition, Telejko et al (2010) reported that there is no associations between circulating apelin or apelin/APJ mRNA expression and GDM and no indices of insulin resistance were noted in their study. Furthermore, Tapan et al (2010) recently reported a significant decrease in plasma apelin and adiponectin levels in pubertal obese children. More work is needed to establish the involvement/or not of apelin and insulin resistance in Every now and then, there will be a new factor, hormone, adipocytokine, etc. which is involved in the development of insulin resistance and GDM. But the precise pathophysiological mechanisms which make the women unable to balance insulin needs and develop GDM, remain unknown. However, a number of future studies could explain Genetic variants might be involved in the defect in B-cell function and/or subcellular insulin signaling which contribute to the development of GDM. Therefore, we suggest a screening genotyping test for a significant number of pregnant women, to identify any genetic variants in those who develop GDM. Extending this genotyping to involve familial studies, to demonstrate any involvement of these genetic variants and whether they run in families. Any positive result will help to give a special care to those women anticipated to develop Certain studies should be done to identify the exact subcellular reactions in the normal insulin resistance that develop during normal pregnancy compared with those subcellular changes that lead to glucose intolerance and GDM. The use of radioactive substances might help to identify the change in phosphorylation of serine instead of tyrosine residues, or in To study certain aspects of the immune system, which could demonstrate any B-cell dysfunction that develops GDM and is related to autoimmunity. Furthermore, if there is any interference of the immune system with insulin-receptor interaction, this might contribute to 30-40 years ago or more, it has been reported that the percent of GDM range from 1-2%, 2- 3% then the percent increased to 1-4%, 2-7%, 5-10%. Until recently using 1-hour glucose GDM, and thus reducing the associated maternal or fetal complications. any other subcellular reaction change which occurs in GDM. normal pregnancy and GDM. some of these mechanisms. 5.2 Subcellular studies 5.3 Autoimmunity the development of GDM. 5.4 Environmental and diet factors 5. The future 5.1 Genotyping The precise mechanisms causing GDM remain unknown. All the previously described maternal and feto-placental factors interact in an integrated manner in the development of insulin resistance and GDM. The most prominent factors, which are involved in the pathogenesis of GDM, are the increase in HPL and TNF-α and the decrease in adiponectin during pregnancy. Most of the women reverting to normal after delivery, will suggest that the placenta is the major contributing organ in the development of GDM. The main cause of insulin resistance during GDM is post-cellular defect manifested by a decreased phosphorylation of tyrosine residues in insulin receptors and insulin receptor substrate-1, while serine phosphorylation is increased which inhibit insulin signaling from activating GLUT4 translocation. Finally, GDM is probably produced by a complex and variable interaction of all the previously mentioned factors - pregnancy-induced factors, genetic, diet, environmental, autoimmunity, etc. #### 7. References Pathophysiology of Gestational Diabetes Mellitus: The Past, the Present and the Future 107 Catalano PM. (2010). Obesity, insulin resistance, and pregnancy outcome. Focus Review on Chan TF, Chen YL, Lee CH et al. (2006). Decreased plasma visfatin concentrations in women with gestational diabetes mellitus. J Soc Gynecol Investig., 13: 364–367. 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J Clin relation to endometrial cancer: a case control study in Greece. J Clin Endocrinol pregnancy and risk of gestational diabetes mellitus. Obstet Gynecol, 103(3): 519-525. 7 Iran The Role of Adipocyte Mediators, in Gestational Diabetes Shahid Sadoughi University of Medical Sciences Sedigheh Soheilykhah Inflammatory Markers and Vitamin D Gestational diabetes mellitus (GDM) usually reveals itself in the latter half of pregnancy and The presence of GDM has implications for both the mother and the baby. Perinatal morbidity includes macrosomia, hypoglycemia, hyperbilirubinaemia and respiratory distress syndrome which lead to the subsequent complications (Hod et al., 1991). Long term outcomes for the offspring may include obesity and diabetes independent of genetic factors (Silverman et al., 1995; Van Assche et al., 1992, 2001). For the mother there is an increased risk of overt type 2 diabetes later in life (Mestman, 1987; O'Sullivan, 1989). Both type 2 diabetes and gestational diabetes have common pathogenic mechanisms where pregnancy tends to expose disease in those women who are at risk of developing type 2 diabetes later in life. Similar to all forms of hyperglycemia, GDM is characterized by insulin levels that are inadequate to proper insulin requirement (Metzger et al.,2007).The pathogenesis of GDM has not been clearly defined. The most common hypothesis is that GDM is caused by decreasing insulin sensitivity and increasing anti-insulin hormones that are secreted by the placenta during pregnancy, such as human placental lactogen, prolactin, glucocorticoid and It has become increasingly evident that endocrine/metabolic hormones such as leptin, adiponectin, resistin, proinflammatory mediators including C-reactive protein ( CRP) are In recent times a number of first trimester studies have shown association of different biomarkers with the development of GDM. These include elevated serum or plasma Creactive protein (Wolf et al., 2003), lower sex hormone-binding globulin (Thadhani et al., 2003), increased placental growth factor (Ong et al., 2004) and elevated leptin (Qiu et al., Some studies have recommended that Vitamin D deficiency could play a role in This chapter will focus on the studies about the role of adipocytes mediators, it is identified by carbohydrate intolerance of variable severity. 1. Introduction progesterone (Xue-lian et al., 2008). pathogenesis of gestational diabetes. strongly linked with abnormal carbohydrate metabolism. proinflammatory factors and vitamin D in gestational diabetes. 2004) and decreasing of adiponectin concentration. ## The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes Sedigheh Soheilykhah Shahid Sadoughi University of Medical Sciences Iran #### 1. Introduction 114 Gestational Diabetes Yamauchi T, Hara K, Kubota N et al. (2003). Dual roles of adiponectin/ Acrp30 in vivo as an Yamauchi T, Kamon J, Ito Y, et al. (2003). Cloning of adiponectin receptors that mediate Yamauchi T, Kamon J, Waki H et al. (2001). The fat driven-hormone adiponectin reverses Yokota T, Oritani K, Takahashi I et al. (2000). Adiponectin, a new member of the family of Yool MJ, Lee GY, Chung J et al. (2006). Adiponectin Increases Fatty Acid Oxidation in Zeghari N, Vidal H, Younsi M, et al. (2000). Adipocyte membrane phospholipids and PPAR- Zhang B, Berger J, Hu E, et al. (1996). Negative regulation of peroxisome proliferator- Zhang Y, Proenca R, Maffei M, et al. (1994). Positional cloning of the mouse ob gene and its Zhang XJ, Wang Z, Li HH, Yu L, Gao S. (2010). Association between both resistin, visfatin diabetes mellitus. Zhonghua Liu Xing Bing Xue Za Zhi, 31(12):1393-1396. Zwick E, Bange J, Ullrich A. (2001). Receptor tyrosine kinase signalling as a target for cancer of tumor necrosis factor-alpha. Mol Endocrinol., 10: 1457–1466. intervention strategies. Endocr. Relat. Cancer, 8 (3): 161–173. progenitors and the functions of macrophages. Blood, 96: 1723-1732. antidiabetic metabolic effects. Nature, 423(6941):762-9. Metabol Disord, 3: 243-254. Receptor a. Diabetes, 55: 2562-2570. Endocrinol Metab., 279: E736–E743. human homologue. Nature. 372: 425– 432. 946. anti-diabetic and anti-atherogenic adipokines. Curr Drug Targets Immune Endocr insulin resistance associated with both lipoatrophy and obesity. Nat Med., 7: 941- soluble defense collagens, negatively regulates the growth of myelomonocytic Skeletal Muscle Cell by Sequential Activation of AMP-Activated Protein Kinase, p38 Mitogene-Activated protein Kinase, and Peroxisome Proliferator- Activated gamma expression in obese women: relationship to hyperinsulinemia. Am J Physiol activated receptor-gamma gene expression contributes to the antiadipogenic effects and insulin resistance as well as β cell function in the first-degree relatives of type 2 Gestational diabetes mellitus (GDM) usually reveals itself in the latter half of pregnancy and it is identified by carbohydrate intolerance of variable severity. The presence of GDM has implications for both the mother and the baby. Perinatal morbidity includes macrosomia, hypoglycemia, hyperbilirubinaemia and respiratory distress syndrome which lead to the subsequent complications (Hod et al., 1991). Long term outcomes for the offspring may include obesity and diabetes independent of genetic factors (Silverman et al., 1995; Van Assche et al., 1992, 2001). For the mother there is an increased risk of overt type 2 diabetes later in life (Mestman, 1987; O'Sullivan, 1989). Both type 2 diabetes and gestational diabetes have common pathogenic mechanisms where pregnancy tends to expose disease in those women who are at risk of developing type 2 diabetes later in life. Similar to all forms of hyperglycemia, GDM is characterized by insulin levels that are inadequate to proper insulin requirement (Metzger et al.,2007).The pathogenesis of GDM has not been clearly defined. The most common hypothesis is that GDM is caused by decreasing insulin sensitivity and increasing anti-insulin hormones that are secreted by the placenta during pregnancy, such as human placental lactogen, prolactin, glucocorticoid and progesterone (Xue-lian et al., 2008). It has become increasingly evident that endocrine/metabolic hormones such as leptin, adiponectin, resistin, proinflammatory mediators including C-reactive protein ( CRP) are strongly linked with abnormal carbohydrate metabolism. In recent times a number of first trimester studies have shown association of different biomarkers with the development of GDM. These include elevated serum or plasma Creactive protein (Wolf et al., 2003), lower sex hormone-binding globulin (Thadhani et al., 2003), increased placental growth factor (Ong et al., 2004) and elevated leptin (Qiu et al., 2004) and decreasing of adiponectin concentration. Some studies have recommended that Vitamin D deficiency could play a role in pathogenesis of gestational diabetes. This chapter will focus on the studies about the role of adipocytes mediators, proinflammatory factors and vitamin D in gestational diabetes. The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes 117 Adiponectin exerts some of its weight reduction effects via the brain. This is similar to the action of leptin, but the two hormones perform complementary actions, and can have Adiponectin affects glucose flux through decreasing gluconeogenesis and increasing glucose uptake (Díez & Iglesias, 2003; Nedvídková et al., 2005; Vasseur et al., 2003). Adiponectin has a role in lipid catabolism (Vasseur et al., 2003) by β-oxidation and triglyceride clearance (Nedvídková et al., 2005). The other metabolic effects of adiponectin are protection against endothelial dysfunction and improvement of insulin sensitivity and weight loss, and control of energy metabolism (Vasseur et al., 2006).Adiponectin has also another effects, including putative insulin-sensitising, anti-atherogenic and anti inflammatory characteristics (Berg et Consistent with the low circulating levels of adiponectin observed in type 2 diabetes, adiponectin concentration is contrarily related to both insulin resistance and central adiposity (Arita et al., 1999; Weyer et al., 2001). Low baseline adiponectin concentration can predict the subsequent development of insulin resistance although elevated baseline levels have been shown to be protective against the succeeding development of type 2 diabetes (Daimon et al., 2003; Lindsay et al., 2002; Snehalatha et al., 2003; Spranger et al. , 2003). Recognition of gestational diabetes mellitus helps in identification of populations of young women at high risk of developing type 2 diabetes. Therefore, reduction in serum Some studies exhibited hypoadiponectinemia in pregnant women with gestational diabetes. A cross sectional study of 180 women in their third trimesters illustrated gestational diabetic women had more hypoadiponectinemia compared with normoglycemic controls after adjustment for covariates including insulin resistance. This study showed adiponectin concentration is an independent correlate of beta cell function in late pregnancy. Adiponectin was correlated with insulin secretion-sensitivity index (ISSI). ISSI was positively correlated with adiponectin and negatively correlated with GDM and IGT. Therefore, adiponectin may play a principal role in mediating insulin resistance and beta cell malfunction in the development of diabetes (Rentakaran et al., 2005).To determine whether adiponectin relate to the postpartum metabolic disturbance linking GDM with type 2 diabetes a cohort study was done on 487 pregnant wemen in pregnancy and at 3 months post partum. This study demonstrated adiponectin was related to postpartum insulin sensitivity and hypoadiponectinemia in pregnancy predicts postpartum insulin resistance, beta cell dysfunction and fasting glycaemia, and hence may be relevant to the pathophysiology relating GDM with type 2 diabetes (Rentakaran et al., 2010). To investigate level of adiponectin and metabolic factors in women with gestational diabetes a cross sectional study was done . The result of this study showed the level of adiponectin was lower in gestational diabetes and adiponectin correlates negatively with insulin resistance in homeostasis model assessment-insulin resistance (HOMA-IR) (Altinova et al., 2007). To evaluate whether adiponectin is a predictive factor for gestational diabetes mellitus (GDM) and is appropriate as a screening test for GDM a study was done and the results adiponectin levels in GDM may be comparable to type 2 diabetes. additive effects (Nedvídková et al., 2005). 2.1.2 Metabolic effect of adiponectin al., 2002; Ukkola & Santaniemi, 2002). 2.1.3 Adiponectin and GDM ### 2. Adipocyte mediators It is becoming obvious that adipose tissue is not just a storage for extra energy but that it secretes a number of biologically active peptides as a group named adipocytokines that control glucose and fatty acid metabolism (Youn et al.,2004). These peptides have similar properties with cytokines, and so referred to as "adipocytokines", e.g. leptin, tumor necrosis factor-α(TNF-α), interleukin 6 (IL-6), adiponectin and resistin. These adipocytokines may influence activity of other tissues (Nedvidkova et al., 2005). #### 2.1 Adiponectin #### 2.1.1 Adiponectin structure Adiponectin is a 244-amino-acid-long polypeptide that regulates a number of metabolic processes, including glucose regulation and fatty acid catabolism. Adiponectin is exclusively secreted from adipose tissue into the circulation and is very plentiful in plasma relative to other hormones. Adiponectin concentrations ranging from 5 to 30mg/ml and accounting for approximately 0.01% of total plasma protein . Females have higher levels of plasma concentration than males (Díez & Iglesias, 2003). Adiponectin was first identified in mice as a transcript overexpressed in preadipocytes (Lara-Castro et al., 2007) differentiating into adipocyte (Matsuzawa et al., 2004). The human homologue was recognized as the most abundant transcript in adipose tissue. As opposed to anticipation, in spite of being produced in a fat tissue, adiponectin was found to decline in obesity (Díez & Iglesias, 2003; Nedvídková et al., 2005; Ukkola & Santaniemi, 2002). This down regulation has not been clearly described. The gene was localised to chromosome 3p27, a region highlighted as affecting genetic vulnerability to obesity and type 2 diabetes. Supplementation by varying forms of adiponectin improved control of insulin, blood glucose and triglyceride levels in mouse models. Berbelin, a herbal folk medicine, has been shown to increase adiponectin expression which partly explains its advantageous effects on metabolic disturbances (Choi et al., 2009). A reduction in adiponectin expression is associated with insulin resistance in animal models. Administration of adiponectin has been accompanied by a reduction in plasma glucose and an increase in insulin sensitivity. In addition, thiazolidinediones, drugs that increase insulin sensitivity by stimulation of the peroxisome proliferator-activated receptorδ, increase plasma adiponectin and mRNA levels in mice. A recent study has shown that phosphorylation and Levels of the hormone are conversely correlated with body fat proportion in adults although the association in infants and young children is less clear. The hormone plays a role in the inhibition of the metabolic disturbance that may end in type 2 diabetes (Ukkola & Santaniemi, 2002), obesity, atherosclerosis (Díez & Iglesias, 2003), non-alcoholic fatty liver diseases (NAFLD) and independent risk factors for metabolic syndrome (Renaldi et al., 2009)., Levels of adiponectin are decreased in diabetics compared to non-diabetics. Weight loss significantly increases circulating levels (Coppola et al., 2009). Adiponectin automatically self-associates into larger structures. Initially, three adiponectin molecules bind together to form a homotrimer. The trimers continue to self-associate and form hexamers or dodecamers. Like the plasma concentration, the relative levels of the higher-order structures are sexually dimorphic, where females have increased proportions of the high-molecular weight forms. Adiponectin exerts some of its weight reduction effects via the brain. This is similar to the action of leptin, but the two hormones perform complementary actions, and can have additive effects (Nedvídková et al., 2005). #### 2.1.2 Metabolic effect of adiponectin 116 Gestational Diabetes It is becoming obvious that adipose tissue is not just a storage for extra energy but that it secretes a number of biologically active peptides as a group named adipocytokines that control glucose and fatty acid metabolism (Youn et al.,2004). These peptides have similar properties with cytokines, and so referred to as "adipocytokines", e.g. leptin, tumor necrosis factor-α(TNF-α), interleukin 6 (IL-6), adiponectin and resistin. These adipocytokines may Adiponectin is a 244-amino-acid-long polypeptide that regulates a number of metabolic processes, including glucose regulation and fatty acid catabolism. Adiponectin is exclusively secreted from adipose tissue into the circulation and is very plentiful in plasma relative to other hormones. Adiponectin concentrations ranging from 5 to 30mg/ml and accounting for approximately 0.01% of total plasma protein . Females have higher levels of plasma Adiponectin was first identified in mice as a transcript overexpressed in preadipocytes (Lara-Castro et al., 2007) differentiating into adipocyte (Matsuzawa et al., 2004). The human homologue was recognized as the most abundant transcript in adipose tissue. As opposed to anticipation, in spite of being produced in a fat tissue, adiponectin was found to decline in obesity (Díez & Iglesias, 2003; Nedvídková et al., 2005; Ukkola & Santaniemi, 2002). This down regulation has not been clearly described. The gene was localised to chromosome 3p27, a region highlighted as affecting genetic vulnerability to obesity and type 2 diabetes. Supplementation by varying forms of adiponectin improved control of insulin, blood Berbelin, a herbal folk medicine, has been shown to increase adiponectin expression which A reduction in adiponectin expression is associated with insulin resistance in animal models. Administration of adiponectin has been accompanied by a reduction in plasma glucose and an increase in insulin sensitivity. In addition, thiazolidinediones, drugs that increase insulin sensitivity by stimulation of the peroxisome proliferator-activated receptor- A recent study has shown that phosphorylation and Levels of the hormone are conversely correlated with body fat proportion in adults although the association in infants and young children is less clear. The hormone plays a role in the inhibition of the metabolic disturbance that may end in type 2 diabetes (Ukkola & Santaniemi, 2002), obesity, atherosclerosis (Díez & Iglesias, 2003), non-alcoholic fatty liver diseases (NAFLD) and independent risk factors for metabolic syndrome (Renaldi et al., 2009)., Levels of adiponectin are decreased in diabetics compared to non-diabetics. Weight loss significantly increases circulating levels Adiponectin automatically self-associates into larger structures. Initially, three adiponectin molecules bind together to form a homotrimer. The trimers continue to self-associate and form hexamers or dodecamers. Like the plasma concentration, the relative levels of the higher-order structures are sexually dimorphic, where females have increased proportions partly explains its advantageous effects on metabolic disturbances (Choi et al., 2009). 2. Adipocyte mediators 2.1.1 Adiponectin structure 2.1 Adiponectin (Coppola et al., 2009). of the high-molecular weight forms. influence activity of other tissues (Nedvidkova et al., 2005). concentration than males (Díez & Iglesias, 2003). glucose and triglyceride levels in mouse models. δ, increase plasma adiponectin and mRNA levels in mice. Adiponectin affects glucose flux through decreasing gluconeogenesis and increasing glucose uptake (Díez & Iglesias, 2003; Nedvídková et al., 2005; Vasseur et al., 2003). Adiponectin has a role in lipid catabolism (Vasseur et al., 2003) by β-oxidation and triglyceride clearance (Nedvídková et al., 2005). The other metabolic effects of adiponectin are protection against endothelial dysfunction and improvement of insulin sensitivity and weight loss, and control of energy metabolism (Vasseur et al., 2006).Adiponectin has also another effects, including putative insulin-sensitising, anti-atherogenic and anti inflammatory characteristics (Berg et al., 2002; Ukkola & Santaniemi, 2002). Consistent with the low circulating levels of adiponectin observed in type 2 diabetes, adiponectin concentration is contrarily related to both insulin resistance and central adiposity (Arita et al., 1999; Weyer et al., 2001). Low baseline adiponectin concentration can predict the subsequent development of insulin resistance although elevated baseline levels have been shown to be protective against the succeeding development of type 2 diabetes (Daimon et al., 2003; Lindsay et al., 2002; Snehalatha et al., 2003; Spranger et al. , 2003). Recognition of gestational diabetes mellitus helps in identification of populations of young women at high risk of developing type 2 diabetes. Therefore, reduction in serum adiponectin levels in GDM may be comparable to type 2 diabetes. #### 2.1.3 Adiponectin and GDM Some studies exhibited hypoadiponectinemia in pregnant women with gestational diabetes. A cross sectional study of 180 women in their third trimesters illustrated gestational diabetic women had more hypoadiponectinemia compared with normoglycemic controls after adjustment for covariates including insulin resistance. This study showed adiponectin concentration is an independent correlate of beta cell function in late pregnancy. Adiponectin was correlated with insulin secretion-sensitivity index (ISSI). ISSI was positively correlated with adiponectin and negatively correlated with GDM and IGT. Therefore, adiponectin may play a principal role in mediating insulin resistance and beta cell malfunction in the development of diabetes (Rentakaran et al., 2005).To determine whether adiponectin relate to the postpartum metabolic disturbance linking GDM with type 2 diabetes a cohort study was done on 487 pregnant wemen in pregnancy and at 3 months post partum. This study demonstrated adiponectin was related to postpartum insulin sensitivity and hypoadiponectinemia in pregnancy predicts postpartum insulin resistance, beta cell dysfunction and fasting glycaemia, and hence may be relevant to the pathophysiology relating GDM with type 2 diabetes (Rentakaran et al., 2010). To investigate level of adiponectin and metabolic factors in women with gestational diabetes a cross sectional study was done . The result of this study showed the level of adiponectin was lower in gestational diabetes and adiponectin correlates negatively with insulin resistance in homeostasis model assessment-insulin resistance (HOMA-IR) (Altinova et al., 2007). To evaluate whether adiponectin is a predictive factor for gestational diabetes mellitus (GDM) and is appropriate as a screening test for GDM a study was done and the results The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes 119 diabetes. Based upon this study (Georgiou et al., 2008) the predictive threshold values for GDM at 11 weeks are >25µU/ml insulin and <3.5µg/ml adiponectin. This study confirmed the another survey (Wiliams et al., 2004) that a low blood concentration of adiponectin early In summary the results of case control and prospective studies demonstrated association of Leptin, a protein product of the obese (ob) gene with a 16 KDA molecular weight, is made of 167 amino acids (Hui-lan et al., 2004 ). It is a circulating hormone that is expressed plentifully in the adipose tissue. Leptin is also produced by non adipose tissue including the stomach, intestine and the placenta in humans (Masuzaki et al., 1997) and acts on the receptors of the hypothalamus to decrease food intake and increase energy consumption (Zhang et al., 1994). Leptin is encoded by the obesity gene. It signals to the brain when there has been enough eating to sustain body weight. It has been exhibited that laboratory mice have a mutation on the ob gene which inhibits the function of leptin causing the mice to become morbidly obese. In addition mutation in the gene that codes the leptin receptor causes obesity in the mice. Mutations in the ob gene affect expression of leptin, which in turn can cause obesity, infertility, and diabetes in lab mice (Zhang et al., 1997). It is hypothesized that alteration in the human ob gene causes some serious cases of early-onset obesity. Other less severe kinds of human obesity are considered not to be caused by mutation, but by changing in regulation or resistance to the action of leptin (Flier et al., 1995). Diminished production of leptin or fewer receptors may also be responsible for those who are overweight but not obese. It seems that the appropriate amount of leptin has to be present in the central nervous system to leptin act Homeostatic regulation of body weight depends on the capacity of the brain to sense and respond to changes in peripheral energy supplies. Insulin receptors were recognized in the brain and concentrated in the hypothalamus. These receptors have binding and signaling properties like peripheral insulin receptors. Leptin and insulin enter the brain and act on special hypothalamic neurons to prevent food intake and alter appetite. Leptin raise energy expenditure by regulating of neurotransmitters. (Porter et al., 2002). The evidence shows that insulin and leptin also act on the hypothalamus to control glucose production from the liver via stimulation of the autonomic nervous system. Thus, communication between leptin, insulin and significant hypothalamic neurons is necessary for normal energy balance and glucose homeostasis, deregulation of which will lead to obesity and type 2 diabetes. Indeed, in many forms of obesity, hypothalamic leptin and insulin resistance develops in which leptin and insulin are less effective in causing anorexia and decreased body weight. Some studies propose that leptin also has a specific effect on the regulation of whole body glucose homeostasis (Al-Dahhri et al., 2002; Ceddia et al., 2002). Numerous metabolic studies have shown a positive association between direct and indirect measures of adiposity with plasma leptin concentrations. Plasma leptin concentration correlated with BMI, percent of body fat and plasma insulin in both overweight/obese and normal weight and decreased in pregnancy is associated with increased risk of subsequent GDM. as weight lowering in human obesity (Zhang et al., 1997). after sustained weight loss (Havel et al., 1996). low adiponectin and GDM. 2.2.1 Leptin structure 2.2.2 Leptin action 2.2 Leptin demonstrated that adiponectin was an independent predictor for GDM. For GDM screening, adiponectin was not as strong a predictor as GCT. In any case, adiponectin could be used to rule out pregnant women at low risk of GDM (Weerakiet et al., 2006). In a cross sectional study serum level of adiponectin in GDM women and impaired glucose test and normal pregnant women was compared. This study showed the serum adiponectin level in gestational diabetes was significantly lower than the impaired glucose test and control groups and a negative correlation between prepregnancy BMI and adiponectin (Soheilykhah et al., 2009). In another study, class A2 and B gestational diabetes are associated with a suppressed level of adiponectin (Thyfault et al., 2005 ). To investigate changes in serum adiponectin during pregnancy and postpartum and assess its relationship with insulin resistance as measured by homeostasis model assessment (HOMA-IR) a study was done and revealed adiponectin was lower in gestational diabetes than in control groups during pregnancy and a significant post reduction in adiponectin was observed in maternal adiponectin after delivery indicating a significant placental contribution to adiponectin production (Vitoratos et al., 2008). The result of the another study identified weight gain as the strongest factor associated with declining β cell compensation for insulin resistance in Hispanic women at high risk for type 2 diabetes. Such effects may be mediated through at least two mechanisms: alteration in adipokine levels and increasing insulin resistance (Xiang et al., 2010). A prospective, nested case control study showed adiponectin concentration in early pregnancy was significantly lower in women with GDM than controls (4.4 vs. 8.1), approximately 73% of women with GDM, compared with 33% of controls, had adiponectin concentration less than 6.4µg/ml. After adjustment for confounding, women with adiponectin concentration in early pregnancy, less than 6.4µg/ml, experienced a 4.6-fold increased risk of GDM as compared with those with higher concentrations (Williams et al., 2004). Postpartum follow-up showed that women developing metabolic syndrome had significantly lower adiponectin and resistin concentration during pregnancy. Nonetheless, after adjustment for age, prepregnancy BMI, logistic regression analysis did not show independent relation between adiponectin and resistin with development of postpartum metabolic syndrome (Hossein-nezhad et al., 2010 ). To evaluate plasma adiponectin levels, insulin resistance and glucose tolerance in women with gestational diabetes mellitus and in normal pregnancies adiponectin measurement were performed two times as in 28–31 weeks of pregnancy and at 3 months after delivery. Insulin resistance was calculated by the homeostasis model assessment. This study demonstrated decreased adiponectin levels in GDM do not normalize instantaneously following the delivery. The difference is more apparent in adiponectin levels than in HOMA-IR (Cavit et al., 2007). Low plasma adiponectin concentration was associated with GDM in another study and adiponectin mRNA levels in adipose tissue biopsies from GDM subjects were reduced (Ranheim et al., 2004). Concentration of adiponectin may change before the appearance of the abnormal glucose level during pregnancy (Xue-lian et al., 2008). To identify potential biomarkers for impending gestational diabetes that appear in the plasma before impaired glucose tolerance a prospectively study was done and the results demonstrated a significant difference in insulin and adiponectin concentration at 11 weeks gestation and, compared to control groups, women with gestational diabetes exhibited elevated plasma insulin and reduced plasma adiponectin at 28 weeks gestation. Bivariate logistic regression analysis showed that both insulin and adiponectin were associated with subsequent development of gestational diabetes. Based upon this study (Georgiou et al., 2008) the predictive threshold values for GDM at 11 weeks are >25µU/ml insulin and <3.5µg/ml adiponectin. This study confirmed the another survey (Wiliams et al., 2004) that a low blood concentration of adiponectin early in pregnancy is associated with increased risk of subsequent GDM. In summary the results of case control and prospective studies demonstrated association of low adiponectin and GDM. #### 2.2 Leptin 118 Gestational Diabetes demonstrated that adiponectin was an independent predictor for GDM. For GDM screening, adiponectin was not as strong a predictor as GCT. In any case, adiponectin could be used to rule out pregnant women at low risk of GDM (Weerakiet et al., 2006). In a cross sectional study serum level of adiponectin in GDM women and impaired glucose test and normal pregnant women was compared. This study showed the serum adiponectin level in gestational diabetes was significantly lower than the impaired glucose test and control groups and a negative correlation between prepregnancy BMI and adiponectin (Soheilykhah et al., 2009). In another study, class A2 and B gestational diabetes are associated with a suppressed level of adiponectin (Thyfault et al., 2005 ). To investigate changes in serum adiponectin during pregnancy and postpartum and assess its relationship with insulin resistance as measured by homeostasis model assessment (HOMA-IR) a study was done and revealed adiponectin was lower in gestational diabetes than in control groups during pregnancy and a significant post reduction in adiponectin was observed in maternal adiponectin after delivery indicating a significant placental contribution to adiponectin production (Vitoratos et al., 2008). The result of the another study identified weight gain as the strongest factor associated with declining β cell compensation for insulin resistance in Hispanic women at high risk for type 2 diabetes. Such effects may be mediated through at least two mechanisms: alteration in adipokine levels and increasing insulin resistance (Xiang et al., 2010). A prospective, nested case control study showed adiponectin concentration in early pregnancy was significantly lower in women with GDM than controls (4.4 vs. 8.1), approximately 73% of women with GDM, compared with 33% of controls, had adiponectin concentration less than 6.4µg/ml. After adjustment for confounding, women with adiponectin concentration in early pregnancy, less than 6.4µg/ml, experienced a 4.6-fold increased risk of GDM as compared with those with higher concentrations (Williams et al., 2004). Postpartum follow-up showed that women developing metabolic syndrome had significantly lower adiponectin and resistin concentration during pregnancy. Nonetheless, after adjustment for age, prepregnancy BMI, logistic regression analysis did not show independent relation between adiponectin and resistin with development of postpartum metabolic syndrome (Hossein-nezhad et al., 2010 ). To evaluate plasma adiponectin levels, insulin resistance and glucose tolerance in women with gestational diabetes mellitus and in normal pregnancies adiponectin measurement were performed two times as in 28–31 weeks of pregnancy and at 3 months after delivery. Insulin resistance was calculated by the homeostasis model assessment. This study demonstrated decreased adiponectin levels in GDM do not normalize instantaneously following the delivery. The difference is more apparent in adiponectin levels than in HOMA-IR (Cavit et al., 2007). Low plasma adiponectin concentration was associated with GDM in another study and adiponectin mRNA levels in adipose tissue biopsies from GDM subjects were reduced (Ranheim et al., 2004). Concentration of adiponectin may change before the appearance of the abnormal glucose level during pregnancy (Xue-lian et al., 2008). To identify potential biomarkers for impending gestational diabetes that appear in the plasma before impaired glucose tolerance a prospectively study was done and the results demonstrated a significant difference in insulin and adiponectin concentration at 11 weeks gestation and, compared to control groups, women with gestational diabetes exhibited elevated plasma insulin and reduced plasma adiponectin at 28 weeks gestation. Bivariate logistic regression analysis showed that both insulin and adiponectin were associated with subsequent development of gestational #### 2.2.1 Leptin structure Leptin, a protein product of the obese (ob) gene with a 16 KDA molecular weight, is made of 167 amino acids (Hui-lan et al., 2004 ). It is a circulating hormone that is expressed plentifully in the adipose tissue. Leptin is also produced by non adipose tissue including the stomach, intestine and the placenta in humans (Masuzaki et al., 1997) and acts on the receptors of the hypothalamus to decrease food intake and increase energy consumption (Zhang et al., 1994). Leptin is encoded by the obesity gene. It signals to the brain when there has been enough eating to sustain body weight. It has been exhibited that laboratory mice have a mutation on the ob gene which inhibits the function of leptin causing the mice to become morbidly obese. In addition mutation in the gene that codes the leptin receptor causes obesity in the mice. Mutations in the ob gene affect expression of leptin, which in turn can cause obesity, infertility, and diabetes in lab mice (Zhang et al., 1997). It is hypothesized that alteration in the human ob gene causes some serious cases of early-onset obesity. Other less severe kinds of human obesity are considered not to be caused by mutation, but by changing in regulation or resistance to the action of leptin (Flier et al., 1995). Diminished production of leptin or fewer receptors may also be responsible for those who are overweight but not obese. It seems that the appropriate amount of leptin has to be present in the central nervous system to leptin act as weight lowering in human obesity (Zhang et al., 1997). #### 2.2.2 Leptin action Homeostatic regulation of body weight depends on the capacity of the brain to sense and respond to changes in peripheral energy supplies. Insulin receptors were recognized in the brain and concentrated in the hypothalamus. These receptors have binding and signaling properties like peripheral insulin receptors. Leptin and insulin enter the brain and act on special hypothalamic neurons to prevent food intake and alter appetite. Leptin raise energy expenditure by regulating of neurotransmitters. (Porter et al., 2002). The evidence shows that insulin and leptin also act on the hypothalamus to control glucose production from the liver via stimulation of the autonomic nervous system. Thus, communication between leptin, insulin and significant hypothalamic neurons is necessary for normal energy balance and glucose homeostasis, deregulation of which will lead to obesity and type 2 diabetes. Indeed, in many forms of obesity, hypothalamic leptin and insulin resistance develops in which leptin and insulin are less effective in causing anorexia and decreased body weight. Some studies propose that leptin also has a specific effect on the regulation of whole body glucose homeostasis (Al-Dahhri et al., 2002; Ceddia et al., 2002). Numerous metabolic studies have shown a positive association between direct and indirect measures of adiposity with plasma leptin concentrations. Plasma leptin concentration correlated with BMI, percent of body fat and plasma insulin in both overweight/obese and normal weight and decreased after sustained weight loss (Havel et al., 1996). The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes 121 prepregnancy adiposity and other confounders, women with leptin concentrations of 31ng/ml or higher experienced a 4.7-fold increased risk of GDM as compared with women who had concentrations of 14.3ng/ml or lower. Each 10-ng/ml increase in the leptin concentration was associated with a 20% increase in GDM risk (Qiu et al., 2004). Serum leptin level is correlated with glucose tolerance during pregnancy (Liu et al., 2003). In a casecontrol study, at 28 weeks of gestation, fasting serum concentration of leptin, insulin and homeostatic model assessment index were measured in three groups, GDM, IGT, and normal control, and compared them with each other. This study demonstrated that the serum leptin level was significantly higher in women with GDM than in the two other groups ( p = 0.03). In women with GDM and IGT, leptin was significantly positively related with insulin and homeostatic model assessment index (r = 0.221, p = 0.03) and (r = 0.246, p = In a case-control study, noted that maternal third trimester leptin concentrations were significantly lower in GDM cases as compared with controls after adjusting for possible confounding factors such as BMI and insulin concentrations (Festa et al., 1999). Concentration of leptin increased before the appearance of the abnormal glucose level during pregnancy (Xue-lian et al., 2008). Some studies evaluated the relationship between leptin concentration and insulin resistance. Leptin concentration was positively associated with insulin level and HOMA index (Maghbooli et al., 2007). A positive and significant correlation between the maternal leptin and fasting insulin levels (Liu et al., 2003) and also has shown that leptin predicts the development of GDM independent of maternal BMI and other risk factors. The findings of Kautzky-Willer et al are generally consistent with the Different reports. (Kautzky-Willer et al., 2001 ,Lappas et al., 2005 Qiu et al., 2004). Several possible explanations are suggested for the disparities in the existing studies. The study design and the confounding factors such as the time of blood sampling (whether blood samples were collected before, after, or during labor) and maternal factors, including whether women were treated with medication or diet before blood was collected for leptin determination might account for differences. Moreover, variations in population characteristics and status of glycemic control could also account for some of the observed In summary, the results of different studies from experimental, clinical, and epidemiological investigations suggest that leptin is an important mediator of glucose homeostasis in pregnancy (Qiu et al., 2004) and measurement of leptin alone, or combined with the assessment of other risk factors, may help identify women at risk of developing GDM. Insulin resistance is strongly associated with obesity, but even among obese subjects insulin sensitivity is different. Recently, a new adipocyte hormone, resistin, was identified, shown to decrease insulin-mediated glucose uptake, and shown to be increased in obese mice Resistin, also known as adipose tissue-specific secretory factor , is a cystein-rich protein that in humans is encoded by the RETN gene (Wang et al., 2002). Resistin was discovered to be produced and released from adipose tissue to provide endocrine functions likely involved in insulin resistance. This thought initially developed from studies exhibiting that serum resistin levels increase with obesity in several species (humans, rats, and mice) . (Yamauchi et al., 2003; Gabriely et al., 2002; Steppan et al., 2001). Resistin is also produced by several 0.03), respectively. (Soheilykhah et al., 2011) differences in the study results. 2.3 Resistin 2.3.1 Resistin structure #### 2.2.3 Leptin and gestational diabetes The increased risk of GDM with increasing maternal plasma concentration of leptin is biologically plausible and is likely accounted for by diverse molecular and biochemical pathways in multiple tissue. Leptin has been shown to regulate peripheral glucose homeostasis through its action in skeletal muscle and its effects on hepatic gene expression of the gluconeogenetic enzymes and phosphoenolpyruvate carboxykinase (Rossetti et al., 1997). In addition, leptin has been shown to directly modulate glucose handling in skeletal muscle by promoting fatty acid oxidation. Investigators have postulated that leptin induced insulin resistance may be secondary to glucose flux via the hexosamine pathway (Mueller et al., 1998). Although the pathophysiology of hyperleptinemia in GDM is unknown, it is clear that leptin has numerous actions on target tissues and is involved in regulation on several endocrine pathways. Although the biologic action of leptin primarily mediated through interactions with receptors expressed in the hypothalamus, leptin receptors are widely distributed across other tissues including the lungs, liver, kidney, pancreas, heart and the placenta (Chen et al., 1999; Hoggard et al., 1997; Kieffer et al., 1996; Schulz et al., 2000). This wide distribution of leptin receptors portends the peptide diverse influence on neuroendocrine, cardiovascular and reproductive functions. Leptin is correlated with a series of endocrine parameters including insulin, insulin-like growth factors, hemoglobin A1c and sex hormone-binding globulin In pregnant women with changes in maternal fat stores and glucose metabolism, leptin increases (Schubring et al., 2000). Maternal leptin concentration increases 2 to 3 times above the non-pregnant concentration with the peak around 28 weeks of gestation (Schubring et al., 2000). The serum leptin level relates to body weight, body mass index, fat accumulation of the pregnant woman, fetal growth and development and fetal fat deposits. In recent years, it was considered that leptin is associated in pregnancy-induced hypertension syndrome, fetal intrauterine growth retardation and gestational diabetes. Increasing maternal plasma leptin levels may result from an upregulation of adipocyte leptin synthesis in the presence of increasing insulin resistance and hyperinsulinemia in the second half of pregnancy (Laivuori et al., 2000; Rossetti et al., 1997). Investigators have shown that leptin directly affects whole body insulin sensitivity through regulating the efficiency of insulinmediated glucose metabolism by skeletal muscle (Cohen et al., 1996) and by hepatic regulation of gluconeogenesis (Rossetti et al., 1997). Some evidence suggests that leptin has an acute inhibitory effect on secretion of insulin (Ceddia et al., 2002). Large epidemiological studies have shown that plasma leptin concentrations were positively associated with insulin resistance in men and non-pregnant women (Donahue et al., 1999). Available data suggest a complex relation between leptin and glucose homeostasis in humans. Two teams of investigators have studied maternal leptin concentrations in GDM women and the related published results are conflicting. On the other hand, the available data do not explain whether the alterations in leptin concentrations are the cause or result of the metabolic disturbances, such as hyperglycemia, that are essential to GDM. In addition, the severity of any possible association of GDM risk with different concentrations of leptin was not assessed in either study (Festa et al., 1999; Kautzky-Willer et al., 2001). In a casecontrol study reported that maternal third-trimester plasma leptin concentrations were higher in GDM women compared with the control group (24.9 versus 18.2 ng/mL; P=0.001) (Kautzky-Willer et al., 2001). Such a relation was also found in other study. (Vitoratus et al., 2000) Hyperleptinemia, independent of maternal adiposity, in early pregnancy appears to be predictive of an increased risk of GDM later in pregnancy. After adjusting for maternal prepregnancy adiposity and other confounders, women with leptin concentrations of 31ng/ml or higher experienced a 4.7-fold increased risk of GDM as compared with women who had concentrations of 14.3ng/ml or lower. Each 10-ng/ml increase in the leptin concentration was associated with a 20% increase in GDM risk (Qiu et al., 2004). Serum leptin level is correlated with glucose tolerance during pregnancy (Liu et al., 2003). In a casecontrol study, at 28 weeks of gestation, fasting serum concentration of leptin, insulin and homeostatic model assessment index were measured in three groups, GDM, IGT, and normal control, and compared them with each other. This study demonstrated that the serum leptin level was significantly higher in women with GDM than in the two other groups ( p = 0.03). In women with GDM and IGT, leptin was significantly positively related with insulin and homeostatic model assessment index (r = 0.221, p = 0.03) and (r = 0.246, p = 0.03), respectively. (Soheilykhah et al., 2011) In a case-control study, noted that maternal third trimester leptin concentrations were significantly lower in GDM cases as compared with controls after adjusting for possible confounding factors such as BMI and insulin concentrations (Festa et al., 1999). Concentration of leptin increased before the appearance of the abnormal glucose level during pregnancy (Xue-lian et al., 2008). Some studies evaluated the relationship between leptin concentration and insulin resistance. Leptin concentration was positively associated with insulin level and HOMA index (Maghbooli et al., 2007). A positive and significant correlation between the maternal leptin and fasting insulin levels (Liu et al., 2003) and also has shown that leptin predicts the development of GDM independent of maternal BMI and other risk factors. The findings of Kautzky-Willer et al are generally consistent with the Different reports. (Kautzky-Willer et al., 2001 ,Lappas et al., 2005 Qiu et al., 2004). Several possible explanations are suggested for the disparities in the existing studies. The study design and the confounding factors such as the time of blood sampling (whether blood samples were collected before, after, or during labor) and maternal factors, including whether women were treated with medication or diet before blood was collected for leptin determination might account for differences. Moreover, variations in population characteristics and status of glycemic control could also account for some of the observed differences in the study results. In summary, the results of different studies from experimental, clinical, and epidemiological investigations suggest that leptin is an important mediator of glucose homeostasis in pregnancy (Qiu et al., 2004) and measurement of leptin alone, or combined with the assessment of other risk factors, may help identify women at risk of developing GDM. #### 2.3 Resistin 120 Gestational Diabetes The increased risk of GDM with increasing maternal plasma concentration of leptin is biologically plausible and is likely accounted for by diverse molecular and biochemical pathways in multiple tissue. Leptin has been shown to regulate peripheral glucose homeostasis through its action in skeletal muscle and its effects on hepatic gene expression of the gluconeogenetic enzymes and phosphoenolpyruvate carboxykinase (Rossetti et al., 1997). In addition, leptin has been shown to directly modulate glucose handling in skeletal muscle by promoting fatty acid oxidation. Investigators have postulated that leptin induced insulin resistance may be secondary to glucose flux via the hexosamine pathway (Mueller et al., 1998). Although the pathophysiology of hyperleptinemia in GDM is unknown, it is clear that leptin has numerous actions on target tissues and is involved in regulation on several endocrine pathways. Although the biologic action of leptin primarily mediated through interactions with receptors expressed in the hypothalamus, leptin receptors are widely distributed across other tissues including the lungs, liver, kidney, pancreas, heart and the placenta (Chen et al., 1999; Hoggard et al., 1997; Kieffer et al., 1996; Schulz et al., 2000). This wide distribution of leptin receptors portends the peptide diverse influence on neuroendocrine, cardiovascular and reproductive functions. Leptin is correlated with a series of endocrine parameters including insulin, insulin-like growth factors, hemoglobin In pregnant women with changes in maternal fat stores and glucose metabolism, leptin increases (Schubring et al., 2000). Maternal leptin concentration increases 2 to 3 times above the non-pregnant concentration with the peak around 28 weeks of gestation (Schubring et al., 2000). The serum leptin level relates to body weight, body mass index, fat accumulation of the pregnant woman, fetal growth and development and fetal fat deposits. In recent years, it was considered that leptin is associated in pregnancy-induced hypertension syndrome, fetal intrauterine growth retardation and gestational diabetes. Increasing maternal plasma leptin levels may result from an upregulation of adipocyte leptin synthesis in the presence of increasing insulin resistance and hyperinsulinemia in the second half of pregnancy (Laivuori et al., 2000; Rossetti et al., 1997). Investigators have shown that leptin directly affects whole body insulin sensitivity through regulating the efficiency of insulinmediated glucose metabolism by skeletal muscle (Cohen et al., 1996) and by hepatic regulation of gluconeogenesis (Rossetti et al., 1997). Some evidence suggests that leptin has an acute inhibitory effect on secretion of insulin (Ceddia et al., 2002). Large epidemiological studies have shown that plasma leptin concentrations were positively associated with Available data suggest a complex relation between leptin and glucose homeostasis in humans. Two teams of investigators have studied maternal leptin concentrations in GDM women and the related published results are conflicting. On the other hand, the available data do not explain whether the alterations in leptin concentrations are the cause or result of the metabolic disturbances, such as hyperglycemia, that are essential to GDM. In addition, the severity of any possible association of GDM risk with different concentrations of leptin was not assessed in either study (Festa et al., 1999; Kautzky-Willer et al., 2001). In a casecontrol study reported that maternal third-trimester plasma leptin concentrations were higher in GDM women compared with the control group (24.9 versus 18.2 ng/mL; P=0.001) (Kautzky-Willer et al., 2001). Such a relation was also found in other study. (Vitoratus et al., 2000) Hyperleptinemia, independent of maternal adiposity, in early pregnancy appears to be predictive of an increased risk of GDM later in pregnancy. After adjusting for maternal insulin resistance in men and non-pregnant women (Donahue et al., 1999). 2.2.3 Leptin and gestational diabetes A1c and sex hormone-binding globulin #### 2.3.1 Resistin structure Insulin resistance is strongly associated with obesity, but even among obese subjects insulin sensitivity is different. Recently, a new adipocyte hormone, resistin, was identified, shown to decrease insulin-mediated glucose uptake, and shown to be increased in obese mice Resistin, also known as adipose tissue-specific secretory factor , is a cystein-rich protein that in humans is encoded by the RETN gene (Wang et al., 2002). Resistin was discovered to be produced and released from adipose tissue to provide endocrine functions likely involved in insulin resistance. This thought initially developed from studies exhibiting that serum resistin levels increase with obesity in several species (humans, rats, and mice) . (Yamauchi et al., 2003; Gabriely et al., 2002; Steppan et al., 2001). Resistin is also produced by several The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes 123 development of gestational diabetes. Resistin levels were significantly higher in normal pregnant women than in nonpregnant controls and showed a negative correlation with gestational age. Resistin was detected in the umbilical venous blood in fetus from 20 to 41 weeks of gestation. Detection of high levels of resistin in cord blood during gestation is consistent with a regulatory action of these adipokines on tissue differentiation and foetal growth (Cortelazzi et al., 2007). Recent reports have measured the level of resistin during pregnancies complicated by gestational diabetes with inconsistent results (Cortelazzi et al., 2007, Chen et al., 2007). The result of a study showed resistin level in gestational diabetes was lower than normal pregnancy(Megia et al.,2008)This result inconsistent with other study that demonstrated serum resistin concentration was significantly higher in women with GDM than in controls before delivery and the serum levels of resistin significantly decreased after delivery in both the GDM group and controls.The serum level of resistin was different on days 1 and 3 but not by day 5 after delivery (Chen et al.,2007). The serum resistin levels were higher in the 1st, 2nd and 3rd trimesters of pregnancy and higher in GDM than in control groups and hyperresistinemia may also be associated with the pregnancy-induced insulin resistance (Palik et al., 2007). The discrepancy of these findings is unclear, but may be related to different populations of the studies, the type of study or the C-reactive protein (CRP) is a sensitive marker of systemic infection and is widely used in clinical settings (Kushner I& Rzewnicki, 1994). CRP was first detected in 1930 by Tillet and Frances (Tillett & Francis, 1930), who identified a substance that formed a precipitate when combined with polysaccharide C of streptococcus pneumonia in the sera of patients acutely infected with pneumococcal pneumonia. Subsequently, it was found that this reaction was not unique to pneumococcal pneumonia but could be found with a variety of the other acute infections. This was early evidence of the body's chemical response to inflammatory states and led to characterization of other so called acute phase proteins (Abernathy & Avery, 1941). CRP is normally present in low levels in serum but increase rapidly and dramatically in response to a variety of infectious or inflammatory conditions (Ballou & Kushner, 1992). Since its discovery, CRP has been studied as a screening device for occult inflammation, as a marker of disease activity and as a diagnostic tool (Pepys, 1981). Recently, more rapid and accurate methods of quantifying CRP have lead to a new interest in its value in clinical medicine (Palosuo et al., 1986) although low-grade inflammatory states not originated from infections and atherosclerosis have also been associated with an increase in CRP levels. For instance, obesity is linked to chronic subclinical inflammation as manifested by mild increases in CRP cytokines and adipocytokines and is the principal risk factor for type 2 diabetes. In addition, patients with increases in CRP are at risk of myocardial infarction and peripheral arterial disease (Engström et al., 2003; Ford, 1999; Ridker et al., 2000; Tracy et al., 1997; Yudkin et al., 1999). Elevated serum concentration of acute-phase proteins, exhibiting chronic subclinical inflammation, has been associated with insulin resistance syndrome in men and non-pregnant women (Festa et al., 2000; Han et al., 2002; Pradhan et al., 2002; Ridker et al., 2003). The molecular basis for the association between the inflammation and diabetes related to the action of cytokines such as interlukin-6 and tumor necrosis factor (TNF)-α which lead to time of sampling during pregnancy. 3.1 C-reactive structure and actions 3. Inflammatory mediators (C-reactive protein) other tissues, including the hypothalamus, pituitary and adrenal glands, pancreas, gastrointestinal tract, myocytes, spleen and white blood cells The role of resistin in obesity and insulin resistance in humans is controversial. #### 2.3.2 Resistin action Resistin causes insulin resistance and glucose intolerance in mice. Serum resistin levels will rise with increased adiposity, particularly central obesity (Degawa-Yamauchi et al., 2003; Vendrell et al., 2004). Conversely, serum resistin levels decrease with lowering adiposity following medical treatment (Valsamakis et al., 2004). The level of tissue resistin is decreased by insulin, cytokines such as tumour necrosis factor, α endothelin-1 and increased by growth and gonadal hormones, hyperglycaemia, interleukin-6 and lipopolysaccharide (Adeghate, 2004). Animal study exhibited that resistin gene expression and protein levels are regulated in parallel with glucose and insulin during fasting and feeding Many researchers have shown positive correlations between resistin levels and insulin resistance.Thus resistin has been suggested to link obesity with type 2 diabetes (Hirosumi et al., 2002; Rajala et al., 2004; Silha et al., 2003; Smith et al., 2003). This discovery is further confirmed by studies which verified a direct correlation between resistin levels and subjects with type 2DM (Asensio et al., 2004; Fujinami et al., 2004; McTernan et al., 2003; Steppan et al., 2001). With the finding that resistin was at least in part a cause of the insulin resistance and T2DM, medications which specifically lead to decreased serum resistin in T2DM subjects were developed(Tjokroprawiro, 2006). The level of circulating resistin is decreased by the anti-diabetic drug such as rosiglitazone and increased by obesity. Administration of the anti resistin antibody decreases blood sugar and improve insulin action in mice with diet induced obesity. Treatment of normal mice with recombinant resistin impairs glucose tolerance and decreases insulin action. Insulin stimulated glucose uptake by adipocyte is increased by neutralization of resistin and is reduced by resistin treatment (Steppan et al., 2001). Because resistin is identical to a protein which had a role in allergic pulmonary infiltration, the effect of resistin in inflammation was studied and these researches demonstrated association of resistin with other physiological systems such as inflammation and energy homeostasis (Adeghate, 2004; Stumvoll & Häring, 2002; Vendrell et al., 2004). #### 2.3.3 Resistin and gestational diabetes Resistin is expressed in human placenta and has been supposed to play a role in regulating energy metabolism in pregnancy. Resistin protein expression in placental tissue was much higher than that in subcutaneous adipose tissue in normal human abdomen, pregnant abdomen and thigh. It was indicated that resistin protein can be secreted from human placental tissue. Resistin might be one of the factors that lead to pregnant physiological insulin resistance and GDM (Yongming et al., 2006).Resistin is secreted by the placenta during human pregnancy (Sagawa et al., 2002) Serum resistin levels are not different among non-pregnant women and women in the first and second trimesters of pregnancy .Serum resistin increases by the third trimester (Chen et al., 2005). Resistin is detectable at 20 weeks of gestation. In newborns, resistin concentrations were two to three-fold higher than those reported in adults regardless of sex, birth weight, pattern of growth or metabolic state of the mother (Yongming et al., 2006). Resistin gene expression is found in placental tissues during pregnancy (Yura et al., 2003) and it has been supposed that it could be involved in the pathogenesis of the insulin resistance state found in the second half of pregnancy and in the development of gestational diabetes. Resistin levels were significantly higher in normal pregnant women than in nonpregnant controls and showed a negative correlation with gestational age. Resistin was detected in the umbilical venous blood in fetus from 20 to 41 weeks of gestation. Detection of high levels of resistin in cord blood during gestation is consistent with a regulatory action of these adipokines on tissue differentiation and foetal growth (Cortelazzi et al., 2007). Recent reports have measured the level of resistin during pregnancies complicated by gestational diabetes with inconsistent results (Cortelazzi et al., 2007, Chen et al., 2007). The result of a study showed resistin level in gestational diabetes was lower than normal pregnancy(Megia et al.,2008)This result inconsistent with other study that demonstrated serum resistin concentration was significantly higher in women with GDM than in controls before delivery and the serum levels of resistin significantly decreased after delivery in both the GDM group and controls.The serum level of resistin was different on days 1 and 3 but not by day 5 after delivery (Chen et al.,2007). The serum resistin levels were higher in the 1st, 2nd and 3rd trimesters of pregnancy and higher in GDM than in control groups and hyperresistinemia may also be associated with the pregnancy-induced insulin resistance (Palik et al., 2007). The discrepancy of these findings is unclear, but may be related to different populations of the studies, the type of study or the time of sampling during pregnancy. #### 3. Inflammatory mediators (C-reactive protein) #### 3.1 C-reactive structure and actions 122 Gestational Diabetes other tissues, including the hypothalamus, pituitary and adrenal glands, pancreas, Resistin causes insulin resistance and glucose intolerance in mice. Serum resistin levels will rise with increased adiposity, particularly central obesity (Degawa-Yamauchi et al., 2003; Vendrell et al., 2004). Conversely, serum resistin levels decrease with lowering adiposity following medical treatment (Valsamakis et al., 2004). The level of tissue resistin is decreased by insulin, cytokines such as tumour necrosis factor, α endothelin-1 and increased by growth and gonadal hormones, hyperglycaemia, interleukin-6 and lipopolysaccharide (Adeghate, 2004). Animal study exhibited that resistin gene expression and protein levels are regulated in parallel with glucose and insulin during fasting and feeding Many researchers have shown positive correlations between resistin levels and insulin resistance.Thus resistin has been suggested to link obesity with type 2 diabetes (Hirosumi et al., 2002; Rajala et al., 2004; Silha et al., 2003; Smith et al., 2003). This discovery is further confirmed by studies which verified a direct correlation between resistin levels and subjects with type 2DM (Asensio et al., 2004; Fujinami et al., 2004; McTernan et al., 2003; Steppan et al., 2001). With the finding that resistin was at least in part a cause of the insulin resistance and T2DM, medications which specifically lead to decreased serum resistin in T2DM subjects were developed(Tjokroprawiro, 2006). The level of circulating resistin is decreased by the anti-diabetic drug such as rosiglitazone and increased by obesity. Administration of the anti resistin antibody decreases blood sugar and improve insulin action in mice with diet induced obesity. Treatment of normal mice with recombinant resistin impairs glucose tolerance and decreases insulin action. Insulin stimulated glucose uptake by adipocyte is increased by neutralization of resistin and is reduced by resistin treatment (Steppan et al., 2001). Because resistin is identical to a protein which had a role in allergic pulmonary infiltration, the effect of resistin in inflammation was studied and these researches demonstrated association of resistin with other physiological systems such as inflammation and energy homeostasis (Adeghate, 2004; Stumvoll & Häring, 2002; Vendrell et al., 2004). Resistin is expressed in human placenta and has been supposed to play a role in regulating energy metabolism in pregnancy. Resistin protein expression in placental tissue was much higher than that in subcutaneous adipose tissue in normal human abdomen, pregnant abdomen and thigh. It was indicated that resistin protein can be secreted from human placental tissue. Resistin might be one of the factors that lead to pregnant physiological insulin resistance and GDM (Yongming et al., 2006).Resistin is secreted by the placenta during human pregnancy (Sagawa et al., 2002) Serum resistin levels are not different among non-pregnant women and women in the first and second trimesters of pregnancy .Serum resistin increases by the third trimester (Chen et al., 2005). Resistin is detectable at 20 weeks of gestation. In newborns, resistin concentrations were two to three-fold higher than those reported in adults regardless of sex, birth weight, pattern of growth or metabolic state of the mother (Yongming et al., 2006). Resistin gene expression is found in placental tissues during pregnancy (Yura et al., 2003) and it has been supposed that it could be involved in the pathogenesis of the insulin resistance state found in the second half of pregnancy and in the The role of resistin in obesity and insulin resistance in humans is controversial. gastrointestinal tract, myocytes, spleen and white blood cells 2.3.2 Resistin action 2.3.3 Resistin and gestational diabetes C-reactive protein (CRP) is a sensitive marker of systemic infection and is widely used in clinical settings (Kushner I& Rzewnicki, 1994). CRP was first detected in 1930 by Tillet and Frances (Tillett & Francis, 1930), who identified a substance that formed a precipitate when combined with polysaccharide C of streptococcus pneumonia in the sera of patients acutely infected with pneumococcal pneumonia. Subsequently, it was found that this reaction was not unique to pneumococcal pneumonia but could be found with a variety of the other acute infections. This was early evidence of the body's chemical response to inflammatory states and led to characterization of other so called acute phase proteins (Abernathy & Avery, 1941). CRP is normally present in low levels in serum but increase rapidly and dramatically in response to a variety of infectious or inflammatory conditions (Ballou & Kushner, 1992). Since its discovery, CRP has been studied as a screening device for occult inflammation, as a marker of disease activity and as a diagnostic tool (Pepys, 1981). Recently, more rapid and accurate methods of quantifying CRP have lead to a new interest in its value in clinical medicine (Palosuo et al., 1986) although low-grade inflammatory states not originated from infections and atherosclerosis have also been associated with an increase in CRP levels. For instance, obesity is linked to chronic subclinical inflammation as manifested by mild increases in CRP cytokines and adipocytokines and is the principal risk factor for type 2 diabetes. In addition, patients with increases in CRP are at risk of myocardial infarction and peripheral arterial disease (Engström et al., 2003; Ford, 1999; Ridker et al., 2000; Tracy et al., 1997; Yudkin et al., 1999). Elevated serum concentration of acute-phase proteins, exhibiting chronic subclinical inflammation, has been associated with insulin resistance syndrome in men and non-pregnant women (Festa et al., 2000; Han et al., 2002; Pradhan et al., 2002; Ridker et al., 2003). The molecular basis for the association between the inflammation and diabetes related to the action of cytokines such as interlukin-6 and tumor necrosis factor (TNF)-α which lead to The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes 125 trimester predicted the development of GDM in the pregnancy. In this study risk of developing GDM among women in the highest CRP tertile compared with the lowest tertile was 3.2 (95% CI 1.2-8.8), after adjusting for age, race, smoking, parity, blood pressure and gestational age at CRP sampling. The risk of developing GDM among women in the highest compared with the lowest tertile was 3.6 (95%CI 1.2-11.4), when BMI was included in the model; however, the association between increased CRP and GDM was reduced (odds ratio for the highest compared with the lowest tertile 1.5 (95% CI 0.4-5.51). Therefore, this association is mediated in part by increasing BMI (Wolf et al., 2003) and pregestational Another prospective study was done to examine the association between CRP and GDM risk. Women were recruited before 16 weeks gestation and were followed until delivery. This study demonstrated elevated CRP was associated with GDM risk. After adjustment of maternal prepregnancy BMI, family history of diabetes and nulliparity, women with CRP in the highest tertile experienced a 3.5-fold increased risk of GDM (95% CI 1.2-9.8) as compared with those in the lowest tertile. The association between CRP and GDM was evident when analyses were restricted to lean women (BMI<25kg/m2). Lean women with CRP ≥5.3mg/l had a 3.7-fold increased risk of GDM (95%CI 1.6-8.7) as compared with women with CRP < 5.3mg/l. This study concluded systemic inflammation is associated with an increased risk of GDM and this association is independent of maternal prepregnancy adiposity (Qiu et al., 2004). Serum CRP in gestational diabetes and pregnant control women was evaluated and showed CRP was positively related with fructose amine hemoglobin A1c, triglyceride and BMI. This study concluded CRP plays a role in pathogenesis of GDM (Li et al., 2010). The association of sex hormone-binding globulin, high sensitive C-reactive protein and fasting glucose and insulin in the late first trimester and early second trimester of pregnancy with the diagnosis of gestational diabetes were also evaluated. In this study sex hormone-binding globulin was lower and high-sensitive CRP was higher among women who subsequently developed gestational diabetes. Multivariate analysis suggested that sex hormone-binding globulin measurement was the best predictor of GDM (Smirnakis et al., 2007). A 180 healthy pregnant women undergoing oral glucose tolerance testing in the late second or early third trimester were evaluated . Based on OGTT and prepregnancy BMI, participants were divided to 4 groups: (1) normal OGTT and BMI<25kg/m2; (2) normal OGTT and BMI >25; (3) impaired glucose tolerance, and (4) GDM. This study showed CRP level was higher in overweight women with normal OGTT, followed by GDM, impaired OGTT groups and lean normal GTT. This study demonstrated that maternal CRP are not related to GDM, but rather obesity as the most prominent risk factors of GDM (Wolf et al., 2003). correlate significantly with prepregnancy obesity (Rentakaran et al., 2003). Vitamin D, or calciferol, is a group of lipid soluble substance with a four-ringed cholesterol backbone. Human obtained Vitamin D from exposure to Sunlight, their diet and from dietary supplement. Ultraviolet light convert provitamin D to vitamin D3 (cholecalciferol) in the skin and afterwards Vitamin D3 was bounded by vitamin D binding proteins (DBP) and transported via blood to target organs for metabolism and activity. Vitamin D hydroxylate to form 25-hydroxy-vitamin-D (25OHD) in the liver. Hydroxylation of 25-hydroxy-vitamin-D to 1, 25-dihydroxy-vitamin D occurs in the mitochondria of the proximal tubules of the kidney.This form of vitamin D (1,25(OH)2-vitamin D) is the physiologically active form. The 4. Vitamin D 4.1 Vitamin D structure and actions insulin resistance and stimulate the acute phase inflammatory response (Fernandez-Real et al., 2001; Kern et al., 2001; Mohamed-Ali et al., 1998; Vozarova et al., 2001). #### 3.2 C-reactive protein and GDM Some evidence supports the theory that chronic inflammation might be a risk factor for developing type 2 diabetes (Freeman et al., 2002; Pradhan et al., 2001; Taniguchi et al., 2002; Thorand et al., 2003). Inflammation may have a role in the pathogenesis of diabetes, suggesting that inflammatory markers may identify patients at risk of diabetes. The issue was investigated in a subset of women (1584 who developed diabetes and 2193 who were normal after 6 years. Women with diabetes had higher median baseline level of interleukin-6 (IL6), high sensitivity CRP and tumor necrosis factor alpha receptor 2 compared with control.Two markers (IL6 and CRP) were significantly associated with diabetes risk in all ethnic groups (Liu et al., 2007).Similar results were obtained in the women's health study (Pradhan et al., 2001) and the nurse health study (Hu et al., 2004). In a population –based study in Mexico City, serum CRP was a predictor of the metabolic syndrome and type 2 diabetes in women but not in men (Han et al., 2002). Among middle aged men in Germany those with a serum CRP in the highest quartile ≥2.91ng/ml had an increased risk of type 2 diabetes compared with men in the lowest quartile (≤0.67ng/ml) (RR 2.7,95%CI1.4-5.2) (Thorand et al., 2003). Epidemiological studies have shown that CRP predicts incident type 2 diabetes and increased cardiovascular disease. In healthy middle-aged women, or young men, CRP levels were associated with a three–fourfold increased risk of developing diabetes (Buchanan,2001; kjos &Buchanan,1999). Very limited attention has been given to the role of inflammation in the etiology of gestational diabetes a condition that is biochemically and epidemiologically similar to type 2 diabetes (kjos & Buchanan,1999).The second and third trimesters of pregnancy represent a physiological type of insulin resistance (Kautzky-Willer et al., 1997). Insulin resistance is associated with dysfunction of endothelial and inflammation as well as increase production of cytokines by adipose tissue(Baalletshofer et al.,2000). Limited available data suggest that proinflammatory cytokines may be predictive of GDM(Winkler et al., 2002; Kirwan et al., 2002). Some studies have measured CRP at various gestational ages in pregnant women and found inconsistent results regarding the association between inflammatory markers and the incidence of GDM and the interdependence with the degree of adiposity (Retnakaran et al., 2003; Wolf et al., 2003,2004). The interpretation of the results was influenced by coexistence of hypertension, preeclampsia, and different race groups or small sample size in some studies. The result of a study demonstrated women with GDM had significantly higher CRP serum levels than normal pregnant women at 37-38 weeks of gestation but at the time of OGTT (24-28 weeks of pregnancy) there was not any significant difference between the two groups (Leipold et al. , 2005 ). This report is inconsistent with the findings of the Massachusetts General Hospital obstetric maternal report, where increased CRP concentration was shown in the first trimester (10 weeks of gestation) and the association between GDM and CRP depended primarily on coexisting obesity (Wolf et al., 2004). The result of another study demonstrated that CRP concentration is not affected by GDM until the end of the second trimester of pregnancy (Retnakaran et al., 2003). In the third trimester, however, these results suggest an up-regulation of inflammatory markers by GDM resulting in elevation of CRP concentration at the end of pregnancy in women with GDM. In a prospective nested case control study Wolf et al found that CRP concentration in the first trimester predicted the development of GDM in the pregnancy. In this study risk of developing GDM among women in the highest CRP tertile compared with the lowest tertile was 3.2 (95% CI 1.2-8.8), after adjusting for age, race, smoking, parity, blood pressure and gestational age at CRP sampling. The risk of developing GDM among women in the highest compared with the lowest tertile was 3.6 (95%CI 1.2-11.4), when BMI was included in the model; however, the association between increased CRP and GDM was reduced (odds ratio for the highest compared with the lowest tertile 1.5 (95% CI 0.4-5.51). Therefore, this association is mediated in part by increasing BMI (Wolf et al., 2003) and pregestational obesity as the most prominent risk factors of GDM (Wolf et al., 2003). Another prospective study was done to examine the association between CRP and GDM risk. Women were recruited before 16 weeks gestation and were followed until delivery. This study demonstrated elevated CRP was associated with GDM risk. After adjustment of maternal prepregnancy BMI, family history of diabetes and nulliparity, women with CRP in the highest tertile experienced a 3.5-fold increased risk of GDM (95% CI 1.2-9.8) as compared with those in the lowest tertile. The association between CRP and GDM was evident when analyses were restricted to lean women (BMI<25kg/m2). Lean women with CRP ≥5.3mg/l had a 3.7-fold increased risk of GDM (95%CI 1.6-8.7) as compared with women with CRP < 5.3mg/l. This study concluded systemic inflammation is associated with an increased risk of GDM and this association is independent of maternal prepregnancy adiposity (Qiu et al., 2004). Serum CRP in gestational diabetes and pregnant control women was evaluated and showed CRP was positively related with fructose amine hemoglobin A1c, triglyceride and BMI. This study concluded CRP plays a role in pathogenesis of GDM (Li et al., 2010). The association of sex hormone-binding globulin, high sensitive C-reactive protein and fasting glucose and insulin in the late first trimester and early second trimester of pregnancy with the diagnosis of gestational diabetes were also evaluated. In this study sex hormone-binding globulin was lower and high-sensitive CRP was higher among women who subsequently developed gestational diabetes. Multivariate analysis suggested that sex hormone-binding globulin measurement was the best predictor of GDM (Smirnakis et al., 2007). A 180 healthy pregnant women undergoing oral glucose tolerance testing in the late second or early third trimester were evaluated . Based on OGTT and prepregnancy BMI, participants were divided to 4 groups: (1) normal OGTT and BMI<25kg/m2; (2) normal OGTT and BMI >25; (3) impaired glucose tolerance, and (4) GDM. This study showed CRP level was higher in overweight women with normal OGTT, followed by GDM, impaired OGTT groups and lean normal GTT. This study demonstrated that maternal CRP are not related to GDM, but rather correlate significantly with prepregnancy obesity (Rentakaran et al., 2003). #### 4. Vitamin D 124 Gestational Diabetes insulin resistance and stimulate the acute phase inflammatory response (Fernandez-Real et al., Some evidence supports the theory that chronic inflammation might be a risk factor for developing type 2 diabetes (Freeman et al., 2002; Pradhan et al., 2001; Taniguchi et al., 2002; Thorand et al., 2003). Inflammation may have a role in the pathogenesis of diabetes, suggesting that inflammatory markers may identify patients at risk of diabetes. The issue was investigated in a subset of women (1584 who developed diabetes and 2193 who were normal after 6 years. Women with diabetes had higher median baseline level of interleukin-6 (IL6), high sensitivity CRP and tumor necrosis factor alpha receptor 2 compared with control.Two markers (IL6 and CRP) were significantly associated with diabetes risk in all ethnic groups (Liu et al., 2007).Similar results were obtained in the women's health study (Pradhan et al., 2001) and the nurse health study (Hu et al., 2004). In a population –based study in Mexico City, serum CRP was a predictor of the metabolic syndrome and type 2 diabetes in women but not in men (Han et al., 2002). Among middle aged men in Germany those with a serum CRP in the highest quartile ≥2.91ng/ml had an increased risk of type 2 diabetes compared with men in the lowest quartile (≤0.67ng/ml) (RR 2.7,95%CI1.4-5.2) (Thorand et al., 2003). Epidemiological studies have shown that CRP predicts incident type 2 diabetes and increased cardiovascular disease. In healthy middle-aged women, or young men, CRP levels were associated with a three–fourfold increased risk of developing diabetes Very limited attention has been given to the role of inflammation in the etiology of gestational diabetes a condition that is biochemically and epidemiologically similar to type 2 diabetes (kjos & Buchanan,1999).The second and third trimesters of pregnancy represent a physiological type of insulin resistance (Kautzky-Willer et al., 1997). Insulin resistance is associated with dysfunction of endothelial and inflammation as well as increase production of cytokines by adipose tissue(Baalletshofer et al.,2000). Limited available data suggest that proinflammatory cytokines may be predictive of GDM(Winkler et al., 2002; Kirwan et al., 2002). Some studies have measured CRP at various gestational ages in pregnant women and found inconsistent results regarding the association between inflammatory markers and the incidence of GDM and the interdependence with the degree of adiposity (Retnakaran et al., 2003; Wolf et al., 2003,2004). The interpretation of the results was influenced by coexistence of hypertension, preeclampsia, and different race groups or small sample size in some studies. The result of a study demonstrated women with GDM had significantly higher CRP serum levels than normal pregnant women at 37-38 weeks of gestation but at the time of OGTT (24-28 weeks of pregnancy) there was not any significant difference between the two groups (Leipold et al. , 2005 ). This report is inconsistent with the findings of the Massachusetts General Hospital obstetric maternal report, where increased CRP concentration was shown in the first trimester (10 weeks of gestation) and the association between GDM and CRP depended primarily on coexisting obesity (Wolf et al., 2004). The result of another study demonstrated that CRP concentration is not affected by GDM until the end of the second trimester of pregnancy (Retnakaran et al., 2003). In the third trimester, however, these results suggest an up-regulation of inflammatory markers by GDM resulting in elevation of CRP concentration at the end of pregnancy in women with GDM. In a prospective nested case control study Wolf et al found that CRP concentration in the first 2001; Kern et al., 2001; Mohamed-Ali et al., 1998; Vozarova et al., 2001). 3.2 C-reactive protein and GDM (Buchanan,2001; kjos &Buchanan,1999). #### 4.1 Vitamin D structure and actions Vitamin D, or calciferol, is a group of lipid soluble substance with a four-ringed cholesterol backbone. Human obtained Vitamin D from exposure to Sunlight, their diet and from dietary supplement. Ultraviolet light convert provitamin D to vitamin D3 (cholecalciferol) in the skin and afterwards Vitamin D3 was bounded by vitamin D binding proteins (DBP) and transported via blood to target organs for metabolism and activity. Vitamin D hydroxylate to form 25-hydroxy-vitamin-D (25OHD) in the liver. Hydroxylation of 25-hydroxy-vitamin-D to 1, 25-dihydroxy-vitamin D occurs in the mitochondria of the proximal tubules of the kidney.This form of vitamin D (1,25(OH)2-vitamin D) is the physiologically active form. The The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes 127 along with the previously demonstrated presence in the pancreas of a vitamin D-dependent calcium-binding protein and cytosol receptor for the hormonal form of vitamin D, 1,25 dihydroxyvitamin D3, indicates an important role for vitamin D in the endocrine functioning of the pancreas. The data demonstrated a positive correlation of 25(OH)D concentration with insulin sensitivity and a negative effect of hypovitaminosis D on function of ß cell. Subjects with hypovitaminosis D are at higher risk of insulin resistance and the metabolic syndrome.Vitamin D repletion in early stages of experimental dietary vitamin D deficiency and in vitamin D deficiency subjects improves glucose intolerance and increases insulin secretion. Some studies demonstrated that vitamin D supplementation increased insulin secretion in response to an oral glucose load in patients with type 2 diabetes but not in patients with established type 2 diabetes (Chiu et al., 2004; Inomata et al., 1986; Orwoll et al., 1994; Gedik & Akalin, 1986). Some evidence indicates that vitamin D increases insulin secretion from β cells by increasing intracellular calcium concentration through nonselective voltage-dependent calcium channels. The main effect of vitamin D on insulin secretion is acquired from conversion of proinsulin to insulin. Calcium is principal not only for insulin exocytosis but also for cell glycolysis ( Boucher, 1998). Vitamin D also activates protein biosynthesis in pancreatic islets. Vitamin D deficiency reduces insulin secretion and action. Variation in the vitamin D receptor or vitamin D-binding protein Vitamin D increases cellular glucose absorption either directly or by increasing insulin sensitivity. Vitamin D may directly or indirectly regulate β cell function and secretion by binding 1,25 dihydroxy vitamin D to β cell vitamin D receptors and controlling the balance between the extracellular and intracellular β cell calcium pools (Norman et al ., 1980). Vitamin D can promote insulin sensitivity by stimulating the expression of insulin receptors and enhancing insulin responsiveness for glucose transport. It also regulates extracellular calcium and thus establishes normal calcium inflow through cell membranes and an adequate intracellular cytosolic calcium pool, which is necessary for the insulin-mediated Data about vitamin D as a risk factor for GDM is spare. Pregnant women with diabetes are known to be more vitamin D deficient compared with normal pregnant women (Bikle, 1992). Intravenous administration of vitamin D to pregnant women with gestational diabetes transiently decreases fasting glucose; however, the level of insulin also decreases (Rudnicki & Molsted-Pedersen, 1997). Vitamin D deficiency was associated with a 2.66-fold increase in GDM risk and each 5 ng/ml decrease in 25-hydroxy D concentrations was related to a 1.29-fold increase in GDM risk (Zhang et al., 2008). Another study demonstrated that the serum concentration of vitamin D during 24-28 weeks of pregnancy in gestational diabetes was lower than normal groups (Maghbooli et al., 2007; Soheilykhah et al., 2009).Women with GDM had a 2.66 fold increased risk of vitamin D deficiency (25-hydroxy D<15ng/ml) compared with control group(Soheilykhah et al.,2009). Maternal hypovitaminosis was reported in diabetic pregnancies in Spain and fasting glycaemia decreased with vitamin D supplementation (Farrant et al., 2008). Vitamin D, insulin and proinsulin were measured at 30 weeks gestation in another study . This study demonstrated that vitamin D insufficiency is common in mothers but is not associated with gestational diabetes. There was no association between maternal 25(OH)D and gestational diabetes.In this study mothers with hypovitaminosis D, higher 25(OH)D concentrations were associated intracellular process in insulin responsive tissues (Draznin et al.,1988). 4.3 Vitamin D deficiency and gestational diabetes causes glucose intolerance. renal production of 1,25-dihydroxy-vitamin D is regulated by plasma parathyroid hormone and serum calcium and phosphorus levels.Vitamin D increases calcium and phosphorus absorption from the gut and reabsorption from the kidneys and increases plasma concentration of these elements. As such, the main effect of vitamin D is maintenance of mineral homeostasis and regulation of bone remodeling (Holick et al., 2006) . Vitamin D deficiency is defined when the level of 25- Hydroxyvitamin D is less than 20 ng/ml(50 nmol/l). Level of 25-29 ng/ml can be considered to indicate a relative insufficiency of vitamin D and a level of 30 ng/ml or more indicate sufficient vitamin D.Vitamin D intoxication is observed when serum level of 25-hydroxyvitamin D are greater than 150 ng/ml( Dawson et al.,2005). Vitamin D deficiency or resistance is caused by different mechanisms including reduced of vitamin D access due to insufficient dietary vitamin D, fat malabsorptive disorders, and/or lack of photoisomerization, Impaired hydroxylation of vitamin D by the liver and kidney to produce 25-OH vitamin and 1,25(OH)2-vitamin D respectively and end organ insensitivity to vitamin D metabolites. #### 4.2 Vitamin D deficiency and diabetes Some human and animal studies have shown a relationship between diabetes type 1and vitamin D deficiency .Vitamin D deficiency make predispose subjects to type 1 and type 2 diabetes, and receptors for its activated form 1, 25-dihydroxy-vitamin D have been recognized in beta cells and immune cells. Vitamin D deficiency impairs insulin synthesis and secretion in humans and animal models of diabetes and some investigations suggested that vitamin D deficiency has a role in the development of type 2 diabetes. Epidemiological studies recommended a link between vitamin D deficiency in early life and the subsequently onset of type 1 diabetes. In some populations, type 1 diabetes is associated with certain polymorphisms within the vitamin D receptor gene. In studies in non obese diabetic mice, pharmacological doses of 1alpha,25-dihydroxyvitamin D3, or its structural analogues, have been shown to delay the onset of diabetes, mainly through immune modulation. Vitamin D deficiency may, therefore, be included in the pathogenesis of both types of diabetes.(Luong et al., 2005; Mathieu et al., 2005). Vitamin D supplementation could improve or prevent diabetes This effect may be due to immunomodulatory action of vitamin D. (Stene et al., 2000; Eva , 1999). There was less data about the association between vitamin D and type 2 diabetes. Some evidences show the role of vitamin D in insulin secretion, for example the presence of vitamin D receptors in β cells and the vitamin D-binding proteins in pancreatic tissue and the association between specific allelic variations in the vitamin D receptor and vitamin Dbinding protein genes with glucose tolerance and insulin secretion have further supported this hypothesis. The mechanism of action of vitamin D in type 2 diabetes is thought to be mediated not only through regulation of plasma calcium levels, which regulate insulin synthesis and secretion, but also through a direct action on pancreatic beta-cell function(Palomer et al.,2008).Vitamin D deficiency decreases insulin secretion without changing in glucagon secretion. The effects of a vitamin D deficiency on insulin and glucagon secretion was obtained in isolated perfused rat pancreas by radioimmunoassay of the secreted proteins. Throughout a 30-minute times of perfusion with glucose and arginine, pancreases from vitamin D-deficient rats showed a 48 percent reduction in insulin secretion compared to that for pancreases from vitamin D-deficient rats that had been resupplied with vitamin D. Vitamin D status had no effect on pancreatic glucagon secretion. This result, along with the previously demonstrated presence in the pancreas of a vitamin D-dependent calcium-binding protein and cytosol receptor for the hormonal form of vitamin D, 1,25 dihydroxyvitamin D3, indicates an important role for vitamin D in the endocrine functioning of the pancreas. The data demonstrated a positive correlation of 25(OH)D concentration with insulin sensitivity and a negative effect of hypovitaminosis D on function of ß cell. Subjects with hypovitaminosis D are at higher risk of insulin resistance and the metabolic syndrome.Vitamin D repletion in early stages of experimental dietary vitamin D deficiency and in vitamin D deficiency subjects improves glucose intolerance and increases insulin secretion. Some studies demonstrated that vitamin D supplementation increased insulin secretion in response to an oral glucose load in patients with type 2 diabetes but not in patients with established type 2 diabetes (Chiu et al., 2004; Inomata et al., 1986; Orwoll et al., 1994; Gedik & Akalin, 1986). Some evidence indicates that vitamin D increases insulin secretion from β cells by increasing intracellular calcium concentration through nonselective voltage-dependent calcium channels. The main effect of vitamin D on insulin secretion is acquired from conversion of proinsulin to insulin. Calcium is principal not only for insulin exocytosis but also for cell glycolysis ( Boucher, 1998). Vitamin D also activates protein biosynthesis in pancreatic islets. Vitamin D deficiency reduces insulin secretion and action. Variation in the vitamin D receptor or vitamin D-binding protein causes glucose intolerance. Vitamin D increases cellular glucose absorption either directly or by increasing insulin sensitivity. Vitamin D may directly or indirectly regulate β cell function and secretion by binding 1,25 dihydroxy vitamin D to β cell vitamin D receptors and controlling the balance between the extracellular and intracellular β cell calcium pools (Norman et al ., 1980). Vitamin D can promote insulin sensitivity by stimulating the expression of insulin receptors and enhancing insulin responsiveness for glucose transport. It also regulates extracellular calcium and thus establishes normal calcium inflow through cell membranes and an adequate intracellular cytosolic calcium pool, which is necessary for the insulin-mediated intracellular process in insulin responsive tissues (Draznin et al.,1988). #### 4.3 Vitamin D deficiency and gestational diabetes 126 Gestational Diabetes renal production of 1,25-dihydroxy-vitamin D is regulated by plasma parathyroid hormone and serum calcium and phosphorus levels.Vitamin D increases calcium and phosphorus absorption from the gut and reabsorption from the kidneys and increases plasma concentration of these elements. As such, the main effect of vitamin D is maintenance of Vitamin D deficiency is defined when the level of 25- Hydroxyvitamin D is less than 20 ng/ml(50 nmol/l). Level of 25-29 ng/ml can be considered to indicate a relative insufficiency of vitamin D and a level of 30 ng/ml or more indicate sufficient vitamin D.Vitamin D intoxication is observed when serum level of 25-hydroxyvitamin D are greater Vitamin D deficiency or resistance is caused by different mechanisms including reduced of vitamin D access due to insufficient dietary vitamin D, fat malabsorptive disorders, and/or lack of photoisomerization, Impaired hydroxylation of vitamin D by the liver and kidney to produce 25-OH vitamin and 1,25(OH)2-vitamin D respectively and end organ insensitivity Some human and animal studies have shown a relationship between diabetes type 1and vitamin D deficiency .Vitamin D deficiency make predispose subjects to type 1 and type 2 diabetes, and receptors for its activated form 1, 25-dihydroxy-vitamin D have been recognized in beta cells and immune cells. Vitamin D deficiency impairs insulin synthesis and secretion in humans and animal models of diabetes and some investigations suggested that vitamin D deficiency has a role in the development of type 2 diabetes. Epidemiological studies recommended a link between vitamin D deficiency in early life and the subsequently onset of type 1 diabetes. In some populations, type 1 diabetes is associated with certain polymorphisms within the vitamin D receptor gene. In studies in non obese diabetic mice, pharmacological doses of 1alpha,25-dihydroxyvitamin D3, or its structural analogues, have been shown to delay the onset of diabetes, mainly through immune modulation. Vitamin D deficiency may, therefore, be included in the pathogenesis of both types of diabetes.(Luong et al., 2005; Mathieu et al., 2005). Vitamin D supplementation could improve or prevent diabetes This effect may be due to immunomodulatory action of There was less data about the association between vitamin D and type 2 diabetes. Some evidences show the role of vitamin D in insulin secretion, for example the presence of vitamin D receptors in β cells and the vitamin D-binding proteins in pancreatic tissue and the association between specific allelic variations in the vitamin D receptor and vitamin Dbinding protein genes with glucose tolerance and insulin secretion have further supported this hypothesis. The mechanism of action of vitamin D in type 2 diabetes is thought to be mediated not only through regulation of plasma calcium levels, which regulate insulin synthesis and secretion, but also through a direct action on pancreatic beta-cell function(Palomer et al.,2008).Vitamin D deficiency decreases insulin secretion without changing in glucagon secretion. The effects of a vitamin D deficiency on insulin and glucagon secretion was obtained in isolated perfused rat pancreas by radioimmunoassay of the secreted proteins. Throughout a 30-minute times of perfusion with glucose and arginine, pancreases from vitamin D-deficient rats showed a 48 percent reduction in insulin secretion compared to that for pancreases from vitamin D-deficient rats that had been resupplied with vitamin D. Vitamin D status had no effect on pancreatic glucagon secretion. This result, mineral homeostasis and regulation of bone remodeling (Holick et al., 2006) . than 150 ng/ml( Dawson et al.,2005). 4.2 Vitamin D deficiency and diabetes vitamin D. (Stene et al., 2000; Eva , 1999). to vitamin D metabolites. Data about vitamin D as a risk factor for GDM is spare. Pregnant women with diabetes are known to be more vitamin D deficient compared with normal pregnant women (Bikle, 1992). Intravenous administration of vitamin D to pregnant women with gestational diabetes transiently decreases fasting glucose; however, the level of insulin also decreases (Rudnicki & Molsted-Pedersen, 1997). Vitamin D deficiency was associated with a 2.66-fold increase in GDM risk and each 5 ng/ml decrease in 25-hydroxy D concentrations was related to a 1.29-fold increase in GDM risk (Zhang et al., 2008). Another study demonstrated that the serum concentration of vitamin D during 24-28 weeks of pregnancy in gestational diabetes was lower than normal groups (Maghbooli et al., 2007; Soheilykhah et al., 2009).Women with GDM had a 2.66 fold increased risk of vitamin D deficiency (25-hydroxy D<15ng/ml) compared with control group(Soheilykhah et al.,2009). Maternal hypovitaminosis was reported in diabetic pregnancies in Spain and fasting glycaemia decreased with vitamin D supplementation (Farrant et al., 2008). Vitamin D, insulin and proinsulin were measured at 30 weeks gestation in another study . This study demonstrated that vitamin D insufficiency is common in mothers but is not associated with gestational diabetes. There was no association between maternal 25(OH)D and gestational diabetes.In this study mothers with hypovitaminosis D, higher 25(OH)D concentrations were associated The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes 129 Abernethy, T. J. and Avery, O. T. (1941). The occurrence during acute infections of a protein Adeghate, E. (2004). An update on the biology and physiology of resistin. Cellular and Al Daghri, N., Bartlett, W. A., Jones, A. F., and Kumar, S. (2002). Role of leptin in glucose Altinova, A. E., Toruner, F., Bozkurt, N., Bukan, N., Karakoc, A., Yetkin, I., Ayvaz, G., Cakir, Arita, Y., Kihara, S., Ouchi, N., Takahashi, M., Maeda, K., Miyagawa, J., Hotta, K., Asensio, C., Cettour-Rose, P., Theander-Carrillo, C., Rohner-Jeanrenaud, F., and Muzzin, P. Ballou, S. P. and Kushner, I. (1992). C-reactive protein and the acute phase response. Berg, A. H., Combs, T. P., and Scherer, P. E. (2002). ACRP30/adiponectin: an adipokine Bikle, D. D. (1992). Clinical counterpoint: vitamin D: new actions, new analogs, new Boucher, B. J. (1998). Inadequate vitamin D status: does it contribute to the disorders Buchanan, T. A. (2001). Pancreatic B-cell defects in gestational diabetes: implications for the Cavit, C., Suheyla, G., Rustu, S., Yavuz, D., Pinar, K., and Yalcin, A. (2007). Gestational Endocrinology 2007, pp.88, Budapest, Hungary, 28 April -2 May, 2007. Ceddia, R. B., Koistinen, H. A., Zierath, J. R., and Sweeney, G. (2002). Analysis of The FASEB journal, Vol. 16, No.10, pp. 1163,ISSN 0892-6638 control of glucose homeostasis. Endocrinology, Vol. 145, No.5, pp. 220. Balletshofer, B. M., Rittig, K., Enderle, M. D., Volk, A., Maerker, E., Jacob, S., Matthaei, S., Communications, Vol. 257, No.1, pp. 79-83,ISSN 0006-291X with insulin resistance. Circulation, Vol. 101, No.15, pp. 1780, Advances in internal medicine, Vol. 37, pp. 313,ISSN 0065-2822 therapeutic potential. Endocrine reviews, Vol. 13, No.4, pp. 765, Vol .13 ,No.2, pp. 84-89, ISSN 1043-2760 Metabolism, Vol. 86, No.3, pp. 989. 327,ISSN 1475-2662. molecular life sciences, Vol. 61, No.19, pp. 2485-2496, ISSN 1420-682X not normally present in the blood. The Journal of experimental medicine, Vol. 73, No.2, metabolism in type 2 diabetes. Diabetes, Obesity and Metabolism, Vol. 4, No.3, pp. N., and Arslan, M. (2007). Circulating concentrations of adiponectin and tumor necrosis factor- in gestational diabetes mellitus. Gynecological Endocrinology, Vol. 23, Shimomura, I., Nakamura, T., and Miyaoka, K. (1999). Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochemical and Biophysical Research (2004). Changes in glycemia by leptin administration or high-fat feeding in rodent models of obesity/type 2 diabetes suggest a link between resistin expression and Rett, K., and Haring, H. U. (2000). Endothelial dysfunction is detectable in young normotensive first-degree relatives of subjects with type 2 diabetes in association regulating glucose and lipid metabolism. Trends in Endocrinology and Metabolism, comprising syndrome X? British Journal of Nutrition, Vol. 79, No.04, pp. 315- pathogenesis and prevention of type 2 diabetes. Journal of Clinical Endocrinology & diabetes mellitus and adiponectin levels. Proceeding of European Congress of paradoxical observations on the association between leptin and insulin resistance. 7. References pp. 173, ISSN 0022-1007 147-155, ISSN 1463-1326. No.3, pp. 161-165,ISSN 0951-3590. with lower 30-min glucose concentrations and higher fasting proinsulin concentrations (Farrant et al., 2009). Clifton-Bligh et al. showed mean serum 25(OH)D concentration in pregnant women was negatively correlated with fasting plasma glucose, fasting insulin and insulin resistance as calculated by homeostasis model assessment. The association between fasting glucose and log-transformed 25OHD concentration was of borderline significance after accounting for ethnicity, age and body mass index in multivariate analyses. The odds ratio of gestational diabetes in women with 25OHD < 50 nmol/L did not reach statistical significance (1.92, 95% confidence interval 0.89-4.17) (Clifton-Bligh et al., 2008). In another study total prevalence of vitamin D deficiency (<25 nmol/L) was 70.6% in pregnant women. Prevalence of severe vitamin D deficiency (<12.5) in GDM patients was higher than in normoglycaemic pregnancies. These results show that a positive correlation of 25(OH) vitamin D concentrations with insulin sensitivity and vitamin D deficiency could be a confirmative sign of insulin resistance (Maghbooli et al., 2007). Several studies suggest that vitamin D supplementation in children reduces the risk of type 1 diabetes. Increasing vitamin D intake during pregnancy reduces the development of islet autoantibodies in offspring (Chiu et al., 2003). In Finland, 10,366 children who were given 2000 IU of vitamin D3 per day during their first year of life were followed for 31 years. The risk of type 1 diabetes was reduced by approximately 80% (relative risk, 0.22; 95% CI, 0.05 to 0.89) ( Hypponen et al., 2001). Among children with vitamin D deficiency, the risk was increased by approximately 200% (relative risk, 3.0; 95% CI, 1.0 to 9.0). In another study, vitamin D deficiency increased insulin resistance, decreased insulin production, and was associated with the metabolic syndrome (Chiu et al., 2004). Another study demonstrated that a combined daily intake of 1200 mg of calcium and 800 IU of vitamin D lowered the risk of type 2 diabetes by 33% (relative risk, 0.67; 95% CI, 0.49 to 0.90) as compared with a daily intake of less than 600 mg of calcium and less than 400 IU of vitamin D (Pittas et al., 2006). 4000 IU vitamin D was administrated for 6 months to women with vitamin D less than 50 nm/Land median serum 25(OH)D3 increased significantly and insulin resistance and fasting insulin decreased (Von Hurst et al., 2010).In summary the result of different studies show high prevalence of vitamin D deficiency in pregnant women and most of these findings demonstrated the relationship between vitamin D status and glucose tolerance in pregnancy. #### 5. Conclusion Early diagnosis of gestational diabetes prevents maternal and fetal complications . Recently a number of studies illustrated association of various biomarkers with subsequent development of GDM . These metabolic mediators are known to be produced in the intrauterine environment . Some studies demonstrated that decreased level of adiponectin and increased level of leptin and resistin preceded the onset of diabetes in pregnancy. Some investigations also have been shown association between C-reactive protein and risk of GDM. Some researches exhibited maternal vitamin D concentration inversely related to fasting glucose and insulin concentration and vitamin D deficiency was associated with increasing risk of GDM. #### 6. Acknowledgement The author would like to thank Dr Mahdieh Mojibian, Mrs Nahid Dara and Mrs Ashraf Ahmadi for their assistance. #### 7. References 128 Gestational Diabetes with lower 30-min glucose concentrations and higher fasting proinsulin concentrations (Farrant et al., 2009). Clifton-Bligh et al. showed mean serum 25(OH)D concentration in pregnant women was negatively correlated with fasting plasma glucose, fasting insulin and insulin resistance as calculated by homeostasis model assessment. The association between fasting glucose and log-transformed 25OHD concentration was of borderline significance after accounting for ethnicity, age and body mass index in multivariate analyses. The odds ratio of gestational diabetes in women with 25OHD < 50 nmol/L did not reach statistical In another study total prevalence of vitamin D deficiency (<25 nmol/L) was 70.6% in pregnant women. Prevalence of severe vitamin D deficiency (<12.5) in GDM patients was higher than in normoglycaemic pregnancies. These results show that a positive correlation of 25(OH) vitamin D concentrations with insulin sensitivity and vitamin D deficiency could be a confirmative sign of insulin resistance (Maghbooli et al., 2007). Several studies suggest that vitamin D supplementation in children reduces the risk of type 1 diabetes. Increasing vitamin D intake during pregnancy reduces the development of islet autoantibodies in offspring (Chiu et al., 2003). In Finland, 10,366 children who were given 2000 IU of vitamin D3 per day during their first year of life were followed for 31 years. The risk of type 1 diabetes was reduced by approximately 80% (relative risk, 0.22; 95% CI, 0.05 to 0.89) ( Hypponen et al., 2001). Among children with vitamin D deficiency, the risk was increased by approximately 200% (relative risk, 3.0; 95% CI, 1.0 to 9.0). In another study, vitamin D deficiency increased insulin resistance, decreased insulin production, and was associated with the metabolic syndrome (Chiu et al., 2004). Another study demonstrated that a combined daily intake of 1200 mg of calcium and 800 IU of vitamin D lowered the risk of type 2 diabetes by 33% (relative risk, 0.67; 95% CI, 0.49 to 0.90) as compared with a daily intake of less than 600 mg of calcium and less than 400 IU of vitamin D (Pittas et al., 2006). 4000 IU vitamin D was administrated for 6 months to women with vitamin D less than 50 nm/Land median serum 25(OH)D3 increased significantly and insulin resistance and fasting insulin decreased (Von Hurst et al., 2010).In summary the result of different studies show high prevalence of vitamin D deficiency in pregnant women and most of these findings demonstrated the relationship Early diagnosis of gestational diabetes prevents maternal and fetal complications . Recently a number of studies illustrated association of various biomarkers with subsequent development of GDM . These metabolic mediators are known to be produced in the intrauterine environment . Some studies demonstrated that decreased level of adiponectin and increased level of leptin and resistin preceded the onset of diabetes in pregnancy. Some investigations also have been shown association between C-reactive protein and risk of GDM. Some researches exhibited maternal vitamin D concentration inversely related to fasting glucose and insulin concentration and vitamin D deficiency was associated with The author would like to thank Dr Mahdieh Mojibian, Mrs Nahid Dara and Mrs Ashraf significance (1.92, 95% confidence interval 0.89-4.17) (Clifton-Bligh et al., 2008). between vitamin D status and glucose tolerance in pregnancy. 5. Conclusion increasing risk of GDM. 6. Acknowledgement Ahmadi for their assistance. The Role of Adipocyte Mediators, Inflammatory Markers and Vitamin D in Gestational Diabetes 131 Draznin,B., Sussman,K.E., Eckel, R.H., Kao, M., Yost,T.,Sherman, N. A. (1988). Possible role Engstrom, G., Stavenow, L., Hedblad, B., Lind, P., Eriksson, K. 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Ray1 and Ravi Retnakaran2 The metabolic syndrome is a clustering of traditional cardiometabolic risk factors that include central obesity, dysglycemia, hypertension, hypertriglyceridemia, and reduced high-density lipoprotein (HDL) cholesterol. In recent years, its clinical utility, diagnostic criteria and underlying etiology have been the subject of continuous debate and controversy. While the debate continues, it remains incontrovertible that those identified with the metabolic syndrome are at high risk for the future development of type 2 diabetes (T2DM) and cardiovascular disease (CVD). In addition, an expanding body of evidence has linked the metabolic syndrome with several emerging non-traditional risk factors, including markers of hepatic fat, chronic inflammation (such as C-reactive protein (CRP)), and adipocyte dysregulation (such as low circulating levels of adiponectin). Interestingly, many of these features of the metabolic syndrome are also common to gestational diabetes mellitus (GDM). Indeed, GDM has also been the subject of longstanding debate throughout its history and it too identifies women who are at high risk of developing T2DM and CVD in the future. Moreover, in recent years, GDM has been similarly linked to an array of non-traditional cardiometabolic risk factors, including CRP and hypoadiponectinemia. A series of studies have demonstrated that women with GDM are at risk of developing the metabolic syndrome in the years following their index pregnancy. Furthermore, emerging evidence shows that components of the metabolic syndrome identified in early gestation and even prior to pregnancy can predict the subsequent development of GDM. Taken together, these findings have raised the intriguing possibility that women who develop GDM may have an underlying latent metabolic syndrome that warrants clinical evaluation and risk factor modification. Though intricate and still incompletely understood, the gradual expansion of knowledge about inter-relationships between the metabolic syndrome, GDM and T2DM may provide us with opportunities to screen for and detect metabolic dysfunction at various stages of disease progression. In this way, GDM represents an important and early "metabolic flag" for an affected mother and, perhaps, her offspring. Thus, in this chapter, we explore the emerging relationship between GDM and the metabolic syndrome. We review the definitions of each condition, their limitations and controversies, and their utility and predictive value in identifying T2DM and CVD risk. The clinical evidence for metabolic syndrome as a precursor to the development of GDM and, in turn, T2DM is also discussed. 1. Introduction 2Mount Sinai Hospital University of Toronto, Toronto, Ontario, Metabolic Syndrome 1St. Michael's Hospital, tissue and placenta. Journal of Huazhong University of Science and Technology--Medical Sciences--, Vol. 26, No.3, pp. 288-291,ISSN 1672-0733 ### Gestational Diabetes and the Metabolic Syndrome Leanne R. De Souza1, Joel G. Ray1 and Ravi Retnakaran2 1St. Michael's Hospital, 2Mount Sinai Hospital University of Toronto, Toronto, Ontario, Canada #### 1. Introduction 140 Gestational Diabetes Youn, B. S., Min, S. S., Park, K. S., Lee, H. W., Yu ,R., and Kwon, B. S. (2004). The role of Yudkin, J. S., Stehouwer, C. D. A., Emeis, J. J., and Coppack, S. W. (1999). C-reactive protein Zhang, C., Qiu, C., Hu, F. B., David, R. M., Van Dam, R .M., Bralley, A., and Williams, M. A. Zhang, F., Basinski, M. B., Beals, J. M., Briggs, S. L., Churgay, L. M., Clawson, D. K., structure of the obese protein Ieptin-E100. Nature , No.387, pp.206 – 209. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J. M. (1994). 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Crystal Positional cloning of the mouse obese gene and its human homologue. Nature, Vol. The metabolic syndrome is a clustering of traditional cardiometabolic risk factors that include central obesity, dysglycemia, hypertension, hypertriglyceridemia, and reduced high-density lipoprotein (HDL) cholesterol. In recent years, its clinical utility, diagnostic criteria and underlying etiology have been the subject of continuous debate and controversy. While the debate continues, it remains incontrovertible that those identified with the metabolic syndrome are at high risk for the future development of type 2 diabetes (T2DM) and cardiovascular disease (CVD). In addition, an expanding body of evidence has linked the metabolic syndrome with several emerging non-traditional risk factors, including markers of hepatic fat, chronic inflammation (such as C-reactive protein (CRP)), and adipocyte dysregulation (such as low circulating levels of adiponectin). Interestingly, many of these features of the metabolic syndrome are also common to gestational diabetes mellitus (GDM). Indeed, GDM has also been the subject of longstanding debate throughout its history and it too identifies women who are at high risk of developing T2DM and CVD in the future. Moreover, in recent years, GDM has been similarly linked to an array of non-traditional cardiometabolic risk factors, including CRP and hypoadiponectinemia. A series of studies have demonstrated that women with GDM are at risk of developing the metabolic syndrome in the years following their index pregnancy. Furthermore, emerging evidence shows that components of the metabolic syndrome identified in early gestation and even prior to pregnancy can predict the subsequent development of GDM. Taken together, these findings have raised the intriguing possibility that women who develop GDM may have an underlying latent metabolic syndrome that warrants clinical evaluation and risk factor modification. Though intricate and still incompletely understood, the gradual expansion of knowledge about inter-relationships between the metabolic syndrome, GDM and T2DM may provide us with opportunities to screen for and detect metabolic dysfunction at various stages of disease progression. In this way, GDM represents an important and early "metabolic flag" for an affected mother and, perhaps, her offspring. Thus, in this chapter, we explore the emerging relationship between GDM and the metabolic syndrome. We review the definitions of each condition, their limitations and controversies, and their utility and predictive value in identifying T2DM and CVD risk. The clinical evidence for metabolic syndrome as a precursor to the development of GDM and, in turn, T2DM is also discussed. Gestational Diabetes and the Metabolic Syndrome 143 other criteria. Elevated triglycerides and/or low HDL-C must fall within the prescribed threshold or can be applied if a person is being treated specifically for the lipid abnormality. In addition, the defining criteria consider those with T2DM, an elevated WC and at least 1 other risk factor as having metabolic syndrome. The WHO requires the presence of diabetes, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) or insulin resistance and at least 2 other criteria. Among the 3 definitions, the IDF and ATP III are more commonly cited within the recent literature. The prevalence of metabolic syndrome in U.S. adults is estimated to be approximately 22% to 34% using the ATP III definition and 39% when the > WC >94 cm- White (M) WC >80 cm- White (F) WC >90cm- Asian (M) WC >80cm- Asian (F) triglycerides >1.7 mmol/L >1.7 mmol/L >1.7 mmol/L >1.7 mmol/L pressure >130/85 mm Hg >130/85 mm Hg >140/90 mm Hg Systolic >130 and/or The debate surrounding the metabolic syndrome stems from disagreement about its definition and diagnostic criteria, alongside questions related to its pathogenesis, origins, and applicability across populations. However, despite this ongoing debate, central obesity and insulin resistance have been widely postulated (Lann & LeRoith, 2007) as comprising the fundamental basis of the metabolic syndrome. Categorically, the syndrome is influenced by the complex genetic, hormonal and nutritional origins of its individual component risk factors. Discrepancies among the commonly used NCEP-ATP III, IDF and WHO definitions of the metabolic syndrome, have contributed substantially to this debate. For example, ATP III and WHO differ in their criteria for blood pressure, and neither definition provides specific guidance on how to implement these diagnostic thresholds (i.e., whether to use abnormal systolic vs. diastolic or both; whether to obtain measures in a particular body position; or whether to calculate an averaged measure). Recently, the American Heart Association/National Heart, Lung and Blood Institute (AHA/NHLBI) and IDF attempted to resolve such discrepancies with new harmonized criteria. These criteria, shown in Table 1, include (i) clarification of the blood pressure measurement to specify elevated levels of systolic and/or diastolic pressure and (ii) elimination of abdominal obesity as a mandatory <1.03 mmol/L- (M) <1.29 mmol/L- (F) creatinine ratio - - > 3.4 mg/mmol - NCEP-ATP III IDF WHO Harmonized Waist-to-hip ratio: >0.90- (M) >0.85- (F) <0.9 mmol/L- (M) <1.0 mmol/L- (F) diabetes or IGT Diabetes, IFG, IGT >5.6 mmol/L IDF/AHA Same as IDF cut points for non-Europeans & either IDF or AHA criteria for Europeans <1.0 mmol/L- (M) <1.3 mmol/L- (F) diastolic >85 mmHg IDF criteria are applied (Ford, 2005). Central obesity WC >102 cm- (M) Elevated Reduced HDL-C Fasting Elevated blood Urine albumin: WC >88 cm- (F) <1.0 mmol/L- (M) <1.3 mmol/L- (F) hyperglycemia >6.1 mmol/L >5.6 mmol/L or Table 1. Various diagnostic criteria for the metabolic syndrome 2.2 Controversy regarding the metabolic syndrome Emerging non-traditional risk factors for both metabolic syndrome and GDM will be described, alongside the evidence for metabolic syndrome as a consequence of GDM and as a potential predictive tool to detect risk for GDM before and during early pregnancy. Finally, we consider the concept that women who develop GDM may have a latent metabolic syndrome. #### 2. Metabolic syndrome #### 2.1 General definition and varying sets of diagnostic criteria The metabolic syndrome, also referred to as the insulin resistance syndrome, was initially proposed as a model for understanding the underlying biology and risk factors for CVD. In his Banting award lecture, Gerald Reaven first described 'Syndrome X' as the clustering of abnormalities related to insulin resistance (Reaven, 1988). The World Health Organization formally proposed the term 'metabolic syndrome' in 1998 (Alberti et al., 1998; DeFronzo & Ferrannini, 1991) to identify those at high risk for metabolic disorders and CVD. Though the syndrome was originally intended to identify individuals at risk for CVD, it has since expanded to capture those at high risk for T2DM, with which it is thought to have a stronger association (Ford et al., 2008). The definition of metabolic syndrome continues to evolve today, and is widely studied as a promising marker of cardiovascular risk. The syndrome is characterized by a clustering of central abdominal (visceral) obesity, glucose intolerance, insulin resistance, dyslipidemia and hypertension (Reaven, 1988). The presence of any one risk factor implies the existence of others, such that their concomitant occurrence collectively describes a positive dysmetabolic risk profile for CVD, or 'cardiometabolic risk' (Despres et al., 2008). While several organizations and authoritative bodies have proposed diagnostic criteria for the metabolic syndrome, the most cited working definitions are those of the International Diabetes Federation (IDF), the World Health Organization (WHO), and the National Cholesterol Education Program – Adult Treatment Panel III (NCEP-ATP III) (Alberti et al., 2005; WHO Expert Consultation, 2004; Alexander et al., 2003). These authorities have synthesized, analyzed and translated information gathered from a vast, globally representative body of research studies, in order to provide a set of diagnostic criteria with clinically relevant thresholds and measurements that can identify the metabolic syndrome and hence the risk of diabetes and CVD. Despite continued efforts, there are variations in the definitions, which have prompted international debate about the actual utility and strength of the metabolic syndrome as a diagnostic tool. Table 1 lists the criteria and diagnostic thresholds defined by the IDF, WHO, NCEP ATP-III, and, lastly, the recently published harmonized criteria (discussed in section 2.2). Although the ATP III and IDF definitions differ in their diagnostic threshold criteria for metabolic syndrome, both include the same 5 components: increased adiposity, hypertriglyceridemia, low levels of high density lipoprotein cholesterol (HDL-C), hypertension and dysglycemia. The WHO definition also includes a urine albumin to creatinine ratio. Meeting the dichotomous cut-off points for an abnormality in 3 or more of the 5 components fulfills the requirements for diagnosis according to the ATP III definition (Hunt et al., 2004). Though all definitions include an obesity criterion, the IDF definition provides ethnicity-specific values for diagnosing abdominal obesity (Reaven, 2009). Moreover, for diagnosing metabolic syndrome, the IDF definition requires the presence of increased waist circumference (WC) as a necessary prerequisite along with any 2 of the Emerging non-traditional risk factors for both metabolic syndrome and GDM will be described, alongside the evidence for metabolic syndrome as a consequence of GDM and as a potential predictive tool to detect risk for GDM before and during early pregnancy. Finally, we consider the concept that women who develop GDM may have a latent The metabolic syndrome, also referred to as the insulin resistance syndrome, was initially proposed as a model for understanding the underlying biology and risk factors for CVD. In his Banting award lecture, Gerald Reaven first described 'Syndrome X' as the clustering of abnormalities related to insulin resistance (Reaven, 1988). The World Health Organization formally proposed the term 'metabolic syndrome' in 1998 (Alberti et al., 1998; DeFronzo & Ferrannini, 1991) to identify those at high risk for metabolic disorders and CVD. Though the syndrome was originally intended to identify individuals at risk for CVD, it has since expanded to capture those at high risk for T2DM, with which it is thought to have a stronger association (Ford et al., 2008). The definition of metabolic syndrome continues to evolve The syndrome is characterized by a clustering of central abdominal (visceral) obesity, glucose intolerance, insulin resistance, dyslipidemia and hypertension (Reaven, 1988). The presence of any one risk factor implies the existence of others, such that their concomitant occurrence collectively describes a positive dysmetabolic risk profile for CVD, or While several organizations and authoritative bodies have proposed diagnostic criteria for the metabolic syndrome, the most cited working definitions are those of the International Diabetes Federation (IDF), the World Health Organization (WHO), and the National Cholesterol Education Program – Adult Treatment Panel III (NCEP-ATP III) (Alberti et al., 2005; WHO Expert Consultation, 2004; Alexander et al., 2003). These authorities have synthesized, analyzed and translated information gathered from a vast, globally representative body of research studies, in order to provide a set of diagnostic criteria with clinically relevant thresholds and measurements that can identify the metabolic syndrome and hence the risk of diabetes and CVD. Despite continued efforts, there are variations in the definitions, which have prompted international debate about the actual utility and strength of the metabolic syndrome as a diagnostic tool. Table 1 lists the criteria and diagnostic thresholds defined by the IDF, WHO, NCEP ATP-III, and, lastly, the recently Although the ATP III and IDF definitions differ in their diagnostic threshold criteria for metabolic syndrome, both include the same 5 components: increased adiposity, hypertriglyceridemia, low levels of high density lipoprotein cholesterol (HDL-C), hypertension and dysglycemia. The WHO definition also includes a urine albumin to creatinine ratio. Meeting the dichotomous cut-off points for an abnormality in 3 or more of the 5 components fulfills the requirements for diagnosis according to the ATP III definition (Hunt et al., 2004). Though all definitions include an obesity criterion, the IDF definition provides ethnicity-specific values for diagnosing abdominal obesity (Reaven, 2009). Moreover, for diagnosing metabolic syndrome, the IDF definition requires the presence of increased waist circumference (WC) as a necessary prerequisite along with any 2 of the 2.1 General definition and varying sets of diagnostic criteria today, and is widely studied as a promising marker of cardiovascular risk. metabolic syndrome. 2. Metabolic syndrome 'cardiometabolic risk' (Despres et al., 2008). published harmonized criteria (discussed in section 2.2). other criteria. Elevated triglycerides and/or low HDL-C must fall within the prescribed threshold or can be applied if a person is being treated specifically for the lipid abnormality. In addition, the defining criteria consider those with T2DM, an elevated WC and at least 1 other risk factor as having metabolic syndrome. The WHO requires the presence of diabetes, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) or insulin resistance and at least 2 other criteria. Among the 3 definitions, the IDF and ATP III are more commonly cited within the recent literature. The prevalence of metabolic syndrome in U.S. adults is estimated to be approximately 22% to 34% using the ATP III definition and 39% when the IDF criteria are applied (Ford, 2005). Table 1. Various diagnostic criteria for the metabolic syndrome #### 2.2 Controversy regarding the metabolic syndrome The debate surrounding the metabolic syndrome stems from disagreement about its definition and diagnostic criteria, alongside questions related to its pathogenesis, origins, and applicability across populations. However, despite this ongoing debate, central obesity and insulin resistance have been widely postulated (Lann & LeRoith, 2007) as comprising the fundamental basis of the metabolic syndrome. Categorically, the syndrome is influenced by the complex genetic, hormonal and nutritional origins of its individual component risk factors. Discrepancies among the commonly used NCEP-ATP III, IDF and WHO definitions of the metabolic syndrome, have contributed substantially to this debate. For example, ATP III and WHO differ in their criteria for blood pressure, and neither definition provides specific guidance on how to implement these diagnostic thresholds (i.e., whether to use abnormal systolic vs. diastolic or both; whether to obtain measures in a particular body position; or whether to calculate an averaged measure). Recently, the American Heart Association/National Heart, Lung and Blood Institute (AHA/NHLBI) and IDF attempted to resolve such discrepancies with new harmonized criteria. These criteria, shown in Table 1, include (i) clarification of the blood pressure measurement to specify elevated levels of systolic and/or diastolic pressure and (ii) elimination of abdominal obesity as a mandatory Gestational Diabetes and the Metabolic Syndrome 145 predictor of CVD. These studies illustrate the controversy over whether a diagnosis of metabolic syndrome provides more useful information about CVD risk than any of its individual components (Reaven, 2009). Furthermore, by the current definitions, it is unclear whether any one risk factor is more predictive than the other, in the form of a weighted To address these criticisms, the American Diabetes Association (ADA) and European Association for the Study of Diabetes (EASD) issued a joint statement about the clinical utility of the metabolic syndrome; they recommended that clinicians evaluate and treat discrete risk factors, without diagnosing the metabolic syndrome, per se (Kahn et al., 2005). Specifically, rather than solely relying on diagnosis of metabolic syndrome, identification of one or more of its component features should prompt investigation for the presence of the other features. For the latter, one may also consider specific emerging risk factors not 2.3 Metabolic syndrome and the identification of future risk of T2DM and CVD predicted CVD incidence five years after the diagnosis of T2DM (Guzder et al., 2006). Showing that dysglycemia predicts metabolic syndrome necessarily identifies a predictive potential for T2DM as well. This is so given that metabolic syndrome -- and especially glucose intolerance -- is central to the development of T2DM. In the GENFIEV study, metabolic syndrome prevalence was 42% in those with IFG, 34% in IGT, and 74% in IFG + IGT (Bianchi et al., 2010). In addition, the prevalence of insulin resistance was higher in those with metabolic syndrome than in its absence. Hypertriglyceridemia (odds ratio [OR] 3.38; 95% confidence interval [CI] 2.29-4.99), abdominal obesity (3.26; 95% CI 2.18-4.89), hyperglycemia (3.02; 95% CI 1.80-5.07) and hypertension (1.69; 95% CI 1.12-2.55) were all associated with insulin resistance. These findings suggest that the prevalence of the metabolic syndrome is high in individuals with dysglycemia, and is generally associated with insulin resistance (Bianchi et al., 2010). Moreover, dysglycemia and insulin resistance are highly predictive of T2DM. Similarly, long-term glycemic excursions will identify those at high risk for metabolic syndrome and T2DM. In their exploratory study, Giuffrida et al. (2010) investigated the relation between glycated hemoglobin (GHb), an indicator of longterm glycemic control, and metabolic syndrome with T2DM. Each 1% increase in GHb was associated with metabolic syndrome (OR 1.31, 95% CI 1.18–1.45), demonstrating a strong relation between chronic hyperglycemia and metabolic syndrome (Giuffrida et al., 2010). Aboriginal Canadians have a 5-fold higher risk of T2DM compared to non-Aboriginals. Among the former, the metabolic syndrome can be readily identified using available clinical measures, and thus, may be a useful clinical tool (Reaven, 2009; Ley et al., 2009). In a prospective study, Ley and colleagues (2009) found that the 10-year cumulative incidence of T2DM in the Aboriginal Canadian population was 17.5%, with an age-dependent gradient ranging from 10.5% among those aged 10–19 years, to 43.3% among those aged 40–49 years. The IDF recommends screening for metabolic syndrome features in those with T2DM (Alberti et al., 2006, Alberti et al., 2005). While current recommendations are subject to criticism and controversy, they nevertheless provide a practical basis upon which to adopt management strategies. Individuals with metabolic syndrome have a 5-fold higher risk of developing T2DM (Alberti et al., 2010). Similarly, in a study from the UK that examined the prognostic impact of metabolic syndrome in T2DM, the investigators modified the ATP III definition to include BMI instead of WC, and found that the metabolic syndrome further included in the existing definition, as outlined below. hierarchy. prerequisite, such that the presence of any 3 of the 5 criteria is sufficient for diagnosis of metabolic syndrome (Alberti et al., 2010). The ATP III and IDF definitions differ in their criteria for increased fasting glucose and central obesity (using WC) (Alberti et al., 2006; NCEP 2001) and while obesity is measured by WC according to the ATP III and IDF definitions, waist-to-hip ratio is used in the WHO definition. Furthermore, urine albumincreatinine ratio is a criterion in the WHO definition, but is not found in the ATP III and IDF definitions, while several risk factors associated with insulin resistance are not considered in any of the definitions, including physical inactivity, family history, sex and age (Kahn et al., 2005). Further complicating the controversy is the practical observation that, despite its centrality to the metabolic syndrome, contrasting evidence suggests that many overweight or obese individuals may, by any guideline, have normal metabolic profiles (Wildman et al., 2008), and are not prone to future development of metabolic syndrome. Similarly, among those who display metabolic syndrome, not all are obese (Bruce & Hanson, 2010). Some lean individuals are insulin resistant and exhibit increased cardiometabolic risk. In a study of otherwise healthy obese individuals and insulin resistant lean individuals with a family history of T2DM, obesity was associated with higher insulin resistance and diastolic blood pressure, but conveyed no difference in other metabolic markers. In addition, within each BMI category, insulin resistance independently predicted metabolic syndrome, while WC did not. Only when age was combined with WC (but not BMI) did obesity independently predict metabolic syndrome, and, even so, WC was less predictive of insulin resistance at higher WC values (Utzschneider et al., 2010). The authors concluded that insulin sensitivity is a stronger predictor of metabolic syndrome than obesity, and is better than WC at identifying obese individuals with an otherwise healthy metabolic profile. They also recommended employing metabolic testing among lean individuals with a first-degree relative with T2DM (Utzschneider et al., 2010). Nevertheless, even when weight is considered, cut-points used to define obesity are not universally agreed upon and may vary by ethnicity (Despres et al., 2008). The use of different definitions of the metabolic syndrome has clouded our ability to compare findings across research studies. In addition, there is the question of whether the diagnostic criteria are too restrictive, missing those at highest risk, or, conversely, are too broad, resulting in an overestimation of the prevalence of metabolic syndrome. Considering its inherently chronic and progressive nature, it is reasonable to infer that indicators of dysmetabolism, especially in younger adults, underestimate its consequences for predicting T2DM and CVD. Indeed, manifestation may even occur at different time-points in the disease trajectory, such that risk factor assessment necessitates systematic evaluation across a spectrum of sub-diagnostic and diagnostic ranges standardized for age. Another criticism of the metabolic syndrome is whether its value extends beyond that of its individual components. The criticism highlights both the redundancy of the classification as a 'syndrome' and the inadvertent undermining of the importance of the individual components. The diagnosis of metabolic syndrome, by any definition, has been studied in relation to the predictive value of the individual criteria. The Framingham study (Wilson et al., 2005) demonstrated no substantial increase in risk associated with clusters of 3 of the 5 metabolic syndrome criteria compared with clusters of only 2 traits. In contrast, data from the Third National Health and Nutrition Examination Survey (Ninomiya et al., 2004) indicated that each of the 5 components of metabolic syndrome was an independent prerequisite, such that the presence of any 3 of the 5 criteria is sufficient for diagnosis of metabolic syndrome (Alberti et al., 2010). The ATP III and IDF definitions differ in their criteria for increased fasting glucose and central obesity (using WC) (Alberti et al., 2006; NCEP 2001) and while obesity is measured by WC according to the ATP III and IDF definitions, waist-to-hip ratio is used in the WHO definition. Furthermore, urine albumincreatinine ratio is a criterion in the WHO definition, but is not found in the ATP III and IDF definitions, while several risk factors associated with insulin resistance are not considered in any of the definitions, including physical inactivity, family history, sex and age (Kahn et al., Further complicating the controversy is the practical observation that, despite its centrality to the metabolic syndrome, contrasting evidence suggests that many overweight or obese individuals may, by any guideline, have normal metabolic profiles (Wildman et al., 2008), and are not prone to future development of metabolic syndrome. Similarly, among those who display metabolic syndrome, not all are obese (Bruce & Hanson, 2010). Some lean individuals are insulin resistant and exhibit increased cardiometabolic risk. In a study of otherwise healthy obese individuals and insulin resistant lean individuals with a family history of T2DM, obesity was associated with higher insulin resistance and diastolic blood pressure, but conveyed no difference in other metabolic markers. In addition, within each BMI category, insulin resistance independently predicted metabolic syndrome, while WC did not. Only when age was combined with WC (but not BMI) did obesity independently predict metabolic syndrome, and, even so, WC was less predictive of insulin resistance at higher WC values (Utzschneider et al., 2010). The authors concluded that insulin sensitivity is a stronger predictor of metabolic syndrome than obesity, and is better than WC at identifying obese individuals with an otherwise healthy metabolic profile. They also recommended employing metabolic testing among lean individuals with a first-degree relative with T2DM (Utzschneider et al., 2010). Nevertheless, even when weight is considered, cut-points used to define obesity are not universally agreed upon and may vary The use of different definitions of the metabolic syndrome has clouded our ability to compare findings across research studies. In addition, there is the question of whether the diagnostic criteria are too restrictive, missing those at highest risk, or, conversely, are too broad, resulting in an overestimation of the prevalence of metabolic syndrome. Considering its inherently chronic and progressive nature, it is reasonable to infer that indicators of dysmetabolism, especially in younger adults, underestimate its consequences for predicting T2DM and CVD. Indeed, manifestation may even occur at different time-points in the disease trajectory, such that risk factor assessment necessitates systematic evaluation across Another criticism of the metabolic syndrome is whether its value extends beyond that of its individual components. The criticism highlights both the redundancy of the classification as a 'syndrome' and the inadvertent undermining of the importance of the individual components. The diagnosis of metabolic syndrome, by any definition, has been studied in relation to the predictive value of the individual criteria. The Framingham study (Wilson et al., 2005) demonstrated no substantial increase in risk associated with clusters of 3 of the 5 metabolic syndrome criteria compared with clusters of only 2 traits. In contrast, data from the Third National Health and Nutrition Examination Survey (Ninomiya et al., 2004) indicated that each of the 5 components of metabolic syndrome was an independent a spectrum of sub-diagnostic and diagnostic ranges standardized for age. 2005). by ethnicity (Despres et al., 2008). predictor of CVD. These studies illustrate the controversy over whether a diagnosis of metabolic syndrome provides more useful information about CVD risk than any of its individual components (Reaven, 2009). Furthermore, by the current definitions, it is unclear whether any one risk factor is more predictive than the other, in the form of a weighted hierarchy. To address these criticisms, the American Diabetes Association (ADA) and European Association for the Study of Diabetes (EASD) issued a joint statement about the clinical utility of the metabolic syndrome; they recommended that clinicians evaluate and treat discrete risk factors, without diagnosing the metabolic syndrome, per se (Kahn et al., 2005). Specifically, rather than solely relying on diagnosis of metabolic syndrome, identification of one or more of its component features should prompt investigation for the presence of the other features. For the latter, one may also consider specific emerging risk factors not included in the existing definition, as outlined below. #### 2.3 Metabolic syndrome and the identification of future risk of T2DM and CVD The IDF recommends screening for metabolic syndrome features in those with T2DM (Alberti et al., 2006, Alberti et al., 2005). While current recommendations are subject to criticism and controversy, they nevertheless provide a practical basis upon which to adopt management strategies. Individuals with metabolic syndrome have a 5-fold higher risk of developing T2DM (Alberti et al., 2010). Similarly, in a study from the UK that examined the prognostic impact of metabolic syndrome in T2DM, the investigators modified the ATP III definition to include BMI instead of WC, and found that the metabolic syndrome further predicted CVD incidence five years after the diagnosis of T2DM (Guzder et al., 2006). Showing that dysglycemia predicts metabolic syndrome necessarily identifies a predictive potential for T2DM as well. This is so given that metabolic syndrome -- and especially glucose intolerance -- is central to the development of T2DM. In the GENFIEV study, metabolic syndrome prevalence was 42% in those with IFG, 34% in IGT, and 74% in IFG + IGT (Bianchi et al., 2010). In addition, the prevalence of insulin resistance was higher in those with metabolic syndrome than in its absence. Hypertriglyceridemia (odds ratio [OR] 3.38; 95% confidence interval [CI] 2.29-4.99), abdominal obesity (3.26; 95% CI 2.18-4.89), hyperglycemia (3.02; 95% CI 1.80-5.07) and hypertension (1.69; 95% CI 1.12-2.55) were all associated with insulin resistance. These findings suggest that the prevalence of the metabolic syndrome is high in individuals with dysglycemia, and is generally associated with insulin resistance (Bianchi et al., 2010). Moreover, dysglycemia and insulin resistance are highly predictive of T2DM. Similarly, long-term glycemic excursions will identify those at high risk for metabolic syndrome and T2DM. In their exploratory study, Giuffrida et al. (2010) investigated the relation between glycated hemoglobin (GHb), an indicator of longterm glycemic control, and metabolic syndrome with T2DM. Each 1% increase in GHb was associated with metabolic syndrome (OR 1.31, 95% CI 1.18–1.45), demonstrating a strong relation between chronic hyperglycemia and metabolic syndrome (Giuffrida et al., 2010). Aboriginal Canadians have a 5-fold higher risk of T2DM compared to non-Aboriginals. Among the former, the metabolic syndrome can be readily identified using available clinical measures, and thus, may be a useful clinical tool (Reaven, 2009; Ley et al., 2009). In a prospective study, Ley and colleagues (2009) found that the 10-year cumulative incidence of T2DM in the Aboriginal Canadian population was 17.5%, with an age-dependent gradient ranging from 10.5% among those aged 10–19 years, to 43.3% among those aged 40–49 years. Gestational Diabetes and the Metabolic Syndrome 147 Nakano et al, (2010) investigated the clinical significance of LDL and CRP in coronary artery disease (CAD) risk (Nakano et al., 2010). Among those without the metabolic syndrome, high CRP was not associated with a higher risk of CAD; however, those with both high CRP and metabolic syndrome had a doubling in their risk of CAD (Ridker et al., 2003; Sattar et al., 2003). Despite uncertainty regarding the utility of adding CRP to the metabolic syndrome definition, investigation of its potential as a predictive tool and meaningful Previous studies have also examined the use of adipose tissue biomarkers, including adiponectin, in predicting CVD risk. Adiponectin is an adipocyte-derived polypeptide -- an adipokine -- that is inversely associated with obesity, insulin resistance and T2DM. As a protective adipokine, it inhibits gluconeogenesis and suppresses lipogenesis. Low levels of adiponectin result in reduced fatty acid oxidation and increased fat accumulation in the liver. Adipose tissue plays a central role to metabolic syndrome, and low adiponectin levels are associated with metabolic syndrome. In addition, the strong association between hypoadiponectinemia and CVD risk implicates adiponectin in the disease trajectory. Compared with lean controls, patients with metabolic syndrome and T2DM have lower circulating levels of total and high molecular weight (HMW) adiponectin, and higher levels of leptin and interleukin-6 (IL-6). Decreased total and HMW adiponectin, and increased levels of leptin and IL-6, are characteristic of patients with metabolic syndrome and T2DM (Lee et al., 2009). There may also be a link between low adiponectin and fatty liver disease Hepatic dysregulation is characterized by insulin resistance -related steatosis and oxidative stress (Kim & Younossi, 2008). Non-alcoholic fatty liver disease (NAFLD) -- ranging from simple steatosis (fatty infiltration) to inflammatory steatohepatitis (NASH), to long-term injury (fibrosis) -- is a strong indicator of insulin resistance in non-pregnant adults (Youssef & McCullough, 2002). The process of NAFLD development is in itself an extension of insulin resistance that reduces free fatty esterification and triglyceride storage in adipose tissue, subsequently resulting in the deposition of free fatty acids in non-adipose tissues, especially the liver (Utzschneider & Kahn, 2006). Hence, NAFLD is considered to be the principal liver manifestation of the metabolic syndrome (Kim & Younossi, 2008), as it requires the presence of insulin resistance and is closely associated with T2DM (Targher et al., 2005). In a recent study of adults with newly diagnosed T2DM, there was significant interplay between T2DM and liver injury, likely explained by NAFLD (Porepa et al., 2010). NAFLD may also be detected with the novel serum marker, Fetuin-A, an endogenous inhibitor of insulin receptor tyrosine-kinase, and a recognized "hepatokine". Elevated plasma Fetuin-A levels positively predict the incidence of T2DM independent of other established risk factors (Ix et al., 2008; Stefan et al., 2008). In a study of 330 adults at risk for T2DM, liver fat was the strongest predictor of prediabetes (Kantartzis et al., 2010) (Kantartzis et al., 2010). Among studied biochemical measures, serum Fetuin-A was a more significant predictor of fasting hyperglycemia than serum adiponectin (Kantartzis et al., 2010). In addition, individual liver enzymes, such as alanine aminotransferase, have varying positive associations with the While hyperuricemia is prevalent among those with metabolic syndrome, its clinical utility remains controversial. Nonetheless, it appears to be a predictor of metabolic syndrome. One hypothesis is that enhanced insulin resistance due to fatty acid synthesis in the liver may be linked to additional purine synthesis, thereby accelerating production of uric acid. Since insulin resistance is considered an underlying mechanism connecting visceral obesity and addition to metabolic syndrome is advocated (Ridker et al., 2004). components of the metabolic syndrome (Zhang et al., 2010). (Matsubara et al., 2004). The authors reported that, at baseline, metabolic syndrome had a low positive predictive value for future diabetes; however, the syndrome predicted incident diabetes to the same degree as IGT, while its high negative predictive value identified disease-free individuals at follow-up (Ley et al., 2009). In addition to identifying those at risk of T2DM, metabolic syndrome also independently predicts risk of CVD. In the joint statement from the ADA and the EASD (2005), the authors emphasized the practical use of the metabolic syndrome, focusing on its predictive value for CVD (Kahn et al., 2005). A meta-analysis of a series of European trials reported that metabolic syndrome raises the hazard ratio for CVD in women from 0.6 to 2.8 (Hu et al., 2004). Moreover, patients with metabolic syndrome are at twice the risk of developing CVD over a 5-10 year period than those without the syndrome (Alberti et al., 2010). Several population studies have described an increased cardiovascular risk in the presence of metabolic syndrome (Alexander et al., 2003; Ford, 2004, 2005; Hunt et al., 2004; Isomaa et al., 2001; McNeill et al., 2005). Alexander and colleagues (2003) used the ATP III criteria to assess the prevalence of coronary heart disease (CHD) among patients with the metabolic syndrome. They reported that those without metabolic syndrome, regardless of diabetes status, had a low CHD prevalence (less than 10%), while those with diabetes but not the metabolic syndrome exhibited no increased risk of CHD (Alexander et al., 2003). Otherwise, metabolic syndrome was a significant predictor of CHD (OR 2.07, 95% CI 1.66-2.59) and conferred a risk beyond that of diabetes alone (Alexander et al., 2003). In the San Antonio Heart Study, metabolic syndrome at baseline was a significant predictor of cardiovascular mortality over a mean follow-up of 13 years (Hunt et al., 2004). Similarly, using the WHO definition, Isomaa and colleagues (2001) found that the risk for CHD and stroke was increased 3-fold in those with the metabolic syndrome (P < 0.001), as was cardiovascular mortality (P < 0.001) (Isomaa et al., 2001). In a study of individuals without diabetes or CVD at baseline, the ATP III– defined metabolic syndrome had an adjusted hazard ratio of CHD of 1.46 (95% CI, 1.23–1.74) for men and 2.05 (95% CI, 1.59–2.64) for women (McNeill et al., 2005). Ford and colleagues (2004) showed a linear association between ATP III-based metabolic syndrome and CVD-related mortality as well as all-cause mortality (Ford et al., 2004). A meta-analysis of worldwide data from studies published between 1998 and 2005, showed pooled relative risks (RR) of 1.27 (95% CI, 0.90–1.78) for all-cause mortality, 1.65 (95% CI, 1.38–1.99) for CVD and 2.99 (95% CI, 1.96–4.57) for T2DM using ATP III-defined metabolic syndrome; in the fewer studies that used the most exact WHO definition, the pooled RRs were 1.37 (95% CI, 1.09–1.74) for all-cause mortality and 1.93 (95% CI, 1.39–2.67) for CVD (Ford, 2005). Thus, there is considerable evidence for the predictive value of the metabolic syndrome for identifying risk of T2DM and CVD. #### 2.4 Metabolic syndrome and emerging non-traditional risk factors As the components of metabolic syndrome continue to be better understood, the syndrome appears to be a promising diagnostic and screening tool. Recent studies have identified chronic low-grade inflammation as a systemic consequence of obesity that is related to both metabolic and vascular disease. For example, the inflammatory nature of atherosclerosis prompted the study of inflammatory proteins, such as high-sensitivity C-reactive protein (CRP), as potential predictors of CVD. Indeed, epidemiological studies have shown the independent relation between CRP and CHD (Ridker, 1997, 1998). The authors reported that, at baseline, metabolic syndrome had a low positive predictive value for future diabetes; however, the syndrome predicted incident diabetes to the same degree as IGT, while its high negative predictive value identified disease-free individuals at In addition to identifying those at risk of T2DM, metabolic syndrome also independently predicts risk of CVD. In the joint statement from the ADA and the EASD (2005), the authors emphasized the practical use of the metabolic syndrome, focusing on its predictive value for CVD (Kahn et al., 2005). A meta-analysis of a series of European trials reported that metabolic syndrome raises the hazard ratio for CVD in women from 0.6 to 2.8 (Hu et al., 2004). Moreover, patients with metabolic syndrome are at twice the risk of developing CVD over a 5-10 year period than those without the syndrome (Alberti et al., 2010). Several population studies have described an increased cardiovascular risk in the presence of metabolic syndrome (Alexander et al., 2003; Ford, 2004, 2005; Hunt et al., 2004; Isomaa et al., 2001; McNeill et al., 2005). Alexander and colleagues (2003) used the ATP III criteria to assess the prevalence of coronary heart disease (CHD) among patients with the metabolic syndrome. They reported that those without metabolic syndrome, regardless of diabetes status, had a low CHD prevalence (less than 10%), while those with diabetes but not the metabolic syndrome exhibited no increased risk of CHD (Alexander et al., 2003). Otherwise, metabolic syndrome was a significant predictor of CHD (OR 2.07, 95% CI 1.66-2.59) and In the San Antonio Heart Study, metabolic syndrome at baseline was a significant predictor of cardiovascular mortality over a mean follow-up of 13 years (Hunt et al., 2004). Similarly, using the WHO definition, Isomaa and colleagues (2001) found that the risk for CHD and stroke was increased 3-fold in those with the metabolic syndrome (P < 0.001), as was cardiovascular mortality (P < 0.001) (Isomaa et al., 2001). In a study of individuals without diabetes or CVD at baseline, the ATP III– defined metabolic syndrome had an adjusted hazard ratio of CHD of 1.46 (95% CI, 1.23–1.74) for men and 2.05 (95% CI, 1.59–2.64) for Ford and colleagues (2004) showed a linear association between ATP III-based metabolic syndrome and CVD-related mortality as well as all-cause mortality (Ford et al., 2004). A meta-analysis of worldwide data from studies published between 1998 and 2005, showed pooled relative risks (RR) of 1.27 (95% CI, 0.90–1.78) for all-cause mortality, 1.65 (95% CI, 1.38–1.99) for CVD and 2.99 (95% CI, 1.96–4.57) for T2DM using ATP III-defined metabolic syndrome; in the fewer studies that used the most exact WHO definition, the pooled RRs were 1.37 (95% CI, 1.09–1.74) for all-cause mortality and 1.93 (95% CI, 1.39–2.67) for CVD (Ford, 2005). Thus, there is considerable evidence for the predictive value of the metabolic As the components of metabolic syndrome continue to be better understood, the syndrome appears to be a promising diagnostic and screening tool. Recent studies have identified chronic low-grade inflammation as a systemic consequence of obesity that is related to both metabolic and vascular disease. For example, the inflammatory nature of atherosclerosis prompted the study of inflammatory proteins, such as high-sensitivity C-reactive protein (CRP), as potential predictors of CVD. Indeed, epidemiological studies have shown the conferred a risk beyond that of diabetes alone (Alexander et al., 2003). 2.4 Metabolic syndrome and emerging non-traditional risk factors independent relation between CRP and CHD (Ridker, 1997, 1998). follow-up (Ley et al., 2009). women (McNeill et al., 2005). syndrome for identifying risk of T2DM and CVD. Nakano et al, (2010) investigated the clinical significance of LDL and CRP in coronary artery disease (CAD) risk (Nakano et al., 2010). Among those without the metabolic syndrome, high CRP was not associated with a higher risk of CAD; however, those with both high CRP and metabolic syndrome had a doubling in their risk of CAD (Ridker et al., 2003; Sattar et al., 2003). Despite uncertainty regarding the utility of adding CRP to the metabolic syndrome definition, investigation of its potential as a predictive tool and meaningful addition to metabolic syndrome is advocated (Ridker et al., 2004). Previous studies have also examined the use of adipose tissue biomarkers, including adiponectin, in predicting CVD risk. Adiponectin is an adipocyte-derived polypeptide -- an adipokine -- that is inversely associated with obesity, insulin resistance and T2DM. As a protective adipokine, it inhibits gluconeogenesis and suppresses lipogenesis. Low levels of adiponectin result in reduced fatty acid oxidation and increased fat accumulation in the liver. Adipose tissue plays a central role to metabolic syndrome, and low adiponectin levels are associated with metabolic syndrome. In addition, the strong association between hypoadiponectinemia and CVD risk implicates adiponectin in the disease trajectory. Compared with lean controls, patients with metabolic syndrome and T2DM have lower circulating levels of total and high molecular weight (HMW) adiponectin, and higher levels of leptin and interleukin-6 (IL-6). Decreased total and HMW adiponectin, and increased levels of leptin and IL-6, are characteristic of patients with metabolic syndrome and T2DM (Lee et al., 2009). There may also be a link between low adiponectin and fatty liver disease (Matsubara et al., 2004). Hepatic dysregulation is characterized by insulin resistance -related steatosis and oxidative stress (Kim & Younossi, 2008). Non-alcoholic fatty liver disease (NAFLD) -- ranging from simple steatosis (fatty infiltration) to inflammatory steatohepatitis (NASH), to long-term injury (fibrosis) -- is a strong indicator of insulin resistance in non-pregnant adults (Youssef & McCullough, 2002). The process of NAFLD development is in itself an extension of insulin resistance that reduces free fatty esterification and triglyceride storage in adipose tissue, subsequently resulting in the deposition of free fatty acids in non-adipose tissues, especially the liver (Utzschneider & Kahn, 2006). Hence, NAFLD is considered to be the principal liver manifestation of the metabolic syndrome (Kim & Younossi, 2008), as it requires the presence of insulin resistance and is closely associated with T2DM (Targher et al., 2005). In a recent study of adults with newly diagnosed T2DM, there was significant interplay between T2DM and liver injury, likely explained by NAFLD (Porepa et al., 2010). NAFLD may also be detected with the novel serum marker, Fetuin-A, an endogenous inhibitor of insulin receptor tyrosine-kinase, and a recognized "hepatokine". Elevated plasma Fetuin-A levels positively predict the incidence of T2DM independent of other established risk factors (Ix et al., 2008; Stefan et al., 2008). In a study of 330 adults at risk for T2DM, liver fat was the strongest predictor of prediabetes (Kantartzis et al., 2010) (Kantartzis et al., 2010). Among studied biochemical measures, serum Fetuin-A was a more significant predictor of fasting hyperglycemia than serum adiponectin (Kantartzis et al., 2010). In addition, individual liver enzymes, such as alanine aminotransferase, have varying positive associations with the components of the metabolic syndrome (Zhang et al., 2010). While hyperuricemia is prevalent among those with metabolic syndrome, its clinical utility remains controversial. Nonetheless, it appears to be a predictor of metabolic syndrome. One hypothesis is that enhanced insulin resistance due to fatty acid synthesis in the liver may be linked to additional purine synthesis, thereby accelerating production of uric acid. Since insulin resistance is considered an underlying mechanism connecting visceral obesity and Gestational Diabetes and the Metabolic Syndrome 149 consequences include increased risks of T2DM and CVD. Neonatal complications include fetal macrosomia and the associated risk of shoulder dystocia (Athukorala et al., 2007) which in turn can lead to neonatal musculoskeletal and brachial plexus injury (Christoffersson & Rydhstroem, 2002), while long-term sequelae are childhood obesity (Metzger, 2007), metabolic syndrome, and higher risk of T2DM and hypertension (Athukorala et al., 2007; Though practices vary, many countries recommend that all pregnant women be screened at 24 to 28 weeks' gestation with a 1- hour 50-g glucose challenge test (GCT), followed by a confirmatory 2-hour 75-g, or 3-hour 100-g oral glucose tolerance test (OGTT). While a strategy of selectively screening only women at high risk of GDM may improve the true positive detection rates, some women without classical risk factors for GDM will be missed, ADA 2003 WHO 1999 IADPSG 2010 (Harmonized) Boney et al., 2005; Joffe et al., 1998; Leon, 1998; Metzger, 2007; Reece et al., 2009). accordingly. Table 2 outlines some commonly used diagnostic criteria for GDM. OGTT 3-h, 100-g 3-h, 100-g 2-h, 75-g 2-h, 75-g OGTT-3 h 8.0 mmol/L 7.8 mmol/L - - Table 2. Diagnostic criteria for GDM according to commonly used definitions. OGTT-Fasting 5.8 mmol/L 5.3 mmol/L 7.0 mmol/L 5.1 mmol/L OGTT-1 h 10.5 mmol/L 10.0 mmol/L - 10.0 mmol/L OGTT-2 h 9.2 mmol/L 8.6 mmol/L 7.8 mmol/L 8.5 mmol/L >2 >2 >1 >1 Despite variations in diagnostic criteria, the utility of these definitions for predicting clinical outcomes has been demonstrated. As an example, while the ADA and WHO diagnostic criteria for GDM differ slightly (International Association of Diabetes and Pregnancy Study Group [IADPSG], 2010), the antepartum 2-hour 75g OGTT predicts adverse pregnancy outcomes based on both criteria: the ADA criteria resulted in an increased risk of macrosomia (RR 1.29, 95% CI 0.73-2.18), preeclampsia (RR 2.28, 95% CI 1.22-4.16) and perinatal death (RR 3.10, 95% CI 1.42-6.47) (Schmidt et al., 2001) and similar results were observed using the WHO criteria. Some speculate that the restrictive diagnostic criteria for GDM may overlook the risks faced by women with lesser degrees of dysglycemia (Ferrara et al., 2007; Vambergue et al., 2000). Others assert that lack of international uniformity and agreement of diagnostic thresholds for GDM limits their utility within clinical settings (Metzger & Coustan, 1998). For example, the UK guidelines recommend that only high-risk groups be screened (IADPSG, 2010). In Canada, screening for GDM is routinely done, but not in a universal manner (Wen et al., 2000). Furthermore, current guidelines do not account for the variable risk attributed to ethnicity, in which there are considerable differences in the prevalence of GDM. In a study of ethnicity and postpartum metabolism in women with prior GDM, South Asian Indian women had higher serum triglycerides and lower HDL-C NDDG (National Diabetes Data Group) Diagnostic Abnormal values needed for diagnosis metabolic syndrome (Matsuura et al., 1998), it follows that insulin resistance is related to elevated serum uric acid levels in those with metabolic syndrome (Borges et al., 2010). While a sex-dependent association between hyperuricemia and metabolic syndrome is apparent, there is no current evidence for its association with sex hormones. Sex hormone binding globulin (SHBG) is a liver-derived glycoprotein regulated by insulin, which inhibits its production in hepatocytes. Low serum SHBG levels are associated with increased insulin resistance and hyperinsulinemia. In a recent review Brand et al. (2011) examined 52 observational studies and found that, for both sexes, metabolic syndrome was associated with low levels of SHBG (Brand et al., 2011). Fig. 1. Traditional & non-traditional (bolded) cardiometabolic risk factors associated with metabolic syndrome. #### 3. Gestational Diabetes Mellitus (GDM) #### 3.1 General definition and varying sets of diagnostic criteria GDM, defined as glucose intolerance of varying severity with first onset in late pregnancy, bears many of the same risk factors as T2DM, including: older maternal age, a family history of T2DM, non-White ethnicity, obesity, sedentary lifestyle, and previous GDM (Dornhorst et al., 1992; Hedderson & Ferrera, 2008; Hillier et al., 2008; Ray et al., 2001). GDM has both short and long-term risks for a mother and her child. Acute maternal effects include pregnancy-induced hypertension and increased risk of Caesarian-section, while long-term metabolic syndrome (Matsuura et al., 1998), it follows that insulin resistance is related to elevated serum uric acid levels in those with metabolic syndrome (Borges et al., 2010). While a sex-dependent association between hyperuricemia and metabolic syndrome is apparent, there is no current evidence for its association with sex hormones. Sex hormone binding globulin (SHBG) is a liver-derived glycoprotein regulated by insulin, which inhibits its production in hepatocytes. Low serum SHBG levels are associated with increased insulin resistance and hyperinsulinemia. In a recent review Brand et al. (2011) examined 52 observational studies and found that, for both sexes, metabolic syndrome was associated Fig. 1. Traditional & non-traditional (bolded) cardiometabolic risk factors associated with GDM, defined as glucose intolerance of varying severity with first onset in late pregnancy, bears many of the same risk factors as T2DM, including: older maternal age, a family history of T2DM, non-White ethnicity, obesity, sedentary lifestyle, and previous GDM (Dornhorst et al., 1992; Hedderson & Ferrera, 2008; Hillier et al., 2008; Ray et al., 2001). GDM has both short and long-term risks for a mother and her child. Acute maternal effects include pregnancy-induced hypertension and increased risk of Caesarian-section, while long-term with low levels of SHBG (Brand et al., 2011). metabolic syndrome. 3. Gestational Diabetes Mellitus (GDM) 3.1 General definition and varying sets of diagnostic criteria consequences include increased risks of T2DM and CVD. Neonatal complications include fetal macrosomia and the associated risk of shoulder dystocia (Athukorala et al., 2007) which in turn can lead to neonatal musculoskeletal and brachial plexus injury (Christoffersson & Rydhstroem, 2002), while long-term sequelae are childhood obesity (Metzger, 2007), metabolic syndrome, and higher risk of T2DM and hypertension (Athukorala et al., 2007; Boney et al., 2005; Joffe et al., 1998; Leon, 1998; Metzger, 2007; Reece et al., 2009). Though practices vary, many countries recommend that all pregnant women be screened at 24 to 28 weeks' gestation with a 1- hour 50-g glucose challenge test (GCT), followed by a confirmatory 2-hour 75-g, or 3-hour 100-g oral glucose tolerance test (OGTT). While a strategy of selectively screening only women at high risk of GDM may improve the true positive detection rates, some women without classical risk factors for GDM will be missed, accordingly. Table 2 outlines some commonly used diagnostic criteria for GDM. Table 2. Diagnostic criteria for GDM according to commonly used definitions. Despite variations in diagnostic criteria, the utility of these definitions for predicting clinical outcomes has been demonstrated. As an example, while the ADA and WHO diagnostic criteria for GDM differ slightly (International Association of Diabetes and Pregnancy Study Group [IADPSG], 2010), the antepartum 2-hour 75g OGTT predicts adverse pregnancy outcomes based on both criteria: the ADA criteria resulted in an increased risk of macrosomia (RR 1.29, 95% CI 0.73-2.18), preeclampsia (RR 2.28, 95% CI 1.22-4.16) and perinatal death (RR 3.10, 95% CI 1.42-6.47) (Schmidt et al., 2001) and similar results were observed using the WHO criteria. Some speculate that the restrictive diagnostic criteria for GDM may overlook the risks faced by women with lesser degrees of dysglycemia (Ferrara et al., 2007; Vambergue et al., 2000). Others assert that lack of international uniformity and agreement of diagnostic thresholds for GDM limits their utility within clinical settings (Metzger & Coustan, 1998). For example, the UK guidelines recommend that only high-risk groups be screened (IADPSG, 2010). In Canada, screening for GDM is routinely done, but not in a universal manner (Wen et al., 2000). Furthermore, current guidelines do not account for the variable risk attributed to ethnicity, in which there are considerable differences in the prevalence of GDM. In a study of ethnicity and postpartum metabolism in women with prior GDM, South Asian Indian women had higher serum triglycerides and lower HDL-C Gestational Diabetes and the Metabolic Syndrome 151 complications. How the IADPSG recommendations will impact the risk of long-term It is estimated that 20 to 60% of women with a history of GDM will eventually develop T2DM. Indeed, the relation between GDM and T2DM is well described, and the two conditions share a similar pathophysiology, characterized by insulin resistance of peripheral tissues and insufficient secretion of insulin by the pancreatic beta cells to compensate for this resistance (Buchanan, 2001; Buchanan & Xiang, 2005; Retnakaran et al., 2010a). Pregnancy itself has been described as a "stress test" for T2DM and CVD (Reece et al., 2009). It necessarily involves a state of severe acquired insulin resistance that is comparable to that of a non-pregnant person with T2DM (Bergman, 1989). Furthermore, adult offspring with prediabetes, born to women with previous GDM, display an 8-times higher risk of T2DM (Clausen et al., 2008), demonstrating the cyclical nature of diabetes (Damm, 2009) and the Since O'Sullivan's early research illustrating high rates of IGT in the years following GDM (O'Sullivan, 1991), many studies have investigated the phenomena of elevated risk of T2DM attributed to previous GDM. In their systematic review and meta-analysis to quantify the risk of T2DM following GDM, Bellamy and colleagues (2009) found that women with GDM had an increased risk of developing T2DM compared to those women who had normoglycemic pregnancies (RR 7.43, 95% CI 4.79-11.51 (Bellamy et al., 2009). Epidemiological evidence shows that, for all populations and ethnic groups, GDM increases the risk of T2DM (Ben-Haroush et al., 2004). While the shared risk factors between GDM and T2DM imply a common etiology, the salient message is that women with a history of GDM represent a highly vulnerable group for the development of T2DM. This is readily evident in the 2% prevalence of T2DM following GDM as early as 6 weeks postpartum, with reported rates of 50-60% at 5-10 years postpartum, and 70% by 28 years postpartum (Kim et al, 2002; Lauenborg et al., 2004). Furthermore, at the population level, the health significance of GDM is apparent in the number of individuals with diabetes preceded by GDM. In their meta-analysis of follow-up studies of women with previous GDM, Cheung and Byth (2003) calculated the population-attributable risk percent (PAR%) for the proportion of cases of T2DM associated with prior GDM. The PAR% ranged from 10-31% (Cheung & Byth, 2003). These data suggest that up to one third of parous women In addition to identifying women at risk for T2DM, GDM also has implications for future risk of CVD. Indeed, women with a history of GDM are at risk for sub-clinical atherosclerosis (Tarim et al., 2006). Studies also show an increased prevalence of cardiovascular risk factors in women with previous GDM (Carr et al., 2006; Lauenborg et al., 2005; Verma et al., 2002). Shah and colleagues (2008) used large population-based administrative databases to examine the CVD risk in women with a history of GDM. They found that, by 11.5 years after delivery, the hazard ratio for CVD in women with GDM was 1.71 (95% CI 1.08-2.69)(Shah et al., 2008). Moreover, even mild glucose intolerance in pregnancy is associated with an increased risk of CVD. Compared with normoglycemic women who did not receive an OGTT, those who had an abnormal GCT followed by an OGTT that was not diagnostic of GDM still had an increased risk of CVD within 12 years of GDM likely increases the risk for developing cardiometabolic dysfunction after an affected pregnancy. In a longitudinal study comprising 12-18 years of follow-up, 45% of women with development of metabolic disease remains unknown at this time (IADPSG, 2010). 3.3 GDM and the Identification of future risk of T2DM and CVD compounding effects of dysmetabolism in pregnancy. with T2DM have a history of GDM. the index pregnancy (Retnakaran & Shah, 2009b). levels, while African-Caribbean women had a higher WC, blood pressure, and insulin levels (Savitz et al., 2008). #### 3.2 Controversy regarding GDM Like the metabolic syndrome, GDM has also been the subject of controversy, especially surrounding the timing of screening, the choice of diagnostic test, and the defining thresholds on these tests for its identification. Existing guidelines used to identify GDM, and hence the high risk of T2DM following pregnancy, were initially adapted from criteria that were applied to the non-pregnant population; they were not designed to identify those at risk for adverse perinatal outcomes (IADPSG, 2010). Extensive research has led to modifications of the definition (Cutchie et al., 2006) following the original publication of the criteria (O'Sullivan & Mahan, 1964). Of note, these original criteria were based on the identification of those women at risk of developing diabetes in the years after the index pregnancy (O'Sullivan & Mahan, 1964). The clinical justification for screening for GDM currently focuses on the prevention of fetal macrosomia and associated obstetrical complications (Retnakaran et al., 2009c). Notably, this focus has resulted in a single set of diagnostic criteria used to identify women at risk for two different adverse outcomes (Retnakaran et al., 2009c), which effectively leads to the assumption that a diagnosis of GDM optimally identifies the risks of both macrosomia and postpartum prediabetes/diabetes. In a study designed to test this assumption, subjects representing the full spectrum of antepartum glucose tolerance underwent a 3-hour OGTT, and the results showed that only fasting glucose emerged as a significant predictor for delivery of a large-for-gestational-age (LGA) infant, with an OR of 2.0 (95% CI 1.20-3.34) per 1 mmol/L incremental increase (Retnakaran et al., 2009c). However, all three post-load measures were significant predictors of postpartum prediabetes/diabetes (1-h glucose: OR 1.37, 95% CI 1.17-1.61; 2-h glucose: OR 1.55, 95% CI 1.32-1.83; 3-h glucose: OR 1.30, 95% CI 1.10-1.53). Thus, fasting glucose values may better predict LGA risk, but post-load values better predict postpartum glucose intolerance (Retnakaran et al., 2009c). Clearly, an additional challenge to the GDM diagnostic definition includes how the results are applied. The prevailing consensus within the existing framework for diagnosing GDM is that hyperglycemia, including levels below those for overt diabetes, is associated with the adverse pregnancy outcomes common to GDM. In addition, most agree that screening for GDM at 24-28 weeks' gestation identifies individuals in whom effective management can reduce glycemic excursions and minimize adverse perinatal outcomes. It remains to be determined, however, whether these current strategies can effectively reduce long-term risks of metabolic syndrome, T2DM and CVD in affected women (Nolan, 2011). Indeed, women who do not meet the prescribed thresholds for GDM may incur glucose-mediated fetal macrosomia (Mello et al., 1997; Rudge et al., 2000; Scholl et al., 2001; Sermer, et al., 1995), and may be at risk for T2DM and CVD (Retnakaran et al., 2008a, 2008b, 2009a, 2009b, 2009c, 2009d, 2009e, 2010c; Shah et al., 2008). The Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study was undertaken to examine the risks associated with glucose values below traditional thresholds used to diagnose GDM. The study findings were translated by the IADPSG, in order to harmonize the existing diagnostic criteria (Table 2). The practical implications of these revised criteria includes the universal adoption of a 2-hour, 75-g OGTT. In doing so, these recommendations may identify an increased number of women at lower risk for levels, while African-Caribbean women had a higher WC, blood pressure, and insulin levels Like the metabolic syndrome, GDM has also been the subject of controversy, especially surrounding the timing of screening, the choice of diagnostic test, and the defining thresholds on these tests for its identification. Existing guidelines used to identify GDM, and hence the high risk of T2DM following pregnancy, were initially adapted from criteria that were applied to the non-pregnant population; they were not designed to identify those at risk for adverse perinatal outcomes (IADPSG, 2010). Extensive research has led to modifications of the definition (Cutchie et al., 2006) following the original publication of the criteria (O'Sullivan & Mahan, 1964). Of note, these original criteria were based on the identification of those women at risk of developing diabetes in the years after the index The clinical justification for screening for GDM currently focuses on the prevention of fetal macrosomia and associated obstetrical complications (Retnakaran et al., 2009c). Notably, this focus has resulted in a single set of diagnostic criteria used to identify women at risk for two different adverse outcomes (Retnakaran et al., 2009c), which effectively leads to the assumption that a diagnosis of GDM optimally identifies the risks of both macrosomia and postpartum prediabetes/diabetes. In a study designed to test this assumption, subjects representing the full spectrum of antepartum glucose tolerance underwent a 3-hour OGTT, and the results showed that only fasting glucose emerged as a significant predictor for delivery of a large-for-gestational-age (LGA) infant, with an OR of 2.0 (95% CI 1.20-3.34) per 1 mmol/L incremental increase (Retnakaran et al., 2009c). However, all three post-load measures were significant predictors of postpartum prediabetes/diabetes (1-h glucose: OR 1.37, 95% CI 1.17-1.61; 2-h glucose: OR 1.55, 95% CI 1.32-1.83; 3-h glucose: OR 1.30, 95% CI 1.10-1.53). Thus, fasting glucose values may better predict LGA risk, but post-load values better predict postpartum glucose intolerance (Retnakaran et al., 2009c). Clearly, an additional challenge to the GDM diagnostic definition includes how the results are applied. The prevailing consensus within the existing framework for diagnosing GDM is that hyperglycemia, including levels below those for overt diabetes, is associated with the adverse pregnancy outcomes common to GDM. In addition, most agree that screening for GDM at 24-28 weeks' gestation identifies individuals in whom effective management can reduce glycemic excursions and minimize adverse perinatal outcomes. It remains to be determined, however, whether these current strategies can effectively reduce long-term risks of metabolic syndrome, T2DM and CVD in affected women (Nolan, 2011). Indeed, women who do not meet the prescribed thresholds for GDM may incur glucose-mediated fetal macrosomia (Mello et al., 1997; Rudge et al., 2000; Scholl et al., 2001; Sermer, et al., 1995), and may be at risk for T2DM and CVD (Retnakaran et al., 2008a, 2008b, 2009a, 2009b, The Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study was undertaken to examine the risks associated with glucose values below traditional thresholds used to diagnose GDM. The study findings were translated by the IADPSG, in order to harmonize the existing diagnostic criteria (Table 2). The practical implications of these revised criteria includes the universal adoption of a 2-hour, 75-g OGTT. In doing so, these recommendations may identify an increased number of women at lower risk for (Savitz et al., 2008). 3.2 Controversy regarding GDM pregnancy (O'Sullivan & Mahan, 1964). 2009c, 2009d, 2009e, 2010c; Shah et al., 2008). complications. How the IADPSG recommendations will impact the risk of long-term development of metabolic disease remains unknown at this time (IADPSG, 2010). #### 3.3 GDM and the Identification of future risk of T2DM and CVD It is estimated that 20 to 60% of women with a history of GDM will eventually develop T2DM. Indeed, the relation between GDM and T2DM is well described, and the two conditions share a similar pathophysiology, characterized by insulin resistance of peripheral tissues and insufficient secretion of insulin by the pancreatic beta cells to compensate for this resistance (Buchanan, 2001; Buchanan & Xiang, 2005; Retnakaran et al., 2010a). Pregnancy itself has been described as a "stress test" for T2DM and CVD (Reece et al., 2009). It necessarily involves a state of severe acquired insulin resistance that is comparable to that of a non-pregnant person with T2DM (Bergman, 1989). Furthermore, adult offspring with prediabetes, born to women with previous GDM, display an 8-times higher risk of T2DM (Clausen et al., 2008), demonstrating the cyclical nature of diabetes (Damm, 2009) and the compounding effects of dysmetabolism in pregnancy. Since O'Sullivan's early research illustrating high rates of IGT in the years following GDM (O'Sullivan, 1991), many studies have investigated the phenomena of elevated risk of T2DM attributed to previous GDM. In their systematic review and meta-analysis to quantify the risk of T2DM following GDM, Bellamy and colleagues (2009) found that women with GDM had an increased risk of developing T2DM compared to those women who had normoglycemic pregnancies (RR 7.43, 95% CI 4.79-11.51 (Bellamy et al., 2009). Epidemiological evidence shows that, for all populations and ethnic groups, GDM increases the risk of T2DM (Ben-Haroush et al., 2004). While the shared risk factors between GDM and T2DM imply a common etiology, the salient message is that women with a history of GDM represent a highly vulnerable group for the development of T2DM. This is readily evident in the 2% prevalence of T2DM following GDM as early as 6 weeks postpartum, with reported rates of 50-60% at 5-10 years postpartum, and 70% by 28 years postpartum (Kim et al, 2002; Lauenborg et al., 2004). Furthermore, at the population level, the health significance of GDM is apparent in the number of individuals with diabetes preceded by GDM. In their meta-analysis of follow-up studies of women with previous GDM, Cheung and Byth (2003) calculated the population-attributable risk percent (PAR%) for the proportion of cases of T2DM associated with prior GDM. The PAR% ranged from 10-31% (Cheung & Byth, 2003). These data suggest that up to one third of parous women with T2DM have a history of GDM. In addition to identifying women at risk for T2DM, GDM also has implications for future risk of CVD. Indeed, women with a history of GDM are at risk for sub-clinical atherosclerosis (Tarim et al., 2006). Studies also show an increased prevalence of cardiovascular risk factors in women with previous GDM (Carr et al., 2006; Lauenborg et al., 2005; Verma et al., 2002). Shah and colleagues (2008) used large population-based administrative databases to examine the CVD risk in women with a history of GDM. They found that, by 11.5 years after delivery, the hazard ratio for CVD in women with GDM was 1.71 (95% CI 1.08-2.69)(Shah et al., 2008). Moreover, even mild glucose intolerance in pregnancy is associated with an increased risk of CVD. Compared with normoglycemic women who did not receive an OGTT, those who had an abnormal GCT followed by an OGTT that was not diagnostic of GDM still had an increased risk of CVD within 12 years of the index pregnancy (Retnakaran & Shah, 2009b). GDM likely increases the risk for developing cardiometabolic dysfunction after an affected pregnancy. In a longitudinal study comprising 12-18 years of follow-up, 45% of women with Gestational Diabetes and the Metabolic Syndrome 153 levels were lower among women who went on to develop GDM compared to their peers In addition to traditional measures for GDM, these emerging risk factors are the same as those described for metabolic syndrome arising outside of pregnancy (Figure 2). Accordingly, they raise the question of whether the metabolic syndrome relates to GDM. Fig. 2. Traditional and non-traditional (bolded boxes) cardiometabolic risk factors associated Evidence that the metabolic syndrome both precedes and follows GDM suggests an increased lifetime risk of T2DM in women with prior GDM. In addition to chronic beta-cell dysfunction, women with GDM have chronic insulin resistance that is apparent after delivery. Indeed, in the decade after pregnancy, many women with previous GDM exhibit features of the metabolic syndrome. Considering the shared risk factors of metabolic syndrome and T2DM, and the similarities between GDM and T2DM, it is not surprising that GDM is likewise associated with metabolic syndrome. Akinci and colleagues (2010) collected antepartum characteristics of women who developed metabolic syndrome in their later years. Using the ATP III and IDF definitions for metabolic syndrome, pre-pregnancy obesity, weight gain, and OGTT fasting glucose levels each predicted the development of 4. Risk of metabolic syndrome and its sequelae following GDM 4.1 Development of metabolic syndrome after GDM (187 nmol/L vs 233 nmol/L) (Thadhani et al., 2003). with GDM. previous GDM went on to develop hypertension compared to only 4% in the control group (Mestman, 1972). Another study demonstrated significantly higher rates of dyslipidemia, hypertension and mortality 26 years after GDM (O'Sullivan, 1991). Further exacerbating the burden of disease is family history of T2DM, which adds to the elevated risk associated with GDM. Carr and colleagues (2006) quantified the increased risk of CVD in women with GDM and a family history of T2DM, compared to women without a history of GDM (OR 1.85, 95% CI: 1.21-2.82) (Carr et al., 2006). It is generally recommended that women with GDM undergo a postpartum OGTT to detect ongoing dysglycemia. If lower thresholds for GDM are adopted, then more women are likely to be screened for T2DM postpartum. One hopes that this will offer a preventive opportunity that would otherwise be missed in these women. #### 3.4 GDM and emerging non-traditional risk factors CVD is described as an inflammatory disease, with analogous findings in diabetes and obesity (Stern, 1995). Studies have also demonstrated the presence of inflammation in GDM, with high concentrations of serum CRP associated with GDM, but which are attenuated by further adjustment for BMI (Winzer et al., 2004; Wolf et al., 2003). In a cross-sectional study examining the role of maternal obesity in the association between CRP and GDM, prepregnancy BMI emerged as the most important determinant of serum CRP concentration, independent of GDM (Retnakaran et al., 2003). It thus emerges that obesity may mediate a systemic inflammatory response that underlies the relation between CRP and GDM. Similar to its potential as a metabolic syndrome risk factor, adiponectin is also a promising marker of GDM. Compared to unaffected women, those with GDM have lower levels in pregnancy of both total and HMW adiponectin (Retnakaran et al., 2004; Retnakaran et al., 2007). These lower levels of total and HMW adiponectin are associated with both insulin resistance and pancreatic beta-cell dysfunction (Retnakaran et al., 2005; Retnakaran et al., 2007). Furthermore, hypoadiponectinemia in pregnancy independently predicts postpartum metabolic dysfunction, including fasting glycemia, insulin resistance and beta-cell dysfunction (Retnakaran et al., 2010d). Thus, hypoadiponectinemia may play a role in the development of T2DM in women with a history of GDM. Another novel marker potentially associated with GDM is the presence of a fatty liver. In non-pregnant women with previous GDM who underwent MRI of the liver, those with high liver fat had elevated fasting serum triglyceride and insulin concentrations and lower whole-body insulin sensitivity than those with low liver fat on MRI (Tiikkainen et al., 2002). Given that NAFLD is common in T2DM, Forbes and colleagues (2011) investigated the prevalence and risk for NAFLD among European women with previous GDM. The prevalence of NAFLD was much higher in women with previous GDM (38%, 95% CI 28-47) than in those without GDM (17%, 95% CI 10-24)(Forbes et al., 2011). Limited evidence exists for the association between uric acid and GDM, although its predictive value in T2DM makes it a promising candidate for studies of GDM. High uric acid levels have been detected in women with GDM (Seghieri et al., 2003), and are considered a marker of preeclampsia (Barden et al., 2004). SHBG (Smirnakis et al., 2007) is another biochemical marker of much interest. Bartha et al. (2000) compared serum SHBG levels between women with and without GDM, and found that SHBG levels were lower in the GDM group (Bartha et al., 2000). Similarly, SHBG, in addition to adiponectin, was shown to be lower in women with GDM than unaffected controls (Nanda et al., 2011). Even when measured in early pregnancy, first-trimester SHBG previous GDM went on to develop hypertension compared to only 4% in the control group (Mestman, 1972). Another study demonstrated significantly higher rates of dyslipidemia, hypertension and mortality 26 years after GDM (O'Sullivan, 1991). Further exacerbating the burden of disease is family history of T2DM, which adds to the elevated risk associated with GDM. Carr and colleagues (2006) quantified the increased risk of CVD in women with GDM and a family history of T2DM, compared to women without a history of GDM (OR 1.85, 95% CI: 1.21-2.82) (Carr et al., 2006). It is generally recommended that women with GDM undergo a postpartum OGTT to detect ongoing dysglycemia. If lower thresholds for GDM are adopted, then more women are likely to be screened for T2DM postpartum. One hopes that this will offer a preventive opportunity that would otherwise be missed in these CVD is described as an inflammatory disease, with analogous findings in diabetes and obesity (Stern, 1995). Studies have also demonstrated the presence of inflammation in GDM, with high concentrations of serum CRP associated with GDM, but which are attenuated by further adjustment for BMI (Winzer et al., 2004; Wolf et al., 2003). In a cross-sectional study examining the role of maternal obesity in the association between CRP and GDM, prepregnancy BMI emerged as the most important determinant of serum CRP concentration, independent of GDM (Retnakaran et al., 2003). It thus emerges that obesity may mediate a Similar to its potential as a metabolic syndrome risk factor, adiponectin is also a promising marker of GDM. Compared to unaffected women, those with GDM have lower levels in pregnancy of both total and HMW adiponectin (Retnakaran et al., 2004; Retnakaran et al., 2007). These lower levels of total and HMW adiponectin are associated with both insulin resistance and pancreatic beta-cell dysfunction (Retnakaran et al., 2005; Retnakaran et al., 2007). Furthermore, hypoadiponectinemia in pregnancy independently predicts postpartum metabolic dysfunction, including fasting glycemia, insulin resistance and beta-cell dysfunction (Retnakaran et al., 2010d). Thus, hypoadiponectinemia may play a role in the Another novel marker potentially associated with GDM is the presence of a fatty liver. In non-pregnant women with previous GDM who underwent MRI of the liver, those with high liver fat had elevated fasting serum triglyceride and insulin concentrations and lower whole-body insulin sensitivity than those with low liver fat on MRI (Tiikkainen et al., 2002). Given that NAFLD is common in T2DM, Forbes and colleagues (2011) investigated the prevalence and risk for NAFLD among European women with previous GDM. The prevalence of NAFLD was much higher in women with previous GDM (38%, 95% CI 28-47) Limited evidence exists for the association between uric acid and GDM, although its predictive value in T2DM makes it a promising candidate for studies of GDM. High uric acid levels have been detected in women with GDM (Seghieri et al., 2003), and are SHBG (Smirnakis et al., 2007) is another biochemical marker of much interest. Bartha et al. (2000) compared serum SHBG levels between women with and without GDM, and found that SHBG levels were lower in the GDM group (Bartha et al., 2000). Similarly, SHBG, in addition to adiponectin, was shown to be lower in women with GDM than unaffected controls (Nanda et al., 2011). Even when measured in early pregnancy, first-trimester SHBG systemic inflammatory response that underlies the relation between CRP and GDM. women. 3.4 GDM and emerging non-traditional risk factors development of T2DM in women with a history of GDM. considered a marker of preeclampsia (Barden et al., 2004). than in those without GDM (17%, 95% CI 10-24)(Forbes et al., 2011). levels were lower among women who went on to develop GDM compared to their peers (187 nmol/L vs 233 nmol/L) (Thadhani et al., 2003). In addition to traditional measures for GDM, these emerging risk factors are the same as those described for metabolic syndrome arising outside of pregnancy (Figure 2). Accordingly, they raise the question of whether the metabolic syndrome relates to GDM. Fig. 2. Traditional and non-traditional (bolded boxes) cardiometabolic risk factors associated with GDM. #### 4. Risk of metabolic syndrome and its sequelae following GDM #### 4.1 Development of metabolic syndrome after GDM Evidence that the metabolic syndrome both precedes and follows GDM suggests an increased lifetime risk of T2DM in women with prior GDM. In addition to chronic beta-cell dysfunction, women with GDM have chronic insulin resistance that is apparent after delivery. Indeed, in the decade after pregnancy, many women with previous GDM exhibit features of the metabolic syndrome. Considering the shared risk factors of metabolic syndrome and T2DM, and the similarities between GDM and T2DM, it is not surprising that GDM is likewise associated with metabolic syndrome. Akinci and colleagues (2010) collected antepartum characteristics of women who developed metabolic syndrome in their later years. Using the ATP III and IDF definitions for metabolic syndrome, pre-pregnancy obesity, weight gain, and OGTT fasting glucose levels each predicted the development of Gestational Diabetes and the Metabolic Syndrome 155 serum HDL cholesterol and higher fasting plasma insulin, triglycerides, free fatty acids and pre-pregnancy BMI. These common features of the metabolic syndrome were each individually predictive of GDM, and persisted after adjustment for differences in BMI In addition to conventional measures of metabolic syndrome, several non-traditional biomarkers have also emerged as possible predictors of GDM. As discussed earlier, low adiponectin is a risk factor for T2DM and an emerging risk factor for metabolic syndrome and GDM. Using a prospective nested case-control study design, Williams et al. (2004) determined whether first trimester hypoadiponectinemia predicts GDM. They found that 73% of those with GDM had a low adiponectin level compared to 33% of controls (adjusted OR 4.6, 95% CI 1.8-11.6) (Williams et al., 2004). Similarly, Lain and colleagues (2008) found that women with low adiponectin concentrations in the first trimester were much more In choosing an optimal early serum marker to predict GDM, Smirnakis and colleagues (2007) compared SHBG, high-sensitive CRP, and the homeostasis model of assessment of insulin resistance (HOMA-IR) in late first trimester and early second trimester of pregnancy (Smirnakis et al., 2007). Serum SHBG was lower, and serum CRP higher, in women who went on to develop GDM, who also had elevated HOMA-IR in the second trimester. After multivariate analysis, SHBG emerged as the best predictor of GDM (Smirnakis et al., 2007). Alternately, Wolf et al. (2003) found that the risk of developing GDM was higher in women in the upper vs. lower tertiles of first-trimester CRP, after adjusting for confounders (OR 3.6, 95% CI 1.2-11.4). Importantly, the association was attenuated when BMI was included in the analysis (OR 1.5, 95% CI 0.4-5.5) (Wolf et al., 2003), suggesting that obesity confounds the Qiu and colleagues (2004) found that, even after adjusting for maternal pre-pregnancy BMI and other confounders, women with CRP in the highest vs. lowest tertiles experienced a 3.5 times increased risk of GDM (95% CI 1.2-9.8) (Qiu et al., 2004). Moreover, even lean women had an OR for GDM of 3.7 (95% CI 1.6-8.7), suggesting that the association between elevated CRP and GDM may not solely depend on the presence of maternal obesity (Qiu et al., 2004). However, Savvidou and colleagues (2010) evaluated various first-trimester conventional and novel biomarkers, including adiponectin and CRP, and found only a low HDL-C and a high tissue plasminogen activator were significant independent predictors of GDM (Savvidou et al., 2010). Laughon et al (2009) reported that a first trimester concentration of uric acid in the highest quartile had an OR for GDM of 3.25 (95% CI 1.35-7.83), after adjusting for BMI and age (Laughon et al., 2009). Together, these emerging risk factors present an opportunity for early detection of GDM, and possibly, the identification of an It is likely that components of metabolic syndrome exist before and after GDM. Similar to T2DM, where persons with IGT and IFG are at significant risk of T2DM, so too may be the case for metabolic syndrome in early pregnancy. Ray and colleagues (2010) coined the term "gestational prediabetes" to describe the absence of diabetes before pregnancy, and the presence of a blood glucose level (or a related marker) in early pregnancy that is higher than normal, but not yet high enough to meet the diagnostic criteria for GDM (Ray et al., 2010). Given the promising findings of using emerging biomarkers to detect dysmetabolism in early pregnancy and predict GDM, the next step is to identify a robust biomarker that can be assayed at a low cost in early pregnancy. Since they are chronic in nature, metabolic likely to be diagnosed with GDM (OR 10.2, 95% CI 1.3, 78.7) (Lain et al., 2008). (Clark et al., 1997). relation between inflammation and GDM. effective tool for long-term prevention of metabolic syndrome. metabolic syndrome. Moreover, even a fasting glucose concentration above 5.5 mmol/L at the antepartum OGTT was an independent predictor of metabolic syndrome (Akinci et al., 2010). Indeed, many studies have demonstrated an increased prevalence of features of the metabolic syndrome following GDM. Egeland and Meltzer (2010) investigated the effects of GDM on future risk of metabolic and cardiovascular abnormalities. The prevalence of glucose intolerance at 15 years follow-up was 44.4% among women with prior GDM, vs. only 13.1% in those without GDM. WC at 15 year follow-up was the strongest predictor of this difference (Egeland & Meltzer, 2010). Similarly, in a U.S. study, the prevalence of the metabolic syndrome was 27.2% 11 years after pregnancy in women with previous GDM, compared to only 8.2% in unaffected controls (Verma et al., 2002). Lauenborg et al. (2005) estimated the risk of metabolic syndrome in a Danish cohort of women 9.8 years after delivery. Women with previous GDM had a 3-fold higher risk of metabolic syndrome compared to non-GDM controls (Lauenborg et al., 2005). A similar study in Europe reported prevalences of metabolic syndrome of 21% and 4.6%, respectively, 8.5 years' postpartum (Bo et al., 2004b). Indeed, prior gestational hyperglycemia in the absence of fulfilling the overt criteria for GDM results in a future risk of metabolic syndrome 2-4 times that of those with normoglycemia in pregnancy. This risk is 10 times higher in women with concomitant pre-pregnancy obesity (Bo et al., 2004a). Thus, even mild gestational hyperglycemia predicts metabolic syndrome (Bo et al., 2004b), and metabolic syndrome is increasingly more likely to develop over time following the index pregnancy (Bo et al., 2006). These studies highlight the chronic nature of the metabolic dysfunction associated with GDM. Furthermore, they raise the possibility that a diagnosis of GDM may indicate the presence of an underlying latent metabolic syndrome (Retnakaran et al., 2010b). #### 4.2 Development of metabolic syndrome in the early postpartum after GDM Recent evidence implicates GDM as an early expression of metabolic syndrome (Haffner & Taegtmeyer, 2003). Indeed, it was recently reported that both GDM (OR 2.05, 95% CI 1.07- 3.94) and the milder state of gestational impaired glucose tolerance (GIGT) (OR 2.16, 95% CI 1.05-4.42) independently predict postpartum metabolic syndrome by 3 months postpartum, even after adjustment for covariates (Retnakaran et al., 2010d). Furthermore, by 3 months postpartum women with GDM and GIGT also exhibit non-traditional risk factors associated with metabolic syndrome, including low levels of adiponectin and increased serum CRP (Retnakaran et al., 2010c). While many of the metabolic disturbances of pregnancy resolve after delivery, growing evidence supports the concept that pregnancy provides an opportunity to observe a pronounced expression of an otherwise subclinical metabolic disorder. Such metabolic disturbances, which include the metabolic syndrome component disorders, may indeed be apparent prior to the diagnosis of GDM. #### 5. Prediction of GDM by metabolic syndrome components and associated risk factors #### 5.1 Prediction of GDM by metabolic syndrome components in early pregnancy It is quite likely that the metabolic syndrome exists prior to the development of GDM. Indeed, GDM has even been proposed to be a component of the metabolic syndrome (Clark et al. 1997). In their study, Clark et al. (1997) showed that, at the time of their antepartum OGTT, women with GDM expressed markers of the metabolic syndrome, including low metabolic syndrome. Moreover, even a fasting glucose concentration above 5.5 mmol/L at the antepartum OGTT was an independent predictor of metabolic syndrome (Akinci et al., 2010). Indeed, many studies have demonstrated an increased prevalence of features of the Egeland and Meltzer (2010) investigated the effects of GDM on future risk of metabolic and cardiovascular abnormalities. The prevalence of glucose intolerance at 15 years follow-up was 44.4% among women with prior GDM, vs. only 13.1% in those without GDM. WC at 15 year follow-up was the strongest predictor of this difference (Egeland & Meltzer, 2010). Similarly, in a U.S. study, the prevalence of the metabolic syndrome was 27.2% 11 years after pregnancy in women with previous GDM, compared to only 8.2% in unaffected controls (Verma et al., 2002). Lauenborg et al. (2005) estimated the risk of metabolic syndrome in a Danish cohort of women 9.8 years after delivery. Women with previous GDM had a 3-fold higher risk of metabolic syndrome compared to non-GDM controls (Lauenborg et al., 2005). A similar study in Europe reported prevalences of metabolic syndrome of 21% and 4.6%, respectively, 8.5 years' postpartum (Bo et al., 2004b). Indeed, prior gestational hyperglycemia in the absence of fulfilling the overt criteria for GDM results in a future risk of metabolic syndrome 2-4 times that of those with normoglycemia in pregnancy. This risk is 10 times higher in women with concomitant pre-pregnancy obesity (Bo et al., 2004a). Thus, even mild gestational hyperglycemia predicts metabolic syndrome (Bo et al., 2004b), and metabolic syndrome is increasingly more likely to develop over time following the index pregnancy (Bo et al., 2006). These studies highlight the chronic nature of the metabolic dysfunction associated with GDM. Furthermore, they raise the possibility that a diagnosis of GDM may indicate the presence of an underlying latent metabolic syndrome 4.2 Development of metabolic syndrome in the early postpartum after GDM disorders, may indeed be apparent prior to the diagnosis of GDM. Recent evidence implicates GDM as an early expression of metabolic syndrome (Haffner & Taegtmeyer, 2003). Indeed, it was recently reported that both GDM (OR 2.05, 95% CI 1.07- 3.94) and the milder state of gestational impaired glucose tolerance (GIGT) (OR 2.16, 95% CI 1.05-4.42) independently predict postpartum metabolic syndrome by 3 months postpartum, even after adjustment for covariates (Retnakaran et al., 2010d). Furthermore, by 3 months postpartum women with GDM and GIGT also exhibit non-traditional risk factors associated with metabolic syndrome, including low levels of adiponectin and increased serum CRP (Retnakaran et al., 2010c). While many of the metabolic disturbances of pregnancy resolve after delivery, growing evidence supports the concept that pregnancy provides an opportunity to observe a pronounced expression of an otherwise subclinical metabolic disorder. Such metabolic disturbances, which include the metabolic syndrome component 5. Prediction of GDM by metabolic syndrome components and associated It is quite likely that the metabolic syndrome exists prior to the development of GDM. Indeed, GDM has even been proposed to be a component of the metabolic syndrome (Clark et al. 1997). In their study, Clark et al. (1997) showed that, at the time of their antepartum OGTT, women with GDM expressed markers of the metabolic syndrome, including low 5.1 Prediction of GDM by metabolic syndrome components in early pregnancy metabolic syndrome following GDM. (Retnakaran et al., 2010b). risk factors serum HDL cholesterol and higher fasting plasma insulin, triglycerides, free fatty acids and pre-pregnancy BMI. These common features of the metabolic syndrome were each individually predictive of GDM, and persisted after adjustment for differences in BMI (Clark et al., 1997). In addition to conventional measures of metabolic syndrome, several non-traditional biomarkers have also emerged as possible predictors of GDM. As discussed earlier, low adiponectin is a risk factor for T2DM and an emerging risk factor for metabolic syndrome and GDM. Using a prospective nested case-control study design, Williams et al. (2004) determined whether first trimester hypoadiponectinemia predicts GDM. They found that 73% of those with GDM had a low adiponectin level compared to 33% of controls (adjusted OR 4.6, 95% CI 1.8-11.6) (Williams et al., 2004). Similarly, Lain and colleagues (2008) found that women with low adiponectin concentrations in the first trimester were much more likely to be diagnosed with GDM (OR 10.2, 95% CI 1.3, 78.7) (Lain et al., 2008). In choosing an optimal early serum marker to predict GDM, Smirnakis and colleagues (2007) compared SHBG, high-sensitive CRP, and the homeostasis model of assessment of insulin resistance (HOMA-IR) in late first trimester and early second trimester of pregnancy (Smirnakis et al., 2007). Serum SHBG was lower, and serum CRP higher, in women who went on to develop GDM, who also had elevated HOMA-IR in the second trimester. After multivariate analysis, SHBG emerged as the best predictor of GDM (Smirnakis et al., 2007). Alternately, Wolf et al. (2003) found that the risk of developing GDM was higher in women in the upper vs. lower tertiles of first-trimester CRP, after adjusting for confounders (OR 3.6, 95% CI 1.2-11.4). Importantly, the association was attenuated when BMI was included in the analysis (OR 1.5, 95% CI 0.4-5.5) (Wolf et al., 2003), suggesting that obesity confounds the relation between inflammation and GDM. Qiu and colleagues (2004) found that, even after adjusting for maternal pre-pregnancy BMI and other confounders, women with CRP in the highest vs. lowest tertiles experienced a 3.5 times increased risk of GDM (95% CI 1.2-9.8) (Qiu et al., 2004). Moreover, even lean women had an OR for GDM of 3.7 (95% CI 1.6-8.7), suggesting that the association between elevated CRP and GDM may not solely depend on the presence of maternal obesity (Qiu et al., 2004). However, Savvidou and colleagues (2010) evaluated various first-trimester conventional and novel biomarkers, including adiponectin and CRP, and found only a low HDL-C and a high tissue plasminogen activator were significant independent predictors of GDM (Savvidou et al., 2010). Laughon et al (2009) reported that a first trimester concentration of uric acid in the highest quartile had an OR for GDM of 3.25 (95% CI 1.35-7.83), after adjusting for BMI and age (Laughon et al., 2009). Together, these emerging risk factors present an opportunity for early detection of GDM, and possibly, the identification of an effective tool for long-term prevention of metabolic syndrome. It is likely that components of metabolic syndrome exist before and after GDM. Similar to T2DM, where persons with IGT and IFG are at significant risk of T2DM, so too may be the case for metabolic syndrome in early pregnancy. Ray and colleagues (2010) coined the term "gestational prediabetes" to describe the absence of diabetes before pregnancy, and the presence of a blood glucose level (or a related marker) in early pregnancy that is higher than normal, but not yet high enough to meet the diagnostic criteria for GDM (Ray et al., 2010). Given the promising findings of using emerging biomarkers to detect dysmetabolism in early pregnancy and predict GDM, the next step is to identify a robust biomarker that can be assayed at a low cost in early pregnancy. Since they are chronic in nature, metabolic Gestational Diabetes and the Metabolic Syndrome 157 Fig. 3. Theoretical framework and conceptual model for latent metabolic syndrome Alberti, KG. & Zimmet, PZ. (1998). Definition, diagnosis and classification of diabetes Alberti, KG.; Zimmet, PZ. & Shaw, J. IDF Epidemiology Task Force Consensus Group. Alberti, KG.; Eckel, RH., Grundy, SM., Zimmet, PZ., Cleeman, JI., Donato, KA., et al. (2010). 9491, (Sept. 2005), pp. 1059-1062, doi:10.1016/S0140-6736(05)67402-8 Alberti, KG.; Zimmet, PZ. & Shaw, J. (2006). Metabolic syndrome--a new world-wide mellitus and its complications. Part 1: Diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabetic Medicine, Vol. 15, No. 7, (July 1998), pp. 539-553, doi:10.1002/(SICI)1096-9136(199807)15:7<539::AID- (2005). The metabolic syndrome--a new worldwide definition. Lancet, Vol. 366, No. definition. A Consensus Statement from the International Diabetes Federation. Diabetic Medicine, Vol. 23, No. 5, (May 2006), pp. 469-480, doi:10.1111/j.1464- Harmonizing the metabolic syndrome: A joint interim statement of the international Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International preceding GDM. 7. References DIA668>3.0.CO;2-S 5491.2006.01858.x abnormalities likely precede pregnancy, which means that they should be detectable in early pregnancy as well. #### 5.2 Prediction of GDM by metabolic syndrome components prior to pregnancy A modest body of literature exists about the existence of metabolic syndrome prior to the detection of GDM. Gunderson and colleagues (2009) examined pre-pregnancy cardiometabolic risk factors and the risk of GDM in subsequent pregnancies. They found that metabolic impairment often predated the onset of GDM, and that 27% of overweight women with one or more cardiometabolic risk factors developed GDM (Gunderson et al., 2009). Normoglycemia with at least one metabolic risk factor (i.e., low plasma HDL-C and/or hyperinsulinemia) was present before pregnancy in 34% of those who developed GDM; among overweight women, the presence of any cardiometabolic feature was associated with an almost 4 times higher risk of GDM. Hedderson and Ferrara (2008) measured blood pressure before pregnancy and in early pregnancy, and found that women with mild hypertension in early pregnancy had a small increased risk of GDM (OR 1.56, 95% CI 1.16-2.10). Those with frank hypertension had a 2-fold increased risk of GDM (OR 2.04, 95% CI 1.14-3.65) compared to normotensive women, even after adjusting for confounders. These findings were paralleled by mild (OR 1.44, 95 CI 0.95-2.19) and frank (OR 2.01, 95% CI 1.01-3.99) hypertension detected before pregnancy (Hedderson & Ferrara, 2008). #### 6. Conclusions We have reviewed the parallels and associations between the metabolic syndrome and GDM. Both conditions have had multiple sets of diagnostic criteria and a history marked by controversies about their definition, clinical utility and significance. Both conditions identify a patient population that has an increased future risk of T2DM and CVD. Furthermore, both conditions have been associated with a similar set of emerging non-traditional risk factors. Consistent with these parallels, GDM predicts an increased risk of metabolic syndrome both in the early postpartum and in the years thereafter. Moreover, it is now becoming apparent that the metabolic syndrome and its associated risk factors may precede the diagnosis of GDM, both in early gestation and prior to the pregnancy. Taken together, these data suggest that GDM may represent a transient 'unmasking' of a latent metabolic syndrome, which may extend in both directions through (i) the pre-gravid state and early pregnancy, and (ii) the early and late postpartum. Figure 3 illustrates this lifetime continuum that may link metabolic syndrome, GDM, T2DM, and CVD. The chronic nature of the features of metabolic syndrome suggests that what we know about the temporal relation between metabolic syndrome, GDM, T2DM, and CVD is limited. The global burden of diabetes has been estimated at more than 171 million individuals with an expected increase to 366 million by 2030 (Wild et al., 2004). The prevalence of obesity and related metabolic dysfunction worldwide is a vivid demonstration of the undiscriminating potential of cardiometabolic diseases across ethnicities and age groups. In this context, the emerging relation between metabolic syndrome and GDM may offer the opportunity for early detection of atrisk individuals, long before the manifestation of overt disease. Ideally, this opportunity may lead to new strategies for early risk modification and ultimately disease prevention. As such, the emerging relation between metabolic syndrome and GDM represents an important area of research that may hold both clinical and public health implications. abnormalities likely precede pregnancy, which means that they should be detectable in early A modest body of literature exists about the existence of metabolic syndrome prior to the detection of GDM. Gunderson and colleagues (2009) examined pre-pregnancy cardiometabolic risk factors and the risk of GDM in subsequent pregnancies. They found that metabolic impairment often predated the onset of GDM, and that 27% of overweight women with one or more cardiometabolic risk factors developed GDM (Gunderson et al., 2009). Normoglycemia with at least one metabolic risk factor (i.e., low plasma HDL-C and/or hyperinsulinemia) was present before pregnancy in 34% of those who developed GDM; among overweight women, the presence of any cardiometabolic feature was associated with an almost 4 times higher risk of GDM. Hedderson and Ferrara (2008) measured blood pressure before pregnancy and in early pregnancy, and found that women with mild hypertension in early pregnancy had a small increased risk of GDM (OR 1.56, 95% CI 1.16-2.10). Those with frank hypertension had a 2-fold increased risk of GDM (OR 2.04, 95% CI 1.14-3.65) compared to normotensive women, even after adjusting for confounders. These findings were paralleled by mild (OR 1.44, 95 CI 0.95-2.19) and frank (OR 2.01, 95% CI 1.01-3.99) hypertension detected We have reviewed the parallels and associations between the metabolic syndrome and GDM. Both conditions have had multiple sets of diagnostic criteria and a history marked by controversies about their definition, clinical utility and significance. Both conditions identify a patient population that has an increased future risk of T2DM and CVD. Furthermore, both conditions have been associated with a similar set of emerging non-traditional risk factors. Consistent with these parallels, GDM predicts an increased risk of metabolic syndrome both in the early postpartum and in the years thereafter. Moreover, it is now becoming apparent that the metabolic syndrome and its associated risk factors may precede the diagnosis of GDM, both in early gestation and prior to the pregnancy. Taken together, these data suggest that GDM may represent a transient 'unmasking' of a latent metabolic syndrome, which may extend in both directions through (i) the pre-gravid state and early pregnancy, and (ii) the early and late postpartum. Figure 3 illustrates this lifetime continuum that may link metabolic syndrome, GDM, T2DM, and CVD. The chronic nature of the features of metabolic syndrome suggests that what we know about the temporal relation between metabolic syndrome, GDM, T2DM, and CVD is limited. The global burden of diabetes has been estimated at more than 171 million individuals with an expected increase to 366 million by 2030 (Wild et al., 2004). The prevalence of obesity and related metabolic dysfunction worldwide is a vivid demonstration of the undiscriminating potential of cardiometabolic diseases across ethnicities and age groups. In this context, the emerging relation between metabolic syndrome and GDM may offer the opportunity for early detection of atrisk individuals, long before the manifestation of overt disease. Ideally, this opportunity may lead to new strategies for early risk modification and ultimately disease prevention. As such, the emerging relation between metabolic syndrome and GDM represents an important area of research that may hold both clinical and public health implications. 5.2 Prediction of GDM by metabolic syndrome components prior to pregnancy pregnancy as well. 6. Conclusions before pregnancy (Hedderson & Ferrara, 2008). Fig. 3. Theoretical framework and conceptual model for latent metabolic syndrome preceding GDM. #### 7. References Gestational Diabetes and the Metabolic Syndrome 159 Boney, CM.; Verma, A., Tucker, R., & Vohr, BR. (2005). Metabolic Syndrome in childhood: Borges, RL.; Ribeiro, AB., Zanella, MT. & Batista, MC. (2010). Uric acid as a factor in the Brand, JS.; van der Tweel, I., Grobbee, DE., Emmelot-Vonk, MH. & van der Schouw, YT. Bruce, KD. & Hanson, MA. (2010). The developmental origins, mechanisms, and Buchanan, TA. (2001). Pancreatic B-cell defects in gestational diabetes: implications for the Buchanan, TA. & Xiang, AH. (2005). Gestational diabetes mellitus. The Journal of Clinical Investigation, Vol. 115, No. 3, (March 2005), pp. 485-491, doi:10.1172/JCI24531 Carr, DB.; Utzschneider, KM., Hull, RL., Tong, J., Wallace, TM., Kodama, K., Shofer, JB., et Cheung, NW. & Byth, K. (2003). Population health significance of gestational diabetes. Christoffersson, M. & Rydhstroem, H. (2002). Shoulder dystocia and brachial plexus injury: Clark, CM.; Qiu, C., Amerman, B., Porter, B., Fineberg, N., Aldasouqi, S. & Golichowski, A. Clausen, TD.; Mathiesen, ER., Hansen, T., Pedersen, O., Jensen, DM., Lauenborg, J. & Cutchie, WA.; Cheung, NW. & Simmons, D. (2006). Comparison of international and New Vol. 23, No. 5, (May 2006), pp. 460-468, doi:10.1111/j.1464-5491.2006.01850.x Damm, P. (2009). 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AJOG, Vol. 189, No. 1, (July 2003), pp. 171-176, doi:10.1067/mob.2003.343 9 Insulin Resistance in the Third Trimester of Mellitus or Impaired Glucose Tolerance Mohammed Chyad Al-Noaemi2 and Salah A.M. Ahmed3 Mohammed Helmy Faris Shalayel1, 2Al-Yarmouk College, Khartoum, 3Nile College - Khartoum, Sudan 1National College for Medical and Technical Studies, Pregnancy Suffering from Gestational Diabetes Pregnancy results in a state of insulin resistance (Dahlgren, 2006) that appears to include a decrease in maximum insulin sensitivity or responsivity (Baban et al., 2010). This insulin resistance abates in the postpartum period (Kuhl, 1991). Insulin resistance is defined as the decrease of the biological action of insulin (Catalano, 2010; Robert, 1995), and it mainly presents as hyperinsulinemia (Baban et al., 2010; Robert, 1995) or decreased ability of insulin The resistance to insulin can be characterized as pre-receptor (insulin antibodies), receptor (decreased number of receptors on the cell surface), or post-receptor (defects in the intracellular insulin signaling pathway). In pregnancy, the decreased insulin sensitivity is best characterized as a post-receptor defect resulting in the decreased ability of insulin to bring about glucose transporter (GLUT4) mobilization from the interior of the cell to the cell Most pregnant women are able to counteract the insulin resistance state by increasing their insulin secretion. However, when the capacity of insulin secretion is not sufficiently large to meet the insulin resistance, glucose intolerance develops and the women develop Gestational diabetes mellitus (GDM) is defined as a carbohydrate intolerance of varying severity with onset or first recognition during the present pregnancy (Kaaja & Rönnemaa, 2008; Damm et al., 1994; Summary and recommendation of the second international workshop conference of gestational diabetes, 1985; Shalayel et al., 2010). GDM has onset or discovery of glucose intolerance during pregnancy (Reece et al., 2009), usually in the second or third trimester (Shalayel et al., 2007). GDM carries long-term implications for the subsequent development of type 2 diabetes in the mother and increased risk for obesity and glucose intolerance in the offspring (Barbour et al., 2007). The world health organization defines diabetes in pregnancy as a fasting glucose ≥ 7.9 mmol/L or a value >11 mmol/L 2- hour after a 75g glucose load (Shalayel et al., 2010; Campbell & Lees, 2000). 1. Introduction surface (Catalano, 2010). gestational diabetes (Kuhl et al., 1985). to regulate glucose utilization (Kim et al., 1996). in women with prior gestational diabetes mellitus. Diabetes Care, Vol. 27, No. 7, (July 2004), pp. 1721-1727, doi:10.2337/diacare.27.7.1721 ## Insulin Resistance in the Third Trimester of Pregnancy Suffering from Gestational Diabetes Mellitus or Impaired Glucose Tolerance Mohammed Helmy Faris Shalayel1, Mohammed Chyad Al-Noaemi2 and Salah A.M. Ahmed3 1National College for Medical and Technical Studies, 2Al-Yarmouk College, Khartoum, 3Nile College - Khartoum, Sudan #### 1. Introduction 168 Gestational Diabetes Wolf, M.; Sandler, L., Hsu, K., et al. (2003). First-trimester C-reactive protein and subsequent Youssef, W. & McCullough, AJ. (2002). Diabetes mellitus, obesity, and hepatic steatosis. Seminars in Gastrointestinal Disease, Vol. 13, No. 1, (Jan. 2002), pp. 17-30. Zhang, Y.; Lu, X., Hong, J., Chao, M., Gu, W., Wang, W. & Ning, G. (2010). Positive (July 2004), pp. 1721-1727, doi:10.2337/diacare.27.7.1721 doi:10.2337/diacare.26.3.819 doi:10.1007/s12020-010-9369-6 in women with prior gestational diabetes mellitus. Diabetes Care, Vol. 27, No. 7, gestational diabetes. Diabetes Care, Vol. 26, No. 3, (July 2003), pp. 819-824, correlations of liver enzymes with metabolic syndrome including insulin resistance in newly diagnosed type 2 diabetes mellitus. Endocrine, Vol. 38, No. 2, pp. 181-187, > Pregnancy results in a state of insulin resistance (Dahlgren, 2006) that appears to include a decrease in maximum insulin sensitivity or responsivity (Baban et al., 2010). This insulin resistance abates in the postpartum period (Kuhl, 1991). Insulin resistance is defined as the decrease of the biological action of insulin (Catalano, 2010; Robert, 1995), and it mainly presents as hyperinsulinemia (Baban et al., 2010; Robert, 1995) or decreased ability of insulin to regulate glucose utilization (Kim et al., 1996). > The resistance to insulin can be characterized as pre-receptor (insulin antibodies), receptor (decreased number of receptors on the cell surface), or post-receptor (defects in the intracellular insulin signaling pathway). In pregnancy, the decreased insulin sensitivity is best characterized as a post-receptor defect resulting in the decreased ability of insulin to bring about glucose transporter (GLUT4) mobilization from the interior of the cell to the cell surface (Catalano, 2010). > Most pregnant women are able to counteract the insulin resistance state by increasing their insulin secretion. However, when the capacity of insulin secretion is not sufficiently large to meet the insulin resistance, glucose intolerance develops and the women develop gestational diabetes (Kuhl et al., 1985). > Gestational diabetes mellitus (GDM) is defined as a carbohydrate intolerance of varying severity with onset or first recognition during the present pregnancy (Kaaja & Rönnemaa, 2008; Damm et al., 1994; Summary and recommendation of the second international workshop conference of gestational diabetes, 1985; Shalayel et al., 2010). GDM has onset or discovery of glucose intolerance during pregnancy (Reece et al., 2009), usually in the second or third trimester (Shalayel et al., 2007). GDM carries long-term implications for the subsequent development of type 2 diabetes in the mother and increased risk for obesity and glucose intolerance in the offspring (Barbour et al., 2007). The world health organization defines diabetes in pregnancy as a fasting glucose ≥ 7.9 mmol/L or a value >11 mmol/L 2 hour after a 75g glucose load (Shalayel et al., 2010; Campbell & Lees, 2000). Insulin Resistance in the Third Trimester of Pregnancy Suffering performed using Statistical Packages for Social Sciences (SPSS) program. 2.3 Statistical analysis 3. Results (p<0.0001). group (46.7% vs 6.7%, z=3.2 and p<0.01). group (53.3% vs 10%, z=3.3 and p<0.001). 3.1 Plasma levels of glucose \* Significant \\ Highly significant \* Significant \\ Highly significant from Gestational Diabetes Mellitus or Impaired Glucose Tolerance 171 Data were expressed as mean, standard deviation (S.D.), standard error of mean (S.E.), and 95% confidence interval (CI) for mean. Comparisons were made using one-way analysis of variance (one-way ANOVA) and the significant differences among mean values were indicated by Scheffe test. Data of anti-insulin antibodies (AIA) of different groups of the study were compared by Kruskal–Wallis one-way ANOVA. The differences among the incidence percentages of family history of diabetes and previous heavy babies in the different groups of the study were assessed by calculation of z-values and their corresponding p-values. The correlation coefficient (r) is used to measure the closeness of the linear relationship between age and 2h-plasma glucose and between parity and 2h-plasma glucose. Calculations were The GDM and the IGT pregnant women were found to be significantly older than the control women [32.8 ±0.93 year (mean ± S.E.) and 31.1±1.1 vs. 23.9 ±0.82 respectively, p<0.0001]. Incidence of previous heavy babies (≥ 4.5 kg at birth) of the IGT group was significantly higher than that of the control group (33.3% vs 6.7%, z=2.25 and p<0.05). Also, incidence of previous heavy babies of the GDM group was significantly higher than that of the control Percentage of first degree family history of diabetes was significantly higher in the IGT group when compared with that of the control group (36.7% vs 10%, z=2.1 and p < 0.05). Much higher significant difference was shown between the GDM group and the control The GDM women were found to have higher mean levels of plasma glucose when compared with the IGT and the control pregnant women as shown in table 1 and table 2 GDM 7.59\\ 1.85 0.34 6.9-8.28 IGT 5.9\* 0.45 0.082 5.73-6.07 Control 2.91 0.43 0.079 2.75-3.07 Table 1. Fasting plasma glucose levels (0 min) in the three studied groups (mmol/L) GDM 12.35\\ 3.11 0.57 11.19-13.51 IGT 7.98\* 0.69 0.13 7.72-8.23 Control 4.35 0.65 0.12 4.11-4.6 Table 2. 2h-plasma glucose levels (120 min) in the three studied groups (mmol/l) Mean S.D. S.E. 95% CI Mean S.D. S.E. 95% CI Impaired Glucose Tolerance (IGT) was previously known as chemical diabetes or subclinical diabetes (Shalayel et al., 2007). It may be defined as an intermediate group of individuals whose carbohydrate metabolism does not constitute diabetes but is not entirely normal (Brudenell, 1993). Thus, IGT designed glucose tolerance results intermediate between normal glucose homeostasis and overt diabetes (Kahn et al., 2005; Bilous & Donnelly, 2010; Burch, 1994). It is diagnosed if fasting glucose ≥ 6 but < 7.8 m mol/L or 2-hour glucose > 7.8 m mol/L and <11.1 mmol/L (Shalayel et al., 2010; Hope et al., 1993). About 25% of patients with IGT eventually become diabetic (Shalayel et al., 2007). In fact there are significant alterations in glucose metabolism during pregnancy (Catalano, 1994). The carbohydrate tolerance is reduced, especially in the last trimester due to reduced sensitivity to insulin action (Hod &, Yogev, 2007). Insulin resistance is defined where a normal or elevated insulin level produces an attenuated biological response classically this refers to impaired sensitivity to insulin mediated glucose disposal (Wilcox, 2005). The objectives of this study were to stand on the state of insulin resistance that occurs in pregnancy and to assess the possible role of cortisol, human placental lactogen and prolactin in augmentation of such state. The study also showed the effect of some maternal risk factors such as age, parity, previous heavy babies and first-degree family history of diabetes in glucose tolerance impairment in pregnancy. #### 2. Subjects and methods The study was carried on Sudanese pregnant women in the third trimester (34.2±0.63, 35.97±0.71 and 36.53 ±0.64 current week of gestation for the GDM, IGT and control groups respectively). #### 2.1 Subjects Thirty pregnant women with the GDM,30 pregnant women with IGT and 30 pregnant women with a normal glucose tolerance (control group) chosen from Khartoum teaching hospital, Khartoum north hospital, Soba hospital, Ibrahim Malik hospital, Maternity hospital and Fath-Elrahman Elbasheer referral center. Oral consent was obtained from all the participants involved in the present study. All subjects overnight fasted before the test. A fasting blood sample was drawn at 6:00 O'clock a.m. then 75 g oral glucose dissolved in 200 cc water was given for each, waiting for 2 hours and then another blood sample was drawn from each. #### 2.2 Parameters analysis The concentration of fasting serum c-peptide, serum cortisol, human placental lactogen and prolactin (2-h after 75-g glucose load) were measured with the specific radio-immunoassay. Insulin: the concentrations of serum insulin in the fasting sample (0 min) and in the 2-hour after 75 g glucose load sample were measured with the specific immunoradiometric assay (IRMA). Anti insulin antibodies (AIA): the presence of circulating anti-insulin antibodies is semiquantitatively estimated by determination of the binding of tracer to AIA in the serum fraction. Plasma levels of glucose were assayed with a glucose oxidase kit while serum levels of cholesterol and triacylglycerols (TAGs) were estimated by the specific enzymatic colorimetric methods. #### 2.3 Statistical analysis 170 Gestational Diabetes Impaired Glucose Tolerance (IGT) was previously known as chemical diabetes or subclinical diabetes (Shalayel et al., 2007). It may be defined as an intermediate group of individuals whose carbohydrate metabolism does not constitute diabetes but is not entirely normal (Brudenell, 1993). Thus, IGT designed glucose tolerance results intermediate between normal glucose homeostasis and overt diabetes (Kahn et al., 2005; Bilous & Donnelly, 2010; Burch, 1994). It is diagnosed if fasting glucose ≥ 6 but < 7.8 m mol/L or 2-hour glucose > 7.8 m mol/L and <11.1 mmol/L (Shalayel et al., 2010; Hope et al., 1993). About In fact there are significant alterations in glucose metabolism during pregnancy (Catalano, 1994). The carbohydrate tolerance is reduced, especially in the last trimester due to reduced Insulin resistance is defined where a normal or elevated insulin level produces an attenuated biological response classically this refers to impaired sensitivity to insulin mediated glucose The objectives of this study were to stand on the state of insulin resistance that occurs in pregnancy and to assess the possible role of cortisol, human placental lactogen and prolactin in augmentation of such state. The study also showed the effect of some maternal risk factors such as age, parity, previous heavy babies and first-degree family history of diabetes The study was carried on Sudanese pregnant women in the third trimester (34.2±0.63, 35.97±0.71 and 36.53 ±0.64 current week of gestation for the GDM, IGT and control groups Thirty pregnant women with the GDM,30 pregnant women with IGT and 30 pregnant women with a normal glucose tolerance (control group) chosen from Khartoum teaching hospital, Khartoum north hospital, Soba hospital, Ibrahim Malik hospital, Maternity hospital and Fath-Elrahman Elbasheer referral center. Oral consent was obtained from all the All subjects overnight fasted before the test. A fasting blood sample was drawn at 6:00 O'clock a.m. then 75 g oral glucose dissolved in 200 cc water was given for each, waiting for The concentration of fasting serum c-peptide, serum cortisol, human placental lactogen and prolactin (2-h after 75-g glucose load) were measured with the specific radio-immunoassay. Insulin: the concentrations of serum insulin in the fasting sample (0 min) and in the 2-hour after 75 g glucose load sample were measured with the specific immunoradiometric assay Anti insulin antibodies (AIA): the presence of circulating anti-insulin antibodies is semiquantitatively estimated by determination of the binding of tracer to AIA in the serum fraction. Plasma levels of glucose were assayed with a glucose oxidase kit while serum levels of cholesterol and triacylglycerols (TAGs) were estimated by the specific enzymatic 25% of patients with IGT eventually become diabetic (Shalayel et al., 2007). sensitivity to insulin action (Hod &, Yogev, 2007). in glucose tolerance impairment in pregnancy. participants involved in the present study. 2 hours and then another blood sample was drawn from each. disposal (Wilcox, 2005). 2. Subjects and methods 2.2 Parameters analysis colorimetric methods. respectively). 2.1 Subjects (IRMA). Data were expressed as mean, standard deviation (S.D.), standard error of mean (S.E.), and 95% confidence interval (CI) for mean. Comparisons were made using one-way analysis of variance (one-way ANOVA) and the significant differences among mean values were indicated by Scheffe test. Data of anti-insulin antibodies (AIA) of different groups of the study were compared by Kruskal–Wallis one-way ANOVA. The differences among the incidence percentages of family history of diabetes and previous heavy babies in the different groups of the study were assessed by calculation of z-values and their corresponding p-values. The correlation coefficient (r) is used to measure the closeness of the linear relationship between age and 2h-plasma glucose and between parity and 2h-plasma glucose. Calculations were performed using Statistical Packages for Social Sciences (SPSS) program. #### 3. Results The GDM and the IGT pregnant women were found to be significantly older than the control women [32.8 ±0.93 year (mean ± S.E.) and 31.1±1.1 vs. 23.9 ±0.82 respectively, p<0.0001]. Incidence of previous heavy babies (≥ 4.5 kg at birth) of the IGT group was significantly higher than that of the control group (33.3% vs 6.7%, z=2.25 and p<0.05). Also, incidence of previous heavy babies of the GDM group was significantly higher than that of the control group (46.7% vs 6.7%, z=3.2 and p<0.01). Percentage of first degree family history of diabetes was significantly higher in the IGT group when compared with that of the control group (36.7% vs 10%, z=2.1 and p < 0.05). Much higher significant difference was shown between the GDM group and the control group (53.3% vs 10%, z=3.3 and p<0.001). 3.1 Plasma levels of glucose The GDM women were found to have higher mean levels of plasma glucose when compared with the IGT and the control pregnant women as shown in table 1 and table 2 (p<0.0001). \* Significant \\ Highly significant Table 1. Fasting plasma glucose levels (0 min) in the three studied groups (mmol/L) \* Significant \\ Highly significant Table 2. 2h-plasma glucose levels (120 min) in the three studied groups (mmol/l) Insulin Resistance in the Third Trimester of Pregnancy Suffering [7.15±0.49Mg/ml (mean ± S.E.), 6.85+0.58 and 5.73±0.24, p> 0.05]. 3.7 Serum hPL results 3.8 Serum prolactin results third trimester of gestation. Haroush et al., 2004). gestational diabetes mellitus incidence. tolerance (IGT & GDM) in Sudanese pregnant women. respectively, p>0.05]. 4. Discussion from Gestational Diabetes Mellitus or Impaired Glucose Tolerance 173 Although the mean HPL levels of the IGT group and that of the GDM group were higher than that of the control group, there were no significant differences among them Although the control group recorded the highest levels of serum prolactin and the GDM recorded the lowest results, there were no significant differences among the studied groups [123.6±9.61 ng/dl (mean ± S.E.), 145±15 and 150.2±9.7 for the GDM, IGT and control groups There is an increased frequency of gestational diabetes in oriental women and those from the Indian subcontinent and the Middle East (Stewart & Taylor, 1994). Pregnancy and diabetes mellitus aggravate each other (Potemkin, 1989). Hormonal changes occur in pregnancy, which profoundly affect carbohydrate metabolism. The levels of estrogen, progesterone, human placental lactogen, free cortisol and prolactin rise progressively as pregnancy advances. Of these a number, notably human placental lactogen and cortisol are insulin antagonists. So, insulin resistance develops in the mother as the pregnancy progresses, and it is most marked in the last trimester. This leads to deterioration in glucose tolerance (Brudenell, 1993). This explains why IGT and GDM were only discovered in the The results showed that many maternal risk factors affect the incidence of abnormal glucose Maternal age is an established risk factor for gestational diabetes mellitus (GDM), but there is no consensus on the age above which there is significantly increased risk of GDM (American Diabetes Association, 2004). The finding that the IGT and the GDM groups were significantly older than those with normal glucose tolerance (control) group, agrees with many previous studies that approved the direct relation between advanced maternal age (<35) and greater risk for incidence of GDM (Solomon et al., 1997; Cianni et al., 2003). Moreover, the presence of a linear relationship between the age and the 2h-plasma glucose ensure that there is an age related deterioration of glucose tolerance and makes the age a The association between parity and diabetes seems consistent in different studies. Women with highest parity are frequently older and heavier. Therefore, no study that evaluates parity could ignore a proper age adjustment (Dode & dos Santos, 2009). Multiparity has been associated with GDM in some studies but not in other ones (Seghieri et al., 2005; Ben- Kumari et al. (2002) found that grand multiparous women with parity > 10 had greater Significant higher mean parity of the IGT and the GDM groups when compared with the mean parity of the control (normal glucose tolerance) group as well as the existence of a linear relationship between parity and the 2h-plasma glucose, make the parity a very important maternal risk factor in impairment of glucose tolerance. This may be explained in terms of the diabetogenicity of the pregnancy, which is related to a pronounced peripheral very important maternal risk factor to affect glucose intolerance incidence. #### 3.2 Serum cholesterol and triacylglycerols results It was found that the mean levels of serum cholesterol of the IGT group and that of the GDM group were significantly higher than that of the control group [252.5±13.17 mg/dl (mean ±S.E.), 239.07±14.82 vs 195.73±8.47 respectively, F=6.22 and p<0.003]. Also, it was found that the mean levels of serum triacylglycerols of the GDM group and that of the IGT group were significant higher than that of the control group [278.47±15.90 mg/dl (mean ±S.E.), 259.37±11.6 vs 188.63 ±8.92 respectively, F=17.81 and p<0.0001]. But, there was no significant difference between the mean levels of TAGs of the GDM group and that of the IGT group although the serum TAGs levels were greater in the GDM group. #### 3.3 Serum c-peptide results Fasting c-peptides mean of the IGT group was not significantly higher than that of the GDM and control groups [0.34±0.04 p mol/l (mean ± S.E.) vs 0.26±0.03 and 0.28±0.04 respectively, p>0.05]. #### 3.4 Serum levels of cortisol There was a highly significant difference (p<0.0003) between serum level of cortisol of the GDM group and the IGT group from one hand and between the GDM group and the control group from other hand (table 3). \* Significant \\ Highly significant Table 3. Serum cortisol (nmol/l) values of the three studied group #### 3.5 Serum levels of insulin There was no significant difference among the mean fasting serum insulin levels of the studied groups [12.29±0.83 MIU/ml (mean ±S.E.), 14.93±2.17 and 2.39±2.25 for the GDM, IGT and control groups respectively, p>0.05]. The men level of 2h-serum insulin of the IGT group was significantly higher than that of the GDM group (68±6.71 vs 36.5±3.06, p<0.002) while, it was not significantly higher than that of the control group (68±6.71 vs 54.88±8.15). #### 3.6 Serum anti-insulin antibodies (AIA) results The results of AIA binding percentage to the tracer (table 4) showed significant difference among the three studied groups [X² (df=2) = 7.34,p<0.025]. \* Significant \\ Highly significant Table 4. Percent bound results of serum AIA #### 3.7 Serum hPL results 172 Gestational Diabetes It was found that the mean levels of serum cholesterol of the IGT group and that of the GDM group were significantly higher than that of the control group [252.5±13.17 mg/dl Also, it was found that the mean levels of serum triacylglycerols of the GDM group and that of the IGT group were significant higher than that of the control group [278.47±15.90 mg/dl But, there was no significant difference between the mean levels of TAGs of the GDM group and that of the IGT group although the serum TAGs levels were greater in the GDM group. Fasting c-peptides mean of the IGT group was not significantly higher than that of the GDM and control groups [0.34±0.04 p mol/l (mean ± S.E.) vs 0.26±0.03 and 0.28±0.04 respectively, There was a highly significant difference (p<0.0003) between serum level of cortisol of the GDM group and the IGT group from one hand and between the GDM group and the control There was no significant difference among the mean fasting serum insulin levels of the studied groups [12.29±0.83 MIU/ml (mean ±S.E.), 14.93±2.17 and 2.39±2.25 for the GDM, The men level of 2h-serum insulin of the IGT group was significantly higher than that of the GDM group (68±6.71 vs 36.5±3.06, p<0.002) while, it was not significantly higher than that of The results of AIA binding percentage to the tracer (table 4) showed significant difference mean Mean rank Mean S.D. S.E. 95%CI for GDM 25.11\\ 31.01 5.66 13.53-36.69 54.25 IGT 7.18\* 2.47 0.45 6.25-8.10 46.23 Control 6.04 1.50 0.27 5.48-6.60 36.02 Mean S.D. S.E. 95% CI GDM 937.2\\ 434.8 79.38 774.9-1099.6 IGT 794.2\* 331.5 60.52 670.4-918 Control 597.3 169.4 30.93 534-660.5 Table 3. Serum cortisol (nmol/l) values of the three studied group (mean ±S.E.), 239.07±14.82 vs 195.73±8.47 respectively, F=6.22 and p<0.003]. (mean ±S.E.), 259.37±11.6 vs 188.63 ±8.92 respectively, F=17.81 and p<0.0001]. 3.2 Serum cholesterol and triacylglycerols results 3.3 Serum c-peptide results 3.4 Serum levels of cortisol group from other hand (table 3). \* Significant \\ Highly significant 3.5 Serum levels of insulin \* Significant \\ Highly significant Table 4. Percent bound results of serum AIA IGT and control groups respectively, p>0.05]. 3.6 Serum anti-insulin antibodies (AIA) results among the three studied groups [X² (df=2) = 7.34,p<0.025]. the control group (68±6.71 vs 54.88±8.15). p>0.05]. Although the mean HPL levels of the IGT group and that of the GDM group were higher than that of the control group, there were no significant differences among them [7.15±0.49Mg/ml (mean ± S.E.), 6.85+0.58 and 5.73±0.24, p> 0.05]. #### 3.8 Serum prolactin results Although the control group recorded the highest levels of serum prolactin and the GDM recorded the lowest results, there were no significant differences among the studied groups [123.6±9.61 ng/dl (mean ± S.E.), 145±15 and 150.2±9.7 for the GDM, IGT and control groups respectively, p>0.05]. #### 4. Discussion There is an increased frequency of gestational diabetes in oriental women and those from the Indian subcontinent and the Middle East (Stewart & Taylor, 1994). Pregnancy and diabetes mellitus aggravate each other (Potemkin, 1989). Hormonal changes occur in pregnancy, which profoundly affect carbohydrate metabolism. The levels of estrogen, progesterone, human placental lactogen, free cortisol and prolactin rise progressively as pregnancy advances. Of these a number, notably human placental lactogen and cortisol are insulin antagonists. So, insulin resistance develops in the mother as the pregnancy progresses, and it is most marked in the last trimester. This leads to deterioration in glucose tolerance (Brudenell, 1993). This explains why IGT and GDM were only discovered in the third trimester of gestation. The results showed that many maternal risk factors affect the incidence of abnormal glucose tolerance (IGT & GDM) in Sudanese pregnant women. Maternal age is an established risk factor for gestational diabetes mellitus (GDM), but there is no consensus on the age above which there is significantly increased risk of GDM (American Diabetes Association, 2004). The finding that the IGT and the GDM groups were significantly older than those with normal glucose tolerance (control) group, agrees with many previous studies that approved the direct relation between advanced maternal age (<35) and greater risk for incidence of GDM (Solomon et al., 1997; Cianni et al., 2003). Moreover, the presence of a linear relationship between the age and the 2h-plasma glucose ensure that there is an age related deterioration of glucose tolerance and makes the age a very important maternal risk factor to affect glucose intolerance incidence. The association between parity and diabetes seems consistent in different studies. Women with highest parity are frequently older and heavier. Therefore, no study that evaluates parity could ignore a proper age adjustment (Dode & dos Santos, 2009). Multiparity has been associated with GDM in some studies but not in other ones (Seghieri et al., 2005; Ben-Haroush et al., 2004). Kumari et al. (2002) found that grand multiparous women with parity > 10 had greater gestational diabetes mellitus incidence. Significant higher mean parity of the IGT and the GDM groups when compared with the mean parity of the control (normal glucose tolerance) group as well as the existence of a linear relationship between parity and the 2h-plasma glucose, make the parity a very important maternal risk factor in impairment of glucose tolerance. This may be explained in terms of the diabetogenicity of the pregnancy, which is related to a pronounced peripheral Insulin Resistance in the Third Trimester of Pregnancy Suffering be responsible for the hypertriglyceridemia of pregnancy (Butte, 2000). rather than glucose such as fat to overcome the state of insulin resistance. diabetes appear to have a greater degree of insulin resistance (Ryan et al., 1985). although many other studies tried to put general points to explain this state. elevated c-peptides may result from increased beta-cells activity. IGT group when compared with the control group. control group. from Gestational Diabetes Mellitus or Impaired Glucose Tolerance 175 Maternal responses to these demands consist of an accelerated switch from carbohydrate to fat utilization that is facilitated by peripheral insulin resistance and by high blood levels of lipolytic hormones. In patients with GDM, insulin resistance is either comparable or greater than in non-diabetic pregnancy whereas insulin secretion appears to be compromised. Changes in hepatic and adipose metabolism alter circulating concentrations of triacylglycerols, fatty acids, cholesterol, and phospholipids. After an initial decrease in the first 8 wk of pregnancy, there is a steady increase in triacylglycerols, fatty acids, cholesterol, lipoproteins, and phospholipids (Butte, 2000). There is a two- to threefold increase in basal triglyceride and cholesterol concentrations with advancing gestation. The increases are more pronounced in the GDM as compared with the normal glucose tolerant pregnant woman (Catalano, 2010). The higher concentration of estrogen and insulin resistance are thought to Thus, it is concluded that in the third trimester of pregnancy, there is a competition between mother and her fetus on glucose uptake. This competition will be directed towards the benefit of the fetus. For this reason, the pregnant women do switch to other energy source The syndrome of insulin resistance is a group of clinically diverse disorders (Catalano, 2010; Flier, 1992). Pregnancy induces complex changes in energy metabolism, manifested clinically by insulin resistance (Bedalov & Balasubramanyam, 1997). Glucose tolerance deteriorates in all pregnant women, but only in 2-3% of all pregnancies is the deterioration sufficiently large to fulfill the diagnostic criteria for gestational diabetes1. Many previous studies demonstrated that pregnancy result in a state of insulin resistance and women with gestational-onset Why pregnancy is capable of inducing the temporary diabetic state is still partly unknown, Bergstrom et al. (1990) revealed that an increased fasting c-peptide reflects insulin resistance. In fact, insulin and c-peptide are secreted in equimolar amount. However, because of its longer half-life, the plasma concentration of c-peptide is higher than that of insulin. Within limits, c-peptides levels can serve as valuable index to insulin secretion. Thus, low c-peptide levels are to be expected when insulin secretion is diminished whereas Regarding our results, the serum fasting c-peptide mean level was the highest in the IGT groups (0.337± 0.038 Pmol/L) when compared with the GDM group (0.262 ± 0.025) and the control group (0.284 ± 0.041) although the differences were not significant (P > 0.05). This gives prediction to the higher insulin resistance in the IGT group when compared with the Most pregnant women are able to counteract the insulin resistance in pregnancy by increasing their insulin secretion. This also explains the highest mean levels of insulin in the Although the insulin levels, (fasting and 2h-insulin) were lower in the control group than that of the IGT group, the glucose tolerance kept normal. This may support the suggestion that during normal pregnancy, the Staub-Traugott effect i.e., improved glucose disposal after successive glucose load administrations occurs and appears to be caused by mechanisms other However, when the capacity of insulin secretion is not sufficiently large to meet the resistance, glucose intolerance develops and the women develop gestational diabetes than enhanced insulin secretion with successive glucose loads (Lewis et al., 1993). resistance to insulin (Kuhl, 1991). Parity reflects the duration of exposure to the insulin resistance (Peters et al., 1995). Thus, one can conclude that higher parity may lead to accumulation of the diabetogenic effect of pregnancy. Consequently, much more glucose impairment occurs. Genetic factors play a part in the development of diabetes although the exact mode of inheritance is not established (Brudenell, 1993). Recent evidence suggests that the gestational diabetes has a strong genetic component and is usually NIDDM. Both GDM and NIDDM are characterized by insulin deficiency and by insulin resistance (Dornhorst et al., 1990). This evidence agrees with that of Csorba and Edwards (1995) who showed that the development of both type II and GDM is probably governed by a complex and variable interaction of genes and environments. Moreover, they suggest that both beta cell dysfunction and insulin resistance is operative in the manifestation of these disorders. This may explain why the incidence of first degree family history of diabetes was significantly higher in the GDM and in the IGT groups when compared with that of the control group. Therefore, family history of diabetes is a very important maternal risk factor from the obstetrician's point of view (Brudenell, 1993). The result that the GDM and the IGT groups have incidence of previous heavy babies significantly higher than that of control group, make previous delivery of a large baby to be a very important maternal risk factor since a tendency to bear heavy babies may precede the development of clinical diabetes by many years (Brudenell, 1993). Changes in lipid metabolism occur during pregnancy. Plasma levels of triglycerides, cholesterol and free fatty acids rise, and there is a greater tendency to ketosis (Campbell & Lees, 2000). Some studies showed that total triglycerides increase with gestational time in pre-gestational diabetic women, GDM women and healthy control women (Montelongo et al., 1992). Every aspect of lipid metabolism is affected by pregnancy. The plasma level of free fatty acids falls from early to mid-pregnancy and thereafter shows a significant rise. The same is true for the plasma level of glycerol. This is in keeping with the accumulation of body fat that occurs during the anabolic phase of pregnancy (first two trimesters). In the catabolic phase of pregnancy (last trimester), raised free fatty acids and glycerol levels are available as fuel to the maternal tissues to offset the increasing diversion to the rapidly growing fetus of glucose and amino acids. As with free fatty, glycerol and triglycerides, plasma levels of cholesterol and phospholipids are increased in pregnancy (Brudenell, 1993) taking in account that plasma triglycerides may be a physiological contributor to infant birth weight (Knopp et al., 1992). Thus, one can expect more increase of these lipid substances when the glucose tolerance deteriorates in pregnancy. Therefore, the mean level of serum cholesterol and triglycerides were significantly higher in the GDM and in the IGT groups when compared with the control (normal glucose tolerance) group. The changes in lipid metabolism are mediated by hormonal changes and fit into the general pattern of an increase in storage of glycogen and fat in most maternal tissues during the metabolic first two trimesters of pregnancy, followed by the mobilization of fuel for the benefit of both mother and fetus in catabolic third trimester (Brudenell, 1993). Boden (1996) demonstrated that in early pregnancy, insulin secretion in response to glucose is increased, peripheral insulin sensitivity is normal or increased and glucose tolerance is normal or slightly enhanced. In addition, there is maternal fat accumulation. During late pregnancy, there is increased fetal growth and increased fetal demand for nutrients. resistance to insulin (Kuhl, 1991). Parity reflects the duration of exposure to the insulin resistance (Peters et al., 1995). Thus, one can conclude that higher parity may lead to accumulation of the diabetogenic effect of pregnancy. Consequently, much more glucose Genetic factors play a part in the development of diabetes although the exact mode of inheritance is not established (Brudenell, 1993). Recent evidence suggests that the gestational diabetes has a strong genetic component and is usually NIDDM. Both GDM and NIDDM are characterized by insulin deficiency and by insulin resistance (Dornhorst et al., 1990). This evidence agrees with that of Csorba and Edwards (1995) who showed that the development of both type II and GDM is probably governed by a complex and variable interaction of genes and environments. Moreover, they suggest that both beta cell dysfunction and insulin resistance is operative in the manifestation of these disorders. This may explain why the incidence of first degree family history of diabetes was significantly higher in the GDM and in the IGT groups when compared with that of the control group. Therefore, family history of diabetes is a very important maternal risk factor from the The result that the GDM and the IGT groups have incidence of previous heavy babies significantly higher than that of control group, make previous delivery of a large baby to be a very important maternal risk factor since a tendency to bear heavy babies may precede the Changes in lipid metabolism occur during pregnancy. Plasma levels of triglycerides, cholesterol and free fatty acids rise, and there is a greater tendency to ketosis (Campbell & Lees, 2000). Some studies showed that total triglycerides increase with gestational time in pre-gestational diabetic women, GDM women and healthy control women (Montelongo et Every aspect of lipid metabolism is affected by pregnancy. The plasma level of free fatty acids falls from early to mid-pregnancy and thereafter shows a significant rise. The same is true for the plasma level of glycerol. This is in keeping with the accumulation of body fat that occurs during the anabolic phase of pregnancy (first two trimesters). In the catabolic phase of pregnancy (last trimester), raised free fatty acids and glycerol levels are available as fuel to the maternal tissues to offset the increasing diversion to the rapidly growing fetus of glucose and amino acids. As with free fatty, glycerol and triglycerides, plasma levels of cholesterol and phospholipids are increased in pregnancy (Brudenell, 1993) taking in account that plasma triglycerides may be a physiological contributor to infant birth weight (Knopp et al., 1992). Thus, one can expect more increase of these lipid substances when the glucose tolerance deteriorates in pregnancy. Therefore, the mean level of serum cholesterol and triglycerides were significantly higher in the GDM and in the IGT groups when The changes in lipid metabolism are mediated by hormonal changes and fit into the general pattern of an increase in storage of glycogen and fat in most maternal tissues during the metabolic first two trimesters of pregnancy, followed by the mobilization of fuel for the Boden (1996) demonstrated that in early pregnancy, insulin secretion in response to glucose is increased, peripheral insulin sensitivity is normal or increased and glucose tolerance is normal or slightly enhanced. In addition, there is maternal fat accumulation. During late pregnancy, there is increased fetal growth and increased fetal demand for nutrients. benefit of both mother and fetus in catabolic third trimester (Brudenell, 1993). impairment occurs. al., 1992). obstetrician's point of view (Brudenell, 1993). development of clinical diabetes by many years (Brudenell, 1993). compared with the control (normal glucose tolerance) group. Maternal responses to these demands consist of an accelerated switch from carbohydrate to fat utilization that is facilitated by peripheral insulin resistance and by high blood levels of lipolytic hormones. In patients with GDM, insulin resistance is either comparable or greater than in non-diabetic pregnancy whereas insulin secretion appears to be compromised. Changes in hepatic and adipose metabolism alter circulating concentrations of triacylglycerols, fatty acids, cholesterol, and phospholipids. After an initial decrease in the first 8 wk of pregnancy, there is a steady increase in triacylglycerols, fatty acids, cholesterol, lipoproteins, and phospholipids (Butte, 2000). There is a two- to threefold increase in basal triglyceride and cholesterol concentrations with advancing gestation. The increases are more pronounced in the GDM as compared with the normal glucose tolerant pregnant woman (Catalano, 2010). The higher concentration of estrogen and insulin resistance are thought to be responsible for the hypertriglyceridemia of pregnancy (Butte, 2000). Thus, it is concluded that in the third trimester of pregnancy, there is a competition between mother and her fetus on glucose uptake. This competition will be directed towards the benefit of the fetus. For this reason, the pregnant women do switch to other energy source rather than glucose such as fat to overcome the state of insulin resistance. The syndrome of insulin resistance is a group of clinically diverse disorders (Catalano, 2010; Flier, 1992). Pregnancy induces complex changes in energy metabolism, manifested clinically by insulin resistance (Bedalov & Balasubramanyam, 1997). Glucose tolerance deteriorates in all pregnant women, but only in 2-3% of all pregnancies is the deterioration sufficiently large to fulfill the diagnostic criteria for gestational diabetes1. Many previous studies demonstrated that pregnancy result in a state of insulin resistance and women with gestational-onset diabetes appear to have a greater degree of insulin resistance (Ryan et al., 1985). Why pregnancy is capable of inducing the temporary diabetic state is still partly unknown, although many other studies tried to put general points to explain this state. Bergstrom et al. (1990) revealed that an increased fasting c-peptide reflects insulin resistance. In fact, insulin and c-peptide are secreted in equimolar amount. However, because of its longer half-life, the plasma concentration of c-peptide is higher than that of insulin. Within limits, c-peptides levels can serve as valuable index to insulin secretion. Thus, low c-peptide levels are to be expected when insulin secretion is diminished whereas elevated c-peptides may result from increased beta-cells activity. Regarding our results, the serum fasting c-peptide mean level was the highest in the IGT groups (0.337± 0.038 Pmol/L) when compared with the GDM group (0.262 ± 0.025) and the control group (0.284 ± 0.041) although the differences were not significant (P > 0.05). This gives prediction to the higher insulin resistance in the IGT group when compared with the control group. Most pregnant women are able to counteract the insulin resistance in pregnancy by increasing their insulin secretion. This also explains the highest mean levels of insulin in the IGT group when compared with the control group. Although the insulin levels, (fasting and 2h-insulin) were lower in the control group than that of the IGT group, the glucose tolerance kept normal. This may support the suggestion that during normal pregnancy, the Staub-Traugott effect i.e., improved glucose disposal after successive glucose load administrations occurs and appears to be caused by mechanisms other than enhanced insulin secretion with successive glucose loads (Lewis et al., 1993). However, when the capacity of insulin secretion is not sufficiently large to meet the resistance, glucose intolerance develops and the women develop gestational diabetes Insulin Resistance in the Third Trimester of Pregnancy Suffering from Gestational Diabetes Mellitus or Impaired Glucose Tolerance 177 chromosome (Davis, 1990). Human PL has only a limited homology with prolactin, but a very high homology with the growth hormone mRNA coding sequence, although in other species there is a more homology with prolactin than growth hormone. The hormone is synthesized in the syncytiotrophoblastic villous epithelium of the placenta and it is secreted into maternal blood (Davis, 1990; Strauss & Barbieri, 2009). Maternal serum hPL levels rise progressively from the first trimester through till term (Strauss & Barbieri, 2009) and this agrees with our results of serum hPL in the control group. Although, there were no significant differences among hPL mean level of the IGT and the GDM groups were higher than that of the control group. This non-significant slight increase in the IGT and the GDM groups may share in Some authors reported that pronounced fetal macrosomia may occur even with adequate maternal blood glucose control. The severe fetal hyperinsulinemia in this case may be imputed to some factor other than excessive glucose load, possibly hPL, which induces proliferation and enhanced function of pancreatic ß-cells. In concert with this hypothesis, maternal serum hPL concentrations measured in the third trimester are higher in diabetic The regulation of hPL secretion is not fully understood. Progesterone exerts stimulatory effects in early gestation, but not in late gestation. Finally the hPL/CS gene contains a binding site for the glucocorticoids receptor, suggesting a potential modulatory role for corticosteroids, and also has binding sites for a pituitary protein factor that is thought to regulate both prolactin and growth hormone gene expression (Davis, 1990). Furthermore, the very high levels of circulating estrogen that occur during pregnancy result in a parallel increase in the circulating levels of prolactin (Schlechte, 2007). This may explain the interconnection between hPL and prolactin since, it seems that prolactin, growth hormone and placental lactogen hormone are phylogenetically ancient hormones which in vertebrates have evolved from common ancestral molecules. These hormones share common effects in growth stimulation and lactation. Hypersomatotropism is associated with disturbance of glucose tolerance and insulin resistance. Hyperprolactinaemia, like hypersomatotropism, is The present study (on control group) demonstrated that prolactin increases progressively from the first trimester through till third trimester. Moreover, our study revealed that there were no significant differences among the levels of serum prolactin in GDM, IGT, and control groups. This agrees with what has been mentioned by Grigorakiz et al. (2000). Consequently, there is no evidence that prolactin may be directly incorporated with the pathogenesis of glucose intolerance in pregnancy. This may agree with the study of Milasinović et al. (1997) who reported that there is no evidence of the functional connection Prolactin is found in large amounts in the amniotic fluid of humans and other primates, and it is now clearly established that the source of this prolactin is the placenta rather than the maternal or fetal pituitary. The endometrial lining of the uterus is greatly modified during pregnancy to form the decidua. This decidual tissue has been confirmed as the site of placental prolactin production by a number of different groups and the mature peptide hormone is immunologically indistinguishable from pituitary prolactin. Immunocytochemical studies have shown that the hormone is predominantly located in the parietal decidual cells and only very rarely in the chorionic cytotrophoblast. Amniotic fluid prolactin levels are very low in ectopic tubal pregnancy, confirming the role of the increasing insulin resistance in these groups when compared with control group. pregnancies complicated by fetal macrosomia (Persson et al., 1995; Reis et al., 2002). associated with decreased insulin sensitivity (Foss et al., 1995). between prolactin and glucose metabolism. decidualized endometrium (Davis, 1990). (Shalayel et al., 2010; Buchanan & Xiang, 2005). This may be due to the presence of high levels of insulin antagonistic hormones as hPL and cortisol (Carr & Gabbe, 1998) as well as high level of circulating anti-insulin antibodies which may make the pregnant women secrete more insulin to overcome the insulin resistance and eventually may lead to exhaustion of beta-cells of the pancreas. This explain the lowest mean levels of fasting cpeptide, fasting serum insulin and 2h-serum insulin in the GDM group when compared with the IGT and the control groups. Damm et al. (1995) demonstrated that women who develop GDM have a relative insulin secretion deficiency, the severity of which is predictive for later development of diabetes. Furthermore, their relatively reduced beta-cells functions may be a significant pathogenic factor in relation to the high incidence of subsequent diabetes in women with GDM. This agrees with Paulus et al. (1995) who showed that the diagnoses of gestational diabetes mellitus are at a greater risk for developing diabetes in later life. Homko et al (2001) reported that patients with GDM during late pregnancy not only had severe deficiencies in insulin secretion rate (ISR) and were more insulin resistant than controls. In addition, the women with GDM had a major ß-cell defect that made it impossible for them to compensate for their increased level of insulin resistance, which occurred during late pregnancy. Our study also showed that the cortisol increases progressively as pregnancy advances and the mean cortisol level was significantly the highest in the GDM group when compared with the IGT and the control groups while its level was the lowest in the control group in the third trimester. This ensures the possibility role of cortisol as an insulin antagonist in the deterioration of the glucose tolerance in pregnancy. Cortisol is bound in the circulation to alpha-globulin called transcortin or corticosteroidbinding globulin (CBG). The bound cortisol functions as circulating reservoir of hormone that keeps a supply of free cortisol available to the tissues. At normal levels of total plasma cortisol (13.5µg/dL), there is very little free cortisol in plasma, but the binding sites on CBG become saturated when the total plasma cortisol exceeds 20µg/dL. CBG is synthesized in the liver, and its production is increased by estrogen and its level is elevated during pregnancy. When the CBG level rises, more cortisol is bound, and initially there is a drop in the free cortisol level which stimulates adrenocorticotrophic hormone (ACTH) secretion. Therefore, more cortisol is secreted until a new equilibrium is reached at which the bound cortisol is elevated but the free cortisol is normal. Changes in the opposite direction occur when the CBG level falls. This explains why pregnant women have higher total plasma levels of cortisol without symptoms of glucocorticoids excess (Barrett et al., 2010). Cortisol is nearly totally (90%) bound to CBG up to concentrations of 25µg/dL. But, when cortisol concentrations rise above this level, as occurred in the IGT group (serum cortisol mean level = 28.57µg/dL) and in the GDM group (serum cortisol mean level = 33.71µg/dL), the binding capacity of CBG is exceeded and the proportion of unbound, free, cortisol rises greatly (Burch, 1994). This free cortisol increases glucose tolerance deterioration therefore confirms the dominating role of cortisol as a regulator of stress dependent insulin resistance (Lehrke et al., 2008). Human placental lactogen (hPL) is a single protein of 191 amino acids, which is encoded by two genes hPL/CS-A and hPL/CS-B, which are identical except for one minor difference in the single sequence coding region. The hPL/CS genes are clustered together with the pituitary growth hormone gene (hGH-N) and a variant GH gene, hGH-V, on the long arm of (Shalayel et al., 2010; Buchanan & Xiang, 2005). This may be due to the presence of high levels of insulin antagonistic hormones as hPL and cortisol (Carr & Gabbe, 1998) as well as high level of circulating anti-insulin antibodies which may make the pregnant women secrete more insulin to overcome the insulin resistance and eventually may lead to exhaustion of beta-cells of the pancreas. This explain the lowest mean levels of fasting cpeptide, fasting serum insulin and 2h-serum insulin in the GDM group when compared Damm et al. (1995) demonstrated that women who develop GDM have a relative insulin secretion deficiency, the severity of which is predictive for later development of diabetes. Furthermore, their relatively reduced beta-cells functions may be a significant pathogenic factor in relation to the high incidence of subsequent diabetes in women with GDM. This agrees with Paulus et al. (1995) who showed that the diagnoses of gestational diabetes Homko et al (2001) reported that patients with GDM during late pregnancy not only had severe deficiencies in insulin secretion rate (ISR) and were more insulin resistant than controls. In addition, the women with GDM had a major ß-cell defect that made it impossible for them to compensate for their increased level of insulin resistance, which Our study also showed that the cortisol increases progressively as pregnancy advances and the mean cortisol level was significantly the highest in the GDM group when compared with the IGT and the control groups while its level was the lowest in the control group in the third trimester. This ensures the possibility role of cortisol as an insulin antagonist in the Cortisol is bound in the circulation to alpha-globulin called transcortin or corticosteroidbinding globulin (CBG). The bound cortisol functions as circulating reservoir of hormone that keeps a supply of free cortisol available to the tissues. At normal levels of total plasma cortisol (13.5µg/dL), there is very little free cortisol in plasma, but the binding sites on CBG become saturated when the total plasma cortisol exceeds 20µg/dL. CBG is synthesized in the liver, and its production is increased by estrogen and its level is elevated during pregnancy. When the CBG level rises, more cortisol is bound, and initially there is a drop in the free cortisol level which stimulates adrenocorticotrophic hormone (ACTH) secretion. Therefore, more cortisol is secreted until a new equilibrium is reached at which the bound cortisol is elevated but the free cortisol is normal. Changes in the opposite direction occur when the CBG level falls. This explains why pregnant women have higher total plasma levels of cortisol without symptoms of glucocorticoids excess (Barrett et al., 2010). Cortisol is nearly totally (90%) bound to CBG up to concentrations of 25µg/dL. But, when cortisol concentrations rise above this level, as occurred in the IGT group (serum cortisol mean level = 28.57µg/dL) and in the GDM group (serum cortisol mean level = 33.71µg/dL), the binding capacity of CBG is exceeded and the proportion of unbound, free, cortisol rises greatly (Burch, 1994). This free cortisol increases glucose tolerance deterioration therefore confirms the dominating role of cortisol as a regulator of stress dependent insulin resistance Human placental lactogen (hPL) is a single protein of 191 amino acids, which is encoded by two genes hPL/CS-A and hPL/CS-B, which are identical except for one minor difference in the single sequence coding region. The hPL/CS genes are clustered together with the pituitary growth hormone gene (hGH-N) and a variant GH gene, hGH-V, on the long arm of with the IGT and the control groups. occurred during late pregnancy. (Lehrke et al., 2008). mellitus are at a greater risk for developing diabetes in later life. deterioration of the glucose tolerance in pregnancy. chromosome (Davis, 1990). Human PL has only a limited homology with prolactin, but a very high homology with the growth hormone mRNA coding sequence, although in other species there is a more homology with prolactin than growth hormone. The hormone is synthesized in the syncytiotrophoblastic villous epithelium of the placenta and it is secreted into maternal blood (Davis, 1990; Strauss & Barbieri, 2009). Maternal serum hPL levels rise progressively from the first trimester through till term (Strauss & Barbieri, 2009) and this agrees with our results of serum hPL in the control group. Although, there were no significant differences among hPL mean level of the IGT and the GDM groups were higher than that of the control group. This non-significant slight increase in the IGT and the GDM groups may share in increasing insulin resistance in these groups when compared with control group. Some authors reported that pronounced fetal macrosomia may occur even with adequate maternal blood glucose control. The severe fetal hyperinsulinemia in this case may be imputed to some factor other than excessive glucose load, possibly hPL, which induces proliferation and enhanced function of pancreatic ß-cells. In concert with this hypothesis, maternal serum hPL concentrations measured in the third trimester are higher in diabetic pregnancies complicated by fetal macrosomia (Persson et al., 1995; Reis et al., 2002). The regulation of hPL secretion is not fully understood. Progesterone exerts stimulatory effects in early gestation, but not in late gestation. Finally the hPL/CS gene contains a binding site for the glucocorticoids receptor, suggesting a potential modulatory role for corticosteroids, and also has binding sites for a pituitary protein factor that is thought to regulate both prolactin and growth hormone gene expression (Davis, 1990). Furthermore, the very high levels of circulating estrogen that occur during pregnancy result in a parallel increase in the circulating levels of prolactin (Schlechte, 2007). This may explain the interconnection between hPL and prolactin since, it seems that prolactin, growth hormone and placental lactogen hormone are phylogenetically ancient hormones which in vertebrates have evolved from common ancestral molecules. These hormones share common effects in growth stimulation and lactation. Hypersomatotropism is associated with disturbance of glucose tolerance and insulin resistance. Hyperprolactinaemia, like hypersomatotropism, is associated with decreased insulin sensitivity (Foss et al., 1995). The present study (on control group) demonstrated that prolactin increases progressively from the first trimester through till third trimester. Moreover, our study revealed that there were no significant differences among the levels of serum prolactin in GDM, IGT, and control groups. This agrees with what has been mentioned by Grigorakiz et al. (2000). Consequently, there is no evidence that prolactin may be directly incorporated with the pathogenesis of glucose intolerance in pregnancy. This may agree with the study of Milasinović et al. (1997) who reported that there is no evidence of the functional connection between prolactin and glucose metabolism. Prolactin is found in large amounts in the amniotic fluid of humans and other primates, and it is now clearly established that the source of this prolactin is the placenta rather than the maternal or fetal pituitary. The endometrial lining of the uterus is greatly modified during pregnancy to form the decidua. This decidual tissue has been confirmed as the site of placental prolactin production by a number of different groups and the mature peptide hormone is immunologically indistinguishable from pituitary prolactin. Immunocytochemical studies have shown that the hormone is predominantly located in the parietal decidual cells and only very rarely in the chorionic cytotrophoblast. Amniotic fluid prolactin levels are very low in ectopic tubal pregnancy, confirming the role of the decidualized endometrium (Davis, 1990). Insulin Resistance in the Third Trimester of Pregnancy Suffering Hill Companies, ISBN: 978-0-07-160567-0, USA. Diabetes Care, 30 (2): S112-S119. Diabetic Care, 20(6): 922-924. 9781405184090, UK. Egypt. 1261S. 365-371. 131-137. overview. Melbourne and Newyork. 340 74082 5, London. Gynecol, 37(1): 25-38. Clin Diabetes, 16(1): 4-11. gynecol. Clin. North Am., 23 (1): 1-10. from Gestational Diabetes Mellitus or Impaired Glucose Tolerance 179 Barbour LA, Carrie E. McCurdy, Teri L. Hernandez, John P. Kirwanet et al. (2007). Cellular Barrett K, Brooks H, Boitano S, Barman S. (2010). The adrenal medulla and adrenal cortex, Bedalov A. and Balasubramanyam A. (1997). Glucocorticoid- induced ketoacidosis in Ben-Haroush, A., Yogev, Y., Hod, M. (2004). Epidemiology of gestational diabetes mellitus and its association with type 2 diabetes. Diabetic Medicine, 21: 103-113. Bergstrom R.W., Newell-Morris L.L., Leonetti D. L., et al. (1990). Association of elevated Boden G. (1992). Fuel metabolism in pregnancy and in gestational diabetes mellitus. Obstet- Brudenell M. (1993) Diabetic pregnancy. In: Obstetrics (2nd edition), Turnbull S. A., Buchanan TA, Xiang AH. (2005). Gestational diabetes mellitus. J Clin Invest, 115(3): 485–491. Burch W. M. (1994). Endocrinology (3rd edition), Mass Publishing Co., ISBN: 977-269-018-7, Butte N.F. (2000). Carbohydrate and lipid metabolism in pregnancy: normal compared with Campbell S. and Lees C. (Eds). (2000). Obstetrics by Ten Teachers, 17th edition. Arnold, ISBN 0 Carr DB, Gabbe S. (1998). Gestational Diabetes: Detection, Management, and Implications. Catalano P. M. (1994). Carbohydrate metabolism and gestational diabetes. Clin. Obstet. Catalano PM. Obesity, insulin resistance, and pregnancy outcome. Reproduction (2010) 140 Cianni GD, Volpe L, Lencioni C, et al. (2003). Prevalence and risk factors for gestational Corenblum B. (2008). Pituitary Disease and Pregnancy, In: eMedicine from WebMD. Article Csorba T. R. and Edwards A. L. (1995). The genetic and pathophysiology of type II and gestational diabetes. Crit. Rev. Clin. Lab. Sci., 32 (5-6): 509-550. diabetes assessed by universal screening. Diabetes research and clinical practice, 62(2): Last Updated: Jun 25, 2008. http://emedicine.medscape.com/article/127650- Mechanisms for Insulin Resistance in Normal Pregnancy and Gestational Diabetes. In: Ganong's Review of Medical Physiology (23rd edition), pp. 346-347, The Mc Grow gestational diabetes: Sequela of the acute treatment of preterm labor. A case report. fasting C-peptide level and increased intra-abdominal fat distribution with development of NIDDM in Japanese- American men. Diabetes, 39 (1): 104-111. Bilous R, Donnelly R. (2010). Hand book of Diabetes (4th edition), Wiley-Blackwell, ISBN: Chamberlain G. (eds). pp. 585-602, Churchill Livingstone, Edinburgh, London, gestational diabetes mellitus. American Journal of Clinical Nutrition, 71(5): 1256S- Amniotic fluid prolactin levels rise progressively after the 14th week human gestation and decline somewhat during the 3rd trimester. Prolactin secretion by the deciduas appears to be regulated quite differently from that in the pituitary gland. The first striking difference in regulation is that dopamine and dopamine agonists' drugs have no inhibitory effect on decidual prolactin secretion or amniotic fluid prolactin levels. Estrogen exerts a strong stimulation on pituitary lactotrophs but appears at most to have only small effects on decidual prolactin production (Davis, 1990). The very high levels of circulating estrogen during pregnancy result in a parallel increase in the circulating levels of prolactin in pregnancy. The prolactin increase is to prepare the breasts for lactation. Prolactin levels begin to rise at 5-8 weeks of gestation and parallel the increase in the size and number of lactotrophs (Schlechte, 2007; Corenblum, 2008). Progesterone appears to stimulate decidual prolactin secretion although it has little or no effect on decidual cells obtained in early pregnancy. Insulin stimulates both acute secretion and de novo synthesis of decidual prolactin (Davis, 1990). This may explain why prolactin mean level was the lowest in the GDM group as the level of insulin, which stimulates prolactin secretion, is the lowest when compared with the other groups (IGT and the control groups). #### 5. Conclusion Now, it is clear that pregnancy is diabetogenic and characterized by increased insulin resistance which may be explained in term of reduced insulin secretion, reduced tissue sensitivity to insulin and increased secretion of hormones with an anti-insulin effect such as human placental lactogen, free cortisol and prolactin. All these characteristics with the incorporation of some maternal risk factors such as age, parity, previous heavy babies and family history of diabetes may lead to the impairment of glucose tolerance in some pregnant women. Most pregnant women are able to counteract the insulin resistance in pregnancy by increasing their insulin secretion or by switching to other energy source rather than glucose such as fat particularly in the third trimester to preserve glucose to the fetus. However, when the capacity of insulin secretion is not sufficiently large to meet the resistance, glucose intolerance develops and women develop gestational diabetes. #### 6. Abbreviation GDM, gestational diabetes mellitus; IGT, impaired glucose tolerance; hPL, human placental lactogen; TAGs, triacylglycerol; NIDDM, non-insulin dependent diabetes mellitus; CBG, corticosteroid-binding globulin; ACTH, adrenocorticotrophic hormone. #### 7. References American Diabetes Association. (2004). Gestational diabetes mellitus. Diabetes Care, 27 (Suppl. 1):S88–S90. Baban RS, Kasar KAK, Al-Karawi IN. (2010). Fasting glucose to leptin ratio as a new diagnostic marker in patients with diabetes mellitus. OMJ, 25(4):269-275. Amniotic fluid prolactin levels rise progressively after the 14th week human gestation and decline somewhat during the 3rd trimester. Prolactin secretion by the deciduas appears to be regulated quite differently from that in the pituitary gland. The first striking difference in regulation is that dopamine and dopamine agonists' drugs have no inhibitory effect on decidual prolactin secretion or amniotic fluid prolactin levels. Estrogen exerts a strong stimulation on pituitary lactotrophs but appears at most to have only small effects on The very high levels of circulating estrogen during pregnancy result in a parallel increase in the circulating levels of prolactin in pregnancy. The prolactin increase is to prepare the breasts for lactation. Prolactin levels begin to rise at 5-8 weeks of gestation and parallel the Progesterone appears to stimulate decidual prolactin secretion although it has little or no effect on decidual cells obtained in early pregnancy. Insulin stimulates both acute secretion and de novo synthesis of decidual prolactin (Davis, 1990). This may explain why prolactin mean level was the lowest in the GDM group as the level of insulin, which stimulates prolactin secretion, is the lowest when compared with the other groups (IGT and the control Now, it is clear that pregnancy is diabetogenic and characterized by increased insulin resistance which may be explained in term of reduced insulin secretion, reduced tissue sensitivity to insulin and increased secretion of hormones with an anti-insulin effect such as human placental lactogen, free cortisol and prolactin. All these characteristics with the incorporation of some maternal risk factors such as age, parity, previous heavy babies and family history of diabetes may lead to the impairment of glucose tolerance in some pregnant women. Most pregnant women are able to counteract the insulin resistance in pregnancy by increasing their insulin secretion or by switching to other energy source rather than glucose such as fat particularly in the third trimester to preserve glucose to the fetus. However, when the capacity of insulin secretion is not sufficiently large to meet the resistance, glucose GDM, gestational diabetes mellitus; IGT, impaired glucose tolerance; hPL, human placental lactogen; TAGs, triacylglycerol; NIDDM, non-insulin dependent diabetes mellitus; CBG, American Diabetes Association. (2004). Gestational diabetes mellitus. Diabetes Care, 27 Baban RS, Kasar KAK, Al-Karawi IN. (2010). Fasting glucose to leptin ratio as a new diagnostic marker in patients with diabetes mellitus. OMJ, 25(4):269-275. intolerance develops and women develop gestational diabetes. corticosteroid-binding globulin; ACTH, adrenocorticotrophic hormone. increase in the size and number of lactotrophs (Schlechte, 2007; Corenblum, 2008). decidual prolactin production (Davis, 1990). groups). 5. Conclusion 6. Abbreviation 7. References (Suppl. 1):S88–S90. Insulin Resistance in the Third Trimester of Pregnancy Suffering Diabetes Care, 16 (12): 1551-1556. women. Diabetes, 41 (12): 1651-1659. 417-422. Metab, 92(8):2861-5. 106. 136-138. acute inflammatory reactions. Critical Care, 12 (6): R157. macrosomia—a case report. Early Hum Dev., 41:203–213. Diabetes, Abstracts book. 44(suppl.1): 14A (abstract no. 45). with the euglycemic clamp technique. Diabetes, 34: 380-389. trimester. Sudan Medical Monitor, 2(2): 59-61. ISSN: 1858-5000. ground. Lancet, 373(9677): 1789-97. [abstract] euglycemique. La press Medicale, 24 (15): 730-734. during pregnancy? Diabetic Medicine, 22: 1574-1580. from Gestational Diabetes Mellitus or Impaired Glucose Tolerance 181 Lehrke M, BroedlUC, Biller-Friedmann IM, et al. (2008). Serum concentrations of cortisol, Lewis G. F., McNally C., Blackman J. D., et al. (1993). Prior feeding alters the response to the Milasinović L, Djurdjević J, Dokmanović-Djordjević M, et al. (1997). Prolactin levels in Montelongo A. Lasunciaon M. A., Pallardo L. F. and Herrera E. (1992). Longitudinal study Paulus W. E., Stoz F., Bugan S. and Kreienberg R. (1995). Disorder of glucose metabolism Persson B, Hanson U, Marcus C. (1995). Gestational diabetes mellitus and paradoxical fetal Peters R., Kjos S., Xiang A. and Buchanan T. (1995). Effect of a second pregnancy on the risk Potemkin V. (1989). Diseases of the Ilet Apparatus of the Pancreas, In: Endocrinology (2nd edition), pp. (185-243), Mir Publishers, ISBN: 5-03-000754-7, Moscow. Reece EA, Leguizamon G, Wiznitzer A. (2009). Gestational diabetes: the need for a common Reis FM, D'Antona D and Petraglia F. (2002). Predictive Value of Hormone Measurements in Maternal and Fetal Complications of Pregnancy. Endocrine Reviews, 23 (2): 230-257. Robert J.J. (1995). Methods de mesure de la resistance a l'insulin-clamp hyperinsulinemique Ryan E. A., O'sullivan M. J. and Skyler J. S. (1985). Insulin action during pregnancy- Studies Schlechte JA. (2007). Long-term management of prolactinomas. J Clin Endocrinol Seghieri, G., De Bellis, A., Anichini, R., et al. (2005). Does parity increase insulin resistance Shalayel MH, Ahmed SA, Khattab AH, Satti GM. (2007). Prevalence of gestational diabetes Shalayel MH, Elrobh MS, Idris SA, Mohammed MS, Ahmed SA. (2010). Prolactin and Solomon GC, Willet WC, Carey VJ. et al. (1997). A Prospective Study of Pregravid Determinants of Gestational Diabetes Mellitus. JAMA, 278(13):1078-1083. Stewart M. and Taylor R. (1994). Gestational diabetes mellitus. Prof. Care-Mother-child, 4(5): mellitus and impaired glucose tolerance in pregnant Sudanese women in the third insulin estimates in pregnancy with glucose intolerance. Pak J Med Sci., 26(1):102- interleukin 6, leptin and adiponectin predict stress induced insulin resistance in 50-g glucose challenge test in pregnancy- The Staub-Traugott effect revisited. pregnant women with glucose intolerance at full-term delivery. Med Pregl, 50:269-73. of plasma lipoproteins and hormones during pregnancy in normal and diabetic and insulin resistance after gestational diabetes. Zentralblatt fur Gynakologie, 117 (8): of non-insulin-dependent diabetes in women with recent gestational diabetes. Dahlgren J. (2006). Pregnancy and insulin resistance. Metabolic Syndrome and Related Damm P., Kuhl C., Buchard K., et al. (1994). Prevalence and predictive value of islet cell Damm P., Kuhl C., Hornnes P. and Molsted-Pedersen L. (1995). A longitudinal study of Davis JRE. (1990). 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Diagnostic criteria for polycystic ovary syndrome 10 Austria The Impact of Polycystic Ovarian Medical University of Vienna, Department of Gynecologic Stefanie Aust and Johannes Ott Endocrinology and Reproductive Medicine, Syndrome on Gestational Diabetes When Stein and Leventhal, in 1935, observed a group of women who suffered from sterility, oligomenorrhoea, or amenorhhoea, hirsutism, and enlarged polycystic ovaries, the disorder that became known as the polycystic ovarian syndrome (PCOS) or the Stein-Leventhal syndrome, was diagnosed for the first time (Stein, 1958). The variability of individual presentation, as well as a varying collection of signs and characteristic features, with no single diagnostic test justify the categorization of the PCOS as a syndrome that affects not only reproductive health, but also the metabolic and cardiovascular systems. Although PCOS is one of the most common endocrinopathies in women, with an incidence of about 5- 10% throughout the reproductive age span (Metalliotakis, 2006), there are divergent opinions about how to define and diagnose PCOS, as well as different types of treatment options throughout Europe and the US (Badawy & Elnashar, 2011). The high prevalence of women with this endocrine disorder highlights the importance of understanding the clinical presentation, pathophysiology, associated disorders, and treatment options. Up to 40% of women of reproductive age with PCOS have Type 2 diabetes or an impaired glucose tolerance (Legro et al., 2005), a form of insulin resistance that occurs equally in obese, normal weight, and thin women with PCOS (Matalliotakis et al., 2006). PCOS has been associated with an increased risk for gestational diabetes mellitus (GDM), but solid evidence confirming PCOS as a risk factor for GDM is still missing (Toulis et al., 2009). GDM, a wellknown state of carbohydrate intolerance with a high, and rising, prevalence, causes not only maternal but also fetal pregnancy complications. GDM has a presentation similar to PCOS, and both are considered risk factors for Type 2 diabetes mellitus (Retnakaran et al., 2008). The aim of this review is to summarize the available evidence about the risk of impaired glucose tolerance and GDM in PCOS women, as well as to review the pathophysiological aspects. In addition, the potential beneficial influence of several PCOS-specific treatment The National Institutes of Health (NIH) has published criteria for diagnosing PCOS, based on an international conference on PCOS held in 1990. Accordingly, diagnostic criteria for the syndrome includes chronic anovulation, combined with clinical or biochemical ## The Impact of Polycystic Ovarian Syndrome on Gestational Diabetes Stefanie Aust and Johannes Ott Medical University of Vienna, Department of Gynecologic Endocrinology and Reproductive Medicine, Austria #### 1. Introduction 182 Gestational Diabetes Strauss JF & Barbieri RL. (2009). The endocrinology of human pregnancy and fetoplacental gestational diabetes. (1985). Diabetes, 34 (suppl.2): 123-126. Wilcox G. (2005). Insulin and Insulin Resistance. Clin Biochem Rev, 26(2): 19–39. neuroendocrine development, In: Yen and Jaffe's Reproductive Endocrinology – Physiology, pathophysiology and clinical management (6th edition), Strauss JF & Barbieri RL, pp. 256, Saunders Elsevier, ISBN: 978-1-4160-4907-4, Philadelphia. Summary and recommendation of the second international workshop conference of > When Stein and Leventhal, in 1935, observed a group of women who suffered from sterility, oligomenorrhoea, or amenorhhoea, hirsutism, and enlarged polycystic ovaries, the disorder that became known as the polycystic ovarian syndrome (PCOS) or the Stein-Leventhal syndrome, was diagnosed for the first time (Stein, 1958). The variability of individual presentation, as well as a varying collection of signs and characteristic features, with no single diagnostic test justify the categorization of the PCOS as a syndrome that affects not only reproductive health, but also the metabolic and cardiovascular systems. Although PCOS is one of the most common endocrinopathies in women, with an incidence of about 5- 10% throughout the reproductive age span (Metalliotakis, 2006), there are divergent opinions about how to define and diagnose PCOS, as well as different types of treatment options throughout Europe and the US (Badawy & Elnashar, 2011). The high prevalence of women with this endocrine disorder highlights the importance of understanding the clinical presentation, pathophysiology, associated disorders, and treatment options. Up to 40% of women of reproductive age with PCOS have Type 2 diabetes or an impaired glucose tolerance (Legro et al., 2005), a form of insulin resistance that occurs equally in obese, normal weight, and thin women with PCOS (Matalliotakis et al., 2006). PCOS has been associated with an increased risk for gestational diabetes mellitus (GDM), but solid evidence confirming PCOS as a risk factor for GDM is still missing (Toulis et al., 2009). GDM, a wellknown state of carbohydrate intolerance with a high, and rising, prevalence, causes not only maternal but also fetal pregnancy complications. GDM has a presentation similar to PCOS, and both are considered risk factors for Type 2 diabetes mellitus (Retnakaran et al., 2008). The aim of this review is to summarize the available evidence about the risk of impaired > glucose tolerance and GDM in PCOS women, as well as to review the pathophysiological aspects. In addition, the potential beneficial influence of several PCOS-specific treatment options on PCOS and GDM will be discussed. #### 2. Diagnostic criteria for polycystic ovary syndrome The National Institutes of Health (NIH) has published criteria for diagnosing PCOS, based on an international conference on PCOS held in 1990. Accordingly, diagnostic criteria for the syndrome includes chronic anovulation, combined with clinical or biochemical The Impact of Polycystic Ovarian Syndrome on Gestational Diabetes 185 to the number of antral follicles and can be correlated to the level of ovarian dysfunction in infertility. Thus, it is possible that the process leading to ovarian aging is delayed in PCOS, which might also lead to a later onset of menopause in these women (Mulders et al., 2004). Three hypotheses are frequently discussed in literature about how defects in primary cellular control mechanisms might result in PCOS: (i) an elevated luteinizing hormone (LH) pulse frequency and amplitude, and relatively low follicle stimulating hormone (FSH) serum levels (LH+/FSH-) lead to anovulation and ovarian hyperandrogenism; (ii) a defect in the sex steroid metabolism within the ovaries (theca cells) causes an exaggerated ovarian androgen secretion; and (iii) a regulatory dysfunction of the insulin pathway results in hyperinsulinemia and insulin resistance and contributes to the development of PCOS (Franks et al., 1998). These pathophysiologic mechanisms might be of special interest regarding the risk for GDM. Indeed, several possible insulin-mediated pathways have been identified that might contribute to hyperandrogenism in PCOS patients (see Figure 1). Fig. 1. How increased insulin levels might contribute to hyperandrogenism These pathophysiologic hypotheses are related in a variety of ways. Elevated hypothalamic gonadotropin-releasing hormone (GnRH) pulsatility influences LH secretion; consequently, dysfunctional pulse frequency and amplitude lead to an increased 24-hour secretion of LH. High LH combined with high levels of insulin result in increased ovarian androgen production. Hypersecretion of LH also affects oocyte development. A defect in androgen synthesis that results in an increased enzymatic activity involved in the synthesis of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and androstenedione leads, consequently, to an inadequate production of testosterone in ovarian theca cells. The synergistic interaction between LH and insulin on the ovarian theca cells leads to stimulation of androgen production. Several studies have provided useful information about a correlation between hyperandrogenism and a state of increased insulin resistance (Balen, 2004) (Baptiste et al., 2010). In women with PCOS, an another cause of high androgen levels can be explained by the influence of compensatory hyperinsulinemia hyperandrogenism, where other causes have been excluded (diagnosis of exclusion) (Huang et al. 2010). An expanded definition can be found in the revised Rotterdam criteria, a consensus on diagnostic criteria of the American Society for Reproductive Medicine and the European Society of Human Reproduction and Embryology. At least two of three criteria must be present: (i) oligoamenorrhoea or amenorrhoea; (ii) hyperandrogenism (clinical/biochemical); and (iii) polycystic ovaries on ultrasound, defined as more than 12 cysts of 2-9mm, or >10ml volume (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004). The revised Rotterdam criteria are considered the current standard diagnostic criteria. In 2006, the Androgen Excess Society (AES) attempted to define evidence-based guidelines for diagnosis and future research in a review (Azziz et al., 2006). The AES task force suggested that androgen excess must be considered a central feature of the disease, and that PCOS should be defined by the presence of hyperandrogenism (clinical and/or biochemical) together with signs of ovarian dysfunction (oligoovulation or anovulation and/or polycystic ovaries), where similar disorders have been excluded (Azziz et al., 2006). Conditions for exclusion must be clarified in the diagnostic procedure. Premature ovarian failure with oligo-/amenorrhea that might be associated with other autoimmune endocrinopathies, hyperprolactinoma, or Cushing's syndrome are a few clinical possibilities that merit attention. #### 3. Pathophysiologic aspects of polycystic ovary syndrome The heterogeneity of the syndrome and the unclear etiology favor the theory of multiple underlying pathophysiologic mechanisms that have yet to be fully elucidated. A heritable etiology for PCOS has been investigated intensively and several associated polymorphisms have been identified. However, to date, none of the possible candidate genes (e.g., regulators of the microbiological action of insulin) could be correlated with the onset of PCOS (Dumesic et al., 2007). In the research field of PCOS, studies on polymorphisms gained on importance within the last years. Moreover, environmental factors (e.g.: lifestyle, nutrition) together with certain genetic mutations might lead to the individual manifestation of PCOS but the diversity of clinical presentations aggravates the identification of genes involved in the origin of PCOS. Although the definition of "polycystic ovary syndrome" might be ambiguous, it is important to emphasize that polycystic ovaries need not be present to diagnose this syndrome. Nevertheless, PCOS patients who demonstrate ovaries with multiple subcortical cysts on ultrasound and an increased proportion of primary follicles (Dumesic et al., 2007) have a greater rate of hyperandrogenism than women with PCOS without abnormal follicle development. The presence of polycystic ovaries might indicate a major clinical alteration of PCOS, and the presence of polycystic ovaries in childhood has been suggested as an indicator of a genetic predisposition (Battaglia et al., 2002). Moreover, an abnormal autoimmune history has been considered in PCOS, in which functional autoantibodies might favor the development of PCOS (Ott et al., 2010, Gleicher et al., 2007). Assuming a relation between insulin resistance, ovarian function, and thyroid function, elevated antiTPO levels have been found to influence treatment response in women with PCOS and infertility (Ott et al., 2010). Notably, PCOS has been called a marker for "reduced ovarian aging," since serum anti-Müller hormone (AMH) levels are higher in anovulatory women and have been found to be elevated in women with PCOS. AMH concentrations correspond hyperandrogenism, where other causes have been excluded (diagnosis of exclusion) (Huang et al. 2010). An expanded definition can be found in the revised Rotterdam criteria, a consensus on diagnostic criteria of the American Society for Reproductive Medicine and the European Society of Human Reproduction and Embryology. At least two of three criteria must be present: (i) oligoamenorrhoea or amenorrhoea; (ii) hyperandrogenism (clinical/biochemical); and (iii) polycystic ovaries on ultrasound, defined as more than 12 cysts of 2-9mm, or >10ml volume (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004). The revised Rotterdam criteria are considered the current standard In 2006, the Androgen Excess Society (AES) attempted to define evidence-based guidelines for diagnosis and future research in a review (Azziz et al., 2006). The AES task force suggested that androgen excess must be considered a central feature of the disease, and that PCOS should be defined by the presence of hyperandrogenism (clinical and/or biochemical) together with signs of ovarian dysfunction (oligoovulation or anovulation and/or polycystic ovaries), where similar disorders have been excluded (Azziz et al., 2006). Conditions for exclusion must be clarified in the diagnostic procedure. Premature ovarian failure with oligo-/amenorrhea that might be associated with other autoimmune endocrinopathies, hyperprolactinoma, or Cushing's syndrome are a few clinical possibilities that merit The heterogeneity of the syndrome and the unclear etiology favor the theory of multiple underlying pathophysiologic mechanisms that have yet to be fully elucidated. A heritable etiology for PCOS has been investigated intensively and several associated polymorphisms have been identified. However, to date, none of the possible candidate genes (e.g., regulators of the microbiological action of insulin) could be correlated with the onset of PCOS (Dumesic et al., 2007). In the research field of PCOS, studies on polymorphisms gained on importance within the last years. Moreover, environmental factors (e.g.: lifestyle, nutrition) together with certain genetic mutations might lead to the individual manifestation of PCOS but the diversity of clinical presentations aggravates the identification of genes Although the definition of "polycystic ovary syndrome" might be ambiguous, it is important to emphasize that polycystic ovaries need not be present to diagnose this syndrome. Nevertheless, PCOS patients who demonstrate ovaries with multiple subcortical cysts on ultrasound and an increased proportion of primary follicles (Dumesic et al., 2007) have a greater rate of hyperandrogenism than women with PCOS without abnormal follicle development. The presence of polycystic ovaries might indicate a major clinical alteration of PCOS, and the presence of polycystic ovaries in childhood has been suggested as an indicator of a genetic predisposition (Battaglia et al., 2002). Moreover, an abnormal autoimmune history has been considered in PCOS, in which functional autoantibodies might favor the development of PCOS (Ott et al., 2010, Gleicher et al., 2007). Assuming a relation between insulin resistance, ovarian function, and thyroid function, elevated antiTPO levels have been found to influence treatment response in women with PCOS and infertility (Ott et al., 2010). Notably, PCOS has been called a marker for "reduced ovarian aging," since serum anti-Müller hormone (AMH) levels are higher in anovulatory women and have been found to be elevated in women with PCOS. AMH concentrations correspond 3. Pathophysiologic aspects of polycystic ovary syndrome diagnostic criteria. attention. involved in the origin of PCOS. to the number of antral follicles and can be correlated to the level of ovarian dysfunction in infertility. Thus, it is possible that the process leading to ovarian aging is delayed in PCOS, which might also lead to a later onset of menopause in these women (Mulders et al., 2004). Three hypotheses are frequently discussed in literature about how defects in primary cellular control mechanisms might result in PCOS: (i) an elevated luteinizing hormone (LH) pulse frequency and amplitude, and relatively low follicle stimulating hormone (FSH) serum levels (LH+/FSH-) lead to anovulation and ovarian hyperandrogenism; (ii) a defect in the sex steroid metabolism within the ovaries (theca cells) causes an exaggerated ovarian androgen secretion; and (iii) a regulatory dysfunction of the insulin pathway results in hyperinsulinemia and insulin resistance and contributes to the development of PCOS (Franks et al., 1998). These pathophysiologic mechanisms might be of special interest regarding the risk for GDM. Indeed, several possible insulin-mediated pathways have been identified that might contribute to hyperandrogenism in PCOS patients (see Figure 1). Fig. 1. How increased insulin levels might contribute to hyperandrogenism These pathophysiologic hypotheses are related in a variety of ways. Elevated hypothalamic gonadotropin-releasing hormone (GnRH) pulsatility influences LH secretion; consequently, dysfunctional pulse frequency and amplitude lead to an increased 24-hour secretion of LH. High LH combined with high levels of insulin result in increased ovarian androgen production. Hypersecretion of LH also affects oocyte development. A defect in androgen synthesis that results in an increased enzymatic activity involved in the synthesis of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and androstenedione leads, consequently, to an inadequate production of testosterone in ovarian theca cells. The synergistic interaction between LH and insulin on the ovarian theca cells leads to stimulation of androgen production. Several studies have provided useful information about a correlation between hyperandrogenism and a state of increased insulin resistance (Balen, 2004) (Baptiste et al., 2010). In women with PCOS, an another cause of high androgen levels can be explained by the influence of compensatory hyperinsulinemia The Impact of Polycystic Ovarian Syndrome on Gestational Diabetes 187 separately, there were largely conflicting results: while most of the studies demonstrated an increased risk for GDM in PCOS women (odds ratios ranging from 1.15 to 22.15) (Wortsman et al., 1991 as cited in Boomsma et al., 2006) (Radon et al., 1999, as cited in Boomsma et al., 2006), a few found odds ratios from 0.31 to 0.96 (Turhan et al., 2003, as cited in Boomsma et al., 2006) (Haakova et al., as cited in Boomsma et al., 2006). A comparison between the study designs revealed that the increased risks were predominantly found in cohort studies rather than in case-control studies. In addition, meta-analyses revealed a significant heterogeneity Conversely, some studies did not seem to support a higher prevalence and previous history of PCOS in women diagnosed with GDM, when compared to pregnancies in women with normal glucose homeostasis (Wijeyaratne et al., 2006) (Kousta et al., 2000). Obesity, PCOS, and diabetes in first-degree relatives have been described as risk factors for developing GDM and gestational impaired glucose tolerance, especially in young women and teenage All in all, there is no solid evidence proving the increased risk for GDM in PCOS patients, but a trend assuming that the risk is, indeed, increased in women with PCOS, is recognizable. Confronted with the wide clinical and pathophysiological spectrum associated The guidelines developed by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) consider a screening for pre-gestational diabetes in high-risk women at the first prenatal visit and universal screening between the 24th and 28th week of gestation (Holt et al., 2011). In daily routine, an OGTT is performed in the third trimester. According to the diagnostic recommendations published in March 2010 by the IADPSG, fasting blood glucose concentrations that exceed 92 mg/dl, one-hour and two-hour glucose levels of more than 180 mg/dl, or 153 mg/dl measured during the OGTT, lead to the diagnosis of GDM (Metzger et al., 2010). Increased measurements in each of the three values raise the possibility of an adverse pregnancy outcome. A benefit of generalized testing to evaluate glycaemia levels in all pregnant women before the usual window is still a subject of controversy in the literature and must be decided individually (Karagiannis et al., 2010, Hadar et al., 2009). If increased levels of glucose are found in urine samples, or if GDM was present in a previous pregnancy, an OGTT in the second trimester should be performed. Since women who suffer from PCOS are considered to be at higher risk for developing GDM, it could be argued that they should undergo a more detailed and earlier screening for 5.2 Pathophysiological hypotheses about the risk of gestational diabetes in women phenomenon of the increased GDM risk associated with PCOS. Several pathophysiologic mechanisms have been discussed that might contribute to the It has been hypothesized that a particular genetic background could contribute to the association between PCOS and GDM. In women with familial partial lipodystrophy due to LMNA (lamin A/C) mutations, a rare disorder characterized by a selective loss of adipose with the syndrome, further studies are warranted to validate the existing data. between the analyzed studies. 5.1 Diagnostic aspects GDM. with polycystic ovary syndrome 5.2.1 Genetic predisposition pregnancies (<20 years) (Karcaaltincaba et al., 2011). on the hepatic synthesis and secretion of the sex hormone-binding globulin (SHBG). SHBG levels are reduced and the blood concentrations of biologically active androgens are thus increased. In addition, it has been suggested that adipose tissue dysfunction also plays a central role in the metabolic and endocrine abnormalities observed in PCOS (Villa J et al., 2011). #### 4. Clinical manifestation of polycystic ovary syndrome A variety of signs and symptoms can be found in women who suffer from PCOS in which ovarian hyperandrogenism is considered the cardinal characteristic. The variable clinical presentation includes gynecological symptoms that include dysfunctional menstrual bleeding, such as oligo- or amenorrhea, or any kind of abnormal uterine bleeding combined with infrequent or absent ovulation, infertility resulting from elevated androgen levels, and polycystic ovaries (Dumesic et al., 2007). With regard to gynecologic malignancies, chronic anovulation over a long period of time is associated with a higher risk of developing endometrial adenocarcinoma and a higher incidence of endometrial hyperplasia, compared to age-matched controls (Badawy & Elnashar, 2011). Elevated serum androgen levels lead to androgenic disorders, such as acne, hirsutism, and alopecia androgenica, where hyperandrogenism is differently expressed in the PCOS phenotypes, resulting in cosmetic issues and psychological effects that can be burdensome and difficult to cope with. Last not least, PCOS has been considered to be associated with a increased risks for type II diabetes mellitus and GDM. When considering the association with GDM, it is notable that both PCOS and GDM share some common characteristic features, including obesity, increased insulin resistance, dyslipidemia, and other metabolic abnormalities. The common presence of lipid abnormalities, such as elevated serum triglyceride- and low-density lipoprotein levels due to negative hormonal influences on the lipid homeostasis coexist with obesity and increased insulin resistance. PCOS shares components with the metabolic syndrome characterized by a combination of insulin resistance, dyslipidemia, and hypertension (Boomsma et al., 2006). Although a high BMI is associated with a higher risk of arterial disease, an increased cardiovascular risk (up to two-fold) in women with PCOS cannot be completely ascribed to a higher BMI (de Groot et al., 2011). Obesity and PCOS show, on the one hand, an independent influence, but, on the other hand, seem to have additive adverse effects on insulin action. Up to 50% of women with PCOS suffer from an imbalance in carbohydrate homeostasis, central fat deposition, and increasing insulin resistance during pregnancy (Huang et al., 2010). It has been estimated that 25–70% of women with PCOS show a rise in insulin resistance and have an increased risk of developing complications during pregnancy, first and foremost of which is GDM (Godoy-Matos et al., 2009). #### 5. The risk of gestational diabetes in women with polycystic ovary syndrome GDM is defined as the onset or first recognition and diagnosis of glucose intolerance during pregnancy. The diagnostic criterion for GDM is the 75g, two-hour oral glucose tolerance test (OGTT). In fact, recent meta-analyses of pregnancy outcomes in women with PCOS demonstrated a significantly higher chance of developing GDM for PCOS women (odds ratios of about 2.90) (Boomsma et al., 2006) (Toulis et al., 2009). However, when analyzing the available evidence separately, there were largely conflicting results: while most of the studies demonstrated an increased risk for GDM in PCOS women (odds ratios ranging from 1.15 to 22.15) (Wortsman et al., 1991 as cited in Boomsma et al., 2006) (Radon et al., 1999, as cited in Boomsma et al., 2006), a few found odds ratios from 0.31 to 0.96 (Turhan et al., 2003, as cited in Boomsma et al., 2006) (Haakova et al., as cited in Boomsma et al., 2006). A comparison between the study designs revealed that the increased risks were predominantly found in cohort studies rather than in case-control studies. In addition, meta-analyses revealed a significant heterogeneity between the analyzed studies. Conversely, some studies did not seem to support a higher prevalence and previous history of PCOS in women diagnosed with GDM, when compared to pregnancies in women with normal glucose homeostasis (Wijeyaratne et al., 2006) (Kousta et al., 2000). Obesity, PCOS, and diabetes in first-degree relatives have been described as risk factors for developing GDM and gestational impaired glucose tolerance, especially in young women and teenage pregnancies (<20 years) (Karcaaltincaba et al., 2011). All in all, there is no solid evidence proving the increased risk for GDM in PCOS patients, but a trend assuming that the risk is, indeed, increased in women with PCOS, is recognizable. Confronted with the wide clinical and pathophysiological spectrum associated with the syndrome, further studies are warranted to validate the existing data. #### 5.1 Diagnostic aspects 186 Gestational Diabetes on the hepatic synthesis and secretion of the sex hormone-binding globulin (SHBG). SHBG levels are reduced and the blood concentrations of biologically active androgens are thus increased. In addition, it has been suggested that adipose tissue dysfunction also plays a central role in the metabolic and endocrine abnormalities observed in PCOS (Villa J et al., A variety of signs and symptoms can be found in women who suffer from PCOS in which ovarian hyperandrogenism is considered the cardinal characteristic. The variable clinical presentation includes gynecological symptoms that include dysfunctional menstrual bleeding, such as oligo- or amenorrhea, or any kind of abnormal uterine bleeding combined with infrequent or absent ovulation, infertility resulting from elevated androgen levels, and polycystic ovaries (Dumesic et al., 2007). With regard to gynecologic malignancies, chronic anovulation over a long period of time is associated with a higher risk of developing endometrial adenocarcinoma and a higher incidence of endometrial hyperplasia, compared to age-matched controls (Badawy & Elnashar, 2011). Elevated serum androgen levels lead to androgenic disorders, such as acne, hirsutism, and alopecia androgenica, where hyperandrogenism is differently expressed in the PCOS phenotypes, resulting in cosmetic issues and psychological effects that can be burdensome and difficult to cope with. Last not least, PCOS has been considered to be associated with a increased risks for type II diabetes mellitus and GDM. When considering the association with GDM, it is notable that both PCOS and GDM share some common characteristic features, including obesity, increased insulin resistance, dyslipidemia, and other metabolic abnormalities. The common presence of lipid abnormalities, such as elevated serum triglyceride- and low-density lipoprotein levels due to negative hormonal influences on the lipid homeostasis coexist with obesity and increased insulin resistance. PCOS shares components with the metabolic syndrome characterized by a combination of insulin resistance, dyslipidemia, and hypertension (Boomsma et al., 2006). Although a high BMI is associated with a higher risk of arterial disease, an increased cardiovascular risk (up to two-fold) in women with PCOS cannot be completely ascribed to a higher BMI (de Groot et al., 2011). Obesity and PCOS show, on the one hand, an independent influence, but, on the other hand, seem to have Up to 50% of women with PCOS suffer from an imbalance in carbohydrate homeostasis, central fat deposition, and increasing insulin resistance during pregnancy (Huang et al., 2010). It has been estimated that 25–70% of women with PCOS show a rise in insulin resistance and have an increased risk of developing complications during pregnancy, first 5. The risk of gestational diabetes in women with polycystic ovary syndrome GDM is defined as the onset or first recognition and diagnosis of glucose intolerance during pregnancy. The diagnostic criterion for GDM is the 75g, two-hour oral glucose tolerance test In fact, recent meta-analyses of pregnancy outcomes in women with PCOS demonstrated a significantly higher chance of developing GDM for PCOS women (odds ratios of about 2.90) (Boomsma et al., 2006) (Toulis et al., 2009). However, when analyzing the available evidence 4. Clinical manifestation of polycystic ovary syndrome additive adverse effects on insulin action. (OGTT). and foremost of which is GDM (Godoy-Matos et al., 2009). 2011). The guidelines developed by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) consider a screening for pre-gestational diabetes in high-risk women at the first prenatal visit and universal screening between the 24th and 28th week of gestation (Holt et al., 2011). In daily routine, an OGTT is performed in the third trimester. According to the diagnostic recommendations published in March 2010 by the IADPSG, fasting blood glucose concentrations that exceed 92 mg/dl, one-hour and two-hour glucose levels of more than 180 mg/dl, or 153 mg/dl measured during the OGTT, lead to the diagnosis of GDM (Metzger et al., 2010). Increased measurements in each of the three values raise the possibility of an adverse pregnancy outcome. A benefit of generalized testing to evaluate glycaemia levels in all pregnant women before the usual window is still a subject of controversy in the literature and must be decided individually (Karagiannis et al., 2010, Hadar et al., 2009). If increased levels of glucose are found in urine samples, or if GDM was present in a previous pregnancy, an OGTT in the second trimester should be performed. Since women who suffer from PCOS are considered to be at higher risk for developing GDM, it could be argued that they should undergo a more detailed and earlier screening for GDM. #### 5.2 Pathophysiological hypotheses about the risk of gestational diabetes in women with polycystic ovary syndrome Several pathophysiologic mechanisms have been discussed that might contribute to the phenomenon of the increased GDM risk associated with PCOS. #### 5.2.1 Genetic predisposition It has been hypothesized that a particular genetic background could contribute to the association between PCOS and GDM. In women with familial partial lipodystrophy due to LMNA (lamin A/C) mutations, a rare disorder characterized by a selective loss of adipose The Impact of Polycystic Ovarian Syndrome on Gestational Diabetes 189 free IGF-1 and IGFBP-1, where higher C-peptide levels were positively associated with the development of GDM (Qiu et al., 2005). It should be emphasized that lower levels were found in obese women with/or without PCOS, compared to normal weight controls, suggesting that body weight has a certain influence on serum IGFBP-1 levels. The physiologic processes underlying the role of IGF-1 in glucose regulation are unclear. Infusions of IGF-1 are known to suppress glucose counter-regulatory hormones, such as glucagon and growth hormone (Jones & Clemmons, 1995). IGF-1 may also contribute to changes in insulin sensitivity and glucose uptake via direct IGF receptor-mediated effects on skeletal muscle (Sjogren et al., 2001). Alterations in IGFBP-1 concentrations may be due to variations in insulin secretion or hepatic insulin sensitivity, both known to be important PCOS is often accompanied by infertility that necessitates ovulation induction, using clomiphene citrate, gonadotropins, or even in vitro fertilization (IVF) (Boomsma et al., 2006). These treatment methods are known to increase the incidence of multiple pregnancies, as well as some negative consequences, including a rise in the risk for GDM (Schwartz et al., 1999). Furthermore, pregnancies established after IVF carry an increased risk for maternal complications (Pinborg et al., 2004) (Shevell et al., 2005). However, the increased risk of developing GDM has been suggested to occur independently of obesity, as well as in populations without assisted reproductive techniques (Boomsma et al., 2006) (Toulis et al., The therapeutic challenges in the treatment of women with PCOS and GDM or impaired glucose tolerance arise from the diversity of recommendations and research conclusions. If adequately managed, the incidence of adverse perinatal outcomes in PCOS patients who develop GDM was found to be not significantly elevated (Li et al., 2010). Several treatment options exist for each of the symptoms of PCOS, and management of these patients depends on the individual symptoms. Clinical management of PCOS involves risk assessment for metabolic disorders, as diabetes and dyslipidemia, and for hypertension, cardiovascular complications, and liver diseases (Setji & Brown, 2007). Insulin-sensitizing drugs, such as metformin and/or oral contraceptives, are thought to improve the clinical features of PCOS, but limited data is available that assesses the true influence and safety of these drugs in this population (Setji & Brown, 2007). Targeting androgen symptoms, such as acne, hirsutism, and alopecia androgenica, estrogen-containing oral contraceptives, antiandrogens and topical agents are considered first-line therapy for women who do not want to conceive, as they effectively reduce serum androgen levels. In addition, treatment with oral contraceptives is associated with an improvement in the menstrual pattern. However, when trying to achieve pregnancy, other treatment options are offered to the patient. All of these Addressing life-style factors before conception should be the first-line approach to reduce any further therapeutic strategies during pregnancy. The appearance of the varying factors in glucose regulation (Lee et al., 1997). 5.2.5 The influence of infertility treatments 6. Therapeutic considerations might also independently influence the risk for GDM. 6.1 Lifestyle modifications 2009). tissue and insulin resistance, the prevalence of PCOS and gestational diabetes was found to be higher than in the general population (Vantyghem et al., 2008). Moreover, mutations of the VNTR (variable number of tandem repeats) locus, upstream of the insulin gene (INS) where insulin expression is regulated, have been found in both women who suffer from PCOS and in women with GDM (Waterworth et al., 1997) (Lambrinoudaki et al., 2010). #### 5.2.2 Preexisting insulin resistance In pregnancy, rising blood levels of lipolytic placental hormones lead to an elevation of free fatty acids that are commonly associated with the development of a dose-dependent insulin resistance. Affecting skeletal muscle glucose uptake, free fatty acids create a state of local insulin resistance and, as concentrations are elevated in late pregnancy, an increase in tissue insulin resistance can be observed during pregnancy (Sivan & Boden, 2003). In the presence of PCOS and a preexisting state of increased insulin resistance accompanying the syndrome, hyperglycemia seems to be induced more easily. The high pre-conception insulin resistance might have a deleterious additive effect on pancreatic beta cells, which are incapable of coping with the additive physiological insulin resistance of pregnancy (Khattab et al., 2011), thereby leading to an increased incidence of GDM (Toulis et al., 2009). #### 5.2.3 SHBG levels Hyperinsulinemia stimulates in much the same way as LH-agonist ovarian testosterone production and decreases the serum sex hormone-binding globulin (SHBG) concentration. SHBG is known to have biologic functions beyond the regulation of free estrogen and testosterone serum levels. It has also been emphasized that low SHBG levels are associated with a minor glucose tolerance, thus, attributing a role to these low SHBG levels in the maintenance of glucose homeostasis. A possible explanation might be a modulation of the biologic effects of both estrogen and testosterone on liver, fat, and muscle tissue, as well as on other peripheral tissues (Ding et al., 2009). Furthermore, low plasma SHBG levels has been suggested as predictive for the risk of developing Type 2 diabetes mellitus (Ding et al., 2009). As an early indicator of GDM risk, low preconception levels of SHBG concentrations have been identified as strong predictors in PCOS women, independently of obesity and measures of insulin resistance. One study suggested that assessment of SHBG levels before conception might be a useful tool to by which to identify PCOS patients at risk for GDM during pregnancy (Veltman-Verhulst et al., 2010). However, the pathophysiologic pathways of this effect are unknown. It remains unclear whether the association between GDM and SHBG is direct or indirect. A lower SHBG results in higher free androgens that have already been demonstrated to be associated with an increased GDM risk (Bartha et al., 2000). #### 5.2.4 Insulin-like growth factor and insulin-like growth factor binding protein-1 The risk for GDM has been connected to maternal plasma concentrations of insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-1 (IGFBP-1), suggesting that these determinants of glucose homeostasis play a potential role in the pathophysiologic process and contribute to the development of GDM (Qiu et al., 2005). Increased serum insulin concentrations lead to an inhibition of IGFBP-1 production, resulting in elevated serum levels of free IGF-1. A recent meta analysis on the interaction between IGFBP-1 and PCOS described lower levels of IGFBP-1 in affected patients (Kelly et al. 2011). Notably, a lower risk for developing GDM was correlated with increased levels of tissue and insulin resistance, the prevalence of PCOS and gestational diabetes was found to be higher than in the general population (Vantyghem et al., 2008). Moreover, mutations of the VNTR (variable number of tandem repeats) locus, upstream of the insulin gene (INS) where insulin expression is regulated, have been found in both women who suffer from PCOS and in women with GDM (Waterworth et al., 1997) (Lambrinoudaki et al., 2010). In pregnancy, rising blood levels of lipolytic placental hormones lead to an elevation of free fatty acids that are commonly associated with the development of a dose-dependent insulin resistance. Affecting skeletal muscle glucose uptake, free fatty acids create a state of local insulin resistance and, as concentrations are elevated in late pregnancy, an increase in tissue insulin resistance can be observed during pregnancy (Sivan & Boden, 2003). In the presence of PCOS and a preexisting state of increased insulin resistance accompanying the syndrome, hyperglycemia seems to be induced more easily. The high pre-conception insulin resistance might have a deleterious additive effect on pancreatic beta cells, which are incapable of coping with the additive physiological insulin resistance of pregnancy (Khattab et al., 2011), Hyperinsulinemia stimulates in much the same way as LH-agonist ovarian testosterone production and decreases the serum sex hormone-binding globulin (SHBG) concentration. SHBG is known to have biologic functions beyond the regulation of free estrogen and testosterone serum levels. It has also been emphasized that low SHBG levels are associated with a minor glucose tolerance, thus, attributing a role to these low SHBG levels in the maintenance of glucose homeostasis. A possible explanation might be a modulation of the biologic effects of both estrogen and testosterone on liver, fat, and muscle tissue, as well as on other peripheral tissues (Ding et al., 2009). Furthermore, low plasma SHBG levels has been suggested as predictive for the risk of developing Type 2 diabetes mellitus (Ding et al., 2009). As an early indicator of GDM risk, low preconception levels of SHBG concentrations have been identified as strong predictors in PCOS women, independently of obesity and measures of insulin resistance. One study suggested that assessment of SHBG levels before conception might be a useful tool to by which to identify PCOS patients at risk for GDM during pregnancy (Veltman-Verhulst et al., 2010). However, the pathophysiologic pathways of this effect are unknown. It remains unclear whether the association between GDM and SHBG is direct or indirect. A lower SHBG results in higher free androgens that have already been demonstrated to be associated with an increased GDM risk (Bartha et al., 2000). 5.2.4 Insulin-like growth factor and insulin-like growth factor binding protein-1 The risk for GDM has been connected to maternal plasma concentrations of insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-1 (IGFBP-1), suggesting that these determinants of glucose homeostasis play a potential role in the pathophysiologic process and contribute to the development of GDM (Qiu et al., 2005). Increased serum insulin concentrations lead to an inhibition of IGFBP-1 production, resulting in elevated serum levels of free IGF-1. A recent meta analysis on the interaction between IGFBP-1 and PCOS described lower levels of IGFBP-1 in affected patients (Kelly et al. 2011). Notably, a lower risk for developing GDM was correlated with increased levels of thereby leading to an increased incidence of GDM (Toulis et al., 2009). 5.2.2 Preexisting insulin resistance 5.2.3 SHBG levels free IGF-1 and IGFBP-1, where higher C-peptide levels were positively associated with the development of GDM (Qiu et al., 2005). It should be emphasized that lower levels were found in obese women with/or without PCOS, compared to normal weight controls, suggesting that body weight has a certain influence on serum IGFBP-1 levels. The physiologic processes underlying the role of IGF-1 in glucose regulation are unclear. Infusions of IGF-1 are known to suppress glucose counter-regulatory hormones, such as glucagon and growth hormone (Jones & Clemmons, 1995). IGF-1 may also contribute to changes in insulin sensitivity and glucose uptake via direct IGF receptor-mediated effects on skeletal muscle (Sjogren et al., 2001). Alterations in IGFBP-1 concentrations may be due to variations in insulin secretion or hepatic insulin sensitivity, both known to be important factors in glucose regulation (Lee et al., 1997). #### 5.2.5 The influence of infertility treatments PCOS is often accompanied by infertility that necessitates ovulation induction, using clomiphene citrate, gonadotropins, or even in vitro fertilization (IVF) (Boomsma et al., 2006). These treatment methods are known to increase the incidence of multiple pregnancies, as well as some negative consequences, including a rise in the risk for GDM (Schwartz et al., 1999). Furthermore, pregnancies established after IVF carry an increased risk for maternal complications (Pinborg et al., 2004) (Shevell et al., 2005). However, the increased risk of developing GDM has been suggested to occur independently of obesity, as well as in populations without assisted reproductive techniques (Boomsma et al., 2006) (Toulis et al., 2009). #### 6. Therapeutic considerations The therapeutic challenges in the treatment of women with PCOS and GDM or impaired glucose tolerance arise from the diversity of recommendations and research conclusions. If adequately managed, the incidence of adverse perinatal outcomes in PCOS patients who develop GDM was found to be not significantly elevated (Li et al., 2010). Several treatment options exist for each of the symptoms of PCOS, and management of these patients depends on the individual symptoms. Clinical management of PCOS involves risk assessment for metabolic disorders, as diabetes and dyslipidemia, and for hypertension, cardiovascular complications, and liver diseases (Setji & Brown, 2007). Insulin-sensitizing drugs, such as metformin and/or oral contraceptives, are thought to improve the clinical features of PCOS, but limited data is available that assesses the true influence and safety of these drugs in this population (Setji & Brown, 2007). Targeting androgen symptoms, such as acne, hirsutism, and alopecia androgenica, estrogen-containing oral contraceptives, antiandrogens and topical agents are considered first-line therapy for women who do not want to conceive, as they effectively reduce serum androgen levels. In addition, treatment with oral contraceptives is associated with an improvement in the menstrual pattern. However, when trying to achieve pregnancy, other treatment options are offered to the patient. All of these might also independently influence the risk for GDM. #### 6.1 Lifestyle modifications Addressing life-style factors before conception should be the first-line approach to reduce any further therapeutic strategies during pregnancy. The appearance of the varying The Impact of Polycystic Ovarian Syndrome on Gestational Diabetes 191 Metformin affects the hepatic glucose output and the insulin-mediated glucose consumption in peripheral tissues, and suppresses the free fatty acid concentrations, which results in a lower substrate level for gluconeogenesis. Metformin improves insulin sensitivity and reduces insulin blood levels by increasing peripheral glucose utilization without negatively influencing normal blood glucose concentrations. With regard to pregnancy, a positive impact on uterine vascularity and blood flow and a reduction in plasma endothelin-1 levels, as well as androgen and LH concentrations, have been mentioned, suggesting that metformin is a possible positive therapeutic drug in the prevention of pregnancy complications in PCOS (Khattab et al., 2011). Thus, it has been assumed that metformin treatment combined with a special diet reduces pregnancy complications, and also prevents Several studies have investigated a possible beneficial effect of metformin in the pregnancies of PCOS patients, particularly with regard to the risk of GDM. Some trials even report about a nine-fold (Begum et al., 2009) or ten-fold reduction (Glueck et al., 2002) of GDM after metformin treatment throughout pregnancy. However, the literature is controversial. The anticipated reduction in the prevalence of GDM after treatment with metformin during pregnancy could not be verified in randomized controlled trials, although metformin therapy had improved insulin sensitivity (Legro et al., 2007) (Fougner et al., 2008). In particular, recent large randomized controlled studies and meta analyses could not verify a relevant reduction of pregnancy complications due to metformin treatment (Vanky et al., 2010). Thus, the general use of metformin during pregnancy in non-diabetic women with Metformin is associated with low gastrointestinal disturbances, but the available trials did not describe any serious adverse events. Classified as a category B drug in pregnancy, metformin appears to be non-teratogenic (Glueck et al., 2004b). However, results from placebo-controlled trials on maternal and fetal health risk have not yet been clarified. The pathogenesis of PCOS has been related to increased intrauterine androgen exposure; thus, the effect of therapeutic investigations on maternal and fetal hormone levels must be considered when treating pregnant women with PCOS. metformin seems to pass the placental barrier and was found to be present in the fetal circulation. However, androgen and estrogen levels did not seem to be influenced and remained within normal range (Carlsen & Vanky, 2010), whereas elevated SHBG levels have been reported in newborns after intrauterine metformin exposure (Vanky et With regard to pregnancy outcome, there are different possibilities of infertility treatment in women with PCOS and ovulatory dysfunction. Pregnancy induction by assisted reproductive techniques (ART) is offered if women fail to conceive spontaneously. Ovulation induction is based on two principles: (i) ovaries are exposed to a higher level of follicle stimulating hormone; and/or (ii) hormonal derangements are corrected. After exclusion of other causes of infertility, the fertility medication, clomiphen citrate (CC), is considered the first-line therapeutic approach to ovulation induction. In this approach, the development of a single ovulatory follicle is the main goal, since the risk for multiple Literature on the risk for GDM as it relates to CC stimulation is scarce. To date, only one retrospective study has been published that compared the effects of CC stimulation and the fetus from elevated androgen concentrations (Glueck et al., 2004a). al., 2005). The clinical relevance of these findings remains unclear. 6.3 Other methods of ovulation induction pregnancies has to be kept as low as possible. PCOS cannot be recommended. phenotypes of PCOS depends, to a great extent, on lifestyle and environmental factors (Garruti et al., 2009). Reducing weight improves the endocrine profile and has the most significant impact on the likelihood of ovulation and pregnancy, the most relevant endpoints in infertile PCOS women. Dietary composition has been considered to improve the initial metabolic and reproductive situation in these patients. It has been suggested that low fat diets be recommended to these patients to produce a decrease in hyperinsulinemia (Reaven, 2005). Since it has been well-established that obesity is not only associated with anovulation, infertility, and early miscarriage, but also with late pregnancy complications in women with PCOS (Badawy & Elnashar, 2011), lifestyle modifications, including dietary recommendations and increased exercise in order to achieve weight reduction, should be recommended to these patients. Bariatric surgery has also been advocated as a possible strategy for weight loss in PCOS women, at least in the morbidly obese, and is effective in restoring ovulation and improving insulin resistance. Reducing weight, along with lifestyle modifications that affect the patients' behavior in a continuing way, is considered the most relevant therapeutic approach in women with PCOS (Hirschberg, 2009). Whether it affects the risk of developing GDM in subsequent pregnancies remains an open question (Escobar-Morreale et al., 2005). #### 6.2 Insulin-sensitizing drugs Elevated insulin resistance plays an important part in the pathophysiology of PCOS and GDM. Insulin-sensitizing drugs, particularly metformin, are thought to reduce androgen symptoms, positively influence reproductive deregulations (oligo-amenorrhoe, anovulation), and increase pregnancy rates in PCOS patients (Dunaif, 2008). Large placebocontrolled trials are available only for metformin, as this is the only insulin-sensitizing drug with extensive clinical use in women with PCOS (De Leo et al., 2003). #### 6.2.1 Metformin as a PCOS-specific therapy Metformin, a biguanide (see Figure 2), is a therapeutic option for restoration of ovulation in PCOS women. Moreover, several studies have suggested metformin as a promising medication in pregnancy in order to reduce the incidence of developing GDM and to minimize the risk for an adverse pregnancy outcome (Carlsen & Vanky, 2010). A variety of studies have been performed to determine the beneficial effects of metformin in PCOS, although the mechanisms of action–a broad spectrum of endocrine, metabolic, vascular, and even anti-inflammatory effects–of this drug have not been completely clarified, as yet (Khattab et al., 2011). Fig. 2. Formula of metformin phenotypes of PCOS depends, to a great extent, on lifestyle and environmental factors (Garruti et al., 2009). Reducing weight improves the endocrine profile and has the most significant impact on the likelihood of ovulation and pregnancy, the most relevant endpoints in infertile PCOS women. Dietary composition has been considered to improve the initial metabolic and reproductive situation in these patients. It has been suggested that low fat diets be recommended to these patients to produce a decrease in hyperinsulinemia (Reaven, 2005). Since it has been well-established that obesity is not only associated with anovulation, infertility, and early miscarriage, but also with late pregnancy complications in women with PCOS (Badawy & Elnashar, 2011), lifestyle modifications, including dietary recommendations and increased exercise in order to achieve weight reduction, should be recommended to these patients. Bariatric surgery has also been advocated as a possible strategy for weight loss in PCOS women, at least in the morbidly obese, and is effective in restoring ovulation and improving insulin resistance. Reducing weight, along with lifestyle modifications that affect the patients' behavior in a continuing way, is considered the most relevant therapeutic approach in women with PCOS (Hirschberg, 2009). Whether it affects the risk of developing GDM in subsequent pregnancies remains an open question (Escobar- Elevated insulin resistance plays an important part in the pathophysiology of PCOS and GDM. Insulin-sensitizing drugs, particularly metformin, are thought to reduce androgen symptoms, positively influence reproductive deregulations (oligo-amenorrhoe, anovulation), and increase pregnancy rates in PCOS patients (Dunaif, 2008). Large placebocontrolled trials are available only for metformin, as this is the only insulin-sensitizing drug Metformin, a biguanide (see Figure 2), is a therapeutic option for restoration of ovulation in PCOS women. Moreover, several studies have suggested metformin as a promising medication in pregnancy in order to reduce the incidence of developing GDM and to minimize the risk for an adverse pregnancy outcome (Carlsen & Vanky, 2010). A variety of studies have been performed to determine the beneficial effects of metformin in PCOS, although the mechanisms of action–a broad spectrum of endocrine, metabolic, vascular, and even anti-inflammatory effects–of this drug have not been completely clarified, as yet with extensive clinical use in women with PCOS (De Leo et al., 2003). 6.2.1 Metformin as a PCOS-specific therapy Morreale et al., 2005). (Khattab et al., 2011). Fig. 2. Formula of metformin 6.2 Insulin-sensitizing drugs Metformin affects the hepatic glucose output and the insulin-mediated glucose consumption in peripheral tissues, and suppresses the free fatty acid concentrations, which results in a lower substrate level for gluconeogenesis. Metformin improves insulin sensitivity and reduces insulin blood levels by increasing peripheral glucose utilization without negatively influencing normal blood glucose concentrations. With regard to pregnancy, a positive impact on uterine vascularity and blood flow and a reduction in plasma endothelin-1 levels, as well as androgen and LH concentrations, have been mentioned, suggesting that metformin is a possible positive therapeutic drug in the prevention of pregnancy complications in PCOS (Khattab et al., 2011). Thus, it has been assumed that metformin treatment combined with a special diet reduces pregnancy complications, and also prevents the fetus from elevated androgen concentrations (Glueck et al., 2004a). Several studies have investigated a possible beneficial effect of metformin in the pregnancies of PCOS patients, particularly with regard to the risk of GDM. Some trials even report about a nine-fold (Begum et al., 2009) or ten-fold reduction (Glueck et al., 2002) of GDM after metformin treatment throughout pregnancy. However, the literature is controversial. The anticipated reduction in the prevalence of GDM after treatment with metformin during pregnancy could not be verified in randomized controlled trials, although metformin therapy had improved insulin sensitivity (Legro et al., 2007) (Fougner et al., 2008). In particular, recent large randomized controlled studies and meta analyses could not verify a relevant reduction of pregnancy complications due to metformin treatment (Vanky et al., 2010). Thus, the general use of metformin during pregnancy in non-diabetic women with PCOS cannot be recommended. Metformin is associated with low gastrointestinal disturbances, but the available trials did not describe any serious adverse events. Classified as a category B drug in pregnancy, metformin appears to be non-teratogenic (Glueck et al., 2004b). However, results from placebo-controlled trials on maternal and fetal health risk have not yet been clarified. The pathogenesis of PCOS has been related to increased intrauterine androgen exposure; thus, the effect of therapeutic investigations on maternal and fetal hormone levels must be considered when treating pregnant women with PCOS. metformin seems to pass the placental barrier and was found to be present in the fetal circulation. However, androgen and estrogen levels did not seem to be influenced and remained within normal range (Carlsen & Vanky, 2010), whereas elevated SHBG levels have been reported in newborns after intrauterine metformin exposure (Vanky et al., 2005). The clinical relevance of these findings remains unclear. #### 6.3 Other methods of ovulation induction With regard to pregnancy outcome, there are different possibilities of infertility treatment in women with PCOS and ovulatory dysfunction. Pregnancy induction by assisted reproductive techniques (ART) is offered if women fail to conceive spontaneously. Ovulation induction is based on two principles: (i) ovaries are exposed to a higher level of follicle stimulating hormone; and/or (ii) hormonal derangements are corrected. After exclusion of other causes of infertility, the fertility medication, clomiphen citrate (CC), is considered the first-line therapeutic approach to ovulation induction. In this approach, the development of a single ovulatory follicle is the main goal, since the risk for multiple pregnancies has to be kept as low as possible. Literature on the risk for GDM as it relates to CC stimulation is scarce. To date, only one retrospective study has been published that compared the effects of CC stimulation and The Impact of Polycystic Ovarian Syndrome on Gestational Diabetes 193 these patients (Alshammari et al., 2010). According to the Barker hypothesis, an altered maternal nutrition and metabolism is thought to lead both to an altered fetal nutrition and to changes in the endocrine and metabolic environment in which the fetus develops (de Boo Maternal glucose levels correlate with fetal birth weight, development of fetal macrosomia, fetal hyperinsulinemia, and fetal body-fat percentage (Yang et al., 2002) (Metzger et al., 2010). Cesarean section is performed more frequently in women with GDM, as the diagnosis "large for gestational age" due to elevated maternal glucose levels is associated with a higher incidence of adverse pregnancy outcomes in spontaneous delivery (e.g., shoulder dystocia). PCOS also seems to correlate with a lower rate of vaginal delivery compared to healthy controls (Bjercke et al., 2002), although the higher incidence of Caesarean sections correlates with the occurrence of obesity, since women with a normal BMI and PCOS have an incidence of Caesarean section equal to that of age-matched controls (Boomsma et al., Accordingly, the perinatal outcome of women with PCOS who develop GDM has been investigated intensively within the last several years. Both PCOS and associated factors, such as obesity or the treatment methods for fertility induction, can be considered responsible for the poorer pregnancy outcomes (Thatcher & Jackson, 2006, Boomsma et al., 2008). However, with regard to the risk of macrosomia, preeclampsia, neonatal complications, neonatal anomalies, and death of the fetus in women with GDM and PCOS, compared to women with GDM alone, no significant differences have been observed (Li et al., 2010). Manifest obesity before pregnancy and total weight gain during pregnancy must be closely monitored and addressed insistently, not only to minimize the risk for GDM, but The diverse observations regarding the elevated risk of GDM in women with PCOS have presented scientists and clinicians with a challenge. Caution is advised when interpreting clinical and statistical heterogeneous studies on PCOS and pregnancy complications, as a variety of contradictory results are present throughout the literature. The diagnosis of PCOS is inconsistent, as some investigators use only ultrasound criteria alone, and others rely on hormonal or clinical parameters, whereas the revised Rotterdam criteria are considered the Women with PCOS who want to have children must be informed about the increased risk for developing GDM in their pregnancies. Metabolic findings in PCOS include increased insulin resistance, dyslipidemia, and elevated androgen levels - often accompanied by infertility and infertility treatments in order to achieve pregnancy. Confounding factors, such as obesity and the diverse ovulation induction treatments in infertile women with PCOS, can be considered potentially risk-increasing variables. Those coexisting factors, together with additional predisposing factors, such as a positive family history for diabetes mellitus, have been suggested to correlate with a generally increased risk for developing Comparable pathophysiological mechanisms of insulin resistance and impaired glucose tolerance can be found in GDM and in women with PCOS who demonstrate an increased tissue resistance to insulin. However, the exact pathophysiologic link between PCOS and GDM has not yet been fully elucidated. Future scientific research could aim to clarify the & Harding 2006). This explains why PCOS complications might affect the fetus. also to achieve better global well-being in women with PCOS. current valid diagnostic criteria for the diagnosis of PCOS. GDM and impaired glucose tolerance (Toulis et al., 2009). 8. Conclusion and future research areas 2006). laparoscopic ovarian drilling in women pre-treated with metformin to those treated with metformin only. For all groups, there was a rate of GDM of about 30%, suggesting that neither CC stimulation nor laparoscopic ovarian drilling exert any effect on the risk for GDM (Ott et al., 2010). For CC-resistant anovulatory PCOS patients, second-line therapeutic approaches include laparoscopic ovarian drilling and gonadotropin stimulation (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004). As mentioned above, only one trial has evaluated whether laparoscopic ovarian drilling would alter the risk for GDM and found no influence (Ott et al., 2010). Whether gonadotropin stimulation is associated with an increased rate of GDM remains controversial. While some studies found no difference in the prevalence of GDM between PCOS patients and controls (Vollenhoven et al., 2000), data from prospective studies suggest that medicamentous ovulation induction leads to an increased risk for GDM (Shevell et al., 2005). When all of these treatment options fail, IVF remains the last option by which to achieve pregnancy (Badawy & Elnashar, 2011). It has been reported that the prevalence of GDM is substantially increased in women after pregnancy induction via ART than in PCOS patients who conceive spontaneously (Bals-Pratsch et al., 2011). Proceeding on the assumption of a possibly genetically determined - highly deranged insulin metabolism that may lead to this state of infertility where women cannot conceive spontaneously or with the help of lowerdose stimulation protocols, the higher rate of GDM could be explained. One might raise the question whether the maternal endocrine status before placentation and/or after IVF stimulation possibly influences glucose tolerance in women with PCOS. Furthermore, elevated estrogen levels lead to an excessive stimulation of estrogen receptor alpha in the pancreatic beta cells, producing exaggerated insulin signaling that may cause an increased state of insulin resistance in peripheral tissues (Nadal et al., 2009). Moreover, IVF is known to lead to an increased risk for maternal complications, including GDM independently of the presence of PCOS (Pinborg et al., 2004) (Shevell et al., 2005). When IVF stimulation is complicated by ovarian hyperstimulation syndrome (OHSS), the risk for GDM increases even more (Raziel et al., 2009). Notably, PCOS is a known risk factor for the development of OHSS. Higher rates of GDM, but also placental abruption prematurity and low birth weight have been reported for pregnancies complicated by severe OHSS. Therefore, these pregnancies should be considered high-risk pregnancies, and followed/treated as such. The best option to prevent OHSS is to use mild stimulation protocols (Raziel et al., (2009). However, metformin has also been mentioned as leading to a significant reduction in OHSS rates (Khattab et al., 2006). #### 7. Perinatal outcome of women with polycystic ovary syndrome and gestational diabetes Hyperglycemia negatively influences not only maternal, but also fetal health. Obesity, fertility treatments, and other characteristics of patients with PCOS are associated with a higher incidence of pregnancy complications, such as hypertension and preeclampsia (Boomsma et al., 2008). Compared to normal pregnancies, PCOS patients demonstrate a higher incidence of early pregnancy loss (Li et al., 2010). Likewise, perinatal mortality seems to be increased among women with PCOS, and neonatal complications are observed more frequently (Boomsma et al., 2006). A potential additive effect of co-existent PCOS and GDM on obstetrical complications advocates for a closer antenatal and intrapartal monitoring of laparoscopic ovarian drilling in women pre-treated with metformin to those treated with metformin only. For all groups, there was a rate of GDM of about 30%, suggesting that neither CC stimulation nor laparoscopic ovarian drilling exert any effect on the risk for For CC-resistant anovulatory PCOS patients, second-line therapeutic approaches include laparoscopic ovarian drilling and gonadotropin stimulation (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004). As mentioned above, only one trial has evaluated whether laparoscopic ovarian drilling would alter the risk for GDM and found no influence (Ott et al., 2010). Whether gonadotropin stimulation is associated with an increased rate of GDM remains controversial. While some studies found no difference in the prevalence of GDM between PCOS patients and controls (Vollenhoven et al., 2000), data from prospective studies suggest that medicamentous ovulation induction leads to an When all of these treatment options fail, IVF remains the last option by which to achieve pregnancy (Badawy & Elnashar, 2011). It has been reported that the prevalence of GDM is substantially increased in women after pregnancy induction via ART than in PCOS patients who conceive spontaneously (Bals-Pratsch et al., 2011). Proceeding on the assumption of a possibly genetically determined - highly deranged insulin metabolism that may lead to this state of infertility where women cannot conceive spontaneously or with the help of lowerdose stimulation protocols, the higher rate of GDM could be explained. One might raise the question whether the maternal endocrine status before placentation and/or after IVF stimulation possibly influences glucose tolerance in women with PCOS. Furthermore, elevated estrogen levels lead to an excessive stimulation of estrogen receptor alpha in the pancreatic beta cells, producing exaggerated insulin signaling that may cause an increased Moreover, IVF is known to lead to an increased risk for maternal complications, including GDM independently of the presence of PCOS (Pinborg et al., 2004) (Shevell et al., 2005). When IVF stimulation is complicated by ovarian hyperstimulation syndrome (OHSS), the risk for GDM increases even more (Raziel et al., 2009). Notably, PCOS is a known risk factor for the development of OHSS. Higher rates of GDM, but also placental abruption prematurity and low birth weight have been reported for pregnancies complicated by severe OHSS. Therefore, these pregnancies should be considered high-risk pregnancies, and followed/treated as such. The best option to prevent OHSS is to use mild stimulation protocols (Raziel et al., (2009). However, metformin has also been mentioned as leading to 7. Perinatal outcome of women with polycystic ovary syndrome and Hyperglycemia negatively influences not only maternal, but also fetal health. Obesity, fertility treatments, and other characteristics of patients with PCOS are associated with a higher incidence of pregnancy complications, such as hypertension and preeclampsia (Boomsma et al., 2008). Compared to normal pregnancies, PCOS patients demonstrate a higher incidence of early pregnancy loss (Li et al., 2010). Likewise, perinatal mortality seems to be increased among women with PCOS, and neonatal complications are observed more frequently (Boomsma et al., 2006). A potential additive effect of co-existent PCOS and GDM on obstetrical complications advocates for a closer antenatal and intrapartal monitoring of GDM (Ott et al., 2010). increased risk for GDM (Shevell et al., 2005). state of insulin resistance in peripheral tissues (Nadal et al., 2009). a significant reduction in OHSS rates (Khattab et al., 2006). gestational diabetes these patients (Alshammari et al., 2010). According to the Barker hypothesis, an altered maternal nutrition and metabolism is thought to lead both to an altered fetal nutrition and to changes in the endocrine and metabolic environment in which the fetus develops (de Boo & Harding 2006). This explains why PCOS complications might affect the fetus. Maternal glucose levels correlate with fetal birth weight, development of fetal macrosomia, fetal hyperinsulinemia, and fetal body-fat percentage (Yang et al., 2002) (Metzger et al., 2010). Cesarean section is performed more frequently in women with GDM, as the diagnosis "large for gestational age" due to elevated maternal glucose levels is associated with a higher incidence of adverse pregnancy outcomes in spontaneous delivery (e.g., shoulder dystocia). PCOS also seems to correlate with a lower rate of vaginal delivery compared to healthy controls (Bjercke et al., 2002), although the higher incidence of Caesarean sections correlates with the occurrence of obesity, since women with a normal BMI and PCOS have an incidence of Caesarean section equal to that of age-matched controls (Boomsma et al., 2006). Accordingly, the perinatal outcome of women with PCOS who develop GDM has been investigated intensively within the last several years. Both PCOS and associated factors, such as obesity or the treatment methods for fertility induction, can be considered responsible for the poorer pregnancy outcomes (Thatcher & Jackson, 2006, Boomsma et al., 2008). However, with regard to the risk of macrosomia, preeclampsia, neonatal complications, neonatal anomalies, and death of the fetus in women with GDM and PCOS, compared to women with GDM alone, no significant differences have been observed (Li et al., 2010). Manifest obesity before pregnancy and total weight gain during pregnancy must be closely monitored and addressed insistently, not only to minimize the risk for GDM, but also to achieve better global well-being in women with PCOS. #### 8. Conclusion and future research areas The diverse observations regarding the elevated risk of GDM in women with PCOS have presented scientists and clinicians with a challenge. Caution is advised when interpreting clinical and statistical heterogeneous studies on PCOS and pregnancy complications, as a variety of contradictory results are present throughout the literature. The diagnosis of PCOS is inconsistent, as some investigators use only ultrasound criteria alone, and others rely on hormonal or clinical parameters, whereas the revised Rotterdam criteria are considered the current valid diagnostic criteria for the diagnosis of PCOS. Women with PCOS who want to have children must be informed about the increased risk for developing GDM in their pregnancies. Metabolic findings in PCOS include increased insulin resistance, dyslipidemia, and elevated androgen levels - often accompanied by infertility and infertility treatments in order to achieve pregnancy. Confounding factors, such as obesity and the diverse ovulation induction treatments in infertile women with PCOS, can be considered potentially risk-increasing variables. Those coexisting factors, together with additional predisposing factors, such as a positive family history for diabetes mellitus, have been suggested to correlate with a generally increased risk for developing GDM and impaired glucose tolerance (Toulis et al., 2009). Comparable pathophysiological mechanisms of insulin resistance and impaired glucose tolerance can be found in GDM and in women with PCOS who demonstrate an increased tissue resistance to insulin. However, the exact pathophysiologic link between PCOS and GDM has not yet been fully elucidated. Future scientific research could aim to clarify the The Impact of Polycystic Ovarian Syndrome on Gestational Diabetes 195 Battaglia, C., Regnani, G., Mancini, F., Iughetti, L., Flamigni, C. & Venturoli, S. (2002). Boomsma, C. M., Eijkemans, M. J., Hughes, E. G., Visser, G. H., Fauser, B. C. & Macklon, N. Boomsma, C. M., Fauser, B. C. & Macklon, N. S. (2008). Pregnancy complications in women Carlsen, S. M. & Vanky, E. (2010). Metformin influence on hormone levels at birth, in PCOS De Groot, P. C., Dekkers, O. M., Romijn, J. A., Dieben, S. W. & Helmerhorst, F. M. (2011). Ding, E. L., Song, Y., Manson, J. E., Hunter, D. J., Lee, C. C., Rifai, N., Buring, J. E., Gaziano, Dumesic, D. A., Abbott, D. H. & Padmanabhan, V. (2007). Polycystic ovary syndrome and Dunaif, A. (2008). Drug insight: insulin-sensitizing drugs in the treatment of polycystic Escobar-Morreale, H. F., Botella-Carretero, J. I., Alvarez-Blasco, F., Sancho, J. & San Millan, J. Fougner, K. J., Vanky, E. & Carlsen, S. M. (2008). Metformin has no major effects on glucose study. Scand J Clin Lab Invest, Vol.68, No.8, pp. 771-6, ISSN: 1502-7686 Franks, S., Gharani, N., Waterworth, D., Batty, S., White, D., Williamson, R. & McCarthy, M. 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Current developments in the molecular genetics of the polycystic ovary syndrome. Trends Endocrinol Metab, Vol.9, No.2, (February 1998), pp. 51-4, ISSN: association between PCOS and GDM and shed some new light on the possible underlying pathomechanisms. Moreover, further investigations to evaluate a potential benefit of early GDM screening are warranted, particularly as GDM is a well-known risk factor for fetal and maternal complications (Tieu et al., 2010). The presence of increased glucose levels might lead to considerable pregnancy complications and stress on the mother and fetus. Accurate screening for GDM, together with regular consultations and monitoring, as well as addressing additional preventable stress factors, reduce the risk for developing GDM. Primary prevention can be further improved by lifestyle modification in women with PCOS. With regard to early diagnosis, screening in PCOS patients with a variety of risk factors might be justified. One might focus on the development of alternative markers to identify a woman at risk for developing GDM, in addition to the available parameters, such as fasting plasma glucose and OGTT, which enable clinicians to select patients at risk even before the manifestation of GDM. Thus, such markers as androgen levels, SHBG levels, fasting insulin levels, baseline proinsulin levels, and the hip-waist ratio might be of future interest. Metformin has been highlighted as a promising substance for reducing the risk of GDM in PCOS patients. However, a relevant reduction of obstetrical complications due to metformin treatment could not be verified in a recent randomized and controlled multicenter study (Vanky et al., 2010). The general use of metformin during pregnancy in non-diabetic women with PCOS cannot be considered a valid recommendation. Even with all the evidence and comparative studies, PCOS, still remains a challenging diagnostic and research issue, particularly with regard to its impact on GDM. #### 9. References association between PCOS and GDM and shed some new light on the possible underlying pathomechanisms. Moreover, further investigations to evaluate a potential benefit of early GDM screening are warranted, particularly as GDM is a well-known risk factor for fetal and The presence of increased glucose levels might lead to considerable pregnancy complications and stress on the mother and fetus. Accurate screening for GDM, together with regular consultations and monitoring, as well as addressing additional preventable stress factors, reduce the risk for developing GDM. Primary prevention can be further improved by lifestyle modification in women with PCOS. With regard to early diagnosis, screening in PCOS patients with a variety of risk factors might be justified. One might focus on the development of alternative markers to identify a woman at risk for developing GDM, in addition to the available parameters, such as fasting plasma glucose and OGTT, which enable clinicians to select patients at risk even before the manifestation of GDM. Thus, such markers as androgen levels, SHBG levels, fasting insulin levels, baseline proinsulin levels, Metformin has been highlighted as a promising substance for reducing the risk of GDM in PCOS patients. However, a relevant reduction of obstetrical complications due to metformin treatment could not be verified in a recent randomized and controlled multicenter study (Vanky et al., 2010). The general use of metformin during pregnancy in non-diabetic women Even with all the evidence and comparative studies, PCOS, still remains a challenging Azziz, R., Carmina, E., Dewailly, D., Diamanti-Kandarakis, E., Escobar-Morreale, H. F., Balen, A. (2004). 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International association of diabetes and pregnancy study groups recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care, Vol.33, No.3, (March 2010), pp.676-82, (2004). Changes in anti-Mullerian hormone serum concentrations over time suggest Garruti, G., Depalo, R., Vita, M. G., Lorusso, F., Giampetruzzi, F., Damato, A. B. & Giorgino, Gleicher, N., Barad, D. & Weghofer, A. (2007). Functional autoantibodies, a new paradigm in Glueck, C. J., Wang, P., Kobayashi, S., Phillips, H. & Sieve-Smith, L. (2002). Metformin Glueck, C. J., Goldenberg, N., Wang, P., Loftspring, M. & Sherman, A. (2004a). Metformin Glueck, C. J., Goldenberg, N., Pranikoff, J., Loftspring, M., Sieve, L. & Wang, P. (2004b). Godoy-Matos, A. F., Vaisman, F., Pedrosa, A. P., Farias, M. L., Mendonca, L. M. & Pinheiro, Holt, R. I., Coleman, M. A. & McCance, D. R. (2011). The implications of the new Huang, A., Brennan, K. & Azziz, R. (2010). 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(2004). Revised Toulis, K. A., Goulis, D. G., Kolibianakis, E. M., Venetis, C. A., Tarlatzis, B. C. & Papadimas, Vanky, E., Zahlsen, K., Spigset, O. & Carlsen, S. M. (2005). Placental passage of metformin in Vanky, E., Stridsklev, S., Heimstad, R., Romundstad, P., Skogoy, K., Kleggetveit, O., Hjelle, Vantyghem, M. C., Vincent-Desplanques, D., Defrance-Faivre, F., Capeau, J., Fermon, C., Veltman-Verhulst, S. M., van Haeften, T. W., Eijkemans, M. J., de Valk, H. W., Fauser, B. C. Vollenhoven, B., Clark, S., Kovacs, G., Burger, H. & Healy, D. (2000). Prevalence of Gynaecol, Vol.40, No.1, (February 2000), pp. 54-8, ISSN: 0004-8666 Isaksson, O. G., Ahren, B., Jansson, J. O. & Ohlsson, C. (2001). Liver-derived IGF-I is of importance for normal carbohydrate and lipid metabolism. Diabetes, Vol.50, polycystic ovary syndrome who were treated with metformin. Fertil Steril, Vol.85, 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril, Vol.81, No.1, pp.19-25, ISSN: 0015-0282 Tieu, J., Middleton, P., McPhee, A. J. & Crowther, C. A. (2010). Screening and subsequent management for gestational diabetes for improving maternal and infant health. I. (2009). Risk of gestational diabetes mellitus in women with polycystic ovary syndrome: a systematic review and a meta-analysis. Fertil Steril, Vol.92, No.2, women with polycystic ovary syndrome. Fertil Steril, Vol. 83, No.5, (May 2005), pp. S., von Brandis, P., Eikeland, T., Flo, K., Berg, K. F., Bunford, G., Lund, A., Bjerke, C., Almas, I., Berg, A. H., Danielson, A., Lahmami, G. & Carlsen, S. M. (2010). Metformin versus placebo from first trimester to delivery in polycystic ovary syndrome: a randomized, controlled multicenter study. J Clin Endocrinol Metab, Valat, A. S., Lascols, O., Hecart, A. C., Pigny, P., Delemer, B., Vigouroux, C. & Wemeau, J. L. (2008). Fertility and obstetrical complications in women with LMNArelated familial partial lipodystrophy. J Clin Endocrinol Metab, Vol.93, No.6, (June & Goverde, A. J. (2010). Sex hormone-binding globulin concentrations before conception as a predictor for gestational diabetes in women with polycystic ovary syndrome. Hum Reprod, Vol.25, No.12, (December 2010), pp. 3123-8, ISSN: 1460- gestational diabetes mellitus in polycystic ovarian syndrome (PCOS) patients pregnant after ovulation induction with gonadotrophins. Aust N Z J Obstet Rep, Vol.3, No.4, (August 2003), pp. 319-22, ISSN: 1534-4827 No.7, (July 2001), 1539-45, ISSN: 0012-1797 No.4, (April 2006), 1002-9, ISSN: 1556-5653 (August 2009), 667-77, ISSN: 1556-5653 2008), pp. 2223-9, ISSN: 0021-972X 2350 1575-8, ISSN: 1556-5653 Med, Vol.259, No.9, pp. 420-423, ISSN: 0028-4793 Cochrane Database Syst Rev, CD007222, ISSN: 1469-493X Vol.95, No.12, (December 2010), pp. 448-55, ISSN: 1945-7197 delayed ovarian ageing in normogonadotrophic anovulatory infertility. 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Pregnancy outcome in women with polycystic ovary syndrome comparing the effects of laparoscopic ovarian drilling and clomiphene citrate stimulation in women pre-treated with metformin: a retrospective study. Reprod Biol Endocrinol, antithyroid peroxidase antibodies indicating Hashimoto's thyroiditis are associated with the treatment response in infertile women with polycystic ovary syndrome. and perinatal outcome in a Danish national cohort of 1005 twin pregnancies: the role of in vitro fertilization. Acta Obstet Gynecol Scand, Vol.83, No.1, (January plasma concentrations of IGF-1, IGFBP-1, and C-peptide in early pregnancy and subsequent risk of gestational diabetes mellitus. Am J Obstet Gynecol, Vol.193, following severe OHSS. Reprod Biomed Online, Vol.19, No.1, (July 2009), 61-5, disease. Panminerva Med, Vol.47, No.4, (December, 2005), pp. 201-10, ISSN: 0031- Glucose intolerance in pregnancy and future risk of pre-diabetes or diabetes. Diabetes Care, Vol.31, No. 10, (Octoberober 2008), pp. 2026-31, ISSN: 1935-5548 Schwartz, D. B., Daoud, Y., Zazula, P., Goyert, G., Bronsteen, R., Wright, D. & Copes, J. (1999). Gestational diabetes mellitus: metabolic and blood glucose parameters in singleton versus twin pregnancies. Am J Obstet Gynecol, Vol.181, No.4, (October E. (2005). Assisted reproductive technology and pregnancy outcome. Obstet 11 France Impact and Mechanisms of Pancreatic Audrey Chavey, Jamileh Movassat and Bernard Portha Laboratoire B2PE (Biologie et Pathologie du Pancréas Endocrine), Unité BFA (Biologie Fonctionnelle et Adaptive), Université Paris-Diderot, Paris, Beta-Cell Mass Programming by Maternal Diabetes - Insight from Animal Model Studies The incidence of type 2 diabetes mellitus (T2D) is growing worldwide. It is now established that interactions between the individual genetic makeup and environment contribute to the development of T2D. In this review, we first discuss the evidence for beta-cell dysfunction in IUED (in utero exposure to maternal diabetes), IUEO (in utero exposure to maternal overnutrition) and IUGR (in utero growth restriction) humans. We then evaluate relevant animal models of IUED, IUEO and IUGR focusing on the strengths and limits of each, in order to define critical periods and types of alterations that can lead to impaired beta-cell function. Finally, we discuss several potential mechanisms dissected in relevant animal There are strong arguments showing that T2D is more prevalent among subjects that were exposed to maternal diabetes in utero (IUED). The role of maternal inheritance in T2D was first suggested by epidemiological studies. A higher susceptibility for diabetes is described in descendents from diabetic great grand mothers via the maternal line than via the paternal line (Dörner et al., 1984). A higher incidence of T2D and of gestational diabetes (GD) is seen in children of diabetic mothers as compared to those of diabetic fathers (Martin et al., 1985). A decreased prevalence of diabetes is seen in children from diabetic mothers, after starting a systematic treatment of diabetic pregnant women (Dörner et al., 1987). Especially interesting in this context are the studies on the Pima Indians, a population with an exceptionally high T2D incidence. The prevalence of impaired glucose tolerance, of T2D and of GD is much higher (7 fold increased) in children from mothers who had diabetes during pregnancy, than in children from mothers who developed diabetes only after their pregnancy. Moreover, paternal diabetes has a much smaller effect on the prevalence of diabetes in these offspring The maternal influence in the development of T2D was reported in a majority of studies (Alcolado et al., 2002). Although some studies did not find a maternal effect, none reported a 2. Perinatal risk factors for diabetes in later life: Human studies 1. Introduction models that begin to explain these outcomes. than maternal diabetes (Pettitt, 1996). ## Impact and Mechanisms of Pancreatic Beta-Cell Mass Programming by Maternal Diabetes - Insight from Animal Model Studies Audrey Chavey, Jamileh Movassat and Bernard Portha Laboratoire B2PE (Biologie et Pathologie du Pancréas Endocrine), Unité BFA (Biologie Fonctionnelle et Adaptive), Université Paris-Diderot, Paris, France #### 1. Introduction 200 Gestational Diabetes Waterworth, D. M., Bennett, S. T., Gharani, N., McCarthy, M. I., Hague, S., Batty, S., Wijeyaratne, C. N., Waduge, R., Arandara, D., Arasalingam, A., Sivasuriam, A., mellitus. BJOG, Vol.113, No.10, (October 2006), pp. 1182-7, ISSN: 1470-0328 6736 Conway, G. S., White, D., Todd, J. A., Franks, S. & Williamson, R. (1997). Linkage and association of insulin gene VNTR regulatory polymorphism with polycystic ovary syndrome. Lancet, Vol.349, No.9057, (April 5 1997), pp. 986-90, ISSN: 0140- Dodampahala, S. H. & Balen, A. H. (2006). Metabolic and polycystic ovary syndromes in indigenous South Asian women with previous gestational diabetes > The incidence of type 2 diabetes mellitus (T2D) is growing worldwide. It is now established that interactions between the individual genetic makeup and environment contribute to the development of T2D. In this review, we first discuss the evidence for beta-cell dysfunction in IUED (in utero exposure to maternal diabetes), IUEO (in utero exposure to maternal overnutrition) and IUGR (in utero growth restriction) humans. We then evaluate relevant animal models of IUED, IUEO and IUGR focusing on the strengths and limits of each, in order to define critical periods and types of alterations that can lead to impaired beta-cell function. Finally, we discuss several potential mechanisms dissected in relevant animal models that begin to explain these outcomes. #### 2. Perinatal risk factors for diabetes in later life: Human studies There are strong arguments showing that T2D is more prevalent among subjects that were exposed to maternal diabetes in utero (IUED). The role of maternal inheritance in T2D was first suggested by epidemiological studies. A higher susceptibility for diabetes is described in descendents from diabetic great grand mothers via the maternal line than via the paternal line (Dörner et al., 1984). A higher incidence of T2D and of gestational diabetes (GD) is seen in children of diabetic mothers as compared to those of diabetic fathers (Martin et al., 1985). A decreased prevalence of diabetes is seen in children from diabetic mothers, after starting a systematic treatment of diabetic pregnant women (Dörner et al., 1987). Especially interesting in this context are the studies on the Pima Indians, a population with an exceptionally high T2D incidence. The prevalence of impaired glucose tolerance, of T2D and of GD is much higher (7 fold increased) in children from mothers who had diabetes during pregnancy, than in children from mothers who developed diabetes only after their pregnancy. Moreover, paternal diabetes has a much smaller effect on the prevalence of diabetes in these offspring than maternal diabetes (Pettitt, 1996). The maternal influence in the development of T2D was reported in a majority of studies (Alcolado et al., 2002). Although some studies did not find a maternal effect, none reported a Impact and Mechanisms of Pancreatic Beta-Cell Mass Programming by Maternal Diabetes - Insight from Animal Model Studies 203 found reduced in those offspring whose mother was diabetic before pregnancy while it remained normal in those whose mother developed diabetes after pregnancy (Gautier et al., 2001). Body fat and insulin sensitivity (euglycemic hyperinsulinemic clamp) were similar in the two groups of subjects (Gautier et al., 2001). In the same study, acute insulin response was found reduced in offspring of parents (mother or father) with early onset of T2D (Gautier et al., 2001), suggesting that gene(s) linked to early onset diabetes is(are) associated with reduced insulin secretory response to glucose (Hanson et al., 1995). Offspring of T1D mothers had reduced insulin secretion, more pronounced in IGT subjects, but similar fat mass and insulin action compared with offspring of T1D fathers (Sobngwi et al., 2003). Also in non-diabetic offspring of mothers with young-onset T2D (diagnosed under age 50), betacell function (early insulin release after oral glucose) was found decreased as compared to Therefore human studies suggest that insulin secretion defect participates to the abnormal glucose tolerance observed in adult offspring exposed to maternal diabetes during fetal life. Importantly, they showed that insulin secretion may be reduced even in normal glucosetolerant offspring. Nevertheless, in children and adolescent offspring, insulin resistance involvement was suggested and may be related, at least in part, to their higher body weight. Besides IUED populations, evidence continues to mount showing that T2D is also more prevalent among subjects that were intrauterine growth restricted (IUGR). The first study to link low birth weight to increased T2D risk was conducted in a group of men born in Hertfordshire, UK, who were 64 years old at the time of the study. Those men who had the lowest birth weight were 6 times more likely to currently have either impaired glucose tolerance or T2D than those men who were heaviest at birth (Hales et al., 1991). These findings have been reproduced in over 40 populations worldwide, including many ethnic groups. In some cohorts where there is a high prevalence of maternal obesity, there is also increased risk of diabetes at the high-birth weight end of the spectrum (U-shaped curve for diabetes risk distribution). This is thought to reflect the increased risk of diabetes in the macrosomic offspring of women with gestational diabetes (Nathanielsz et al., 2007). Some of the strongest evidence in support of the role for environment in underlying the relationship between fetal growth and T2D had come from the study of twins. Studies of adult twins in Denmark revealed that, in both monozygotic (identical) and dizygotic (nonidentical) twin pairs who were discordant for T2D, the diabetic twin had a significantly lower birth weight than the normoglycemic cotwin (Poulsen et al., 1997). If it is assumed that the monozygotic twins are genetically identical, then, the difference in birth weight must be related to the fetal environment. These studies thus provide strong evidence for the importance of a nongenetic intrauterine factor in the development of type 2 diabetes in later life. Assessing the impact of maternal nutrition on the health of offspring in humans is difficult. Investigations involving individuals conceived during conditions of famine have provided direct evidence of the effects exerted by maternal nutrition during gestation and lactation on the overall health of the adult offspring. The Dutch famine, which occurred in the western part of The Netherlands at the end of World War II, was a short period of famine lasting from December 1944 to May 1945. Prior to the onset of the famine, the affected area of The Netherlands consisted of a reasonably well-nourished population. The occurrence of this abrupt famine therefore granted researchers a unique opportunity to retrospectively study the effect of maternal nutrition on offspring's glucose tolerance. Compared with individuals born the year before the famine, those who were in utero during the famine had higher plasma glucose levels, 2 h after a standard oral glucose tolerance test (Ravelli et al., 1998). that of offspring of fathers with young-onset T2D ( Singh et al., 2006). higher paternal transmission (Mitchell et al., 1993; Mc Carthy et al., 1996; Viswanathan et al., 1996; Frayling et al., 1999). The prospective Framingham Offspring Study in which all offspring and parents were formally tested for diabetes, demonstrated that the risk of impaired glucose tolerance or T2D was greater in offspring of mothers with early diabetes onset (before 50), suggesting the role of fetal environment (Meigs et al., 2000). However, the effects of fetal exposure to diabetes may be confounded by genetic factors. Mothers who had T2D before or during pregnancy have, by definition, early diabetes. Therefore, they may carry T2D susceptibility genes, which are transmitted to their offspring. To determine the role of the intrauterine diabetic environment per se, the prevalence of diabetes was compared in Pima nuclear families in which at least one sibling was born before and one after the mother was diagnosed with T2D. Offspring born after their mother displayed diabetes had a 4-fold higher risk of diabetes and a higher body mass index (BMI) than their full siblings born before their mother developed diabetes (Dabelea et al., 2000). These findings indicate that intrauterine exposure to a diabetic environment increases risk of obesity and T2D beyond that attributable to genetic factors, at least in Pima Indians. In Caucasians, offspring whose mothers had pregestational diabetes (type 1 or 2) or gestational diabetes had a higher frequency of impaired glucose tolerance (IGT). (Plagemann et al., 1997). Carriers of mutations in the MODY gene hepatocyte nuclear factor 1 alpha whose mothers had diabetes when they were in utero, were diagnosed with diabetes 8 years earlier than those who inherited the mutation from the father (Stride et al. 2002). To circumvent the confounding effect of genes linked to early onset T2D and transmitted by the pregnant T2D mother, the effect of fetal exposure to T1D was evaluated in adult offspring lacking T1D immunological markers. A 33% prevalence of IGT was reported in offspring of T1D mothers compared with none in offspring of T1D fathers (control group) (Sobngwi et al., 2003). Altogether, these findings suggest that fetal exposure to maternal diabetes is indeed associated with abnormal glucose homeostasis in offspring and may participate in the excess of maternal transmission in T2D. There are some studies related to the metabolic defects associated with fetal exposure to maternal diabetes in human offspring. The typical feature of infants of diabetic mothers is fetal macrosomia and high birth weight. A correlation between high birth weight and later impairment of glucose tolerance therefore is expected. Among Pima Indians high birth weight increases the risk of developing diabetes in later life (Pettitt, 1996). In the population of Malta, with a high prevalence of type-2 diabetes, woman with high birth weight have an increased risk of developing gestational diabetes. Birth weight is significantly higher in mothers with a family history of diabetes from the maternal side than from the paternal side, and than in those with no family history of diabetes (Savona-Ventura & Chircop, 2002). The relation between large babies and later impaired glucose tolerance in these studies appears to be related to the macrosomia of babies from diabetic mothers. Two studies reported that IGT in offspring exposed to intrauterine diabetic environment resulted from decreased insulin action based on finding of a high insulin-to-glucose ratio during oral glucose challenge (Plagemann et al., 1997; Silverman et al., 1995). However, fetal exposure to maternal T1D was not associated with reduced insulin sensitivity (minimal model) in young offspring (Hunter et al., 2004). Offspring of T2D mothers tended to have decreased insulin action, but were heavier compared with offspring of healthy mothers (Hunter et al., 2004). In adult Pima Indians with normal glucose tolerance and who had been exposed to an intrauterine diabetic environment, acute insulin response to iv glucose was higher paternal transmission (Mitchell et al., 1993; Mc Carthy et al., 1996; Viswanathan et al., 1996; Frayling et al., 1999). The prospective Framingham Offspring Study in which all offspring and parents were formally tested for diabetes, demonstrated that the risk of impaired glucose tolerance or T2D was greater in offspring of mothers with early diabetes onset (before 50), suggesting the role of fetal environment (Meigs et al., 2000). However, the effects of fetal exposure to diabetes may be confounded by genetic factors. Mothers who had T2D before or during pregnancy have, by definition, early diabetes. Therefore, they may carry T2D susceptibility genes, which are transmitted to their offspring. To determine the role of the intrauterine diabetic environment per se, the prevalence of diabetes was compared in Pima nuclear families in which at least one sibling was born before and one after the mother was diagnosed with T2D. Offspring born after their mother displayed diabetes had a 4-fold higher risk of diabetes and a higher body mass index (BMI) than their full siblings born before their mother developed diabetes (Dabelea et al., 2000). These findings indicate that intrauterine exposure to a diabetic environment increases risk of obesity and T2D beyond that attributable to genetic factors, at least in Pima Indians. In Caucasians, offspring whose mothers had pregestational diabetes (type 1 or 2) or gestational diabetes had a higher frequency of impaired glucose tolerance (IGT). (Plagemann et al., 1997). Carriers of mutations in the MODY gene hepatocyte nuclear factor 1 alpha whose mothers had diabetes when they were in utero, were diagnosed with diabetes 8 years earlier than those who inherited the mutation from the father (Stride et al. 2002). To circumvent the confounding effect of genes linked to early onset T2D and transmitted by the pregnant T2D mother, the effect of fetal exposure to T1D was evaluated in adult offspring lacking T1D immunological markers. A 33% prevalence of IGT was reported in offspring of T1D mothers compared with none in offspring of T1D fathers (control group) (Sobngwi et al., 2003). Altogether, these findings suggest that fetal exposure to maternal diabetes is indeed associated with abnormal glucose homeostasis in offspring and may participate in the excess There are some studies related to the metabolic defects associated with fetal exposure to maternal diabetes in human offspring. The typical feature of infants of diabetic mothers is fetal macrosomia and high birth weight. A correlation between high birth weight and later impairment of glucose tolerance therefore is expected. Among Pima Indians high birth weight increases the risk of developing diabetes in later life (Pettitt, 1996). In the population of Malta, with a high prevalence of type-2 diabetes, woman with high birth weight have an increased risk of developing gestational diabetes. Birth weight is significantly higher in mothers with a family history of diabetes from the maternal side than from the paternal side, and than in those with no family history of diabetes (Savona-Ventura & Chircop, 2002). The relation between large babies and later impaired glucose tolerance in these studies Two studies reported that IGT in offspring exposed to intrauterine diabetic environment resulted from decreased insulin action based on finding of a high insulin-to-glucose ratio during oral glucose challenge (Plagemann et al., 1997; Silverman et al., 1995). However, fetal exposure to maternal T1D was not associated with reduced insulin sensitivity (minimal model) in young offspring (Hunter et al., 2004). Offspring of T2D mothers tended to have decreased insulin action, but were heavier compared with offspring of healthy mothers (Hunter et al., 2004). In adult Pima Indians with normal glucose tolerance and who had been exposed to an intrauterine diabetic environment, acute insulin response to iv glucose was appears to be related to the macrosomia of babies from diabetic mothers. of maternal transmission in T2D. found reduced in those offspring whose mother was diabetic before pregnancy while it remained normal in those whose mother developed diabetes after pregnancy (Gautier et al., 2001). Body fat and insulin sensitivity (euglycemic hyperinsulinemic clamp) were similar in the two groups of subjects (Gautier et al., 2001). In the same study, acute insulin response was found reduced in offspring of parents (mother or father) with early onset of T2D (Gautier et al., 2001), suggesting that gene(s) linked to early onset diabetes is(are) associated with reduced insulin secretory response to glucose (Hanson et al., 1995). Offspring of T1D mothers had reduced insulin secretion, more pronounced in IGT subjects, but similar fat mass and insulin action compared with offspring of T1D fathers (Sobngwi et al., 2003). Also in non-diabetic offspring of mothers with young-onset T2D (diagnosed under age 50), betacell function (early insulin release after oral glucose) was found decreased as compared to that of offspring of fathers with young-onset T2D ( Singh et al., 2006). Therefore human studies suggest that insulin secretion defect participates to the abnormal glucose tolerance observed in adult offspring exposed to maternal diabetes during fetal life. Importantly, they showed that insulin secretion may be reduced even in normal glucosetolerant offspring. Nevertheless, in children and adolescent offspring, insulin resistance involvement was suggested and may be related, at least in part, to their higher body weight. Besides IUED populations, evidence continues to mount showing that T2D is also more prevalent among subjects that were intrauterine growth restricted (IUGR). The first study to link low birth weight to increased T2D risk was conducted in a group of men born in Hertfordshire, UK, who were 64 years old at the time of the study. Those men who had the lowest birth weight were 6 times more likely to currently have either impaired glucose tolerance or T2D than those men who were heaviest at birth (Hales et al., 1991). These findings have been reproduced in over 40 populations worldwide, including many ethnic groups. In some cohorts where there is a high prevalence of maternal obesity, there is also increased risk of diabetes at the high-birth weight end of the spectrum (U-shaped curve for diabetes risk distribution). This is thought to reflect the increased risk of diabetes in the macrosomic offspring of women with gestational diabetes (Nathanielsz et al., 2007). Some of the strongest evidence in support of the role for environment in underlying the relationship between fetal growth and T2D had come from the study of twins. Studies of adult twins in Denmark revealed that, in both monozygotic (identical) and dizygotic (nonidentical) twin pairs who were discordant for T2D, the diabetic twin had a significantly lower birth weight than the normoglycemic cotwin (Poulsen et al., 1997). If it is assumed that the monozygotic twins are genetically identical, then, the difference in birth weight must be related to the fetal environment. These studies thus provide strong evidence for the importance of a nongenetic intrauterine factor in the development of type 2 diabetes in later life. Assessing the impact of maternal nutrition on the health of offspring in humans is difficult. Investigations involving individuals conceived during conditions of famine have provided direct evidence of the effects exerted by maternal nutrition during gestation and lactation on the overall health of the adult offspring. The Dutch famine, which occurred in the western part of The Netherlands at the end of World War II, was a short period of famine lasting from December 1944 to May 1945. Prior to the onset of the famine, the affected area of The Netherlands consisted of a reasonably well-nourished population. The occurrence of this abrupt famine therefore granted researchers a unique opportunity to retrospectively study the effect of maternal nutrition on offspring's glucose tolerance. Compared with individuals born the year before the famine, those who were in utero during the famine had higher plasma glucose levels, 2 h after a standard oral glucose tolerance test (Ravelli et al., 1998). Impact and Mechanisms of Pancreatic Beta-Cell Mass may confer T2D risk. 3.1 IUED models Programming by Maternal Diabetes - Insight from Animal Model Studies 205 possible, a number of points support the hypothesis that early impairment in beta-cell development leads to fetal growth and predispose individuals to development of T2D later in life. Indeed, beta-cell mass deficit has been increasingly recognized as a central cause of T2D over the years (Meier, 2008). The importance of beta-cell mass for T2D risk has been further highlighted by the results of recent genome-wide scans that have linked the likelihood of developing T2D to genetic defects in insulin secretion (Saxena et al., 2007; Scott et al., 2007; Zeggini et al., 2007). Importantly, among these T2D loci, two of them (CDKAL1 and HHEX-IDE) which are associated with significant impairments in beta-cell function (Pascoe et al., 2008; Groenewoud et al., 2008), have been related to low birth weight (Freathy et al., 2009). In this case, the most likely explanation for the association between low birth weight and T2D risk seems to be a genetically determined defective development of betacells leading to insufficient insulin secretion. The intrauterine insulin deficiency may then impair fetal growth (Terauchi et al., 2000), while insufficient insulin secretion later in life 3. Perinatal risk factors for diabetes in later life - Animal studies developmental programming of glucose intolerance and T2D. Aerts et al., 1990; Oh et al., 1988; Gauguier et al., 1991). Thank to abundant studies, mostly in rodents in which the foetal environment can be manipulated, a substantial body of data now addresses the mechanisms involved in the In rat, maternal diabetes may be induced experimentally by streptozotocin (STZ) injection that selectively destroys beta-cells. Mild or severe diabetes ensue depending on the dose used. At birth, the progeny of mild diabetic mothers had normal weight or slight macrosomia and an enhanced percentage of pancreatic endocrine tissue due to hyperplasia and hypertrophy of the islet cells (Aerts et al., 1990 ; Reusens-Billen et al., 1984), leading to a higher beta-cell mass that was hyper-vascularized (Reusens & Remacle, 2001). The pancreatic insulin content and insulin secretion were raised in these fetuses (Kervran, et al., 1978). On the other hand, fetuses from severe diabetic dams were small at birth and had decreased pancreatic weight (Aerts et al., 1997). Their beta-cells were almost degranulated, leading to low pancreatic insulin content and low plasma insulin (Kervran, et al., 1978). Similar endocrine pancreas/beta-cell alterations with low beta-cell mass have been reported in fetuses from spontaneous diabetic BB rats (Verhaeghe et al., 1989) or spontaneous diabetic GK rats (Serradas et al., 1998; Miralles & Portha, 2001). The long-term consequences have been evaluated in the progeny of these models. Impaired glucose tolerance was observed in the offspring of mild STZ diabetic rats due to lower insulin secretion in response to glucose, while insulin resistance was reported in the offspring of the severe STZ diabetic mothers (Aerts & Van Assche, 2006; Han et al., 2007; Grill et al., 1991). Glucose tolerance was also impaired in offspring of normal mothers receiving glucose infusion during late gestation, and it was associated to decreased glucose-induced insulin secretion (Ktorza et al., 1990; The greatest difficulty in most animal models of diabetic pregnancy has been the attainment of a stable degree of mild hyperglycemia during gestation. Though useful, most techniques used to achieve models of diabetes in pregnancy have some drawbacks. Maternal glucose infusions limited to the last trimester of pregnancy result in hyperglycemia and However this association was not observed in the Leningrad Siege Study (Stanner et al., 1997). The inconsistent results might be due to differences in postnatal environmental life exposures. Although the Dutch population rapidly developed into a wealthy and rich population after the famine, the Leningrad people remained relatively poor. In a more recent study of a large sample of Chinese adults, a significant association was found between severe famine exposure during the fetal period and an increased risk of hyperglycemia in adulthood (Li et al., 2010). The association was stronger in subjects with a Western dietary pattern or higher economic status in adulthood. No consistent association was observed between famine exposure during childhood and hyperglycemia. These studies therefore provided direct evidence that poor maternal nutrition leads to increased susceptibility to T2D in the offspring. The mechanisms that underlie the association between poor maternal nutrition and T2D are unclear. Several studies in children and adults have shown that non-diabetic and prediabetic subjects with low birth weight are insulin resistant and, thus, predisposed to develop T2D (Barker, 2004; Valdez et al., 1994; Bhargawa et al., 2004; Li et al., 2001; Boney et al., 2005; Clausen et al., 1997; Flanagan et al., 2000). Adults born small for gestational age showed a significantly higher percentage of body fat (Jaquet et al., 2000) and their insulin sensitivity adjusted for either BMI or total fat mass, was markedly decreased. In fact in was first thought that the adverse effect of IUGR on glucose homeostasis is mediated through programming of the fetal endocrine pancreas (Hales et al., 1991), since IUGR infants have reduced plasma concentrations of insulin (Economides et al., 1989) and beta-cell numbers (Van Assche et al., 1977). However, several studies found no impact of low birth weight on insulin secretion in humans (Barker, 2004; Clausen et al., 1997; Flanagan et al., 2000). To address this discrepancy and since insulin sensitivity per se has a profound impact on insulin secretion, Jensen et al. (2002) measured both insulin secretion and insulin sensitivity in well-matched Caucasian glucose-tolerant men either IUGR or controls. To eliminate the major confounders, such as 'diabetes genes', none of the participants had a family history of diabetes, hypertension and ischemic heart disease. There was no difference between the groups with regard to current weight, body mass index (BMI), body composition and lipid profile. When adjusted for insulin sensitivity, insulin secretion was found reduced by 30%. However insulin sensitivity was found normal in the IUGR subjects. The authors hypothesized that defects in insulin secretion might precede defects in insulin action and that when IUGR individuals accumulate body fat, they develop insulin resistance (Jensen et al., 2002). This is entirely consistent with the concept that despite insulin resistance being a crucial component of T2D in humans, the failure of beta-cell function and growth determines progression to the diabetic phenotype (Weir et al., 2001). Thus, decreased substrate availability to the IUGR fetus caused by uteroplacental insufficiency might have permanently impaired pancreatic beta-cell growth by neogenesis and proliferation processes which take place mostly during the fetal-neonatal period. This is consistent with the observation that pancreatic tissue taken from human fetuses with severe IUGR is characterized by a reduction in endocrine cell mass (Van Assche & Aerts, 1979). However this has not been confirmed since no difference was also found between IUGR and control human fetuses in insulin-positive area or islet organization during the last two months of pregnancy (Beringue et al., 2002). To summarize, what are the more common factors that confer coincident risk of T2D and low birth weight? Although alterations in both insulin secretion and insulin action are possible, a number of points support the hypothesis that early impairment in beta-cell development leads to fetal growth and predispose individuals to development of T2D later in life. Indeed, beta-cell mass deficit has been increasingly recognized as a central cause of T2D over the years (Meier, 2008). The importance of beta-cell mass for T2D risk has been further highlighted by the results of recent genome-wide scans that have linked the likelihood of developing T2D to genetic defects in insulin secretion (Saxena et al., 2007; Scott et al., 2007; Zeggini et al., 2007). Importantly, among these T2D loci, two of them (CDKAL1 and HHEX-IDE) which are associated with significant impairments in beta-cell function (Pascoe et al., 2008; Groenewoud et al., 2008), have been related to low birth weight (Freathy et al., 2009). In this case, the most likely explanation for the association between low birth weight and T2D risk seems to be a genetically determined defective development of betacells leading to insufficient insulin secretion. The intrauterine insulin deficiency may then impair fetal growth (Terauchi et al., 2000), while insufficient insulin secretion later in life may confer T2D risk. #### 3. Perinatal risk factors for diabetes in later life - Animal studies Thank to abundant studies, mostly in rodents in which the foetal environment can be manipulated, a substantial body of data now addresses the mechanisms involved in the developmental programming of glucose intolerance and T2D. #### 3.1 IUED models 204 Gestational Diabetes However this association was not observed in the Leningrad Siege Study (Stanner et al., 1997). The inconsistent results might be due to differences in postnatal environmental life exposures. Although the Dutch population rapidly developed into a wealthy and rich population after the famine, the Leningrad people remained relatively poor. In a more recent study of a large sample of Chinese adults, a significant association was found between severe famine exposure during the fetal period and an increased risk of hyperglycemia in adulthood (Li et al., 2010). The association was stronger in subjects with a Western dietary pattern or higher economic status in adulthood. No consistent association was observed between famine exposure during childhood and hyperglycemia. These studies therefore provided direct evidence that poor maternal nutrition leads to increased The mechanisms that underlie the association between poor maternal nutrition and T2D are unclear. Several studies in children and adults have shown that non-diabetic and prediabetic subjects with low birth weight are insulin resistant and, thus, predisposed to develop T2D (Barker, 2004; Valdez et al., 1994; Bhargawa et al., 2004; Li et al., 2001; Boney et al., 2005; Clausen et al., 1997; Flanagan et al., 2000). Adults born small for gestational age showed a significantly higher percentage of body fat (Jaquet et al., 2000) and their insulin sensitivity adjusted for either BMI or total fat mass, was markedly decreased. In fact in was first thought that the adverse effect of IUGR on glucose homeostasis is mediated through programming of the fetal endocrine pancreas (Hales et al., 1991), since IUGR infants have reduced plasma concentrations of insulin (Economides et al., 1989) and beta-cell numbers (Van Assche et al., 1977). However, several studies found no impact of low birth weight on insulin secretion in humans (Barker, 2004; Clausen et al., 1997; Flanagan et al., 2000). To address this discrepancy and since insulin sensitivity per se has a profound impact on insulin secretion, Jensen et al. (2002) measured both insulin secretion and insulin sensitivity in well-matched Caucasian glucose-tolerant men either IUGR or controls. To eliminate the major confounders, such as 'diabetes genes', none of the participants had a family history of diabetes, hypertension and ischemic heart disease. There was no difference between the groups with regard to current weight, body mass index (BMI), body composition and lipid profile. When adjusted for insulin sensitivity, insulin secretion was found reduced by 30%. However insulin sensitivity was found normal in the IUGR subjects. The authors hypothesized that defects in insulin secretion might precede defects in insulin action and that when IUGR individuals accumulate body fat, they develop insulin resistance (Jensen et al., 2002). This is entirely consistent with the concept that despite insulin resistance being a crucial component of T2D in humans, the failure of beta-cell function and growth determines progression to the diabetic phenotype (Weir et al., 2001). Thus, decreased substrate availability to the IUGR fetus caused by uteroplacental insufficiency might have permanently impaired pancreatic beta-cell growth by neogenesis and proliferation processes which take place mostly during the fetal-neonatal period. This is consistent with the observation that pancreatic tissue taken from human fetuses with severe IUGR is characterized by a reduction in endocrine cell mass (Van Assche & Aerts, 1979). However this has not been confirmed since no difference was also found between IUGR and control human fetuses in insulin-positive area or islet organization during the last two months of To summarize, what are the more common factors that confer coincident risk of T2D and low birth weight? Although alterations in both insulin secretion and insulin action are susceptibility to T2D in the offspring. pregnancy (Beringue et al., 2002). In rat, maternal diabetes may be induced experimentally by streptozotocin (STZ) injection that selectively destroys beta-cells. Mild or severe diabetes ensue depending on the dose used. At birth, the progeny of mild diabetic mothers had normal weight or slight macrosomia and an enhanced percentage of pancreatic endocrine tissue due to hyperplasia and hypertrophy of the islet cells (Aerts et al., 1990 ; Reusens-Billen et al., 1984), leading to a higher beta-cell mass that was hyper-vascularized (Reusens & Remacle, 2001). The pancreatic insulin content and insulin secretion were raised in these fetuses (Kervran, et al., 1978). On the other hand, fetuses from severe diabetic dams were small at birth and had decreased pancreatic weight (Aerts et al., 1997). Their beta-cells were almost degranulated, leading to low pancreatic insulin content and low plasma insulin (Kervran, et al., 1978). Similar endocrine pancreas/beta-cell alterations with low beta-cell mass have been reported in fetuses from spontaneous diabetic BB rats (Verhaeghe et al., 1989) or spontaneous diabetic GK rats (Serradas et al., 1998; Miralles & Portha, 2001). The long-term consequences have been evaluated in the progeny of these models. Impaired glucose tolerance was observed in the offspring of mild STZ diabetic rats due to lower insulin secretion in response to glucose, while insulin resistance was reported in the offspring of the severe STZ diabetic mothers (Aerts & Van Assche, 2006; Han et al., 2007; Grill et al., 1991). Glucose tolerance was also impaired in offspring of normal mothers receiving glucose infusion during late gestation, and it was associated to decreased glucose-induced insulin secretion (Ktorza et al., 1990; Aerts et al., 1990; Oh et al., 1988; Gauguier et al., 1991). The greatest difficulty in most animal models of diabetic pregnancy has been the attainment of a stable degree of mild hyperglycemia during gestation. Though useful, most techniques used to achieve models of diabetes in pregnancy have some drawbacks. Maternal glucose infusions limited to the last trimester of pregnancy result in hyperglycemia and Impact and Mechanisms of Pancreatic Beta-Cell Mass (Holemans et al. 2003; Stoffers et al., 2003). 3.3 IUEO models Programming by Maternal Diabetes - Insight from Animal Model Studies 207 male offspring undergo an age-dependent loss in glucose tolerance, such that by 17 months of age they develop T2D and insulin resistance (Petry et al., 2001). Female offspring only develop hyperinsulinemia and impaired glucose tolerance at a much later age (21 months) (Fernandez-Twinn et al., 2005). Studies in this model have also demonstrated reductions in beta-cell mass (Snoeck et al., 1990;), skeletal muscle mass (Desai et al., 1996), central adipose deposit weights (Shepherd et al., 1997; Ozanne et al., 2000) and insulin signalling defects in muscle, adipocytes and liver (Ozanne et al., 1996; Ozanne et al., 2000; Ozanne et al., 2005). This IUGR model has also been associated with the development of hypertension with the kidney and the renin–angiotensin system as playing a role (Langley-Evans et al., 2003). Administration of either dexamethasone or carbenoxolone (to inhibit 11 betahydroxysteroid dehydrogenase type 2) to normal pregnant rats also causes fetal growth retardation and the adult offspring are hypertensive and hyperglycemic, with hyperactive hypothalamic-pituitary-adrenal axis (Seckl, 2004). Fetal growth retardation may also result from experimental uteroplacental insufficiency (UPI). Fetal UPI rats have decreased levels of glucose, insulin, IGF1, amino acids and oxygen (Ogata et al., 1986; Simmons et al., 1992; Unterman et al., 1990). UPI offspring develop diabetes in later life (Simmons et al. 2001; Boloker et al., 2002) with a phenotype that is similar to that observed in T2D humans with alterations in insulin secretion and action and a failure of beta-cell function and growth There are several reports on the consequences of a high fat diet (during gestation only or both gestation and lactation) on the adult progeny. High fat diet consumption by female rats malprograms the male offspring for glucose intolerance and increased body weight in adulthood (Srinivasan et al., 2006). Some of the observed consequences include reduced whole body insulin sensitivity, impaired insulin secretion and changes in the structure of pancreas (Guo & Jen, 1995; Taylor et al., 2005), defective mesenteric artery endothelial function (Khan et al., 2005), hypertension (Khan et al., 2003; Langley-Evans et al., 1996), alterations in renal functions (Armitage et al., 2005), increased body adiposity (Guo & Jen, 1995; Khan et al., 2005), deranged blood lipid profile (Guo & Jen, 1995; Karnik et al., 1989; Khan et al., 2003), hyperleptinemia (Taylor et al., 2005), and proatherogenic lesions (Palinski et al., 2001). There are not many reports on fetal islet adaptations due to a high fat dietary modification in the dam. Cerf et al. (2005) demonstrated that feeding female rats with a high fat diet throughout gestation resulted in significant decreases in beta-cell volume and number resulting in hyperglycemia in 1-day-old newborn rat pups without changes in serum insulin concentrations. However, the report of fetal hyperinsulinemia in the high fat Also male mice whose mothers consumed a high fat diet were heavier, glucose intolerant and insulin resistant, and produced second-generation offspring who were insulin resistant, although not obese (Dunn & Bale, 2009). Whether this is a consequence of paternal in utero exposure or their adult sequelaes of obesity and diabetes, is unclear. It was recently reported that chronic high fat diet consumption in father rats induced increased body weight, adiposity, impaired glucose tolerance and insulin sensitivity in their offspring (Ng et al., 2010). Relative to controls, their female offspring had an early onset of impaired insulin secretion and glucose tolerance that worsened with time, and normal adiposity. Among the differentially expressed islet genes, hypomethylation of the Il13ra2 gene was demonstrated. term rat fetus (Srinivasan et al., 2006) is not consistent with this finding. hyperinsulinemia, and do not mimic the relative insulin deficiency of gestational diabetes (Bihoreau et al., 1986). The multiple lipid and protein abnormalities associated with diabetes may be as important in the induction of fetal abnormalities as hyperglycemia, but they are not replicated by the maternal glucose infusion model. A concern of studies using STZ during pregnancy is the possibility that the toxin might cross the placenta and be directly harmful to the fetal pancreas and other fetal tissues, and thus make any analysis of the longterm effects of hyperglycemia in utero difficult (Ryan et al., 1995). The problem may be circumvented by giving STZ to female neonates who will later become pregnant: this will result in moderate gestational hyperglycemia (Triadou et al., 1982). Finally it must be recognized that none of the previously mentioned models will serve directly as a model of human gestational diabetes. An ideal animal model to test the isolated impact of diabetic pregnancy would enter the pregnancy in a euglycemic state, become exposed to hyperglycaemic during whole pregnancy and return postpartum to normoglycemic environment. Such a model also would allow study of the long-term effects of diabetes independent of any genetic influence. It was recently proposed that the pregnant GK rat being transferred normal Wistar (W) rat embryo represents a more relevant paradigm in such a perspective (Gill-Randall et al., 2004). Using the GK/Par rat we have transferred W rat oocytes to diabetic GK/Par females and at their birth the W neonates were suckled by non-diabetic W foster mothers. Under these unique conditions, we have found that maternal diabetes negatively imprints the growth of a genetically normal (Wistar) beta-cell mass in a way as the insult is still present later at adult age as a decreased beta-cell population (Chavey et al., 2008; Portha et al., 2009). #### 3.2 IUGR models Not only maternal diabetes but also intrauterine undernutrition induced by several means such as protein or calorie restriction, or alteration in the availability of the nutrients by placental insufficiency alter early islet development and provoke lasting consequences in rodents. Global restrictions (to 40-50% of normal intake) in the last week of rat pregnancy results in low birth weight offspring with decreased beta-cell mass. Although these animals can regain their body and pancreatic weights upon normal postnatal feeding, they still demonstrate a reduced beta-cell mass and insulin content in adulthood (Garofano et al., 1997; Bertin et al., 2002). Extending this level of nutrient restriction during suckling results in a permanent reduction of beta-cell mass (Martin et al., 1997; Garofano et al., 1998) and subsequent agedependent loss of glucose tolerance in the offspring (Garofano et al., 1999). Underfeeding the rat mothers during the first two trimesters of gestation exerts no adverse effect upon insulin secretion and insulin action in the adult male offspring (Portha et al., 1995). The maternal protein restriction (5-8% as compared to 20% in normal diet) model has been one of the most extensively studied models of IUGR. The low-protein-fed mothers give birth to growth-restricted offspring (Snoeck et al., 1990; Dahri et al., 1991; Langley-Evans et al., 1998; Desai et al., 1996; Fernandez-Twinn et al., 2005), and when suckled by their mothers maintained on the same low-protein diet, they remain permanently growth restricted, despite being weaned on a normal diet (Desai et al., 1996). Reduced placental weight and endocrine and metabolic abnormalities are also observed (Dahri et al., 1991; Fernandez-Twinn et al., 2003; Ozanne et al., 1998). Despite young offspring of low-protein-fed dams demonstrating improved glucose tolerance (Ozanne et al., 1998; Shepherd et al., 1997), the male offspring undergo an age-dependent loss in glucose tolerance, such that by 17 months of age they develop T2D and insulin resistance (Petry et al., 2001). Female offspring only develop hyperinsulinemia and impaired glucose tolerance at a much later age (21 months) (Fernandez-Twinn et al., 2005). Studies in this model have also demonstrated reductions in beta-cell mass (Snoeck et al., 1990;), skeletal muscle mass (Desai et al., 1996), central adipose deposit weights (Shepherd et al., 1997; Ozanne et al., 2000) and insulin signalling defects in muscle, adipocytes and liver (Ozanne et al., 1996; Ozanne et al., 2000; Ozanne et al., 2005). This IUGR model has also been associated with the development of hypertension with the kidney and the renin–angiotensin system as playing a role (Langley-Evans et al., 2003). Administration of either dexamethasone or carbenoxolone (to inhibit 11 betahydroxysteroid dehydrogenase type 2) to normal pregnant rats also causes fetal growth retardation and the adult offspring are hypertensive and hyperglycemic, with hyperactive hypothalamic-pituitary-adrenal axis (Seckl, 2004). Fetal growth retardation may also result from experimental uteroplacental insufficiency (UPI). Fetal UPI rats have decreased levels of glucose, insulin, IGF1, amino acids and oxygen (Ogata et al., 1986; Simmons et al., 1992; Unterman et al., 1990). UPI offspring develop diabetes in later life (Simmons et al. 2001; Boloker et al., 2002) with a phenotype that is similar to that observed in T2D humans with alterations in insulin secretion and action and a failure of beta-cell function and growth (Holemans et al. 2003; Stoffers et al., 2003). #### 3.3 IUEO models 206 Gestational Diabetes hyperinsulinemia, and do not mimic the relative insulin deficiency of gestational diabetes (Bihoreau et al., 1986). The multiple lipid and protein abnormalities associated with diabetes may be as important in the induction of fetal abnormalities as hyperglycemia, but they are not replicated by the maternal glucose infusion model. A concern of studies using STZ during pregnancy is the possibility that the toxin might cross the placenta and be directly harmful to the fetal pancreas and other fetal tissues, and thus make any analysis of the longterm effects of hyperglycemia in utero difficult (Ryan et al., 1995). The problem may be circumvented by giving STZ to female neonates who will later become pregnant: this will result in moderate gestational hyperglycemia (Triadou et al., 1982). Finally it must be recognized that none of the previously mentioned models will serve directly as a model of An ideal animal model to test the isolated impact of diabetic pregnancy would enter the pregnancy in a euglycemic state, become exposed to hyperglycaemic during whole pregnancy and return postpartum to normoglycemic environment. Such a model also would allow study of the long-term effects of diabetes independent of any genetic influence. It was recently proposed that the pregnant GK rat being transferred normal Wistar (W) rat embryo represents a more relevant paradigm in such a perspective (Gill-Randall et al., 2004). Using the GK/Par rat we have transferred W rat oocytes to diabetic GK/Par females and at their birth the W neonates were suckled by non-diabetic W foster mothers. Under these unique conditions, we have found that maternal diabetes negatively imprints the growth of a genetically normal (Wistar) beta-cell mass in a way as the insult is still present later at adult age as a decreased beta-cell population (Chavey et al., 2008; Portha et al., 2009). Not only maternal diabetes but also intrauterine undernutrition induced by several means such as protein or calorie restriction, or alteration in the availability of the nutrients by placental insufficiency alter early islet development and provoke lasting consequences in Global restrictions (to 40-50% of normal intake) in the last week of rat pregnancy results in low birth weight offspring with decreased beta-cell mass. Although these animals can regain their body and pancreatic weights upon normal postnatal feeding, they still demonstrate a reduced beta-cell mass and insulin content in adulthood (Garofano et al., 1997; Bertin et al., 2002). Extending this level of nutrient restriction during suckling results in a permanent reduction of beta-cell mass (Martin et al., 1997; Garofano et al., 1998) and subsequent agedependent loss of glucose tolerance in the offspring (Garofano et al., 1999). Underfeeding the rat mothers during the first two trimesters of gestation exerts no adverse effect upon The maternal protein restriction (5-8% as compared to 20% in normal diet) model has been one of the most extensively studied models of IUGR. The low-protein-fed mothers give birth to growth-restricted offspring (Snoeck et al., 1990; Dahri et al., 1991; Langley-Evans et al., 1998; Desai et al., 1996; Fernandez-Twinn et al., 2005), and when suckled by their mothers maintained on the same low-protein diet, they remain permanently growth restricted, despite being weaned on a normal diet (Desai et al., 1996). Reduced placental weight and endocrine and metabolic abnormalities are also observed (Dahri et al., 1991; Fernandez-Twinn et al., 2003; Ozanne et al., 1998). Despite young offspring of low-protein-fed dams demonstrating improved glucose tolerance (Ozanne et al., 1998; Shepherd et al., 1997), the insulin secretion and insulin action in the adult male offspring (Portha et al., 1995). human gestational diabetes. 3.2 IUGR models rodents. There are several reports on the consequences of a high fat diet (during gestation only or both gestation and lactation) on the adult progeny. High fat diet consumption by female rats malprograms the male offspring for glucose intolerance and increased body weight in adulthood (Srinivasan et al., 2006). Some of the observed consequences include reduced whole body insulin sensitivity, impaired insulin secretion and changes in the structure of pancreas (Guo & Jen, 1995; Taylor et al., 2005), defective mesenteric artery endothelial function (Khan et al., 2005), hypertension (Khan et al., 2003; Langley-Evans et al., 1996), alterations in renal functions (Armitage et al., 2005), increased body adiposity (Guo & Jen, 1995; Khan et al., 2005), deranged blood lipid profile (Guo & Jen, 1995; Karnik et al., 1989; Khan et al., 2003), hyperleptinemia (Taylor et al., 2005), and proatherogenic lesions (Palinski et al., 2001). There are not many reports on fetal islet adaptations due to a high fat dietary modification in the dam. Cerf et al. (2005) demonstrated that feeding female rats with a high fat diet throughout gestation resulted in significant decreases in beta-cell volume and number resulting in hyperglycemia in 1-day-old newborn rat pups without changes in serum insulin concentrations. However, the report of fetal hyperinsulinemia in the high fat term rat fetus (Srinivasan et al., 2006) is not consistent with this finding. Also male mice whose mothers consumed a high fat diet were heavier, glucose intolerant and insulin resistant, and produced second-generation offspring who were insulin resistant, although not obese (Dunn & Bale, 2009). Whether this is a consequence of paternal in utero exposure or their adult sequelaes of obesity and diabetes, is unclear. It was recently reported that chronic high fat diet consumption in father rats induced increased body weight, adiposity, impaired glucose tolerance and insulin sensitivity in their offspring (Ng et al., 2010). Relative to controls, their female offspring had an early onset of impaired insulin secretion and glucose tolerance that worsened with time, and normal adiposity. Among the differentially expressed islet genes, hypomethylation of the Il13ra2 gene was demonstrated. Impact and Mechanisms of Pancreatic Beta-Cell Mass programmed. 4.1.2 Post-implantation 4.1.3 Postnatal versus prenatal intolerance, impaired insulin secretion). Programming by Maternal Diabetes - Insight from Animal Model Studies 209 protein diet displayed significantly reduced cell numbers, within the inner cell mass and trophectoderm lineages, apparently induced by a slower rate of cellular proliferation. The low protein diet significantly reduced insulin and essential amino acid levels, and increased glucose levels within maternal serum by day 4 of development. These data indicate that the mildly hyperglycemic and amino acid-depleted maternal environment generated by undernutrition may act as an early mechanism of programming and initiate conditions of 'metabolic stress', restricting early embryonic proliferation and the generation of appropriately sized stem-cell lineages. In chemically or genetically obtained rat diabetes models in which maternal serum insulin depletion and hyperglycemia are induced, proliferation of inner cell mass or total cell numbers within blastocysts is inhibited (Lea et al., 1996; Pampfer et al., 1997). Therefore the preimplantation embryo is particularly sensitive to metabolic modifications that may have programming consequences (Reik et al., 1993; Dean et al., 1998), and one possibility is that the preimplantation embryo itself is Embryo transfer experiments may also help to dissociate the impact of the maternal environment into early (pre-implantation) versus late gestation (postimplantation). We recently found that embryos (blastocysts) from a nondiabetic Wistar strain, placed into a diabetic GK/Par uterus, develop a reduced beta-cell mass which remains low on the long term (Chavey et al., 2008). Data with rat models of prenatal undernutrition (Dumortier et al., 2007) also illustrate that low-energy and low-protein diets that reduce the development of the beta-cell mass in both cases, act at different critical time-windows. The beta-cell mass is deficient in the low-energy pancreas because this diet reduces neogenesis, probably because of high glucocorticoid levels, rather than by impairing vascularisation and proliferation. Early gestation is thus a very sensitive period in this model. By contrast, pancreatic alterations take place at a later fetal stage in the low-protein model and the beta-cell mass is deficient in this case because this diet reduces beta-cell vascularisation and proliferation Further support for the crucial impact of prenatal nutritional environment is the recent report that prenatal nutrient restriction in both male and female rats led to an inappropriate postnatal beta-cell mass formation attributed to a decrease in the rate of beta-cell replication and beta-cell neogenesis (Matveyenko et al., 2010). In contrast, male and female rats exposed to postnatal nutrient restriction alone (with normal prenatal nutrient exposure) were characterized by decreased pancreatic and body weights, but a weight-adjusted beta-cell mass higher than control animals (Matveyenko et al., 2010). Another illustration is offered by observations in normal rat pups reared artificially on a high carbohydrate milk formula (Patel & Srinivasan, 2002): such alteration of nutrition during the suckling period only, induced persistent adaptation of energy metabolism in adulthood (obesity, glucose 4.2 Molecular mechanisms mediating the perinatal beta-cell adaptive response Molecular mechanisms responsible for impaired beta-cell mass formation after IUGR have come under investigation. First, it has been proposed that IUGR can result in a reduction of without altering beta-cell differentiation (Dumortier et al., 2007). This is a proof of concept that paternal high-fat-diet exposure programs beta-cell dysfunction in rat F1 female offspring. This is the first report in mammals of non-genetic, intergenerational transmission of metabolic sequelae of a high fat diet from father to offspring (Ng et al., 2010). Among the many types of maternal metabolic stress used to produce IUGR, hypercholesterolemia combined to high fat diet was recently added since feeding LDL receptor null (LDLR-/-) mice a high fat resulted in litters with significant growth retardation. The LDLR-/- high fat diet offspring developed significantly larger atherosclerotic lesions by 90 days compared with chow diet offspring (Bhasin et al., 2009). Importantly, maternal hypoaminoacidemia proved to be an important antecedent in this hypercholesterolemic IUGR mouse (Bhasin et al., 2009) as in a protein-deficient IUGR mouse model (Bhasin et al., 2009) and a IUED rat model (Aerts et al., 1989). An important between these mechanisms may contribute to adult glucose intolerance onset, obesity, and atherosclerosis. In this study beta-cell mass was not investigated. To sum-up, it turns to be manifest that despite differences in the type, timing, and duration of intrauterine insult, most animal models of IUED, IUEO or IUGR have outcomes of impaired glucose tolerance or T2D, similar to IUED, IUEO or IUGR humans. #### 4. Various early life stressors, the same target: The developing beta-cell mass As abundantly illustrated in animal models, many early life stressors such as maternal hyperglycaemia, undernutrition, overnutrition, hypercholesterolemia, corticosteroid therapy, uteroplacental insufficiency, or hypoxia, trigger a beta-cell mass adaptive response in the fetus. #### 4.1 Critical windows for beta-cell adaptive response to early life stressors The development of the endocrine pancreas starts from a pool of common precursor cells that become progressively committed to the endocrine lineage under the control of a hierarchical network of transcription factors. During late fetal and early postnatal life , the beta cell mass is determined by the recruitment of undifferentiated precursors, as well as the replication and apoptosis rates of the beta cells. Obviously, any disturbance of the environment of the endocrine cells at a specific developmental time-point, as it occurs in a perturbed intra-uterine milieu, may modify the balance of controlling factors, thereby contributing to an adaptive beta-cell growth response which is metabolically appropriate on the short term. However this adaptive response may turn to be detrimental if maintained on the long term, as it may foster beta-cell failure and diabetes later in life. We are largely ignorant of when programming may be initiated during development. #### 4.1.1 Pre-implantation An early onset for programming was indicated, as maternal low protein diet during only the preimplantation period of rat development (0-4 days after mating), before return to control diet for the remainder of the gestation, induced blastocyst abnormalities and programming of postnatal growth rate and hypertension (Kwong et al., 2000). More specifically it was shown that preimplantation embryos collected from dams after 0-4 days of maternal low protein diet displayed significantly reduced cell numbers, within the inner cell mass and trophectoderm lineages, apparently induced by a slower rate of cellular proliferation. The low protein diet significantly reduced insulin and essential amino acid levels, and increased glucose levels within maternal serum by day 4 of development. These data indicate that the mildly hyperglycemic and amino acid-depleted maternal environment generated by undernutrition may act as an early mechanism of programming and initiate conditions of 'metabolic stress', restricting early embryonic proliferation and the generation of appropriately sized stem-cell lineages. In chemically or genetically obtained rat diabetes models in which maternal serum insulin depletion and hyperglycemia are induced, proliferation of inner cell mass or total cell numbers within blastocysts is inhibited (Lea et al., 1996; Pampfer et al., 1997). Therefore the preimplantation embryo is particularly sensitive to metabolic modifications that may have programming consequences (Reik et al., 1993; Dean et al., 1998), and one possibility is that the preimplantation embryo itself is programmed. #### 4.1.2 Post-implantation 208 Gestational Diabetes This is a proof of concept that paternal high-fat-diet exposure programs beta-cell dysfunction in rat F1 female offspring. This is the first report in mammals of non-genetic, intergenerational transmission of metabolic sequelae of a high fat diet from father to Among the many types of maternal metabolic stress used to produce IUGR, hypercholesterolemia combined to high fat diet was recently added since feeding LDL receptor null (LDLR-/-) mice a high fat resulted in litters with significant growth retardation. The LDLR-/- high fat diet offspring developed significantly larger atherosclerotic lesions by 90 days compared with chow diet offspring (Bhasin et al., 2009). Importantly, maternal hypoaminoacidemia proved to be an important antecedent in this hypercholesterolemic IUGR mouse (Bhasin et al., 2009) as in a protein-deficient IUGR mouse model (Bhasin et al., 2009) and a IUED rat model (Aerts et al., 1989). An important between these mechanisms may contribute to adult glucose intolerance onset, obesity, and To sum-up, it turns to be manifest that despite differences in the type, timing, and duration of intrauterine insult, most animal models of IUED, IUEO or IUGR have outcomes of As abundantly illustrated in animal models, many early life stressors such as maternal hyperglycaemia, undernutrition, overnutrition, hypercholesterolemia, corticosteroid therapy, uteroplacental insufficiency, or hypoxia, trigger a beta-cell mass adaptive response The development of the endocrine pancreas starts from a pool of common precursor cells that become progressively committed to the endocrine lineage under the control of a hierarchical network of transcription factors. During late fetal and early postnatal life , the beta cell mass is determined by the recruitment of undifferentiated precursors, as well as the replication and apoptosis rates of the beta cells. Obviously, any disturbance of the environment of the endocrine cells at a specific developmental time-point, as it occurs in a perturbed intra-uterine milieu, may modify the balance of controlling factors, thereby contributing to an adaptive beta-cell growth response which is metabolically appropriate on the short term. However this adaptive response may turn to be detrimental if maintained on the long term, as it may foster beta-cell failure and diabetes later in life. We are largely An early onset for programming was indicated, as maternal low protein diet during only the preimplantation period of rat development (0-4 days after mating), before return to control diet for the remainder of the gestation, induced blastocyst abnormalities and programming of postnatal growth rate and hypertension (Kwong et al., 2000). More specifically it was shown that preimplantation embryos collected from dams after 0-4 days of maternal low atherosclerosis. In this study beta-cell mass was not investigated. impaired glucose tolerance or T2D, similar to IUED, IUEO or IUGR humans. 4. Various early life stressors, the same target: The developing 4.1 Critical windows for beta-cell adaptive response to early life stressors ignorant of when programming may be initiated during development. offspring (Ng et al., 2010). beta-cell mass 4.1.1 Pre-implantation in the fetus. Embryo transfer experiments may also help to dissociate the impact of the maternal environment into early (pre-implantation) versus late gestation (postimplantation). We recently found that embryos (blastocysts) from a nondiabetic Wistar strain, placed into a diabetic GK/Par uterus, develop a reduced beta-cell mass which remains low on the long term (Chavey et al., 2008). Data with rat models of prenatal undernutrition (Dumortier et al., 2007) also illustrate that low-energy and low-protein diets that reduce the development of the beta-cell mass in both cases, act at different critical time-windows. The beta-cell mass is deficient in the low-energy pancreas because this diet reduces neogenesis, probably because of high glucocorticoid levels, rather than by impairing vascularisation and proliferation. Early gestation is thus a very sensitive period in this model. By contrast, pancreatic alterations take place at a later fetal stage in the low-protein model and the beta-cell mass is deficient in this case because this diet reduces beta-cell vascularisation and proliferation without altering beta-cell differentiation (Dumortier et al., 2007). #### 4.1.3 Postnatal versus prenatal Further support for the crucial impact of prenatal nutritional environment is the recent report that prenatal nutrient restriction in both male and female rats led to an inappropriate postnatal beta-cell mass formation attributed to a decrease in the rate of beta-cell replication and beta-cell neogenesis (Matveyenko et al., 2010). In contrast, male and female rats exposed to postnatal nutrient restriction alone (with normal prenatal nutrient exposure) were characterized by decreased pancreatic and body weights, but a weight-adjusted beta-cell mass higher than control animals (Matveyenko et al., 2010). Another illustration is offered by observations in normal rat pups reared artificially on a high carbohydrate milk formula (Patel & Srinivasan, 2002): such alteration of nutrition during the suckling period only, induced persistent adaptation of energy metabolism in adulthood (obesity, glucose intolerance, impaired insulin secretion). #### 4.2 Molecular mechanisms mediating the perinatal beta-cell adaptive response Molecular mechanisms responsible for impaired beta-cell mass formation after IUGR have come under investigation. First, it has been proposed that IUGR can result in a reduction of Impact and Mechanisms of Pancreatic Beta-Cell Mass and telomere length in islets (Tarry-Adkins et al., 2009). simple change of Igf2 gene imprinting in the GK rat. Programming by Maternal Diabetes - Insight from Animal Model Studies 211 regulation and gene expression (Chen et al., 2007). While DNA is being targeted throughout by ROS, there are particular regions that are known to be more sensitive to ROS-mediated damage, for example telomeres. Telomeres comprise GC-rich repeats and are found at the ends of each chromosome. They are known to shorten with each cellular division and, hence, can act as a mitotic clock, registering the number of replicative divisions to have taken place within the cell. Investigations using a low protein IUGR model have indeed reported a decrease in longevity in the offspring (Jennings et al., 1999; Chen et al., 2009) accompanied by reduction in mitochondrial antioxidant defences (Tarry-Adkins et al., 2009) Pancreatic islet development has been shown to be influenced by a number of growth factors including the insulin-like growth factors, IGF-I and IGF-II whose expression in utero is regulated by nutrient and hormone concentrations. IUGR modify expression of both IGF genes in a variety of fetal tissues. In a low protein IUGR rat model with a decreased beta-cell mass and beta-cell replication and an increased rate of beta-cell apoptosis, gene expression for IGF-II but not IGF-I was found reduced in the fetal pancreas and this was (Petrik et al., 1999). In a different IUGR model with more severe global food restriction which induced hyperinsulinemia and an increase in beta-cell mass in their fetuses (Alvarez et al., 1997), the fetal phenotype was associated with an increase in pancreatic IGF-I expression, islet IGF-1R (Martn et al., 2005) and IRS-2 (Fernandez et al., 2007). In the fetal GK/Par rat exposed to mild hyperglycemia during gestation (a model of IUED), data from our group suggest that the beta-cell deficit (reduced by more than 50%) starts as early as fetal age 16 days E16 and reflects decreased beta-cell proliferation, a limitation of beta-cell neogenesis from precursors and increased apoptosis of both beta-cells and their precursors (Calderari et al., 2007). Notably, Pdx1 and Ngn3 expression were decreased on E18 but normally expressed on E13 (Calderari et al., 2007). Defective signalling through the IGF2/IGF1-R pathway may represents the primary instrumental anomaly since IGF2 and IGF1-R protein expressions are already decreased within the GK/Par pancreatic rudiment at E13, at a time when beta-cell mass (first wave of beta cell expansion) is in fact normal (Miralles & Portha, 2001). Low levels of pancreatic IGF2, associated with beta-cell mass deficiency, are maintained thereafter within the fetal pancreas (Serradas et al., 2002). Crossbreeding protocols between non-diabetic W and diabetic GK rats showed that in late gestation (E18), pancreatic IGF2 protein expression was as low in GKmother/GKfather and Wmother/GKfather crosses than in GKmother/GK father crosses (Serradas et al., 2002). These findings rather support the hypothesis that the pancreatic IGF2 anomaly in the GK diabetic model is linked to a genetic determinism. This view is also consistent with the results of genetic analyses that linked a locus containing the gene encoding IGF2 to diabetes in the GK rat (Gauguier et al., 1996). The Igf2 gene is subjected to paternal genomic imprinting. However, because the Igf2 expression is similarly affected in fetuses, regardless of whether the father is W or GK (Serradas et al., 2002), we cannot conclude to a Finally, studies have demonstrated that the maintenance of methylated histone H3 Lys4 by Set7/9 a member of the SET methyltransferase family, is crucial to Pdx1 activity in beta-cell lines (Chakrabarti et al., 2003; Francis et al., 2005; Deering et al., 2009). This led to the hypothesis that Set7/9 may represent a novel chromatin-modifying protein that functions in part through its recruitment to target genes by cell-specific transcription factors such as Pdx1. Since, a role of histone methyl transferases, particularly set7, has also been demonstrated in the sustained deleterious effects of chronic hyperglycemia on human the embryonic beta-cell progenitor pool, leading to inappropriate postnatal beta-cell formation. Stanger et al. (2007) demonstrated that selective genetic reduction in the size of Pdx1+ pancreatic progenitors during the fetal period results in impaired beta-cell formation during the postnatal period with consequent development of glucose intolerance during adulthood. Consistent with this, maternal food restriction leads to significant reduction in Pdx1+ and Ngn3+ (Neurogenin 3) pancreatic precursors during embryonic development in rats, decreased postnatal beta-cell formation, and inability to expand beta-cell mass in response to pregnancy (Garofano et al., 1998; Blondeau et al., 2002). Another mechanism proposed to explain reduced beta-cell formation after IUGR is related to prenatal glucocorticoid exposure. Maternal undernutrition significantly increased both fetal and maternal corticosterone concentrations in rats (Blondeau et al., 2001). Subsequently, maternal and/or fetal overexposure to glucocorticoids (via administration of dexamethasone) impairs both fetal and postnatal beta-cell formation in rodents and nonhuman primates (Blondeau et al., 2002; Bréant et al, 2006; De Vries et al., 2007; Gesina et al., 2004). Blondeau et al. (2001) have shown that fetal corticosterone concentrations are inversely correlated with fetal insulin content and postnatal beta-cell formation in rats. Evidence suggests that glucocorticoids can exert a direct effect on the developing fetal pancreas via transcriptional modulation of transcription factors involved in beta-cell formation and differentiation (Bréant et al., 2006). Glucocorticoid receptors are present in the pancreas during embryonic development of rodents and humans (Bréant et al., 2006) and glucocorticoids can bind to the Pdx1 promoter and thus suppress fetal endocrine cell differentiation (Bréant et al., 2006). Glucocorticoid treatment has been shown to significantly reduce fetal expression of key endocrine transcription factors such as Pdx1 and Pax6 but simultaneously increase expression of transcription factors that regulate development of the exocrine pancreas (Gesina et al., 2006). The UPI model of IUGR (due to bilateral uterine artery ligation) is also characterized by a permanent decrease in islet Pdx1 mRNA expression. This decrease has recently been shown to be due to progressive epigenetic silencing of the Pdx1 gene locus secondary to proximal promoter methylation (Stoffers et al., 2003; Park et al., 2008) and it may be responsible for the decreased rate of beta-cell replication and inappropriate postnatal beta-cell mass development (Stoffers et al., 2003; Kulkarni et al., 2004). It has also been demonstrated that the UPI or the low protein IUGR offspring experience increased oxidative stress and impaired mitochondrial function (Simmons et al., 2005). The mitochondrial dysfunction was not limited to just the beta-cell, as mitochondria from both the liver and skeletal muscle exhibit decreased oxidation of pyruvate, subsequently leading to the development of features commonly found in T2D (Peterside et al., 2003; Selak et al., 2003). Also exposure to a Western-style diet before and during pregnancy (an IUEO model) alters the redox state as early as preimplantation development, leading to mild oxidative stress associated with inflammation. The finding that administration of antioxidants to the dam reverses oxidative stress and completely prevents the development of glucose intolerance and increased adiposity in the adult offspring suggests that oxidative stress plays an important role in the development of adiposity in this case (Sen & Simmons, 2010). Some studies in the low protein IUGR model have demonstrated that oxidative stress is not limited to just mitochondrial DNA damage, but also genomic DNA, impacting on cell-cycle the embryonic beta-cell progenitor pool, leading to inappropriate postnatal beta-cell formation. Stanger et al. (2007) demonstrated that selective genetic reduction in the size of Pdx1+ pancreatic progenitors during the fetal period results in impaired beta-cell formation during the postnatal period with consequent development of glucose intolerance during adulthood. Consistent with this, maternal food restriction leads to significant reduction in Pdx1+ and Ngn3+ (Neurogenin 3) pancreatic precursors during embryonic development in rats, decreased postnatal beta-cell formation, and inability to expand beta-cell mass in response to pregnancy (Garofano et al., 1998; Blondeau et al., 2002). Another mechanism proposed to explain reduced beta-cell formation after IUGR is related to prenatal glucocorticoid exposure. Maternal undernutrition significantly increased both fetal and maternal corticosterone concentrations in rats (Blondeau et al., 2001). Subsequently, maternal and/or fetal overexposure to glucocorticoids (via administration of dexamethasone) impairs both fetal and postnatal beta-cell formation in rodents and nonhuman primates (Blondeau et al., 2002; Bréant et al, 2006; De Vries et al., 2007; Gesina et al., 2004). Blondeau et al. (2001) have shown that fetal corticosterone concentrations are inversely correlated with fetal insulin content and postnatal beta-cell formation in rats. Evidence suggests that glucocorticoids can exert a direct effect on the developing fetal pancreas via transcriptional modulation of transcription factors involved in beta-cell formation and differentiation (Bréant et al., 2006). Glucocorticoid receptors are present in the pancreas during embryonic development of rodents and humans (Bréant et al., 2006) and glucocorticoids can bind to the Pdx1 promoter and thus suppress fetal endocrine cell differentiation (Bréant et al., 2006). Glucocorticoid treatment has been shown to significantly reduce fetal expression of key endocrine transcription factors such as Pdx1 and Pax6 but simultaneously increase expression of transcription factors that regulate development of the The UPI model of IUGR (due to bilateral uterine artery ligation) is also characterized by a permanent decrease in islet Pdx1 mRNA expression. This decrease has recently been shown to be due to progressive epigenetic silencing of the Pdx1 gene locus secondary to proximal promoter methylation (Stoffers et al., 2003; Park et al., 2008) and it may be responsible for the decreased rate of beta-cell replication and inappropriate postnatal beta-cell mass It has also been demonstrated that the UPI or the low protein IUGR offspring experience increased oxidative stress and impaired mitochondrial function (Simmons et al., 2005). The mitochondrial dysfunction was not limited to just the beta-cell, as mitochondria from both the liver and skeletal muscle exhibit decreased oxidation of pyruvate, subsequently leading to the development of features commonly found in T2D (Peterside et al., 2003; Selak et al., 2003). Also exposure to a Western-style diet before and during pregnancy (an IUEO model) alters the redox state as early as preimplantation development, leading to mild oxidative stress associated with inflammation. The finding that administration of antioxidants to the dam reverses oxidative stress and completely prevents the development of glucose intolerance and increased adiposity in the adult offspring suggests that oxidative stress plays an important role in the development of adiposity in Some studies in the low protein IUGR model have demonstrated that oxidative stress is not limited to just mitochondrial DNA damage, but also genomic DNA, impacting on cell-cycle exocrine pancreas (Gesina et al., 2006). this case (Sen & Simmons, 2010). development (Stoffers et al., 2003; Kulkarni et al., 2004). regulation and gene expression (Chen et al., 2007). While DNA is being targeted throughout by ROS, there are particular regions that are known to be more sensitive to ROS-mediated damage, for example telomeres. Telomeres comprise GC-rich repeats and are found at the ends of each chromosome. They are known to shorten with each cellular division and, hence, can act as a mitotic clock, registering the number of replicative divisions to have taken place within the cell. Investigations using a low protein IUGR model have indeed reported a decrease in longevity in the offspring (Jennings et al., 1999; Chen et al., 2009) accompanied by reduction in mitochondrial antioxidant defences (Tarry-Adkins et al., 2009) and telomere length in islets (Tarry-Adkins et al., 2009). Pancreatic islet development has been shown to be influenced by a number of growth factors including the insulin-like growth factors, IGF-I and IGF-II whose expression in utero is regulated by nutrient and hormone concentrations. IUGR modify expression of both IGF genes in a variety of fetal tissues. In a low protein IUGR rat model with a decreased beta-cell mass and beta-cell replication and an increased rate of beta-cell apoptosis, gene expression for IGF-II but not IGF-I was found reduced in the fetal pancreas and this was (Petrik et al., 1999). In a different IUGR model with more severe global food restriction which induced hyperinsulinemia and an increase in beta-cell mass in their fetuses (Alvarez et al., 1997), the fetal phenotype was associated with an increase in pancreatic IGF-I expression, islet IGF-1R (Martn et al., 2005) and IRS-2 (Fernandez et al., 2007). In the fetal GK/Par rat exposed to mild hyperglycemia during gestation (a model of IUED), data from our group suggest that the beta-cell deficit (reduced by more than 50%) starts as early as fetal age 16 days E16 and reflects decreased beta-cell proliferation, a limitation of beta-cell neogenesis from precursors and increased apoptosis of both beta-cells and their precursors (Calderari et al., 2007). Notably, Pdx1 and Ngn3 expression were decreased on E18 but normally expressed on E13 (Calderari et al., 2007). Defective signalling through the IGF2/IGF1-R pathway may represents the primary instrumental anomaly since IGF2 and IGF1-R protein expressions are already decreased within the GK/Par pancreatic rudiment at E13, at a time when beta-cell mass (first wave of beta cell expansion) is in fact normal (Miralles & Portha, 2001). Low levels of pancreatic IGF2, associated with beta-cell mass deficiency, are maintained thereafter within the fetal pancreas (Serradas et al., 2002). Crossbreeding protocols between non-diabetic W and diabetic GK rats showed that in late gestation (E18), pancreatic IGF2 protein expression was as low in GKmother/GKfather and Wmother/GKfather crosses than in GKmother/GK father crosses (Serradas et al., 2002). These findings rather support the hypothesis that the pancreatic IGF2 anomaly in the GK diabetic model is linked to a genetic determinism. This view is also consistent with the results of genetic analyses that linked a locus containing the gene encoding IGF2 to diabetes in the GK rat (Gauguier et al., 1996). The Igf2 gene is subjected to paternal genomic imprinting. However, because the Igf2 expression is similarly affected in fetuses, regardless of whether the father is W or GK (Serradas et al., 2002), we cannot conclude to a simple change of Igf2 gene imprinting in the GK rat. Finally, studies have demonstrated that the maintenance of methylated histone H3 Lys4 by Set7/9 a member of the SET methyltransferase family, is crucial to Pdx1 activity in beta-cell lines (Chakrabarti et al., 2003; Francis et al., 2005; Deering et al., 2009). This led to the hypothesis that Set7/9 may represent a novel chromatin-modifying protein that functions in part through its recruitment to target genes by cell-specific transcription factors such as Pdx1. Since, a role of histone methyl transferases, particularly set7, has also been demonstrated in the sustained deleterious effects of chronic hyperglycemia on human Impact and Mechanisms of Pancreatic Beta-Cell Mass (Gauguier et al., 1994). cell mass programming gene expression (Waterland & Michels, 2007). growth and reduce weight in their progeny (Gluckman et al., 2007). Programming by Maternal Diabetes - Insight from Animal Model Studies 213 metabolic phenotypes, as recently observed at the Pdx1 and GLUT4 loci in UPI rats (Park et al., 2008; Raychaudhuri et al., 2008). If these epigenetic changes occur in the germ line, they can be inherited through meiosis (Chong et al., 2007), thus providing a plausible explanation for intergenerational effects, transmitted via either maternal or paternal lines. In addition, other indirect biological processes may influence phenotypes in subsequent generations. For example, physical constraints may alter birth size through the maternal lineage: since uterine size is reduced in girls that are born small and remain short, this may influence fetal Furthermore, maternal metabolism may also influence cross-generational phenotypes (Aerts & Van Assche, 2006). Maternal undernutrition during pregnancy (F0) increases risk for developing diabetes and obesity in her offspring (F1). When these high-risk adult F1 females become pregnant, the metabolic stress of pregnancy may result in hyperglycemia and/or overt gestational diabetes that may, in turn, contribute to defective beta-cell mass and increased diabetes risk in F2 offspring (Aerts & Van Assche, 2006). By this mechanism gestational diabetes may pass from one generation to the next one. In these last examples, intergenerational transmission of phenotypes would occur exclusively through the maternal lineage, as opposed to the epigenetic mechanisms mentioned above. Such a scenario is relevant to the GK/Par rat, since the GK/Par mothers are mildly hyperglycemic through their gestation and during the suckling period. It offers a rationale to elucidate several clues: 1/ the initiation of pancreas programming in the F1 offspring of the first founders (F0), since the GK line is issued from intercrosses between Wistar females and males with borderline IGT but otherwise normal basal blood glucose level (Goto et al., 1975); 2/ the progression of the IGT phenotype until a stable mild diabetic phenotype was reached among the generations (n=35) (Goto et al., 1975); 3/ the lack of attenuation of the diabetic GK phenotype overtime (along more than 20 years and 80 generations), since offspring of GK female/W male crosses were more hyperglycemic than those of W female/GK male crosses 7. Epigenetic mechanisms mediating the diabetes risk associated with beta- Several lines of evidence indicate that epigenetic modification may be a key unifying mechanism mediating risk associated with a perturbed intrauterine environment. First, disruption of physiologic responses and functional capacity as observed in multiple tissues in of IUED or IUGR animals and humans, including muscle, adipose, pancreas, liver, and CNS may be related to histone modification and DNA methylation thereby altering related The preimplantation embryo is particularly sensitive to epigenetic modifications that might permanently alter the phenotype in the adult (Reik et al., 1993; Doherty et al., 2000). For example, in the agouti mouse model, folate supplementation of the maternal diet at conception increases DNA methylation of the agouti gene and increases longevity of the offspring (Cooney et al., 2002). Maternal protein restriction has been shown to alter the methylation status of the promoters of the glucocorticoid receptor (Lillycrop et al., 2005), PPARa (Lillycrop et al., 2008), and the angiotensin receptor (Bogdarina et al., 2007) with parallel changes in gene expression. More recent studies have shown that histone modifications can also be influenced by the early environment. Alterations in histone modifications have also been implicated in mediating the effect of caloric restriction during microvascular endothelial cells (Siebel et al., 2010), such an epigenetic change could potentially be involved in the deleterious effect of high glucose upon the fetal pancreas in the IUED models. #### 5. Various early life stressors, one ultimate programming agent: Perinatal hyperglycemia As abundantly illustrated in animal models, early life stressors such as maternal undernutrition, overnutrition, hypercholesterolemia, corticosteroid therapy, uteroplacental insufficiency, or hypoxia, program metabolic adaptations that favor survival initially, but are ultimately detrimental to adult health. Interestingly, a crucial commonality exists between these models with quite different etiologies: in most of the cases, the altered maternal/fetal metabolism appears to be associated with a diabetogenic effect in the adult offspring either male or female, resulting in a permanent deficiency of the endocrine pancreatic function (F1). In females, the combination of a latent diabetogenic tendency (low insulin response) and the metabolic stress of pregnancy promotes gestational diabetes. F1 gestational diabetes per se is an inducing factor for impaired glucose tolerance and gestational diabetes again in the next female generation (F2). Finally, the relevant message is that programming of the endocrine pancreas ultimately originates from hyperglycemia experienced during the fetal and/or early postnatal life, whatever the etiology of maternal hyperglycemia is primary (in F0 diabetic mothers) or secondary (in F1 diabetic mothers issued from F0 mothers exposed to undernutrition, UPI, or high glucocorticoid). #### 6. Transgenerational inheritance of beta-cell mass programming While a large number of animal studies have shown the effects of undernutrition during foetal/perinatal development on the glucose metabolism of offspring (F1) in adulthood, several studies have shown that glucose metabolism is also altered in the offspring (F2) as well as grand offspring (F3) of fetally malnourished F1 females, even when the F1 and F2 females have been well nourished since weaning (Aerts & Van Assche, 2006; Benyshek et al., 2006). With a aim to dissect the relative parental contributions that lead to F2 offspring outcomes in these models of maternal (F0) undernutrition, it was recently reported that F1 males exhibit moderate hyperglycemia and IGT with aging and impaired glucose-stimulated insulin secretion and that all F2 offspring of F1 males or F1 females develop glucose intolerance (Jimenez-Chillaron et al., 2005). Therefore, intergenerational progression of glucose intolerance can derive from both the maternal and paternal lines. This is an experimental proof that transgenerational transmission of IGT may also occur through the paternal lineage, besides the more widely accepted maternal and grand maternal inheritance of diabetes (Zambrano et al., 2005; Drake et al., 2005; Blondeau et al., 2002; Benyshek et al., 2006). Conceptually, transgenerational inheritance of disease risk may be mediated by nongenomic mechanisms, including either 1) epigenetic mechanisms (Ozanne & Constancia, 2007; Pinney & Simmons, 2009; Drake & Liu, 2009; Waterland & Michels, 2007) or 2) other broader indirect mechanisms associated with parental physiology (Gluckman et al., 2007). First, alterations in nutrition during development can alter epigenetic marks, thus regulating gene expression through DNA methylation and/or histone modifications. Interestingly, such epigenetic modifications may progress with aging during postnatal life, in association with microvascular endothelial cells (Siebel et al., 2010), such an epigenetic change could potentially be involved in the deleterious effect of high glucose upon the fetal pancreas in 5. Various early life stressors, one ultimate programming agent: Perinatal gestational diabetes again in the next female generation (F2). As abundantly illustrated in animal models, early life stressors such as maternal undernutrition, overnutrition, hypercholesterolemia, corticosteroid therapy, uteroplacental insufficiency, or hypoxia, program metabolic adaptations that favor survival initially, but are ultimately detrimental to adult health. Interestingly, a crucial commonality exists between these models with quite different etiologies: in most of the cases, the altered maternal/fetal metabolism appears to be associated with a diabetogenic effect in the adult offspring either male or female, resulting in a permanent deficiency of the endocrine pancreatic function (F1). In females, the combination of a latent diabetogenic tendency (low insulin response) and the metabolic stress of pregnancy promotes gestational diabetes. F1 gestational diabetes per se is an inducing factor for impaired glucose tolerance and Finally, the relevant message is that programming of the endocrine pancreas ultimately originates from hyperglycemia experienced during the fetal and/or early postnatal life, whatever the etiology of maternal hyperglycemia is primary (in F0 diabetic mothers) or secondary (in F1 diabetic mothers issued from F0 mothers exposed to undernutrition, UPI, While a large number of animal studies have shown the effects of undernutrition during foetal/perinatal development on the glucose metabolism of offspring (F1) in adulthood, several studies have shown that glucose metabolism is also altered in the offspring (F2) as well as grand offspring (F3) of fetally malnourished F1 females, even when the F1 and F2 females have been well nourished since weaning (Aerts & Van Assche, 2006; Benyshek et al., 2006). With a aim to dissect the relative parental contributions that lead to F2 offspring outcomes in these models of maternal (F0) undernutrition, it was recently reported that F1 males exhibit moderate hyperglycemia and IGT with aging and impaired glucose-stimulated insulin secretion and that all F2 offspring of F1 males or F1 females develop glucose intolerance (Jimenez-Chillaron et al., 2005). Therefore, intergenerational progression of glucose intolerance can derive from both the maternal and paternal lines. This is an experimental proof that transgenerational transmission of IGT may also occur through the paternal lineage, besides the more widely accepted maternal and grand maternal inheritance of diabetes (Zambrano et al., Conceptually, transgenerational inheritance of disease risk may be mediated by nongenomic mechanisms, including either 1) epigenetic mechanisms (Ozanne & Constancia, 2007; Pinney & Simmons, 2009; Drake & Liu, 2009; Waterland & Michels, 2007) or 2) other broader indirect mechanisms associated with parental physiology (Gluckman et al., 2007). First, alterations in nutrition during development can alter epigenetic marks, thus regulating gene expression through DNA methylation and/or histone modifications. Interestingly, such epigenetic modifications may progress with aging during postnatal life, in association with 6. Transgenerational inheritance of beta-cell mass programming 2005; Drake et al., 2005; Blondeau et al., 2002; Benyshek et al., 2006). the IUED models. hyperglycemia or high glucocorticoid). metabolic phenotypes, as recently observed at the Pdx1 and GLUT4 loci in UPI rats (Park et al., 2008; Raychaudhuri et al., 2008). If these epigenetic changes occur in the germ line, they can be inherited through meiosis (Chong et al., 2007), thus providing a plausible explanation for intergenerational effects, transmitted via either maternal or paternal lines. In addition, other indirect biological processes may influence phenotypes in subsequent generations. For example, physical constraints may alter birth size through the maternal lineage: since uterine size is reduced in girls that are born small and remain short, this may influence fetal growth and reduce weight in their progeny (Gluckman et al., 2007). Furthermore, maternal metabolism may also influence cross-generational phenotypes (Aerts & Van Assche, 2006). Maternal undernutrition during pregnancy (F0) increases risk for developing diabetes and obesity in her offspring (F1). When these high-risk adult F1 females become pregnant, the metabolic stress of pregnancy may result in hyperglycemia and/or overt gestational diabetes that may, in turn, contribute to defective beta-cell mass and increased diabetes risk in F2 offspring (Aerts & Van Assche, 2006). By this mechanism gestational diabetes may pass from one generation to the next one. In these last examples, intergenerational transmission of phenotypes would occur exclusively through the maternal lineage, as opposed to the epigenetic mechanisms mentioned above. Such a scenario is relevant to the GK/Par rat, since the GK/Par mothers are mildly hyperglycemic through their gestation and during the suckling period. It offers a rationale to elucidate several clues: 1/ the initiation of pancreas programming in the F1 offspring of the first founders (F0), since the GK line is issued from intercrosses between Wistar females and males with borderline IGT but otherwise normal basal blood glucose level (Goto et al., 1975); 2/ the progression of the IGT phenotype until a stable mild diabetic phenotype was reached among the generations (n=35) (Goto et al., 1975); 3/ the lack of attenuation of the diabetic GK phenotype overtime (along more than 20 years and 80 generations), since offspring of GK female/W male crosses were more hyperglycemic than those of W female/GK male crosses (Gauguier et al., 1994). #### 7. Epigenetic mechanisms mediating the diabetes risk associated with betacell mass programming Several lines of evidence indicate that epigenetic modification may be a key unifying mechanism mediating risk associated with a perturbed intrauterine environment. First, disruption of physiologic responses and functional capacity as observed in multiple tissues in of IUED or IUGR animals and humans, including muscle, adipose, pancreas, liver, and CNS may be related to histone modification and DNA methylation thereby altering related gene expression (Waterland & Michels, 2007). The preimplantation embryo is particularly sensitive to epigenetic modifications that might permanently alter the phenotype in the adult (Reik et al., 1993; Doherty et al., 2000). For example, in the agouti mouse model, folate supplementation of the maternal diet at conception increases DNA methylation of the agouti gene and increases longevity of the offspring (Cooney et al., 2002). Maternal protein restriction has been shown to alter the methylation status of the promoters of the glucocorticoid receptor (Lillycrop et al., 2005), PPARa (Lillycrop et al., 2008), and the angiotensin receptor (Bogdarina et al., 2007) with parallel changes in gene expression. More recent studies have shown that histone modifications can also be influenced by the early environment. Alterations in histone modifications have also been implicated in mediating the effect of caloric restriction during Impact and Mechanisms of Pancreatic Beta-Cell Mass "programmed" diabetes risk. 9. Acknowledgments 10. References 38, 1–19. 19:89–98. Programming by Maternal Diabetes - Insight from Animal Model Studies 215 Several interventions (dietary or pharmacological) to reduce the long-term sequelae of early life programming effects have been used in animal models. For example, the administration of folic acid with a low protein diet during pregnancy prevents the altered phenotype and epigenotype in rat offspring (Lillycrop et al., 2005), and administration of a diet rich in methyl donors prevents the transgenerational increase in obesity in agouti yellow mice (Waterland et al., 2008). Importantly, the timing of such interventions can be crucial. Examples include neonatal leptin treatment which reverses the programming effects of prenatal undernutrition (Vickers et al., 2005). In the UPI rat model, epigenetic silencing of the Pdx1 gene can be reversed during a critical developmental window in the neonatal period, using trichostatin A which inhibit HDACs (Park et al., 2008). In the same model, exposure to Exendin-4 in the neonatal period reversed the detrimental fetal programming of the beta-cell mass and prevented the development of diabetes in adulthood: this was closely related to restoration of pdx1 expression and beta-cell proliferation rate (Stoffers Diabetes 2003). A GLP-1 or Exendin-4 treatment limited to the neonatal pre-diabetic period was also shown to delay the installation and limit the severity of T2D in the GK/Par model (Tourrel et al., 2002). In such context, it is important to note that GLP1 derived drugs that are currently used to treat patients with T2D may target chromatin remodelling. Treating beta-cells from the INS1 cell line or dispersed mouse islet cells with GLP-1 increased global acetylation of histone H3 and increased its phosphorylation in a concentration-dependent manner (Kim et al., 2009). Such histone modifications increased association with the transcription factor phospho- CREB and with cAMP-response CREB coactivator 2. Taken as a whole, these data may provoke optimism - that there may be a window for potential postnatal therapeutic interventions to prevent/modify the The studies from our lab and referred to in this review have been funded by the French ANR (programme Physio 2006 - Prograbeta - ref ANR-06-PHYSIO-028), EGIDE (PHC franco-espagnol PICASSO 2008-2009) and NEB Association. A. Chavey received a CNRS Aerts, A & Van Assche, FA (2006). Animal evidence for the transgenerational development Aerts, L, Vercruysse, L & Van Assche, FA (1997). The endocrine pancreas in virgin and Aerts, L, Holemans, K & Van Assche, FA (1990). Maternal diabetes during pregnancy: Consequences for the offspring. Diabetes Metabolic Review, 16, 147–197. Aerts, L et al. (1989). Plasma amino-acids in diabetic pregnant rats and in their fetal and Alcolado JC, Laji K & Gill-Randall R (2002). Maternal transmission of diabetes. Diabet Med pregnant offspring of diabetic pregnant rats. Diabetes Research and Clinical Practice, postdoctoral fellowship and was granted by NESTLE-France and SFD/Lilly. of diabetes mellitus. Int J Biochem Cell Biol 38 : 894–903. adult offspring. Biol. Neonate, 56, 31–39. the second half of pregnancy on the programmed reduction of GLUT4 expression in the offspring (Raychaudhuri et al., 2008). In the case of the UPI rat model and the pancreatic tissue, Simmons and colleagues have reported a progressive reduction in expression of Pdx1, a key transcription factor regulating pancreatic development and function (Stoffers et al., 2003). Pdx1 expression is reduced by 50% in UPI fetuses and by 80% in adult UPI offspring. Notably, these changes precede the onset of beta-cell dysfunction, suggesting a primary pathogenic role. Since the Pdx1 promoter is a target for epigenetic modification, as it contains a conserved CpG islands and is associated with high levels of histone acetylation. Interestingly, binding of both acetylated histone H3/H4 and the transcription factor USF1 was found abolished in UPI fetuses (Park et al., 2008). While there was methylation at multiple CpGs in UPI adult offspring, no methylation was detected in UPI neonates, indicating that methylation was unlikely to explain Pdx1 repression early in life. Together, these data indicate that progressive silencing of gene expression is largely initiated by early epigenetic changes and is maintained thereafter even in the absence of further experimental insults during postnatal life. UPI also increases histone acetylation of the PPARalpha coactivator PGC-1 and carnitine–palmitoyltransferase I (CPT1) promoters in newborn and young rats, and these changes are associated with increased PGC-1 and CPT1 mRNAs (Fu et al., 2004). Finally, there is now little doubt that epigenetic regulation of gene expression also occurs in humans as a response to early nutritional insult: a recent study has revealed that individuals who were exposed to famine in utero during the Dutch Hunger Winter had altered methylation of the Igf2 gene in white blood cells in adulthood (Heijmans et al., 2008). #### 8. Implications for human health Although the focus of most studies in the metabolic programming field has been on delineating the effects of reduced maternal nutrition, there is now a growing interest in the role of maternal overnutrition in the programming of diabetes risk. The worldwide prevalence of obesity continues to increase, in association with an increase in the risk of metabolic T2D. Indeed, a recent study estimated that the number of people worldwide with diabetes would increase from 171 million in 2000 to 366 million by 2030 if the prevalence of obesity remained constant (Wild et al., 2004), which has major implications for public health strategies worldwide (WHO, 2000). This global trend to increasing obesity is reflected in the increasing numbers of women who are obese during pregnancy (Kanagalingam et al., 2005). Given that the offspring of obese mothers have an increased risk of developing obesity and T2D themselves (Boney et al., 2005; Catalano, 2003; Catalano et al., 2009), the potential impact of the intergenerational consequences of maternal obesity is of great concern for public health policy makers. Moreover, maternal hyperglycemia per se increases the probability of adolescent obesity and future T2D. To what extent maternal hyperglycemia is fuelling the global rise in obesity and T2D is unknown, but its contribution is highly significant. The exact degree of hyperglycemia that has this effect and the exact timing in pregnancy at which hyperglycemia is impressionable on fetal programming is unknown. The need to identify and treat all women with gestational diabetes is very much dependent on us knowing this. Meanwhile, achieving rigorous glycemic control in women with diabetic pregnancy has to remain a major therapeutic goal. Several interventions (dietary or pharmacological) to reduce the long-term sequelae of early life programming effects have been used in animal models. For example, the administration of folic acid with a low protein diet during pregnancy prevents the altered phenotype and epigenotype in rat offspring (Lillycrop et al., 2005), and administration of a diet rich in methyl donors prevents the transgenerational increase in obesity in agouti yellow mice (Waterland et al., 2008). Importantly, the timing of such interventions can be crucial. Examples include neonatal leptin treatment which reverses the programming effects of prenatal undernutrition (Vickers et al., 2005). In the UPI rat model, epigenetic silencing of the Pdx1 gene can be reversed during a critical developmental window in the neonatal period, using trichostatin A which inhibit HDACs (Park et al., 2008). In the same model, exposure to Exendin-4 in the neonatal period reversed the detrimental fetal programming of the beta-cell mass and prevented the development of diabetes in adulthood: this was closely related to restoration of pdx1 expression and beta-cell proliferation rate (Stoffers Diabetes 2003). A GLP-1 or Exendin-4 treatment limited to the neonatal pre-diabetic period was also shown to delay the installation and limit the severity of T2D in the GK/Par model (Tourrel et al., 2002). In such context, it is important to note that GLP1 derived drugs that are currently used to treat patients with T2D may target chromatin remodelling. Treating beta-cells from the INS1 cell line or dispersed mouse islet cells with GLP-1 increased global acetylation of histone H3 and increased its phosphorylation in a concentration-dependent manner (Kim et al., 2009). Such histone modifications increased association with the transcription factor phospho- CREB and with cAMP-response CREB coactivator 2. Taken as a whole, these data may provoke optimism - that there may be a window for potential postnatal therapeutic interventions to prevent/modify the "programmed" diabetes risk. #### 9. Acknowledgments 214 Gestational Diabetes the second half of pregnancy on the programmed reduction of GLUT4 expression in the offspring (Raychaudhuri et al., 2008). In the case of the UPI rat model and the pancreatic tissue, Simmons and colleagues have reported a progressive reduction in expression of Pdx1, a key transcription factor regulating pancreatic development and function (Stoffers et al., 2003). Pdx1 expression is reduced by 50% in UPI fetuses and by 80% in adult UPI offspring. Notably, these changes precede the onset of beta-cell dysfunction, suggesting a primary pathogenic role. Since the Pdx1 promoter is a target for epigenetic modification, as it contains a conserved CpG islands and is associated with high levels of histone acetylation. Interestingly, binding of both acetylated histone H3/H4 and the transcription factor USF1 was found abolished in UPI fetuses (Park et al., 2008). While there was methylation at multiple CpGs in UPI adult offspring, no methylation was detected in UPI neonates, indicating that methylation was unlikely to explain Pdx1 repression early in life. Together, these data indicate that progressive silencing of gene expression is largely initiated by early epigenetic changes and is maintained thereafter even in the absence of further experimental insults during postnatal life. UPI also increases histone acetylation of the PPARalpha coactivator PGC-1 and carnitine–palmitoyltransferase I (CPT1) promoters in newborn and young rats, and these changes are associated with increased PGC-1 and CPT1 mRNAs (Fu et al., 2004). Finally, there is now little doubt that epigenetic regulation of gene expression also occurs in humans as a response to early nutritional insult: a recent study has revealed that individuals who were exposed to famine in utero during the Dutch Hunger Winter had altered methylation of the Igf2 gene in white blood cells in adulthood Although the focus of most studies in the metabolic programming field has been on delineating the effects of reduced maternal nutrition, there is now a growing interest in the role of maternal overnutrition in the programming of diabetes risk. The worldwide prevalence of obesity continues to increase, in association with an increase in the risk of metabolic T2D. Indeed, a recent study estimated that the number of people worldwide with diabetes would increase from 171 million in 2000 to 366 million by 2030 if the prevalence of obesity remained constant (Wild et al., 2004), which has major implications for public health strategies worldwide (WHO, 2000). This global trend to increasing obesity is reflected in the increasing numbers of women who are obese during pregnancy (Kanagalingam et al., 2005). Given that the offspring of obese mothers have an increased risk of developing obesity and T2D themselves (Boney et al., 2005; Catalano, 2003; Catalano et al., 2009), the potential impact of the intergenerational consequences of maternal obesity is of great concern for Moreover, maternal hyperglycemia per se increases the probability of adolescent obesity and future T2D. To what extent maternal hyperglycemia is fuelling the global rise in obesity and T2D is unknown, but its contribution is highly significant. The exact degree of hyperglycemia that has this effect and the exact timing in pregnancy at which hyperglycemia is impressionable on fetal programming is unknown. The need to identify and treat all women with gestational diabetes is very much dependent on us knowing this. 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Omu Frcog Between Gestational Diabetes Mellitus and Butyrylcholinesterase Gestational Diabetes Mellitus (GDM) is glucose intolerance first diagnosed during pregnancy (Metzger and Coustan 1998, World Health Organization 1999). Gestational diabetes is a condition that complicates 3-12.% of pregnancies (Gabbe and Graves 2003, Omu et al 2010 ) with wide variation in the incidence of gestational diabetes reported among ethnic groups.This could be newly diagnosed type 1 or type 2 Diabetes Mellitus or a new onset of hyperglycemia secondary to metabolic changes related to pregnancy (Yogev and Visser 2009). The rates of Gestational diabetes mellitus are increasing with the epidemic of obesity worldwide. Risk factors for GDM include advanced maternal age, multiparity, and South Asian (specifically women whose country of family origin is India, Middle Eastern (specifically women whose country of family origin is Saudi Arabia, United Arab Emirates, Iraq, Jordan, Syria, Oman, Qatar, Kuwait, Reproduced from the National Institute for Health and Clinical Excellence guideline for diabetes in pregnancy (12) by RCOG Scientific Advisory Committee Opinion Paper 23: Diagnosis and Treatment of 1. Introduction racial or ethnic minority status (Table 1). Previous gestational diabetes Pakistan or Bangladesh) Black Caribbean Gestational Diabetes (RCOG 2011). Lebanon or Egypt) Body mass index more than 30 kg/m² Previous macrosomic baby weighing 4.5 kg or more Family origin with a high prevalence of diabetes: Table 1. Risk factors for Gestational diabetes mellitus Family history of diabetes (first-degree relative with diabetes) ## Unravelling the Connection Between Gestational Diabetes Mellitus and Butyrylcholinesterase Alexander E. Omu Frcog Department of Obstetrics and Gynaecology, Faculty of Medicine, Kuwait University, Kuwait ### 1. Introduction 226 Gestational Diabetes Zambranon E, et al. (2005): Sex differences in transgenerational alterations of growth and Zeggini, E, et al. (2007). Replication of genome-wide association signals in UK samples during pregnancy and lactation. J Physiol 566:225–236. reveals risk loci for type 2 diabetes. Science; 316:1336–1341. metabolism in progeny (F2) of female offspring (F1) of rats fed a low protein diet Gestational Diabetes Mellitus (GDM) is glucose intolerance first diagnosed during pregnancy (Metzger and Coustan 1998, World Health Organization 1999). Gestational diabetes is a condition that complicates 3-12.% of pregnancies (Gabbe and Graves 2003, Omu et al 2010 ) with wide variation in the incidence of gestational diabetes reported among ethnic groups.This could be newly diagnosed type 1 or type 2 Diabetes Mellitus or a new onset of hyperglycemia secondary to metabolic changes related to pregnancy (Yogev and Visser 2009). The rates of Gestational diabetes mellitus are increasing with the epidemic of obesity worldwide. Risk factors for GDM include advanced maternal age, multiparity, and racial or ethnic minority status (Table 1). Reproduced from the National Institute for Health and Clinical Excellence guideline for diabetes in pregnancy (12) by RCOG Scientific Advisory Committee Opinion Paper 23: Diagnosis and Treatment of Gestational Diabetes (RCOG 2011). Table 1. Risk factors for Gestational diabetes mellitus Unravelling the Connection Between Gestational Diabetes Mellitus and Butyrylcholinesterase 229 as NF-kB, p38 MAPK, JNK/SAPK, and hexosamine, by elevations in glucose and possibly FFA levels leads to both insulin resistance and impaired insulin secretion through ß-cell dysfunction (Kyriakis et al 1992). Circulating serum levels of lipid peroxidation product malonedialdehyde (MDA) and protein oxidation markers are elevated in GDM compared to healthy normal pregnancy and give rise to a negatively strong correlation between MDA and BuChE in serum and placenta (Omu et al 2010). A third causal pathway may be through the induction of apoptosis of the beta cells by advanced glycation end-products. This would explain the varying severity of GDM among the patients. BuChE deficiency results in delayed metabolism of a number of compounds of clinical significance, including glucose, thus contributing to the pathogenesis of diabetes mellitus. Glucose metabolism is controlled by the hormone insulin produced in the pancreas. BuChE deficiency in pregnancy may be as a result The estrogen receptor ER-alpha is emerging as a key molecule involved in glucose and lipid metabolism. The activation of ER-alpha by physiological concentrations of E2 may play an important role in the adaptation of the endocrine pancreas to pregnancy. However, if ERalpha is over stimulated by an excess of E2 or the action of an environmental estrogen such as Biphenol A (Nadal et al 2009, Paloma et al 2008, Ropero et al 2008), it can result in an excessive insulin signaling. This may provoke insulin resistance in the liver and muscle, as well as beta-cell exhaustion and therefore, contribute to the development of Gestational Diabetes. An association between oestrogen receptor alpha and BuChE has been reported The increase of endocrine-disrupting chemicals (EDCs) in the environment has been implicated in the aetiology of GDM (Elobeid and Allison 2008, Newbold et al 2009, Rubin and Sato 2009). A connection at the epidemiologic level in humans has been recently proposed for dioxin, an environmental contaminant that acts through other than estrogen Autoimmune phenomena associated with type 1 diabetes mellitus (DM) can also be detected in a subgroup of women with GDM. Islet autoantibodies are present in sera from women with GDM with variable frequency. Distinct phenotypic and genotypic features may be recognised in this subset of women with GDM, which are representative of a distinct clinical entity. Women with previous autoimmune GDM may be candidates for potential immune intervention strategies (Mauricio et al 2001). Normal activity of Treg subpopulations are disrupted in GDM by mechanisms that threaten pregnancy and may contribute to other features of the disorder, with higher percentages of activated T cells than a matched population of healthy pregnant women. BuChE detoxifies anticholinesterases (AC) that are known to threaten pregnancy and one or more of these fetotoxins adversely impacts pregnancy outcome through a mechanism that may include Treg cells. In normal pregnancy, there is correlation between Treg activity and BuChE, whereas in women in whom adequate correlation between Treg cells and BuChE activity is not achieved (Mahmoud et al 2003, Mahmoud et al 2006, Mahmoud et al 2008, Saito et al 2005), there is receptors (ERs) as an endocrine disruptor (Bertazzi et al 2001, Remillard et al 2002). of hereditary deficiency and haemodilution in second half of pregnancy. 3.2 Role of estrogen receptor alpha in glucose and lipid metabolism (Combarros et al 2007). 3.3 Environmental factors 3.4 Autoimmunity and Treg in GDM There are health implications for both the mother and infant who remain at risk for complications such as embryopathies, spontaneous abortion and perinatal mortality and morbidity (Loeken 2006). There has been considerable controversy surrounding the screening and diagnosis, natural history, management and outcome of women with gestational diabetes. The 2008 NICE guidelines (NICE 2008) on diabetes in pregnancy detailed a screening programme targeting biochemical screening to women with risk factors. Women with a history of gestational diabetes mellitus (GDM) have an increased risk for recurrence in subsequent pregnancies, according to the results of a population-based, retrospective cohort study (HAPO Study Cooperative Research Group 2008). Oxidative stress has been implicated in the pathogenesis and development of complications of diabetes in pregnancy (King and Loeken 2004, Loeken 2004, Marfella et al 2001, Morgan et al 2008, Rosen et al 2001, Wender-Ozegowska et al 2004). The role of Butyrylcholinesterase (BuChE) in the aetiology, screening and monitoring, complications and future drug development of Gestational Diabetes Mellitus has become an interesting area of speculative research (Mahmoud et al 2003, Mohmoud etal 2006, Mahmoud et al 2008, Rustemeijer et al 2001, Serlin et al 2009, Sternfield et al 1997). #### 2. The objective The objective of this chapter is to elucidate the relationship between gestational diabetes mellitus (GDM) and BuChE in the pathogenesis, monitoring and future drug development. #### 3. Pathogenesis of GDM The cornerstones of development of gestational diabetes mellitus are related to modern lifestyle, principally, a lack of exercise and an unhealthy diet, the environment and some degree of genetic profile (American Diabetic Association 2003, Hollander et al 2007, Serlin et al 2009). Elevated glucose in pregnancy may be caused by increased levels of diabetogenic factors of pregnancy such as glucocorticoids, human placental lactogen and oestrogens. Hyperglycaemia causes oxidative stress due to increased production of mitochondrial ROS, nonenzymatic glycation of proteins, and glucose autoxidation (Brownlee 2001). Elevated FFA can also cause oxidative stress due to increased mitochondrial uncoupling and ßoxidation, leading to the increased production of ROS. In addition, hyperglycemia- and FFAinduced oxidative stress leads to the activation of stress-sensitive signaling pathways (Evans et al 2003). This, in turn, worsens both insulin secretion and action, leading to overt Gestational Diabetes Mellitus. Administration of glucocorticoids significantly decreases the catalytic activity of BuChE in plasma and liver regardless of sex (Vrdoljaki et al 2005). #### 3.1 Hyperglycemia and oxidative stress The pathogenic effect of hyperglycaemia is mediated to a significant extent via increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and subsequent oxidative stress (King and Loeken 2004, Marfella2001, Rosen et al 2001, Wender-Ozegowska et al 2004). ROS and RNS directly oxidize and damage DNA, proteins, and lipids and thus adversely affect the pancreas especially the Langerhan cells that produce insulin. Similarly, there is disruption of the alpha cells that produce glucagon. The paracrine relationship between the pancreatic beta and alpha cells is disrupted to cause -cell dysfunction. There is also growing evidence that activation of stress-sensitive pathways, such as NF-kB, p38 MAPK, JNK/SAPK, and hexosamine, by elevations in glucose and possibly FFA levels leads to both insulin resistance and impaired insulin secretion through ß-cell dysfunction (Kyriakis et al 1992). Circulating serum levels of lipid peroxidation product malonedialdehyde (MDA) and protein oxidation markers are elevated in GDM compared to healthy normal pregnancy and give rise to a negatively strong correlation between MDA and BuChE in serum and placenta (Omu et al 2010). A third causal pathway may be through the induction of apoptosis of the beta cells by advanced glycation end-products. This would explain the varying severity of GDM among the patients. BuChE deficiency results in delayed metabolism of a number of compounds of clinical significance, including glucose, thus contributing to the pathogenesis of diabetes mellitus. Glucose metabolism is controlled by the hormone insulin produced in the pancreas. BuChE deficiency in pregnancy may be as a result of hereditary deficiency and haemodilution in second half of pregnancy. #### 3.2 Role of estrogen receptor alpha in glucose and lipid metabolism The estrogen receptor ER-alpha is emerging as a key molecule involved in glucose and lipid metabolism. The activation of ER-alpha by physiological concentrations of E2 may play an important role in the adaptation of the endocrine pancreas to pregnancy. However, if ERalpha is over stimulated by an excess of E2 or the action of an environmental estrogen such as Biphenol A (Nadal et al 2009, Paloma et al 2008, Ropero et al 2008), it can result in an excessive insulin signaling. This may provoke insulin resistance in the liver and muscle, as well as beta-cell exhaustion and therefore, contribute to the development of Gestational Diabetes. An association between oestrogen receptor alpha and BuChE has been reported (Combarros et al 2007). #### 3.3 Environmental factors 228 Gestational Diabetes There are health implications for both the mother and infant who remain at risk for complications such as embryopathies, spontaneous abortion and perinatal mortality and There has been considerable controversy surrounding the screening and diagnosis, natural history, management and outcome of women with gestational diabetes. The 2008 NICE guidelines (NICE 2008) on diabetes in pregnancy detailed a screening programme targeting biochemical screening to women with risk factors. Women with a history of gestational diabetes mellitus (GDM) have an increased risk for recurrence in subsequent pregnancies, according to the results of a population-based, retrospective cohort study (HAPO Study Cooperative Research Group 2008). Oxidative stress has been implicated in the pathogenesis and development of complications of diabetes in pregnancy (King and Loeken 2004, Loeken 2004, Marfella et al 2001, Morgan et al 2008, Rosen et al 2001, Wender-Ozegowska et al 2004). The role of Butyrylcholinesterase (BuChE) in the aetiology, screening and monitoring, complications and future drug development of Gestational Diabetes Mellitus has become an interesting area of speculative research (Mahmoud et al 2003, Mohmoud etal 2006, Mahmoud et al 2008, Rustemeijer et al 2001, Serlin et al 2009, Sternfield et al 1997). The objective of this chapter is to elucidate the relationship between gestational diabetes mellitus (GDM) and BuChE in the pathogenesis, monitoring and future drug development. The cornerstones of development of gestational diabetes mellitus are related to modern lifestyle, principally, a lack of exercise and an unhealthy diet, the environment and some degree of genetic profile (American Diabetic Association 2003, Hollander et al 2007, Serlin et al 2009). Elevated glucose in pregnancy may be caused by increased levels of diabetogenic factors of pregnancy such as glucocorticoids, human placental lactogen and oestrogens. Hyperglycaemia causes oxidative stress due to increased production of mitochondrial ROS, nonenzymatic glycation of proteins, and glucose autoxidation (Brownlee 2001). Elevated FFA can also cause oxidative stress due to increased mitochondrial uncoupling and ßoxidation, leading to the increased production of ROS. In addition, hyperglycemia- and FFAinduced oxidative stress leads to the activation of stress-sensitive signaling pathways (Evans et al 2003). This, in turn, worsens both insulin secretion and action, leading to overt Gestational Diabetes Mellitus. Administration of glucocorticoids significantly decreases the catalytic activity of BuChE in plasma and liver regardless of sex (Vrdoljaki et al 2005). The pathogenic effect of hyperglycaemia is mediated to a significant extent via increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and subsequent oxidative stress (King and Loeken 2004, Marfella2001, Rosen et al 2001, Wender-Ozegowska et al 2004). ROS and RNS directly oxidize and damage DNA, proteins, and lipids and thus adversely affect the pancreas especially the Langerhan cells that produce insulin. Similarly, there is disruption of the alpha cells that produce glucagon. The paracrine relationship between the pancreatic beta and alpha cells is disrupted to cause -cell dysfunction. There is also growing evidence that activation of stress-sensitive pathways, such morbidity (Loeken 2006). 2. The objective 3. Pathogenesis of GDM 3.1 Hyperglycemia and oxidative stress The increase of endocrine-disrupting chemicals (EDCs) in the environment has been implicated in the aetiology of GDM (Elobeid and Allison 2008, Newbold et al 2009, Rubin and Sato 2009). A connection at the epidemiologic level in humans has been recently proposed for dioxin, an environmental contaminant that acts through other than estrogen receptors (ERs) as an endocrine disruptor (Bertazzi et al 2001, Remillard et al 2002). #### 3.4 Autoimmunity and Treg in GDM Autoimmune phenomena associated with type 1 diabetes mellitus (DM) can also be detected in a subgroup of women with GDM. Islet autoantibodies are present in sera from women with GDM with variable frequency. Distinct phenotypic and genotypic features may be recognised in this subset of women with GDM, which are representative of a distinct clinical entity. Women with previous autoimmune GDM may be candidates for potential immune intervention strategies (Mauricio et al 2001). Normal activity of Treg subpopulations are disrupted in GDM by mechanisms that threaten pregnancy and may contribute to other features of the disorder, with higher percentages of activated T cells than a matched population of healthy pregnant women. BuChE detoxifies anticholinesterases (AC) that are known to threaten pregnancy and one or more of these fetotoxins adversely impacts pregnancy outcome through a mechanism that may include Treg cells. In normal pregnancy, there is correlation between Treg activity and BuChE, whereas in women in whom adequate correlation between Treg cells and BuChE activity is not achieved (Mahmoud et al 2003, Mahmoud et al 2006, Mahmoud et al 2008, Saito et al 2005), there is Unravelling the Connection Between Gestational Diabetes Mellitus and Butyrylcholinesterase 231 named "pseudocholinesterase" by Mendel and Rudney in 1943 (1943). Human plasma BuChE (EC 3.1.1.8) is a globular, tetrameric serine esterase with a molecular mass of ≈340 kDa that is stable in plasma with a half-life of 12 days (Lockridge et al 1987, Ostergaard et al 1988). BuChE acts on hydrophilic and hydrophobic choline esters, and that it hydrolyzes a variety of xenobiotics as shown in Table 2. Previous studies have reported a significant association between the serum BuChE activity and obesity, coronary artery disease, serum levels of triglycerides (TG), very low-density lipoprotein, low-density lipoprotein and Apo lipoprotein B, type 2 diabetes mellitus and the hepatic fat content (Alcantara et al 2005, Cucuianau et al 1999, Randell et 2005, Sridhar et al 2005). At variance with AChE-S, BuChE attenuates the fibril-formation process by the aromatic W8 residue. This residue can form heteroaromatic complexes with soluble monomeric or low-oligomeric Aβ conformers. That replacement of tryptophan to a polar residue abolishes the attenuation of Aβ fibril formation is fully compatible with this hypothesis. AChE mRNA is 20-fold more abundant than BuChE mRNA. In human blood, however, BuChE, at 50 nM, is 3-fold more abundant than BuChE protein or mRNA has been found in almost every tissue of the body, showing that The complete amino acid sequence of human serum BuChE have been described (Daresh et al 2003). The human butyrylcholinesterase (BuChE; EC 3.1.1.8) is encoded by a single gene which corresponds to the E1 locus BuCHE gene (3q26.1-q26.2) which presents four exons (Arpagaus et al 1993), with more than 70 already-described variants (Pantuck 1993, Souza et al 2005). Data from dizygotic twin pairs has shown linkage on chromosome 3 at the location of the BuChE gene and also on chromosome 5. BuChE is found in human plasma, either in homomeric viz. monomers (G1), dimers (G2), trimers (G3) and tetramers (G4), or heteromeric forms associated with other substances, such as albumin (G1-ALB) (Masson et AChE (Daresh et al 2003). 2. Protection from neurotoxins - OP nerve agents - OP pesticides Table 2. Functions of BuChE 5.1 Genetics of BuChE 4. Not clear yet 1. Acetylcholine and butyrylthiocholine hydrolysis. it has a function. failure of effective clearance of toxicants which adversely affect maternal immunomodulation in ways that can lead to GDM and other pregnancy-threatening conditions (Baccarelli et al 2002, Bertazzi et al 2001, Eskenazi et al 2004, Lappas et al 2010, Maussolie et al 1992, Remillard et al 2002). #### 3.5 Genetics of gestational diabetes mellitus There is very little published data about the genetic basis for gestational diabetes mellitus (GDM) (Watanabe et al 2007). However, there is evidence for clustering of type 2 diabetes and impaired glucose tolerance in families with a GDM (McLellan et al 1995) and evidence for higher prevalence of type 2 diabetes in mothers of women with GDM (Martin et al 1985). HLA DR3 and DR4 antigens are in higher frequency in women with GDM than in women with normal pregnancies. Furthermore, an association between variation in the insulin receptor (INSR) in Caucasian and African-American women with GDM has been reported (Ober et al 1989). A β-cell defect is one of the primary characteristics of GDM and β-cell function is a highly heritable trait(Watanabe et al 2007). #### 4. Association between GDM and oxidative stress and diabetic complications Pregnancy is susceptible to oxidative stress and antioxidant defenses that can be altered in response to elevated levels of oxidative stress (Chen and Scholl 2005. Marfella et al 2001, Maxwell et al 1997). In GDM products of lipid peroxidation may be increased and antioxidant enzyme activities decreased and the oxygen free radicals may be involved in severe damage of cellular structure (Osawa andKato 2005, Twardowska-Saucha et al 1994) and pregnancy complicated by poor glycemic control is associated with a higher risk of embryopathies, spontaneous abortion and perinatal morbidity and mortality (Loeken 2006). Recently, Karacay et al (2010) demonstrated that plasma and serum maternal total antioxidant status (TAS) was decreased, while circulating levels of lipid peroxidation breakdown products (MDA) were increased between 24 and 36 weeks of gestation, thus showing that increased oxidative stress and reduction in antioxidant defense mechanisms may contribute to disease processes in GDM (Bertazzi et al 2001, Karacy et al 2010, Rustemeijer et al 2001). Carine et al (1993) and Zachara et al (1993) found no differences in glutathione peroxidase (GPX) levels between pregnant women at third trimester and non-pregnant women, but recent studies have demonstrated an association between GDM and impaired SOD activities and enhanced circulating lipid metabolite levels such as MDA (Grissa et al 2007). Catalase, the main regulator of hydrogen peroxide metabolism is involved in Glut 4 expression, insulin secretion, insulin signaling, protein tyrosine phosphatase regulation, and glucose transport stimulation (Goth et al 2005, Mueller et al 1997). Catalase is important in antioxidant defense against hydrogen peroxide and increased risk of diabetes has been reported in hereditary catalase deficiency (Goth and Eaton 2000, Sindhu et al 2004). #### 5. Biology of BuChE The enzyme cholinesterase is present in all mammals and two classes have been identified :acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE); AChE exists in the central nervous system, platelets and the erythrocyte membrane, while BChE is more abundant in the serum and is synthesized by the liver (Daresh et al 2003). BuChE was named "pseudocholinesterase" by Mendel and Rudney in 1943 (1943). Human plasma BuChE (EC 3.1.1.8) is a globular, tetrameric serine esterase with a molecular mass of ≈340 kDa that is stable in plasma with a half-life of 12 days (Lockridge et al 1987, Ostergaard et al 1988). BuChE acts on hydrophilic and hydrophobic choline esters, and that it hydrolyzes a variety of xenobiotics as shown in Table 2. Previous studies have reported a significant association between the serum BuChE activity and obesity, coronary artery disease, serum levels of triglycerides (TG), very low-density lipoprotein, low-density lipoprotein and Apo lipoprotein B, type 2 diabetes mellitus and the hepatic fat content (Alcantara et al 2005, Cucuianau et al 1999, Randell et 2005, Sridhar et al 2005). At variance with AChE-S, BuChE attenuates the fibril-formation process by the aromatic W8 residue. This residue can form heteroaromatic complexes with soluble monomeric or low-oligomeric Aβ conformers. That replacement of tryptophan to a polar residue abolishes the attenuation of Aβ fibril formation is fully compatible with this hypothesis. AChE mRNA is 20-fold more abundant than BuChE mRNA. In human blood, however, BuChE, at 50 nM, is 3-fold more abundant than AChE (Daresh et al 2003). BuChE protein or mRNA has been found in almost every tissue of the body, showing that it has a function. Table 2. Functions of BuChE #### 5.1 Genetics of BuChE 230 Gestational Diabetes failure of effective clearance of toxicants which adversely affect maternal immunomodulation in ways that can lead to GDM and other pregnancy-threatening conditions (Baccarelli et al 2002, Bertazzi et al 2001, Eskenazi et al 2004, Lappas et al 2010, There is very little published data about the genetic basis for gestational diabetes mellitus (GDM) (Watanabe et al 2007). However, there is evidence for clustering of type 2 diabetes and impaired glucose tolerance in families with a GDM (McLellan et al 1995) and evidence for higher prevalence of type 2 diabetes in mothers of women with GDM (Martin et al 1985). HLA DR3 and DR4 antigens are in higher frequency in women with GDM than in women with normal pregnancies. Furthermore, an association between variation in the insulin receptor (INSR) in Caucasian and African-American women with GDM has been reported (Ober et al 1989). A β-cell defect is one of the primary characteristics of GDM and β-cell 4. Association between GDM and oxidative stress and diabetic complications Pregnancy is susceptible to oxidative stress and antioxidant defenses that can be altered in response to elevated levels of oxidative stress (Chen and Scholl 2005. Marfella et al 2001, In GDM products of lipid peroxidation may be increased and antioxidant enzyme activities decreased and the oxygen free radicals may be involved in severe damage of cellular structure (Osawa andKato 2005, Twardowska-Saucha et al 1994) and pregnancy complicated by poor glycemic control is associated with a higher risk of embryopathies, spontaneous abortion and perinatal morbidity and mortality (Loeken 2006). Recently, Karacay et al (2010) demonstrated that plasma and serum maternal total antioxidant status (TAS) was decreased, while circulating levels of lipid peroxidation breakdown products (MDA) were increased between 24 and 36 weeks of gestation, thus showing that increased oxidative stress and reduction in antioxidant defense mechanisms may contribute to disease processes in GDM (Bertazzi et al 2001, Karacy et al 2010, Rustemeijer et al 2001). Carine et al (1993) and Zachara et al (1993) found no differences in glutathione peroxidase (GPX) levels between pregnant women at third trimester and non-pregnant women, but recent studies have demonstrated an association between GDM and impaired SOD activities and enhanced circulating lipid metabolite levels such as MDA (Grissa et al 2007). Catalase, the main regulator of hydrogen peroxide metabolism is involved in Glut 4 expression, insulin secretion, insulin signaling, protein tyrosine phosphatase regulation, and glucose transport stimulation (Goth et al 2005, Mueller et al 1997). Catalase is important in antioxidant defense against hydrogen peroxide and increased risk of diabetes has been reported in hereditary The enzyme cholinesterase is present in all mammals and two classes have been identified :acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE); AChE exists in the central nervous system, platelets and the erythrocyte membrane, while BChE is more abundant in the serum and is synthesized by the liver (Daresh et al 2003). BuChE was Maussolie et al 1992, Remillard et al 2002). 3.5 Genetics of gestational diabetes mellitus function is a highly heritable trait(Watanabe et al 2007). catalase deficiency (Goth and Eaton 2000, Sindhu et al 2004). Maxwell et al 1997). 5. Biology of BuChE The complete amino acid sequence of human serum BuChE have been described (Daresh et al 2003). The human butyrylcholinesterase (BuChE; EC 3.1.1.8) is encoded by a single gene which corresponds to the E1 locus BuCHE gene (3q26.1-q26.2) which presents four exons (Arpagaus et al 1993), with more than 70 already-described variants (Pantuck 1993, Souza et al 2005). Data from dizygotic twin pairs has shown linkage on chromosome 3 at the location of the BuChE gene and also on chromosome 5. BuChE is found in human plasma, either in homomeric viz. monomers (G1), dimers (G2), trimers (G3) and tetramers (G4), or heteromeric forms associated with other substances, such as albumin (G1-ALB) (Masson et Unravelling the Connection Between Gestational Diabetes Mellitus and Butyrylcholinesterase 233 Another reason for continued interest in serum cholinesterase is its extraordinary sensitivity to organophosphate ester. Systemic administration of BuChE, at a dose sufficient to increase plasma BuChE levels 400-fold (5000 I.U.; i.v.), has been shown to significantly decrease cocaine-induced locomotor activity in rats over a 120-min session (Carmona et al 1997). The identification of BuChE variants that exhibit increased cocaine hydrolysis activity provides treatment options for cocaine-induced conditions such as cocaine overdose and addiction Serum levels of BuChE are affected by dietary fat, obesity, hyperlipidemia and diabetes mellitus, alcohol and many drugs are known to increase BuChE activity (Alcantara et al 2005, Stefanello et al 2005, Vrdoljaki et al 2005). Therefore, BuChE may have a role in the altered lipoprotein metabolism in hypertriglyceridaemia associated with diabetes mellitus and insulin resistance. BuChE is synthesized in the liver, and is present in plasma and to a lesser extent in adipose tissue, small intestine and smooth muscle. Sridhar et al (2005) measured the serum level of BuChE levels in persons with type 2 diabetes mellitus and demonstrated a negative correlation between BuChE and serum total cholesterol and LDL cholesterol, thus further confirming that BuChE may be involved in lipid metabolism. The application of the techniques of molecular genetics has permitted precise identification of plasma cholinesterase variants and has resulted in the discovery of previously unrecognized variants. Serum BuChE activity has been determined by the method of Ellman et al (1961). In addition to colorimetric methods, HPLC, Electrophoresis, Immunoassay Human BuChE has been obtained from human plasma by a large scale purification technique (Lockridge et al 2005). This procedure is severely limited by the volume of human plasma needed and may not be cost effective and it may not yield a sufficient amount of enzyme purified commercially. Large quantities of BuChE are needed for effective prophylaxis and treatment of exposure. BuChE has a broad spectrum of activity, a relatively long half-life, and few physiological side effects. Producing recombinant BuChE (rBuChE) is an alternative to purification of the enzyme from human plasma. A number of studies have shown the feasibility of producing large quantities of BuChE in transgenic animals (goats) and transgenic edible plants for prophylaxis or treatment of humans exposed to OP agents (Lockridge et al 2005, Podoly et al 2008, Protexia 2011) and cocaine overdose or addiction (Om et al 1993). Stefanello et al. (2005) investigated the effect of homocysteine administration on BuChE activity in the serum of rats. Acute and chronic administration of homocysteine significantly decreased BuChE activity but administration of vitamins A and C prevented the reduction of the activity. Delwing et al (2005) observed that acute proline administration provoked a 22% increase in BuChE activity in the serum of rats. In a similar study, Wyse et al (2004) 6.2 BuChE and Organophosphatase (OP) and cocaine hydrolysis (Arkhypova et al 2004, Lockridge et al 2005), Lynch et al 1997). methods (ELIZA) and Biosensor methods have been used. 7. Association between BuChE and oxidative stress 6.4 Serum determination of BuChE 6.5 Production of human BuChE 6.3 BuChE activity and dyslipidemia and metabolic syndrome al 1989, Pantuck 1993). The BCHE-K variant has been reported to show allelic association with Alzheimer disease (AD) in subjects who are also carriers of the e4 allele of apolipoprotein E (APOE), especially in subjects over the age of 75 years. The K variant, is carried on one allele by one of four persons (Rao et al 2006). As BuChE is found common to both Alzheimer's disease and diabetes; it may play an etiological role via influencing insulin resistance and lipid metabolism (Arpagaus et al 1990, Lockridge et al 1987, Rao et al 2006). Similarly patients with Alzheimer's disease are more vulnerable to developing impaired fasting glucose and type 2 diabetes mellitus (Janson et al 2004, Johansen et al 1991) #### 5.2 BuChE and placental development In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous absorption and placental malfunction. The major protein interacting with these compounds is butyrylcholinesterase (BuChE), which attenuates the effects of such xenobiotics by their hydrolysis or sequestration. Sternfeld and Associates (1997) studied BuChE expression during placental development. RT-PCR revealed both BuChE mRNA and acetylcholinesterase (AChE) mRNA throughout gestation. Maximum butyrylcholinesterase activity has shown in week 12. In rat placenta, BuChE activity on gestational day 21 reached 150% of the level on gestational day 16. BuChE detoxifies anticholinesterases (AC) and other toxins including free radicals that are known to threaten pregnancy (Hollander et al 2007, Maxwell et al 1997, Osawa and Kato 2005, Twardowska-Saucha et al 1994). There is evidence that Kuwaiti women experiencing disorders of pregnancy like preeclampsia and diabetes mellitus in pregnancy exhibited lower serum activity of BuChE (Mahmoud et al 2003, Mahmoud et al 2006, Mahmoud et al 2008). #### 6. Clinical role of BuChE BuChE (BuChE; EC 3.1.1.1.8) has well-defined pharmacologic functions: BuChE and anaesthetic muscle relaxants: Mivacurium and succinylcholine are short-acting neuromuscular blocking drugs ideal for short surgical procedures as muscle relaxants used in anesthetic practice. The brief duration of action depends on rapid hydrolysis by plasma cholinesterase (Jensen et al 1991Pantuck 1993). An inherited or acquired deficiency of plasma BuChE can prolong the effect of mivacurium. When there is a deficiency of this enzyme due to the presence of one or more atypical alleles, mivacurium and succinylcholine are not properly metabolized and thus muscle paralysis can last for several hours (Davis et al 1997, Goudsouzian et al 1993, Petersen et al 1993, Savarese et al 1997). #### 6.1 Factors affecting BuChE activity Different disease states and/or drug administrations may decrease BuChE activity.; such as extremes of age, pregnancy, renal and liver disease, malignancy, burns, chronic debility/malnutrition, myocardial infarction/cardiac failure, collagen diseases, myxedema, poisoning and protein energy malnutrition. Drugs that inhibit the enzyme's activity include acetylcholinesterase inhibitors (neostigmine, pyridostigmine, physostigmine, and edrophonium), anticholinesterases (especially echothlophate), cytotoxic agents (such as cyclophosphamide), steroids, ester-type local anesthetics, hexafluorenium, pancuronium, oral contraceptives and sertraline (Klein-Schwatz and Anderson 1996, MacQueen et al 2001, Muller et al 2002). #### 6.2 BuChE and Organophosphatase (OP) and cocaine hydrolysis Another reason for continued interest in serum cholinesterase is its extraordinary sensitivity to organophosphate ester. Systemic administration of BuChE, at a dose sufficient to increase plasma BuChE levels 400-fold (5000 I.U.; i.v.), has been shown to significantly decrease cocaine-induced locomotor activity in rats over a 120-min session (Carmona et al 1997). The identification of BuChE variants that exhibit increased cocaine hydrolysis activity provides treatment options for cocaine-induced conditions such as cocaine overdose and addiction (Arkhypova et al 2004, Lockridge et al 2005), Lynch et al 1997). #### 6.3 BuChE activity and dyslipidemia and metabolic syndrome Serum levels of BuChE are affected by dietary fat, obesity, hyperlipidemia and diabetes mellitus, alcohol and many drugs are known to increase BuChE activity (Alcantara et al 2005, Stefanello et al 2005, Vrdoljaki et al 2005). Therefore, BuChE may have a role in the altered lipoprotein metabolism in hypertriglyceridaemia associated with diabetes mellitus and insulin resistance. BuChE is synthesized in the liver, and is present in plasma and to a lesser extent in adipose tissue, small intestine and smooth muscle. Sridhar et al (2005) measured the serum level of BuChE levels in persons with type 2 diabetes mellitus and demonstrated a negative correlation between BuChE and serum total cholesterol and LDL cholesterol, thus further confirming that BuChE may be involved in lipid metabolism. #### 6.4 Serum determination of BuChE 232 Gestational Diabetes al 1989, Pantuck 1993). The BCHE-K variant has been reported to show allelic association with Alzheimer disease (AD) in subjects who are also carriers of the e4 allele of apolipoprotein E (APOE), especially in subjects over the age of 75 years. The K variant, is carried on one allele by one of four persons (Rao et al 2006). As BuChE is found common to both Alzheimer's disease and diabetes; it may play an etiological role via influencing insulin resistance and lipid metabolism (Arpagaus et al 1990, Lockridge et al 1987, Rao et al 2006). Similarly patients with Alzheimer's disease are more vulnerable to developing impaired In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous absorption and placental malfunction. The major protein interacting with these compounds is butyrylcholinesterase (BuChE), which attenuates the effects of such xenobiotics by their hydrolysis or sequestration. Sternfeld and Associates (1997) studied BuChE expression during placental development. RT-PCR revealed both BuChE mRNA and acetylcholinesterase (AChE) mRNA throughout gestation. Maximum butyrylcholinesterase activity has shown in week 12. In rat placenta, BuChE activity on gestational day 21 reached 150% of the level on gestational day 16. BuChE detoxifies anticholinesterases (AC) and other toxins including free radicals that are known to threaten pregnancy (Hollander et al 2007, Maxwell et al 1997, Osawa and Kato 2005, Twardowska-Saucha et al 1994). There is evidence that Kuwaiti women experiencing disorders of pregnancy like preeclampsia and diabetes mellitus in pregnancy exhibited lower serum fasting glucose and type 2 diabetes mellitus (Janson et al 2004, Johansen et al 1991) activity of BuChE (Mahmoud et al 2003, Mahmoud et al 2006, Mahmoud et al 2008). BuChE and anaesthetic muscle relaxants: Mivacurium and succinylcholine are short-acting neuromuscular blocking drugs ideal for short surgical procedures as muscle relaxants used in anesthetic practice. The brief duration of action depends on rapid hydrolysis by plasma cholinesterase (Jensen et al 1991Pantuck 1993). An inherited or acquired deficiency of plasma BuChE can prolong the effect of mivacurium. When there is a deficiency of this enzyme due to the presence of one or more atypical alleles, mivacurium and succinylcholine are not properly metabolized and thus muscle paralysis can last for several hours (Davis et Different disease states and/or drug administrations may decrease BuChE activity.; such as extremes of age, pregnancy, renal and liver disease, malignancy, burns, chronic debility/malnutrition, myocardial infarction/cardiac failure, collagen diseases, myxedema, poisoning and protein energy malnutrition. Drugs that inhibit the enzyme's activity include acetylcholinesterase inhibitors (neostigmine, pyridostigmine, physostigmine, and edrophonium), anticholinesterases (especially echothlophate), cytotoxic agents (such as cyclophosphamide), steroids, ester-type local anesthetics, hexafluorenium, pancuronium, oral contraceptives and sertraline (Klein-Schwatz and Anderson 1996, MacQueen et al 2001, BuChE (BuChE; EC 3.1.1.1.8) has well-defined pharmacologic functions: al 1997, Goudsouzian et al 1993, Petersen et al 1993, Savarese et al 1997). 5.2 BuChE and placental development 6. Clinical role of BuChE 6.1 Factors affecting BuChE activity Muller et al 2002). The application of the techniques of molecular genetics has permitted precise identification of plasma cholinesterase variants and has resulted in the discovery of previously unrecognized variants. Serum BuChE activity has been determined by the method of Ellman et al (1961). In addition to colorimetric methods, HPLC, Electrophoresis, Immunoassay methods (ELIZA) and Biosensor methods have been used. #### 6.5 Production of human BuChE Human BuChE has been obtained from human plasma by a large scale purification technique (Lockridge et al 2005). This procedure is severely limited by the volume of human plasma needed and may not be cost effective and it may not yield a sufficient amount of enzyme purified commercially. Large quantities of BuChE are needed for effective prophylaxis and treatment of exposure. BuChE has a broad spectrum of activity, a relatively long half-life, and few physiological side effects. Producing recombinant BuChE (rBuChE) is an alternative to purification of the enzyme from human plasma. A number of studies have shown the feasibility of producing large quantities of BuChE in transgenic animals (goats) and transgenic edible plants for prophylaxis or treatment of humans exposed to OP agents (Lockridge et al 2005, Podoly et al 2008, Protexia 2011) and cocaine overdose or addiction (Om et al 1993). #### 7. Association between BuChE and oxidative stress Stefanello et al. (2005) investigated the effect of homocysteine administration on BuChE activity in the serum of rats. Acute and chronic administration of homocysteine significantly decreased BuChE activity but administration of vitamins A and C prevented the reduction of the activity. Delwing et al (2005) observed that acute proline administration provoked a 22% increase in BuChE activity in the serum of rats. In a similar study, Wyse et al (2004) Unravelling the Connection Between Gestational Diabetes Mellitus and Butyrylcholinesterase 235 in response to paternal antigens adversely affect maternal health in ways that increase susceptibility to diabetes, thereby leaving women with naturally lower BuChE levels at greater risk for gestational and possibly Type 2 diabetes. The relationship between size of activated lymphocyte cohorts and BuChE activity in the RPL versus healthy cohorts provides additional support for the hypothesis that both immune activation and BuChE activity may be tied to some as-yet-unidentified systemic effector. For example, our observation of positive correlation between the frequency of CD3+CD16+CD56+ cells and BuChE activity in healthy individuals but not RPL-afflicted subjects would be expected if these cells which are often pathogenic, were expanded in response to environmental toxins. In the type 2 diabetes mellitus population serum BuChE activity has been correlated with insulin sensitivity (r = -0.51, P < 0.001). BuChE activity was elevated in the serum and placenta in normal pregnancy versus diabetic cohorts (p < 0.01) and there was a higher activity level in gestational and type 2 diabetes on insulin (p < 0.05) compared with diet controlled (Omu et al 2010, Mahmoud et al 2003, Mahmoud et al 2006, Mahmoud et al 2008, BuChE may also play a significant role in congenital anomalies. Dupont et al. (1995) have reported that fetuses with anencephaly and open spinal bifida and gastroschisis revealed clearly dense band of BuChE in the amniotic fluid. A causative role for elevated free fatty acid (FFA) levels in the development of microvascular complications remains to be established, however. Increased levels of FFAs are positively correlated with both insulin resistance and the deterioration of ß-cell function in the context of concomitant hyperglycemia. These latter effects may result from oxidative stress (Evans et al 2003). 9.1 Role of estrogen alpha receptor and BuChE in pathogenesis of gestational 9. Future directions and hypotheses of connection between BuChE and GDM There is need to explore a number of hypotheses to fully unravel the connection between High levels of estrogens in the second half of pregnancy with high estrogen receptor alpha (ER ) lead to deterioration of glucose metabolism. Estrogens may reduce the risk of AD through enhancing or preserving cholinergic neurotransmission, and aromatase, the product of the CYP19 gene, is a critical enzyme in the peripheral synthesis of estrogens. There is evidence to suggest that the CYP19 and BuChE polymorphisms may interact in determining the risk of AD. Carriers of both the ER-a P/P genotype and the BuChE K variant would have decreased risk of developing AD (Conbarros et al 2007). ER alpha signaling activity and glucose metabolism may therefore be affected by CYP19 and BuChE Advanced glycation end products may inhibit BuChE activities, probably as a result of the Rustemeijer et al 2001) diabetes polymorphisms. 8.1 BuChE and congenital anomalies GDM and BuChE through aggressive research efforts. 9.2 Advanced glycation end products hydrolysis by BuChE hydrolyzing effect of the latter, upstream before they cause oxidative stress. demonstrated that vitamins E and C reversed the inhibition of BuChE activities provoked by arginine in the serum of rats, thus indicating that the reduction of BuChE activities caused by arginine was probably mediated by oxidative stress. In a similar fashion, Cederberg et al. (2001) have shown that combined treatment with vitamins E and C decreased oxidative stress and improved fetal outcome in experimental pregnancy. From the foregoing, BuChE may yet be another mechanism in the fight against oxidative stress. #### 7.1 Mechanisms of the association between BuChE and oxidative stress in GDM In a recent report, we (Omu et al 2010) showed that BuChE activity was elevated in the serum and placenta in normal pregnancy as compared to diabetic cohorts (p < 0.01) and there was a higher activity level in gestational and type 2 diabetes on insulin (p<0.05) compared with diet controlled. Conversely, there was higher MDA and lower antioxidant activity in diet versus insulin controlled diabetes (p < 0.01). Both serum and placental BuChE activity showed a strong inverse correlation with MDA (r = -0.876, p < 0.001) and (r = - 0.542, p < 0.01), but strong positive correlation with total antioxidant activity in serum (r = 0.764, p < 0.001) and placenta (r = 0.642, p < 0.01). These results are therefore consistent with a mechanism in which BuChE acts to scavenge free radicals in the presence of oxidative stress. An interesting finding in the study was the higher BuChE activity in the two groups of insulin-treated diabetics compared with their counterparts on diet. This led to the speculation that the diabetic patients on diet only might not have had satisfactory glycogenic control. However, BuChE did not show any correlation with enzymatic antioxidants SOD and GPX (Omu et al 2010); indirectly showing that BuChE was not inhibiting MDA through the antioxidants pathway. While this is mere speculation, it has important clinical implication if the association between BuChE and glycemic control is confirmed by future research. HbA1c has been used for monitoring diabetic control of the last 3 months, maybe BuChE could be used for short term or immediate monitoring of glycemic control. BuChE is already a known marker of metabolic syndrome (Sridhar et al 2005), and its activity is high in human term placenta (Hahn et al 1993, Lappas et al 2010, Omu et al 2010, Simone et al 1994, Sternfield et al 1997). The lower level of placental BuChE activity compared with serum, shown in the study may be as a result of a high level of fetotoxic agents, including free radicals (oxidative stress), in the placenta that BuChE metabolises by hydrolysis. #### 7.2 Advanced glycation end-products (AGE), reactive oxygen species (ROS) and BuChE Glycation reactions lead to the production of reactive oxygen species (ROS), which are harmful to cellular metabolism and cause cell damage. There are no research data of any relationship between AGE and BuChE activity. While it is highly speculative, BuChE may protect pregnancy from the effect of oxidative stress by preventing the (formation) of reactive oxygen species formation by hydrolyzing and inactivating advanced glycation end products upstream. This hypothesis is consistent with the finding of lack of correlation between BuChE and SOD and GPX (Omu et al 2010). #### 8. Gestational diabetes mellitus and BuChE Another contributor to toxicant-induced immune dysregulation as a contributor to GDM might be that reactive products of inflammation expressed by the maternal immune system in response to paternal antigens adversely affect maternal health in ways that increase susceptibility to diabetes, thereby leaving women with naturally lower BuChE levels at greater risk for gestational and possibly Type 2 diabetes. The relationship between size of activated lymphocyte cohorts and BuChE activity in the RPL versus healthy cohorts provides additional support for the hypothesis that both immune activation and BuChE activity may be tied to some as-yet-unidentified systemic effector. For example, our observation of positive correlation between the frequency of CD3+CD16+CD56+ cells and BuChE activity in healthy individuals but not RPL-afflicted subjects would be expected if these cells which are often pathogenic, were expanded in response to environmental toxins. In the type 2 diabetes mellitus population serum BuChE activity has been correlated with insulin sensitivity (r = -0.51, P < 0.001). BuChE activity was elevated in the serum and placenta in normal pregnancy versus diabetic cohorts (p < 0.01) and there was a higher activity level in gestational and type 2 diabetes on insulin (p < 0.05) compared with diet #### 8.1 BuChE and congenital anomalies Rustemeijer et al 2001) 234 Gestational Diabetes demonstrated that vitamins E and C reversed the inhibition of BuChE activities provoked by arginine in the serum of rats, thus indicating that the reduction of BuChE activities caused by arginine was probably mediated by oxidative stress. In a similar fashion, Cederberg et al. (2001) have shown that combined treatment with vitamins E and C decreased oxidative stress and improved fetal outcome in experimental pregnancy. From the foregoing, BuChE 7.1 Mechanisms of the association between BuChE and oxidative stress in GDM In a recent report, we (Omu et al 2010) showed that BuChE activity was elevated in the serum and placenta in normal pregnancy as compared to diabetic cohorts (p < 0.01) and there was a higher activity level in gestational and type 2 diabetes on insulin (p<0.05) compared with diet controlled. Conversely, there was higher MDA and lower antioxidant activity in diet versus insulin controlled diabetes (p < 0.01). Both serum and placental BuChE activity showed a strong inverse correlation with MDA (r = -0.876, p < 0.001) and (r = - 0.542, p < 0.01), but strong positive correlation with total antioxidant activity in serum (r = 0.764, p < 0.001) and placenta (r = 0.642, p < 0.01). These results are therefore consistent with a mechanism in which BuChE acts to scavenge free radicals in the presence of oxidative stress. An interesting finding in the study was the higher BuChE activity in the two groups of insulin-treated diabetics compared with their counterparts on diet. This led to the speculation that the diabetic patients on diet only might not have had satisfactory glycogenic control. However, BuChE did not show any correlation with enzymatic antioxidants SOD and GPX (Omu et al 2010); indirectly showing that BuChE was not inhibiting MDA through the antioxidants pathway. While this is mere speculation, it has important clinical implication if the association between BuChE and glycemic control is confirmed by future research. HbA1c has been used for monitoring diabetic control of the last 3 months, maybe BuChE could be used for short term or immediate monitoring of glycemic control. BuChE is already a known marker of metabolic syndrome (Sridhar et al 2005), and its activity is high in human term placenta (Hahn et al 1993, Lappas et al 2010, Omu et al 2010, Simone et al 1994, Sternfield et al 1997). The lower level of placental BuChE activity compared with serum, shown in the study may be as a result of a high level of fetotoxic agents, including free radicals (oxidative stress), in the placenta that BuChE 7.2 Advanced glycation end-products (AGE), reactive oxygen species (ROS) and Glycation reactions lead to the production of reactive oxygen species (ROS), which are harmful to cellular metabolism and cause cell damage. There are no research data of any relationship between AGE and BuChE activity. While it is highly speculative, BuChE may protect pregnancy from the effect of oxidative stress by preventing the (formation) of reactive oxygen species formation by hydrolyzing and inactivating advanced glycation end products upstream. This hypothesis is consistent with the finding of lack of correlation between BuChE Another contributor to toxicant-induced immune dysregulation as a contributor to GDM might be that reactive products of inflammation expressed by the maternal immune system may yet be another mechanism in the fight against oxidative stress. metabolises by hydrolysis. and SOD and GPX (Omu et al 2010). 8. Gestational diabetes mellitus and BuChE BuChE BuChE may also play a significant role in congenital anomalies. Dupont et al. (1995) have reported that fetuses with anencephaly and open spinal bifida and gastroschisis revealed clearly dense band of BuChE in the amniotic fluid. A causative role for elevated free fatty acid (FFA) levels in the development of microvascular complications remains to be established, however. Increased levels of FFAs are positively correlated with both insulin resistance and the deterioration of ß-cell function in the context of concomitant hyperglycemia. These latter effects may result from oxidative stress (Evans et al 2003). controlled (Omu et al 2010, Mahmoud et al 2003, Mahmoud et al 2006, Mahmoud et al 2008, #### 9. Future directions and hypotheses of connection between BuChE and GDM There is need to explore a number of hypotheses to fully unravel the connection between GDM and BuChE through aggressive research efforts. #### 9.1 Role of estrogen alpha receptor and BuChE in pathogenesis of gestational diabetes High levels of estrogens in the second half of pregnancy with high estrogen receptor alpha (ER ) lead to deterioration of glucose metabolism. Estrogens may reduce the risk of AD through enhancing or preserving cholinergic neurotransmission, and aromatase, the product of the CYP19 gene, is a critical enzyme in the peripheral synthesis of estrogens. There is evidence to suggest that the CYP19 and BuChE polymorphisms may interact in determining the risk of AD. Carriers of both the ER-a P/P genotype and the BuChE K variant would have decreased risk of developing AD (Conbarros et al 2007). ER alpha signaling activity and glucose metabolism may therefore be affected by CYP19 and BuChE polymorphisms. #### 9.2 Advanced glycation end products hydrolysis by BuChE Advanced glycation end products may inhibit BuChE activities, probably as a result of the hydrolyzing effect of the latter, upstream before they cause oxidative stress. Unravelling the Connection Between Gestational Diabetes Mellitus and Butyrylcholinesterase 237 Cederberg J, Siman CM, Eriksson UJ.. Combined treatment with vitamins E and C decrease Chen X, Scholl TO. Oxidative stress: changes in pregnancy and with gestational diabetes Combarros O, Riancho JA, Arozamena J, Mateo I, Llorca J, Infante J et al. Interaction Cucuianu M. Serum gamma glutamyltransferase and/or serum cholinesterase as markers of Darvesh S, Hopkins D A. & Geula C. 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Concluding remarks Unraveling the connection between GDM and BuChE has become a veritable area of research in the pathogenesis, screening, prevention and management. With the large scale purification of BuChE from human plasma, milk of transgenic goats and edible transgenic plants and its suitability for prophylactic and therapeutic protection against cocaine and nerve agent toxicity, the way for therapeutic use in humans, especially during complicated pregnancy needs urgent scientific exploration as BuChE may have an important protective role in normal and diabetic pregnancy by reducing oxidative stress and therefore reduce diabetes induced complications. Mechanisms for attenuation of the effects of oxidative stress by BuChE should be investigated. Heritable factors may be an underlying biological thread in the connection between GDM and BuChE. In addition, genetic variants of BuChE exist, which may play a role in biological manifestation of individuals. Identification of such sequences would provide leads for further understanding of aetiological, therapeutic or prognostic aspects of Gestational diabetes mellitus. If future studies reveal that immune dysregulation is a contributor to the pathogenesis of GD or DM, characterization of the mechanisms will open additional avenues to development of therapeutic approaches to both disorders. #### 11. References Unraveling the connection between GDM and BuChE has become a veritable area of research in the pathogenesis, screening, prevention and management. With the large scale purification of BuChE from human plasma, milk of transgenic goats and edible transgenic plants and its suitability for prophylactic and therapeutic protection against cocaine and nerve agent toxicity, the way for therapeutic use in humans, especially during complicated pregnancy needs urgent scientific exploration as BuChE may have an important protective role in normal and diabetic pregnancy by reducing oxidative stress and therefore reduce diabetes induced complications. Mechanisms for attenuation of the effects of oxidative stress by BuChE should be investigated. Heritable factors may be an underlying biological thread in the connection between GDM and BuChE. In addition, genetic variants of BuChE exist, which may play a role in biological manifestation of individuals. Identification of such sequences would provide leads for further understanding of aetiological, therapeutic or prognostic aspects of Gestational diabetes mellitus. 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Environ Health Perspect 2002; 110:853-858. 2006, 5:28-34. 212, 6:559–56. 13 USA Gestational Hyperglycemia, Xinhua Chen, Theresa O. Scholl, Robert A. Steer and T. Peter Stein University of Medicine and Dentistry of New Jersey – School of Osteopathic Medicine Excessive Pregnancy Weight Gain and Risk of Fetal Overgrowth Between 1989 and 2004 the prevalence of gestational diabetes mellitus (GDM) in the US increased from 1.9% to 4.2% in parallel with the well documented obesity epidemic (Getahun et al., 2008; Mokdad et al., 2001). However, an additional 9-20% of pregnant women, with a milder form of glucose intolerance which does not meet the diagnostic criteria for GDM, may also be at risk of type 2 diabetes, cardiovascular disease and problems while pregnant (Stamilio et al., 2004; Mello et al., 1997; Bo et al., 2004; Nordin Fetal overgrowth, defined as macrosomia (birth weight >4000g) or large-for-gestational-age birth (LGA, birth weight >90th percentile for a given gestational age) increases maternal morbidity from operative delivery and also causes serious consequences to the offspring including birth trauma, obesity during childhood and type 2 diabetes and metabolic GDM and excessive pregnancy weight gain, especially in obese women are known risk factors for fetal overgrowth (Ray et al., 2001; Hillier et al., 2008). Although previous research has suggested that metabolic abnormalities are also present in pregnant women with less severe hyperglycemia, the clinical implications of milder maternal hyperglycemia are poorly The primary objective of this chapter is to use prospective data from a population of low income and minority women to examine (i) the influence of maternal hyperglycemia including GDM and less severe maternal hyperglycemia on risk of fetal overgrowth; (ii) the association of longitudinally measured excessive pregnancy weight gain with risk of fetal overgrowth (IOM, 2009); (iii) the independent contribution of gestational hyperglycemia Because gestational hyperglycemia and excess weight gain during pregnancy are preventable risk factors, early detection and treatment of mild hyperglycemia and monitoring pregnancy weight gain may be important for reducing the risk of LGA, for reducing childhood and later syndrome in adult life (Boney et al., 2005; Zhang & Bowes, 1995; Langer, 2000). described (Chen et al., 2010; Cheng et al., 2006; Nordin et al., 2006). and excessive pregnancy weight gain to fetal overgrowth. obesity and for improving the long term risk for metabolic disease. 1. Introduction et al., 2006). ## Gestational Hyperglycemia, Excessive Pregnancy Weight Gain and Risk of Fetal Overgrowth Xinhua Chen, Theresa O. Scholl, Robert A. Steer and T. Peter Stein University of Medicine and Dentistry of New Jersey – School of Osteopathic Medicine USA #### 1. Introduction 242 Gestational Diabetes Wyse ATS, Stefanello FM, Chiarani F, Delwing D, Wannmacher CMD, Wajner M. Arginine Yogev, Y. & Visser, G. Obesity, gestational diabetes and pregnancy outcome. Seminars in Zachara BA, Wardak C, Didkowski W, Maciage A, Marchaluk E. Changes in blood selenium .29:385–389. Fetal & Neonatal Medicine 2009, 14, 77-84 human pregnancy. Gynecol Obstet Invest 1993; 35: 12-17. administration decreases cerebral cortex acetylcholinesterase and serum butyrylcholinesterase by oxidative stress induction. Neurochemical Research 2004; and glutathione peroxidase concentrations and glutathione peroxidase activity in Between 1989 and 2004 the prevalence of gestational diabetes mellitus (GDM) in the US increased from 1.9% to 4.2% in parallel with the well documented obesity epidemic (Getahun et al., 2008; Mokdad et al., 2001). However, an additional 9-20% of pregnant women, with a milder form of glucose intolerance which does not meet the diagnostic criteria for GDM, may also be at risk of type 2 diabetes, cardiovascular disease and problems while pregnant (Stamilio et al., 2004; Mello et al., 1997; Bo et al., 2004; Nordin et al., 2006). Fetal overgrowth, defined as macrosomia (birth weight >4000g) or large-for-gestational-age birth (LGA, birth weight >90th percentile for a given gestational age) increases maternal morbidity from operative delivery and also causes serious consequences to the offspring including birth trauma, obesity during childhood and type 2 diabetes and metabolic syndrome in adult life (Boney et al., 2005; Zhang & Bowes, 1995; Langer, 2000). GDM and excessive pregnancy weight gain, especially in obese women are known risk factors for fetal overgrowth (Ray et al., 2001; Hillier et al., 2008). Although previous research has suggested that metabolic abnormalities are also present in pregnant women with less severe hyperglycemia, the clinical implications of milder maternal hyperglycemia are poorly described (Chen et al., 2010; Cheng et al., 2006; Nordin et al., 2006). The primary objective of this chapter is to use prospective data from a population of low income and minority women to examine (i) the influence of maternal hyperglycemia including GDM and less severe maternal hyperglycemia on risk of fetal overgrowth; (ii) the association of longitudinally measured excessive pregnancy weight gain with risk of fetal overgrowth (IOM, 2009); (iii) the independent contribution of gestational hyperglycemia and excessive pregnancy weight gain to fetal overgrowth. Because gestational hyperglycemia and excess weight gain during pregnancy are preventable risk factors, early detection and treatment of mild hyperglycemia and monitoring pregnancy weight gain may be important for reducing the risk of LGA, for reducing childhood and later obesity and for improving the long term risk for metabolic disease. Gestational Hyperglycemia, Excessive Pregnancy Weight Gain and Risk of Fetal Overgrowth 245 Conflicting data on mild hyperglycemia and fetal overgrowth can be, at least in part, due to the different population studied and the criteria used for mild hyperglycemia. Although the HAPO study included multiple countries and populations and had a large sample size, it was focused on developing an international consensus for the diagnosis and treatment of carbohydrate intolerance during pregnancy, and was not designed to compare the difference in LGA between GDM and less severe hyperglycemic pregnancies (HAPO, 2008). Thus, to establish the risk of adverse fetal outcomes in relation to milder degrees of maternal Camden Study is a prospective cohort study of pregnancy outcome and complications in young, generally healthy women residing in one of the poorest cities in the continental United States (Webster & Bishaw, 2006). Women with serious non-obstetric problems (e.g., Lupus, type 1 or 2 diabetes, seizure disorders, malignancies, acute or chronic liver disease, drug or alcohol abuse and psychiatric problems) were excluded from participation. The sample for this analysis totaled 2,373 pregnant women with live births who enrolled The diagnosis of GDM was made using a two-step approach. All participants were initially screened by measuring the plasma glucose concentration 1h after a 50-g oral glucose challenge test (GCT) at 28±0.1 (mean±SE) weeks' gestation. A diagnostic OGTT was performed on that subset of women exceeding the glucose threshold value (>140 mg/dl). The diagnostic criteria for GDM was the Carpenter/Coustan conversion as recommended by the American Diabetes Association (ADA) which defines GDM by two or more glucose values over the cut points of 95,180,155,140 mg/dl at fasting, 1, 2, and 3 hours during a 100g OGTT (American Diabetes Association, 2000). Women with a positive GCT and fewer than two abnormal glucose values were identified as having an impaired GCT without GDM (impaired GCT non-GDM). All of patients diagnosed with GDM obtained dietary counseling and/or insulin treatment for their We identified 2,092 women (88.1%) with normal GCT, 182 women (7.7%) with impaired GCT non-GDM and 99 women (4.2%) with GDM (table 1). Ethnic composition of the cohort was 46% Hispanic, 37.6% African American and 16.6% Caucasian plus others. When the maternal characteristics were rank ordered with respect to glucose tolerance status, older maternal age, higher pre-pregnant body mass index (BMI), obesity and prior history of GDM, all were found to be positively associated with maternal glycemic status (normal GCT, impaired GCT non-GDM or GDM) and represented a linear increasing trend in terms LGA is defined as a neonatal birth weight greater than the 90th percentile for gestational age that has been adjusted for ethnicity, parity and infant sex (Zhang & Bowes, 1995). Women with GDM or impaired GCT without GDM had a ~2-fold increased risk for bearing an LGA infant as compared to women with normal GCT (table 2, model 1) after controlling for maternal confounding variables and using a standard which adjusted LGA for maternal ethnicity, parity and fetal gender. This increased risk persisted after additional adjustments hyperglycemia is clinically important, particularly in high risk US populations. 2.4.1 Definition of GDM and mild hyperglycemia non-GDM in Camden study glycemic control; patients with impaired CGT non-GDM were untreated. of severity (p for trend <0.001 for each). 2.4.2 Maternal hyperglycemia and LGA birth 2.4 The Camden study between 1996-2006. ### 2. Maternal hyperglycemia and fetal overgrowth #### 2.1 Fetal overgrowth Fetal growth is dependent on the capacity of mother to supply nutrients and also on the capacity of the placenta to transfer these nutrients to the fetus (Oken & Gillman., 2003; Ehrenberg et al., 2004). Maternal factors including parity, length of gestation, mother's adult size, and mother's own birth weight are strongly related to fetal growth and development (Langer, 2000). The prevalence of fetal overgrowth was reported as 8-16% from women without GDM and 15-40% from women with gestational hyperglycemia (Mello et al., 1997; Ostlund et al., 2003; Bo et al., 2004; Gruendhammer et al., 2003). #### 2.2 GDM and fetal overgrowth Despite different diagnostic criteria, many studies confirmed that GDM increases the risk of macrosomia or LGA birth (Ricart et al., 2005; Ray et al., 2001; Ehrenberg et al., 2004). Maternal hyperglycemia increases fetal growth via delivery of excess maternal plasma glucose to the fetus, which results in fetal hyperinsulinemia and promotes fetal overgrowth (HAPO, 2008; Oken & Gillman., 2003). GDM women undergoing intensive diabetic care had similar neonatal birth weights and macrosomia rates compared to non-GDM women (Ogonowski et al., 2008) and a clinical trial of continuous glucose monitoring in GDM resulted in a significant improvement in infant birth weight with a reduced risk of macrosomia (Murphy et al., 2008). #### 2.3 Significance of mild gestational hyperglycemia and LGA birth 2.3.1 Prevalence of mild gestational hyperglycemia Recent studies have paid more attention to pregnancy outcomes of women with gestational hyperglycemia less severe than overt GDM. The prevalence of mild hyperglycemia (defined as abnormal plasma glucose concentration during glucose challenge test with a diagnostic oral glucose tolerance test (OGTT) that did not meet the criteria of GDM) in women screened for GDM was 32% in Caucasian women (Mello et al, 1997), 25% in Mexican-Americans (Yogev et al, 2004) and 9% in multiethnic US population where 70% were African American (Stamilio et al., 2004). The relatively high prevalence of less severe maternal hyperglycemia raises important questions about effects on the fetus since these women are not provided the usual diabetic care for GDM. #### 2.3.2 The impact of mild gestational hyperglycemia on fetal overgrowth is inconsistent Studies suggest that an impaired maternal OGTT is associated with adverse maternal-fetal outcomes especially risk of macrosomia and LGA birth (Vambergue et al., 2000; Ostlund et al., 2003). The degree of maternal glucose intolerance was associated with a graded increase in the incidence of fetal macrosomia (Sermer et al., 1995). Moreover, the recent hyperglycemia and adverse pregnancy outcome study (HAPO) found a strong association of maternal glucose concentrations below levels diagnostic of diabetes with increased infant birth weight (HAPO, 2008), but others found no significant increase in the risk of LGA in mildly hyperglycemic women (Nordin et al., 2006; Gruendhammer et al., 2003). Bo et al suggested that metabolic syndrome in mid-pregnancy might be an independent predictor of macrosomia in women with some degree of gestational hyperglycemia, including GDM and mild hyperglycemia without GDM (Bo et al., 2004). Conflicting data on mild hyperglycemia and fetal overgrowth can be, at least in part, due to the different population studied and the criteria used for mild hyperglycemia. Although the HAPO study included multiple countries and populations and had a large sample size, it was focused on developing an international consensus for the diagnosis and treatment of carbohydrate intolerance during pregnancy, and was not designed to compare the difference in LGA between GDM and less severe hyperglycemic pregnancies (HAPO, 2008). Thus, to establish the risk of adverse fetal outcomes in relation to milder degrees of maternal hyperglycemia is clinically important, particularly in high risk US populations. #### 2.4 The Camden study 244 Gestational Diabetes Fetal growth is dependent on the capacity of mother to supply nutrients and also on the capacity of the placenta to transfer these nutrients to the fetus (Oken & Gillman., 2003; Ehrenberg et al., 2004). Maternal factors including parity, length of gestation, mother's adult size, and mother's own birth weight are strongly related to fetal growth and development (Langer, 2000). The prevalence of fetal overgrowth was reported as 8-16% from women without GDM and 15-40% from women with gestational hyperglycemia (Mello et al., 1997; Despite different diagnostic criteria, many studies confirmed that GDM increases the risk of macrosomia or LGA birth (Ricart et al., 2005; Ray et al., 2001; Ehrenberg et al., 2004). Maternal hyperglycemia increases fetal growth via delivery of excess maternal plasma glucose to the fetus, which results in fetal hyperinsulinemia and promotes fetal overgrowth (HAPO, 2008; Oken & Gillman., 2003). GDM women undergoing intensive diabetic care had similar neonatal birth weights and macrosomia rates compared to non-GDM women (Ogonowski et al., 2008) and a clinical trial of continuous glucose monitoring in GDM resulted in a significant improvement in infant birth weight with a reduced risk of Recent studies have paid more attention to pregnancy outcomes of women with gestational hyperglycemia less severe than overt GDM. The prevalence of mild hyperglycemia (defined as abnormal plasma glucose concentration during glucose challenge test with a diagnostic oral glucose tolerance test (OGTT) that did not meet the criteria of GDM) in women screened for GDM was 32% in Caucasian women (Mello et al, 1997), 25% in Mexican-Americans (Yogev et al, 2004) and 9% in multiethnic US population where 70% were African American (Stamilio et al., 2004). The relatively high prevalence of less severe maternal hyperglycemia raises important questions about effects on the fetus since these women are Studies suggest that an impaired maternal OGTT is associated with adverse maternal-fetal outcomes especially risk of macrosomia and LGA birth (Vambergue et al., 2000; Ostlund et al., 2003). The degree of maternal glucose intolerance was associated with a graded increase in the incidence of fetal macrosomia (Sermer et al., 1995). Moreover, the recent hyperglycemia and adverse pregnancy outcome study (HAPO) found a strong association of maternal glucose concentrations below levels diagnostic of diabetes with increased infant birth weight (HAPO, 2008), but others found no significant increase in the risk of LGA in mildly hyperglycemic women (Nordin et al., 2006; Gruendhammer et al., 2003). Bo et al suggested that metabolic syndrome in mid-pregnancy might be an independent predictor of macrosomia in women with some degree of gestational hyperglycemia, including GDM and 2. Maternal hyperglycemia and fetal overgrowth Ostlund et al., 2003; Bo et al., 2004; Gruendhammer et al., 2003). 2.3 Significance of mild gestational hyperglycemia and LGA birth 2.3.2 The impact of mild gestational hyperglycemia on fetal overgrowth is 2.3.1 Prevalence of mild gestational hyperglycemia not provided the usual diabetic care for GDM. mild hyperglycemia without GDM (Bo et al., 2004). inconsistent 2.1 Fetal overgrowth 2.2 GDM and fetal overgrowth macrosomia (Murphy et al., 2008). Camden Study is a prospective cohort study of pregnancy outcome and complications in young, generally healthy women residing in one of the poorest cities in the continental United States (Webster & Bishaw, 2006). Women with serious non-obstetric problems (e.g., Lupus, type 1 or 2 diabetes, seizure disorders, malignancies, acute or chronic liver disease, drug or alcohol abuse and psychiatric problems) were excluded from participation. The sample for this analysis totaled 2,373 pregnant women with live births who enrolled between 1996-2006. #### 2.4.1 Definition of GDM and mild hyperglycemia non-GDM in Camden study The diagnosis of GDM was made using a two-step approach. All participants were initially screened by measuring the plasma glucose concentration 1h after a 50-g oral glucose challenge test (GCT) at 28±0.1 (mean±SE) weeks' gestation. A diagnostic OGTT was performed on that subset of women exceeding the glucose threshold value (>140 mg/dl). The diagnostic criteria for GDM was the Carpenter/Coustan conversion as recommended by the American Diabetes Association (ADA) which defines GDM by two or more glucose values over the cut points of 95,180,155,140 mg/dl at fasting, 1, 2, and 3 hours during a 100g OGTT (American Diabetes Association, 2000). Women with a positive GCT and fewer than two abnormal glucose values were identified as having an impaired GCT without GDM (impaired GCT non-GDM). All of patients diagnosed with GDM obtained dietary counseling and/or insulin treatment for their glycemic control; patients with impaired CGT non-GDM were untreated. We identified 2,092 women (88.1%) with normal GCT, 182 women (7.7%) with impaired GCT non-GDM and 99 women (4.2%) with GDM (table 1). Ethnic composition of the cohort was 46% Hispanic, 37.6% African American and 16.6% Caucasian plus others. When the maternal characteristics were rank ordered with respect to glucose tolerance status, older maternal age, higher pre-pregnant body mass index (BMI), obesity and prior history of GDM, all were found to be positively associated with maternal glycemic status (normal GCT, impaired GCT non-GDM or GDM) and represented a linear increasing trend in terms of severity (p for trend <0.001 for each). #### 2.4.2 Maternal hyperglycemia and LGA birth LGA is defined as a neonatal birth weight greater than the 90th percentile for gestational age that has been adjusted for ethnicity, parity and infant sex (Zhang & Bowes, 1995). Women with GDM or impaired GCT without GDM had a ~2-fold increased risk for bearing an LGA infant as compared to women with normal GCT (table 2, model 1) after controlling for maternal confounding variables and using a standard which adjusted LGA for maternal ethnicity, parity and fetal gender. This increased risk persisted after additional adjustments Gestational Hyperglycemia, Excessive Pregnancy Weight Gain and Risk of Fetal Overgrowth 247 Complicating the interpretation of previous studies has been the inability to adjust the observations for important factors related to fetal growth such as pre-pregnant obesity or gestational weight gain, often because the data were not available (Hillier et al., 2008; Rosenberg et al., 2005; Aberg et al., 2001). Our study in Camden also differs because of the inclusion of several ethnic groups (Vambergue et al., 2000; Bo et al., 2004; Mello et al., 1997). With our large cohort, we had the power to control for known potential confounders that influence the fetal overgrowth, including pre-pregnant BMI and net gestational weight gain. We confirmed that untreated maternal mild hyperglycemia (impaired GCT with one abnormal or no abnormal glucose value at OGTT) in otherwise young and healthy women in the US was associated with a 2-3 fold increased risk of delivering an LGA infant (table 2). These observations are consistent with previous findings from populations studied worldwide by the HAPO group and other studies (HAPO, 2008; Stamilio et al 2004). In addition, increased risk of LGA birth in GDM women (all of whom were treated by diet or To optimize gestational weight gain for both mother and fetus is critical and remains controversial. A large number of studies have found that excess gestational weight gain is associated with decreased risk of small-for-gestational age birth and with increased risk for LGA birth (Oken et al., 2009; Jensen et al., 2005; Hinkle et a., 2010), regardless the definition or the scales used for excess pregnancy weight gain (Kiel et al., 2007; Jain et al., 2007). The independent contribution of excess gestational weight gain and maternal obesity on the risk of LGA or macrosomia has been extensively investigated (Jensen et al., 2005; Jain et al., 2007), because obesity is a common problem during pregnancy (Chu et al., 2007; Sebire et al., 2001; Rosenberg et al., 2005). Even in women with a normal pre-pregnant BMI, a higher pregnancy weight gain was associated with an increased risk of LGA, while a normal weight gain by the 1990 The Institute of Medicine (IOM) guidelines was associated with a decreased risk of LGA (DeVader et al., 2007). The definition of an optimal gestational weight gain in obese pregnancy remains controversial (Nohr et al., 2008). Jain et al observed that overweight/obese women who gain weight within the IOM recommendation achieve better fetal outcomes (Jain et al., 2007), while others found weight gain within IOM ranges did not reduce the risk of LGA among the obese (Dietz et al., 2009). It was suggested that limited or no weight gain in more severely obese pregnant women may increase favorable neonatal outcomes (Kiel et al., 2007; Jensen et al., 2005). Thus, an optimal gestational weight gain may need to be further defined by obesity severity (Hinkle et al., 2010; Oken et al., 2009). pregnancy to prevent obesity in the post-partum for mother and child (IOM, 2009). There are limited data that link excess pregnancy weight gain measured prior to screening for GDM and /or longitudinal gestational weight gain measures through out the pregnancy to adverse pregnancy outcomes. In 2009, the IOM published revised gestational weight gain guidelines for how much weight a woman should gain during pregnancy to optimize both maternal and child outcomes. The report highlighted the importance of intervention in 3.3 What are problems in the research of weight gain during pregnancy? 2.4.3 What do we learn from the Camden study? with insulin) was associated with high maternal pre-pregnant BMI. 3.1 Significance of optimal pregnancy weight gain and fetal growth 3. Pregnancy weight gain and LGA birth 3.2 Weight gain and maternal obesity Data are mean ± SE or n (%). Normal GCT, during a glucose challenge test, glucose ≤140mg/dl; Impaired GCT non-GDM, glucose >140mg/dl during a glucose challenge test and non-GDM; GDM, gestational diabetes mellitus (same in tables 2,4,5). 1. p for linear contrasts among groups <0.001 from ANOVA, or Mantel-Haenszel chi-square test; 2. p for linear contrasts among groups <0.05 from Mantel-Haenszel chi-square test. Table 1. Characteristics of Study Participants for pre-pregnant BMI and net maternal weight gain (net weight gain=total weight gaininfant birth weight) in women with impaired GCT non-GDM (Table 2, model 2), but it was non-significant in women with GDM. LGA, large-for-gestational age infant; AOR, adjusted odds ratio; 95% CI, 95% confidence interval (same as in tables 4-5). 1 p for linear trend <0.01. 2 Model 1 was adjusted for age and cigarette smoking in addition to using a standard which adjusted LGA for maternal ethnicity, parity and fetal gender. 3 Model 2 was adjusted for all variables in model 1 with additional adjustment for pre-pregnant BMI and net of weight gain (kg) during pregnancy. Table 2. Maternal Hyperglycemia and LGA birth #### 2.4.3 What do we learn from the Camden study? 246 Gestational Diabetes N (%) 2373 (100) 2092 (88.1) 182 (7.7) 99 (4.2) Age (yr)1 22.1±0.1 21.7±0.1 24.4±0.4 26.3±0.6 Pre-pregnant BMI (kg/m2)1 25.7±0.1 25.4±0.1 26.6±0.5 29.8±0.8 <18.5 146 (6.2) 141 (6.7) 5 (2.8) 0 18.5-24.9 1157 (48.8) 1050 (50.2) 82 (45.1) 25 (25.3) 25-29.9 587 (24.7) 503 (24.0) 50 (27.5) 34 (34.3) ≥30 483 (20.4) 398 (19.0) 45 (24.7) 40 (40.0) Cigarette smoking 440 (18.5) 381(18.2) 39 (21.4) 20 (20.2) Hispanic 1089 (45.9) 949 (45.4) 82 (45.0) 58 (58.6) African American 891(37.6) 803 (38.4) 67 (36.8) 21 (21.2) Caucasian & other 393 (16.6) 340 (16.3) 33 (18.1) 20 (20.2) Medicaid 2323 (97.9) 2047 (97.9) 178 (97.8) 98 (99.0) Prior GDM in multiparas1 33 (1.4) 15 (0.7) 8 (4.4) 10 (10.0) Primiparas2 923 (38.9) 833 (39.8) 60 (33.0) 30 (30.3) Normal GCT, during a glucose challenge test, glucose ≤140mg/dl; Impaired GCT non-GDM, glucose >140mg/dl during a glucose challenge test and non-GDM; GDM, gestational diabetes mellitus (same in for pre-pregnant BMI and net maternal weight gain (net weight gain=total weight gaininfant birth weight) in women with impaired GCT non-GDM (Table 2, model 2), but it was GDM 99 10.1 2.05 (1.03, 4.10) 1.52 (0.74, 3.13) Impaired GCT non-GDM 182 12.1 2.48 (1.50, 4.09) 2.50 (1.50, 4.14) Normal GCT 2092 5.3 Reference Reference LGA, large-for-gestational age infant; AOR, adjusted odds ratio; 95% CI, 95% confidence interval (same 1 p for linear trend <0.01. 2 Model 1 was adjusted for age and cigarette smoking in addition to using a standard which adjusted 3 Model 2 was adjusted for all variables in model 1 with additional adjustment for pre-pregnant BMI LGA AOR (95% CI)1 % Model 12 Model 23 1. p for linear contrasts among groups <0.001 from ANOVA, or Mantel-Haenszel chi-square test; 2. p for linear contrasts among groups <0.05 from Mantel-Haenszel chi-square test. Impaired GCT non-GDM GDM Characteristics All patients Normal GCT BMI categories1 Ethnicity2 tables 2,4,5). as in tables 4-5). Data are mean ± SE or n (%). Table 1. Characteristics of Study Participants <sup>N</sup>Unadjusted LGA for maternal ethnicity, parity and fetal gender. Table 2. Maternal Hyperglycemia and LGA birth and net of weight gain (kg) during pregnancy. non-significant in women with GDM. Complicating the interpretation of previous studies has been the inability to adjust the observations for important factors related to fetal growth such as pre-pregnant obesity or gestational weight gain, often because the data were not available (Hillier et al., 2008; Rosenberg et al., 2005; Aberg et al., 2001). Our study in Camden also differs because of the inclusion of several ethnic groups (Vambergue et al., 2000; Bo et al., 2004; Mello et al., 1997). With our large cohort, we had the power to control for known potential confounders that influence the fetal overgrowth, including pre-pregnant BMI and net gestational weight gain. We confirmed that untreated maternal mild hyperglycemia (impaired GCT with one abnormal or no abnormal glucose value at OGTT) in otherwise young and healthy women in the US was associated with a 2-3 fold increased risk of delivering an LGA infant (table 2). These observations are consistent with previous findings from populations studied worldwide by the HAPO group and other studies (HAPO, 2008; Stamilio et al 2004). In addition, increased risk of LGA birth in GDM women (all of whom were treated by diet or with insulin) was associated with high maternal pre-pregnant BMI. ### 3. Pregnancy weight gain and LGA birth #### 3.1 Significance of optimal pregnancy weight gain and fetal growth To optimize gestational weight gain for both mother and fetus is critical and remains controversial. A large number of studies have found that excess gestational weight gain is associated with decreased risk of small-for-gestational age birth and with increased risk for LGA birth (Oken et al., 2009; Jensen et al., 2005; Hinkle et a., 2010), regardless the definition or the scales used for excess pregnancy weight gain (Kiel et al., 2007; Jain et al., 2007). #### 3.2 Weight gain and maternal obesity The independent contribution of excess gestational weight gain and maternal obesity on the risk of LGA or macrosomia has been extensively investigated (Jensen et al., 2005; Jain et al., 2007), because obesity is a common problem during pregnancy (Chu et al., 2007; Sebire et al., 2001; Rosenberg et al., 2005). Even in women with a normal pre-pregnant BMI, a higher pregnancy weight gain was associated with an increased risk of LGA, while a normal weight gain by the 1990 The Institute of Medicine (IOM) guidelines was associated with a decreased risk of LGA (DeVader et al., 2007). The definition of an optimal gestational weight gain in obese pregnancy remains controversial (Nohr et al., 2008). Jain et al observed that overweight/obese women who gain weight within the IOM recommendation achieve better fetal outcomes (Jain et al., 2007), while others found weight gain within IOM ranges did not reduce the risk of LGA among the obese (Dietz et al., 2009). It was suggested that limited or no weight gain in more severely obese pregnant women may increase favorable neonatal outcomes (Kiel et al., 2007; Jensen et al., 2005). Thus, an optimal gestational weight gain may need to be further defined by obesity severity (Hinkle et al., 2010; Oken et al., 2009). #### 3.3 What are problems in the research of weight gain during pregnancy? There are limited data that link excess pregnancy weight gain measured prior to screening for GDM and /or longitudinal gestational weight gain measures through out the pregnancy to adverse pregnancy outcomes. In 2009, the IOM published revised gestational weight gain guidelines for how much weight a woman should gain during pregnancy to optimize both maternal and child outcomes. The report highlighted the importance of intervention in pregnancy to prevent obesity in the post-partum for mother and child (IOM, 2009). Gestational Hyperglycemia, Excessive Pregnancy Weight Gain and Risk of Fetal Overgrowth 249 variables with the exception of pre-pregnant BMI (p for trend <0.0001 for each model). Similar results were obtained when additional adjustment for pre-pregnant BMI. In addition, women with inadequate weight gain had a reduced risk of LGA birth at week 28 These data confirmed that excess pregnancy weight gain throughout the 2ndand 3rd trimesters and at delivery significantly increased risk of LGA, whereas inadequate pregnancy weight gain reduced LGA risk. The current study has several important strengths. Firstly, we used the most recent IOM recommendations (2009) for the estimation of excess weight gain by BMI categories and the analysis models were fully adjusted for potential confounding, including pre-pregnant BMI. In contrast, more arbitrary criteria were used to define excess weight gain in previous studies (Herring et al., 2009; Rosenberg et al., 2005), and pre-pregnant BMI either was not available or was not adjusted for in the analysis (Hillier et al., 2008; Rosenberg et al., 2005). Secondly, there are limited data on longitudinal measures of pregnancy weight gain (Herring et al., 2009; Saldana et al., 2006). We used multiple measures of pregnancy weight gain starting prior to the screen for GDM. The relationship between excess pregnancy weight gain assessed relatively early in pregnancy and risk of LGA could be important for preventive strategies. Total weight gain is not an ideal measure to evaluate in relation to fetal growth in GDM patients, because most of Week 24 Week 28 Week 32 At delivery LGA (%) (0.27, 0.95) 2.3 0.49 (1.05, 2.61) 7.6 1.58 AOR (95% CI) LGA (%) (0.24, 0.98) 2.3 0.62 (1.00, 2.50) 9.1 2.66 AOR (95% CI) (0.31, 1.25) (1.69, 4.19) AOR (95% CI) Adequate 3.5 Reference 5.1 Reference 6.1 Reference 3.6 Reference 1 Models were adjusted for age and cigarette smoking in addition to using a standard which adjusted 4. Association of various degree of gestational hyperglycemia, excess 4.1 Does excess gestational weight gain prior to screening GDM predict risk of GDM? High gestational weight gain between early and mid pregnancy was positively associated with risk of GDM or impaired glucose tolerance (Hedderson et al., 2010; Herring et al., 2009). In the Camden study, we found a positive association between excess weight gain prior to or at the time of screening for GDM (weeks 24 and 28) with increased risk for GDM (AOR 1.57, 95% CI 1.01, 2.44 for week 24, AOR 1.94, 95% CI 1.28, 2.95 for week 28). This association was not significant at week 32 or at delivery which may suggest an effect of treatment and weight monitoring after the diagnosis of GDM. We did not observe a patients are treated and their weight gain is monitored after the diagnosis. LGA (%) (0.47,1.71) 2.6 0.50 (1.55, 4.43) 8.0 1.65 2 p for trend <0.0001 for week 24, 28, 32 and delivery respectively. Table 4. Excess pregnancy weight gain and LGA birth1,2 and 32 (p<0.05). Weight gain LGA (%) Inadequate 3.2 0.90 Excessive 8.7 2.62 LGA for parity, ethnicity and infant gender. pregnancy weight gain with LGA birth AOR (95% CI) 3.7 Contribution of Camden data #### 3.4 The US Institute of medicine new guidelines for gestational weight gain The new 2009 IOM guidelines are based on the WHO BMI (kg/m2) categories (WHO, 1998) and include the recommended total pregnancy weight gain (kg) or rates of weight gain during the 2nd and 3rd trimester (kg/week). Weight gain below or above the recommended range is defined as inadequate or excessive gain, respectively. 1 IOM, Institute of Medicine in US. 2 Calculations assume a 0.5-2kg (1.1-4.4 lbs) weight gain in the first trimester (based on Siega-Riz et. al., 1994; Abrams et al 1995, Carmichael et al 1997). Table 3. New recommendations for total and rate of weight gain during pregnancy, by prepregnancy BMI (IOM, 2009)1 #### 3.5 Assessment of pregnancy weight gain We explored the associations between gestational weight gain assessed throughout pregnancy with LGA using the 2009 IOM guidelines for weight gain during pregnancy. Inadequate, adequate and excessive pregnancy weight gain at weeks 24, 28, 32 and at delivery was categorized according to IOM recommendations (table 3). Maternal obesity was defined as BMI ≥30 (WHO, 1998); height was measured with a stadiometer at entry. Gestational duration was based upon gestation from participants' last normal menstrual period confirmed or modified by ultrasound. Pregnancy weight was measured at week 12, 24, 28, 32 and at delivery. Total gestational weight gain was computed as the difference between weight at delivery and recalled pre-pregnant weight. Gestational weight gain during the 2nd and 3rd trimester was computed as the difference between weights measured at each visit minuses the weight measured at week 12. The rate of weight gain was the weight gain (kg) divided by gestational age (weeks). #### 3.6 Excess pregnancy weight gain measured mid to late gestations and LGA birth The proportion of women with excess pregnancy weight gain at weeks 24, 28, and 32 was similar to delivery (48.5%, 52%, 54% and 50% respectively). Depending on the gestation, 21- 27% had an adequate weight gain and 22-30% had an inadequate gain throughout the four time points. Compared to women with adequate weight gain, excessive weight gain was associated with a 1.58-2.66 fold increased risk of LGA birth (table 4) after controlling for all the confounding variables with the exception of pre-pregnant BMI (p for trend <0.0001 for each model). Similar results were obtained when additional adjustment for pre-pregnant BMI. In addition, women with inadequate weight gain had a reduced risk of LGA birth at week 28 and 32 (p<0.05). #### 3.7 Contribution of Camden data 248 Gestational Diabetes The new 2009 IOM guidelines are based on the WHO BMI (kg/m2) categories (WHO, 1998) and include the recommended total pregnancy weight gain (kg) or rates of weight gain during the 2nd and 3rd trimester (kg/week). Weight gain below or above the recommended > Total weight gain range Under weight <18.5 28-40 12.5-18.0 1.0 (1.0-1.3) 0.51 (0.44- Normal weight 18.5-24.9 25-35 11.5-16.0 1.0 (0.8-1.0) 0.42 (0.35- Overweight 25.0-29.9 15-25 7.0-11.5 0.6 (0.5-0.7) 0.28 (0.23- all classes) ≥30.0 11-20 5.0-9.0 0.5 (0.4-0.6) 0.22 (0.17- 2 Calculations assume a 0.5-2kg (1.1-4.4 lbs) weight gain in the first trimester (based on Siega-Riz et. al., 1994; We explored the associations between gestational weight gain assessed throughout pregnancy with LGA using the 2009 IOM guidelines for weight gain during pregnancy. Inadequate, adequate and excessive pregnancy weight gain at weeks 24, 28, 32 and at delivery was categorized according to IOM recommendations (table 3). Maternal obesity was defined as BMI ≥30 (WHO, 1998); height was measured with a stadiometer at entry. Gestational duration was based upon gestation from participants' last normal menstrual period confirmed or modified by ultrasound. Pregnancy weight was measured at week 12, 24, 28, 32 and at delivery. Total gestational weight gain was computed as the difference between weight at delivery and recalled pre-pregnant weight. Gestational weight gain during the 2nd and 3rd trimester was computed as the difference between weights measured at each visit minuses the weight measured at week 12. The rate of weight gain was the 3.6 Excess pregnancy weight gain measured mid to late gestations and LGA birth The proportion of women with excess pregnancy weight gain at weeks 24, 28, and 32 was similar to delivery (48.5%, 52%, 54% and 50% respectively). Depending on the gestation, 21- 27% had an adequate weight gain and 22-30% had an inadequate gain throughout the four Compared to women with adequate weight gain, excessive weight gain was associated with a 1.58-2.66 fold increased risk of LGA birth (table 4) after controlling for all the confounding Table 3. New recommendations for total and rate of weight gain during pregnancy, by (lbs) (kg) (lbs/wk) (kg/wk) Rate of weight gain 2nd and 3rd trimester (mean, range)2 0.58) 0.50) 0.33) 0.27) 3.4 The US Institute of medicine new guidelines for gestational weight gain range is defined as inadequate or excessive gain, respectively. BMI (kg/m2) (WHO) Prepregnancy Obese (includes time points. 1 IOM, Institute of Medicine in US. prepregnancy BMI (IOM, 2009)1 Abrams et al 1995, Carmichael et al 1997). 3.5 Assessment of pregnancy weight gain weight gain (kg) divided by gestational age (weeks). BMI These data confirmed that excess pregnancy weight gain throughout the 2ndand 3rd trimesters and at delivery significantly increased risk of LGA, whereas inadequate pregnancy weight gain reduced LGA risk. The current study has several important strengths. Firstly, we used the most recent IOM recommendations (2009) for the estimation of excess weight gain by BMI categories and the analysis models were fully adjusted for potential confounding, including pre-pregnant BMI. In contrast, more arbitrary criteria were used to define excess weight gain in previous studies (Herring et al., 2009; Rosenberg et al., 2005), and pre-pregnant BMI either was not available or was not adjusted for in the analysis (Hillier et al., 2008; Rosenberg et al., 2005). Secondly, there are limited data on longitudinal measures of pregnancy weight gain (Herring et al., 2009; Saldana et al., 2006). We used multiple measures of pregnancy weight gain starting prior to the screen for GDM. The relationship between excess pregnancy weight gain assessed relatively early in pregnancy and risk of LGA could be important for preventive strategies. Total weight gain is not an ideal measure to evaluate in relation to fetal growth in GDM patients, because most of patients are treated and their weight gain is monitored after the diagnosis. 1 Models were adjusted for age and cigarette smoking in addition to using a standard which adjusted LGA for parity, ethnicity and infant gender. 2 p for trend <0.0001 for week 24, 28, 32 and delivery respectively. Table 4. Excess pregnancy weight gain and LGA birth1,2 #### 4. Association of various degree of gestational hyperglycemia, excess pregnancy weight gain with LGA birth 4.1 Does excess gestational weight gain prior to screening GDM predict risk of GDM? High gestational weight gain between early and mid pregnancy was positively associated with risk of GDM or impaired glucose tolerance (Hedderson et al., 2010; Herring et al., 2009). In the Camden study, we found a positive association between excess weight gain prior to or at the time of screening for GDM (weeks 24 and 28) with increased risk for GDM (AOR 1.57, 95% CI 1.01, 2.44 for week 24, AOR 1.94, 95% CI 1.28, 2.95 for week 28). This association was not significant at week 32 or at delivery which may suggest an effect of treatment and weight monitoring after the diagnosis of GDM. We did not observe a Gestational Hyperglycemia, Excessive Pregnancy Weight Gain and Risk of Fetal Overgrowth 251 These data confirmed that hyperglycemia and excess weight gain are independently associated with risk of fetal overgrowth. Women with excess weight gain at any of four times in gestation and hyperglycemia, even a mild hyperglycemia, had a substantially increased risk of LGA. Risk of LGA was not increased among gestational diabetics without an excessive weight gain but only among the group with impaired GCT non-GDM. Thus, excess pregnancy weight gain and hyperglycemia are independent risk factors for LGA. By using the most updated IOM guidelines (2009) on pregnancy weight gain, we found that healthy, young women with mild but untreated hyperglycemia were at increased risk for fetal overgrowth (LGA). The risk of LGA birth in women with GDM, who are treated by diet or with insulin after diagnosis, was dependent on maternal pre-pregnant weight. Excess pregnancy weight gain assessed longitudinally through out mid to late gestation consistently showed strong associations with LGA risk. In addition, excess pregnancy weight gain and hyperglycemia appeared to be independent risk factors for LGA. The risk for LGA was increased still further in women with excess weight gain, even in those with mild hyperglycemia. Because prepregnancy obesity, mild gestational hyperglycemia, and excess weight gain during pregnancy are preventable risk factors, these findings suggest that early detection and treatment of mild hyperglycemia and monitoring pregnancy weight gain during early gestation are both important for reducing the risk of LGA, for reducing childhood and later obesity and for improving the long term risk for metabolic disease in This work was supported by grants from the National Institute of Child Health and Human Development (HD18269 and HD38329 to T.O.S.) and National Institute of Diabetes and Aberg, A., Rydhstroem H., & Frid, A. (2001). Impaired glucose tolerance associated with American Diabetes Association. (2000). Gestational diabetes mellitus. Diabetes Care Vol.23, Bo, S., Menato, G., Gallo, M., Bardelli, C., Lezo, A., Signorile, A. Gambino, R., Cassader, M., Boney, C., Verma, A., Tucker, R., & Vohr, B. (2005). Metabolic syndrome in childhood: Pediatrics Vol.115, No.3, (March 2005), pp.e290-6, ISSN 1098-4275. Obstet Gynecol Vol.184, No.2, (January 2001), pp.77-83, ISSN 1097-6868. Abrams, B., Carmichael, S., & Selvin, S. (1995). Factors associated with the pattern of adverse pregnancy outcome: A population-based study in southern Sweden. Amer J maternal weight gain during pregnancy. Obstet Gynecol Vol.86, No.2, (August Massobrio, M., & Pagano, G. (2004). Mild gestational hyperglycemia, the metabolic syndrome and adverse neonatal outcomes. Acta Obstet Gynecol Scand Vol.83, No.4, association with birth weight, maternal obesity, and gestational diabetes mellitus. Digestive and Kidney Diseases (1R21DK078865-01 and 5R21DK078865-02 to X.C.). 4.3 What do we learn? 5. Conclusion the offspring. 7. References 6. Acknowledgements 1995), pp.170-6, ISSN 0029-7844. No. 1, (January 2000), pp. S77-9, ISSN 0149-5992. (March 2004), pp.335-40, ISSN 0001-6349. significant association between excess weight gain and impaired GCT non-GDM (p>0.05 for all time points). #### 4.2 The association of gestational hyperglycemia and excess weight gain with LGA Combined association of gestational hyperglycemia and excess weight gain on the risk of LGA has not been examined extensively. Hillier et al observed at GDM screening that macrosomia risk was increased across the spectrum of maternal glucose levels and that this relationship was further modified by excessive maternal weight gain (Hillier et al., 2008). A weight gain of 40lbs or more nearly doubled the risk of fetal macrosomia among glucose intolerant women including those with GDM. However, results were not adjusted for pre-pregnancy BMI which can complicate the relationship between maternal hyperglycemia and pregnancy weight gain. Thus, our next goal was to examine the independent and combined contributions of hyperglycemia and excess weight gain on risk of LGA. An analysis stratified by excessive weight gain was used to index the influence of various degrees of maternal glycemic status on LGA risk, using women with non-excess pregnancy weight gain (adequate and inadequate gain combined) and a normal GCT as the reference group. Our results showed that excess weight gain (at week 24, 28, 32 or at delivery) was positively associated with a 2- 6 fold increased risks for delivering an LGA infant (table 5) regardless of whether the women were diagnosed with GDM, impaired GCT non-GDM or normal GCT. Furthermore, women with non-excess weight gain and an impaired GCT non-GDM also had a 2-3 fold increased risk of LGA birth during all of four time points, whereas risk of LGA was not increased in the GDM group with non-excess weight gain. 1 Model was adjusted for age, pre-pregnant BMI and cigarette smoking in addition to using a standard which adjusted LGA for maternal ethnicity, parity and fetal gender. 2 Adequate and inadequate weight gain is combined. 3 p for trend <0.0001. Table 5. Maternal hyperglycemia and LGA infant: Stratified by excess weight gain1 #### 4.3 What do we learn? 250 Gestational Diabetes significant association between excess weight gain and impaired GCT non-GDM (p>0.05 for 4.2 The association of gestational hyperglycemia and excess weight gain with LGA Combined association of gestational hyperglycemia and excess weight gain on the risk of LGA has not been examined extensively. Hillier et al observed at GDM screening that macrosomia risk was increased across the spectrum of maternal glucose levels and that this relationship was further modified by excessive maternal weight gain (Hillier et al., 2008). A weight gain of 40lbs or more nearly doubled the risk of fetal macrosomia among glucose intolerant women including those with GDM. However, results were not adjusted for pre-pregnancy BMI which can complicate the relationship between maternal hyperglycemia and pregnancy weight gain. Thus, our next goal was to examine the independent and combined contributions of hyperglycemia and excess weight gain on risk of LGA. An analysis stratified by excessive weight gain was used to index the influence of various degrees of maternal glycemic status on LGA risk, using women with non-excess pregnancy weight gain (adequate and inadequate gain combined) and a normal GCT as the reference group. Our results showed that excess weight gain (at week 24, 28, 32 or at delivery) was positively associated with a 2- 6 fold increased risks for delivering an LGA infant (table 5) regardless of whether the women were diagnosed with GDM, impaired GCT non-GDM or normal GCT. Furthermore, women with non-excess weight gain and an impaired GCT non-GDM also had a 2-3 fold increased risk of LGA birth during all of four time points, whereas risk of LGA was not > Weight gain at week 28 > > AOR (95% CI)3 LGA (%) (1.67, 9.73) 10.5 3.31 (3.16, 11.90) 14.6 4.97 (1.88, 4.48) 7.2 2.43 (0.37, 7.21) 6.7 1.69 (1.17, 6.59) 9.5 2.98 Table 5. Maternal hyperglycemia and LGA infant: Stratified by excess weight gain1 Normal GCT 2.8 Reference 3.1 Reference 3.6 Reference 2.5 Reference 1 Model was adjusted for age, pre-pregnant BMI and cigarette smoking in addition to using a standard Weight gain at week 32 > AOR (95% CI)3 LGA (%) (1.39, 7.90) 11.3 3.20 (2.53, 9.71) 14.1 4.62 (1.58, 3.73) 6.8 2.02 (0.38, 7.50) 5.9 1.31 (1.31, 6.77) 9.9 2.69 Weight gain at delivery > AOR (95% CI)3 (2.58, 14.70) (3.20, 12.88) ( 2.29, 5.60) (0.37, 7.11) (1.65, 8.68) LGA (%) (1.34, 7.63) 14.3 6.07 (2.18, 8.31) 14.9 6.42 (1.33, 3.10) 8.2 3.58 (0.30, 5.71) 4.7 1.62 (1.19, 6.08) 9.1 3.79 increased in the GDM group with non-excess weight gain. AOR (95% CI)3 which adjusted LGA for maternal ethnicity, parity and fetal gender. 2 Adequate and inadequate weight gain is combined. Weight gain at week 24 LGA (%) GDM 11.5 4.03 non-GDM 16.0 6.14 Normal GCT 7.8 2.91 GDM 5.6 1.64 non-GDM 8.1 2.78 Excess weight gain Impaired GCT Non-excess weight gain2 Impaired GCT 3 p for trend <0.0001. all time points). These data confirmed that hyperglycemia and excess weight gain are independently associated with risk of fetal overgrowth. Women with excess weight gain at any of four times in gestation and hyperglycemia, even a mild hyperglycemia, had a substantially increased risk of LGA. Risk of LGA was not increased among gestational diabetics without an excessive weight gain but only among the group with impaired GCT non-GDM. Thus, excess pregnancy weight gain and hyperglycemia are independent risk factors for LGA. #### 5. Conclusion By using the most updated IOM guidelines (2009) on pregnancy weight gain, we found that healthy, young women with mild but untreated hyperglycemia were at increased risk for fetal overgrowth (LGA). The risk of LGA birth in women with GDM, who are treated by diet or with insulin after diagnosis, was dependent on maternal pre-pregnant weight. Excess pregnancy weight gain assessed longitudinally through out mid to late gestation consistently showed strong associations with LGA risk. In addition, excess pregnancy weight gain and hyperglycemia appeared to be independent risk factors for LGA. The risk for LGA was increased still further in women with excess weight gain, even in those with mild hyperglycemia. Because prepregnancy obesity, mild gestational hyperglycemia, and excess weight gain during pregnancy are preventable risk factors, these findings suggest that early detection and treatment of mild hyperglycemia and monitoring pregnancy weight gain during early gestation are both important for reducing the risk of LGA, for reducing childhood and later obesity and for improving the long term risk for metabolic disease in the offspring. #### 6. Acknowledgements This work was supported by grants from the National Institute of Child Health and Human Development (HD18269 and HD38329 to T.O.S.) and National Institute of Diabetes and Digestive and Kidney Diseases (1R21DK078865-01 and 5R21DK078865-02 to X.C.). #### 7. References Gestational Hyperglycemia, Excessive Pregnancy Weight Gain and Risk of Fetal Overgrowth 253 Jain, N., Denk, C., Kruse, L., & Dandolu, V. (2007). Maternal obesity: can pregnancy weight Jensen, D., Ovesen, P., Beck-Nielsen, H., Mølsted-Pedersen, L., Sørensen, B., Vinter, C., & Kiel, D., Dodson, E., Artal, R., Boehmer, T., & Leet, T. (2007). Gestational weight gain and Langer, O. (2000). Fetal macrosomia: Etiologic factors. Clinical Obstetrics Gynecology Vol.43, Mello, G., Parretti, E., Mecacci, F., Lucchetti, R., Lagazio, C., Pratesi, M., & Scarselli, G. 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Obstet Gynecol Vol.115, No.3, (March 2010), pp.597- Gillman, M. (2009). Weight gain in pregnancy and risk of maternal hyperglycemia. Excess gestational weight gain: modifying fetal macrosomia risk associated with maternal glucose. Obstet Gynecol Vol.112, No.5, (November 2008), pp.1007-14, ISSN associations with fetal growth. Am J Clin Nutr Vol.92, No.3, (September 2010), (April 2009). The National Academies Press, Washington DC. Available from: 14 Brazil Congenital Cardiopathies Screening Zilma Silveira Nogueira Reis and Gabriel Costa Osanan Obstetrics and Gynecology Department, Universidade Federal de Minas Gerais Associated with Diabetes Mellitus Using Maternal Fructosamine Plasma Concentration The consequences of uncontrolled diabetes mellitus during pregnancy are severe for both mother and fetus. The risk of congenital malformations among infants of diabetic mothers is related to the quality of the diabetic control (Allen et al., 2007; American Diabetes Association [ADA], 2004). In diabetic pregnancy, unsatisfactory glycemic control at the moment of diagnosis or delivery of care was associated with an increased risk of anomalies (Allen et al., 2007; Schaefer-Graf at al., 2000). The most common anomalies involve the cardiac, musculoskeletal, and central nervous systems (Sheffield at al., 2002). Despite this knowledge, it has been disappointing that so few diabetic women receive preconception counseling, plan their pregnancies or are early referred to tertiary centers (American Diabetes Association [ADA], 2004; Reis at al., 2010a). Considering this reality, it is important to study second trimester markers of congenital cardiopathies, in order to decrease fetal If maternal hyperglycemia is present during organogenesis there is an increased risk of congenital anomalies and miscarriage (American Diabetes Association [ADA], 2004). Experimental studies suggest that hyperglycemia is the major teratogen in diabetic pregnancies, but other diabetes-related factors may also affect fetal outcomes (Aberg at al., 2001; Buchanan & Kjos, 1999; Leonard et al., 1989; Ren et al., 2011). It is a fact that an uncontrolled diabetes mellitus in early gestation is associated with a teratogenic effect, causing primary cardiogenesis defects. Most types of cardiac structural lesions have been associated with diabetes mellitus, ranging from small septal defects to major heart disease The exact teratogenic mechanism of maternal diabetes is not fully defined and is likely multifactorial (Hornberger, 2006; Kumar et al., 2007). Diabetes mellitus affects the fetal heart both structurally and functionally. In late gestation, it causes a unique form of hypertrophic cardiomyopathy (Ren et al., 2011; Hornberger, 2006; Chaudhari et al., 2008; Russell et al., 2008), illustrated at Fig. 1. Cardiomegaly is a common finding in stillborn infants of mothers 1. Introduction morbidity and mortality, at birth. 2. Congenital cardiopathies and diabetes mellitus (Sekhavat et al., 2010; Abu-Sulaiman & Subaih, 2004). Ampudia, J., Fernández-Real, J., & Corcoy, R. (2005). Potential impact of American Diabetes Association (2000) criteria for diagnosis of gestational diabetes mellitus in Spain. Diabetologia Vol.48, No.6, (June 2005), pp.1135-41, ISSN 0012-186X. ## Congenital Cardiopathies Screening Associated with Diabetes Mellitus Using Maternal Fructosamine Plasma Concentration Zilma Silveira Nogueira Reis and Gabriel Costa Osanan Obstetrics and Gynecology Department, Universidade Federal de Minas Gerais Brazil #### 1. Introduction 254 Gestational Diabetes Spain. Diabetologia Vol.48, No.6, (June 2005), pp.1135-41, ISSN 0012-186X. Rosenberg, T., Garbers, S., Lipkind, H., & Chiasson, M. (2005). Maternal obesity and Saldana, T., Siega-Riz, A., Adair, L., & Suchindran, C. (2006). The relationship between Sebire, N., Jolly, M., Harris, J., Wadsworth, J., Joffe, M., & Beard, R., Regan, L. & Robinson, S. Sermer, M., Naylor, D., Gare, D., Kenshole, A., Ritchie, J., Farine, D., Cohen, H., McArthur, Siega-Riz, A., Adair, L., & Hobel, C. (1994). Institute of Medicine maternal weight gain Stamilio, D., Olsen, T., Ratcliffe, S., Sehdev, H., & Macones, G. (2004). False-positive 1-hour The HAPO Study Cooperative Research Group. (2008). Hyperglycemia and adverse Vambergue, A., Nuttens, M., Verier-Mine, O., Dognin, C., & Cappoen, J., & Fontaine P. Webster, B., & Bishaw, A. (2006). Income, earnings, and poverty data from the 2005 American Available from: <https://www.census.gov/prod/2007pubs/acs-08.pdf> World Health Organization (WHO). (1998). Obesity: preventing and managing the global epidemic. WHO Consultation on Obesity Report, Geneva. Geneva, June, 1997. Yogev, Y., Langer, O., Xenakis, E., & Rosenn, B. (2004). Glucose screening in Mexican-American women. Obstet Gynecol Vol 103, No.6, (June 2004), pp. 1241-5, ISSN 0029-7844. Zhang, J. & Bowes, W. (1995). Birth-weight-for-gestational-age patterns by race, sex, and Vol.173, No.1, (July 1995), pp. 146-56, ISSN 0002-9378. No.1, (January 2004), pp. 148-56, ISSN 0029-7844. ISSN 0090-0036. ISSN 0307-0565. 0029-7844. 1533-4406. 8, ISSN 1464-5491. pp. 200-8, ISSN 0029-7844. 2006), pp. 1629-35, ISSN 0002-9378. Ampudia, J., Fernández-Real, J., & Corcoy, R. (2005). Potential impact of American Diabetes Association (2000) criteria for diagnosis of gestational diabetes mellitus in diabetes as risk factors for adverse pregnancy outcomes: differences among 4 racial/ethnic groups. Am J Publ Hlth Vol.95, No.9, (September 2005), pp.1544-51, pregnancy weight gain and glucose tolerance status among black and white women in central North Carolina. Am J Obstet Gynecol Vol.195, No.6, (December (2001). Maternal obesity and pregnancy outcome: a study of 287 213 pregnancies in London. 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Obstet Gynecol Vol.86, No.2, (August 1995), The consequences of uncontrolled diabetes mellitus during pregnancy are severe for both mother and fetus. The risk of congenital malformations among infants of diabetic mothers is related to the quality of the diabetic control (Allen et al., 2007; American Diabetes Association [ADA], 2004). In diabetic pregnancy, unsatisfactory glycemic control at the moment of diagnosis or delivery of care was associated with an increased risk of anomalies (Allen et al., 2007; Schaefer-Graf at al., 2000). The most common anomalies involve the cardiac, musculoskeletal, and central nervous systems (Sheffield at al., 2002). Despite this knowledge, it has been disappointing that so few diabetic women receive preconception counseling, plan their pregnancies or are early referred to tertiary centers (American Diabetes Association [ADA], 2004; Reis at al., 2010a). Considering this reality, it is important to study second trimester markers of congenital cardiopathies, in order to decrease fetal morbidity and mortality, at birth. #### 2. Congenital cardiopathies and diabetes mellitus If maternal hyperglycemia is present during organogenesis there is an increased risk of congenital anomalies and miscarriage (American Diabetes Association [ADA], 2004). Experimental studies suggest that hyperglycemia is the major teratogen in diabetic pregnancies, but other diabetes-related factors may also affect fetal outcomes (Aberg at al., 2001; Buchanan & Kjos, 1999; Leonard et al., 1989; Ren et al., 2011). It is a fact that an uncontrolled diabetes mellitus in early gestation is associated with a teratogenic effect, causing primary cardiogenesis defects. Most types of cardiac structural lesions have been associated with diabetes mellitus, ranging from small septal defects to major heart disease (Sekhavat et al., 2010; Abu-Sulaiman & Subaih, 2004). The exact teratogenic mechanism of maternal diabetes is not fully defined and is likely multifactorial (Hornberger, 2006; Kumar et al., 2007). Diabetes mellitus affects the fetal heart both structurally and functionally. In late gestation, it causes a unique form of hypertrophic cardiomyopathy (Ren et al., 2011; Hornberger, 2006; Chaudhari et al., 2008; Russell et al., 2008), illustrated at Fig. 1. Cardiomegaly is a common finding in stillborn infants of mothers Congenital Cardiopathies Screening Associated with the poorly-controlled diabetic pregnancies. Diabetes Mellitus Diabetes Mellitus Using Maternal Fructosamine Plasma Concentration 257 Given the increased risk of congenital abnormalities among infants of diabetic mothers, an appropriate biochemical, ultrasonographic screening process and a detailed evaluation of fetal cardiac structure should be offered to all pregnant women with diabetes (Allen et al., 2007). A prenatal cardiac screening is purposed to identify defects that may require further evaluation and treatment, and to provide appropriate counselling to the family in a timely manner (American Diabetes Association [ADA], 2004; Sekhavat et al., 2010). Detailed fetal anatomic surveys in the early second trimester are common practice and typically include The occurrence of congenital cardiopathies at echocardiography in fetuses whose mothers had preexisting diabetes mellitus was investigated in our tertiary university medical center (Reis at al., 2010a, 2010b). The most frequent conditions were hypertrophic cardiomyopathy (70%), pericardial effusion (15%), followed by intermittent or persistent bradycardia (15%). The most frequent structural congenital cardiopathies at echocardiography was interventricular communication (85.7%) associated or not to another heart malformations (Fig. 2). Functional findings at echocardiography were significantly more frequent among Fig. 2. Image of fetal ultrasonography: interventricular comunication associated with the interventricular septum (Abu-Sulaiman & Subaih, 2004). In other reports, the malformations found in neonates, born of insulin-dependent diabetes mellitus gestations, included endocardial cushion defects, persistent truncus arteriosus and ventricular septal defects which appear to result from aberrant cardiac neural crest development (Sekhavat et al., 2010; Abu-Sulaiman & Subaih, 2004; Hornberger, 2006; Kumar et al., 2007; Russell et al., 2008). Hypertrophic cardiomyopathy was observed in 38% of neonates from insulin-dependent diabetes mellitus pregnant women, mainly hypertrophy of examination of both four-chamber and outflow tract views of the fetal heart. with diabetes mellitus and may contribute to the risk of fetal death in these pregnancies (Russell et al., 2008). Hypertrophic cardiomyopathy observed in the infant of the diabetic mother is characterized by thickening of the interventricular septum, and to a lesser extent the ventricular free walls (Hornberger, 2006). The presence of this pathology whether is associated with fetal hyperinsulinaemia and general somatic growth in maternal diabetes (Buchanan & Kjos, 1999; Ren et al., 2011). But, the wide variety of cardiac abnormalities suggests a complex pathogenesis. Experimental study proposed that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects (Kumar et al., 2007). Fig. 1. Image of fetal ultrasonography: hypertrophic cardiomyopathy associated with Diabetes Mellitus #### 3. The strategies for prevention and management of the cardiac teratogenic effects in diabetes mellitus The prevalence of pregestational diabetes among women early in their reproductive years is increasing. Thus, identifying women with diabetes is important because the diagnosis and appropriate therapy can decrease fetal and maternal morbidity (Crowtherl et al., 2005). Preconceptional evaluation and counseling of women with diabetes mellitus (type 1 or type 2) is fundamental point to minimize the risk to the fetus and mother. It is known that pregnancies in diabetes mellitus women should be planned, but that condition was not so frequent (American Diabetes Association [ADA], 2004; Reis at al., 2010a; Chaudhari et al., 2008). Unplanned pregnancy occurs in about two-thirds of women with diabetes leading to a persistent excess of malformations in their infants (American Diabetes Association [ADA], 2004). with diabetes mellitus and may contribute to the risk of fetal death in these pregnancies (Russell et al., 2008). Hypertrophic cardiomyopathy observed in the infant of the diabetic mother is characterized by thickening of the interventricular septum, and to a lesser extent the ventricular free walls (Hornberger, 2006). The presence of this pathology whether is associated with fetal hyperinsulinaemia and general somatic growth in maternal diabetes (Buchanan & Kjos, 1999; Ren et al., 2011). But, the wide variety of cardiac abnormalities suggests a complex pathogenesis. Experimental study proposed that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects (Kumar et al., 2007). Fig. 1. Image of fetal ultrasonography: hypertrophic cardiomyopathy associated with 3. The strategies for prevention and management of the cardiac teratogenic The prevalence of pregestational diabetes among women early in their reproductive years is increasing. Thus, identifying women with diabetes is important because the diagnosis and appropriate therapy can decrease fetal and maternal morbidity (Crowtherl et al., 2005). Preconceptional evaluation and counseling of women with diabetes mellitus (type 1 or type 2) is fundamental point to minimize the risk to the fetus and mother. It is known that pregnancies in diabetes mellitus women should be planned, but that condition was not so frequent (American Diabetes Association [ADA], 2004; Reis at al., 2010a; Chaudhari et al., 2008). Unplanned pregnancy occurs in about two-thirds of women with diabetes leading to a persistent excess of malformations in their infants (American Diabetes Association [ADA], Diabetes Mellitus 2004). effects in diabetes mellitus Given the increased risk of congenital abnormalities among infants of diabetic mothers, an appropriate biochemical, ultrasonographic screening process and a detailed evaluation of fetal cardiac structure should be offered to all pregnant women with diabetes (Allen et al., 2007). A prenatal cardiac screening is purposed to identify defects that may require further evaluation and treatment, and to provide appropriate counselling to the family in a timely manner (American Diabetes Association [ADA], 2004; Sekhavat et al., 2010). Detailed fetal anatomic surveys in the early second trimester are common practice and typically include examination of both four-chamber and outflow tract views of the fetal heart. The occurrence of congenital cardiopathies at echocardiography in fetuses whose mothers had preexisting diabetes mellitus was investigated in our tertiary university medical center (Reis at al., 2010a, 2010b). The most frequent conditions were hypertrophic cardiomyopathy (70%), pericardial effusion (15%), followed by intermittent or persistent bradycardia (15%). The most frequent structural congenital cardiopathies at echocardiography was interventricular communication (85.7%) associated or not to another heart malformations (Fig. 2). Functional findings at echocardiography were significantly more frequent among the poorly-controlled diabetic pregnancies. Fig. 2. Image of fetal ultrasonography: interventricular comunication associated with Diabetes Mellitus In other reports, the malformations found in neonates, born of insulin-dependent diabetes mellitus gestations, included endocardial cushion defects, persistent truncus arteriosus and ventricular septal defects which appear to result from aberrant cardiac neural crest development (Sekhavat et al., 2010; Abu-Sulaiman & Subaih, 2004; Hornberger, 2006; Kumar et al., 2007; Russell et al., 2008). Hypertrophic cardiomyopathy was observed in 38% of neonates from insulin-dependent diabetes mellitus pregnant women, mainly hypertrophy of the interventricular septum (Abu-Sulaiman & Subaih, 2004). Congenital Cardiopathies Screening Associated with Diabetes Mellitus Using Maternal Fructosamine Plasma Concentration 259 Fig. 3. Receiver–operating characteristics (ROC) curve for prediction of congenital cardiopathies at echocardiography for fetuses whose mothers had preexisting diabetes 2.23 mmol/L 88.2% 54.8% 11.8% 2.0 2.68 mmol/L 58.8% 87.1% 41.2% 4.6 2.90 mmol/L 44.1% 96.8% 55.9% 13.7 Table 1. Accuracy of the maternal fructosamine levels for the prediction of fetal cardiac anomalies in gestations complicated by diabetes mellitus. Reference: Reis et al., 2010b If it is considered the cut-off value of fructosamine recommended for our local population (2.68mmol\L), this test will have a good specificity (87.1%) but a low sensitivity (58.8%), with a false negative rate of 41.2%. Even though, for an abnormal exam, the risk of fetal cardiac anomaly is increased 4.6-fold. If it is considered the cut-off value recommended by the manufacturer of the test (2.90mmol/L), the exam will have a high specificity (96.8%) but a low sensitivity (44.1%) with a false negative rate of 55.9%. In this case, for an abnormal test, the risk of fetal cardiac defects would be increased 13.7-fold. Finally considering the use of a cut-off point of 2.23mmol\L, the test will show a low specificity (54.8%) but a high sensitivity (88.2%), with a false negative rate of 11.8%. Thus, the cut-off point of 2.23 mmol/L is better than the other values tested, since it has the greatest sensitivity and lowest Sensitivity Specificity False negative Positive Likelihood ratio mellitus (n=65). AUC 0.78 (95% CI, 0.66–0.89), from: Reis et al., 2010 Cut-off points fructosamine ) (maternal plasmatic levels of false negative rate among them. #### 4. The fructosamine level as a late marker (beyond the first trimester) for congenital cardiopathies The measurement of glycated hemoglobin (HbAlc) and serum fructosamine in order to assess the recent glycemic control of diabetic patients has become well established. Maternal HbAlc at the beginning of pregnancy and maternal age at the onset of diabetes were associated with congenital malformations (Aberg at al., 2001). Fructosamines are keto-amines formed by a non-enzymatic reaction between glucose and a protein (60-70% of which is glycosylated with albumin in serum), depending upon the severity and the duration of the hyperglycemia. Therefore, serum fructosamine directly reflects the dynamics of blood glucose concentration and correlates significantly with the mean plasma glucose levels from the preceding 1 to 3 weeks (Roberts et al., 1988; Weerasekera, 2000; Jenkins et al., 2007). Fructosamine testing has been available since the 1980s. Both fructosamine and HbA1c are used primarily as monitoring tools to help diabetics control their blood sugar, but the A1C test is much more popular and more widely accepted. However, the American Diabetes Association recognizes both tests and says that fructosamine may be useful in situations where the A1C cannot be reliably measured (Goldstein et al., 2004). In addition, the measurement of fructosamine can be a helpful adjunct to HbA1c glycaemic control monitoring during pregnancy (Chaudry et al., 2007). The role of fructosamine levels as a teratogenic marker is less studied. Fetuses presenting a normal and abnormal echocardiography were compared using plasma fructosamine level means (Reis at al., 2010a). An association between congenital cardiopathies at echocardiography (functional and structural including isolated hypertrophic cardiomiopathy) and types of diabete mellitus (insulin-depending or not), was evaluated. An abnormal plasma fructosamine level at 20.4±8.0 weeks of gestation was associated with congenital cardiopathies at echocardiography, whether or not the cardiac embryogenesis happened in the first trimester. The congenital cardiopathies at echocardiography odds ratio was 9.6 (95% CI: 2.8 – 33.7) for abnormal plasma fructosamine (≥2.68mmol/L) and 10.9 (95% CI: 2.7 – 45.2) when adjusted for maternal age and insulin usage. There was also an increased chance (3.1, 95% CI: 1.1 - 8.8) of fetal heart anomaly with insulin usage, but only when evaluated individually by crude odds ratio. In many underdeveloped countries, women do not have access or do not attend medical care early in pregnancy (Reis at al., 2010a, 2010b). Therefore, they are especially subject to the teratogenic effects of hyperglycemia. Analyzing results of our universitary referral center, it was disappointing that so few diabetic women receive preconception counseling and plan their pregnancies (Reis at al., 2010a). Therefore, considering this reality, without HbA1c early values, it was important to determine the correlation between fructosamine maternal levels and fetal malformations. Based on our previous study, abnormal maternal fructosamine levels, even at the second trimester of pregnancy, predicts a high risk of fetal cardiac anomalies (Reis at al., 2010a). In this way, a fetal echocardiographic exam should be, routinely, performed in all diabetic mothers whose fructosamine levels are above 2.23 mmol/L (Reis et al., 2010b). This recommendation was based on the significant capacity of maternal fructosamine levels to predict fetal heart anomaly in diabetic patients (Area Under Curve: 0.78 p-valor <0.0001), as shown in Fig. 3. However, different cut-off values from which fructosamine could indicate these malformations and they should be cautiously defined and discussed as shown in Table 1. The measurement of glycated hemoglobin (HbAlc) and serum fructosamine in order to assess the recent glycemic control of diabetic patients has become well established. Maternal HbAlc at the beginning of pregnancy and maternal age at the onset of diabetes were Fructosamines are keto-amines formed by a non-enzymatic reaction between glucose and a protein (60-70% of which is glycosylated with albumin in serum), depending upon the severity and the duration of the hyperglycemia. Therefore, serum fructosamine directly reflects the dynamics of blood glucose concentration and correlates significantly with the mean plasma glucose levels from the preceding 1 to 3 weeks (Roberts et al., 1988; Weerasekera, 2000; Jenkins et al., 2007). Fructosamine testing has been available since the 1980s. Both fructosamine and HbA1c are used primarily as monitoring tools to help diabetics control their blood sugar, but the A1C test is much more popular and more widely accepted. However, the American Diabetes Association recognizes both tests and says that fructosamine may be useful in situations where the A1C cannot be reliably measured (Goldstein et al., 2004). In addition, the measurement of fructosamine can be a helpful adjunct to HbA1c glycaemic control monitoring during pregnancy (Chaudry et al., 2007). Fetuses presenting a normal and abnormal echocardiography were compared using plasma fructosamine level means (Reis at al., 2010a). An association between congenital cardiopathies at echocardiography (functional and structural including isolated hypertrophic cardiomiopathy) and types of diabete mellitus (insulin-depending or not), was evaluated. An abnormal plasma fructosamine level at 20.4±8.0 weeks of gestation was associated with congenital cardiopathies at echocardiography, whether or not the cardiac embryogenesis happened in the first trimester. The congenital cardiopathies at echocardiography odds ratio was 9.6 (95% CI: 2.8 – 33.7) for abnormal plasma fructosamine (≥2.68mmol/L) and 10.9 (95% CI: 2.7 – 45.2) when adjusted for maternal age and insulin usage. There was also an increased chance (3.1, 95% CI: 1.1 - 8.8) of fetal heart anomaly with In many underdeveloped countries, women do not have access or do not attend medical care early in pregnancy (Reis at al., 2010a, 2010b). Therefore, they are especially subject to the teratogenic effects of hyperglycemia. Analyzing results of our universitary referral center, it was disappointing that so few diabetic women receive preconception counseling and plan their pregnancies (Reis at al., 2010a). Therefore, considering this reality, without HbA1c early values, it was important to determine the correlation between fructosamine Based on our previous study, abnormal maternal fructosamine levels, even at the second trimester of pregnancy, predicts a high risk of fetal cardiac anomalies (Reis at al., 2010a). In this way, a fetal echocardiographic exam should be, routinely, performed in all diabetic This recommendation was based on the significant capacity of maternal fructosamine levels to predict fetal heart anomaly in diabetic patients (Area Under Curve: 0.78 p-valor <0.0001), as shown in Fig. 3. However, different cut-off values from which fructosamine could indicate these malformations and they should be cautiously defined and discussed as shown in Table 1. mothers whose fructosamine levels are above 2.23 mmol/L (Reis et al., 2010b). 4. The fructosamine level as a late marker (beyond the first trimester) for associated with congenital malformations (Aberg at al., 2001). The role of fructosamine levels as a teratogenic marker is less studied. insulin usage, but only when evaluated individually by crude odds ratio. maternal levels and fetal malformations. congenital cardiopathies Fig. 3. Receiver–operating characteristics (ROC) curve for prediction of congenital cardiopathies at echocardiography for fetuses whose mothers had preexisting diabetes mellitus (n=65). AUC 0.78 (95% CI, 0.66–0.89), from: Reis et al., 2010 Table 1. Accuracy of the maternal fructosamine levels for the prediction of fetal cardiac anomalies in gestations complicated by diabetes mellitus. Reference: Reis et al., 2010b If it is considered the cut-off value of fructosamine recommended for our local population (2.68mmol\L), this test will have a good specificity (87.1%) but a low sensitivity (58.8%), with a false negative rate of 41.2%. Even though, for an abnormal exam, the risk of fetal cardiac anomaly is increased 4.6-fold. If it is considered the cut-off value recommended by the manufacturer of the test (2.90mmol/L), the exam will have a high specificity (96.8%) but a low sensitivity (44.1%) with a false negative rate of 55.9%. In this case, for an abnormal test, the risk of fetal cardiac defects would be increased 13.7-fold. Finally considering the use of a cut-off point of 2.23mmol\L, the test will show a low specificity (54.8%) but a high sensitivity (88.2%), with a false negative rate of 11.8%. Thus, the cut-off point of 2.23 mmol/L is better than the other values tested, since it has the greatest sensitivity and lowest false negative rate among them. Congenital Cardiopathies Screening Associated with 1130, (May 2006), ISSN 1355-6037. pp.2995-3014, (May 2007), ISSN 0009-7322. ISSN 1935-5548. Diabetes Mellitus Using Maternal Fructosamine Plasma Concentration 261 Goldstein, D.E.; Little, R.R.; Lorenz, R.A.; Malone, J.I.; Nathan, D.M.; Peterson, C.M. (2004) Hornberger, L.K. (2006). Maternal diabetes and the fetal heart. Heart, Vol.92, No.8, pp.1125- Jenkins, K.J.: Correa, A.: Feinstein, J.A.; Botto, L.: Britt, A.E.; Daniels, S.R.; Elixson, M.; Kumar, S.D.; Dheen, S.T. & Tay, S.S.W. (2007). Maternal diabetes induces congenital heart Leonard, C.L.; Bergman, M.; Frenz, D.A.; Macreery, L.A.; Newman, S.A. (1989). Abnormal Reis, Z.S.N.; Miranda, A.P.B.; Rezende, C.A.; Bragança, R.D.; Ribeiro, C.C.; Cabral, A.C.V. Ren, Y.; Zhou1, Q.; Yan, Y.; Chu, C.; Gui, Y. Li, X. (2011). Characterization of fetal cardiac Roberts, A.B.; Baker, J.R.; James, A.G.; Henley, P. (1988). Fructosamine in the management Russell, N.E.; Holloway, P.; Quinn, S.; Foley, M.: Kelehan, P.; McAuliffe, F.M. (2008). Schaefer-Graf, U.M.; Buchanan, T.A.; Xiang, A.N.; Songster, G. Montoro, M.; Kjos, S.L. Dev Pathol, Vol.11, No.1, pp.10-14, (Jan-Fev 2008), ISSN 1615-5742. Obst Gynecol, Vol.182, No.2, pp.313–320, (Feb 2000), ISSN 0002-9378. Sekhavat, S.; Kishore, N.; Levine J.C. (2010). Screening fetal echocardiography in diabetic Gynecol, Vol.35, No.2, pp.178–182, (Feb 2010), ISSN 1471-0528. Fetal Neonatal Med. (Early on line, Dec 2010), ISSN 1476-7058. Vol.32, No.2, pp. 66-71, (Feb 2010), ISSN 0100-7203. 2011). ISSN 1097-0223. 0002-9378. Tests of glycemia in diabetes. Diabetes Care, Vol.27, No.7, pp.1761-1763, (Jul 2004), Warnes, C.A.; Webb, C.L. (2007). Noninherited risk factors and congenital cardiovascular defects: current knowledge: a scientific statement from the American Heart Association Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation, Vol.115, No.23, defects in mice by altering the expression of genes involved in cardiovascular development. Cardiovascular Diabetology, Vol. 6, No. 34, (Oct 2007), ISSN 1475-2840. ambient glucose levels inhibit proteoglycan core protein gene expression and reduce proteoglycan accumulation during chondrogenesis: Possible mechanism for teratogenic effects of maternal diabetes (extracellular matrix/skeletal birth defects). Proc. Nati. Acad. Sci, Vol.86, No.24, pp.10113-10117, (Dec 1989), ISSN 0027-8424. Reis, Z.S.; Miranda, A.P.; Lage, E.M.; Bragança, R.D.; Costa, C.R.; Cabral, A.C.V. (2010a). Echocardiographic findings of congenital cardiopathies among fetuses of diabetic pregnant women and their relationship with plasma fructosamine levels. J Matern (2010b). [Congenital cardiopathies screening associated with diabetes mellitus using maternal fructosamine plasma concentration]. Rev. Bras. Ginecol. Obstet, structure and function detected by echocardiography in women with normal pregnancy and gestational diabetes mellitus. Prenat Diagn. (Early on line, Mar of gestational diabetes. Am J Obstet Gynecol, Vol.159, No.1, pp.66-71, (Jul 1988), ISSN Cardiomyopathy and cardiomegaly in stillborn infants of diabetic mothers. Pediatr (2000). Patterns of congenital anomalies and relationship to initial maternal fasting glucose levels in pregnancies complicated by type 2 and gestational diabetes. Am J mothers with normal findings on detailed anatomic survey. Ultrasound Obstet #### 5. Conclusions Abnormal echocardiographic findings were associated with the first maternal plasma fructosamine levels in referral pregnancies complicated by diabetes mellitus. Hyperglycemia seems to be the most important determinant of these risks. Many pregnant diabetic women are referred at a late stage to a tertiary level of care. At this context, an abnormal plasma fructosamine level increases the chances of abnormalities at fetal echocardiography. It is possible to use a second trimester plasma fructosamine level to refer women with pregestational diabetes mellitus to a center of maternal-fetal medicine in order to offer them an appropriated assistance at birth. These findings are important for the management of women with diabetes mellitus and late prenatal care. #### 6. Acknowledgment CAPES Foundation, Ministry of Education of Brazil, BEX 3105/10-5 process #### 7. References Abnormal echocardiographic findings were associated with the first maternal plasma fructosamine levels in referral pregnancies complicated by diabetes mellitus. Hyperglycemia seems to be the most important determinant of these risks. Many pregnant diabetic women are referred at a late stage to a tertiary level of care. At this context, an abnormal plasma fructosamine level increases the chances of abnormalities at fetal echocardiography. It is possible to use a second trimester plasma fructosamine level to refer women with pregestational diabetes mellitus to a center of maternal-fetal medicine in order to offer them an appropriated assistance at birth. These findings are important for the management of Aberg, A.: Westbom, L. & Kallen, B. (2001). Congenital malformations among infants whose Abu-Sulaiman, R.M. & Subaih, B: (2004). Congenital heart disease in infants of diabetic Allen, V.M.; Armson, B.A.; Wilson, R.D.; Allen, V.M.; Blight, C.; Gagnon, A.; Johnson, J.A.; Buchanan, T.A. & Kjos, S.L. (1999). Gestational Diabetes: Risk or Myth?. The Journal of Chaudhari, M.; Brodlie, M. & Hasan, A. (2008). Hypertrophic cardiomyopathy and Chaudry, R.; Gilby, P. & Carroll, V. (2007). Pre-existing (type 1 and type 2) diabetes in Crowther, C.A.; Hiller, J.E.; Moss, J.R., et al. (2005). Effect of treatment of gestational diabetes Gynaecol Can, Vol.29, No.11, (Nov 2007), pp.927-944, ISSN 1701-2163. American Diabetes Association [ADA]. (2004). Preconception care of women with diabetes. Diabetes Care, Vol.27, Suppl 1, pp. 76, (Jan 2004). ISSN 1935-5548. mothers had gestational diabetes or preexisting diabetes. Early Human Development, mothers: echocardiographic study. Pediatr Cardiol, Vol.25, No.2, (Mar-Apr 2004), Langlois, S.; Summers, A.; Wyatt, P.; Farine, D.; Armson, B.A.: Crane, J.; Delisle, M.F.; Keenan-Lindsay, L.; Morin, V.; Schneider, C.E.; Van Aerde, J. (2007) Teratogenicity associated with pre-existing and gestational diabetes. J Obstet Clinical Endocrinology & Metabolism, Vol.84, No.6, (1999), pp.1854-1857, ISSN 1945- transposition of great arteries associated with maternal diabetes and presumed gestational diabetes. Acta Paediatr, Vol.97, No.12, pp.1755-1757, (2008), ISSN 1651- pregnancy. Obstetrics, Gynaecology and Reproductive Medicine, Vol.17, No.12, (Dec mellitus on pregnancy outcomes. N Engl J Med, Vol.352, No.24, pp.2477, (Jun 2005), 5. Conclusions 6. Acknowledgment 7197. 2227. ISSN 1533-4406. 7. References women with diabetes mellitus and late prenatal care. pp.137-140, ISSN 1551-4439. 2007), pp.339-334. ISSN 1751-7214. CAPES Foundation, Ministry of Education of Brazil, BEX 3105/10-5 process Vol.61, No.2, (Mar 2001), pp.85–95, ISSN 0378-3782. 15 China William WK To Department of Obstetrics & Gynaecology United Christian Hospital, Hong Kong Applications of Doppler Studies for Fetal Surveillance in Diabetic Pregnancies Diabetes mellitus complicating pregnancy is one of the most common antenatal complications that are associated with significant perinatal mortality and morbidity (Magee et al., 1993; Platt et al., 2002; Schmidt et al., 2001). Diabetic pregnancies can be divided into two categories: those with pre-gestational or pre-existing diabetes mellitus in which the diagnosis is made in the pre-pregnancy state, and those with gestational diabetes mellitus (GDM). Pre-existing diabetes consists of type I (insulin-dependent) diabetes mellitus (IDDM)with an incidence of around 0.5%, and type 2 ( non-insulin-dependent) diabetes with an incidence of 2-3% ( Kapoor et al., 2007). The incidence of gestational diabetes mellitus differs in different populations (Gunton et al., 2001) and ethnic groups, and was shown to be as high as 13% in Chinese populations (Ko et al., 2002). Effective treatment of pre-existing as well as gestational diabetes mellitus was shown to improve outcome and reduce perinatal mortality, as compared to untreated patients The pathological conditions encountered in fetuses of diabetic pregnancies differ in those with pre-existing diabetes mellitus and those with gestational diabetes. Pre-existing diabetics with persistent hyperglycaemia in the perinatal period are at higher risks of congenital malformations (Reece et al., 2007). In addition, those women with long-standing pre-existing diabetes before the index pregnancy run a higher risk of having diabetic vasculopathy that may affect various organ-systems in the body. Involvement of the uterine arteries will affect the development of an effective utero-placenta blood flow, which would be vital in maintaining normal growth and development in the fetus. The clinical manifestation of fetal growth restriction (also called intrauterine growth restriction) is thus more common in these pregnancies. The presence of significant congenital abnormalities and severe fetal growth restriction resulting from such conditions are logically directly related to increase in perinatal mortality and morbidity. On the other hand, gestational diabetes is usually only diagnosable either in a screening protocol or from clinical risk factors, by oral glucose tolerance test from mid trimester onwards, due to the effects of diabetogenic hormones from the placenta. The carbohydrate intolerance is thus short-lived and should last only from mid trimester to term. The hyperglycaemic states of the diabetes should revert to normal shortly after delivery with the removal of the placenta. Thus, chronic complications such as vasculopathy in the pregnant women will not have time to evolve during the course of pregnancy. On the contrary, the diabetic hyperglycaemic states 1. Introduction (Lao et al., 2001; Langer et al., 2005). ### Applications of Doppler Studies for Fetal Surveillance in Diabetic Pregnancies #### William WK To Department of Obstetrics & Gynaecology United Christian Hospital, Hong Kong China #### 1. Introduction 262 Gestational Diabetes Sheffield, J.S.; Butler-Koster, E.L.; Casey, B.M.; McIntire, D.D.; Leveno, K.J. (2002). Maternal Weerasekera, D.S. & Peiris H. (2000). The value of serum fructosamine in comparison with pp.925–930, (Dec 2002), ISSN 1873-233X. (Mar 2000), ISSN 0144-3615. Diabetes Mellitus and Infant Malformations. Obstet Gynecol, Vol.100, No.5,pt1, oral glucose tolerance test (OGTT) as a screening test for detection of gestational diabetes mellitus. Journal of Obstetrics and Gynaecology, Vol.20, No.2, pp.136-138, > Diabetes mellitus complicating pregnancy is one of the most common antenatal complications that are associated with significant perinatal mortality and morbidity (Magee et al., 1993; Platt et al., 2002; Schmidt et al., 2001). Diabetic pregnancies can be divided into two categories: those with pre-gestational or pre-existing diabetes mellitus in which the diagnosis is made in the pre-pregnancy state, and those with gestational diabetes mellitus (GDM). Pre-existing diabetes consists of type I (insulin-dependent) diabetes mellitus (IDDM)with an incidence of around 0.5%, and type 2 ( non-insulin-dependent) diabetes with an incidence of 2-3% ( Kapoor et al., 2007). The incidence of gestational diabetes mellitus differs in different populations (Gunton et al., 2001) and ethnic groups, and was shown to be as high as 13% in Chinese populations (Ko et al., 2002). Effective treatment of pre-existing as well as gestational diabetes mellitus was shown to improve outcome and reduce perinatal mortality, as compared to untreated patients (Lao et al., 2001; Langer et al., 2005). > The pathological conditions encountered in fetuses of diabetic pregnancies differ in those with pre-existing diabetes mellitus and those with gestational diabetes. Pre-existing diabetics with persistent hyperglycaemia in the perinatal period are at higher risks of congenital malformations (Reece et al., 2007). In addition, those women with long-standing pre-existing diabetes before the index pregnancy run a higher risk of having diabetic vasculopathy that may affect various organ-systems in the body. Involvement of the uterine arteries will affect the development of an effective utero-placenta blood flow, which would be vital in maintaining normal growth and development in the fetus. The clinical manifestation of fetal growth restriction (also called intrauterine growth restriction) is thus more common in these pregnancies. The presence of significant congenital abnormalities and severe fetal growth restriction resulting from such conditions are logically directly related to increase in perinatal mortality and morbidity. On the other hand, gestational diabetes is usually only diagnosable either in a screening protocol or from clinical risk factors, by oral glucose tolerance test from mid trimester onwards, due to the effects of diabetogenic hormones from the placenta. The carbohydrate intolerance is thus short-lived and should last only from mid trimester to term. The hyperglycaemic states of the diabetes should revert to normal shortly after delivery with the removal of the placenta. Thus, chronic complications such as vasculopathy in the pregnant women will not have time to evolve during the course of pregnancy. On the contrary, the diabetic hyperglycaemic states Applications of Doppler Studies for Fetal Surveillance in Diabetic Pregnancies 265 born non-large- for- gestational- age when abdominal circumference was < 90th percentile at 24-27 weeks and 28-32 weeks respectively; and 88% were born non-large-for-gestational-age when both scans showed normal growth. For those women who had no risk factors for fetal overgrowth, such as body mass index > 30 kg/m2, history of macrosomia, and fasting glucose > 100 mg/dl, the accuracy of prediction of a non-large-for-gestational-age neonate was 90.0, 89.5 and 95.2%. The predictive ability did not increase with more than two normal scans. It was concluded that the yield of sonographic diagnosis of a large fetus dropped markedly after the finding if a large fetal abdominal circumference < 90th percentile on tow sonograms, which excludes with a high reliability the risk of a large for gestational age newborn. The ability was enhanced in women who had no risk factors for neonatal macrosomia (Schaefer-Graf et al., 2011). Thus, sonographic evaluation of fetal growth parameters is a crucial part of ultrasound assessment of diabetic pregnancies. The application of Doppler examination should take into account the data from such growth Various Doppler parameters have conventionally been utilized for fetal surveillance. The use maternal uterine artery Doppler as a screening tool to predict subsequent development of pre-eclampsia and fetal growth restriction in later gestation have gained acceptance in recent years. Umbilical artery Doppler, middle cerebral artery Doppler, or the combination of the two to produce a cerebral-placental ratio, have been widely used for the assessment of growth restricted fetuses. Less commonly used arterial Doppler parameters include fetal thoracic aorta or renal arteries. Venous Doppler assessment has been extensively investigated in recent years, including umbilical venous waveforms, intrahepatic venous Doppler and ductus venosus Doppler. The following sections attempt to review the application of the more commonly used Doppler parameters in diabetic pregnancies. adverse outcomes for either pre-existing or gestational diabetes mellitus alone. When uterine artery Doppler were examined in a cohort of 43 pregnancies complicated by insulin-dependent diabetes, it was found that the uterine artery resistance indices were Maternal uterine artery remodelling is the hallmark sign of successful placentation, which can be demonstrated by progressive alterations in Doppler waveforms derived from the immediate extrauterine portion of the uterine artery. Pre-pregnancy uterine arteries show high resistance and elastic recoil in the form of early diastolic notches and low diastolic flow (Schulman et al., 1986). Successful placental invasion removes intimal muscle and reduces vascular resistance and elastic properties, giving more rigorous diastolic flow. In pregnancies with normal placentation, Doppler studies show that this remodelling is rapid, with loss of notching by 12 weeks and low resistance indices by 20 weeks or sooner. When placentation is deficient, notching of waveforms remains with high resistance (Papageorhiou et al., 2001). Such a picture of deficient placentation strongly predicts maternal hypertension, including proteinuric pre-eclampsia, fetal growth restriction and fetal demise (Coleman et al., 2000). Uterine artery Doppler screening using high resistance, persistent notching, or both, identifies such risks with a sensitivity up to 85% for severe proteinuric pre-eclampsia and for severe fetal growth restriction (Papageorghiou et al., 2002). However, there is so far no well established evidence of using such screening for assessment parameters. 3.1 Maternal uterine artery 3. Use of Doppler studies for fetal surveillance in gestational diabetes would stimulate fetal hyperinsulinemia, which would in turn lead to over-secretion of insulin-like growth factors (IGF), leading to overgrowth of the fetus (Kapoor et al., 2007). Thus, in contrast to pre-gestational diabetes mellitus, where vasculopathy is rife and the incidences of pre-eclampsia and fetal growth restriction are commonly encountered, GDM pregnancies pose a different category of pathophysiology and clinical risks. The most common fetal problems for gestational diabetes will be macrosomia with associated polyhydramnios, as well as increased risks of near term stillbirths and neonatal metabolic complications in livebirths (Sacks, 2007). Indeed, recent data borne out by the HAPO study has demonstrated that in maternal hyperglycaemic states less severe than in diabetes mellitus is also associated with increased risks of adverse pregnancy outcomes (HAPO Study Cooperative Research Group, 2008) A lot of emphasis has been put on fetal surveillance in these high-risk pregnancies in the attempt to optimise pregnancy outcome. There is, however, no consensus as to the best methods of antepartum surveillance (American College of Obstetricians & Gynecologists, 2001). The use of Doppler studies of the umbilical artery has been demonstrated to reduce perinatal adverse outcome in non-diabetic pregnancies (Alfirevic et al., 1995), but its use in diabetic pregnancies have shown conflicting results. While umbilical artery Doppler studies have been shown to be more predictive of adverse outcome than cardiotocography and biophysical profile in diabetic pregnancies (Bracero et al., 1996), fetal compromise would occasionally still be observed despite normal Doppler studies (Johnstone et al., 1992). Thus, the precise value of Doppler studies in the monitoring of GDM pregnancies remains controversial. #### 2. Serial growth scans for diabetic pregnancies The recommendations for diagnosis and treatment of GDM of the Fifth International Workshop-Conference on Gestational Diabetes Mellitus (Metzger et al., 2007) suggest consideration of fetal growth patterns to guide metabolic management of pregnant women with GDM. However, estimation of fetal weight, particularly at term and in fetuses with high neonatal weight, is not as precise as desirable (Sacks et al, 2000). Fetal overgrowth and accelerated growth velocity of the abdominal circumference in the third trimester is known to predict large for gestational age babies (Kehl et al., 1996). Fetal overgrowth with macrosomia and associated polyhydramnios is associated with higher risks of near term stillbirth, as well as various neonatal metabolic derangements, including neonatal hypoglycaemia, electrolyte disturbances and neonatal jaundice. Previous randomized studies have demonstrated that measurement of fetal abdominal circumference throughout pregnancy in women with GDM is useful to identify pregnancies at high risk for fetal overgrowth and thus in need of more vigilant treatment including insulin therapy (Bonomo et al., 2004). Serial measurements of the fetal abdominal circumference have been used to guide metabolic management of pregnancies complicated by gestational diabetes mellitus. There is at present no consensus as to the optimal protocol for such growth scans. A recent study evaluated the number of sonograms needed to reliably predict the absence of fetal overgrowth in GDM pregnancies. A total of 4478 ultrasound examinations were performed on 1914 subjects. Of 518 women with fetal abdominal circumference > 90th percentile, the diagnosis was detected in 73.9% with the first ultrasound examination at entry and in 13.1% with the second ultrasound examination. Of the fetuses, 85.9 and 86.9% of the fetuses were born non-large- for- gestational- age when abdominal circumference was < 90th percentile at 24-27 weeks and 28-32 weeks respectively; and 88% were born non-large-for-gestational-age when both scans showed normal growth. For those women who had no risk factors for fetal overgrowth, such as body mass index > 30 kg/m2, history of macrosomia, and fasting glucose > 100 mg/dl, the accuracy of prediction of a non-large-for-gestational-age neonate was 90.0, 89.5 and 95.2%. The predictive ability did not increase with more than two normal scans. It was concluded that the yield of sonographic diagnosis of a large fetus dropped markedly after the finding if a large fetal abdominal circumference < 90th percentile on tow sonograms, which excludes with a high reliability the risk of a large for gestational age newborn. The ability was enhanced in women who had no risk factors for neonatal macrosomia (Schaefer-Graf et al., 2011). Thus, sonographic evaluation of fetal growth parameters is a crucial part of ultrasound assessment of diabetic pregnancies. The application of Doppler examination should take into account the data from such growth assessment parameters. #### 3. Use of Doppler studies for fetal surveillance Various Doppler parameters have conventionally been utilized for fetal surveillance. The use maternal uterine artery Doppler as a screening tool to predict subsequent development of pre-eclampsia and fetal growth restriction in later gestation have gained acceptance in recent years. Umbilical artery Doppler, middle cerebral artery Doppler, or the combination of the two to produce a cerebral-placental ratio, have been widely used for the assessment of growth restricted fetuses. Less commonly used arterial Doppler parameters include fetal thoracic aorta or renal arteries. Venous Doppler assessment has been extensively investigated in recent years, including umbilical venous waveforms, intrahepatic venous Doppler and ductus venosus Doppler. The following sections attempt to review the application of the more commonly used Doppler parameters in diabetic pregnancies. #### 3.1 Maternal uterine artery 264 Gestational Diabetes in gestational diabetes would stimulate fetal hyperinsulinemia, which would in turn lead to over-secretion of insulin-like growth factors (IGF), leading to overgrowth of the fetus (Kapoor et al., 2007). Thus, in contrast to pre-gestational diabetes mellitus, where vasculopathy is rife and the incidences of pre-eclampsia and fetal growth restriction are commonly encountered, GDM pregnancies pose a different category of pathophysiology and clinical risks. The most common fetal problems for gestational diabetes will be macrosomia with associated polyhydramnios, as well as increased risks of near term stillbirths and neonatal metabolic complications in livebirths (Sacks, 2007). Indeed, recent data borne out by the HAPO study has demonstrated that in maternal hyperglycaemic states less severe than in diabetes mellitus is also associated with increased risks of adverse A lot of emphasis has been put on fetal surveillance in these high-risk pregnancies in the attempt to optimise pregnancy outcome. There is, however, no consensus as to the best methods of antepartum surveillance (American College of Obstetricians & Gynecologists, 2001). The use of Doppler studies of the umbilical artery has been demonstrated to reduce perinatal adverse outcome in non-diabetic pregnancies (Alfirevic et al., 1995), but its use in diabetic pregnancies have shown conflicting results. While umbilical artery Doppler studies have been shown to be more predictive of adverse outcome than cardiotocography and biophysical profile in diabetic pregnancies (Bracero et al., 1996), fetal compromise would occasionally still be observed despite normal Doppler studies (Johnstone et al., 1992). Thus, the precise value of Doppler studies in the monitoring of GDM pregnancies remains The recommendations for diagnosis and treatment of GDM of the Fifth International Workshop-Conference on Gestational Diabetes Mellitus (Metzger et al., 2007) suggest consideration of fetal growth patterns to guide metabolic management of pregnant women with GDM. However, estimation of fetal weight, particularly at term and in fetuses with high neonatal weight, is not as precise as desirable (Sacks et al, 2000). Fetal overgrowth and accelerated growth velocity of the abdominal circumference in the third trimester is known to predict large for gestational age babies (Kehl et al., 1996). Fetal overgrowth with macrosomia and associated polyhydramnios is associated with higher risks of near term stillbirth, as well as various neonatal metabolic derangements, including neonatal hypoglycaemia, electrolyte disturbances and neonatal jaundice. Previous randomized studies have demonstrated that measurement of fetal abdominal circumference throughout pregnancy in women with GDM is useful to identify pregnancies at high risk for fetal overgrowth and thus in need of more vigilant treatment including insulin therapy (Bonomo Serial measurements of the fetal abdominal circumference have been used to guide metabolic management of pregnancies complicated by gestational diabetes mellitus. There is at present no consensus as to the optimal protocol for such growth scans. A recent study evaluated the number of sonograms needed to reliably predict the absence of fetal overgrowth in GDM pregnancies. A total of 4478 ultrasound examinations were performed on 1914 subjects. Of 518 women with fetal abdominal circumference > 90th percentile, the diagnosis was detected in 73.9% with the first ultrasound examination at entry and in 13.1% with the second ultrasound examination. Of the fetuses, 85.9 and 86.9% of the fetuses were pregnancy outcomes (HAPO Study Cooperative Research Group, 2008) 2. Serial growth scans for diabetic pregnancies controversial. et al., 2004). Maternal uterine artery remodelling is the hallmark sign of successful placentation, which can be demonstrated by progressive alterations in Doppler waveforms derived from the immediate extrauterine portion of the uterine artery. Pre-pregnancy uterine arteries show high resistance and elastic recoil in the form of early diastolic notches and low diastolic flow (Schulman et al., 1986). Successful placental invasion removes intimal muscle and reduces vascular resistance and elastic properties, giving more rigorous diastolic flow. In pregnancies with normal placentation, Doppler studies show that this remodelling is rapid, with loss of notching by 12 weeks and low resistance indices by 20 weeks or sooner. When placentation is deficient, notching of waveforms remains with high resistance (Papageorhiou et al., 2001). Such a picture of deficient placentation strongly predicts maternal hypertension, including proteinuric pre-eclampsia, fetal growth restriction and fetal demise (Coleman et al., 2000). Uterine artery Doppler screening using high resistance, persistent notching, or both, identifies such risks with a sensitivity up to 85% for severe proteinuric pre-eclampsia and for severe fetal growth restriction (Papageorghiou et al., 2002). However, there is so far no well established evidence of using such screening for adverse outcomes for either pre-existing or gestational diabetes mellitus alone. When uterine artery Doppler were examined in a cohort of 43 pregnancies complicated by insulin-dependent diabetes, it was found that the uterine artery resistance indices were Applications of Doppler Studies for Fetal Surveillance in Diabetic Pregnancies 267 occurs in women with pre-existing diabetes mellitus with vasculopathy. In pregnancies with suspected fetal growth restriction, the use of umbilical artery Doppler has been demonstrated to reduce the number of perinatal deaths and unnecessary obstetric interventions. In the context of diabetic pregnancies, however, the applications of umbilical To investigate the vascular resistance by Doppler ultrasound in the umbilical artery of 53 IDDM pregnancies longitudinally over the course of pregnancy, the resistance index of the Doppler waveform was correlated with the mean value of a 24 hour blood glucose profile and the concentrations of glycosylated haemoglobin (HbA1C), which represented parameters of metabolic control. Regression analysis, however, showed no significant correlation between vascular resistance and mean blood glucose levels or HbA1C In another study that included 67 normotensive women with pregnancies complicated by IDDM, the umbilical artery pulsatility index was compared with both pregnancy complications and perinatal outcome. The last umbilical pulsatility index value before delivery was used for analysis, and Doppler results were not used for patient management. Out of this cohort, 44 (66%) had pulsatility index values within the normal range between the 5th to 95th percentile, while 23 (34%) had abnormally high pulsatility index values. Among the group with pathologically abnormal umbilical pulsatility indices, analysis of the data revealed a significantly higher incidence of both caesarean section for acute fetal distress and perinatal complications. These complications include respiratory distress syndrome, hyperbilirubinemia, neonatal hypoglycaemia, and the need for neonatal intensive care unit admission. The authors concluded that in at least one third of IDDM patients, increased vascular resistance in the umbilical arteries were found, and these also A retrospective study of 146 patients with gestational diabetes noted that Doppler examination of the umbilical arteries seemed to be of little clinical value unless pregnancy was complicated by pre-eclampsia or intrauterine growth restriction. The study included 227 patients with diabetes, and umbilical artery Doppler velocimetry and glycaemic control were examined in the third trimester. An elevated systolic/diastolic ratio and an abnormal glycosylated haemoglobin level were associated with adverse pregnancy outcome, but there was no stratification for vascular disease or fetal growth restriction in the data (Bracero et al., 1996). In a prospective study of 65 well controlled diabetic pregnancies, Doppler measurements of uterine arteries, umbilical artery, the fetal descending thoracic aorta, and the middle cerebral artery (MCA) were performed together with cordocentesis for measurement of umbilical venous blood pH, pO2 and haematocrit. It was found that the mean umbilical venous blood pH was significantly lower and the haematocrit significantly higher than the appropriate normal mean for gestation for these diabetic pregnancies. However, the Doppler indices of the placental and fetal circulations were essentially normal, except in some of the cases complicated by pre-eclampsia or intrauterine growth restriction (Salvesen et al., 1993). It was thus concluded that maternal diabetes mellitus was not associated with abnormalities To evaluate a random single Doppler study of the systolic/diastolic ratio of the umbilical artery as a predictor of perinatal outcome in diabetic pregnancies, a prospective doubleblind study was performed in 92 diabetic pregnancies between 28 and 40 weeks gestation, and the results were associated with perinatal outcome parameters. The sensitivity and specificity of the Doppler studies as a predictor of poor perinatal outcome were 39% and suffered from higher incidences of perinatal complications (Fadda et al., 2001). in Doppler indices of the placental or fetal circulations. artery Doppler remain controversial. concentrations (Zimmermann et al., 1992). slightly higher in the presence of evident morphological vasculopathy, but could not predict diabetic specific fetal morbidity. It was concluded that in patients with diabetic vasculopathy, the uterine artery was also affected, but there was no relationship with long or short term parameters of glycaemic control. Based on the data, it was concluded that Doppler flow velocimetry of the uterine artery was a poor predictor of diabetes-specific fetal morbidity (Zimmermann et al., 1994). In another prospective survey, 24 well controlled insulin dependent pregestational diabetic pregnancies were compared with 25 healthy pregnant women and Doppler ultrasound was performed on two occasions in the third trimester separated by a one-month interval. At the first examination, the median pulsatility index in the fetal thoracic aorta and in the uterine artery was significantly lower in the diabetic group as compared to healthy controls, whereas umbilical and uterine pulsatility indices were similar. At the second examination at a more advanced gestation, a significant physiological decrease in the median pulsatility index of the fetal aorta and uterine artery was observed in the controls, but not in the diabetic group. There was no correlation between the glycosylated haemoglobin or random blood glucose levels and the Doppler indices. An increased incidence of neonatal morbidity was noted in the diabetic group. Thus, the normal physiological third trimester fall in resistance indices in the uteroplacental and fetal placental indices was apparently absent in the diabetic group, and this was associated with increased neonatal morbidity. The pulsatility indices were not influenced by blood glucose regulation (Grunewald et al., 1996). A similar retrospective study of 155 pre-gestational diabetic women between 22 weeks gestation and term also found that abnormal uterine artery Doppler with increased pulsatility index was related to pre-existing diabetic states with vasculopathy and adverse pregnancy outcome. There was an increased incidence of abnormal umbilical artery Doppler in those with abnormal uterine artery Doppler, indicating that the vasculopathy might influence placental perfusion and fetal well-being (Pietryga et al., 2006). Therefore, it is logical to expect that such screening by uterine artery Doppler will likely be effective only if the diabetic pregnancy is also complicated by gestational hypertension or fetal growth restriction. Given the higher incidence of such obstetric complications in pre-existing diabetes mellitus as compared to GDM, screening of diabetic pregnancies using uterine #### 3.2 Fetal umbilical artery Doppler The use of Doppler studies of the umbilical artery has been demonstrated to reduce perinatal adverse outcome in non-diabetic pregnancies (Alfirevic et al.,1995), but its use in diabetic pregnancies have shown conflicting results. Umbilical artery angle independent indices (systolic/diastolic ratio or pulsatility index) decrease with advancing gestation because of decreases in placenta vascular resistance, which physiologically occurs with advancing gestation. In pathologic conditions, such as intrauterine/ fetal growth restriction, the umbilical artery waveform change and the angle-independent indices become abnormal, giving values above their reference ranges. End-diastolic velocity may thus change from normal to reduced, absent or reversed. These changes represent an increased placental vascular resistance, and could pathologically be associated with a decrease in the number of placental arteries per high power field (Giles et al., 1985). The most common scenario for fetal growth restriction include placental insufficiency in singleton or multiple pregnancies from various aetiologies, which may or may not be associated with pre-eclampsia or other maternal conditions, or in fetuses with congenital malformations. Such a picture commonly artery Doppler in mid trimester would probably produce a higher yield in the former group. slightly higher in the presence of evident morphological vasculopathy, but could not predict diabetic specific fetal morbidity. It was concluded that in patients with diabetic vasculopathy, the uterine artery was also affected, but there was no relationship with long or short term parameters of glycaemic control. Based on the data, it was concluded that Doppler flow velocimetry of the uterine artery was a poor predictor of diabetes-specific fetal In another prospective survey, 24 well controlled insulin dependent pregestational diabetic pregnancies were compared with 25 healthy pregnant women and Doppler ultrasound was performed on two occasions in the third trimester separated by a one-month interval. At the first examination, the median pulsatility index in the fetal thoracic aorta and in the uterine artery was significantly lower in the diabetic group as compared to healthy controls, whereas umbilical and uterine pulsatility indices were similar. At the second examination at a more advanced gestation, a significant physiological decrease in the median pulsatility index of the fetal aorta and uterine artery was observed in the controls, but not in the diabetic group. There was no correlation between the glycosylated haemoglobin or random blood glucose levels and the Doppler indices. An increased incidence of neonatal morbidity was noted in the diabetic group. Thus, the normal physiological third trimester fall in resistance indices in the uteroplacental and fetal placental indices was apparently absent in the diabetic group, and this was associated with increased neonatal morbidity. The pulsatility indices were not influenced by blood glucose regulation (Grunewald et al., 1996). A similar retrospective study of 155 pre-gestational diabetic women between 22 weeks gestation and term also found that abnormal uterine artery Doppler with increased pulsatility index was related to pre-existing diabetic states with vasculopathy and adverse pregnancy outcome. There was an increased incidence of abnormal umbilical artery Doppler in those with abnormal uterine artery Doppler, indicating that the vasculopathy might influence placental perfusion and fetal well-being (Pietryga et al., 2006). Therefore, it is logical to expect that such screening by uterine artery Doppler will likely be effective only if the diabetic pregnancy is also complicated by gestational hypertension or fetal growth restriction. Given the higher incidence of such obstetric complications in pre-existing diabetes mellitus as compared to GDM, screening of diabetic pregnancies using uterine artery Doppler in mid trimester would probably produce a higher yield in the former group. The use of Doppler studies of the umbilical artery has been demonstrated to reduce perinatal adverse outcome in non-diabetic pregnancies (Alfirevic et al.,1995), but its use in diabetic pregnancies have shown conflicting results. Umbilical artery angle independent indices (systolic/diastolic ratio or pulsatility index) decrease with advancing gestation because of decreases in placenta vascular resistance, which physiologically occurs with advancing gestation. In pathologic conditions, such as intrauterine/ fetal growth restriction, the umbilical artery waveform change and the angle-independent indices become abnormal, giving values above their reference ranges. End-diastolic velocity may thus change from normal to reduced, absent or reversed. These changes represent an increased placental vascular resistance, and could pathologically be associated with a decrease in the number of placental arteries per high power field (Giles et al., 1985). The most common scenario for fetal growth restriction include placental insufficiency in singleton or multiple pregnancies from various aetiologies, which may or may not be associated with pre-eclampsia or other maternal conditions, or in fetuses with congenital malformations. Such a picture commonly morbidity (Zimmermann et al., 1994). 3.2 Fetal umbilical artery Doppler occurs in women with pre-existing diabetes mellitus with vasculopathy. In pregnancies with suspected fetal growth restriction, the use of umbilical artery Doppler has been demonstrated to reduce the number of perinatal deaths and unnecessary obstetric interventions. In the context of diabetic pregnancies, however, the applications of umbilical artery Doppler remain controversial. To investigate the vascular resistance by Doppler ultrasound in the umbilical artery of 53 IDDM pregnancies longitudinally over the course of pregnancy, the resistance index of the Doppler waveform was correlated with the mean value of a 24 hour blood glucose profile and the concentrations of glycosylated haemoglobin (HbA1C), which represented parameters of metabolic control. Regression analysis, however, showed no significant correlation between vascular resistance and mean blood glucose levels or HbA1C concentrations (Zimmermann et al., 1992). In another study that included 67 normotensive women with pregnancies complicated by IDDM, the umbilical artery pulsatility index was compared with both pregnancy complications and perinatal outcome. The last umbilical pulsatility index value before delivery was used for analysis, and Doppler results were not used for patient management. Out of this cohort, 44 (66%) had pulsatility index values within the normal range between the 5th to 95th percentile, while 23 (34%) had abnormally high pulsatility index values. Among the group with pathologically abnormal umbilical pulsatility indices, analysis of the data revealed a significantly higher incidence of both caesarean section for acute fetal distress and perinatal complications. These complications include respiratory distress syndrome, hyperbilirubinemia, neonatal hypoglycaemia, and the need for neonatal intensive care unit admission. The authors concluded that in at least one third of IDDM patients, increased vascular resistance in the umbilical arteries were found, and these also suffered from higher incidences of perinatal complications (Fadda et al., 2001). A retrospective study of 146 patients with gestational diabetes noted that Doppler examination of the umbilical arteries seemed to be of little clinical value unless pregnancy was complicated by pre-eclampsia or intrauterine growth restriction. The study included 227 patients with diabetes, and umbilical artery Doppler velocimetry and glycaemic control were examined in the third trimester. An elevated systolic/diastolic ratio and an abnormal glycosylated haemoglobin level were associated with adverse pregnancy outcome, but there was no stratification for vascular disease or fetal growth restriction in the data (Bracero et al., 1996). In a prospective study of 65 well controlled diabetic pregnancies, Doppler measurements of uterine arteries, umbilical artery, the fetal descending thoracic aorta, and the middle cerebral artery (MCA) were performed together with cordocentesis for measurement of umbilical venous blood pH, pO2 and haematocrit. It was found that the mean umbilical venous blood pH was significantly lower and the haematocrit significantly higher than the appropriate normal mean for gestation for these diabetic pregnancies. However, the Doppler indices of the placental and fetal circulations were essentially normal, except in some of the cases complicated by pre-eclampsia or intrauterine growth restriction (Salvesen et al., 1993). It was thus concluded that maternal diabetes mellitus was not associated with abnormalities in Doppler indices of the placental or fetal circulations. To evaluate a random single Doppler study of the systolic/diastolic ratio of the umbilical artery as a predictor of perinatal outcome in diabetic pregnancies, a prospective doubleblind study was performed in 92 diabetic pregnancies between 28 and 40 weeks gestation, and the results were associated with perinatal outcome parameters. The sensitivity and specificity of the Doppler studies as a predictor of poor perinatal outcome were 39% and Applications of Doppler Studies for Fetal Surveillance in Diabetic Pregnancies 269 partly pressure-dependent, reflecting structural placental deficiencies, whereas brainsparing is attributed to hypoxia induced cerebrovascular dilatation (Baschat, 2003). In those pregnancies over 34 weeks gestation, placental functional decline may be more dominant that structural decline. Thus MCA changes in brain-sparing may appear in small fetuses with near-normal umbilical artery Doppler (Severi et al., 2002; To et al., 2005). Such findings are again more likely to occur in women with pre-existing diabetes mellitus with Another use for examining the MCA is to detect fetal anaemia. The MCA can be insonated at an angle between zero and 15 degrees to measure the actual flow velocity in the vessel. The lowest intra- and inter-observer variability is obtained when the MCA proximal to the transducer is sampled near its origin from the internal carotid artery without the use of angle correction using a 1-2 mm sample volume (Mari et al., 2005). A peak systolic velocity (PSV) of 1.50 MoM in fetuses at risk of anaemia has a sensitivity for detecting anaemia of up to 100% (confidence interval 86-100%) in red cell alloimmunization as well as other cases of anaemia (Mari et al., 2000). In the context of diabetic pregnancies, maternal hyperglycemia is thought to cause an increase in fetal haematocrit, as cordocentesis has demonstrated a positive relationship between maternal hyperglycaemia and fetal polycythemia (Salvesen et al., 1992). Theoretically, the increase in blood viscosity due to polycythemia might be reflected by a corresponding decrease in blood flow velocity in the fetal circulation, which is opposite to the fetal anaemia model. This would be particularly prominent in affected macrosomic fetuses in gestational diabetes, who often suffer prolonged neonatal jaundice resulting from the polycythemia. However, such findings have not been consistently In a prospective study of 138 singleton pregnancies with GDM, umbilical artery pulsatility index and middle cerebral artery pulsatility index were measured serially every 4 weeks from the diagnosis of GDM until delivery. A total of 305 Doppler examinations were performed with one to four examinations for each woman. About 27% ( 38) had one or more abnormal pregnancy outcomes: placental abruption, pre-eclampsia, preterm delivery, smallfor-gestational-age, low Apgar scores, neonatal jaundice requiring treatment, sepsis, birth trauma, meconium aspiration syndrome, respiratory and neurological complications, However, there was extensive overlapping of the umbilical artery and MCA pulsatility indices, as well as MCA PSV values between those with normal and abnormal pregnancy outcomes. It was thus concluded that that Doppler studies of the umbilical and cerebral vessels were not useful for predicting outcome in these GDM pregnancies (Leung et al., 2004). In another cohort of 84 GDM pregnancies, it was found that stratifying the fetuses into appropriate, small- and large-for-gestational-age did not give any better correlation between the umbilical or middle cerebral impedance indices, but did apparently show that the bigger fetuses had lower MCA PSV and higher mean umbilical venous flow velocity than the smaller fetuses (To et al, 2009). It has been proposed that the pathophysiological basis of altered placental vascular flow patterns in diabetic pregnancies was functional, related to hyperglycemia induced thromboxane/ prostacyclin ratio imbalance (Saldeen et al., 2002), rather than to structural abnormalities related to trophoblastic invasion during development of the placental vascular bed. Thus, the abnormal umbilical waveforms and abnormal placental-cerebral Doppler ratios that were observable in non-diabetic pregnancies with fetal growth restriction would not be applicable to gestational diabetic pregnancies without significant growth restriction. The observation of lower MCA PSV in vasculopathy than in those with gestational diabetes mellitus. demonstrated in published reports. 92% respectively. The positive and negative predictive values were 54% and 86% respectively. The authors suggested that the systolic/diastolic ratio of the umbilical artery offer no advantage over other well established tests in management of diabetic pregnancies (Ben-Ami et al., 1995). In a cohort of 104 women with both type I and type II pre-existing diabetes mellitus, umbilical artery Doppler was performed at 28, 32, 36 and 38 weeks gestation. Overall, 22% had an elevated pulsatility index. If the Doppler examination was carried out within 2 weeks of delivery, 71% with abnormal umbilical artery Doppler had adverse perinatal outcome (likelihood ratio 4.2). However, the sensitivity of umbilical artery Doppler to predict such adverse outcome was only 35%, while specificity was 94%. The positive Predictive value was 80% and negative predictive value was 68%. Only 30% of women with adverse perinatal outcome had abnormal umbilical artery Doppler measurements. The authors thus concluded that the performance of umbilical artery Doppler was not satisfactory even in this group of high risk women with pre-existing diabetes, and that it was not a good predictor of adverse perinatal outcome (Wong et al., 2003). To investigate whether complications were higher in diabetic pregnancies with cardiac maladaptation, fetal, uteroplacental and echocardiographic examinations were compared in the second and third trimester between diabetic and healthy pregnant women. Physiological cardiac hypertrophy was found in healthy women but was less prominent in patients with diabetes. While the majority of patients studied have normal Doppler results, the abnormal uteroplacental flow group consisted entirely of women with pregestational diabetes, especially IDDM patients. Neonatal complications were also more common in this subgroup. No relationship was found between echocardiographic findings, Doppler waveforms and perinatal outcome. Similar to previous studies, the findings confirmed that umbilical and uteroplacental Doppler were useful only in pre-existing diabetes, but not in GDM patients (Parlakgumus et al., 2010). From the above studies, it can be seen that both observational data and randomized control data have failed to show any consistent association between maternal diabetes and abnormal umbilical artery Doppler indices. It is apparent that the current evidence supports the use of umbilical artery Doppler only in those patients with diabetes who have pregnancies complicated by hypertensive diseases, fetal growth restriction, or vasculopathy. Umbilical Doppler studies cannot be recommended as a routine screening for fetal surveillance especially in patients without pre-existing diabetes mellitus (Pietryga et al., 2006). #### 3.3 Middle cerebral artery Doppler The middle cerebral artery (MCA) is the most studied cerebral artery because it is simple to sample, consistent and reproducible, and provides information on the cerebral blood flow in normal and growth restricted fetuses (Mari et al., 2008). In addition, the MCA can be sampled at an angle of near zero degrees between the ultrasound beam and the direction of the blood flow, so that the genuine velocity of the blood flow can be measured. In growth restricted foetuses, there is a redistribution of the blood flow from the fetal periphery to the brain, commonly known as the brain-sparing effect. In severe fetal growth restriction with abnormal umbilical artery, MCA Doppler is a valuable adjunct, with abnormal findings signifying the onset of compromise that should soon require delivery (Dubiel et al, 2002).Overt MCA changes appear as increased diastolic velocity, and an altered cerebralplacental ratio will thus be observed. Changes in cerebral-placental ratio may be at least 92% respectively. The positive and negative predictive values were 54% and 86% respectively. The authors suggested that the systolic/diastolic ratio of the umbilical artery offer no advantage over other well established tests in management of diabetic pregnancies In a cohort of 104 women with both type I and type II pre-existing diabetes mellitus, umbilical artery Doppler was performed at 28, 32, 36 and 38 weeks gestation. Overall, 22% had an elevated pulsatility index. If the Doppler examination was carried out within 2 weeks of delivery, 71% with abnormal umbilical artery Doppler had adverse perinatal outcome (likelihood ratio 4.2). However, the sensitivity of umbilical artery Doppler to predict such adverse outcome was only 35%, while specificity was 94%. The positive Predictive value was 80% and negative predictive value was 68%. Only 30% of women with adverse perinatal outcome had abnormal umbilical artery Doppler measurements. The authors thus concluded that the performance of umbilical artery Doppler was not satisfactory even in this group of high risk women with pre-existing diabetes, and that it To investigate whether complications were higher in diabetic pregnancies with cardiac maladaptation, fetal, uteroplacental and echocardiographic examinations were compared in the second and third trimester between diabetic and healthy pregnant women. Physiological cardiac hypertrophy was found in healthy women but was less prominent in patients with diabetes. While the majority of patients studied have normal Doppler results, the abnormal uteroplacental flow group consisted entirely of women with pregestational diabetes, especially IDDM patients. Neonatal complications were also more common in this subgroup. No relationship was found between echocardiographic findings, Doppler waveforms and perinatal outcome. Similar to previous studies, the findings confirmed that umbilical and uteroplacental Doppler were useful only in pre-existing diabetes, but not in From the above studies, it can be seen that both observational data and randomized control data have failed to show any consistent association between maternal diabetes and abnormal umbilical artery Doppler indices. It is apparent that the current evidence supports the use of umbilical artery Doppler only in those patients with diabetes who have pregnancies complicated by hypertensive diseases, fetal growth restriction, or vasculopathy. Umbilical Doppler studies cannot be recommended as a routine screening for fetal surveillance especially in patients without pre-existing diabetes mellitus (Pietryga et al., The middle cerebral artery (MCA) is the most studied cerebral artery because it is simple to sample, consistent and reproducible, and provides information on the cerebral blood flow in normal and growth restricted fetuses (Mari et al., 2008). In addition, the MCA can be sampled at an angle of near zero degrees between the ultrasound beam and the direction of the blood flow, so that the genuine velocity of the blood flow can be measured. In growth restricted foetuses, there is a redistribution of the blood flow from the fetal periphery to the brain, commonly known as the brain-sparing effect. In severe fetal growth restriction with abnormal umbilical artery, MCA Doppler is a valuable adjunct, with abnormal findings signifying the onset of compromise that should soon require delivery (Dubiel et al, 2002).Overt MCA changes appear as increased diastolic velocity, and an altered cerebralplacental ratio will thus be observed. Changes in cerebral-placental ratio may be at least was not a good predictor of adverse perinatal outcome (Wong et al., 2003). (Ben-Ami et al., 1995). GDM patients (Parlakgumus et al., 2010). 3.3 Middle cerebral artery Doppler 2006). partly pressure-dependent, reflecting structural placental deficiencies, whereas brainsparing is attributed to hypoxia induced cerebrovascular dilatation (Baschat, 2003). In those pregnancies over 34 weeks gestation, placental functional decline may be more dominant that structural decline. Thus MCA changes in brain-sparing may appear in small fetuses with near-normal umbilical artery Doppler (Severi et al., 2002; To et al., 2005). Such findings are again more likely to occur in women with pre-existing diabetes mellitus with vasculopathy than in those with gestational diabetes mellitus. Another use for examining the MCA is to detect fetal anaemia. The MCA can be insonated at an angle between zero and 15 degrees to measure the actual flow velocity in the vessel. The lowest intra- and inter-observer variability is obtained when the MCA proximal to the transducer is sampled near its origin from the internal carotid artery without the use of angle correction using a 1-2 mm sample volume (Mari et al., 2005). A peak systolic velocity (PSV) of 1.50 MoM in fetuses at risk of anaemia has a sensitivity for detecting anaemia of up to 100% (confidence interval 86-100%) in red cell alloimmunization as well as other cases of anaemia (Mari et al., 2000). In the context of diabetic pregnancies, maternal hyperglycemia is thought to cause an increase in fetal haematocrit, as cordocentesis has demonstrated a positive relationship between maternal hyperglycaemia and fetal polycythemia (Salvesen et al., 1992). Theoretically, the increase in blood viscosity due to polycythemia might be reflected by a corresponding decrease in blood flow velocity in the fetal circulation, which is opposite to the fetal anaemia model. This would be particularly prominent in affected macrosomic fetuses in gestational diabetes, who often suffer prolonged neonatal jaundice resulting from the polycythemia. However, such findings have not been consistently demonstrated in published reports. In a prospective study of 138 singleton pregnancies with GDM, umbilical artery pulsatility index and middle cerebral artery pulsatility index were measured serially every 4 weeks from the diagnosis of GDM until delivery. A total of 305 Doppler examinations were performed with one to four examinations for each woman. About 27% ( 38) had one or more abnormal pregnancy outcomes: placental abruption, pre-eclampsia, preterm delivery, smallfor-gestational-age, low Apgar scores, neonatal jaundice requiring treatment, sepsis, birth trauma, meconium aspiration syndrome, respiratory and neurological complications, However, there was extensive overlapping of the umbilical artery and MCA pulsatility indices, as well as MCA PSV values between those with normal and abnormal pregnancy outcomes. It was thus concluded that that Doppler studies of the umbilical and cerebral vessels were not useful for predicting outcome in these GDM pregnancies (Leung et al., 2004). In another cohort of 84 GDM pregnancies, it was found that stratifying the fetuses into appropriate, small- and large-for-gestational-age did not give any better correlation between the umbilical or middle cerebral impedance indices, but did apparently show that the bigger fetuses had lower MCA PSV and higher mean umbilical venous flow velocity than the smaller fetuses (To et al, 2009). It has been proposed that the pathophysiological basis of altered placental vascular flow patterns in diabetic pregnancies was functional, related to hyperglycemia induced thromboxane/ prostacyclin ratio imbalance (Saldeen et al., 2002), rather than to structural abnormalities related to trophoblastic invasion during development of the placental vascular bed. Thus, the abnormal umbilical waveforms and abnormal placental-cerebral Doppler ratios that were observable in non-diabetic pregnancies with fetal growth restriction would not be applicable to gestational diabetic pregnancies without significant growth restriction. The observation of lower MCA PSV in Applications of Doppler Studies for Fetal Surveillance in Diabetic Pregnancies 271 poor maternal glycemic control, and macrosomic fetuses would demonstrate such measurements, it might be argued that the total umbilical venous flow would still indirectly reflect fetal conditions, or the presence or absence of macrosomia. However, it is obvious that direct measurement of fetal growth parameters would be more precise in defining fetal The mean total umbilical venous flow has been shown in previous studies to be related to the total cardiac output of the fetus, so that the larger fetus with a higher cardiac output would have higher flow volumes (Boito et al., 2003). It was not surprising that when controlled for birthweight, the mean total umbilical venous flow differences between largeand small-for-gestational-age fetuses were greatly attenuated. Recent data have shown that in growth restricted fetuses, there would be a preferential distribution of umbilical venous flow to the ductus venosus rather than via the fetal liver (Bellotti et al, 2004). However, whether such venous shunting mechanisms would be responsible for the observed higher mean umbilical venous flow per unit weight in small-for –gestational-age fetuses as compared to larger ones would require further evaluation. In diabetic pregnancies, on the other hand, it has been found that fetal liver volume could be associated with accelerated growth in these fetuses, though only marginal differences could be shown in the umbilical venous volume flow. Further studies to compare growth and TUVF volumes in fetuses within a large non-diabetic population would be of value to study the differences in The atrioventricular values (mitral and tricuspid) are characterized by two peaks – the "E" wave corresponding to the rapid filling of the ventricles and the "A" wave that corresponds to the atrial contraction. The "A" wave is taller than the "E" wave, and may reflect the stiffness of the fetal cardiac chambers. With advancing gestation, the E/A wave ratio increases. By contrast, after birth, the "E" wave will be taller than the "A" wave. In growth restricted fetuses, the two waves become abnormal (the E/A ratio increases) and in severe cases, there will be tricuspid and mitral regurgitation (Rizzo et al., 1988). Thus, in the study of diabetic pregnancies, such studies would be of value only if the pregnancy is complicated The ductus venosus provides a unique combination of data, as it is the primary regulator of venous return in both normal and abnormal fetuses and is also a direct conduit of right atrial retrograde pulse waves (Harman et al., 2003). The ductus waveform is responsive to changes in oxygenation independent of cardiac function, and it is readily imaged because of its very high focal velocity on colour Doppler from early second trimester onwards. Ductus venosus waveforms are characterized by two peaks, the S and D, followed by a nadir, the atrial wave. Haemodynamically, these phases reflect the rapid chronologic change in pressure gradients between the umbilical vein and the right atrium. In normally grown fetuses, there is forward flow at the ductus venosus, and the pulsatility index for veins (S-D/a) decreases with advancing gestation. In growth restricted fetuses, the pulsatility index increases, and in the most severe cases, there will be reverse flow in the atrial wave. Ductus venosus waveform deterioration precedes and predicts changes in biophysical profile score that indicate need for delivery (Baschat et al., 2003). This deterioration is overgrowth. circulatory volumes in relation to size of the fetus. 3.5 Artioventricular valves by severe fetal growth restriction. 3.6 Ductus venosus Doppler waveforms the larger or macrosomic fetuses might be compatible with this hypothesis. In short, in the absence of pre-eclampsia or significant fetal growth abnormalities, the use MCA Doppler in diabetic pregnancies appears to have limited value. #### 3.4 Umbilical Vein waveform and umbilical venous flow volume Arterial waveforms describe downstream resistance in critical vascular beds, in which disease or response to pathological conditions causes blood flow alterations. Venous Doppler, however, provides important cardiac data about stressed fetal circulations. Potential targets include the umbilical vein, inferior vena cava and the ductus venosus, while regional networks such as the hepatic, superior vena cava, intracranial and pulmonary veins have not provided clinically relevant cardiac indicators (Harman et al., 2003). By mid second trimester, the fetal umbilical vein normally has a continuous blood flow pattern, but this pattern can become pulsatile in pathological conditions, such as in significant fetal growth restriction and in hydropic fetuses. Thus, for umbilical venous waveforms, it has been advocated that a qualitative assessment of continuous versus pulsatile blood flow is used (Mari et al., 2008). Such venous pulsations most likely represent severe and critical fetal myocardial dysfunction and usually only appear at very late stages of fetal compromise. Umbilical venous waveforms are therefore not useful as an early sign for assessment of fetal well being for timing delivery. The use of umbilical venous volume flow based on calculations of the cross-sectional area of the umbilical vein has been used and reported in previous studies to have a high degree of reproducibility (Boito et al., 2003). There have been suggestions for using the intraabdominal portion of the umbilical vein (Haugen et al., 2004), as the latter would be less mobile than a free cord loop. Empirical experience showed that the demand on technical expertise between the two sites were largely similar, though the variations in the diameter of the intra-hepatic umbilical vein along its length could be somewhat higher than that of the free cord loop, and calculations of its cross-sectional diameter more complicated. In addition, since routine umbilical arterial Doppler would be performed on a free cord loop, it would be practical and convenient to extend the measurements to the adjacent vein within the cord segment (To & Mok, 2009). To evaluate whether umbilical and middle cerebral arterial Doppler indices and umbilical venous volume flow are reflective of maternal gestational diabetic states, and whether such indices would be associated with the size of the fetus, a prospective observational study was performed in a cohort of 84 GDM pregnancies and compared with 62 non-diabetic controls. It was found that the mean pulsatility index values for the umbilical artery and the mean total umbilical venous flow (TUVF) and TUVF per unit birth weight did not differ significantly between diabetic and non-diabetic pregnancies. Large-for-gestational-age fetuses showed higher TUVF than normal size fetuses, but the TUVF per unit birth weight was higher for small-gestational-age fetuses. These differences were independent of their diabetic status. The only significant differences between non-diabetic and diabetic pregnancies in the data appeared to be the difference in the diameter and the mean flow volume of the umbilical vein, which were again probably more likely to be related to the size of the fetus. It was thus concluded that umbilical venous Doppler measurements near term were unable to distinguish between diabetic and non-diabetic pregnancies, and that umbilical venous flow volume was apparently more sensitive to the size of the fetus than to the maternal diabetic state (To & Mok, 2009). As fetal size variations could be secondary to poor maternal glycemic control, and macrosomic fetuses would demonstrate such measurements, it might be argued that the total umbilical venous flow would still indirectly reflect fetal conditions, or the presence or absence of macrosomia. However, it is obvious that direct measurement of fetal growth parameters would be more precise in defining fetal overgrowth. The mean total umbilical venous flow has been shown in previous studies to be related to the total cardiac output of the fetus, so that the larger fetus with a higher cardiac output would have higher flow volumes (Boito et al., 2003). It was not surprising that when controlled for birthweight, the mean total umbilical venous flow differences between largeand small-for-gestational-age fetuses were greatly attenuated. Recent data have shown that in growth restricted fetuses, there would be a preferential distribution of umbilical venous flow to the ductus venosus rather than via the fetal liver (Bellotti et al, 2004). However, whether such venous shunting mechanisms would be responsible for the observed higher mean umbilical venous flow per unit weight in small-for –gestational-age fetuses as compared to larger ones would require further evaluation. In diabetic pregnancies, on the other hand, it has been found that fetal liver volume could be associated with accelerated growth in these fetuses, though only marginal differences could be shown in the umbilical venous volume flow. Further studies to compare growth and TUVF volumes in fetuses within a large non-diabetic population would be of value to study the differences in circulatory volumes in relation to size of the fetus. #### 3.5 Artioventricular valves 270 Gestational Diabetes the larger or macrosomic fetuses might be compatible with this hypothesis. In short, in the absence of pre-eclampsia or significant fetal growth abnormalities, the use MCA Doppler in Arterial waveforms describe downstream resistance in critical vascular beds, in which disease or response to pathological conditions causes blood flow alterations. Venous Doppler, however, provides important cardiac data about stressed fetal circulations. Potential targets include the umbilical vein, inferior vena cava and the ductus venosus, while regional networks such as the hepatic, superior vena cava, intracranial and pulmonary veins have not provided clinically relevant cardiac indicators (Harman et al., 2003). By mid second trimester, the fetal umbilical vein normally has a continuous blood flow pattern, but this pattern can become pulsatile in pathological conditions, such as in significant fetal growth restriction and in hydropic fetuses. Thus, for umbilical venous waveforms, it has been advocated that a qualitative assessment of continuous versus pulsatile blood flow is used (Mari et al., 2008). Such venous pulsations most likely represent severe and critical fetal myocardial dysfunction and usually only appear at very late stages of fetal compromise. Umbilical venous waveforms are therefore not useful as an early sign The use of umbilical venous volume flow based on calculations of the cross-sectional area of the umbilical vein has been used and reported in previous studies to have a high degree of reproducibility (Boito et al., 2003). There have been suggestions for using the intraabdominal portion of the umbilical vein (Haugen et al., 2004), as the latter would be less mobile than a free cord loop. Empirical experience showed that the demand on technical expertise between the two sites were largely similar, though the variations in the diameter of the intra-hepatic umbilical vein along its length could be somewhat higher than that of the free cord loop, and calculations of its cross-sectional diameter more complicated. In addition, since routine umbilical arterial Doppler would be performed on a free cord loop, it would be practical and convenient to extend the measurements to the adjacent vein within To evaluate whether umbilical and middle cerebral arterial Doppler indices and umbilical venous volume flow are reflective of maternal gestational diabetic states, and whether such indices would be associated with the size of the fetus, a prospective observational study was performed in a cohort of 84 GDM pregnancies and compared with 62 non-diabetic controls. It was found that the mean pulsatility index values for the umbilical artery and the mean total umbilical venous flow (TUVF) and TUVF per unit birth weight did not differ significantly between diabetic and non-diabetic pregnancies. Large-for-gestational-age fetuses showed higher TUVF than normal size fetuses, but the TUVF per unit birth weight was higher for small-gestational-age fetuses. These differences were independent of their diabetic status. The only significant differences between non-diabetic and diabetic pregnancies in the data appeared to be the difference in the diameter and the mean flow volume of the umbilical vein, which were again probably more likely to be related to the size of the fetus. It was thus concluded that umbilical venous Doppler measurements near term were unable to distinguish between diabetic and non-diabetic pregnancies, and that umbilical venous flow volume was apparently more sensitive to the size of the fetus than to the maternal diabetic state (To & Mok, 2009). As fetal size variations could be secondary to diabetic pregnancies appears to have limited value. for assessment of fetal well being for timing delivery. the cord segment (To & Mok, 2009). 3.4 Umbilical Vein waveform and umbilical venous flow volume The atrioventricular values (mitral and tricuspid) are characterized by two peaks – the "E" wave corresponding to the rapid filling of the ventricles and the "A" wave that corresponds to the atrial contraction. The "A" wave is taller than the "E" wave, and may reflect the stiffness of the fetal cardiac chambers. With advancing gestation, the E/A wave ratio increases. By contrast, after birth, the "E" wave will be taller than the "A" wave. In growth restricted fetuses, the two waves become abnormal (the E/A ratio increases) and in severe cases, there will be tricuspid and mitral regurgitation (Rizzo et al., 1988). Thus, in the study of diabetic pregnancies, such studies would be of value only if the pregnancy is complicated by severe fetal growth restriction. #### 3.6 Ductus venosus Doppler waveforms The ductus venosus provides a unique combination of data, as it is the primary regulator of venous return in both normal and abnormal fetuses and is also a direct conduit of right atrial retrograde pulse waves (Harman et al., 2003). The ductus waveform is responsive to changes in oxygenation independent of cardiac function, and it is readily imaged because of its very high focal velocity on colour Doppler from early second trimester onwards. Ductus venosus waveforms are characterized by two peaks, the S and D, followed by a nadir, the atrial wave. Haemodynamically, these phases reflect the rapid chronologic change in pressure gradients between the umbilical vein and the right atrium. In normally grown fetuses, there is forward flow at the ductus venosus, and the pulsatility index for veins (S-D/a) decreases with advancing gestation. In growth restricted fetuses, the pulsatility index increases, and in the most severe cases, there will be reverse flow in the atrial wave. Ductus venosus waveform deterioration precedes and predicts changes in biophysical profile score that indicate need for delivery (Baschat et al., 2003). This deterioration is Applications of Doppler Studies for Fetal Surveillance in Diabetic Pregnancies 273 parameters Primary endpoint PDM or abnormalities P/GDM FGR PGD/ aneuploidy; congenital cardiac Uterine Notching, PI FGR PGD UA, MCA PI, CPR FGR PGD 22-24 Uterine Notching, PI FGR PGD PDM: pre-existing diabetes mellitus, GDM: gestational diabetes mellitus, DV: ductus venosus PSV: peak systolic velocity, AV: atrioventricular, TR: tricuspid regurgitation, MR: mitral regurgitation association of Doppler parameters to maternal diabetic state was particularly true when the pregnancy was not complicated by fetal growth restriction or pre-eclampsia. While higher incidences of adverse outcome in diabetic pregnancies were related to the occurrence of poor glycemic control with macrosomia and polyhydramnios, conventional arterial Doppler indexes and cerebral /placental Doppler ratios have not been shown to be effective in picking up these high risk fetuses. The use of umbilical venous Doppler and venous volume flow based on calculations of the cross-sectional area of the umbilical vein has been reported in various studies to have a high degree of reproducibility. Venous volume flow measurements have not been found to be consistently reflective of maternal gestational diabetic states. Such volume flow measurements apparently reflected well the fetal growth and size and thus indirectly the glycemic control and the risks of perinatal complications. Doppler studies of other venous sites, including the intraabdominal/ intrahepatic portion of the umbilical vein, or the ductus venosus have also been studied with variable results. Whether such measurements could be used directly for monitoring fetal well being requires American College of Obstetricians & Gynecologists Practice Bulletin (2001). Clinical Alfirevic Z, Neilson JP (1995). Doppler ultrasonography in high-risk pregnancies: systematic Baschat AA (2003). Integrated fetal testing in growth restriction: combining multi-vessel Doppler and biophysical parameters. Ultrasound Obstet Gynecol , 21: 1-8 Bellotti M, Pennati G, De Gasperi C, Bozzo M, Battaglia FC, Ferrazzi E (2004). Simultaneous growth-restricted human fetuses. Am J Obstet Gynecol, 190: 1347-1358. review with meta-analysis. Am J Obstet Gynecol , 172: 1379-1387. 2001. Gestational Diabetes. Obstet Gynecol, 98: 525-528. management guidelines for obstetrician-gynecologists. Number 30, September measurements of umbilical venous, fetal hepatic, and ductus venosus blood flow in GDM GDM Gestation 3rd trimester further evaluation. 6. References (weeks) Vessel Doppler UA, MCA UV AV valves DV 12 DV Retrograde atrial waves valves PI, CRP, PSV PI, TUVF AV flow, TR, MR Retrograde atrial PI: pulsatility index, CRP: cerebral placental ratio, FGR: fetal growth restriction, Table 1. Sequential Doppler applications foe diabetic pregnancies hypothesised to be the result of a volume/pressure effect, in which excess afterload is transmitted through the heart, and myocardial dysfunction appears as an end-stage compromise. A second indication of ductus venosus Doppler occurs at 12-14 weeks in conjunction with nuchal translucency screening and uterine artery screening. Abnormal retrograde atrial waves are a strong predictor of fetal cardiac abnormality, and also a good predictor of Down syndrome (Bilardo et al., 2001). Given the higher risks of congenital cardiac abnormalities in pregnancies complicated by pre-existing diabetes mellitus, ductus venosus screening at this gestation may have a role. There is as yet, scanty data in the literature that describe the use of ductus Doppler specific to diabetic pregnancies, and the application of ductus Doppler largely refers to that of growth restricted fetuses in general. To evaluate the ability of the ductus venosus Doppler to predict adverse perinatal outcome in pregnancies complicated by pre-existing diabetes mellitus, a prospective study that included 82 women with pre-existing diabetes mellitus was performed. The ductus venosus Doppler index was defined as abnormal if the ductus venosus peak velocity index for veins was equal to or greater than the 95th percentile for gestation. Abnormal ductus venosus index was identified in 30.5% (n=25). Adverse perinatal outcome was identified in around one-third of these with abnormal indices (8/25) compared to 12.3% (5/57) with a normal ductus index. The sensitivity of the ductus venosus index in predicting adverse perinatal outcome in pre-existing diabetic pregnancies was thus 53.3% and specificity was 74.5%, with a positive predictive value of 32% and negative predictive value of 87.7%. The authors concluded that it should be useful to include ductus venosus Doppler indices as part of antenatal screening of pregnancies complicated by pre-existing diabetes mellitus (Wong et al., 2010). #### 4. Practical application of Doppler studies to diabetic pregnancies It has thus been postulated that the degree of glycemic control would have more impact on the Doppler study results rather than directly related to the diabetic state (Bracero et al., 1991). The lack of association of Doppler parameters to maternal diabetic state was particularly true when the pregnancy was not complicated by fetal growth restriction or pre-eclampsia. While higher incidences of adverse outcome in diabetic pregnancies were related to the occurrence of such complications or to poor glycemic control with macrosomia, Doppler studies have apparently only limited effectiveness in predicting adverse perinatal outcome in these fetuses. Summing up the available data from the literature, a basic protocol for the sequential use of Doppler studies in both pre-existing and gestational diabetic pregnancies can be proposed (Table 1). Nevertheless, the clinical effectiveness of such a protocol still remains to be evaluated. #### 5. Summary Available randomized control data and observational data have failed to demonstrate any consistent association between maternal diabetes and abnormal umbilical arterial Doppler indices. Doppler measurements of other fetal vessels apart from the umbilical arteries, such as the fetal descending aorta and the middle cerebral artery resistance indexes, or the peak systolic velocity of the middle cerebral arteries have also been studied in GDM pregnancies, and a similar lack of predictability for adverse outcome was generally found. The lack of PDM: pre-existing diabetes mellitus, GDM: gestational diabetes mellitus, DV: ductus venosus PI: pulsatility index, CRP: cerebral placental ratio, FGR: fetal growth restriction, PSV: peak systolic velocity, AV: atrioventricular, TR: tricuspid regurgitation, MR: mitral regurgitation Table 1. Sequential Doppler applications foe diabetic pregnancies association of Doppler parameters to maternal diabetic state was particularly true when the pregnancy was not complicated by fetal growth restriction or pre-eclampsia. While higher incidences of adverse outcome in diabetic pregnancies were related to the occurrence of poor glycemic control with macrosomia and polyhydramnios, conventional arterial Doppler indexes and cerebral /placental Doppler ratios have not been shown to be effective in picking up these high risk fetuses. The use of umbilical venous Doppler and venous volume flow based on calculations of the cross-sectional area of the umbilical vein has been reported in various studies to have a high degree of reproducibility. Venous volume flow measurements have not been found to be consistently reflective of maternal gestational diabetic states. Such volume flow measurements apparently reflected well the fetal growth and size and thus indirectly the glycemic control and the risks of perinatal complications. Doppler studies of other venous sites, including the intraabdominal/ intrahepatic portion of the umbilical vein, or the ductus venosus have also been studied with variable results. Whether such measurements could be used directly for monitoring fetal well being requires further evaluation. #### 6. References 272 Gestational Diabetes hypothesised to be the result of a volume/pressure effect, in which excess afterload is transmitted through the heart, and myocardial dysfunction appears as an end-stage compromise. A second indication of ductus venosus Doppler occurs at 12-14 weeks in conjunction with nuchal translucency screening and uterine artery screening. Abnormal retrograde atrial waves are a strong predictor of fetal cardiac abnormality, and also a good predictor of Down syndrome (Bilardo et al., 2001). Given the higher risks of congenital cardiac abnormalities in pregnancies complicated by pre-existing diabetes mellitus, ductus venosus screening at this gestation may have a role. There is as yet, scanty data in the literature that describe the use of ductus Doppler specific to diabetic pregnancies, and the application of ductus Doppler largely refers to that of growth restricted fetuses in general. To evaluate the ability of the ductus venosus Doppler to predict adverse perinatal outcome in pregnancies complicated by pre-existing diabetes mellitus, a prospective study that included 82 women with pre-existing diabetes mellitus was performed. The ductus venosus Doppler index was defined as abnormal if the ductus venosus peak velocity index for veins was equal to or greater than the 95th percentile for gestation. Abnormal ductus venosus index was identified in 30.5% (n=25). Adverse perinatal outcome was identified in around one-third of these with abnormal indices (8/25) compared to 12.3% (5/57) with a normal ductus index. The sensitivity of the ductus venosus index in predicting adverse perinatal outcome in pre-existing diabetic pregnancies was thus 53.3% and specificity was 74.5%, with a positive predictive value of 32% and negative predictive value of 87.7%. The authors concluded that it should be useful to include ductus venosus Doppler indices as part of antenatal screening of pregnancies complicated by pre-existing diabetes mellitus (Wong et 4. Practical application of Doppler studies to diabetic pregnancies effectiveness of such a protocol still remains to be evaluated. It has thus been postulated that the degree of glycemic control would have more impact on the Doppler study results rather than directly related to the diabetic state (Bracero et al., 1991). The lack of association of Doppler parameters to maternal diabetic state was particularly true when the pregnancy was not complicated by fetal growth restriction or pre-eclampsia. While higher incidences of adverse outcome in diabetic pregnancies were related to the occurrence of such complications or to poor glycemic control with macrosomia, Doppler studies have apparently only limited effectiveness in predicting adverse perinatal outcome in these fetuses. Summing up the available data from the literature, a basic protocol for the sequential use of Doppler studies in both pre-existing and gestational diabetic pregnancies can be proposed (Table 1). Nevertheless, the clinical Available randomized control data and observational data have failed to demonstrate any consistent association between maternal diabetes and abnormal umbilical arterial Doppler indices. Doppler measurements of other fetal vessels apart from the umbilical arteries, such as the fetal descending aorta and the middle cerebral artery resistance indexes, or the peak systolic velocity of the middle cerebral arteries have also been studied in GDM pregnancies, and a similar lack of predictability for adverse outcome was generally found. The lack of al., 2010). 5. Summary Applications of Doppler Studies for Fetal Surveillance in Diabetic Pregnancies 275 Ko GTC, Tam WH, Chan JCN, Rogers M (2002). 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Br J Obstet Gynaecol, 110: 1007-1013. randomized clinical trial. Diabetes Metab, 30: 237-244 by diabetes. J Ultrasound Med , 15: 301-308. Ultrasound Obstet Gynecol, 15: 7-12. 804-808. 99: 135-140 273-280 Perinatal, 12: 437-438. umbilical artery in the evaluation of pregnancies complicated by diabetes. Am J fetuses at high risk for chromosomal or heart abnormalities: relationship with nuchal translucency measurement and fetal outcome. Ultrasound Obstet Gynecol, 17: 288-294 Boito S, Struijk PC, Ursem NTC, Stijnen T, Wladimiroff JW. (2003) Assessment of fetal liver volume and umbilical venous volume flow in pregnancies complicated by insulin- Motta G, Costa M, Solerte L, Morabito A (2004). Flexible treatment of gestaional diabetes modulated on ultrasound evaluation of intrauterine growth: a controlled velocimetry, nonstress testing and biophysical profile in pregnancies complicated screening as a predictor of adverse pregnancy outcome in high-risk women. (2001). Umbilical artery pulsatility index in pregnancies complicated by insulindependent diabetes mellitus without hypertension. Gynecol Obstet Invest, 51: 173-177 Giles WB, Trudinger BJ, Baird PJ (1985). Fetal umbilical artery flow velocity waveforms and placental resistance: pathological correlation. Br J Obstet Gynaecol, 92: 31-38 Grunewald C, Divon M, Lunell NO (1996). Doppler velocimetry in last trimester pregnancy complicated by insulin-dependent diabetes mellitus. Acta Obstet Gynecol Scand, 75: resistance and beta cell function in 223 women with an abnormal glucose challenge umbilical artery flow velocity waveform in diabetic pregnancy. Br J Obstet Gynaecol, infants of women with diabetes mellitus during pregnancy. J Matern Fetal Med, 5: 16 Mexico 2IMSS-48, HMI-SSG The Influence of Diet to Control the Rebeca Monroy-Torres1 and Jaime Naves-Sanchez2 1University of Guanajuato, Department Medicine and Nutrition; Metabolism in Gestational Diabetes Mellitus Gestational diabetes mellitus (GDM) is the intolerance to carbohydrates, first recognized during pregnancy. The prevalence of GDM has been increasing in the world and it affects This chapter emphasizes and discusses the role of the dietary and nutritional aspects of the GDM. First, we will do a general review about the transitional changes in food that are experienced worldwide, their phases-dietary habits, industrialization, globalization, culture, the media as television, video games which have led to changes in the eating patterns and how this diet style promotes a metabolic disease, wich will be supported with Mexican evidence. Second, we will do a general literature review of the most important nutritional and dietetic recommendations, findings, calculation and prescription of a correct diet (Distribution of: proteins, lipids and carbohydrates), in combination with counseling nutritional importance. Next, we will compare two recommended diets for GDM with the evidence we got from our research. Moreover, we will discuss the advantages and disadvantages of the glucemyc index and how a low and moderate one allows the control of blood glucose. The glycemic index is defined as the area under the curve of glucose response after eating a recomended amount of carbohydrates from a test food after a control food (white bread or glucose), and these had been considered part of the control of blood glucose. Besides, we present the methods for collecting diet information as a complement to improve the follow up and the adherence to GDM. Other important aspects to discuss are the importance of the preventive and promotional strategies in the medical nutrition therapy, physical activity, benefits, risk and the type of exercise for GDM, education, psychological support, insuline and drugs . The prevention and promotion should consider economical and social aspects because pregnant women use to change their food consumption because of economical influence. The health costs and long management of GDM mothers have a wide range of possible complications. These recommendations should result in adequate weight gain for the fetus and the woman. A deficit in the weight gain is associated with intrauterine growth restriction for the fetus. Dietary control is defined as a part of the comprehensive treatment of GDM and the diets low in carbohydrates, lipids, and proteins have demonstrated to reduce hyperglycemia and to prevent macrosomia compared 1. Introduction more than 200,000 women every year. with diets high in carbohydrates carbon. ### The Influence of Diet to Control the Metabolism in Gestational Diabetes Mellitus Rebeca Monroy-Torres1 and Jaime Naves-Sanchez2 1University of Guanajuato, Department Medicine and Nutrition; 2IMSS-48, HMI-SSG Mexico #### 1. Introduction 276 Gestational Diabetes Saldeen P, Olofsson P, Laurini RN (2002). Structural, functional and circulatory placental Salvesen DR, Brudenell MJ, Nicolaides KH (1992). Fetal polycythemia and Salvesen DR, Higueras MT, Mansur CA, Freeman J, Brudenell JM, Nicolaides KH (1993). Schaefer-Graf UM, Gaber B, Wendt L, Metzner S, Sacks DA, Vetter K, Kilavuz O, Abou- Schmidt MT, Duncan BB, Reichelt AJ, Branchtein L, Matos MC, Forti AC, Spichler ER, Schulam H, Fleischer A, Farmakides G, Bracero L, Rochelson B, Grunfeld L (1986). Severi FM, Bocchi C, Visentin A, Severi FM, Bocchi C, Visentin A, Falco P, Cobellis L, Florio To WWK, Chan AMY, Mok KM (2005). Use of umbilical-cerebral Doppler ratios in predicting fetal growth restriction in near term fetuses. Aust NZ J Obstet Gynaecol , 45: 130-136 To WWK, Mok KM (2009). Fetal umbilical arterial and venous Doppler measurements in Wong SF, Chan FY, Cincotta RB, McIntyre DH, Stone M (2003). Use of umbilical artery Wong SF, Petersen SG, Idris N, Thomae M, McIntyre HD (2010). Ductus venosus Zimmermann P, Kujansuu E, Tuimala R (1992). Doppler velocimetry of umbilical artery in Zimmermann P, Kujansuu E, Tuimala R (1994). Doppler flow velocimetry of the uterine Biol, 105: 136-142 Obstet Gynecol, 166: 1287-1293 outcomes. Diabetes Care, 24: 1151-1155 Neonatal Med, 22: 1176-1182 Gynecol Reprod Biol, 19: 47: 85-93 ultrasound. Am J Obstet Gynecol, 155: 1031-1036 Doppler. Ultrasound Obstet Gynecol, 19: 225-228 diabetes. Aust NZ J Obstet Gynaecol, 43: 302-306 mellitus. Ultrasound Obstet Gynecol, 36: 350-354 diabetes mellitus. J Perinatal Med, 22: 137-147 diabetes mellitus. Am J Obstet Gynecol, 168: 645-652 changes associated with impaired glucose metabolism. Eur J Obstet Gynecol Reprod thrombocytopenia in pregnancies complicated by maternal diabetes mellitus. Am J Placental and fetal Doppler velocimetry in pregnancies complicated by maternal Dakn M (2011). How many sonograms are needed to reliably predict the absence of fetal overgrowth in gestational diabetes mellitus pregnancies? Diabet Care, 34: 39-43 Pousada JMD, Teixeira MM, YamashitaT (2001). Gestational diabetes mellitus diagnosed with a 2-h 75 g oral glucose tolerance test and adverse pregnancy Development of uterine artery compliance in pregnancy as detected by Doppler P, Zagonari S, Pilu G (2002). Uterine and fetal cerebral Doppler predict the outcome of third trimester small-for gestational age fetuses with normal umbilical artery gestational diabetic and non-diabetic pregnancies near term. J Maternal Fetal Doppler velocimetry in the monitoring of pregnancy in women with pre-existing velocimetry in monitoring pregnancy in women with pre-gestational diabetes pregnancies complicated by insulin-dependent diabetes mellitus. Eur J Obstet and uteroplacental circulation in pregnancies complicated by insulin-dependent Gestational diabetes mellitus (GDM) is the intolerance to carbohydrates, first recognized during pregnancy. The prevalence of GDM has been increasing in the world and it affects more than 200,000 women every year. This chapter emphasizes and discusses the role of the dietary and nutritional aspects of the GDM. First, we will do a general review about the transitional changes in food that are experienced worldwide, their phases-dietary habits, industrialization, globalization, culture, the media as television, video games which have led to changes in the eating patterns and how this diet style promotes a metabolic disease, wich will be supported with Mexican evidence. Second, we will do a general literature review of the most important nutritional and dietetic recommendations, findings, calculation and prescription of a correct diet (Distribution of: proteins, lipids and carbohydrates), in combination with counseling nutritional importance. Next, we will compare two recommended diets for GDM with the evidence we got from our research. Moreover, we will discuss the advantages and disadvantages of the glucemyc index and how a low and moderate one allows the control of blood glucose. The glycemic index is defined as the area under the curve of glucose response after eating a recomended amount of carbohydrates from a test food after a control food (white bread or glucose), and these had been considered part of the control of blood glucose. Besides, we present the methods for collecting diet information as a complement to improve the follow up and the adherence to GDM. Other important aspects to discuss are the importance of the preventive and promotional strategies in the medical nutrition therapy, physical activity, benefits, risk and the type of exercise for GDM, education, psychological support, insuline and drugs . The prevention and promotion should consider economical and social aspects because pregnant women use to change their food consumption because of economical influence. The health costs and long management of GDM mothers have a wide range of possible complications. These recommendations should result in adequate weight gain for the fetus and the woman. A deficit in the weight gain is associated with intrauterine growth restriction for the fetus. Dietary control is defined as a part of the comprehensive treatment of GDM and the diets low in carbohydrates, lipids, and proteins have demonstrated to reduce hyperglycemia and to prevent macrosomia compared with diets high in carbohydrates carbon. The Influence of Diet to Control the Metabolism in Gestational Diabetes Mellitus 279 cheaper to be obese. The same phenomenon operates in fast food restaurants and The industrialization of a city has as a consequence that some products contain raw poor materials disguised with flavors that result in products of low nutritional value but tasty. They are also well supported with good marketing strategies and are aimed especially to children. Another important finding is that some Latin American countries spend from 20 to 30% of their income in food and they have the higest consumption of soft drinks in the world; hence, soft drinks have replaced water consumption. In additin to this, the presence of obesity in the family is an important factor to be considered because if both parents are obese then, the risk of obesity in children is 80%. Television and video games are other factors of great influence. In countries where the habit of reading is replaced by television and video games, it has been observed that there is an adjustement in children's behavior and habit consumptions of some products. For example, 85% of the commercials on TV Sciences such as demography and epidemiology help to understand the phenomenology behind food and nutrition in a sistematic way. Where the diet habits are related with the culture, traditional habits, climate, available foods, etc. These habits do not change inmediately. Transitional nutrition has five phases or periods that the majority of societies a. Stage of "food gathering". The diet is high in carbohydrates and fiber but low in fat. This b. Stage of "starvation". It occurred with the early development of agriculture when food c. Stage of "increased consumption of fruits, vegetables and animal products". Starches begin to be less important in the basic diet. This stage is related to the industrial and the second d. Stage of "Presence of chronic degenerative diseases". It increases with the prevalence of obesity as a result of the consumption of diets high in fat, cholesterol, refined carbohydrates, small amounts of polyunsaturated fatty acids and fiber and physical inactivity. In this stage there is an increase in the population that migrate to big cities e. Stage of "behavioral change". This stage is characterized by the adoption of healthy diets, physical activity and the abandonment of traditional diets. Here, the consumption of cereals and tubers is very important. In this stage, there is a tendency to a global homogenization of the type of food that is consumed. Then, an intake of high energy, total fat, saturated fat and simple sugars is required. Moreover, physical activity decreases due to changes in occupational activities, transportation facilities, these contributed to increase the chronic diseases (diabetes mellitus, hypertension and Finally, it is necessary to say that each country goes through these periods of transition at The main characteristic of transactional nutrition is that these changes occur more rapidly in middle or low income countries than in the high-income ones. In low income countries, there is a negative impact in their economical growth due to that the fact that they spend more money in getting food of low nutritional value such as oils and fat of poor nutritional quality. promote soft drinks, desserts and fried food (Ramirez JA, et al., 2003). stage is characterized by the hunting and gathering of food. was less varied and linked to periods of extreme food shortages. where home and work are located far away from traditional markets. atherosclerosis) related to overweight and other nutrition aspects. experienced and these are (Nielsen, S & Popkin, B, 2003): convenience stores, among others. agricultural revolution. different times and with a different speed. The main goal of the dietary treatment is to maintain the maternal and fetal health and the diet with low carbohydrates help to the management of GDM, where the main goal is to achieve and to maintain blood glucose and glycated hemoglobin according to the practice guidelines for GDM and to avoid ketonuria. The adhesion to dietary treatment is difficult in most patients when they intake lower amount of carbohydrates. The findings reported in the control of the GDM such as changes in weight gain, energy intake, and macronutrients are part of a basic treatment to prevent complications for the fetus and the pregnant mother. We will review of promotion and prevention activities as part of the dietary treatment. Finally, we hope that this information let discuss new alternatives for GDM. #### 2. Diet: Transitional changes Since the last century, many countries have experienced changes in health and food patterns that are characterized by an increasing prevalence of chronic diseases. These chronic diseases are the principal cause of death and commonly happen particularly particularly in pregnant women and children. There are many factors that may explain these changes and one of the most important one to consider is the dietary habits because they explain the metabolic diseases (Avila A, et al., 1995; ENSANUT, 2006). As mentioned above, there are other factors that might explain these changes and the principal ones are the alterations in dietary habits that are considered important to explain the metabolic diseases (Avila A, et al., 1995; ENSANUT, 2006). For example, México is living an epidemiological transition that has occurred in recent decades. For instance, the most common causes of death in this transition are cardiovascular diseases, cancer, accidents and diabetes. Fifty years ago, most of the causes of death in the Mexican population were related to diarrhea and respiratory diseases. The research done at the Mexican National Institute of Nutrition supports the idea that the proportion of animal products in the diet of Mexicans has increased sharply. In addition, fast food, ,in most countries, is considered as a frequent choice for many people who live in the city. The epidemiological changes that have occurred in many countries are not homogeneous throughout the world; for instance, they vary according to geographical, regional and socioeconomical factors. Based on the studies carried by Romieu, et al., it is possible to observe the coexistence of over nutrition and under nutrition in the same population. In underdeveloped countries, it is possible to observe that about one-third of children under 5 years are stunted, and 20% of women are obese. (Romieu I, et al., 1997). The dynamic of the epidemiological aspects in many countries provides challenges and opportunities for studying dietary habits. These dietary habits can serve as the base for conducting studies about how the diet can modify the occurrence of chronic diseases, and the impact on the nutritional health can be investigated as well (Romieu I, et al., 1997; Ávila A, et al., 1995). Other then dietary habits, there are more factors to be considered at risk for developing obesity such as: excessive consumption, physical activity, sedentary and hereditary factors. Many countries have had an apparent economical development during the last decade and has contributed to the lifestyle, eating habits, customs, behaviors, etc. The diet of developed countries consist of cereals, legumes, fruits and vegetables. These changes are similar to those of the industrialized countries (high-energy diet, protein, animal fat and low fiber). This is the price we have had to pay for the globalization that our world is facing because this is the result of the free trade agreement in developed countries. Moreover, the powerful influence of the North American culture has affected the entire world. One example of this is the payment-saving culture "pay less, get more" so it is The main goal of the dietary treatment is to maintain the maternal and fetal health and the diet with low carbohydrates help to the management of GDM, where the main goal is to achieve and to maintain blood glucose and glycated hemoglobin according to the practice guidelines for GDM and to avoid ketonuria. The adhesion to dietary treatment is difficult in most patients when they intake lower amount of carbohydrates. The findings reported in the control of the GDM such as changes in weight gain, energy intake, and macronutrients are part of a basic treatment to prevent complications for the fetus and the pregnant mother. We will review of promotion and prevention activities as part of the dietary treatment. Since the last century, many countries have experienced changes in health and food patterns that are characterized by an increasing prevalence of chronic diseases. These chronic diseases are the principal cause of death and commonly happen particularly particularly in pregnant women and children. There are many factors that may explain these changes and one of the most important one to consider is the dietary habits because they explain the metabolic diseases (Avila A, et al., 1995; ENSANUT, 2006). As mentioned above, there are other factors that might explain these changes and the principal ones are the alterations in dietary habits that are considered important to explain the metabolic diseases (Avila A, et al., 1995; ENSANUT, 2006). For example, México is living an epidemiological transition that has occurred in recent decades. For instance, the most common causes of death in this transition are cardiovascular diseases, cancer, accidents and diabetes. Fifty years ago, most of the causes of death in the Mexican population were related to diarrhea and respiratory diseases. The research done at the Mexican National Institute of Nutrition supports the idea that the proportion of animal products in the diet of Mexicans has increased sharply. In addition, fast food, ,in most countries, is considered as a frequent choice for many people who live in the city. The epidemiological changes that have occurred in many countries are not homogeneous throughout the world; for instance, they vary according to geographical, regional and socioeconomical factors. Based on the studies carried by Romieu, et al., it is possible to observe the coexistence of over nutrition and under nutrition in the same population. In underdeveloped countries, it is possible to observe that about one-third of children under 5 years are stunted, and 20% of women are obese. (Romieu I, et al., 1997). The dynamic of the epidemiological aspects in many countries provides challenges and opportunities for studying dietary habits. These dietary habits can serve as the base for conducting studies about how the diet can modify the occurrence of chronic diseases, and the impact on the nutritional health can be investigated as well (Romieu I, et al., 1997; Ávila A, et al., 1995). Other then dietary habits, there are more factors to be considered at risk for developing obesity such as: excessive consumption, physical activity, sedentary and hereditary factors. Many countries have had an apparent economical development during the last decade and has contributed to the lifestyle, eating habits, customs, behaviors, etc. The diet of developed countries consist of cereals, legumes, fruits and vegetables. These changes are similar to those of the industrialized countries (high-energy diet, protein, animal fat and low fiber). This is the price we have had to pay for the globalization that our world is facing because this is the result of the free trade agreement in developed countries. Moreover, the powerful influence of the North American culture has affected the entire world. One example of this is the payment-saving culture "pay less, get more" so it is Finally, we hope that this information let discuss new alternatives for GDM. 2. Diet: Transitional changes cheaper to be obese. The same phenomenon operates in fast food restaurants and convenience stores, among others. The industrialization of a city has as a consequence that some products contain raw poor materials disguised with flavors that result in products of low nutritional value but tasty. They are also well supported with good marketing strategies and are aimed especially to children. Another important finding is that some Latin American countries spend from 20 to 30% of their income in food and they have the higest consumption of soft drinks in the world; hence, soft drinks have replaced water consumption. In additin to this, the presence of obesity in the family is an important factor to be considered because if both parents are obese then, the risk of obesity in children is 80%. Television and video games are other factors of great influence. In countries where the habit of reading is replaced by television and video games, it has been observed that there is an adjustement in children's behavior and habit consumptions of some products. For example, 85% of the commercials on TV promote soft drinks, desserts and fried food (Ramirez JA, et al., 2003). Sciences such as demography and epidemiology help to understand the phenomenology behind food and nutrition in a sistematic way. Where the diet habits are related with the culture, traditional habits, climate, available foods, etc. These habits do not change inmediately. Transitional nutrition has five phases or periods that the majority of societies experienced and these are (Nielsen, S & Popkin, B, 2003): Finally, it is necessary to say that each country goes through these periods of transition at different times and with a different speed. The main characteristic of transactional nutrition is that these changes occur more rapidly in middle or low income countries than in the high-income ones. In low income countries, there is a negative impact in their economical growth due to that the fact that they spend more money in getting food of low nutritional value such as oils and fat of poor nutritional quality. The Influence of Diet to Control the Metabolism in Gestational Diabetes Mellitus 281 consists of an integral component of diabetes prevention, management, and self- A recommended diet for the glucemia control in GDM must be low in carbohydrates,with a percentage between 35% and 50%; lipids from 30% to 40% and 20% of protein, of the total calories. This distribution has proved to reduce hyperglycemia and prevent macrosomia, These recommendations must include a nutritional counseling because the excessive restriction of carbohydrates (< 120g) increases the risk of ketonuria, low birth weight and defective supply of glucose to the fetus impact on the neurological development (Pastor JG , et al, 2002; ADA, 2007;ADA, 2003, 2010; Major CA, et al, 1998). Besides the aforementioned , it is necessary to say that low carbohydrates diets make it difficult for patients to adher to According to the American Diabetes Association (ADA 2010), a good diet control must, appart from getting glycated hemoglobin values (HbA1c) less than 6% and avoiding ketonuria (Standards of Medical Care in Diabetes-2008) maintain the capillary blood glucose concentrations in ≤95 mg/dl (Preprandial); ≤140 mg/dl (1-h postmeal); ≤120 mg/dl 2-h (postmeal). Regarding to plasma-referenced capillary blood glucose, the values suggested are ≤105 mg/dl (Preprandial), ≤155 mg/dl (1-h postmeal), ≤130 mg/dl (2-h postmeal)(Standards of Medical Care in Diabetes-2008). With these recommendations, patients with GDM should have an adequate weight gain for them and the fetus as well. The weight gain recomendation is calculated based on prepregnancy weight or with the body mass index (BMI). A deficit in Monroy-Torres R. et al. studied the influence of an individualized diet of low glycemic index to control GDM in woman between 24 and 26 gestational weeks. The diet was structured with 52% of complex carbohydrates and food of low and moderate glycemic index with 30% fat and 18% protein (plus 10 grams). The findings from the study were that the pregnant women with GDM did not have adverse effects related to blood glucose, glycosylated hemoglobin and weight gain. The newborns did not development macrosomia. According to Jenkins,the glycemic index (GI) is defined as the changes in blood glucose concentration after consuming food wich then has to be compared with standard amount of carbohydrate of a control food (white bread or glucose) (Jenkins DJA, 1984). The food with low and moderate GI has been considered as a part of a glycemic control; where the increased in the percentage of carbohydrates up to 60% of total calories is allowd when and if food of low and moderate glicemic control is given (Fraser RB, et al, 1988). The consumption of processed food or food with low fiber conten increases the glycemic index while the GI decreases with food high in fiber, fats, proteins and it depends on the cooking process. Moses R et al., 2006, compared the effects of a diet of low glycemic index with a diet of high GI. Both diets had 55% of carbohydrates. Women with GDM who had a diet with high glycemic index gave as a result that they had a newborn with high weight (large for gestational age) and higher ponderal index. Another important concept to discuss is the load Glycemic (LG) - that is the amount of carbohydrates in a specific food. The GI of food is not always easy to predict; for example, we could think that an ice cream has a high GI but it has low GI according to published charts. The reason is that an ice cream has fat and weight gain is associated with intrauterine growth restriction (Pastor, et al, 2002). The adherence observed was higher for calorie and macronutrient intake. proteins, this combination decreases the digestion of glucose. 3.1 Glycemic index and glycemic load management education (Goldhaber-Fiebert JD, et al., 2003). the treatment. when compared with diets high in carbohydrates (Franz M, et al., 2002). This is done with the purpose of getting cheaper food that can be obtained faster by people. However, the use of this kind of food promotes the appearance of cardiovascular diseases. Changing of food consumption has altered in many countries because, they prefer to buy prepared food than to cooked it at home. As a result, the consumption of oilseeds, vegetable oils, fish and seafood has been decreasing but there is an increment in the consumption of animal fat, alcoholic beverages, meat and eggs consumption. These habits increase the costs and the quality of ingredients in food (Ortiz-Hernández, L, et al., 2006; Monroy-Torres R, et al., 2010). The changes mentioned above are part of the evidence that has been gathered from the population that lives in urban areas, people employ in the tertiary sectors and the actual role of woman (Ortiz-Hernández L, et al., 2006). The determinants of the are located mainly in both, the social organization and the technological progress of the society (Ortiz-Hernández, et al., 2006). Another determinant that is also important to consider is the educational level. During the periods of 1988–1994 and, 2007-2008, the prevalence of childhood obesity in United State increased at all income and educational levels (CDC, 2010). According to diet and disease, 20 years ago, it was discovered that low birthweight was associated with an increased risk of adult diabetes and cardiovascular disease (CVD). A hypothesis was formulated with this information,wich states that that the exposure to undernutrition in early life increased the risk to develop these disorders in the metabolic programation. In order to solve the problem caused in the metabolic programation, it has been proposed another hypothesis that claims that it would be important to improve the nutritional state from the pre gestational stage to the gestational stage and finally the nutritional attention to the new born to prevent common chronic diseases in the future. The research done with low birth weight children in many countries shows that they have increased CVD risk factors. The scientific findings gotten from maternal nutrition have contribute to understand the role of specific nutrients in the maternal diet, like low maternal vitamin B12 status,wich predicted the increased of adiposity and insulin resistance in children, especially if the mother had a folate deficiency. Both maternal undernutrition and gestational diabetes cause problems (glucose excess) They have also been associated with increased adiposity and insulin resistance in children. In underdeveloped countries, it has been noticed that undernutrition and overnutrition coexist. Recent intervention studies in developed countries have shown that CVD risk factors in the offspring can be improved by supplementing undernourished mothers during pregnancy. Of course, results differ according to the population, the intervention and the post-natal environment (Fall C., 2009). #### 3. Evidence in the diet management of gestational diabetes mellitus Dietary therapy is the most important factor to be consider in the treatment of GDM. Therefore all women with GDM must receive counseling from a dietitian. Recommendations have to be individualized after a dietary evaluation of each patient. The two main objectives for the treatment of GDM are to achieve normoglycemia and to provide the required nutriments for normal fetal growth and maternal health. A third objective that is also considered to be important, is to prevent excessive maternal weight gain, particularly in women who are overweight or have gained excess weight in pregnancy. Few trials have analyzed the efficacy of dietary therapy for GDM. A cluster randomized controlled study supports that Medical Nutrition Therapy (MNT) for GDM is recommended by the American Diabetes Association (ADA, 2010). The MNT is a lifestyle intervention that This is done with the purpose of getting cheaper food that can be obtained faster by people. However, the use of this kind of food promotes the appearance of cardiovascular diseases. Changing of food consumption has altered in many countries because, they prefer to buy prepared food than to cooked it at home. As a result, the consumption of oilseeds, vegetable oils, fish and seafood has been decreasing but there is an increment in the consumption of animal fat, alcoholic beverages, meat and eggs consumption. These habits increase the costs and the quality of ingredients in food (Ortiz-Hernández, L, et al., 2006; Monroy-Torres R, et al., 2010). The changes mentioned above are part of the evidence that has been gathered from the population that lives in urban areas, people employ in the tertiary sectors and the The determinants of the are located mainly in both, the social organization and the technological progress of the society (Ortiz-Hernández, et al., 2006). Another determinant that is also important to consider is the educational level. During the periods of 1988–1994 and, 2007-2008, the prevalence of childhood obesity in United State increased at all income According to diet and disease, 20 years ago, it was discovered that low birthweight was associated with an increased risk of adult diabetes and cardiovascular disease (CVD). A hypothesis was formulated with this information,wich states that that the exposure to undernutrition in early life increased the risk to develop these disorders in the metabolic programation. In order to solve the problem caused in the metabolic programation, it has been proposed another hypothesis that claims that it would be important to improve the nutritional state from the pre gestational stage to the gestational stage and finally the nutritional attention to the new born to prevent common chronic diseases in the future. The research done with low birth weight children in many countries shows that they have increased CVD risk factors. The scientific findings gotten from maternal nutrition have contribute to understand the role of specific nutrients in the maternal diet, like low maternal vitamin B12 status,wich predicted the increased of adiposity and insulin resistance in children, especially if the mother had a folate deficiency. Both maternal undernutrition and gestational diabetes cause problems (glucose excess) They have also been associated with increased adiposity and insulin resistance in children. In underdeveloped countries, it has been noticed that undernutrition and overnutrition coexist. Recent intervention studies in developed countries have shown that CVD risk factors in the offspring can be improved by supplementing undernourished mothers during pregnancy. Of course, results differ according to the population, the intervention and the post-natal environment (Fall C., 2009). 3. Evidence in the diet management of gestational diabetes mellitus overweight or have gained excess weight in pregnancy. Dietary therapy is the most important factor to be consider in the treatment of GDM. Therefore all women with GDM must receive counseling from a dietitian. Recommendations have to be individualized after a dietary evaluation of each patient. The two main objectives for the treatment of GDM are to achieve normoglycemia and to provide the required nutriments for normal fetal growth and maternal health. A third objective that is also considered to be important, is to prevent excessive maternal weight gain, particularly in women who are Few trials have analyzed the efficacy of dietary therapy for GDM. A cluster randomized controlled study supports that Medical Nutrition Therapy (MNT) for GDM is recommended by the American Diabetes Association (ADA, 2010). The MNT is a lifestyle intervention that actual role of woman (Ortiz-Hernández L, et al., 2006). and educational levels (CDC, 2010). consists of an integral component of diabetes prevention, management, and selfmanagement education (Goldhaber-Fiebert JD, et al., 2003). A recommended diet for the glucemia control in GDM must be low in carbohydrates,with a percentage between 35% and 50%; lipids from 30% to 40% and 20% of protein, of the total calories. This distribution has proved to reduce hyperglycemia and prevent macrosomia, when compared with diets high in carbohydrates (Franz M, et al., 2002). These recommendations must include a nutritional counseling because the excessive restriction of carbohydrates (< 120g) increases the risk of ketonuria, low birth weight and defective supply of glucose to the fetus impact on the neurological development (Pastor JG , et al, 2002; ADA, 2007;ADA, 2003, 2010; Major CA, et al, 1998). Besides the aforementioned , it is necessary to say that low carbohydrates diets make it difficult for patients to adher to the treatment. According to the American Diabetes Association (ADA 2010), a good diet control must, appart from getting glycated hemoglobin values (HbA1c) less than 6% and avoiding ketonuria (Standards of Medical Care in Diabetes-2008) maintain the capillary blood glucose concentrations in ≤95 mg/dl (Preprandial); ≤140 mg/dl (1-h postmeal); ≤120 mg/dl 2-h (postmeal). Regarding to plasma-referenced capillary blood glucose, the values suggested are ≤105 mg/dl (Preprandial), ≤155 mg/dl (1-h postmeal), ≤130 mg/dl (2-h postmeal)(Standards of Medical Care in Diabetes-2008). With these recommendations, patients with GDM should have an adequate weight gain for them and the fetus as well. The weight gain recomendation is calculated based on prepregnancy weight or with the body mass index (BMI). A deficit in weight gain is associated with intrauterine growth restriction (Pastor, et al, 2002). Monroy-Torres R. et al. studied the influence of an individualized diet of low glycemic index to control GDM in woman between 24 and 26 gestational weeks. The diet was structured with 52% of complex carbohydrates and food of low and moderate glycemic index with 30% fat and 18% protein (plus 10 grams). The findings from the study were that the pregnant women with GDM did not have adverse effects related to blood glucose, glycosylated hemoglobin and weight gain. The newborns did not development macrosomia. The adherence observed was higher for calorie and macronutrient intake. #### 3.1 Glycemic index and glycemic load According to Jenkins,the glycemic index (GI) is defined as the changes in blood glucose concentration after consuming food wich then has to be compared with standard amount of carbohydrate of a control food (white bread or glucose) (Jenkins DJA, 1984). The food with low and moderate GI has been considered as a part of a glycemic control; where the increased in the percentage of carbohydrates up to 60% of total calories is allowd when and if food of low and moderate glicemic control is given (Fraser RB, et al, 1988). The consumption of processed food or food with low fiber conten increases the glycemic index while the GI decreases with food high in fiber, fats, proteins and it depends on the cooking process. Moses R et al., 2006, compared the effects of a diet of low glycemic index with a diet of high GI. Both diets had 55% of carbohydrates. Women with GDM who had a diet with high glycemic index gave as a result that they had a newborn with high weight (large for gestational age) and higher ponderal index. Another important concept to discuss is the load Glycemic (LG) - that is the amount of carbohydrates in a specific food. The GI of food is not always easy to predict; for example, we could think that an ice cream has a high GI but it has low GI according to published charts. The reason is that an ice cream has fat and proteins, this combination decreases the digestion of glucose. The Influence of Diet to Control the Metabolism in Gestational Diabetes Mellitus 283 We can observe in table 1 that the range of carbohydrates varies from less than 40% to over 55%. The American Diabetes Association of the United States recommends that all pregnant women with overweight and/or GDM must receive a diet with 35-40% of carbohydrates and food with low GI. The percentage of calories, that depends on carbohydrates , is reduced, a 20% of proteins and 30% of fat is recommended. This is done to keep the balance of macronutrients. In fact, it is advisable that the distribution to be similar to a normal diet but it is importan to have considered the quality of food and macronutrients (ADA 2004; 2010). It is important to control the intake of carbohydrates because they are the first nutrient that affects During pregnancy, hormone levels of placental lactogen, cortisol, progesterone and prolactine increase and this affects the insuline to lower blood glucose levels. Therefore, breakfast carbohydrate load of 15 to 30 g is recommended. The total daily carbohydrates and calorie intake should be individualized according to glucose control. When a dietitian designs a meal plan, carbohydrates intake should be distributed during the day in a three time meal and two or three small meals (snacks) (Sheard, N.F., et al., 2004). For example, a diet of 2000 calories where the carbohydrates can represent a 40% of total calories; that in grams is equivalent to 200 grams, the total calories should be distributed during day as The minimum amount of carbohydrates required to prevent the starvation activation systems (ketosis) of fasting is between 100 and 150 grams per day. According to the Food and Nutrition Board in 2002, 130 grams per day of carbohydrate is enough to satisfy the glucose requirement of the brain (FNB, 2005). Moreover, this recommendation should provide a list of food of low and moderate glycemic index (≤ 55 and from 59 to 69, respectively). Each country has different patterns of food exchange that are based on the consumer habits and customs of each population. For example, Mexico has the Mexican Food System Equivalents (Marvan L, et al., 2008), it is a educational system that is provided to patients with GDM to explain the amount of food intake from different food groups. To understand better the amounts and real food portions, the dietitians often used food replicas. Based on what it was meal times explained above, food should be distributed depending on the glucose control in six or seven meals (three main meals and three or four snacks) (Jovanovic L, 2000, Monroy-Torres R, et al., 2008). Dietary and nutritional advice, lifestyle counseling and restriced food should be provided to patients in a written list. Adding proteins and fat to the meal plan for woman with GDM will not raise the postmeal glucose levels and to satisfy the woman´s hunger during the The methods to collect dietary information vary in their accurancy and ease use. The 24 hour recall is one of the easiest methods to collect information from the patient´s intake. It consists of obtaining information from food and fluid intake from a previous day ( 24 hours) and it is based on the assumption that the intake described is typical of a daily intake. However, the method has important problems; for example, the patient may not be able to the postprandial glucose levels in addition to breakfast carbohydrate load. follow: • Lunch 35% • Dinner 20% • Evening Snack 10% • Breakfast 7.5- 10%(15- 20g) day (Monroy-Torres R, et al., 2008). • Snack 10% (between breakfast and lunch) • Snack 15% (between lunch and dinner) While the World Health Organization, the American Diabetes Association, Diabetes UK, and the Canadian Diabetes Association support both concepts ( IG and LG),and many other health professionals consider that to use GI and GL is complex because both have many variables that change their values and responses in the blood glucose. This is why their use in the clinical practice is debatable (Franz M, 2003). Other explanation is that different tables; with GI and LG values for different food, show different values for GI among the existing tables which gives as a result that their use is consider controversial specially with new data has become available since the first tables were published in 2002 (Atkinson F, 2008; Foster-Powell K, 2002). However, the most important recommendation is to follow up the advice of the Standards of Medical Care in Diabetes 2008: choose a variety of grains, fruits, and vegetables, with an emphasis on whole grains and other high-fiber foods, to work with dietary and behavioral changes #### 3.2 Calculation and prescription of the diet The American Diabetes Association consensus to manage GDM recommends to take into account the dietary calculation (ADA 2010) to consider the value of BMI. According to the clinical experience the diet prescription must be individualized (Jovanovic L, 2000). The diet may be calculated in a range between 1700 and 2000 kcal/day. It is important to avoid diets with caloric content lower than 1500 Kcal because the risk of ketonuria can be increased. The calculation and distribution of macronutrients can be as follows: The American Diabetes Association (ADA, 2004) recommends to add 25 kcal per kilogram during pregnancy. A strategy to evaluate the adherence to energy consumption is to monitor weight gain which should not exceed 400 grams per week from the second trimester of pregnancy. According to the table 1, ,we can see how the range of carbohydrates varies between 40% and 55% or more of the total calories. The American Diabetes Association of the United States recommends that all pregnant women with overweight and / or GDM should receive a diet with 35-40 grams of carbohydrates ( simple carbohydrates), from food with low glycemic index. However, a consumption of carbohydrates less than 40% is risky because it results in ketonuria (ADA, 2010). \* Relationship of BMI based on the percentage of body fat Table 1. Comparison of diets recommended in GDM We can observe in table 1 that the range of carbohydrates varies from less than 40% to over 55%. The American Diabetes Association of the United States recommends that all pregnant women with overweight and/or GDM must receive a diet with 35-40% of carbohydrates and food with low GI. The percentage of calories, that depends on carbohydrates , is reduced, a 20% of proteins and 30% of fat is recommended. This is done to keep the balance of macronutrients. In fact, it is advisable that the distribution to be similar to a normal diet but it is importan to have considered the quality of food and macronutrients (ADA 2004; 2010). It is important to control the intake of carbohydrates because they are the first nutrient that affects the postprandial glucose levels in addition to breakfast carbohydrate load. During pregnancy, hormone levels of placental lactogen, cortisol, progesterone and prolactine increase and this affects the insuline to lower blood glucose levels. Therefore, breakfast carbohydrate load of 15 to 30 g is recommended. The total daily carbohydrates and calorie intake should be individualized according to glucose control. When a dietitian designs a meal plan, carbohydrates intake should be distributed during the day in a three time meal and two or three small meals (snacks) (Sheard, N.F., et al., 2004). For example, a diet of 2000 calories where the carbohydrates can represent a 40% of total calories; that in grams is equivalent to 200 grams, the total calories should be distributed during day as follow: 282 Gestational Diabetes While the World Health Organization, the American Diabetes Association, Diabetes UK, and the Canadian Diabetes Association support both concepts ( IG and LG),and many other health professionals consider that to use GI and GL is complex because both have many variables that change their values and responses in the blood glucose. This is why their use in the clinical practice is debatable (Franz M, 2003). Other explanation is that different tables; with GI and LG values for different food, show different values for GI among the existing tables which gives as a result that their use is consider controversial specially with new data has become available since the first tables were published in 2002 (Atkinson F, 2008; Foster-Powell K, 2002). However, the most important recommendation is to follow up the advice of the Standards of Medical Care in Diabetes 2008: choose a variety of grains, fruits, and vegetables, with an emphasis on whole grains and other high-fiber foods, to work with The American Diabetes Association consensus to manage GDM recommends to take into account the dietary calculation (ADA 2010) to consider the value of BMI. According to the clinical experience the diet prescription must be individualized (Jovanovic L, 2000). The diet may be calculated in a range between 1700 and 2000 kcal/day. It is important to avoid diets with caloric content lower than 1500 Kcal because the risk of ketonuria can be increased. The The American Diabetes Association (ADA, 2004) recommends to add 25 kcal per kilogram during pregnancy. A strategy to evaluate the adherence to energy consumption is to monitor weight gain which should not exceed 400 grams per week from the second According to the table 1, ,we can see how the range of carbohydrates varies between 40% and 55% or more of the total calories. The American Diabetes Association of the United States recommends that all pregnant women with overweight and / or GDM should receive a diet with 35-40 grams of carbohydrates ( simple carbohydrates), from food with low glycemic index. However, a consumption of carbohydrates less than 40% is risky because it \BMI: 80 -120 % 35 Kcal / Kg (ideal weight ) 30 Kcal / Kg (ideal weight ) \BMI: 121 –150% 35 Kcal / Kg (ideal weight ) 24 Kcal / Kg (ideal weight ) \BMI: >= 151 % 35 Kcal / Kg (ideal weight ) 12 Kcal / Kg (ideal weight ) Variables ADA 2010 Euglycemic diets* Protein requirement 20 % of total calories 20 % of total calories Fat intake < 25 % of total calories >= 40 % of total calories Carbohydrates requirements > 55 % of total calories < 40 % of total calories Cholesterol Requirements 300 mg/d < 800 mg / d dietary and behavioral changes lowers LDL cholesterol results in ketonuria (ADA, 2010). trimester of pregnancy. 3.2 Calculation and prescription of the diet calculation and distribution of macronutrients can be as follows: Saturated fat intake <7% of total calories \* Relationship of BMI based on the percentage of body fat Table 1. Comparison of diets recommended in GDM The minimum amount of carbohydrates required to prevent the starvation activation systems (ketosis) of fasting is between 100 and 150 grams per day. According to the Food and Nutrition Board in 2002, 130 grams per day of carbohydrate is enough to satisfy the glucose requirement of the brain (FNB, 2005). Moreover, this recommendation should provide a list of food of low and moderate glycemic index (≤ 55 and from 59 to 69, respectively). Each country has different patterns of food exchange that are based on the consumer habits and customs of each population. For example, Mexico has the Mexican Food System Equivalents (Marvan L, et al., 2008), it is a educational system that is provided to patients with GDM to explain the amount of food intake from different food groups. To understand better the amounts and real food portions, the dietitians often used food replicas. Based on what it was meal times explained above, food should be distributed depending on the glucose control in six or seven meals (three main meals and three or four snacks) (Jovanovic L, 2000, Monroy-Torres R, et al., 2008). Dietary and nutritional advice, lifestyle counseling and restriced food should be provided to patients in a written list. Adding proteins and fat to the meal plan for woman with GDM will not raise the postmeal glucose levels and to satisfy the woman´s hunger during the day (Monroy-Torres R, et al., 2008). The methods to collect dietary information vary in their accurancy and ease use. The 24 hour recall is one of the easiest methods to collect information from the patient´s intake. It consists of obtaining information from food and fluid intake from a previous day ( 24 hours) and it is based on the assumption that the intake described is typical of a daily intake. However, the method has important problems; for example, the patient may not be able to The Influence of Diet to Control the Metabolism in Gestational Diabetes Mellitus 285 The ADA (2010) mentions that all women should receive individualized counseling to provide adequate calories and nutrients during pregnancy. This counseling must help achieve the goal and maintain the blood glucose (fasting 105 mg/dl , 1 hr 155 mg/dl , and 2 hrs 130 mg/dl ) and glycated hemoglobin (HbA1c )in 6%. For obese women, the treatment must be a 30%–33% of caloric restriction, it means near to 25 cal/kg per day is recommended, wich should be calculated with the current weight. Carbohydrate should be restricted to 35%–40% of calories. There are also data that support the use of low carbohydrate diets in pregnancy, and for carbohydrates to be low, the glycemic index. In a nonrandomized study, there was evidence that women with GDM on a diet comprising less than 42% carbohydrate, had lower post-prandial glucose levels, were less likely to require insulin, and had a lower incidence of large for gestational age. A small study with randomized pregnant women with low GI or high GI diets found that the former resulted in lower glucose levels, a blunting of the pregnancy associated rise in insulin resistance, and lower birthweight. In another study of GI, women assigned to a low GI diet during pregnancy gave birth to infants who were lighter and had a lower incidence of large for gestational age, compared to women given a high GI diet. Additional dietary measures are usually based upon the general recommendations for diabetes mellitus. A reduction in simple carbohydrates and fat intake is advisable. Emphasis is given to spreading the dietary intake over six meals daily, with three main meals and three snacks in order to avoid large carbohydrate loads at any time. Except for saccharin, which can cross the placenta and is therefore not recommended, other noncaloric sweeteners may be used in moderation • Psychological support to improve acceptance and adherence to disease treatment We must consider that if GDM is not diagnosed at the early stage, mother' dietary habits and overall health in this period will have consequences in the short, medium and long term effecting the child as well. Regarding to the educational intervention, patients with GDM Weight loss is not recommended during pregnancy, even though the woman is obese. For during the GDM is necessary that weight gain should be in the lower ranks recommended, (Moses, R., et al, 2006). • Education • Diet should know: • What is GDM? • Physical activity • Where justified, Insulin 4. Preventive and promotional strategies Medical nutrition therapy (MNT), including: • The importance of metabolic control and risk of ketonuria • Types of insulin, application techniques (where insulin is required) • Importance of early intervention, monitoring and control postpartum • Characteristics of recommended diet plan • Prevention of future type 2 diabetes mellitus • Self-monitoring techniques • Goals of the blood glucose control recall the eaten food or not to estimate the amount of food eaten. On the other hand, the method offers the advantage of analizing the average consumption of energy, carbohydrates, fats and proteins from the diet. This allows to analyze to the amount of food and the adhesion to the diet from the beginning and to follow it up. Another method is the Food-frequencyquestionnaire that is often used in combination with the 24-hour recall. The food-frequencyquestionnaire provides a list of food or food group where the patient can have different options to answer like: rarely, never, frecuently, occasionally, daily, weekly and monthly. The Food-frequency- questionnaire is only recommended to use only at the beginning and the end of GDM treatment and this method is useful to evaluate changes in the eating habits and to analize the diet of patients within the next parameters: adequate, complete, balanced, enough and varied - these define a recommended diet. A diet is recommended for GDM when it has all these parameters: a) Enough: means to cover the energy requirements according to the individual characteristics like age, weight, physical activity and physiological conditions, b) balanced: means to intake nutrients based on references amounts (52% of complex carbohydrates, protein in 18%, fat in 30%); c) Complete: means to include at least three food groups at every meal and, d) varied: means to include different types of food within the same group, in a day (Marvan L, et al., 2008). Food records and postmeal monitoring of blood glucose can help to identify food that is less tolerated and to let individual nutrition and food recommendations changes (Sheard, N.F., et al., 2004). If the modification of the food plan alone does not prove to achieve and mantain normoglicemya, then insuline therapy is needed. To achieve the goals with the use of insulin, women must eat the corrcect amounts of carbohydrates and they must eat at regular meal times (ADA, 2010), all above information is important to avoid unnecessary hypoglycemia risk. Exchange lists or counting carbohydrates are the methods that help patients with GDM select and decide themselves what to eat according their glucose levels and their insulin therapy. Since insulin is the therapy of choice for most diabetic women, several authors suggest to apply the method of carbohydrate counting or exchange lists during the first nutrition interview. In table 2, you can see the recommendations of a dietary treatment in combination with insulin that depend on the response to the glycemic control (Standards of Medical Care in Diabetes-2008). Adapted: Monroy-Torres-Sanchez Naves R & J, 2011 Table 2. Recommendations of dietary treatment in combination with insulin in GDM The ADA (2010) mentions that all women should receive individualized counseling to provide adequate calories and nutrients during pregnancy. This counseling must help achieve the goal and maintain the blood glucose (fasting 105 mg/dl , 1 hr 155 mg/dl , and 2 hrs 130 mg/dl ) and glycated hemoglobin (HbA1c )in 6%. For obese women, the treatment must be a 30%–33% of caloric restriction, it means near to 25 cal/kg per day is recommended, wich should be calculated with the current weight. Carbohydrate should be restricted to 35%–40% of calories. There are also data that support the use of low carbohydrate diets in pregnancy, and for carbohydrates to be low, the glycemic index. In a nonrandomized study, there was evidence that women with GDM on a diet comprising less than 42% carbohydrate, had lower post-prandial glucose levels, were less likely to require insulin, and had a lower incidence of large for gestational age. A small study with randomized pregnant women with low GI or high GI diets found that the former resulted in lower glucose levels, a blunting of the pregnancy associated rise in insulin resistance, and lower birthweight. In another study of GI, women assigned to a low GI diet during pregnancy gave birth to infants who were lighter and had a lower incidence of large for gestational age, compared to women given a high GI diet. Additional dietary measures are usually based upon the general recommendations for diabetes mellitus. A reduction in simple carbohydrates and fat intake is advisable. Emphasis is given to spreading the dietary intake over six meals daily, with three main meals and three snacks in order to avoid large carbohydrate loads at any time. Except for saccharin, which can cross the placenta and is therefore not recommended, other noncaloric sweeteners may be used in moderation (Moses, R., et al, 2006). #### 4. Preventive and promotional strategies Medical nutrition therapy (MNT), including: 284 Gestational Diabetes recall the eaten food or not to estimate the amount of food eaten. On the other hand, the method offers the advantage of analizing the average consumption of energy, carbohydrates, fats and proteins from the diet. This allows to analyze to the amount of food and the adhesion to the diet from the beginning and to follow it up. Another method is the Food-frequencyquestionnaire that is often used in combination with the 24-hour recall. The food-frequencyquestionnaire provides a list of food or food group where the patient can have different options to answer like: rarely, never, frecuently, occasionally, daily, weekly and monthly. The Food-frequency- questionnaire is only recommended to use only at the beginning and the end of GDM treatment and this method is useful to evaluate changes in the eating habits and to analize the diet of patients within the next parameters: adequate, complete, balanced, enough and varied - these define a recommended diet. A diet is recommended for GDM when it has all these parameters: a) Enough: means to cover the energy requirements according to the individual characteristics like age, weight, physical activity and physiological conditions, b) balanced: means to intake nutrients based on references amounts (52% of complex carbohydrates, protein in 18%, fat in 30%); c) Complete: means to include at least three food groups at every meal and, d) varied: means to include different types of food within the same group, in a day (Marvan L, et al., 2008). Food records and postmeal monitoring of blood glucose can help to identify food that is less tolerated and to let individual nutrition and food If the modification of the food plan alone does not prove to achieve and mantain normoglicemya, then insuline therapy is needed. To achieve the goals with the use of insulin, women must eat the corrcect amounts of carbohydrates and they must eat at regular meal times (ADA, 2010), all above information is important to avoid unnecessary hypoglycemia risk. Exchange lists or counting carbohydrates are the methods that help patients with GDM select and decide themselves what to eat according their glucose levels and their insulin therapy. Since insulin is the therapy of choice for most diabetic women, several authors suggest to apply the method of carbohydrate counting or exchange lists during the first nutrition interview. In table 2, you can see the recommendations of a dietary treatment in combination with insulin that depend on the response to the glycemic control Preconception stage First strategy Second strategy hypoglycemic drugs Diet +Insulin Diet+ Insulin GDM controlled with diet Strict Diet Diet+ Insulin Table 2. Recommendations of dietary treatment in combination with insulin in GDM Normal Diet Diet + Insulin recommendations changes (Sheard, N.F., et al., 2004). (Standards of Medical Care in Diabetes-2008). GDM controlled GDM controlled with insulin Diet + Insulin Adapted: Monroy-Torres-Sanchez Naves R & J, 2011 We must consider that if GDM is not diagnosed at the early stage, mother' dietary habits and overall health in this period will have consequences in the short, medium and long term effecting the child as well. Regarding to the educational intervention, patients with GDM should know: Weight loss is not recommended during pregnancy, even though the woman is obese. For during the GDM is necessary that weight gain should be in the lower ranks recommended, The Influence of Diet to Control the Metabolism in Gestational Diabetes Mellitus 287 In people with type 2 diabetes, there is a lot of evidence that suggests that regular physical activity improves insulin sensitivity, weight loss, thereby improves glucose control. Several studies have examined whether regular exercise is also beneficial in the management of GDM, for example, Jovanovic L, (Jovanovic L, 2000) randomized 19 women with GDM to a regime of diet alone, or diet with 20 minutes of supervised aerobic training three days per week for six weeks. This modest amount of physical activity resulted in lower fasting glucose levels, lower glucose responses to a glucose challenge, and a lower HbA1c . Another study randomized 29 women with GDM to 30 minutes of exercise (70% of maximal heart rate) of three to four times per week. In this case the glucose levels did not improve in those who exercised. The action might be through the activation of AMPK, a kinase that is activated during exercise, it is possible that exercise may act through the same molecular cellular pathway and therefore there are not additive effects of these two treatment Another study found that women with a prepregnancy value BMI of 25 and who were in the exercise program were less likely to require insulin. Therefore, it seems reasonable to recommend that when there is not medical or obstetric contraindication, women with GDM should maintain a sensible level of light and moderate intensity if there is physical activity during the pregnancy. The above studies provide enough evidence that moderate physical activity such as walking between 20 and 30 minutes each day, or three to four times per week, through this they can achieve the glycemic control. The diet should provide with adequate nutrition for pregnancy. Carbohydrates should be distributed throughout the day over main meals and snacks. Limiting carbohydrates at 40% of the total caloric intake and having a higher proportion of carbohydrates of lower glycaemic index decreases postprandial glucose levels and reduces the need for insulin therapy (Smith, C.S. & Van There is not enough evidence to support dietary or drug treatment in patients with gestational diabetes. Gestational diabetes and impaired glucose tolerance are associated with macrosomia and may be associated with an increased risk for cesarean delivery, shoulder dystocia, and birth trauma. Although preexisting diabetes has been shown to increase the risk of poor perinatal outcomes, it is not clear that data relating to preexisting diabetes can be extrapolated to patients with gestational Tuffnell and colleagues (Olwan N, 2009)researched the Cochrane Pregnancy and Childbirth Group trials register, the Cochrane Central Register of Controlled Trials, and bibliographies of relevant articles. They identified three studies of 223 women with impaired glucose tolerance; none of these studies was a randomized controlled trial comparing management strategies. Intensive management of gestational diabetes is timeconsuming and resource-intensive. Overall, the evidence is insufficient to support the therapy for gestational diabetes. However, universal screening is the standard of care in most communities. When faced with abnormal results, most family physicians will opt to follow the consensus opinion of our specialist colleagues. For example, a study that analyzed the higher egg and cholesterol intakes found that they are associated with increased risk of type 2 diabetes mellitus. It was also observed that the higher egg and cholesterol intakes before and during pregnancy are associated with an increased risk of regimens (Hardie DG, 2004). diabetes. GDM. (Qiu C, et al., 2011). Andel, R., 2001; Artal, R. & Toole, M.O ., 2003). according to the start value of BMI except in the adolescent woman , where the weight increment must be higher. The objectives of a diet plan are: Some studies recommend energy consumption between 1800 and 2200 kcal, bearing in mind that weight gain should be among the lower ranks. In adolescents, low weight or great complexion is recommended to increase energy requirements. The exercise is useful to help control metabolism. The type of exercise for pregnant women is with the work that can be done with the upper extremities. However it is contraindicated in the following cases: #### 4.1 Physical activity Currently more pregnant women want to have their pregnancy in natural and healthy conditions. Exercising in pregnant women is controversial as to the changes presented in their body and the hormonal levels. A long time ago, pregnancy was experienced as an illness, and had several myths related to it, including the exercise. The scientific arguments to restrict physical activity during pregnancy suggest that exercise causes an increment in maternal body temperature and therefore in the fetus too, with increased release of catecholamines, decreased circulating glucose and decreased blood flow to the placenta. However, there are other scientific studies that show the benefits of a regular physical activity during pregnancy to maintain health. When it is a normal pregnancy is recommended that pregnant women continue to perform normal physical effort. For athletes, it is recommended to decrease the intensity, especially during the second trimester of pregnancy and especially during the last month. Pregnancy increases the elasticity of the ligaments by the effect of hormones released during pregnancy. For this reason, it is advisable to practice sports that do not require jumping, excessive stretching and to use appropriate footwear. Some reported benefits of regular moderate physical activity during pregnancy are (Artal, R. & Toole, M.O ., 2003): according to the start value of BMI except in the adolescent woman , where the weight Some studies recommend energy consumption between 1800 and 2200 kcal, bearing in mind that weight gain should be among the lower ranks. In adolescents, low weight or great The exercise is useful to help control metabolism. The type of exercise for pregnant women is with the work that can be done with the upper extremities. However it is contraindicated Currently more pregnant women want to have their pregnancy in natural and healthy conditions. Exercising in pregnant women is controversial as to the changes presented in their body and the hormonal levels. A long time ago, pregnancy was experienced as an illness, and had several myths related to it, including the exercise. The scientific arguments to restrict physical activity during pregnancy suggest that exercise causes an increment in maternal body temperature and therefore in the fetus too, with increased release of catecholamines, decreased circulating glucose and decreased blood flow to the placenta. However, there are other scientific studies that show the benefits of a regular physical activity during pregnancy to maintain health. When it is a normal pregnancy is recommended that pregnant women continue to perform normal physical For athletes, it is recommended to decrease the intensity, especially during the second trimester of pregnancy and especially during the last month. Pregnancy increases the elasticity of the ligaments by the effect of hormones released during pregnancy. For this reason, it is advisable to practice sports that do not require jumping, excessive stretching and to use appropriate footwear. Some reported benefits of regular moderate physical • Get optimal metabolic control and to prevent hypoglycemia complexion is recommended to increase energy requirements. activity during pregnancy are (Artal, R. & Toole, M.O ., 2003): • Adequate weight gain during pregnancy • During hyperglycemia and hypoglycemia increment must be higher. The objectives of a diet plan are: in the following cases: • Multiple pregnancy • Hypertension effort. weight. benefits. 4.1 Physical activity • Increased uterine contractions • history of stroke or arrhythmia In people with type 2 diabetes, there is a lot of evidence that suggests that regular physical activity improves insulin sensitivity, weight loss, thereby improves glucose control. Several studies have examined whether regular exercise is also beneficial in the management of GDM, for example, Jovanovic L, (Jovanovic L, 2000) randomized 19 women with GDM to a regime of diet alone, or diet with 20 minutes of supervised aerobic training three days per week for six weeks. This modest amount of physical activity resulted in lower fasting glucose levels, lower glucose responses to a glucose challenge, and a lower HbA1c . Another study randomized 29 women with GDM to 30 minutes of exercise (70% of maximal heart rate) of three to four times per week. In this case the glucose levels did not improve in those who exercised. The action might be through the activation of AMPK, a kinase that is activated during exercise, it is possible that exercise may act through the same molecular cellular pathway and therefore there are not additive effects of these two treatment regimens (Hardie DG, 2004). Another study found that women with a prepregnancy value BMI of 25 and who were in the exercise program were less likely to require insulin. Therefore, it seems reasonable to recommend that when there is not medical or obstetric contraindication, women with GDM should maintain a sensible level of light and moderate intensity if there is physical activity during the pregnancy. The above studies provide enough evidence that moderate physical activity such as walking between 20 and 30 minutes each day, or three to four times per week, through this they can achieve the glycemic control. The diet should provide with adequate nutrition for pregnancy. Carbohydrates should be distributed throughout the day over main meals and snacks. Limiting carbohydrates at 40% of the total caloric intake and having a higher proportion of carbohydrates of lower glycaemic index decreases postprandial glucose levels and reduces the need for insulin therapy (Smith, C.S. & Van Andel, R., 2001; Artal, R. & Toole, M.O ., 2003). There is not enough evidence to support dietary or drug treatment in patients with gestational diabetes. Gestational diabetes and impaired glucose tolerance are associated with macrosomia and may be associated with an increased risk for cesarean delivery, shoulder dystocia, and birth trauma. Although preexisting diabetes has been shown to increase the risk of poor perinatal outcomes, it is not clear that data relating to preexisting diabetes can be extrapolated to patients with gestational diabetes. Tuffnell and colleagues (Olwan N, 2009)researched the Cochrane Pregnancy and Childbirth Group trials register, the Cochrane Central Register of Controlled Trials, and bibliographies of relevant articles. They identified three studies of 223 women with impaired glucose tolerance; none of these studies was a randomized controlled trial comparing management strategies. Intensive management of gestational diabetes is timeconsuming and resource-intensive. Overall, the evidence is insufficient to support the therapy for gestational diabetes. However, universal screening is the standard of care in most communities. When faced with abnormal results, most family physicians will opt to follow the consensus opinion of our specialist colleagues. For example, a study that analyzed the higher egg and cholesterol intakes found that they are associated with increased risk of type 2 diabetes mellitus. It was also observed that the higher egg and cholesterol intakes before and during pregnancy are associated with an increased risk of GDM. (Qiu C, et al., 2011). The Influence of Diet to Control the Metabolism in Gestational Diabetes Mellitus 289 Teacher Gabriela Ramirez-Tavares and Joseph Gerald Phillips for their contributions to this English version. Medical and Nutrition Department, Health and Science Division , Campus Alwan, N; Tuffnell, DJ; West J. (2009). Tratamiento para la diabetes gestacional: (Revision http://www.bibliotecacochrane.com/BCPGetDocument.asp?SessionID=%2016594 American diabetes association. (2003). Position statement: Gestational diabetes mellitus. American Diabetes Association. (2004). Gestational diabetes mellitus (Position Statement). American Diabetes Association. (2007). Standards of medical care in diabetes 2007. Diabetes American Diabetes Association. (2010). Standards of Medical Care in Diabetes—2010. Arora, SK & Mcfarlane, SI (2005). The case for low carbohydrate diets in diabetes Artal, R. & Toole, M.O . (2003). Guidelines of the American College of Obstetricians and Atkinson, F; Foster-Powell, K; Brand-Miller, JC. (2008). International Tables of Glycemic Avila, A; Shamah, T; Chávez, A. (1995). Encuesta urbana de alimentación y nutrición en la Briefel, RR; Wilson, A; Gleason PM. (2009). Consumption of Low-Nutrient, Energy- Centers for Disease Control and Prevention. National Center for Health Statistics. National. Goldhaber-Fiebert, JD; Goldhaber-Fiebert, SN; Tristán, ML; Nathan DM.(2003). Randomized Health and Nutrition Examination Survey. Available from: Diabetes Care; 26: 24– 29. ISSN: 0149-5992 http://www.cdc.gov/nchs/nhanes/nhanes\_questionnaires.htm. Accessed Gynecologists for exercise during pregnancy and the postpartum period. Br J Sports Index and Glycemic Load Values: 2008. Diabetes Care; 31:2281–2283. ISSN: 0149- zona metropolitana de la cuidad de México. (Urban food and nutrition survey in the metropolitan area of Mexico City.) México City. Instituto Nacional de la Dense Foods and Beverages at School, Home, and Other Locations among School Lunch Participants and Nonparticipants. J Am Diet Assoc; 109:S79-S90. controlled community-based nutrition and exercise intervention improves glycemia and cardiovascular risk factors in type 2 diabetic patients in rural Costa Rica. Ltd. Available in: http://www.update-software.com; 02&DocumentID=CD003395#CD003395-fig-00120 Diabetes Care; 26(suppl 1): S103-S5. ISSN: 0149-5992 Diabetes Care; 27( Suppl. 1): S88– S90. ISSN: 0149-5992 Diabetes care;33 (suppl 1): 11s-61s. ISSN: 0149-5992 managerment. Nutr Metab; 2:16. ISSN: 1743-7075 care; 30: suppl 1: S4-S41. ISSN: 0149-5992 Med ;37:6-12. ISNN: 1473-0480 5992 Nutrición. November, 2010. ISSN: 0002-8223 Cochrane traducida). En: Biblioteca Cochrane Plus; Num 3. Oxford: Update Software 6. Acknowledgment 7. References Leon, University of Guanajuato. Several lines of evidence indicate that realistic modifications of diet and lifestyle can prevent diabetes type 2. Some of the main determinants include lack of physical activity, hours of TV watching, low quality and energy dense diets, and high caloric sweetener intake. Caloric beverages have been recognized as an important source of energy and have been associated with an increased risk of overweight. (Schulze MB, 2005; Ludwig DS, 2001). Lifestyle modification, to increase exercise and to modify diet habits are an effective way of to prevent and to delay the onset of type 2 diabetes in the future in women with GDM. One important aspect of combating the epidemics of obesity and type 2 diabetes has been through dietary strategies. Recently, very-low-carbohydrate diets have gained much popularity. Recently the effects of a diet lower in carbohydrates and higher in protein has been evaluated in obese and over-weight patients with type 2 diabetes for analyze the changes in blood glucose levels and in the insulin resistance. Because glucose is the major insulin secretagogue carbohydrate, its reduction would be expected to be beneficial in type 2 diabetes and the use of such diets. Although, as noted above, official recommendations generally continue to suggest low fat and high carbohydrate intake (Arora & McFarlane , 2005; Fioster GD, 2003) . Efforts to change diets, physical activity patterns, and other aspects of lifestyle have traditionally attempted to educate individuals through schools, health care providers, worksites, and general media. These efforts will continue to play an important role, but they can be strongly reinforced by policy and environmental changes. #### 5. Conclusion The control and prevention of GDM must be the principal goal. The dietary treatment must be based on the design and prescription of diets according to cultural habits, economical aspects and the diet must be accessible. The family, the health team and the society should be into the treatment of the GDM, mainly the prevention of diabetes mellitus type 2. To achieve this goal the authors suggest: Finally, it is important to consider the economical and social influence (Wilson C, 2003). Pregnant women usually modify their food intake according to the influence of their nutritional orientation . The full costs to achieve a behavioral change and the policies are complex and difficult to estimate. The efforts to reduce the increment of diabetes mellitus type 2, is screening pregnant women. In sum, the benefits will be for the fetus growth and health for both ### 6. Acknowledgment Teacher Gabriela Ramirez-Tavares and Joseph Gerald Phillips for their contributions to this English version. Medical and Nutrition Department, Health and Science Division , Campus Leon, University of Guanajuato. #### 7. References 288 Gestational Diabetes Several lines of evidence indicate that realistic modifications of diet and lifestyle can prevent diabetes type 2. Some of the main determinants include lack of physical activity, hours of TV watching, low quality and energy dense diets, and high caloric sweetener intake. Caloric beverages have been recognized as an important source of energy and have been associated with an increased risk of overweight. (Schulze MB, 2005; Ludwig Lifestyle modification, to increase exercise and to modify diet habits are an effective way of to prevent and to delay the onset of type 2 diabetes in the future in women with GDM. One important aspect of combating the epidemics of obesity and type 2 diabetes has been through dietary strategies. Recently, very-low-carbohydrate diets have gained much popularity. Recently the effects of a diet lower in carbohydrates and higher in protein has been evaluated in obese and over-weight patients with type 2 diabetes for analyze the changes in blood glucose levels and in the insulin resistance. Because glucose is the major insulin secretagogue carbohydrate, its reduction would be expected to be beneficial in type 2 diabetes and the use of such diets. Although, as noted above, official recommendations generally continue to suggest low fat and high carbohydrate intake (Arora & McFarlane , Efforts to change diets, physical activity patterns, and other aspects of lifestyle have traditionally attempted to educate individuals through schools, health care providers, worksites, and general media. These efforts will continue to play an important role, but they The control and prevention of GDM must be the principal goal. The dietary treatment must be based on the design and prescription of diets according to cultural habits, economical aspects and the diet must be accessible. The family, the health team and the society should be into the treatment of the GDM, mainly the prevention of diabetes mellitus type 2. To Finally, it is important to consider the economical and social influence (Wilson C, 2003). Pregnant women usually modify their food intake according to the influence of their nutritional orientation . The full costs to achieve a behavioral change and the policies are complex and difficult to estimate. The efforts to reduce the increment of diabetes mellitus type 2, is screening pregnant women. In sum, the benefits will be for the fetus growth and can be strongly reinforced by policy and environmental changes. order to adapt and correctly identify the nutritional treatment. appointment, for increasing the adherence to the diet. DS, 2001). 2005; Fioster GD, 2003) . 5. Conclusion GDM. health for both achieve this goal the authors suggest: design programs that involve motivation. Alwan, N; Tuffnell, DJ; West J. (2009). Tratamiento para la diabetes gestacional: (Revision Cochrane traducida). En: Biblioteca Cochrane Plus; Num 3. Oxford: Update Software Ltd. Available in: http://www.update-software.com; http://www.bibliotecacochrane.com/BCPGetDocument.asp?SessionID=%2016594 02&DocumentID=CD003395#CD003395-fig-00120 The Influence of Diet to Control the Metabolism in Gestational Diabetes Mellitus 291 Marvan, L; Pérez, AB; Palacios, B. (2008). Mexican Food System Equivalents. Third edition. Fomento de Nutrición y Salud; México D.F. ISBN: 978-970-94523-1-0 Major, CA; Henry, JM; De Veciana, M; Morgan, MA. (1998). The effects of carbohydrate McLeroy, KR; Bibeau, D; Steckler, A; Glanz, K. (1998). An Ecological Perspective for Health Promotion Programs. Health Educ Q ; 15(4):351-378. ISSN: 0195-8402 Medical nutrition therapy: nutrition practice guidelines for gestational diabetes.(2001). Am Monroy-Torres, R; Reeves-Aguirre, CC; Naves-Sánchez, J; Macias, AE. (2008). Influencia Monroy-Torres, R; Velásquez, A; Ortíz, A. (2010). Programa oportunidades sobre la Moses, R; Luebcke, M; Davis, W; Coleman, K; Tapsell,L; Petocz, P; Brand-Miller. (2006), Pastor, JG; Warshaw, H; Daly, A; Franz, M; Kulkarni, K. (2002). The evidence for the Qiu, C; Frederick, IO; Zhang, C; Sorensen, TK; Enquobahrie, DA; Williams, MA. (2011). Risk Ramírez, JA; García, M; Cervantes, R; Mata, N; Zárate, F; Mason, T; Villarreal, A. Romieu, I; Hernandez-Avila, M; Rivera, JA; Ruel, MT; Parra, S. (1997).Dietary studies in Sheard, N.F.; Clark, N.G.; Brand-Miller, J.C.; Franz, M.J.; Pi-Sunyer, F.X.; Mayer-Davis, E.; Schulze, MB & Hu, FB. (2005). Primary prevention of diabetes: what can be done and Smith, C.S. & Van Andel, R. (2001). Pregnancy and North American lifestyles: exercise, work, and diet in pregnancy. Clin Fam Pract ;3:167-181. ISNN: 00943509 Standards of Medical Care in Diabetes—2008. (2008). Diabetes Care ; 31: (Suppl 1): S12- S54. 91: 600-3. ISSN: 0029-7844 Dietetic assoc. ISSN, 0002-8223 Clin Nutr; 84, 807-812. ISSN: 1938-3207 Am J Epidemiol ;173(6):649-58 ISSN 1476-6256 Nutr; 65(suppl): 1159s-65s. ISSN: 1938-3207 association. Diabetes Care ;27(9):2266-71. ISSN: 0149-5992 25: 608-13 . ISSN: 0149-5992 3062 326X. 4033 7525 ISSN: 0149-5992 restriction in patients with diet-controlled gestacional diabetes. Obstet and Gynecol; de una dieta individualizada de bajo en índice glucémico en el control de la diabetes mellitus gestacional. Rev Ginecol obstetr Mex; (76)12:722-729. ISSN 1561- seguridad alimentaria y nutricional en Atarjea: desde la percepción de sus participantes. Avances en seguridad alimentaria y nutricional; 1(1): 63-73 ISSN: 1659- "Effect of a low glycemic index diet during pregnancy on obstetric outcome". Am J effectiveness of medical nutrition therapy in diabetes management. Diabetes care; of gestational diabetes mellitus in relation to maternal egg and cholesterol intake. (2005).Transición alimentaria en México. An Pediatr ;58(6):568-73. ISSN: 1695- countries experiencing a health transition:Mexico and Central America. Am J Clin Kulkarni, K.; Geil, P. (2004). Dietary carbohydrate (amount and type) in the prevention and management of diabetes: a statement by the american diabetes how much can be prevented? Annu Rev Public Health; 26:445-467. ISSN: 0163- Fall C. (2009). Maternal nutrition: effects on health in the next generation. Indian J Med Food and Nutrition Board (FNB). 2005. Dietary Reference Intakes for Energy, Carbohydrate, Foster, GD; Wyatt, HR; Hill, JO; McGuckin, BG; Brill, C; Mohammed, BS; Szapary, PO; Franz, M; Bantle, JP; Beebe, CA; Brunzell, JD; Chiasson, JL; Garg, A; Holzmeister, LA; Franz M.(2003). The glycemic index: not the most effective nutrition therapy intervention. Foster-Powell, K; Holt, SH; Brand-Miller, JC. (2002). International table of glycemic index and glycemic load values: 2002. Am J Clin Nutr; 76:5–56. ISSN: 1938-3207 Fraser, RB; Ford, FA; Lawrence, GF. (1998).Insulin sensitivity in third trimester of Hardie DG. (2004). AMP-activated protein kinase: a key system mediating metabolic Institución Nacional de Salud Pública, Secretaría de Salud, Instituto Nacional de Estadística, Geografía e Informática. (2006). Encuesta Nacional de Salud y Nutrición. International Association of Diabetes and Pregnancy Study Groups Consensus Panel.(2010). Jenkins, DJA; Wolever, TMS; Jenkins, AL; Josse, RG; Wong, GS. (1984).The glycaemic Jovanovic, L. (2000).Medical nutritional therapy in pregnant women with pregestational diabetes mellitus. J Matern Fetal Neonatal Med; 9: 21-8 . ISSN: 1476-7058 Kant, AK. (2000). Consumption of energy-dense, nutrient-poor foods by adult Kim, C; Newton, KM; Knopp, RH. (2002). Gestational diabetes and the incidence of type 2 diabetes: a systematic review. Diabetes Care; 25: 1862– 1868. ISSN: 0149-5992 Ludwig, DS; Peterson, KE; Gortmaker, SL. (2001). Relation between consumption of sugar- Nielsen, S & Popkin, B. (2003). Patterns and Trends in Food Portion Sizes, 1977-1998. JAMA response to carbohydrate foods. Lancet; 2:388-91. ISSN 0140-6736 responses to exercise. Med Sci Sports Exerc; 36:28-34. ISSN 1530-0315 Pregnancy. Diabetes Care;33:676-82. ISSN: 0149-5992 Lancet ; 357(9255):505-508. ISSN 0140-6736 ;289(4):450-453. ISSN: 00987484 This report may be accessed via www.nap.edu and is available in: http://books.nap.edu/openbook.php?record\_id=10490&page=275 Engl J Med; 348(21):2082-2090. ISSN 1533-4406. Diabetes Care 26:2466–2468. ISSN: 0149-5992 Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids (Macronutrients). Rader DJ; et al. ( 2003). A randomized trial of a low-carbohydrate diet for obesity. N Hoogwerf, B; Mayer-Davis, E; Mooradian, AD; Purnell, JQ; Wheeler, M. (2002). Evidence based nutrition principles and recommendations for the treatment and prevention of diabetes and related complications. Diabetes Care; 25:148-98. ISSN: pregnancy. A randomized study of dietary effects. Br J Obstet Gynaecol; 95: 223-9. International Association of Diabetes and Pregnancy Study Groups Recommendations on the Diagnosis and Classification of Hyperglycemia in Americans: nutritional and health implications. The third National Health and Nutrition Examination survey, 1988-1994. Am J Clin Nutr; 72:929-936. ISSN: sweetened drinks and childhood obesity: a prospective, observational analysis. Res;130(5):593-9. ISSN: 0971-5916 0149-5992 ISSN 0306-5456 1938-3207 17 Canada Insulin Use in Gestational McGill University Health Centre, Montreal, QC Sara J Meltzer and Rima Alsayari Diabetes – Pragmatic Protocols for Self-Management and for Labour and Delivery The expectation of treatment goals in the management of gestational diabetes are the most stringent and precise for control of diabetes in adults (American Diabetes Association, 2011; Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008). It is well accepted that the achievement of these goals requires an effective collaboration between a well-trained physician, a nurse educator and a qualified dietician experienced in working with pregnancy and, most importantly the woman with gestational diabetes (Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; Murphy, et al., 2010; Jacqueminet S, 2010; Kim, 2010; Kjos S, 1999). The more involved the woman becomes in her own treatment plan, the more likely that she will succeed (Meltzer S. Once a diagnosis of gestational diabetes has been made, the initial step is always an assessment of the woman's present diet and the introduction of changes which would optimize her glucose control. Without an understanding and application of the concepts of medical nutrition therapy, further attempts at controlling glucose will always be limited. Individual dietary counselling with a trained dietician is preferable, whenever possible (Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; The major concepts of the diet consist of identifying fast and more slowly absorbed carbohydrate sources: fruits and vegetables, starchy foods such as bread, pastas, rice, noodles, grains, and milk and milk products as the major carbohydrate in the diet. In any one meal, optimally there is a small portion of fruits and/or vegetables which will be absorbed quickly, a portion of starches/grains which will be absorbed more slowly, accompanied by a portion of protein and a small portion of fat. The mix of two forms of carbohydrate absorbed over different time periods, as well as the presence of fat and protein which, apart from being essential nutrients, slow further the absorption of glucose from the gut permits a more gradual and slower rise of glucose post-meal. This makes glucose much easier to control with either endogenous insulin or injected forms – presented conceptually 2. Never underestimate the value of a well-planned, balanced diet International Diabetes Federation Writing Group, 2009). 1. Introduction J., 2010). Wilson, C; Brown, T; Acton, K; Gilliland, S. (2003). Effects of clinical nutrition education and educator discipline on glycemic control outcomes in the Indian Health Service. Diabetes Care; 26:S23-S30. ISSN: 0149-5992 ## Insulin Use in Gestational Diabetes – Pragmatic Protocols for Self-Management and for Labour and Delivery Sara J Meltzer and Rima Alsayari McGill University Health Centre, Montreal, QC Canada #### 1. Introduction 292 Gestational Diabetes Wilson, C; Brown, T; Acton, K; Gilliland, S. (2003). Effects of clinical nutrition education and Diabetes Care; 26:S23-S30. ISSN: 0149-5992 educator discipline on glycemic control outcomes in the Indian Health Service. The expectation of treatment goals in the management of gestational diabetes are the most stringent and precise for control of diabetes in adults (American Diabetes Association, 2011; Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008). It is well accepted that the achievement of these goals requires an effective collaboration between a well-trained physician, a nurse educator and a qualified dietician experienced in working with pregnancy and, most importantly the woman with gestational diabetes (Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; Murphy, et al., 2010; Jacqueminet S, 2010; Kim, 2010; Kjos S, 1999). The more involved the woman becomes in her own treatment plan, the more likely that she will succeed (Meltzer S. J., 2010). #### 2. Never underestimate the value of a well-planned, balanced diet Once a diagnosis of gestational diabetes has been made, the initial step is always an assessment of the woman's present diet and the introduction of changes which would optimize her glucose control. Without an understanding and application of the concepts of medical nutrition therapy, further attempts at controlling glucose will always be limited. Individual dietary counselling with a trained dietician is preferable, whenever possible (Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; International Diabetes Federation Writing Group, 2009). The major concepts of the diet consist of identifying fast and more slowly absorbed carbohydrate sources: fruits and vegetables, starchy foods such as bread, pastas, rice, noodles, grains, and milk and milk products as the major carbohydrate in the diet. In any one meal, optimally there is a small portion of fruits and/or vegetables which will be absorbed quickly, a portion of starches/grains which will be absorbed more slowly, accompanied by a portion of protein and a small portion of fat. The mix of two forms of carbohydrate absorbed over different time periods, as well as the presence of fat and protein which, apart from being essential nutrients, slow further the absorption of glucose from the gut permits a more gradual and slower rise of glucose post-meal. This makes glucose much easier to control with either endogenous insulin or injected forms – presented conceptually Insulin Use in Gestational Diabetes – Pragmatic Author's statement: Sara J. Meltzer, MD, FRCPC, FACP Fig. 2. them helpful. 2009; Egeland & Meltzer, 2010). Protocols for Self-Management and for Labour and Delivery 295 Figure 1 and 2 were developed by the author for use in clinical work teaching women with gestational diabetes the basic concepts of diet and are included here for those who may find To be sure that the diet has been effectively instituted – using food diaries and encouraging measuring and weighing can be very helpful to help the women understand successes and problems with her choices of foods in her normal environment (Burke, Wang, & Sevick, 2011). Unfortunately, some women will limit their food choices dramatically leaving out foods typical of their normal diet in an effort to reach glucose targets. Since it is important for the woman to understand how to incorporate the foods normally present in her diet including ethnically diverse foods while she has the help and time of a dietician's advice and can see the effects on her glycemic control with frequent testing, excessive limitation of her food choices should be discouraged. The effectiveness of long-term dietary adjustments to decrease insulin need is more likely if she understands the effects of her preferred foods on her glucose control. Since the mother is often in charge of family meal-planning and cooking, her application of learned knowledge should help her avoid future diabetes and reduce the risks to her offspring whose eating habits develop at home (Ratner, et al., 2008; Tuomilehto, et al., 2001; Verier-Mine, 2010; Diabetes Prevention Program Research Group, in Figure 1. Additionally, use of foods with low glycemic index (higher in fibre and producing a lower post-ingestion glucose elevation) has been shown to be of help in minimizing effects of excess glucose in the pregnancy (Grant, Wolever, O'Connor, Nissenbaum, & Josse, 2011; Tzanetakou, Mikhailidis, & Perrea, 2011; Thomas D, 2009). Foetal growth, particularly asymmetric growth with increased central fat patterning relates to the effectiveness of glucose control of post-meal elevations and in general terms, excess post-prandial hyperglycaemia has negative effects on maternal and foetal well-being (Standl, Schnell, & Ceriello, 2011; Parretti, et al., 2001; DeVeciana, et al., 1995). In gestational diabetes, as with early type 2 diabetes, the first phase of insulin release is impaired (Ornoy, 2011; Catalano, Kirwan, Haugel-de Mouzon, & King, 2003), thus more concentrated forms of carbohydrate (sugar added to coffee, juices, candies, cakes etc.) will be absorbed too quickly for the slow insulin response, allowing the glucose to go up quickly to above normal levels which may then affect the baby. The stimulation of insulin release can occur in response to certain amino acids. There may still be an intact protein-stimulated insulin release response even if the glucose-stimulated insulin release is compromised which can help avoid elevated glucose values (Catalano, Kirwan, Haugel-de Mouzon, & King, 2003). These concepts are relatively simple to understand and when explained to the woman with GDM helps her to understand the basis of her dietary efforts and concerns. Providing an explanation to the woman with visual tools suggesting the relative absorption times and how different foods work will make it easier for her to understand what she is trying to do when she goes home and plans her meals. (Figures 1 and 2 developed for this purpose). Fig. 1. Fig. 2. 294 Gestational Diabetes in Figure 1. Additionally, use of foods with low glycemic index (higher in fibre and producing a lower post-ingestion glucose elevation) has been shown to be of help in minimizing effects of excess glucose in the pregnancy (Grant, Wolever, O'Connor, Nissenbaum, & Josse, 2011; Tzanetakou, Mikhailidis, & Perrea, 2011; Thomas D, 2009). Foetal growth, particularly asymmetric growth with increased central fat patterning relates to the effectiveness of glucose control of post-meal elevations and in general terms, excess post-prandial hyperglycaemia has negative effects on maternal and foetal well-being In gestational diabetes, as with early type 2 diabetes, the first phase of insulin release is impaired (Ornoy, 2011; Catalano, Kirwan, Haugel-de Mouzon, & King, 2003), thus more concentrated forms of carbohydrate (sugar added to coffee, juices, candies, cakes etc.) will be absorbed too quickly for the slow insulin response, allowing the glucose to go up quickly to above normal levels which may then affect the baby. The stimulation of insulin release can occur in response to certain amino acids. There may still be an intact protein-stimulated insulin release response even if the glucose-stimulated insulin release is compromised which can help avoid elevated glucose values (Catalano, Kirwan, Haugel-de Mouzon, & King, 2003). These concepts are relatively simple to understand and when explained to the woman with GDM helps her to understand the basis of her dietary efforts and concerns. Providing an explanation to the woman with visual tools suggesting the relative absorption times and how different foods work will make it easier for her to understand what she is trying to do when she goes home and plans her meals. (Figures 1 and 2 developed for this (Standl, Schnell, & Ceriello, 2011; Parretti, et al., 2001; DeVeciana, et al., 1995). purpose). Fig. 1. Author's statement: Sara J. Meltzer, MD, FRCPC, FACP Figure 1 and 2 were developed by the author for use in clinical work teaching women with gestational diabetes the basic concepts of diet and are included here for those who may find them helpful. To be sure that the diet has been effectively instituted – using food diaries and encouraging measuring and weighing can be very helpful to help the women understand successes and problems with her choices of foods in her normal environment (Burke, Wang, & Sevick, 2011). Unfortunately, some women will limit their food choices dramatically leaving out foods typical of their normal diet in an effort to reach glucose targets. Since it is important for the woman to understand how to incorporate the foods normally present in her diet including ethnically diverse foods while she has the help and time of a dietician's advice and can see the effects on her glycemic control with frequent testing, excessive limitation of her food choices should be discouraged. The effectiveness of long-term dietary adjustments to decrease insulin need is more likely if she understands the effects of her preferred foods on her glucose control. Since the mother is often in charge of family meal-planning and cooking, her application of learned knowledge should help her avoid future diabetes and reduce the risks to her offspring whose eating habits develop at home (Ratner, et al., 2008; Tuomilehto, et al., 2001; Verier-Mine, 2010; Diabetes Prevention Program Research Group, 2009; Egeland & Meltzer, 2010). Insulin Use in Gestational Diabetes – Pragmatic reasons… Protocols for Self-Management and for Labour and Delivery 297 terms of weight gain, macrosomia and relative risks for the offspring, lower achieved values are often possible and near-normal values can often be reached using the right insulin protocol and self-adjustment of insulin by the patient. There is no proven value of running blood glucose values at the top end of normal (or near the abnormal range) rather than the middle, so going for truly normal would appear to make sense if hypoglycemia does not occur. In a study of a Pima Indian cohort, diabetes conveyed excess offspring risk of obesity and glucose intolerance independent of genetic factors and other studies have explored the role of diet type and intake quantity in future offspring risks (Dabalea, et al., 2000; Reusens & Remacle, 2001). In fact, results of glucose targets achieved by any means in the MiG's study (metformin in gestational diabetes) suggest that outcomes are optimal in terms of appropriate fetal weight for dates with values closer to the mean of normal (Rowan, Gao, Hague, & McIntyre, 2010) and a long-term follow-up study (15 years) attaining similar lower mean values led to a very low incidence (1%) of teenage offspring glucose intolerance (Egeland & Meltzer, 2010). In the HAPO study, there was an association with maternal postload glucose and clinical neonatal hypoglycemia; for the one hour glucose value (adjusted OR, 1.13; 95%CI; 1.03-1.26) and a weak association for the two hour glucose value (adjusted OR, 1.13; 95% CI 1.00-1.12) (HAPO Study Cooperative Research Group., 2009). Even within normal ranges, maternal glucose values have been linked to offspring insulin sensitivity and beta cell function (Reusens & Remacle, 2001; Bush, Chandler-Laney, Rouse, Granger, Oster, & Gower, 2011). In obese women particularly, achievement of the optimal glucose targets improves fetal outcomes (Langer, Yogev, Xenakis, & Brustman, 2005). minority of women with gestational diabetes. teach and effective… 4. Physical activity should be encouraged unless precluded for obstetric The effectiveness of a physically active lifestyle has been shown to help prevent the development of type 2 diabetes in those at risk such as women diagnosed with gestational diabetes (Ratner, et al., 2008; Sanz, Gautier, & Hanaire, 2010). Most guidelines recommend some form of regular exercise in pregnancy although only a few studies address this issue in detail (American Diabetes Association, 2011; Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; Division of Nutrition PA, and Obesity, Centers for Disease Control; ACOG Committee Opinion, 2002; Bung, Artal, Khodiguian, & Kjos, 1991; Artal, Lockwood, & Brown, 2010). At the very least, encouraging women to walk after meals often for a total of 30 minutes per day is the recommendation and this can improve glucose results and will help the women understand during pregnancy how effective small amounts of exercise can be on glucose control. Care must be used in women with evidence of pre-term labour or any previous suggestions of cervical incompetence; however this is the 5. Insulin adjustment algorithms with the patient self-adjusting are easy to Initiation of insulin, if needed, is no longer a reason to hospitalize a patient. Insulin is often needed in about 10-50% of women, depending on the population and the criteria used for diagnosis (Langer, Berkus, Brustman, Anyaegbunum, & Mazze, 1991). Once the effect of diet on initial testing has been evaluated and if the woman's glucose values exceed the upper limits of glucose targets, it is a simple matter to initiate insulin in a logical and pragmatic way. Pre-pregnancy BMI and actual weight gain are potentially modifiable risk factors so attention to both weight control pre-pregnancy and rate and total amount of weight gain in the pregnancy are important (Hutcheon, Platt, Meltzer, & Egeland, 2006; Radesky, Oken, Rifas-Shirman, Kleinman, Rich-Edwards, & Gillman, 2008). Recommended weight gain is often based on the Institute of Medicine recommendations, relate to pre-pregnancy BMI and intake will often be 25-30 kcal/kg (Institute of Medicine and National Research Council Committee to Re-examine IOM Pregnancy Weight Guidelines, 2009). #### 3. Teach self-monitoring and establish glucose targets Another important facet of achieving glucose control will be to determine if the glucose values are appropriately controlled at various times of the day. In this situation, use of self-blood glucose monitoring (SBGM) is very effective. Avoiding starvation is also important so many centres will routinely test at some point for urine ketonuria to determine if the women are receiving adequate calories for their needs. The basic concepts of testing are easy to convey and ideally a qualified diabetes educator or pharmacist would review techniques in the use of the meter and lancet device chosen. In situations where this is not available, peer-to peer teaching may be equally effective. It is valuable to verify the accuracy of the woman's technique and device by asking her to do a comparative test against the laboratory value, ideally in a fasting state which provides a better comparator than in post-meal values which are in a state of flux and may differ in the capillary and venous system physiologically. This is important to clarify since the glucose targets required are within the normal range and many meters may have a small degree of consistent error which, if known, can be considered in defining her at-home meter glucose targets. The usefulness of the testing is markedly enhanced if the woman is instructed on how to use this information to adjust her therapy (Hawkins J. S., Casey, Lo, Moss, McIntyre, & Leveno, 2009). The more pragmatic and useful the testing, the more likely compliance with management will be. Thus self-blood glucose monitoring is often done before breakfast and at 60-90 minutes after meals, since studies suggest that this is the highest level of glucose seen in the post-meal phase for the majority of women (Jovanovic L. G., 2008). The peak post-prandial glucose level in obese women tends to be higher and later - more around 90 minutes after the meal (Ben-Haroush, Yogev, Chen, Rosenn, Hod, & Langer, 2004). In many parts of the world, the post-meal test timing recommended may be closer to 120 minutes or 2 hours. Health providers must be aware that only testing before or after main meals may miss other parts of the day with significant increases in glucose so random testing at other times may also need to be suggested (Montaner, Ripolles, Pamies, & Corcoy, 2011)(29). In most national guidelines, the goal of therapy is to achieve values which fall into the normal range, thus under 5.1 or 5.3 mmol/L (92-95mg/dl) fasting, under 7.8mmol/L (140mg/dl) at 1h post load, and under 6.7mmol/L (120mg/dl) at 2h post-load (American Diabetes Association, 2011; Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008). It must be realized that these numbers indicate the mean plus two standard deviations above the mean… the actual mean is lower…usually between 3.8 – 4.4 mmol/L in the fasting state and rarely above 6.5mmol/L at any point after the meal (Gillmer, Beard, Brooke, & Oakley, 1975; Parretti, et al., 2001; Rowan, Gao, Hague, & McIntyre, 2010; Yogev, Ben-Haroush, Chen, Rosenn, Hod, & Langer, 2004). Depending on the reliability of the individual, her meter accuracy and the evaluation of the pregnancy in Pre-pregnancy BMI and actual weight gain are potentially modifiable risk factors so attention to both weight control pre-pregnancy and rate and total amount of weight gain in the pregnancy are important (Hutcheon, Platt, Meltzer, & Egeland, 2006; Radesky, Oken, Rifas-Shirman, Kleinman, Rich-Edwards, & Gillman, 2008). Recommended weight gain is often based on the Institute of Medicine recommendations, relate to pre-pregnancy BMI and intake will often be 25-30 kcal/kg (Institute of Medicine and National Research Council Another important facet of achieving glucose control will be to determine if the glucose values are appropriately controlled at various times of the day. In this situation, use of self-blood glucose monitoring (SBGM) is very effective. Avoiding starvation is also important so many centres will routinely test at some point for urine ketonuria to determine if the women are receiving adequate calories for their needs. The basic concepts of testing are easy to convey and ideally a qualified diabetes educator or pharmacist would review techniques in the use of the meter and lancet device chosen. In situations where this is not available, peer-to peer teaching may be equally effective. It is valuable to verify the accuracy of the woman's technique and device by asking her to do a comparative test against the laboratory value, ideally in a fasting state which provides a better comparator than in post-meal values which are in a state of flux and may differ in the capillary and venous system physiologically. This is important to clarify since the glucose targets required are within the normal range and many meters may have a small degree of consistent error which, if known, can be considered in defining her at-home The usefulness of the testing is markedly enhanced if the woman is instructed on how to use this information to adjust her therapy (Hawkins J. S., Casey, Lo, Moss, McIntyre, & Leveno, 2009). The more pragmatic and useful the testing, the more likely compliance with management will be. Thus self-blood glucose monitoring is often done before breakfast and at 60-90 minutes after meals, since studies suggest that this is the highest level of glucose seen in the post-meal phase for the majority of women (Jovanovic L. G., 2008). The peak post-prandial glucose level in obese women tends to be higher and later - more around 90 minutes after the meal (Ben-Haroush, Yogev, Chen, Rosenn, Hod, & Langer, 2004). In many parts of the world, the post-meal test timing recommended may be closer to 120 minutes or 2 hours. Health providers must be aware that only testing before or after main meals may miss other parts of the day with significant increases in glucose so random testing at other times may also need to be suggested (Montaner, Ripolles, Pamies, & Corcoy, 2011)(29). In most national guidelines, the goal of therapy is to achieve values which fall into the normal range, thus under 5.1 or 5.3 mmol/L (92-95mg/dl) fasting, under 7.8mmol/L (140mg/dl) at 1h post load, and under 6.7mmol/L (120mg/dl) at 2h post-load (American Diabetes Association, 2011; Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008). It must be realized that these numbers indicate the mean plus two standard deviations above the mean… the actual mean is lower…usually between 3.8 – 4.4 mmol/L in the fasting state and rarely above 6.5mmol/L at any point after the meal (Gillmer, Beard, Brooke, & Oakley, 1975; Parretti, et al., 2001; Rowan, Gao, Hague, & McIntyre, 2010; Yogev, Ben-Haroush, Chen, Rosenn, Hod, & Langer, 2004). Depending on the reliability of the individual, her meter accuracy and the evaluation of the pregnancy in Committee to Re-examine IOM Pregnancy Weight Guidelines, 2009). 3. Teach self-monitoring and establish glucose targets meter glucose targets. terms of weight gain, macrosomia and relative risks for the offspring, lower achieved values are often possible and near-normal values can often be reached using the right insulin protocol and self-adjustment of insulin by the patient. There is no proven value of running blood glucose values at the top end of normal (or near the abnormal range) rather than the middle, so going for truly normal would appear to make sense if hypoglycemia does not occur. In a study of a Pima Indian cohort, diabetes conveyed excess offspring risk of obesity and glucose intolerance independent of genetic factors and other studies have explored the role of diet type and intake quantity in future offspring risks (Dabalea, et al., 2000; Reusens & Remacle, 2001). In fact, results of glucose targets achieved by any means in the MiG's study (metformin in gestational diabetes) suggest that outcomes are optimal in terms of appropriate fetal weight for dates with values closer to the mean of normal (Rowan, Gao, Hague, & McIntyre, 2010) and a long-term follow-up study (15 years) attaining similar lower mean values led to a very low incidence (1%) of teenage offspring glucose intolerance (Egeland & Meltzer, 2010). In the HAPO study, there was an association with maternal postload glucose and clinical neonatal hypoglycemia; for the one hour glucose value (adjusted OR, 1.13; 95%CI; 1.03-1.26) and a weak association for the two hour glucose value (adjusted OR, 1.13; 95% CI 1.00-1.12) (HAPO Study Cooperative Research Group., 2009). Even within normal ranges, maternal glucose values have been linked to offspring insulin sensitivity and beta cell function (Reusens & Remacle, 2001; Bush, Chandler-Laney, Rouse, Granger, Oster, & Gower, 2011). In obese women particularly, achievement of the optimal glucose targets improves fetal outcomes (Langer, Yogev, Xenakis, & Brustman, 2005). #### 4. Physical activity should be encouraged unless precluded for obstetric reasons… The effectiveness of a physically active lifestyle has been shown to help prevent the development of type 2 diabetes in those at risk such as women diagnosed with gestational diabetes (Ratner, et al., 2008; Sanz, Gautier, & Hanaire, 2010). Most guidelines recommend some form of regular exercise in pregnancy although only a few studies address this issue in detail (American Diabetes Association, 2011; Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; Division of Nutrition PA, and Obesity, Centers for Disease Control; ACOG Committee Opinion, 2002; Bung, Artal, Khodiguian, & Kjos, 1991; Artal, Lockwood, & Brown, 2010). At the very least, encouraging women to walk after meals often for a total of 30 minutes per day is the recommendation and this can improve glucose results and will help the women understand during pregnancy how effective small amounts of exercise can be on glucose control. Care must be used in women with evidence of pre-term labour or any previous suggestions of cervical incompetence; however this is the minority of women with gestational diabetes. #### 5. Insulin adjustment algorithms with the patient self-adjusting are easy to teach and effective… Initiation of insulin, if needed, is no longer a reason to hospitalize a patient. Insulin is often needed in about 10-50% of women, depending on the population and the criteria used for diagnosis (Langer, Berkus, Brustman, Anyaegbunum, & Mazze, 1991). Once the effect of diet on initial testing has been evaluated and if the woman's glucose values exceed the upper limits of glucose targets, it is a simple matter to initiate insulin in a logical and pragmatic way. Insulin Use in Gestational Diabetes – Pragmatic breakfast, lunch and dinner respectively. Protocols for Self-Management and for Labour and Delivery 299 values (as much as 6 units at once) and gradually reduce to as little as 1 unit more insulin for the evening dose as the target range is reached (See Figure 3a). In someone who is markedly obese or may have evidence of acanthosis nigricans, often an initial dose may be 0.1-0.2 units/kg administered at bedtime again with an increase in dose the following day if the fasting glucose has not achieved the desired range. An important part of the algorithm is a glucose value below which she MUST REDUCE her evening dose of NPH. This avoids hypoglycaemia before it happens and discourages overzealous increases in insulin dosages. Our experience has been very effective and safe using the value of 4.2mmol/L below which a woman will reduce that night's dose as this glucose value is far away from the values of 3.2 or 2.8mmol/L which would be felt as a hypoglycemic reaction and require treatment (Snyder, Gray-Donald, & Koski, 1994; Meltzer, Snyder, Penrod, Nudi, & Morin, 2010). In situations where women have markedly elevated fasting and post-meal glucose levels, an overall dose of 0.5 – 0.7u/kg can be used in the proportion of about 40% as bedtime NPH insulin and the remainder split over the day with a bit more at breakfast and less at lunch ie. 25% - 15% - 20% of the total calculated dose given as regular or rapid-acting insulin prior to On very rare occasions likely explaining the lack of literature related to it, women develop a local allergy to NPH at the injection site with swelling and redness developing up to 12 hours after the last injection of NPH at that site and lasting for about one to two days. In virtually every situation where this has been seen and is deemed intolerable by the woman, switching to regular human insulin as a 10pm injection and retesting glucose at 0400 to adjust the 10 pm dose as well as adding a 0400h injection of human (preferably) or pork regular insulin which will be adjusted based on the pre-breakfast result will often correct the allergy problem and continue to effectively control the glucose. Usually the dose can be reduced from the previous insulin dose and given as one third at 2200h and about one third or a bit less at 0400h. Subsequent self-adjustment protocols can still be used with an added time of 0400h and will allow for safe correction of insulin to optimal doses. Unfortunately, this approach does interfere with the woman's sleep. The use of detemir or glargine insulin If the fasting glucose is normal and the post-meal is the glucose which is elevated (often related to ethnic differences), the insulin administered will be regular insulin or a rapidacting analogue administered prior to the meal (See Figure 3b). If Regular insulin is used, the time it should be taken before the meal is often 20-30 minutes, which is why the rapidacting analogues are often preferred, as they can be take much closer to the meal (0-15 minutes) and still effectively control the post-prandial 60-90 minute peak (Pettitt, Ospina, Kolaczynski, & Jovanovic, 2003). The effectiveness of the dose is evaluated by the woman using her post-meal glucose value for that meal and the following day, the dosage for that meal will be adjusted up or down in order to achieve the desired values. Since at meals, occasionally, even the "best" patient may change her food from the recommended meal plan, it is often appropriate to wait for 2 abnormal values before increasing the dose, however only one value lower than the desired goal requires that the dose be reduced the following day. Allowing the woman to be responsible for the gradual and persistent adjustments of insulin dose regularly seen in GDM as the placenta continues to grow and her insulin needs rise can facilitate her care. Occasional additional tests in relation to snacks or prior to meals may be necessary to determine glucose control is always good (Montaner, Ripolles, Pamies, & Corcoy, 2011). Visits may be further apart because the woman is making the appropriate adjustments, thus the medical team does not need to. Use of colour-coding may be alternatives; however it has yet to be reported in this situation. Consideration of the degree of macrosomia seen on ultrasound, particularly if increased abdominal girth is seen in the foetus may be used as a moderating factor in the need for initiation of insulin and potentially in the target glucose values aimed for (Buchanan, et al., 1998)(44). The HAPO study showed a clear correlation with macrosomia and the glucose values attained on the oral glucose tolerance test result at 28-32 weeks [fasting glucose adjusted OR, 1.38; 95% CI:1.32-1.44, one-hour adjusted OR, 1.46 ; 95% CI 1.39-1.53, two-hour adjusted OR, 1. 38 ; 95% CI: 1.32-1.44] (HAPO Study Cooperative Research Group, Metger BE; Lowe LR; Dyer AR et al, 2008). If there is no evidence of macrosomia on ultrasound and the initial A1c is below a pregnancy normal mean of 5.3%, it may be possible to accept slightly higher glucose values and still obtain a good foetal outcome. The use of insulin pens has greatly simplified the teaching of insulin injection; however it is also relatively easy to teach injections using insulin syringes in a short teaching session, possibly in small groups. It is important that any woman being initiated on insulin (or any oral agent which is a secretogogue for that matter) be taught how to recognize and treat a hypoglycemic episode. The use of written and picture educational material often available from insulin producing companies will facilitate the educational essentials that must be explained. The concept of site use and rotation is another issue which should be addressed by the teaching nurse. There have been studies using both metformin and glyburide as oral agents for the management of gestational diabetes showing reasonably comparative composite outcomes. There were 46% of the women in the metformin study who still required insulin and there was inadequate power in the glyburide randomized trial to determine macrosomia and neonatal hypoglycemia outcomes (Rowan, Hague, Gao, Battin, Moore, & and MiG Trial Investigators, 2008; Langer, Conway, Berkis, Xenakis, & Gonzales, 2000). At present in most countries, it remains "off-label" and most guidelines do not suggest its routine use or only if for some reason insulin cannot be used (American Diabetes Association, 2011; Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; Jacqueminet S, 2010). For these reasons, it will not be addressed in detail in this chapter. In terms of the actual insulin prescription, the concept of using a famous train robber's recommendation - Sutton's law "Go where the money is!" can be very helpful. In other words, pick the part of the day which is most abnormal from the point of view of glucose control. In general, improvement to absolutely normal of the fasting glucose value will facilitate the release of insulin by the woman and improve glucose much of the day (Pennartz, Schenker, Menge, Schmidt, Nauck, & Meier, 2011). In the majority of cases, bedtime insulin is the most effective first step although the women from South East Asia and Asia may have post-meal glucose which tends to be higher and may have normal fasting glucose values. If the fasting glucose is the value which is above range, initiation of intermediate insulin at bedtime (neutral protamine Hagedorn or NPH in human or pork form) at a low dose should be done (can be as low as 2 units in a very nervous woman who is not too obese, but the usual starting dose would be 8 – 10 units minimum or 0.1 units/kg). In many clinics, the women return on a regular basis for adjustment of their insulin doses. As insulin needs increase progressively in response to placental growth and production of anti-insulin hormones, this often means that the initial period after adjustment is well controlled but over the two weeks the glucose control may deteriorate. It is very easy to have the women do the progressive adjustment of her own insulin based on her morning blood glucose (and thus her body's response to the insulin given); she can then increase the dose by specified increments until she achieves the desired range. The increments are largest for high glucose Consideration of the degree of macrosomia seen on ultrasound, particularly if increased abdominal girth is seen in the foetus may be used as a moderating factor in the need for initiation of insulin and potentially in the target glucose values aimed for (Buchanan, et al., 1998)(44). The HAPO study showed a clear correlation with macrosomia and the glucose values attained on the oral glucose tolerance test result at 28-32 weeks [fasting glucose adjusted OR, 1.38; 95% CI:1.32-1.44, one-hour adjusted OR, 1.46 ; 95% CI 1.39-1.53, two-hour adjusted OR, 1. 38 ; 95% CI: 1.32-1.44] (HAPO Study Cooperative Research Group, Metger BE; Lowe LR; Dyer AR et al, 2008). If there is no evidence of macrosomia on ultrasound and the initial A1c is below a pregnancy normal mean of 5.3%, it may be possible to accept slightly The use of insulin pens has greatly simplified the teaching of insulin injection; however it is also relatively easy to teach injections using insulin syringes in a short teaching session, possibly in small groups. It is important that any woman being initiated on insulin (or any oral agent which is a secretogogue for that matter) be taught how to recognize and treat a hypoglycemic episode. The use of written and picture educational material often available from insulin producing companies will facilitate the educational essentials that must be explained. The concept of site use and rotation is another issue which should be addressed There have been studies using both metformin and glyburide as oral agents for the management of gestational diabetes showing reasonably comparative composite outcomes. There were 46% of the women in the metformin study who still required insulin and there was inadequate power in the glyburide randomized trial to determine macrosomia and neonatal hypoglycemia outcomes (Rowan, Hague, Gao, Battin, Moore, & and MiG Trial Investigators, 2008; Langer, Conway, Berkis, Xenakis, & Gonzales, 2000). At present in most countries, it remains "off-label" and most guidelines do not suggest its routine use or only if for some reason insulin cannot be used (American Diabetes Association, 2011; Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; Jacqueminet S, In terms of the actual insulin prescription, the concept of using a famous train robber's recommendation - Sutton's law "Go where the money is!" can be very helpful. In other words, pick the part of the day which is most abnormal from the point of view of glucose control. In general, improvement to absolutely normal of the fasting glucose value will facilitate the release of insulin by the woman and improve glucose much of the day (Pennartz, Schenker, Menge, Schmidt, Nauck, & Meier, 2011). In the majority of cases, bedtime insulin is the most effective first step although the women from South East Asia and Asia may have post-meal If the fasting glucose is the value which is above range, initiation of intermediate insulin at bedtime (neutral protamine Hagedorn or NPH in human or pork form) at a low dose should be done (can be as low as 2 units in a very nervous woman who is not too obese, but the usual starting dose would be 8 – 10 units minimum or 0.1 units/kg). In many clinics, the women return on a regular basis for adjustment of their insulin doses. As insulin needs increase progressively in response to placental growth and production of anti-insulin hormones, this often means that the initial period after adjustment is well controlled but over the two weeks the glucose control may deteriorate. It is very easy to have the women do the progressive adjustment of her own insulin based on her morning blood glucose (and thus her body's response to the insulin given); she can then increase the dose by specified increments until she achieves the desired range. The increments are largest for high glucose 2010). For these reasons, it will not be addressed in detail in this chapter. glucose which tends to be higher and may have normal fasting glucose values. higher glucose values and still obtain a good foetal outcome. by the teaching nurse. values (as much as 6 units at once) and gradually reduce to as little as 1 unit more insulin for the evening dose as the target range is reached (See Figure 3a). In someone who is markedly obese or may have evidence of acanthosis nigricans, often an initial dose may be 0.1-0.2 units/kg administered at bedtime again with an increase in dose the following day if the fasting glucose has not achieved the desired range. An important part of the algorithm is a glucose value below which she MUST REDUCE her evening dose of NPH. This avoids hypoglycaemia before it happens and discourages overzealous increases in insulin dosages. Our experience has been very effective and safe using the value of 4.2mmol/L below which a woman will reduce that night's dose as this glucose value is far away from the values of 3.2 or 2.8mmol/L which would be felt as a hypoglycemic reaction and require treatment (Snyder, Gray-Donald, & Koski, 1994; Meltzer, Snyder, Penrod, Nudi, & Morin, 2010). In situations where women have markedly elevated fasting and post-meal glucose levels, an overall dose of 0.5 – 0.7u/kg can be used in the proportion of about 40% as bedtime NPH insulin and the remainder split over the day with a bit more at breakfast and less at lunch ie. 25% - 15% - 20% of the total calculated dose given as regular or rapid-acting insulin prior to breakfast, lunch and dinner respectively. On very rare occasions likely explaining the lack of literature related to it, women develop a local allergy to NPH at the injection site with swelling and redness developing up to 12 hours after the last injection of NPH at that site and lasting for about one to two days. In virtually every situation where this has been seen and is deemed intolerable by the woman, switching to regular human insulin as a 10pm injection and retesting glucose at 0400 to adjust the 10 pm dose as well as adding a 0400h injection of human (preferably) or pork regular insulin which will be adjusted based on the pre-breakfast result will often correct the allergy problem and continue to effectively control the glucose. Usually the dose can be reduced from the previous insulin dose and given as one third at 2200h and about one third or a bit less at 0400h. Subsequent self-adjustment protocols can still be used with an added time of 0400h and will allow for safe correction of insulin to optimal doses. Unfortunately, this approach does interfere with the woman's sleep. The use of detemir or glargine insulin may be alternatives; however it has yet to be reported in this situation. If the fasting glucose is normal and the post-meal is the glucose which is elevated (often related to ethnic differences), the insulin administered will be regular insulin or a rapidacting analogue administered prior to the meal (See Figure 3b). If Regular insulin is used, the time it should be taken before the meal is often 20-30 minutes, which is why the rapidacting analogues are often preferred, as they can be take much closer to the meal (0-15 minutes) and still effectively control the post-prandial 60-90 minute peak (Pettitt, Ospina, Kolaczynski, & Jovanovic, 2003). The effectiveness of the dose is evaluated by the woman using her post-meal glucose value for that meal and the following day, the dosage for that meal will be adjusted up or down in order to achieve the desired values. Since at meals, occasionally, even the "best" patient may change her food from the recommended meal plan, it is often appropriate to wait for 2 abnormal values before increasing the dose, however only one value lower than the desired goal requires that the dose be reduced the following day. Allowing the woman to be responsible for the gradual and persistent adjustments of insulin dose regularly seen in GDM as the placenta continues to grow and her insulin needs rise can facilitate her care. Occasional additional tests in relation to snacks or prior to meals may be necessary to determine glucose control is always good (Montaner, Ripolles, Pamies, & Corcoy, 2011). Visits may be further apart because the woman is making the appropriate adjustments, thus the medical team does not need to. Use of colour-coding Insulin Use in Gestational Diabetes – Pragmatic dose in units per hour. copy to present on arrival in the delivery room. or if the chart was missing vital maternal or neonatal data. insulin need in patients with type 2 or gestational diabetes. Protocols for Self-Management and for Labour and Delivery 301 patients. In the diabetes and pregnancy clinics today, many of the patients have type 2 diabetes with significantly more insulin resistance and a high total daily dose (TDD). Additionally, many women with gestational diabetes picked up in pregnancy may have glucose abnormalities outside of pregnancy and significant underlying insulin resistance. The majority of protocols presume that the hourly needs for insulin are always the same in labour; however, it may be more appropriate to use a gradually adjusting protocol based on For these reasons, we have developed a protocol based on the patient's total daily dose to provide the initial insulin infusion rate. As a compromise for fluid use, not only for glucose provision, but potentially for oxytocin induction or other fluids needed for obstetric reasons, the protocol developed provides 5g/h of glucose in the form of dextrose 10% at 50 ml/hour beginning on the morning of induction or Caesarean section, or when the patient arrives in labour. Since labour is a significant activity, the obstetricians are encouraged to provide a consistent amount of at least 5g of glucose to be delivered per hour to avoid fatigue and ketosis. If the glucose value exceeds 4.5mmol/L (81mg/dl), an insulin infusion is begun. For women with gestational diabetes, an insulin infusion protocol is not provided if the total daily dose is less than 30 units per day. If above 30 units, the total number of units taken in the day is divided in half (since about half of the insulin she takes will be to cover meals) and the remaining insulin dose is divided by 24 to permit the determination of a starting The insulin dose is adjusted hourly keeping the amount glucose infused stable and realizing that insulin requirements usually fall in labour. If there is a fall of glucose to under 4.0mmol/L (72mg/dl), the dose is reduced quickly, below 3.5mmol/L (63mg/dl)…even more and at 3.0mmol/L, the insulin is stopped – glucose in the form of 50% dextrose is given to provide 10 gm of immediate glucose (i.e. 20 ml.) and repeated every 10 minutes until the glucose rises above 4.5mmol/L (81mg/dl). If the glucose rises, the dose is increased incrementally until a steady state is reached. In our hospital protocol, we aim for a glucose between 4.0 – 5.5mmol/L (72 – 100 mg/dl) during labour using this protocol, with success and minimal hypoglycaemia, however this is due to intensive in-house review of the protocol on a regular basis and consistency over 20 years. The entire protocol is pre-printed in the clinic prior to labour using dosages at about 36-37 weeks and the women is given a As part of a quality assessment program, we retrospectively evaluated the effectiveness of this protocol for labour and delivery in use 20 years in our institution for glucose control in women with type 1, type 2 and gestational diabetes. Ethical approval of the assessment and chart review was obtained from the McGill University Health Center Ethics Board. Women who delivered for the years 2004-2006 and were treated with an insulin dose ≥ 30units/day prior to labour and managed with an intra-partum protocol were included. Patients were excluded if they did not receive the insulin protocol due to precipitated labor of urgent CS The protocol includes a glucose infusion of 5g/h as 10% dextrose in water and an insulin infusion using ½ of the TDD/24 as the initial hourly rate was begun if CBGM was ≥4.5mmol/L and adjusted to maintain glucose between 4.5-5.5mmol/L. At placental delivery, insulin is held and glucose increased to 10g/h until glucose goes above 5.5mmol/L. A total of 80 women were evaluated in 86 pregnancies. Of those, 31(39%) had type 1 DM with mean BMI 21.7, 9 with microvascular complications, mean duration of DM 14.6 years, 43% had Caesarian sections. The mean A1C by trimester was: T1 6.3%, T2 5.5%, has been useful in women whose understanding of the language may be limited so that even women not easily able to read and write have been able to make use of this protocol (Figure 3c). Occasionally, there are women who feel much too insecure to adjust, or simply cannot seem to understand the algorithm. In cases like these, the medical team (diabetes nurse educator and physician) will, of necessity, need to make the adjustments and thus, likely require more frequent visits. This is the exception rather than the rule. Thus, the patient can continue to adjust her insulin appropriately, gradually increasing the insulin doses until between 34-37 weeks, where some decrease in insulin requirements is often seen as the baby is now bigger and eats more, so siphons off glucose most obviously overnight when the patient is not eating but the baby still is. In addition, the placenta is no longer growing and may be aging, so the placental hormones which have been increasing insulin resistance are gradually decreasing. In fact, an early or dramatic fall in insulin requirements may be an early manifestation of a placental problem and may help alert the health professionals. Fig. 3. #### 6. Approaching the finish line – what to do for labour and delivery There are few papers determining the effectiveness of specific protocols for use in labour where the protocols specifics are detailed (Palmer & Inturissi, 1992; Ramanathan, Khoo, & Arismendy, 1991; Leparcq, et al., 2008; Jovanovic & Petersen, 1983; Hawkins & Casey, 2007). In 1983, using a Biostator®, Jovanovic and Peterson determined that the average hourly need for glucose to cover the needs of labour was 2.55 mg/kg/min (Jovanovic & Petersen, 1983). When translated into the approximate hourly rate for a 70kg woman, this would mean 9.45g/hour as an infusion rate. This would require very large amounts of fluids or perhaps a central line to provide such glucose-intense amounts in a normal clinical setting. The majority of the recommended and detailed protocols have been used in type 1 diabetic has been useful in women whose understanding of the language may be limited so that even women not easily able to read and write have been able to make use of this protocol (Figure 3c). Occasionally, there are women who feel much too insecure to adjust, or simply cannot seem to understand the algorithm. In cases like these, the medical team (diabetes nurse educator and physician) will, of necessity, need to make the adjustments and thus, Thus, the patient can continue to adjust her insulin appropriately, gradually increasing the insulin doses until between 34-37 weeks, where some decrease in insulin requirements is often seen as the baby is now bigger and eats more, so siphons off glucose most obviously overnight when the patient is not eating but the baby still is. In addition, the placenta is no longer growing and may be aging, so the placental hormones which have been increasing insulin resistance are gradually decreasing. In fact, an early or dramatic fall in insulin requirements may be an early manifestation of a placental problem and may help alert the likely require more frequent visits. This is the exception rather than the rule. 6. Approaching the finish line – what to do for labour and delivery There are few papers determining the effectiveness of specific protocols for use in labour where the protocols specifics are detailed (Palmer & Inturissi, 1992; Ramanathan, Khoo, & Arismendy, 1991; Leparcq, et al., 2008; Jovanovic & Petersen, 1983; Hawkins & Casey, 2007). In 1983, using a Biostator®, Jovanovic and Peterson determined that the average hourly need for glucose to cover the needs of labour was 2.55 mg/kg/min (Jovanovic & Petersen, 1983). When translated into the approximate hourly rate for a 70kg woman, this would mean 9.45g/hour as an infusion rate. This would require very large amounts of fluids or perhaps a central line to provide such glucose-intense amounts in a normal clinical setting. The majority of the recommended and detailed protocols have been used in type 1 diabetic health professionals. Fig. 3. patients. In the diabetes and pregnancy clinics today, many of the patients have type 2 diabetes with significantly more insulin resistance and a high total daily dose (TDD). Additionally, many women with gestational diabetes picked up in pregnancy may have glucose abnormalities outside of pregnancy and significant underlying insulin resistance. The majority of protocols presume that the hourly needs for insulin are always the same in labour; however, it may be more appropriate to use a gradually adjusting protocol based on insulin need in patients with type 2 or gestational diabetes. For these reasons, we have developed a protocol based on the patient's total daily dose to provide the initial insulin infusion rate. As a compromise for fluid use, not only for glucose provision, but potentially for oxytocin induction or other fluids needed for obstetric reasons, the protocol developed provides 5g/h of glucose in the form of dextrose 10% at 50 ml/hour beginning on the morning of induction or Caesarean section, or when the patient arrives in labour. Since labour is a significant activity, the obstetricians are encouraged to provide a consistent amount of at least 5g of glucose to be delivered per hour to avoid fatigue and ketosis. If the glucose value exceeds 4.5mmol/L (81mg/dl), an insulin infusion is begun. For women with gestational diabetes, an insulin infusion protocol is not provided if the total daily dose is less than 30 units per day. If above 30 units, the total number of units taken in the day is divided in half (since about half of the insulin she takes will be to cover meals) and the remaining insulin dose is divided by 24 to permit the determination of a starting dose in units per hour. The insulin dose is adjusted hourly keeping the amount glucose infused stable and realizing that insulin requirements usually fall in labour. If there is a fall of glucose to under 4.0mmol/L (72mg/dl), the dose is reduced quickly, below 3.5mmol/L (63mg/dl)…even more and at 3.0mmol/L, the insulin is stopped – glucose in the form of 50% dextrose is given to provide 10 gm of immediate glucose (i.e. 20 ml.) and repeated every 10 minutes until the glucose rises above 4.5mmol/L (81mg/dl). If the glucose rises, the dose is increased incrementally until a steady state is reached. In our hospital protocol, we aim for a glucose between 4.0 – 5.5mmol/L (72 – 100 mg/dl) during labour using this protocol, with success and minimal hypoglycaemia, however this is due to intensive in-house review of the protocol on a regular basis and consistency over 20 years. The entire protocol is pre-printed in the clinic prior to labour using dosages at about 36-37 weeks and the women is given a copy to present on arrival in the delivery room. As part of a quality assessment program, we retrospectively evaluated the effectiveness of this protocol for labour and delivery in use 20 years in our institution for glucose control in women with type 1, type 2 and gestational diabetes. Ethical approval of the assessment and chart review was obtained from the McGill University Health Center Ethics Board. Women who delivered for the years 2004-2006 and were treated with an insulin dose ≥ 30units/day prior to labour and managed with an intra-partum protocol were included. Patients were excluded if they did not receive the insulin protocol due to precipitated labor of urgent CS or if the chart was missing vital maternal or neonatal data. The protocol includes a glucose infusion of 5g/h as 10% dextrose in water and an insulin infusion using ½ of the TDD/24 as the initial hourly rate was begun if CBGM was ≥4.5mmol/L and adjusted to maintain glucose between 4.5-5.5mmol/L. At placental delivery, insulin is held and glucose increased to 10g/h until glucose goes above 5.5mmol/L. A total of 80 women were evaluated in 86 pregnancies. Of those, 31(39%) had type 1 DM with mean BMI 21.7, 9 with microvascular complications, mean duration of DM 14.6 years, 43% had Caesarian sections. The mean A1C by trimester was: T1 6.3%, T2 5.5%, Insulin Use in Gestational Diabetes – Pragmatic the neonate in this sample size. (Figure 5) therapy with oral agents or insulin on leaving the hospital. Deierlein, Siega-Riz, Chantala, & Herring, 2011). Protocols for Self-Management and for Labour and Delivery 303 Mean insulin dose in labour was 1.73units/h for DM1, 2.2unit/h in DM2. Maternal hypoglycemia (CBG <3.3 mmol/L) occurred in 16% of labour occurring equally in DM1 or DM2, however there were only 4 episodes of maternal hypoglycemia <2.5mmol/L. Mean glucose achieved overall was 5.8mmol/L (6.1mmol/L for DM1; 5.6mmol/L for DM2). Of the 863 CBG readings, there were 31% between 4.5-5.5mmol/L, 24% lower, and 43% higher with 9% ≥7mmol/l (See Figure 5). Neonatal hypoglycaemic events (BS ≤2.2mmol/L occurred in 32 neonates (37% - 46% in DM1 offspring, 40% in DM2 offspring (p=0.047) and 4(12%) in babies whose mother did not receive maternal IV insulin. No significant relationship was seen between glucose control in labour, nor in any trimester in labour and neonatal hypoglycemia. The results of the glucose control can be seen in Figure 6. The findings suggest that this relatively simple protocol which can be prepared by house-staff based on total daily dose was able to safely control both DM1 and DM2 /gestational diabetic women with minimal hypo or hyper-glycemic risk for mother or offspring. The degree of glucose control in labour did not appear to relate to the risk of hypoglycemia in Once the baby has been delivered, the needs for insulin fall faster than the insulin is metabolized, so the women is at risk of hypoglycaemia in the first 1-2 hours after delivery. Thus, the protocol emphasizes stopping the insulin at delivery and increasing the glucose infusion to 10g/hour immediately after the delivery of the baby and for the next 2 hours. If the glucose remains above 5.5mmol/L, the infusion rate can be reduced to 5g an hour again or discontinued and the woman would be allowed to eat if she underwent a vaginal delivery. As most women with gestational diabetes have normal glucose post-partum, this can be checked with capillary blood glucose monitoring (CBGM) the following day pre and post breakfast. If there is evidence of abnormal glucose intolerance (particularly in someone who may have had type 2 diabetes only diagnosed in pregnancy) in the initial post-partum period, depending on its severity, the woman may be instructed to continue with medical nutrition therapy and testing and re-assess in 1 month, or she may require some form of If her glucose appears normal prior to discharge, she should undergo an OGTT at 6 weeks to 6 months post-partum (or before she next conceives) to verify her glucose tolerance status – the actual timing varies related to guidelines established in various countries and related to the ethnic risks present in that country (Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; Reinblatt, Morin, & Meltzer, 2006; McClean, Farrar, Kelly, Tuffnell, & Whitelaw, 2010). Initial post-partum testing within the first year is most effective if an oral glucose tolerance test is done; however, of those with any abnormality, further regular follow-up is likely adequate with a fasting plasma glucose and potentially an A1C (Lee, Mak, Lao, & Chung, 2011; Kim, Herman, & Vijan, 2007). Many women with GDM will have evidence of some form of dysglycemia or impaired glucose tolerance which would be amenable to preventive therapy. Additionally, even for women who tested normal, they must be reminded that their long term risks of developing diabetes remain elevated as does their cardiovascular risks (Egeland & Meltzer, 2010; Bellamy, Casas, Hingorani, & Williams, 2009; Ratnakaran, Qi, Connelly, Sermer, Hanley, & Zinman, 2010). It should not be forgotten that the presence of gestational diabetes in the mother appears to confer future risk for the offspring in terms of obesity and glucose intolerance (Nolan, Damm, & Prentki, 2011; T3 5.3%. The mean FPG by trimester of T1 7.0, 6.0, 5.6 mmol/L respectively; mean 1hPC T1 7.3 T2 6.2 T3 6.0 mmol/L and insulin was administered in 90% of labours. The 49(61%) women with type 2 DM had a mean BMI of 33kg/m2, a mean duration of DM of 3.3years, 2 with microvascular complications, and 76% had Caesarian sections. The mean A1C by trimester was T1 8.5%, T2 7% and T3 6.7%. The mean FPG by trimester was: T1 7.7, T2 6.3, T3 5.5mmol/L, and the mean 1hPC T1 9.6, T2 7.0, T3 7.2 mmol/L and insulin infusion was used in 72% of cases. Fig. 4. T3 5.3%. The mean FPG by trimester of T1 7.0, 6.0, 5.6 mmol/L respectively; mean 1hPC T1 7.3 T2 6.2 T3 6.0 mmol/L and insulin was administered in 90% of labours. The 49(61%) women with type 2 DM had a mean BMI of 33kg/m2, a mean duration of DM of 3.3years, 2 with microvascular complications, and 76% had Caesarian sections. The mean A1C by trimester was T1 8.5%, T2 7% and T3 6.7%. The mean FPG by trimester was: T1 7.7, T2 6.3, T3 5.5mmol/L, and the mean 1hPC T1 9.6, T2 7.0, T3 7.2 mmol/L and insulin infusion was used in 72% of cases. Fig. 4. Mean insulin dose in labour was 1.73units/h for DM1, 2.2unit/h in DM2. Maternal hypoglycemia (CBG <3.3 mmol/L) occurred in 16% of labour occurring equally in DM1 or DM2, however there were only 4 episodes of maternal hypoglycemia <2.5mmol/L. Mean glucose achieved overall was 5.8mmol/L (6.1mmol/L for DM1; 5.6mmol/L for DM2). Of the 863 CBG readings, there were 31% between 4.5-5.5mmol/L, 24% lower, and 43% higher with 9% ≥7mmol/l (See Figure 5). Neonatal hypoglycaemic events (BS ≤2.2mmol/L occurred in 32 neonates (37% - 46% in DM1 offspring, 40% in DM2 offspring (p=0.047) and 4(12%) in babies whose mother did not receive maternal IV insulin. No significant relationship was seen between glucose control in labour, nor in any trimester in labour and neonatal hypoglycemia. The results of the glucose control can be seen in Figure 6. The findings suggest that this relatively simple protocol which can be prepared by house-staff based on total daily dose was able to safely control both DM1 and DM2 /gestational diabetic women with minimal hypo or hyper-glycemic risk for mother or offspring. The degree of glucose control in labour did not appear to relate to the risk of hypoglycemia in the neonate in this sample size. (Figure 5) Once the baby has been delivered, the needs for insulin fall faster than the insulin is metabolized, so the women is at risk of hypoglycaemia in the first 1-2 hours after delivery. Thus, the protocol emphasizes stopping the insulin at delivery and increasing the glucose infusion to 10g/hour immediately after the delivery of the baby and for the next 2 hours. If the glucose remains above 5.5mmol/L, the infusion rate can be reduced to 5g an hour again or discontinued and the woman would be allowed to eat if she underwent a vaginal delivery. As most women with gestational diabetes have normal glucose post-partum, this can be checked with capillary blood glucose monitoring (CBGM) the following day pre and post breakfast. If there is evidence of abnormal glucose intolerance (particularly in someone who may have had type 2 diabetes only diagnosed in pregnancy) in the initial post-partum period, depending on its severity, the woman may be instructed to continue with medical nutrition therapy and testing and re-assess in 1 month, or she may require some form of therapy with oral agents or insulin on leaving the hospital. If her glucose appears normal prior to discharge, she should undergo an OGTT at 6 weeks to 6 months post-partum (or before she next conceives) to verify her glucose tolerance status – the actual timing varies related to guidelines established in various countries and related to the ethnic risks present in that country (Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2008; Reinblatt, Morin, & Meltzer, 2006; McClean, Farrar, Kelly, Tuffnell, & Whitelaw, 2010). Initial post-partum testing within the first year is most effective if an oral glucose tolerance test is done; however, of those with any abnormality, further regular follow-up is likely adequate with a fasting plasma glucose and potentially an A1C (Lee, Mak, Lao, & Chung, 2011; Kim, Herman, & Vijan, 2007). Many women with GDM will have evidence of some form of dysglycemia or impaired glucose tolerance which would be amenable to preventive therapy. Additionally, even for women who tested normal, they must be reminded that their long term risks of developing diabetes remain elevated as does their cardiovascular risks (Egeland & Meltzer, 2010; Bellamy, Casas, Hingorani, & Williams, 2009; Ratnakaran, Qi, Connelly, Sermer, Hanley, & Zinman, 2010). It should not be forgotten that the presence of gestational diabetes in the mother appears to confer future risk for the offspring in terms of obesity and glucose intolerance (Nolan, Damm, & Prentki, 2011; Deierlein, Siega-Riz, Chantala, & Herring, 2011). Insulin Use in Gestational Diabetes – Pragmatic Protocols for Self-Management and for Labour and Delivery 305 Fig. 6. Mean capillary blood glucose in labour in women with type 1 or type 2 DM management and for insulin in labour protocol. who have taught us much of this information. 7. Conclusion 8. Acknowledgements choices. In the appendix there are copies of the insulin adjustment algorithm, document sheets for management in English and French and in-hospital insulin algorithms for day by day Management of gestational diabetes can be very rewarding. This chapter has tried to raise the important issue that implication of the woman enthusiastically in her own self-care can facilitate care and become a tool to sensitize her to her future role in the family lifestyle The authors would like to acknowledge the health care team members that have been integral in development and implementation of the protocols in our clinical care - in particular: A. Benjamin, MD, FRCPS; Lucie Morin, MD, FRCPS, Louise Bastien, RN, and Jennifer Snyder, PtD. MSc. As well, we would like to signal our appreciations to our patients Fig. 5. Glucose values achieved in labour with insulin adjustment protocol and incidence of neonatal hypoglycemia. Fig. 5. Glucose values achieved in labour with insulin adjustment protocol and incidence of neonatal hypoglycemia. Fig. 6. Mean capillary blood glucose in labour in women with type 1 or type 2 DM In the appendix there are copies of the insulin adjustment algorithm, document sheets for management in English and French and in-hospital insulin algorithms for day by day management and for insulin in labour protocol. #### 7. Conclusion Management of gestational diabetes can be very rewarding. This chapter has tried to raise the important issue that implication of the woman enthusiastically in her own self-care can facilitate care and become a tool to sensitize her to her future role in the family lifestyle choices. #### 8. Acknowledgements The authors would like to acknowledge the health care team members that have been integral in development and implementation of the protocols in our clinical care - in particular: A. Benjamin, MD, FRCPS; Lucie Morin, MD, FRCPS, Louise Bastien, RN, and Jennifer Snyder, PtD. MSc. As well, we would like to signal our appreciations to our patients who have taught us much of this information. Insulin Use in Gestational Diabetes – Pragmatic 10. Appendix 2 French Protocols for Self-Management and for Labour and Delivery 307 Documentation sheet for capillary blood glucose and insulin adjustment in English and ### 9. Appendix 1 Insulin adjustment protocol in English ### 10. Appendix 2 306 Gestational Diabetes 9. Appendix 1 Insulin adjustment protocol in English Documentation sheet for capillary blood glucose and insulin adjustment in English and French Insulin Use in Gestational Diabetes – Pragmatic 12. Appendix 4 Protocols for Self-Management and for Labour and Delivery 309 #### 11. Appendix 3 Insulin adjustment protocol for use in hospital ### 12. Appendix 4 308 Gestational Diabetes 11. 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The Lancet, 378, 169-181. Ornoy, A. (2011). Prenatal origin of obesity and their complications: Gestational diabetes, maternal overweight and the paradoxical effects of fetal growth restriction and Palmer, D. G., & Inturissi, M. (1992). Intravenous insulin infusion therapy in the intrapartum 1. Introduction and correctly managed. 2. Non-pharmacological treatment 18 Serbia Miroslav Radenković Treatment Considerations for Gestational Diabetes Mellitus and Long-Term Postpartum Options Department of Pharmacology, Clinical Pharmacology and Toxicology School of Medicine – University of Belgrade Gestational diabetes mellitus is commonly defined as hyperglycemia with onset or first recognition during pregnancy. However, this definition of gestational diabetes does not exclude pregnant women with undiagnosed pre-existing diabetes that now accounts around 1% of diabetes mellitus cases in pregnancy. Prompt identification of pre-existing diabetes, if compared with women with gestational diabetes mellitus, is essential, for the reason that women with pre-existing diabetes are at risk of giving birth to infants with serious malformations, and adverse pregnancy outcomes are increased in this cluster, too. These include serious injury at birth, increased probability of cesarean delivery, and increased The incidence of gestational diabetes is unfortunately increasing, it accounts for 90% of cases of diabetes mellitus in pregnancy, and it has strong association with adverse pregnancy outcomes. Risk factors connected to gestational diabetes mellitus include older age, family history and previous history of gestational diabetes mellitus, obesity, polycystic ovary syndrome and high blood pressure (American Diabetes Association, 2009; Hedderson and Ferrara, 2008). If untreated, it may lead to diverse complications, such as fetal hyperinsulinemia, increased weight at birth, higher rates of cesarian deliveries, shoulder dystocia, more neonatal hypoglycemia, and is associated with concomitant preeclampsia in pregnant women. Therefore, given that gestational diabetes may have long-term pathological consequences for both mother and the child, it is important that it is recognized Treatment of gestational diabetes is aimed to maintain euglycemia and it involves regular glucose monitoring, dietary modification, life style changes, exercise, and, when necessary, pharmacotherapy. Insulin therapy is the first choice of treatment, although glyburide and metformin may be indicated, too. In women receiving pharmacotherapy scheduled Self-monitoring of blood glucose is considered to be essential during pregnancy. This is supported, for example by the fact, that self-monitoring of blood glucose in women with monitoring of fetal well-being with antenatal tests should be pursued. incidence of newborn admission in intensive care unit. ## Treatment Considerations for Gestational Diabetes Mellitus and Long-Term Postpartum Options ### Miroslav Radenković Department of Pharmacology, Clinical Pharmacology and Toxicology School of Medicine – University of Belgrade Serbia ### 1. Introduction 314 Gestational Diabetes Sanz, C., Gautier, J. F., & Hanaire, H. (Epub 2010 Aug 2. Nov 2010). Physical exercise for the prevention and treatment of type 2 diabetes. Diabetes Metab., 36(5), 346-351. Shareef, A., Zhu, Y., & Musavi, M. (2008). Comparison of Neural Networks for Localization Snyder, J., Gray-Donald, K., & Koski, K. (1994). Predictors of birthweight in gestational Standl, E., Schnell, O., & Ceriello, A. (2011). Postprandial Hyperglycemia and Glycemic Variability - Should we care? Diabetes CAre, Supplement 2, S120-S127. Thomas D, E. E. (2009). Low glycaemic index, or low glycaemic load, diets for diabetes Tuomilehto, J., Lindstrom, J., Eriksson, J. G., Valle, T. T., Hamaiainen, H., Ilanne-Parikka, P., subjects with impaired glucose tolerance. N Engl J Med, 344, 1343-1350. Tzanetakou, I. P., Mikhailidis, D. P., & Perrea, D. N. (2011). Nutrition During Pregnancy and "Perfect Maternal Diet". Open Cardiovascular Medicine Journal, 5, 103-109. Verier-Mine, O. (Dec 2010). Outcomes in women with a history of gestational diabetes. Yogev, Y., Ben-Haroush, A., Chen, R., Rosenn, B., Hod, M., & Langer, O. (Sep 2004). Diurnal et al. (2001). Prevention of type 2 diabetes mellitus by changes in lifestyle among the Effect of Carbohydrates on the Offspring's Metabolic Profile: In search of the Screening and prevention of type 2 diabetes. Literature review. Diabetes Metab., glycemic profile in obese and normal weight nondiabetic pregnant women. Am J (Mobileware'08). Innsbruck: ICST. 36((6 Pt2) Review.), 595-616. Obstet Gynecol., 191(3), 949-953. diabetes. Am J Clin Nutrition, 1409-1414. mellitus. Cochrane Database Syst Rev., 21(1), CD006296. in Wireless Sensor Networks. Proceedings of ACM First International Conference on Mobile Wireless Middleware, Operating Systems, and Applications > Gestational diabetes mellitus is commonly defined as hyperglycemia with onset or first recognition during pregnancy. However, this definition of gestational diabetes does not exclude pregnant women with undiagnosed pre-existing diabetes that now accounts around 1% of diabetes mellitus cases in pregnancy. Prompt identification of pre-existing diabetes, if compared with women with gestational diabetes mellitus, is essential, for the reason that women with pre-existing diabetes are at risk of giving birth to infants with serious malformations, and adverse pregnancy outcomes are increased in this cluster, too. These include serious injury at birth, increased probability of cesarean delivery, and increased incidence of newborn admission in intensive care unit. > The incidence of gestational diabetes is unfortunately increasing, it accounts for 90% of cases of diabetes mellitus in pregnancy, and it has strong association with adverse pregnancy outcomes. Risk factors connected to gestational diabetes mellitus include older age, family history and previous history of gestational diabetes mellitus, obesity, polycystic ovary syndrome and high blood pressure (American Diabetes Association, 2009; Hedderson and Ferrara, 2008). If untreated, it may lead to diverse complications, such as fetal hyperinsulinemia, increased weight at birth, higher rates of cesarian deliveries, shoulder dystocia, more neonatal hypoglycemia, and is associated with concomitant preeclampsia in pregnant women. Therefore, given that gestational diabetes may have long-term pathological consequences for both mother and the child, it is important that it is recognized and correctly managed. > Treatment of gestational diabetes is aimed to maintain euglycemia and it involves regular glucose monitoring, dietary modification, life style changes, exercise, and, when necessary, pharmacotherapy. Insulin therapy is the first choice of treatment, although glyburide and metformin may be indicated, too. In women receiving pharmacotherapy scheduled monitoring of fetal well-being with antenatal tests should be pursued. #### 2. Non-pharmacological treatment Self-monitoring of blood glucose is considered to be essential during pregnancy. This is supported, for example by the fact, that self-monitoring of blood glucose in women with Treatment Considerations for Gestational Diabetes Mellitus and Long-Term Postpartum Options 317 nutrition plan. During pregnancy, the main objective of insulin therapy will be to attain glucose levels similar to those before pregnancy. If indicated, at the beginning small doses of insulin are to be administered, and then insulin doses should be gradually increased. This should be accompanied by the appropriate administration intervals until target glucose levels are attained. Taking into account that insulin resistance rises during the whole pregnancy period, the insulin regimens must be continuously monitored, reviewed and modified. This is particularly significant during the third trimester of pregnancy when the required dosage of insulin usually increases. Hypoglycemia prevention measurements should be clearly explained to all pregnant women on insulin therapy. Sill, insulin therapy is considered to be effective and safe, and it is regarded as the gold standard of pharmacotherapy for gestational diabetes. It has been well determined that the use of insulin to achieve glycemic targets reduces fetal and maternal morbidities. In this way, daily glucose control and diet that were associated with insulin treatment and additional obstetric interventions have been confirmed to reduce the incidence of shoulder dystocia and A diversity of protocols can be used, but multiple injections are considered to be the most effective. The majority insulin protocols include intermediate-acting insulins, such as isophane, and short-acting insulins, such as regular recombinant, as well the insulin analogues aspart and lispro. Although isophane is the intermediate-acting insulin of the first choice for women with gestational diabetes, evidences also support the use of short-acting insulin analogues in women who require pharmacological treatment of gestational diabetes. Moreover, the use of insulin analogs in pregnancy presents the potential benefits of more closely mimicking biological pancreatic insulin secretion compared to regular insulin Insulin lispro is insulin analog with fast absorption rate and a short duration of action that improves postprandial glucose levels and reduces hypoglycemic episodes when injected immediately prior to meals (Anderson et al., 1997). In the study conducted by Jovanovic et al., (1999) it was of interest to compare the immunologic response to insulin lispro with that to regular human insulin, thereby assuring its safety for use in women with gestational diabetes, and to verify that it is effective. Anti-insulin antibody levels were similar in the two groups. Insulin lispro was not detectable in the cord blood. During a meal test, areas under the curve for glucose, insulin, and C-peptide were significantly lower in the lispro group. Mean fasting and postprandial glucose concentrations and end point HbA1c were similar in the two groups but the lispro group demonstrated fewer hypoglycemic episodes. Accordingly, in women with gestational diabetes mellitus, the use of insulin lispro enabled the attainment of near-normal glucose levels at the one hour post-prandial time point and was associated with normal anthropometric characteristics; whereas use of regular insulin was not able to blunt the one hour peak post-prandial response to a near-normal extent and resulted in infants with a tendency toward the disproportionate growth (Mecacci et al., 2003). Bhattacharyya et al. (2001) reported no increase in adverse outcome using lispro insulin in diabetic pregnancies, in either gestational or pre-gestational diabetes. Likewise, there was no difference in respect to congenital anomalies of gestational diabetic groups, which used either insulin lispro or regular human insulin (Aydin et al., 2008). In consideration to insulin aspart use, it has been demonstrated that effective postprandial glycemic control in women with gestational diabetes mellitus who required insulin was brought about by insulin aspart through higher insulin peak and lower demand on macrosomia (Horvath et al., 2010). (Klieger et al., 2008). mild gestational diabetes positively correlates with reduced rate of fetal overgrowth and gestational weight gain (Hawkins, 2010). Moreover, taking into account that excessive gestational weight gain correlates with postpartum weight retention, regular monitoring of blood glucose during pregnancy might certainly have long-term benefits (Siega-Riz et al., 2009). Common fasting, preprandial and postprandial glucose tests are all recommended in order to attain adequate glycemic targets and reduce overall rate of large-for-gestational-age infant births (Aschwald et al., 2009; Jovanovic, 2008; Riskin-Mashiah et al., 2009). The frequency and timing of home glucose monitoring for gaining and maintaining target glucose levels should be individualized. Generally, the monitoring of blood glucose level is performed in the fasting state, and 1–2 hours after meals, too. Taking into account the increased risk of nocturnal hypoglycemia that may be present during the course of pregnancy; in pregnant women receiving insulin night testing or even continuous glucose monitoring might be suggested (McLachlan et al., 2007). It is recommended that women diagnosed with gestational diabetes should have expert dietitian counseling to ensure that medical nutrition therapy supports euglycemia, adequate nutritional intake and controlled weight gain. Dietary recommendations should be individualized for each patient. Moderate carbohydrate restriction and proper distribution of daily meals should be emphasized. Namely, six meals per day are recommendable, including three major meals and three smaller ones - snacks. As a result, it has been shown that restriction of carbohydrates to 35–40% will decrease maternal glucose levels and improve maternal and fetal outcomes (Major et al., 1998). For pregnant women with body mass index >30 kg/m2, a 30–33% calorie restriction is expected to reduce hyperglycemia and plasma triglycerides (Moore, 2010). Moreover, fetal-based strategy to govern maternal glucose control may notably improve outcomes for the fetus given that increased fetal abdominal circumference on an ultrasound (conducted between 28-34 weeks) has been found to be connected to increased insulin in amniotic fluid, thus directly revealing poor maternal glycemic control. Assessing the fetal response to maternal gestational diabetes mellitus by ultrasound measurement of fetal abdominal circumference starting in the second and early third trimesters and repeated every 2– 4 weeks can provide useful information (in combination with maternal self-monitoring of blood glucose levels) to guide management decisions (Metzger et al., 2007). Individually adjusted physical activity should be promoted, especially in overweight or obese women who are often insulin resistant and at risk for preeclampsia. Thus, the amount of physical activity consisting of 20 minutes aerobic training three days weekly for six weeks, has been shown to result in lower fasting glucose levels, lower glucose responses to a glucose challenge, and a lower glycated hemoglobin - HbA1c (Jovanovic-Peterson et al., 1989). Avery et al. (1997) have also observed improved glucose levels in women who exercised 30 minutes 3-4 times per week. The delay in requirement of insulin was reported in another study involving resistance training three times per week (Brankston et al., 2004). Exercises may not be advisable if obstetrical contraindications exist, or in cases in which physical activity actually worsens glycemic control. #### 3. Pharmacological treatment Insulin is the first-line pharmacological intervention for gestational diabetes and it should be initiated in women diagnosed with gestational diabetes or impaired glucose tolerance who did not achieve glycemic control within two weeks by the sole application of individualized mild gestational diabetes positively correlates with reduced rate of fetal overgrowth and gestational weight gain (Hawkins, 2010). Moreover, taking into account that excessive gestational weight gain correlates with postpartum weight retention, regular monitoring of blood glucose during pregnancy might certainly have long-term benefits (Siega-Riz et al., 2009). Common fasting, preprandial and postprandial glucose tests are all recommended in order to attain adequate glycemic targets and reduce overall rate of large-for-gestational-age infant births (Aschwald et al., 2009; Jovanovic, 2008; Riskin-Mashiah et al., 2009). The frequency and timing of home glucose monitoring for gaining and maintaining target glucose levels should be individualized. Generally, the monitoring of blood glucose level is performed in the fasting state, and 1–2 hours after meals, too. Taking into account the increased risk of nocturnal hypoglycemia that may be present during the course of pregnancy; in pregnant women receiving insulin night testing or even continuous glucose It is recommended that women diagnosed with gestational diabetes should have expert dietitian counseling to ensure that medical nutrition therapy supports euglycemia, adequate nutritional intake and controlled weight gain. Dietary recommendations should be individualized for each patient. Moderate carbohydrate restriction and proper distribution of daily meals should be emphasized. Namely, six meals per day are recommendable, including three major meals and three smaller ones - snacks. As a result, it has been shown that restriction of carbohydrates to 35–40% will decrease maternal glucose levels and improve maternal and fetal outcomes (Major et al., 1998). For pregnant women with body mass index >30 kg/m2, a 30–33% calorie restriction is expected to reduce hyperglycemia and plasma triglycerides (Moore, 2010). Moreover, fetal-based strategy to govern maternal glucose control may notably improve outcomes for the fetus given that increased fetal abdominal circumference on an ultrasound (conducted between 28-34 weeks) has been found to be connected to increased insulin in amniotic fluid, thus directly revealing poor maternal glycemic control. Assessing the fetal response to maternal gestational diabetes mellitus by ultrasound measurement of fetal abdominal circumference starting in the second and early third trimesters and repeated every 2– 4 weeks can provide useful information (in combination with maternal self-monitoring of blood glucose levels) to guide management Individually adjusted physical activity should be promoted, especially in overweight or obese women who are often insulin resistant and at risk for preeclampsia. Thus, the amount of physical activity consisting of 20 minutes aerobic training three days weekly for six weeks, has been shown to result in lower fasting glucose levels, lower glucose responses to a glucose challenge, and a lower glycated hemoglobin - HbA1c (Jovanovic-Peterson et al., 1989). Avery et al. (1997) have also observed improved glucose levels in women who exercised 30 minutes 3-4 times per week. The delay in requirement of insulin was reported in another study involving resistance training three times per week (Brankston et al., 2004). Exercises may not be advisable if obstetrical contraindications exist, or in cases in which Insulin is the first-line pharmacological intervention for gestational diabetes and it should be initiated in women diagnosed with gestational diabetes or impaired glucose tolerance who did not achieve glycemic control within two weeks by the sole application of individualized monitoring might be suggested (McLachlan et al., 2007). decisions (Metzger et al., 2007). 3. Pharmacological treatment physical activity actually worsens glycemic control. nutrition plan. During pregnancy, the main objective of insulin therapy will be to attain glucose levels similar to those before pregnancy. If indicated, at the beginning small doses of insulin are to be administered, and then insulin doses should be gradually increased. This should be accompanied by the appropriate administration intervals until target glucose levels are attained. Taking into account that insulin resistance rises during the whole pregnancy period, the insulin regimens must be continuously monitored, reviewed and modified. This is particularly significant during the third trimester of pregnancy when the required dosage of insulin usually increases. Hypoglycemia prevention measurements should be clearly explained to all pregnant women on insulin therapy. Sill, insulin therapy is considered to be effective and safe, and it is regarded as the gold standard of pharmacotherapy for gestational diabetes. It has been well determined that the use of insulin to achieve glycemic targets reduces fetal and maternal morbidities. In this way, daily glucose control and diet that were associated with insulin treatment and additional obstetric interventions have been confirmed to reduce the incidence of shoulder dystocia and macrosomia (Horvath et al., 2010). A diversity of protocols can be used, but multiple injections are considered to be the most effective. The majority insulin protocols include intermediate-acting insulins, such as isophane, and short-acting insulins, such as regular recombinant, as well the insulin analogues aspart and lispro. Although isophane is the intermediate-acting insulin of the first choice for women with gestational diabetes, evidences also support the use of short-acting insulin analogues in women who require pharmacological treatment of gestational diabetes. Moreover, the use of insulin analogs in pregnancy presents the potential benefits of more closely mimicking biological pancreatic insulin secretion compared to regular insulin (Klieger et al., 2008). Insulin lispro is insulin analog with fast absorption rate and a short duration of action that improves postprandial glucose levels and reduces hypoglycemic episodes when injected immediately prior to meals (Anderson et al., 1997). In the study conducted by Jovanovic et al., (1999) it was of interest to compare the immunologic response to insulin lispro with that to regular human insulin, thereby assuring its safety for use in women with gestational diabetes, and to verify that it is effective. Anti-insulin antibody levels were similar in the two groups. Insulin lispro was not detectable in the cord blood. During a meal test, areas under the curve for glucose, insulin, and C-peptide were significantly lower in the lispro group. Mean fasting and postprandial glucose concentrations and end point HbA1c were similar in the two groups but the lispro group demonstrated fewer hypoglycemic episodes. Accordingly, in women with gestational diabetes mellitus, the use of insulin lispro enabled the attainment of near-normal glucose levels at the one hour post-prandial time point and was associated with normal anthropometric characteristics; whereas use of regular insulin was not able to blunt the one hour peak post-prandial response to a near-normal extent and resulted in infants with a tendency toward the disproportionate growth (Mecacci et al., 2003). Bhattacharyya et al. (2001) reported no increase in adverse outcome using lispro insulin in diabetic pregnancies, in either gestational or pre-gestational diabetes. Likewise, there was no difference in respect to congenital anomalies of gestational diabetic groups, which used either insulin lispro or regular human insulin (Aydin et al., 2008). In consideration to insulin aspart use, it has been demonstrated that effective postprandial glycemic control in women with gestational diabetes mellitus who required insulin was brought about by insulin aspart through higher insulin peak and lower demand on Treatment Considerations for Gestational Diabetes Mellitus and Long-Term Postpartum Options 319 women on diet therapy. Other identified predictors of glyburide treatment failure were advanced maternal age, earlier diagnosis of gestational diabetes mellitus, higher gravidity or higher parity (Kahn et al., 2006). Otherwise, glyburide can be recommended for women in whom insulin cannot be used. In that way, it has been confirmed that this oral hypoglycemic can be used safely and effectively during the second and the third trimester of pregnancy without increasing maternal or fetal complications when compared with insulin (Langer et al., 2000). Glyburide has been shown to be safe in breastfeeding, too (Feig et al., 2005). However, it has to be emphasized that in some investigations a glyburide-related increased risk of preeclampsia, macrosomia, neonatal hypoglycemia, admission to a neonatal intensive care unit; as well a need for phototherapy have been reported (Jacobson The second oral antidiabetic drug used in gestational diabetes mellitus, as a monotherapy or with supplemental insulin, is metformin, a biguanide. It lowers blood glucose levels by decreasing hepatic gluconeogenesis, increasing peripheral glucose disposal and reducing intestinal glucose absorption (Hundal & Inzucchi, 2003). In average, up to half the women using metformin may require supplemental insulin. It has been demonstrated that women requiring supplemental insulin had a higher body mass index and had higher baseline glucose levels (Rowan et al. 2008). If compared to insulin, metformin was not associated with increased perinatal complications except of higher incidence of perinatal mortality if administered during the third trimester (Hellmuth et al., 2000). Although it may appear that metformin is safe alternative to insulin therapy, it does cross the placenta (Vanky et al., 2005) and scientific data are still not conclusive enough to recommend the standard use of metformin during pregnancy beyond the first trimester. Data that are more recent suggested that in women with gestational diabetes mellitus, not controlled with diet and exercise, who were then randomized to the metformin or the insulin arm, metformin has been shown to be an effective alternative to insulin in the treatment of gestational diabetes mellitus (Moore et al., 2007). This was substantiated by findings showing that difference in the rate of cesarean delivery was not statistically significant between the two groups, neither the neonatal statistics involving birth weight, Apgar score at 5 minutes, respiratory distress syndrome, hyperbilirubinemia, neonatal hypoglycemia or neonatal intensive care unit admission. Likewise, in investigation of Rowan et al. (2008) women with gestational diabetes mellitus at 20 to 33 weeks of gestation were randomly assigned to open treatment with metformin (with supplemental insulin if required) or insulin. The primary outcome was a composite of neonatal hypoglycemia, respiratory distress, need for phototherapy, birth trauma, 5-minute Apgar score less than 7, or prematurity. The rate of the primary composite outcome was comparable between the group assigned to metformin and the insulin group, suggesting that in women with gestational diabetes mellitus, metformin (alone or with supplemental insulin) was not associated with increased perinatal complications as compared with insulin. The retrospective data of Tertti et al. (2008) have been also indicative for the assumption that metformin was effective in controlling gestational diabetes and was not associated with a higher risk of maternal or neonatal complications compared with insulin. Namely, there were no differences between the metformin-treated group and the other two investigated groups (women treated with insulin and women with no pharmacological treatment) in terms of maternal outcomes (total weight gain during pregnancy or after the diagnosis of gestational diabetes mellitus, pre-pregnancy hypertension, pregnancy induced et al., 2005; Ramos et al., 2007). endogenous insulin secretion (Pettitt et al., 2003). In particular, the peak insulin concentration was higher and the peak glucose and C-peptide concentrations were lower with both insulin preparations than with no exogenous insulin. Moreover, glucose areas under the curve above baseline were significantly lower with insulin aspart, but not with regular insulin, than with no insulin. In another randomized, parallel, open-label, controlled, multicenter and multinational study of type 1 diabetes pregnancy the fetal outcome using insulin aspart was comparable with human insulin, with a tendency toward fewer fetal losses and preterm deliveries (Hod at al., 2008). In another study, insulin aspart was more effective than regular human isulin in decreasing postprandial glucose concentrations (Pettitt et al., 2007). The authors of this investigation found out that duration of insulin aspart injection 5 min before a meal rather than 30 min prior to meals offered a more convenient therapy for subjects with gestational diabetes mellitus. Moreover, overall safety and effectiveness of insulin aspart were comparable to regular human insulin in pregnant women with gestational diabetes mellitus. Glyburide and metformin are oral antidiabetics that may be considered as second line agents in cases of gestational diabetes with poor glycemic control with insulin, or in women who refuse insulin. This is supported by the fact that when compared with insulin, administration of oral hypoglycemic agents was not associated with risk of neonatal hypoglycemia, caesarean section, or large-for-gestational-age babies births (Dhulkotia et al., 2010). No significant differences were found in maternal fasting or postprandial glycemic control, too. It appears that glyburide may be preferred, as metformin use is more likely to need supplemental insulin for glycemic control and in addition metformin crosses the placenta with possible long-term effects. It has been estimated that fetal levels of metformin may reach approximately half of maternal levels (Vanky et al., 2005). The sulfonylurea glyburide is safe and effective at controlling glucose levels in majority of pregnant women with gestational diabetes mellitus. The study of Langer et al. (2005) was aimed to investigate the association between glyburide dose, degree of severity in gestational diabetes mellitus, level of glycemic control, and pregnancy outcome in insulinand glyburide-treated patients. It has been reported that glyburide and insulin were equally efficient for treatment of gestational diabetes mellitus in all levels of disease severity. In earlier investigation it was also demonstrated that there were no significant differences between the glyburide and insulin groups in the percentage of infants who were large for gestational age, who had macrosomia, who had lung complications, who had hypoglycemia, who were admitted to a neonatal intensive care unit; or who had fetal anomalies (Langer et al., 2000). Glyburide is regarded as a pharmacologically active substance that minimally crosses placenta, as supported by different in vitro and in vivo investigations that demonstrated very low transplacental transport of glyburide to the fetal circulation. This is due to high plasma protein binding, short half-life, as well as its active transport from the fetus to the mother (Bertini et al., 2005; Koren 2001; Kraemer et al., 2006). In the study aimed to identify placental transporters potentially involved in limiting the transplacental transfer of glyburide to the fetus it was demonstrated that glyburide is preferentially transported by the breast cancer resistance protein pump and multidrug resistance-associated protein 3, that are highly expressed in placental tissues and limit the passage of therapeutic or toxic xenobiotics to the fetus (Gedeon et al., 2006). Unfortunately, poor clinical response to glyburide has been reported in women with higher fasting and postprandial glucose values on their oral glucose tolerance test or in the group of diabetic endogenous insulin secretion (Pettitt et al., 2003). In particular, the peak insulin concentration was higher and the peak glucose and C-peptide concentrations were lower with both insulin preparations than with no exogenous insulin. Moreover, glucose areas under the curve above baseline were significantly lower with insulin aspart, but not with regular insulin, than with no insulin. In another randomized, parallel, open-label, controlled, multicenter and multinational study of type 1 diabetes pregnancy the fetal outcome using insulin aspart was comparable with human insulin, with a tendency toward fewer fetal losses and preterm deliveries (Hod at al., 2008). In another study, insulin aspart was more effective than regular human isulin in decreasing postprandial glucose concentrations (Pettitt et al., 2007). The authors of this investigation found out that duration of insulin aspart injection 5 min before a meal rather than 30 min prior to meals offered a more convenient therapy for subjects with gestational diabetes mellitus. Moreover, overall safety and effectiveness of insulin aspart were comparable to regular human insulin in Glyburide and metformin are oral antidiabetics that may be considered as second line agents in cases of gestational diabetes with poor glycemic control with insulin, or in women who refuse insulin. This is supported by the fact that when compared with insulin, administration of oral hypoglycemic agents was not associated with risk of neonatal hypoglycemia, caesarean section, or large-for-gestational-age babies births (Dhulkotia et al., 2010). No significant differences were found in maternal fasting or postprandial glycemic control, too. It appears that glyburide may be preferred, as metformin use is more likely to need supplemental insulin for glycemic control and in addition metformin crosses the placenta with possible long-term effects. It has been estimated that fetal levels of metformin The sulfonylurea glyburide is safe and effective at controlling glucose levels in majority of pregnant women with gestational diabetes mellitus. The study of Langer et al. (2005) was aimed to investigate the association between glyburide dose, degree of severity in gestational diabetes mellitus, level of glycemic control, and pregnancy outcome in insulinand glyburide-treated patients. It has been reported that glyburide and insulin were equally efficient for treatment of gestational diabetes mellitus in all levels of disease severity. In earlier investigation it was also demonstrated that there were no significant differences between the glyburide and insulin groups in the percentage of infants who were large for gestational age, who had macrosomia, who had lung complications, who had hypoglycemia, who were admitted to a neonatal intensive care unit; or who had fetal anomalies (Langer et al., 2000). Glyburide is regarded as a pharmacologically active substance that minimally crosses placenta, as supported by different in vitro and in vivo investigations that demonstrated very low transplacental transport of glyburide to the fetal circulation. This is due to high plasma protein binding, short half-life, as well as its active transport from the fetus to the mother (Bertini et al., 2005; Koren 2001; Kraemer et al., 2006). In the study aimed to identify placental transporters potentially involved in limiting the transplacental transfer of glyburide to the fetus it was demonstrated that glyburide is preferentially transported by the breast cancer resistance protein pump and multidrug resistance-associated protein 3, that are highly expressed in placental tissues and limit the passage of therapeutic or toxic xenobiotics to the fetus (Gedeon et al., 2006). Unfortunately, poor clinical response to glyburide has been reported in women with higher fasting and postprandial glucose values on their oral glucose tolerance test or in the group of diabetic pregnant women with gestational diabetes mellitus. may reach approximately half of maternal levels (Vanky et al., 2005). women on diet therapy. Other identified predictors of glyburide treatment failure were advanced maternal age, earlier diagnosis of gestational diabetes mellitus, higher gravidity or higher parity (Kahn et al., 2006). Otherwise, glyburide can be recommended for women in whom insulin cannot be used. In that way, it has been confirmed that this oral hypoglycemic can be used safely and effectively during the second and the third trimester of pregnancy without increasing maternal or fetal complications when compared with insulin (Langer et al., 2000). Glyburide has been shown to be safe in breastfeeding, too (Feig et al., 2005). However, it has to be emphasized that in some investigations a glyburide-related increased risk of preeclampsia, macrosomia, neonatal hypoglycemia, admission to a neonatal intensive care unit; as well a need for phototherapy have been reported (Jacobson et al., 2005; Ramos et al., 2007). The second oral antidiabetic drug used in gestational diabetes mellitus, as a monotherapy or with supplemental insulin, is metformin, a biguanide. It lowers blood glucose levels by decreasing hepatic gluconeogenesis, increasing peripheral glucose disposal and reducing intestinal glucose absorption (Hundal & Inzucchi, 2003). In average, up to half the women using metformin may require supplemental insulin. It has been demonstrated that women requiring supplemental insulin had a higher body mass index and had higher baseline glucose levels (Rowan et al. 2008). If compared to insulin, metformin was not associated with increased perinatal complications except of higher incidence of perinatal mortality if administered during the third trimester (Hellmuth et al., 2000). Although it may appear that metformin is safe alternative to insulin therapy, it does cross the placenta (Vanky et al., 2005) and scientific data are still not conclusive enough to recommend the standard use of metformin during pregnancy beyond the first trimester. Data that are more recent suggested that in women with gestational diabetes mellitus, not controlled with diet and exercise, who were then randomized to the metformin or the insulin arm, metformin has been shown to be an effective alternative to insulin in the treatment of gestational diabetes mellitus (Moore et al., 2007). This was substantiated by findings showing that difference in the rate of cesarean delivery was not statistically significant between the two groups, neither the neonatal statistics involving birth weight, Apgar score at 5 minutes, respiratory distress syndrome, hyperbilirubinemia, neonatal hypoglycemia or neonatal intensive care unit admission. Likewise, in investigation of Rowan et al. (2008) women with gestational diabetes mellitus at 20 to 33 weeks of gestation were randomly assigned to open treatment with metformin (with supplemental insulin if required) or insulin. The primary outcome was a composite of neonatal hypoglycemia, respiratory distress, need for phototherapy, birth trauma, 5-minute Apgar score less than 7, or prematurity. The rate of the primary composite outcome was comparable between the group assigned to metformin and the insulin group, suggesting that in women with gestational diabetes mellitus, metformin (alone or with supplemental insulin) was not associated with increased perinatal complications as compared with insulin. The retrospective data of Tertti et al. (2008) have been also indicative for the assumption that metformin was effective in controlling gestational diabetes and was not associated with a higher risk of maternal or neonatal complications compared with insulin. Namely, there were no differences between the metformin-treated group and the other two investigated groups (women treated with insulin and women with no pharmacological treatment) in terms of maternal outcomes (total weight gain during pregnancy or after the diagnosis of gestational diabetes mellitus, pre-pregnancy hypertension, pregnancy induced Treatment Considerations for Gestational Diabetes Mellitus and Long-Term Postpartum Options 321 recommendations to promote postpartum weight adjustments and decrease the incidence of type 2 diabetes include breastfeeding, exercising at a moderate intensity, and modifications of nutrition for specific weight-loss objectives (National Collaborating Centre for Women's and Children's Health, 2008). It has been determined that breastfeeding itself promotes weight loss for the mother, decreases possibility of maternal progression to type 2 diabetes, reduces insulin resistance in mothers and decreases likelihood of obesity in the child. Children born to mothers who had poor glycemic control should undergo regular evaluations of height, weight and blood glucose concentration, as well as monitoring for appropriate physical activity and diet to minimize the likelihood of obesity (Elchalal, 2004). The incidence of gestational diabetes is increasing and this pathological condition has strong association with adverse pregnancy outcomes. If untreated, gestational diabetes may lead to diverse complications, such as fetal hyperinsulinemia, increased weight at birth, higher rates of cesarian deliveries, shoulder dystocia, neonatal hypoglycemia, and it is also associated with concomitant preeclampsia in pregnant women. Therefore, given that gestational diabetes may have long-term pathological consequences for both mother and the child, it is important that it is recognized and correctly managed. Moreover, preconceptional screening and medical informing of women with diabetes type 1 or 2 would be significant in order to reduce risk to the fetus and mother connected to gestational diabetes. Treatment of gestational diabetes is aimed to maintain euglycemia and it should involve regular glucose monitoring, dietary modification, life style changes, exercise, and, when necessary, pharmacotherapy. Insulin therapy is the first choice of treatment, although glyburide and metformin could be indicated, too. In a long-term view, in order to prevent development of diabetes later in life, as well as different cardiovascular complications, an adequate education on lifestyle modifications should start in pregnancy and continue postpartum. American Diabetes Association. (2009). Standards of medical care in diabetes. Diabetes Care, Anderson, JH Jr.; Brunelle, R.L.; Koivisto, V.A.; Pfutzner, A.; Trautmann, M.E.; Vignati, L. & Aschwald, C.L.; Catanzaro, R.B., Weiss, E.P Gavard, JA; Steitz, KA; Mostello, D.J. (2009). Aydin, Y.; Berker, D.; Direktör, N.; Ustün, I.; Tütüncü, Y.A.; Işik, S.; Delibaşi, T. & Guler, S. DiMarchi, R. (1997). Reduction of postprandial hyperglycemia and frequency of hypoglycemia in IDDM patients on insulin-analog treatment. Diabetes, Vol. 46, No. Large-for-gestationalage infants of type 1 diabetic mothers: an effect of preprandial hyperglycemia? Gynecological Endocrinology, Vol. 25, pp.653–660, ISSN. 0951-3590 Avery, M.D.; Leon, A.S. & Kopher, R.A. (1997). Effects of a partially home-based exercise program for women with gestational diabetes. Obstetrics and Gynecology, Vol.89, (2008). Is insulin lispro safe in pregnant women: Does it cause any adverse outcomes on infants or mothers? Diabetes Research and Clinical Practice, Vol.80, No.3, Vol. 32, Suppl 1, pp. S13 –S61, ISSN 0149-5992. 2, pp. 265–270, ISSN. 0012-1797 pp.10–15. ISSN 0029-7844 pp. 444-448, ISSN. 0168-8227 5. Conclusion 6. References hypertension, pre-eclampsia etc.). In this investigation, no differences between the metformin-treated group and the other two groups were observed in relation to mean birth weights, prevalence of macrosomia, or gestational weeks at delivery. Finally, there were no differences between the groups in relation to other neonatal outcomes (small for gestational age, Apgar scores, umbilical artery pH or base excess, etc.). This drug is contraindicated in the case of preeclampsia, intrauterine growth restriction or placental insufficiency. Moreover, given that metformin crosses placenta, it could increase insulin sensitivity in the fetus, thus probably affecting growth and fetal hepatic glucose production. #### 4. Postpartum considerations After delivery, it is fundamental that women receive the appropriate postpartum counseling, testing, and follow-up. In a long-term view, most women with gestational diabetes do not require insulin therapy following delivery. Nevertheless, glucose levels should be regularly checked after discharge, since it has been confirmed that the progression of gestational diabetes mellitus to type 2 diabetes increased steeply within the first 5 years after delivery and appeared to plateau after 10 years (Kim et al., 2002). It has been determined that progressive beta-cell failure to compensate for the ongoing insulin resistance correlates with progression from gestational diabetes mellitus to type 2 diabetes. Insulin resistance that presents as a high serum insulin concentrations in association with blood glucose concentrations that are normal or high, results from defects in insulin responsiveness in muscle, fat and liver. Therefore, screening for diabetes at regular intervals should be of paramount importance. In addition, among women with a family history of type 2 diabetes, those with prior gestational diabetes mellitus were even more likely not only to have cardiovascular disease risk factors, including metabolic syndrome and type 2 diabetes, but also to have experienced cardiovascular disease events, which occurred at a younger age (Carr et al., 2006). Moreover, the development of metabolic syndrome in children with increasing age is known to be related to maternal gestational diabetes mellitus, maternal glycemia in the third trimester, maternal obesity, neonatal macrosomia, and childhood obesity (Vohr & Boney, 2008). Consequently, post partum evaluation and management of reversible cardiovascular risk factors such as smoking, obesity, hypertension, and hyperlipidemia should be undertaken (Cheung, 2009). It is confirmed that a good predictor of early postpartum development of diabetes is elevated fasting plasma glucose during pregnancy and, in women having positive tests to specific autoantibodies [anti-glutamic acid decarboxylase (anti-GAD); anti-protein tyrosine phosphatase ICA 512 (anti–IA-2)], higher incidence of diabetes by six months postpartum has been shown, too. In addition, it should be pointed out that some women with gestational diabetes mellitus, especially lean ones under 30 years of age who required insulin during pregnancy, could progress to type 1 diabetes. Therefore, women diagnosed with gestational diabetes should be screened for diabetes 6 to 12 weeks postpartum and should have subsequent screening for the development of diabetes or prediabetes (American Diabetes Association, 2009). An oral glucose tolerance test at three-year intervals has been also shown to be a beneficial approach for screening. All women with gestational diabetes should be encouraged on a healthy lifestyle and in order to prevent diabetes and cardiovascular complications education on lifestyle modification should start in pregnancy and continue postpartum. In that way, usual recommendations to promote postpartum weight adjustments and decrease the incidence of type 2 diabetes include breastfeeding, exercising at a moderate intensity, and modifications of nutrition for specific weight-loss objectives (National Collaborating Centre for Women's and Children's Health, 2008). It has been determined that breastfeeding itself promotes weight loss for the mother, decreases possibility of maternal progression to type 2 diabetes, reduces insulin resistance in mothers and decreases likelihood of obesity in the child. Children born to mothers who had poor glycemic control should undergo regular evaluations of height, weight and blood glucose concentration, as well as monitoring for appropriate physical activity and diet to minimize the likelihood of obesity (Elchalal, 2004). ### 5. Conclusion 320 Gestational Diabetes hypertension, pre-eclampsia etc.). In this investigation, no differences between the metformin-treated group and the other two groups were observed in relation to mean birth weights, prevalence of macrosomia, or gestational weeks at delivery. Finally, there were no differences between the groups in relation to other neonatal outcomes (small for gestational age, Apgar scores, umbilical artery pH or base excess, etc.). This drug is contraindicated in the case of preeclampsia, intrauterine growth restriction or placental insufficiency. Moreover, given that metformin crosses placenta, it could increase insulin sensitivity in the After delivery, it is fundamental that women receive the appropriate postpartum counseling, testing, and follow-up. In a long-term view, most women with gestational diabetes do not require insulin therapy following delivery. Nevertheless, glucose levels should be regularly checked after discharge, since it has been confirmed that the progression of gestational diabetes mellitus to type 2 diabetes increased steeply within the first 5 years after delivery and appeared to plateau after 10 years (Kim et al., 2002). It has been determined that progressive beta-cell failure to compensate for the ongoing insulin resistance correlates with progression from gestational diabetes mellitus to type 2 diabetes. Insulin resistance that presents as a high serum insulin concentrations in association with blood glucose concentrations that are normal or high, results from defects in insulin responsiveness in muscle, fat and liver. Therefore, screening for diabetes at regular intervals should be of paramount importance. In addition, among women with a family history of type 2 diabetes, those with prior gestational diabetes mellitus were even more likely not only to have cardiovascular disease risk factors, including metabolic syndrome and type 2 diabetes, but also to have experienced cardiovascular disease events, which occurred at a younger age (Carr et al., 2006). Moreover, the development of metabolic syndrome in children with increasing age is known to be related to maternal gestational diabetes mellitus, maternal glycemia in the third trimester, maternal obesity, neonatal macrosomia, and childhood obesity (Vohr & Boney, 2008). Consequently, post partum evaluation and management of reversible cardiovascular risk factors such as smoking, obesity, fetus, thus probably affecting growth and fetal hepatic glucose production. hypertension, and hyperlipidemia should be undertaken (Cheung, 2009). has been also shown to be a beneficial approach for screening. It is confirmed that a good predictor of early postpartum development of diabetes is elevated fasting plasma glucose during pregnancy and, in women having positive tests to specific autoantibodies [anti-glutamic acid decarboxylase (anti-GAD); anti-protein tyrosine phosphatase ICA 512 (anti–IA-2)], higher incidence of diabetes by six months postpartum has been shown, too. In addition, it should be pointed out that some women with gestational diabetes mellitus, especially lean ones under 30 years of age who required insulin during pregnancy, could progress to type 1 diabetes. Therefore, women diagnosed with gestational diabetes should be screened for diabetes 6 to 12 weeks postpartum and should have subsequent screening for the development of diabetes or prediabetes (American Diabetes Association, 2009). An oral glucose tolerance test at three-year intervals All women with gestational diabetes should be encouraged on a healthy lifestyle and in order to prevent diabetes and cardiovascular complications education on lifestyle modification should start in pregnancy and continue postpartum. In that way, usual 4. Postpartum considerations The incidence of gestational diabetes is increasing and this pathological condition has strong association with adverse pregnancy outcomes. If untreated, gestational diabetes may lead to diverse complications, such as fetal hyperinsulinemia, increased weight at birth, higher rates of cesarian deliveries, shoulder dystocia, neonatal hypoglycemia, and it is also associated with concomitant preeclampsia in pregnant women. Therefore, given that gestational diabetes may have long-term pathological consequences for both mother and the child, it is important that it is recognized and correctly managed. Moreover, preconceptional screening and medical informing of women with diabetes type 1 or 2 would be significant in order to reduce risk to the fetus and mother connected to gestational diabetes. Treatment of gestational diabetes is aimed to maintain euglycemia and it should involve regular glucose monitoring, dietary modification, life style changes, exercise, and, when necessary, pharmacotherapy. Insulin therapy is the first choice of treatment, although glyburide and metformin could be indicated, too. In a long-term view, in order to prevent development of diabetes later in life, as well as different cardiovascular complications, an adequate education on lifestyle modifications should start in pregnancy and continue postpartum. #### 6. References Treatment Considerations for Gestational Diabetes Mellitus and Long-Term Postpartum Options 323 Jacobson, G.F.; Ramos, G.A.; Ching, J.Y.; Kirby, R.S.; Ferrara, A.; Field, D.R. (2005). Jovanovic, L.; Ilic, S.; Pettitt ,D.J.; Hugo, K.; Gutierrez, M.; Bowsher, R.R.; Bastyr, E.J. 3rd. Jovanovic, L.G. (2008). Using meal-based self-monitoring of blood glucose as a tool to Kahn, B.F.; Davies, J.K.; Lynch, A.M.; Reynolds, R.M.; Barbour, L.A. (2006). Predictors of Kim, C.; Newton, K.M.; Knopp, R.H. (2002). Gestational diabetes and the incidence of type 2 Klieger, C.; Pollex, E.; Koren, G. (2008). Treating the mother--protecting the unborn: the Koren, G. (2001). Glyburide and fetal safety; trans-placental pharmacokinetic considerations. Kraemer, J.; Klein, J.; Lubetsky, A.; Koren, G. (2006). Perfusion studies of glyburide transfer Langer, O.; Yogev, Y.; Xenakis, E.M.; Rosenn, B. (2005). Insulin and glyburide therapy: McLachlan, K.; Jenkins, A.; O'Neal, D. (2007) .The role of continuous glucose monitoring in Metzger, B.E.; Buchanan, T.A.; Coustan, D.R.; de Leiva, A.; Dunger, D.B.; Hadden, D.R.; Journal of Obstetrics and Gynaecology, Vol.47, pp.186–190. ISSN 0004-8666 Mecacci, F.; Carignani, L.; Cioni, R.; Bartoli, E.; Parretti, E.; La Torre, P; Scarselli, G.; Mello G. Obstetrics and Gynecology, Vol.195, No.1, pp.270-274. ISSN. 0002-9378 Langer, O.; Conway, D.L.; Berkus, M.D.; Xenakis, E.M.; Gonzales, O. (2000). A comparison Journal of Medicine, vol.343, No.16, pp.1134-1138. ISSN 0028-4793 Diabetes Care , Vol.22, No.9, pp.1422–1427. ISSN 0149-5992. Vol.193, No.1, pp.118-124. ISSN. 0002-9378 Vol.107, No.6, pp.1303-1309. ISSN 0029-7844 Medicine, Vol.21, No.3, pp.191-196. ISSN 1476-7058 Reproductive Toxicology, Vol.15, 227–229. ISSN 0890-6238 No.239–247. ISSN 1530-891X 1998;91(4):600-4. ISSN 0029-7844 pp.S251-S260. ISSN 0149-5992. Biology, Vol.111, No.1, pp. 19-24. ISSN 0301-2115 5992. Comparison of glyburide and insulin for the management of gestational diabetes in a large managed care organization. American Journal of Obstetrics and Gynecology, (1999). Metabolic and immunologic effects of insulin lispro in gestational diabetes. improve outcomes in pregnancy complicated by diabetes. Endocrine Practice, Vol.14, glyburide failure in the treatment of gestational diabetes. Obstetrics and Gynecology, diabetes: a systematic review. Diabetes Care, Vol.25, No.10, pp.1862-1868. ISSN 0149- safety of hypoglycemic drugs in pregnancy. Journal of Maternal-Fetal and Neonatal across the human placenta: implications for fetal safety. American Journal of of glyburide and insulin in women with gestational diabetes mellitus. New England dosage, severity level of gestational diabetes, and pregnancy outcome. American Journal of Obstetrics and Gynecology, Vol.192, No.1, pp.134-139. ISSN. 0002-9378 Major CA, Henry MJ, De Veciana M, Morgan MA. The effects of carbohydrate restriction in patients with diet-controlled gestational diabetes. Obstetrics and Gynecology. Apr clinical decision-making in diabetes in pregnancy. Australian and New Zealand (2003). Maternal metabolic control and perinatal outcome in women with gestational diabetes treated with regular or lispro insulin: comparison with nondiabetic pregnant women. European Journal of Obstetrics Gynecology and Reproductive Hod, M.; Kitzmiller, J.L.; Kjos, SL; Oats, JN; Pettitt, DJ; Sacks, DA; Zoupas, C. (2007). Summary and recommendations of the Fifth International Workshop-Conference on Gestational Diabetes Mellitus. Diabetes Care, Vol.30, Suppl 2, Bhattacharyya, A.; Brown, S.; Hughes, S.; Vice, P.A. (2001). Insulin lispro and regular insulin Bertini, A.M.; Silva, J.C.; Taborda, W.; Becker, F.; Lemos Bebber, F.R.; Zucco Viesi, J.M.; Brankston, G.N.; Mitchell, B.F. & Ryan, E.A. (2004). Resistance exercise decreases the need Journal of Obstetrics and Gynecology, Vol. 190, pp.188–193, ISSN. 0002-9378 Carr, D.B.; Utzschneider, K.M.; Hull, R.L.; Tong, J.; Wallace, T.M.; Kodama, K.; Shofer, J.B.; of type 2 diabetes. Diabetes Care, Vol.29, No.9, pp.2078-2083. ISSN 0149-5992. Cheung, N.W. (2009).The management of gestational diabetes. Vascular Health and Risk Dhulkotia, J.S.; Ola, B.; Fraser, R.; Farrell, T. (2010). Oral hypoglycemic agents vs insulin in Elchalal, U. (2004). Gestational diabetes mellitus: what else is new? Israel Medical Association Feig, D.S.; Briggs, G.G.; Kraemer, J.M.; Ambrose, P.J.; Moskovitz, D.N.; Nageotte, M.; Donat, Hawkins, J.S. (2010). Glucose monitoring during pregnancy. Current Diabetes Reports, Vol.10, Hellmuth, E.; Damm, P.; Mølsted-Pedersen, L. (2000). Oral hypoglycaemic agents in 118 diabetic pregnancies. Diabetic Medicine, Vol.17, No.7, pp.507-511. ISSN 0742-3071 Hedderson, M.M.; Ferrara, A. (2008). High blood pressure before and during early Hod, M.; Damm, P.; Kaaja, R.; Visser, G.H.; Dunne, F.; Demidova, I.; Hansen, A.S.; Horvath, K.; Koch, K.; Jeitler, K.; Matyas, E.; Bender, R.; Bastian, H.; Lange, S.; Siebenhofer, Hundal, R.S.; Inzucchi, S.E. (2003). Metformin: New understandings, new uses. Drugs, Medicine, Vol.94, pp.255–260, ISSN. 1460-2725 Management, Vol.5, No.1, pp.153-164. ISSN 1176-6344. Journal / IMAJ. Vol.6, No.5, pp.268-270. ISSN 1565-1088 Diabetes Care, Vol.31, pp.2362–2367. ISSN 0149-5992. Gynecology, Vol.198, No.2, pp.186.e1-7. ISSN. 0002-9378 Vol.63, No.18, pp.1879–1894. ISSN 0012-6667 No.11– 12, pp.1096–1102. ISSN 0143-4004 No.3, pp.229-234. ISSN 1534-4827 ISSN 0959-535X 0300-5577 9378 in pregnancy. QJM: An International Journal of Medicine / Quarterly Journal of Aquim, G.; Engel Ribeiro, T. (2005). Perinatal outcomes and the use of oral hypoglycemic agents. Journal of Perinatal Medicine, Vol.33, No.6, pp.519-523, ISSN. for insulin in overweight women with gestational diabetes mellitus. American Heckbert, S.R.; Boyko, E.J.; Fujimoto, W.Y.; Kahn, S.E. (2006). Gestational diabetes mellitus increases the risk of cardiovascular disease in women with a family history management of gestational diabetes: a systematic review and metaanalysis. American Journal of Obstetrics and Gynecology. Vol.203, No.5, pp.457.e1-9. ISSN. 0002- D.J.; Padilla, G.; Wan, S.; Klein, J.; Koren, G. (2005). Transfer of glyburide and glipizide into breast milk. Diabetes Care, Vol. 28, pp. 1851–1855. ISSN 0149-5992. Gedeon, C.; Behravan, J.; Koren, G.; Piquette-Miller, M. (2006). Transport of glyburide by placental ABC transporters: Implications in fetal drug exposure. Placenta, Vol.27, pregnancy is associated with an increased risk of gestational diabetes mellitus. Mersebach, H.; Insulin Aspart Pregnancy Study Group. (2008). Fetal and perinatal outcomes in type 1 diabetes pregnancy: a randomized study comparing insulin aspart with human insulin in 322 subjects. American Journal of Obstetrics and A. (2010). Effects of treatment in women with gestational diabetes mellitus: systematic review and meta-analysis. British Medical Journal, Vol.340, pp.c1395. 19 1Department of Pharmaceutics, University of Washington United States of America 2Department of Pharmacy School of Pharmacy, Glyburide Disposition During Pregnancy During pregnancy, 5-14% of women are diagnosed with gestational diabetes mellitus (GDM) and the incidence has been increasing (Jovanovic & Pettitt, 2001; Paglia & Coustan, 2011). While insulin treatment is still the "gold standard" therapy for controlling maternal glycemia, the increasing use of oral anti-diabetic agents such as glyburide and metformin has begun to change standard care (Maymone et al., 2011). Anti-diabetic drugs are often titrated over a prolonged period of time to achieve glycemic control. Prolonged hyperglycemia increases the likelihood of adverse fetal/neonatal and maternal outcomes. Thus, quickly achieving glycemic control during pregnancy can significantly reduce the occurrence of certain adverse perinatal and maternal outcomes (Karakash & Einstein, 2011). Glyburide is a second generation oral sulfonylurea (Feldman, 1985). Glyburide lowers blood sugar levels by stimulating the pancreas to secrete insulin and by helping the body use insulin efficiently. Considerable data in the literature suggest that glyburide may be a safe alternative to insulin for the treatment of GDM due to its similar efficacy to insulin and its low fetal distribution (Nicholson & Baptiste-Roberts, 2011; Maymone et al., 2011). Physiological and biochemical changes that occur during pregnancy alter the pharmacokinetics of glyburide, thus affecting the safety and efficacy of the drug for both the mother and the fetus. Understanding pregnancy-induced changes in the disposition of glyburide (including fetal exposure) will be important for optimizing dosage guidelines during pregnancy. In this chapter, current knowledge on the safety and efficacy of glyburide for the treatment of GDM, pregnancy-related effects on maternal disposition as well as The American College of Obstetricians and Gynecologists (ACOG) committee on practice defines GDM as "carbohydrate intolerance that begins or is first recognized during pregnancy" (2001). Similar to type II diabetes mellitus, GDM is the result of an inability to compensate for the degree of insulin resistance. Insulin resistance is normal to some extent during pregnancy as a means of ensuring that glucose is freely available to the developing fetus; however, in women predisposed to diabetes, the degree of insulin resistance can be so placental transport and metabolism of the drug will be summarized. 2. Gestational diabetes mellitus and treatment options high that treatment is necessary to maintain euglycemia. 1. Introduction Diana L. Shuster1, Mary F. Hebert2 and Qingcheng Mao1 ## Glyburide Disposition During Pregnancy Diana L. Shuster1, Mary F. Hebert2 and Qingcheng Mao1 1Department of Pharmaceutics, 2Department of Pharmacy School of Pharmacy, University of Washington United States of America #### 1. Introduction 324 Gestational Diabetes Moore, L.E.; Briery, C.M.; Clokey, D.; Martin, R.W; Williford, NJ; Bofill, JA; Morrison, J.C. Moore, T.R. (2010). Diabetes mellitus and pregnancy. http://emedicine.medscape.com/ National Collaborating Centre for Women's and Children's Health (2008). Postnatal care. In: of Obstetricians and Gynaecologists, ISBN 978-1-904752-47-9, London. Pettitt, D.J.; Ospina, P.; Kolaczynski, J.W.; Jovanovic, L. (2003). Comparison of an insulin diabetes mellitus. Diabetes Care, Vol.26, No.1, pp.183-186. ISSN 0149-5992. Pettitt, D.J.; Ospina, P.; Howard, C.; Zisser, H.; Jovanovic, L. (2007). Efficacy, safety and lack Ramos, G.A.; Jacobson, G.F.; Kirby, R.S.; Ching, J.Y.; Field, D.R. (2007). Comparison of Riskin-Mashiah, S.; Younes, G.; Damti, A.; Auslender, R. (2009). First trimester fasting Rowan, J.A.; Hague, W.M.; Gao, W.; Battin, M.R.; Moore, M.P.; MiG Trial Investigators. England Journal of Medicine, Vol.358, No.19, pp.2003-2015. ISSN 0028-4793 Siega-Riz, A.M.; Viswanathan, M.; Moos, M.K.; Deierlein, A.; Mumford, S.; Knaack, J.; Obstetrics and Gynecology , Vol.201, pp.339.e1–339.e14. ISSN. 0002-9378 Vanky, E.; Zahlsen, K.; Spigset, O., Carlsen, S.M. (2005). Placental passage of metformin in and Neonatal Medicine, Vol.21, No.3, pp.149-157. ISSN 1476-7058. Vohr, B.R.; Boney, C.M. (2008). Gestational diabetes: the forerunner for the development of Perinatology, Vol.27, No.5, pp.262-267. ISSN 0743-8346 11, pp.1011-1015. ISSN 0024-7758 article/127547. 1135. ISSN 0742-3071 1643. ISSN 0149-5992. pp.1575-1578. ISSN 0015-0282 (2007). Metformin and insulin in the management of gestational diabetes mellitus: preliminary results of a comparison. Journal of Reproductive Medicine, Vol.52, No. Diabetes in pregnancy. Welsh, A (Ed.), pp. 143-152, RCOG Press at the Royal College analog, insulin aspart, and regular human insulin with no insulin in gestational of immunogenicity of insulin aspart compared with regular human insulin for women with gestational diabetes mellitus. Diabetic Medicine, Vol.24, No.10, pp.1129- glyburide and insulin for the management of gestational diabetics with markedly elevated oral glucose challenge test and fasting hyperglycemia. Journal of hyperglycemia and adverse pregnancy outcomes. Diabetes Care, Vol.32, pp.1639– (2008). Metformin versus insulin for the treatment of gestational diabetes. New Thieda, P.; Lux, L.J.; Lohr, K.N. (2009). A systematic review of outcomes of maternal weight gain according to the Institute of Medicine recommendations: birthweight, fetal growth, and postpartum weight retention. American Journal of women with polycystic ovary syndrome. Fertility and Sterility, Vol.83, No.5, maternal and childhood obesity and metabolic syndrome? Journal of Maternal-Fetal During pregnancy, 5-14% of women are diagnosed with gestational diabetes mellitus (GDM) and the incidence has been increasing (Jovanovic & Pettitt, 2001; Paglia & Coustan, 2011). While insulin treatment is still the "gold standard" therapy for controlling maternal glycemia, the increasing use of oral anti-diabetic agents such as glyburide and metformin has begun to change standard care (Maymone et al., 2011). Anti-diabetic drugs are often titrated over a prolonged period of time to achieve glycemic control. Prolonged hyperglycemia increases the likelihood of adverse fetal/neonatal and maternal outcomes. Thus, quickly achieving glycemic control during pregnancy can significantly reduce the occurrence of certain adverse perinatal and maternal outcomes (Karakash & Einstein, 2011). Glyburide is a second generation oral sulfonylurea (Feldman, 1985). Glyburide lowers blood sugar levels by stimulating the pancreas to secrete insulin and by helping the body use insulin efficiently. Considerable data in the literature suggest that glyburide may be a safe alternative to insulin for the treatment of GDM due to its similar efficacy to insulin and its low fetal distribution (Nicholson & Baptiste-Roberts, 2011; Maymone et al., 2011). Physiological and biochemical changes that occur during pregnancy alter the pharmacokinetics of glyburide, thus affecting the safety and efficacy of the drug for both the mother and the fetus. Understanding pregnancy-induced changes in the disposition of glyburide (including fetal exposure) will be important for optimizing dosage guidelines during pregnancy. In this chapter, current knowledge on the safety and efficacy of glyburide for the treatment of GDM, pregnancy-related effects on maternal disposition as well as placental transport and metabolism of the drug will be summarized. #### 2. Gestational diabetes mellitus and treatment options The American College of Obstetricians and Gynecologists (ACOG) committee on practice defines GDM as "carbohydrate intolerance that begins or is first recognized during pregnancy" (2001). Similar to type II diabetes mellitus, GDM is the result of an inability to compensate for the degree of insulin resistance. Insulin resistance is normal to some extent during pregnancy as a means of ensuring that glucose is freely available to the developing fetus; however, in women predisposed to diabetes, the degree of insulin resistance can be so high that treatment is necessary to maintain euglycemia. in vivo. Glyburide Disposition During Pregnancy 327 Glyburide is extensively metabolized in the liver with a low hepatic extraction ratio. One enzyme involved in glyburide metabolism is CYP2C9, which is highly polymorphic. The CYP2C9 variant, CYP2C9\3, exhibits lower catalytic activity than wild-type CYP2C9\1 (Cavallari & Limdi, 2009). Kirchheriner et al. showed that the oral clearance of glyburide in the CYP2C9\3/\3 subjects (n = 3) was ~40% of that in CYP2C9\1/\1 subjects (n = 4) (Kirchheiner et al., 2002). Niemi et al. reported that the area under plasma concentrationtime curve (plasma AUC) of glyburide in subjects heterozygous for CYP2C9\3 (CYP2C9\1/\3 or CYP2C9\2/\3, n = 2) was 280% of that in the CYP2C9\1/\1 subjects (n = 5) (Niemi et al., 2002). Yin et al. demonstrated that the oral plasma AUC of glyburide in CYP2C9\1/\3 subjects (n = 6) of the Chinese population was higher by ~100% as compared with that in the CYP2C9\1/\1 subjects (n = 12) (Yin et al., 2005). These clinical studies appear to suggest that CYP2C9 contributes significantly to glyburide metabolism On the other hand, in vitro* studies using human liver microsomes have shown that CYP3A4 contributes greater than 50% of glyburide metabolism, while CYP2C9 contributes a much smaller percentage (Naritomi et al., 2004; Zharikova et al., 2009; Zhou et al., 2010a). Additionally, Lilja et al. showed that oral administration of clarithromycin, an inhibitor of CYP3A but not CYP2C9, significantly increased Cmax and the plasma AUC of glyburide (Lilja et al., 2007). The epidemiological study (Schelleman et al., 2010) and case reports (Bussing & Gende, 2002; Leiba et al., 2004) all indicated that the concomitant use of glyburide with clarithromycin was associated with severe hypoglycemia. Thus, CYP3A also appears to contribute to glyburide metabolism in vivo. It is possible that glyburide is In vitro metabolism studies using human liver microsomes or recombinant systems revealed that, besides CYP3A4 and CYP2C9, glyburide was also metabolized by other cytochrome P450 enzymes such as CYP3A5, CYP2C8 and CYP2C19, but to a much lesser extent (Naritomi et al., 2004; Zharikova et al., 2009; Zhou et al., 2010a). Zharikova et al. determined five metabolites of glyburide formed in human liver microsomes: M1 (4-transhydrocyclohexyl glyburide), M2a (4-cis-hydrocyclohexyl glyburide), M2b (3-cishydrocyclohexyl glyburide), M3 (3-trans-hydrocyclohexyl glyburide), M4 (2-transhydrocyclohexyl glyburide) and M5 (ethylene-hydroxylated glyburide) (Zharikova et al., 2009; Zharikova et al., 2007). The chemical structures of these glyburide metabolites are shown in Figure 1. CYP3A4 catalyzes the formation of M1-M5. CYP2C9 catalyzes the formation of M1-M3. CYP2C8 catalyzes the formation of M1, M2b, M3 and M4. CYP2C19 catalyzes the formation of M2a, M2b and M3 (Zharikova et al., 2009; Zharikova et al., 2007). The two major metabolites of glyburide, M1 and M2b, which account for approximately half of all the metabolites formed in vitro by human liver microsomes (Zharikova et al., 2007), are excreted into the bile and urine (~50% each) (Feldman, 1985). M1 and M2b are not likely to contribute significantly to hypoglycemic action in humans since they are only weakly active (1/400th and 1/40th as active, respectively, as glyburide in preclinical models, as described in the FDA labeling). However, there were also studies indicating that M1 and M2b retain 75% and 50%, respectively, of the hypoglycemic activity of glyburide in humans (Rydberg et al., 1994). The systemic exposure of M1 is only 2 – 4% of that of glyburide (Zheng et al., 2009). The pharmacological activity of other metabolized in vivo through the joint actions of hepatic CYP3A and CYP2C9. glyburide metabolites is currently not known. Properly treating GDM is of great concern as the condition complicates 5-14% of pregnancies. If untreated, GDM presents a danger to both the mother and baby, particularly the risk of hypertension, preeclampsia, urinary tract infections, cesarean delivery and development of type II diabetes mellitus later in life in mothers, as well as macrosomia, neonatal hypoglycemia, childhood obesity and type II diabetes mellitus in the offspring (2002; 2009; 2010; Paglia & Coustan, 2011). Diet therapy is the first line of treatment for GDM and is adequate for controlling glucose concentrations in the majority of patients. Those failing diet therapy are managed with the addition of pharmacotherapy (Landon et al., 2007). The American Diabetes Association (ADA) suggests that women with GDM should seek nutritional counseling by a dietician in order to individualize diet therapy by patient height and weight (2001). To prevent ketonuria, which can hinder the cognitive development of children ages 3 – 9, the ACOG recommends caloric restrictions that are not to exceed 33% of current diet (2001). There are two general options of pharmacotherapy for the treatment of GDM. Traditionally, insulin therapy has been the "gold standard" for the management of GDM, when diet therapy and exercise fail to achieve maternal glycemic control. Pregnant women have difficulty adhering to insulin therapy regimens because of the challenges with route of administration and schedule. Therefore, oral hypoglycemic agents such as glyburide and metformin are being increasingly used to treat GDM, and have been shown to have similar efficacy and safety as insulin, as well as lower cost and easier route of administration (Maymone et al., 2011; Nicholson & Baptiste-Roberts, 2011). The safety of insulin for use in pregnancy has been well established without the risk of transfer across the placenta. The FDA has not approved the use of glyburide or metformin for the treatment of women with GDM. This chapter will focus its discussion on glyburide. #### 3. Glyburide and its clinical pharmacokinetics Glyburide is a second generation oral sulfonylurea, and its chemical structure is shown in Figure 1. Glyburide is indicated as an adjunct to diet therapy and serves to lower blood glucose levels in patients with type II diabetes mellitus (Feldman, 1985). Glyburide exerts its pharmacological effect by stimulating insulin secretion from pancreatic β-islet cells. It inhibits ATP-sensitive potassium channels on the surface of pancreatic β-islet cells, leading to cellular membrane depolarization. Depolarization at the cellular membrane prompts voltage-gated calcium channels to open, increasing the intracellular calcium concentration, which stimulates the release of insulin into the portal vein. Glyburide is administered in 1.25, 2.5 or 5 mg tablets. The FDA approved dosage range is 1.25 mg up to 20 mg per day. When higher dosages of glyburide are required, patients are typically switched to insulin. Glyburide is a small lipophilic molecule (LogP = 4.8, MW = 494 Da) that is highly bound to plasma proteins (99.8% plasma protein binding). Glyburide is well absorbed with an oral bioavailability of approximately 95% for micronized tablets (Jonsson et al., 1994). It exhibits biphasic elimination kinetics with an initial distribution half-life (T1/2α) of roughly 30 min and a terminal elimination half-life (T1/2β) of approximately 10 hours (Feldman, 1985; Jonsson et al., 1994). Thus, the overall elimination half-life of glyburide is approximately 4 hours. Glyburide has a small volume of distribution (0.2 L/kg), despite its lipophilic nature, and has negligible renal clearance. Properly treating GDM is of great concern as the condition complicates 5-14% of pregnancies. If untreated, GDM presents a danger to both the mother and baby, particularly the risk of hypertension, preeclampsia, urinary tract infections, cesarean delivery and development of type II diabetes mellitus later in life in mothers, as well as macrosomia, neonatal hypoglycemia, childhood obesity and type II diabetes mellitus in the offspring (2002; 2009; 2010; Paglia & Coustan, 2011). Diet therapy is the first line of treatment for GDM and is adequate for controlling glucose concentrations in the majority of patients. Those failing diet therapy are managed with the addition of pharmacotherapy (Landon et al., 2007). The American Diabetes Association (ADA) suggests that women with GDM should seek nutritional counseling by a dietician in order to individualize diet therapy by patient height and weight (2001). To prevent ketonuria, which can hinder the cognitive development of children ages 3 – 9, the ACOG recommends caloric restrictions that are not There are two general options of pharmacotherapy for the treatment of GDM. Traditionally, insulin therapy has been the "gold standard" for the management of GDM, when diet therapy and exercise fail to achieve maternal glycemic control. Pregnant women have difficulty adhering to insulin therapy regimens because of the challenges with route of administration and schedule. Therefore, oral hypoglycemic agents such as glyburide and metformin are being increasingly used to treat GDM, and have been shown to have similar efficacy and safety as insulin, as well as lower cost and easier route of administration (Maymone et al., 2011; Nicholson & Baptiste-Roberts, 2011). The safety of insulin for use in pregnancy has been well established without the risk of transfer across the placenta. The FDA has not approved the use of glyburide or metformin for the treatment of women with Glyburide is a second generation oral sulfonylurea, and its chemical structure is shown in Figure 1. Glyburide is indicated as an adjunct to diet therapy and serves to lower blood glucose levels in patients with type II diabetes mellitus (Feldman, 1985). Glyburide exerts its pharmacological effect by stimulating insulin secretion from pancreatic β-islet cells. It inhibits ATP-sensitive potassium channels on the surface of pancreatic β-islet cells, leading to cellular membrane depolarization. Depolarization at the cellular membrane prompts voltage-gated calcium channels to open, increasing the intracellular calcium concentration, which stimulates the release of insulin into the portal vein. Glyburide is administered in 1.25, 2.5 or 5 mg tablets. The FDA approved dosage range is 1.25 mg up to 20 mg per day. When higher dosages of glyburide are required, patients are typically switched to insulin. Glyburide is a small lipophilic molecule (LogP = 4.8, MW = 494 Da) that is highly bound to plasma proteins (99.8% plasma protein binding). Glyburide is well absorbed with an oral bioavailability of approximately 95% for micronized tablets (Jonsson et al., 1994). It exhibits biphasic elimination kinetics with an initial distribution half-life (T1/2α) of roughly 30 min and a terminal elimination half-life (T1/2β) of approximately 10 hours (Feldman, 1985; Jonsson et al., 1994). Thus, the overall elimination half-life of glyburide is approximately 4 hours. Glyburide has a small volume of distribution (0.2 L/kg), despite its lipophilic nature, to exceed 33% of current diet (2001). and has negligible renal clearance. GDM. This chapter will focus its discussion on glyburide. 3. Glyburide and its clinical pharmacokinetics Glyburide is extensively metabolized in the liver with a low hepatic extraction ratio. One enzyme involved in glyburide metabolism is CYP2C9, which is highly polymorphic. The CYP2C9 variant, CYP2C9\3, exhibits lower catalytic activity than wild-type CYP2C9\1 (Cavallari & Limdi, 2009). Kirchheriner et al. showed that the oral clearance of glyburide in the CYP2C9\3/\3 subjects (n = 3) was ~40% of that in CYP2C9\1/\1 subjects (n = 4) (Kirchheiner et al., 2002). Niemi et al. reported that the area under plasma concentrationtime curve (plasma AUC) of glyburide in subjects heterozygous for CYP2C9\3 (CYP2C9\1/\3 or CYP2C9\2/\3, n = 2) was 280% of that in the CYP2C9\1/\1 subjects (n = 5) (Niemi et al., 2002). Yin et al. demonstrated that the oral plasma AUC of glyburide in CYP2C9\1/\3 subjects (n = 6) of the Chinese population was higher by ~100% as compared with that in the CYP2C9\1/\1 subjects (n = 12) (Yin et al., 2005). These clinical studies appear to suggest that CYP2C9 contributes significantly to glyburide metabolism in vivo. On the other hand, in vitro* studies using human liver microsomes have shown that CYP3A4 contributes greater than 50% of glyburide metabolism, while CYP2C9 contributes a much smaller percentage (Naritomi et al., 2004; Zharikova et al., 2009; Zhou et al., 2010a). Additionally, Lilja et al. showed that oral administration of clarithromycin, an inhibitor of CYP3A but not CYP2C9, significantly increased Cmax and the plasma AUC of glyburide (Lilja et al., 2007). The epidemiological study (Schelleman et al., 2010) and case reports (Bussing & Gende, 2002; Leiba et al., 2004) all indicated that the concomitant use of glyburide with clarithromycin was associated with severe hypoglycemia. Thus, CYP3A also appears to contribute to glyburide metabolism in vivo. It is possible that glyburide is metabolized in vivo through the joint actions of hepatic CYP3A and CYP2C9. In vitro metabolism studies using human liver microsomes or recombinant systems revealed that, besides CYP3A4 and CYP2C9, glyburide was also metabolized by other cytochrome P450 enzymes such as CYP3A5, CYP2C8 and CYP2C19, but to a much lesser extent (Naritomi et al., 2004; Zharikova et al., 2009; Zhou et al., 2010a). Zharikova et al. determined five metabolites of glyburide formed in human liver microsomes: M1 (4-transhydrocyclohexyl glyburide), M2a (4-cis-hydrocyclohexyl glyburide), M2b (3-cishydrocyclohexyl glyburide), M3 (3-trans-hydrocyclohexyl glyburide), M4 (2-transhydrocyclohexyl glyburide) and M5 (ethylene-hydroxylated glyburide) (Zharikova et al., 2009; Zharikova et al., 2007). The chemical structures of these glyburide metabolites are shown in Figure 1. CYP3A4 catalyzes the formation of M1-M5. CYP2C9 catalyzes the formation of M1-M3. CYP2C8 catalyzes the formation of M1, M2b, M3 and M4. CYP2C19 catalyzes the formation of M2a, M2b and M3 (Zharikova et al., 2009; Zharikova et al., 2007). The two major metabolites of glyburide, M1 and M2b, which account for approximately half of all the metabolites formed in vitro by human liver microsomes (Zharikova et al., 2007), are excreted into the bile and urine (~50% each) (Feldman, 1985). M1 and M2b are not likely to contribute significantly to hypoglycemic action in humans since they are only weakly active (1/400th and 1/40th as active, respectively, as glyburide in preclinical models, as described in the FDA labeling). However, there were also studies indicating that M1 and M2b retain 75% and 50%, respectively, of the hypoglycemic activity of glyburide in humans (Rydberg et al., 1994). The systemic exposure of M1 is only 2 – 4% of that of glyburide (Zheng et al., 2009). The pharmacological activity of other glyburide metabolites is currently not known. Glyburide Disposition During Pregnancy 329 and showed no statistically significant difference in fasting, preprandial, 2-hour postprandial and mean blood glucose concentrations between the glyburide and insulin groups (~106 mg/dL, 105 mg/dL, 130 mg/dL, and 115 mg/dL, respectively). Langer concluded that glyburide was as effective as insulin for the treatment of GDM and therefore can be used as a clinically effective alternative to insulin therapy. These authors reported eight women in the glyburide group (4%) who required insulin therapy. These authors also performed a subsequent analysis of the data from this clinical study to determine if the severity of GDM was linked to the dosage of glyburide required to achieve adequate glycemic control (Langer et al., 2000). It was found that glyburide doses were increased and the success rate of glyburide therapy decreased as disease severity increased. At each level of disease severity, there was no difference in maternal and neonatal outcomes between the Two smaller clinical studies also compared the efficacy of glyburide and insulin in the treatment of GDM, and no difference was observed in fasting and 2-hour postprandial blood glucose concentrations (Anjalakshi et al., 2007; Bertini et al., 2005). In contrast, Ogunyemi et al. reported significantly higher fasting and 2-hour postprandial blood glucose concentrations in patients receiving glyburide than in patients receiving insulin (Ogunyemi et al., 2007). Bertini et al. found that glucose control was not achieved in 5 patients in the glyburide group (20.8% failure rate) who had to switch to insulin therapy, despite similarities in maternal demographics across groups (age, weight and parity) (Bertini et al., 2005). Since the sample size of this randomized-controlled trial was relatively small, this In addition, retrospective studies have been conducted to assess the efficacy of glyburide. Jacobson et al. performed a retrospective study of 584 women with GDM who had failed diet therapy and were treated with glyburide or insulin between the years of 1999-2002 (Jacobson et al., 2005). Patients from both groups were similar in age and nulliparity; however, women in the insulin group weighed more on average and had a higher mean fasting blood glucose level (105.4 versus 102.4 mg/dL). Women in the glyburide group had significantly lower post-treatment fasting and postprandial blood glucose levels. The failure rate of glyburide was reported to be 12%. The glyburide group did experience a higher rate of preeclampsia (12% versus 6%), despite controlling for body mass index and ethnicity. Jacobson et al. observed no statistical difference in neonatal outcomes such as birth weight and macrosomia, as well as incidence of cesarean delivery between groups. Ramos et al. retrospectively examined the effectiveness of glyburide (n = 44) versus insulin (n = 78) in women with GDM who had a 50 g 3-hour oral glucose challenge test of ≥200 mg/dL and a pretreatment fasting plasma glucose level of ≥105 mg/dL (Ramos et al., 2007). There were no significant differences between the two groups with respect to blood glucose levels. The failure rate of glyburide was 16%. There were no significant differences in fetal outcomes between the two treatment groups; however, the incidence of neonatal hypoglycemia was higher in glyburide-treated women (34% versus 15%, respectively) (Ramos et al., 2007). It is worth noting that retrospective studies were often not adequately powered and were without adequate controls. Therefore, making general conclusions regarding the efficacy of To predict the treatment failure rate for glyburide in women with GDM, Kahn et al. conducted a prospective cohort study (n = 75) which demonstrated that fasting blood glucose levels ≥ 110 mg/dL, in women with GDM, were associated with higher glyburide may not accurately reflect the true failure rate of glyburide therapy. insulin and glyburide groups. glyburide is difficult. Fig. 1. The chemical structures of glyburide and its metabolites. M1, 4-trans-hydrocyclohexyl glyburide; M2a, 4-cis-hydrocyclohexyl glyburide; M2b, 3-cis-hydrocyclohexyl glyburide; M3, 3-trans-hydrocyclohexyl glyburide; M4, 2-trans-hydrocyclohexyl glyburide; and M5, ethylene-hydroxylated glyburide. #### 4. The efficacy and safety of glyburide during pregnancy Although glyburide is not currently approved by the FDA to treat GDM, the pathophysiology of GDM is similar to type II diabetes mellitus and consequently, glyburide has been increasingly prescribed to women with GDM. There are obvious benefits to using oral hypoglycemic agents such as glyburide and metformin rather than insulin. Oral agents are less expensive, easier to administer and demonstrate improved patient compliance as compared to insulin. Clinical studies have been conducted to compare the efficacy and safety of such oral agents with those of insulin for the treatment of GDM. In this section, the results of clinical studies comparing the efficacy and safety of glyburide versus insulin will be summarized. #### 4.1 Clinical efficacy during pregnancy There are several randomized controlled clinical trials that examined the efficacy of glyburide during pregnancy (Langer et al., 2000; Anjalakshi et al., 2007; Bertini et al., 2005; Ogunyemi et al., 2007). Langer et al. performed the largest clinical study that compared the efficacy of glyburide to insulin for the treatment of GDM (Langer et al., 2000). This group randomly assigned 404 women with GDM into two treatment groups (insulin or glyburide), Fig. 1. The chemical structures of glyburide and its metabolites. M1, 4-trans-hydrocyclohexyl glyburide; M2a, 4-cis-hydrocyclohexyl glyburide; M2b, 3-cis-hydrocyclohexyl glyburide; M3, Although glyburide is not currently approved by the FDA to treat GDM, the pathophysiology of GDM is similar to type II diabetes mellitus and consequently, glyburide has been increasingly prescribed to women with GDM. There are obvious benefits to using oral hypoglycemic agents such as glyburide and metformin rather than insulin. Oral agents are less expensive, easier to administer and demonstrate improved patient compliance as compared to insulin. Clinical studies have been conducted to compare the efficacy and safety of such oral agents with those of insulin for the treatment of GDM. In this section, the results of clinical studies comparing the efficacy and safety of glyburide versus insulin will There are several randomized controlled clinical trials that examined the efficacy of glyburide during pregnancy (Langer et al., 2000; Anjalakshi et al., 2007; Bertini et al., 2005; Ogunyemi et al., 2007). Langer et al. performed the largest clinical study that compared the efficacy of glyburide to insulin for the treatment of GDM (Langer et al., 2000). This group randomly assigned 404 women with GDM into two treatment groups (insulin or glyburide), 3-trans-hydrocyclohexyl glyburide; M4, 2-trans-hydrocyclohexyl glyburide; and M5, 4. The efficacy and safety of glyburide during pregnancy ethylene-hydroxylated glyburide. 4.1 Clinical efficacy during pregnancy be summarized. and showed no statistically significant difference in fasting, preprandial, 2-hour postprandial and mean blood glucose concentrations between the glyburide and insulin groups (~106 mg/dL, 105 mg/dL, 130 mg/dL, and 115 mg/dL, respectively). Langer concluded that glyburide was as effective as insulin for the treatment of GDM and therefore can be used as a clinically effective alternative to insulin therapy. These authors reported eight women in the glyburide group (4%) who required insulin therapy. These authors also performed a subsequent analysis of the data from this clinical study to determine if the severity of GDM was linked to the dosage of glyburide required to achieve adequate glycemic control (Langer et al., 2000). It was found that glyburide doses were increased and the success rate of glyburide therapy decreased as disease severity increased. At each level of disease severity, there was no difference in maternal and neonatal outcomes between the insulin and glyburide groups. Two smaller clinical studies also compared the efficacy of glyburide and insulin in the treatment of GDM, and no difference was observed in fasting and 2-hour postprandial blood glucose concentrations (Anjalakshi et al., 2007; Bertini et al., 2005). In contrast, Ogunyemi et al. reported significantly higher fasting and 2-hour postprandial blood glucose concentrations in patients receiving glyburide than in patients receiving insulin (Ogunyemi et al., 2007). Bertini et al. found that glucose control was not achieved in 5 patients in the glyburide group (20.8% failure rate) who had to switch to insulin therapy, despite similarities in maternal demographics across groups (age, weight and parity) (Bertini et al., 2005). Since the sample size of this randomized-controlled trial was relatively small, this may not accurately reflect the true failure rate of glyburide therapy. In addition, retrospective studies have been conducted to assess the efficacy of glyburide. Jacobson et al. performed a retrospective study of 584 women with GDM who had failed diet therapy and were treated with glyburide or insulin between the years of 1999-2002 (Jacobson et al., 2005). Patients from both groups were similar in age and nulliparity; however, women in the insulin group weighed more on average and had a higher mean fasting blood glucose level (105.4 versus 102.4 mg/dL). Women in the glyburide group had significantly lower post-treatment fasting and postprandial blood glucose levels. The failure rate of glyburide was reported to be 12%. The glyburide group did experience a higher rate of preeclampsia (12% versus 6%), despite controlling for body mass index and ethnicity. Jacobson et al. observed no statistical difference in neonatal outcomes such as birth weight and macrosomia, as well as incidence of cesarean delivery between groups. Ramos et al. retrospectively examined the effectiveness of glyburide (n = 44) versus insulin (n = 78) in women with GDM who had a 50 g 3-hour oral glucose challenge test of ≥200 mg/dL and a pretreatment fasting plasma glucose level of ≥105 mg/dL (Ramos et al., 2007). There were no significant differences between the two groups with respect to blood glucose levels. The failure rate of glyburide was 16%. There were no significant differences in fetal outcomes between the two treatment groups; however, the incidence of neonatal hypoglycemia was higher in glyburide-treated women (34% versus 15%, respectively) (Ramos et al., 2007). It is worth noting that retrospective studies were often not adequately powered and were without adequate controls. Therefore, making general conclusions regarding the efficacy of glyburide is difficult. To predict the treatment failure rate for glyburide in women with GDM, Kahn et al. conducted a prospective cohort study (n = 75) which demonstrated that fasting blood glucose levels ≥ 110 mg/dL, in women with GDM, were associated with higher glyburide Glyburide Disposition During Pregnancy 331 Bertini et al. found just the opposite to be true in a clinical study with 70 patients diagnosed with GDM (Bertini et al., 2005). Patients were placed on insulin therapy (n = 27), glyburide therapy (n = 24) or acarbose therapy (n = 19). The authors reported that neonatal hypoglycemia was observed in 8 newborns, 6 of which were from the glyburide group. Likewise, in the study with 97 women (n = 49 in the insulin group and n = 48 in the glyburide group), Ogunyemi et al. reported that 28% of infants in the glyburide group experienced an episode of hypoglycemia versus 13% in the insulin group, and the difference was statistically significant (Ogunyemi et al., 2007). In contrast, Langer et al. showed no difference in the incidence rate of hypoglycemia for infants between the insulin and glyburide treatment groups (Langer et al., 2000). Bertini et al. also demonstrated that a significantly higher percentage of fetuses were large for gestational age (LGA) infants in the glyburide group compared with the insulin group (25% versus 3.7%, respectively) (Bertini et al., 2005); however, Langer et al. reported comparable incidence rates of LGA between the two groups (n = 404) (Langer et al., 2000). All the clinical studies consistently reported higher average infant birth weights in the glyburide group than the insulin group, but the difference was small (an average of ~100 g) and not statistically significant (Anjalakshi et al., 2007; Bertini et al., 2005; Langer et al., 2000; Ogunyemi et al., 2007). Few congenital malformations or anomalies were reported in either group. It is worth noting that the study by Langer et al. investigated significantly more subjects than any other study, and hence the Overall, in women with GDM, glyburide achieved similar efficacy of glycemic control as insulin therapy. The maternal and neonatal safety of glyburide does not substantially differ from insulin therapy. However, it should be noted that, at present, there is no long-term safety data for infants whose mothers were treated with glyburide. Thus, further studies are needed to assess the long-term effects of maternal glyburide administration on child and adolescent development (neurologic and behavioral) as well as the incidence rate of type II Physiological and biochemical changes that occur in pregnancy may affect the pharmacokinetics of drugs, namely absorption, distribution, metabolism and elimination (Anderson, 2005; Klieger et al., 2009; Loebstein et al., 1997). Such changes include, among others, changes in volume of distribution of drugs and plasma protein binding (Loebstein et al., 1997; Mendenhall, 1970), induction or down-regulation of cytochrome P450 enzyme expression and activity (Hebert et al., 2008; Tracy et al., 2005), and increase in renal blood flow and glomerular filtration (Dunlop & Davison, 1987). The effects of such pregnancyinduced pharmacokinetic changes may be such that a dosage adjustment is required to accommodate increased potency of a drug or decreased efficacy. However, the balance of treating the mother and protecting the fetus may likely present challenges specifically for drug compounds that require increased dosages in order to be effective during pregnancy. To evaluate pregnancy-induced changes in the pharmacokinetics of glyburide, Hebert et al. compared parameter estimates for steady-state pharmacokinetics of glyburide in pregnant women with GDM (n = 40) and non-pregnant women with type II diabetes mellitus (n = 26) results obtained could be more adequately powered and reliable. 5. Pregnancy-induced pharmacokinetic changes of glyburide diabetes mellitus and obesity. Glyburide is such a case. 5.1 Clinical pharmacokinetic studies failure rates (Kahn et al., 2006). The authors also reported that women who were older, had more than one child and were diagnosed with GDM earlier in their pregnancy were more likely to fail glyburide therapy. On the other hand, Rochon et al. suggested that only higher mean blood glucose levels (≥ 200 mg/dL in the 50 g 1-hour oral glucose challenge test) were indicators of glyburide failure (Rochon et al., 2006). #### 4.2 Maternal and neonatal safety Several studies have been conducted to investigate the adverse effects of glyburide versus insulin (Anjalakshi et al., 2007; Bertini et al., 2005; Langer et al., 2000; Ogunyemi et al., 2007; Yogev et al., 2004). Among these studies, Langer et al. conducted the largest randomized controlled trial with 404 pregnant women to receive glyburide or insulin, and found a significantly higher percentage of women with a blood glucose level <40 mg/dL in the insulin group compared with the glyburide group (20% versus 4%) (Langer et al., 2000). Yogev et al. demonstrated that 19 of 30 insulin-treated patients with GDM (63%) experienced asymptomatic hypoglycemia versus 7 of 25 (28%) glyburide-treated patients (Yogev et al., 2004). On the other hand, in the study with 97 pregnant women, Ogunyemi et al. did not report a significant difference in hypoglycemia between the insulin and glyburide groups (31% verse 38%, respectively) (Ogunyemi et al., 2007). Other studies reported no hypoglycemic events (Anjalakshi et al., 2007; Bertini et al., 2005). Although the results varied, possibly due to difference in definition of hypoglycemia, these studies appear to support the notion that glyburide therapy generally causes fewer hypoglycemic events than insulin therapy. Langer et al. also showed no difference in the incidence of preeclampsia among women treated with insulin or glyburide (Langer et al., 2000). Bertini et al. found no significant difference in changes in maternal weight of women treated with insulin as compared to glyburide (Bertini et al., 2005). Likewise, no significant differences were reported in the percentage of women with cesarean delivery in the insulin group compared with the glyburide group (Anjalakshi et al., 2007; Bertini et al., 2005; Langer et al., 2000; Ogunyemi et al., 2007). Various clinical studies have also analyzed the effects of glyburide and insulin on neonatal adverse outcomes. In an earlier study, Coetzee and Jackson treated over 600 pregnant women suffering from GDM or type II diabetes mellitus with glyburide/metformin combination therapy (Coetzee and Jackson, 1985). Patients were classified as new diabetics, known diabetics or untreated diabetics. The untreated diabetic group was made up of pregnant women with type II diabetes mellitus or GDM who were not seen in the clinic until term. Each class of patients was further organized into four treatment groups: (1) diet therapy, (2) diet plus metformin therapy, (3) diet plus glyburide therapy, (4) diet plus metformin/glyburide combination therapy, and (5) treatment group (4) with the addition of insulin therapy due to inadequate glucose control (Coetzee and Jackson, 1985). Metformin therapy appeared to be the safest (0 still births, 1 neonatal death, and 33 per 1,000 perinatal morbidities, i.e. large for gestational age, low birthweight, hypoglycemia, jaundice and congenital abnormalities), followed by glyburide (1 still birth, 0 neonatal deaths, and 43 per 1,000 perinatal morbidities) and the emergency insulin group (1 still birth, 4 neonatal deaths, and 59 per 1,000 perinatal morbidities). In women with newly diagnosed GDM, insulin therapy appeared to be the safest (no adverse birth outcomes), followed by metformin (1 neonatal death and 16 per 1,000 perinatal morbidities) and glyburide (1 still birth and 42 per 1,000 perinatal morbidities). The authors also reported a decrease in perinatal morbidities among the glyburide group compared with the diet therapy group as well as zero cases of serious neonatal hypoglycemia. failure rates (Kahn et al., 2006). The authors also reported that women who were older, had more than one child and were diagnosed with GDM earlier in their pregnancy were more likely to fail glyburide therapy. On the other hand, Rochon et al. suggested that only higher mean blood glucose levels (≥ 200 mg/dL in the 50 g 1-hour oral glucose challenge test) were Several studies have been conducted to investigate the adverse effects of glyburide versus insulin (Anjalakshi et al., 2007; Bertini et al., 2005; Langer et al., 2000; Ogunyemi et al., 2007; Yogev et al., 2004). Among these studies, Langer et al. conducted the largest randomized controlled trial with 404 pregnant women to receive glyburide or insulin, and found a significantly higher percentage of women with a blood glucose level <40 mg/dL in the insulin group compared with the glyburide group (20% versus 4%) (Langer et al., 2000). Yogev et al. demonstrated that 19 of 30 insulin-treated patients with GDM (63%) experienced asymptomatic hypoglycemia versus 7 of 25 (28%) glyburide-treated patients (Yogev et al., 2004). On the other hand, in the study with 97 pregnant women, Ogunyemi et al. did not report a significant difference in hypoglycemia between the insulin and glyburide groups (31% verse 38%, respectively) (Ogunyemi et al., 2007). Other studies reported no hypoglycemic events (Anjalakshi et al., 2007; Bertini et al., 2005). Although the results varied, possibly due to difference in definition of hypoglycemia, these studies appear to support the notion that glyburide therapy generally causes fewer hypoglycemic events than insulin therapy. Langer et al. also showed no difference in the incidence of preeclampsia among women treated with insulin or glyburide (Langer et al., 2000). Bertini et al. found no significant difference in changes in maternal weight of women treated with insulin as compared to glyburide (Bertini et al., 2005). Likewise, no significant differences were reported in the percentage of women with cesarean delivery in the insulin group compared with the glyburide group (Anjalakshi et Various clinical studies have also analyzed the effects of glyburide and insulin on neonatal adverse outcomes. In an earlier study, Coetzee and Jackson treated over 600 pregnant women suffering from GDM or type II diabetes mellitus with glyburide/metformin combination therapy (Coetzee and Jackson, 1985). Patients were classified as new diabetics, known diabetics or untreated diabetics. The untreated diabetic group was made up of pregnant women with type II diabetes mellitus or GDM who were not seen in the clinic until term. Each class of patients was further organized into four treatment groups: (1) diet therapy, (2) diet plus metformin therapy, (3) diet plus glyburide therapy, (4) diet plus metformin/glyburide combination therapy, and (5) treatment group (4) with the addition of insulin therapy due to inadequate glucose control (Coetzee and Jackson, 1985). Metformin therapy appeared to be the safest (0 still births, 1 neonatal death, and 33 per 1,000 perinatal morbidities, i.e. large for gestational age, low birthweight, hypoglycemia, jaundice and congenital abnormalities), followed by glyburide (1 still birth, 0 neonatal deaths, and 43 per 1,000 perinatal morbidities) and the emergency insulin group (1 still birth, 4 neonatal deaths, and 59 per 1,000 perinatal morbidities). In women with newly diagnosed GDM, insulin therapy appeared to be the safest (no adverse birth outcomes), followed by metformin (1 neonatal death and 16 per 1,000 perinatal morbidities) and glyburide (1 still birth and 42 per 1,000 perinatal morbidities). The authors also reported a decrease in perinatal morbidities among the glyburide group compared with the diet therapy group as well as zero cases of serious neonatal hypoglycemia. indicators of glyburide failure (Rochon et al., 2006). al., 2007; Bertini et al., 2005; Langer et al., 2000; Ogunyemi et al., 2007). 4.2 Maternal and neonatal safety Bertini et al. found just the opposite to be true in a clinical study with 70 patients diagnosed with GDM (Bertini et al., 2005). Patients were placed on insulin therapy (n = 27), glyburide therapy (n = 24) or acarbose therapy (n = 19). The authors reported that neonatal hypoglycemia was observed in 8 newborns, 6 of which were from the glyburide group. Likewise, in the study with 97 women (n = 49 in the insulin group and n = 48 in the glyburide group), Ogunyemi et al. reported that 28% of infants in the glyburide group experienced an episode of hypoglycemia versus 13% in the insulin group, and the difference was statistically significant (Ogunyemi et al., 2007). In contrast, Langer et al. showed no difference in the incidence rate of hypoglycemia for infants between the insulin and glyburide treatment groups (Langer et al., 2000). Bertini et al. also demonstrated that a significantly higher percentage of fetuses were large for gestational age (LGA) infants in the glyburide group compared with the insulin group (25% versus 3.7%, respectively) (Bertini et al., 2005); however, Langer et al. reported comparable incidence rates of LGA between the two groups (n = 404) (Langer et al., 2000). All the clinical studies consistently reported higher average infant birth weights in the glyburide group than the insulin group, but the difference was small (an average of ~100 g) and not statistically significant (Anjalakshi et al., 2007; Bertini et al., 2005; Langer et al., 2000; Ogunyemi et al., 2007). Few congenital malformations or anomalies were reported in either group. It is worth noting that the study by Langer et al. investigated significantly more subjects than any other study, and hence the results obtained could be more adequately powered and reliable. Overall, in women with GDM, glyburide achieved similar efficacy of glycemic control as insulin therapy. The maternal and neonatal safety of glyburide does not substantially differ from insulin therapy. However, it should be noted that, at present, there is no long-term safety data for infants whose mothers were treated with glyburide. Thus, further studies are needed to assess the long-term effects of maternal glyburide administration on child and adolescent development (neurologic and behavioral) as well as the incidence rate of type II diabetes mellitus and obesity. #### 5. Pregnancy-induced pharmacokinetic changes of glyburide Physiological and biochemical changes that occur in pregnancy may affect the pharmacokinetics of drugs, namely absorption, distribution, metabolism and elimination (Anderson, 2005; Klieger et al., 2009; Loebstein et al., 1997). Such changes include, among others, changes in volume of distribution of drugs and plasma protein binding (Loebstein et al., 1997; Mendenhall, 1970), induction or down-regulation of cytochrome P450 enzyme expression and activity (Hebert et al., 2008; Tracy et al., 2005), and increase in renal blood flow and glomerular filtration (Dunlop & Davison, 1987). The effects of such pregnancyinduced pharmacokinetic changes may be such that a dosage adjustment is required to accommodate increased potency of a drug or decreased efficacy. However, the balance of treating the mother and protecting the fetus may likely present challenges specifically for drug compounds that require increased dosages in order to be effective during pregnancy. Glyburide is such a case. #### 5.1 Clinical pharmacokinetic studies To evaluate pregnancy-induced changes in the pharmacokinetics of glyburide, Hebert et al. compared parameter estimates for steady-state pharmacokinetics of glyburide in pregnant women with GDM (n = 40) and non-pregnant women with type II diabetes mellitus (n = 26) Glyburide Disposition During Pregnancy 333 protect the fetus by expelling drugs, xenobiotics, and metabolites from the fetal compartment to the maternal circulation (Behravan & Piquette-Miller, 2007; Ceckova-Novotna et al., 2006; Mao, 2008; Ni & Mao, 2010). Glyburide has been shown to be a substrate of such ABC efflux transporters. Thus, it is likely that ABC efflux transporters in To test this hypothesis, Gedeon et al. examined the role of P-gp, BCRP and MRPs in the efflux of glyburide using stable cell lines expressing these transporters in the presence or absence of selective inhibitors (Gedeon et al., 2006). Their results suggested that glyburide was preferentially transported by BCRP and MRP3, but not by P-gp, MRP1 or MRP2. Likewise, Gedeon et al. demonstrated that inhibition of P-gp or MRP1 did not affect accumulation of [3H]-glyburide in inside-out brush border human placental membrane vesicles; however, inhibition of BCRP did (Gedeon et al., 2008b). Using the dually perfused human placental cotyledon model, Gedeon et al. further showed that the rate of transfer of glyburide across the placenta in the presence of indomethacin (an inhibitor of MRP1, MRP2 or MRP3) was not different from the rate of transfer in the absence of inhibitor (Gedeon et al., 2008a), suggesting that MRP1, 2, or 3 may be only minimally involved in the transport of glyburide across the human placenta. On the other hand, the role of BCRP in the transfer of glyburide across the human placenta was confirmed in a similar human placental perfusion study (Pollex et al., 2008). In contrast, Hemauer et al showed that MRP1 appears to play a greater role in the efflux of glyburide than P-gp or BCRP (Hemauer et al., 2010). Using inside-out brush border membrane vesicles isolated from human term placentas in the presence or absence of selective inhibitors (verapamil for P-gp, Ko143 for BCRP, and indomethacin for MRP1), Hemauer et al. determined the relative contribution of P-gp, BCRP, and MRP1 to the uptake of glyburide into the inside-out membrane vesicles to be 9 ± Zhou et al. confirmed that glyburide is a substrate for human BCRP and mouse Bcrp1 using Madin Darby canine kidney (MDCK) cell transwell transport experiments (Zhou et al., 2008). Zhou et al. also characterized glyburide disposition in wild-type and Bcrp1-/ pregnant mice to elucidate the role of Bcrp1 in limiting glyburide transfer across the placenta to the fetal compartment. The results showed that the maternal plasma concentration-time profiles remained the same between wild-type and knockout mice; however, the fetal area under the concentration-time curve (AUC) of glyburide in Bcrp1-/ pregnant mice was two times greater than that in wild-type mice (Zhou et al., 2008). It is worth noting that the amount of glyburide entering the fetus only accounts for a small fraction of the total amount of glyburide in the body (Zhou et al., 2008). These results confirm that BCRP and Bcrp1 are important determinants of fetal exposure to glyburide (Zhou et al., 2008). All these in vitro, ex vivo, and in vivo studies suggest that ABC efflux transporters, particularly BCRP, are important in protecting the fetus from exposure to glyburide. Thus, if a drug known to be a BCRP inhibitor is co-administered with glyburide, fetal exposure to glyburide may be increased through inhibition of placental BCRP. The placenta may also protect the fetus by metabolizing drugs or xenobiotics ingested by the mother. Human term placentas have been used in several studies to characterize placental metabolism of glyburide (Jain et al., 2008; Zharikova et al., 2009; Zharikova et al., 2007). Zharikova et al. reported that placental microsomes converted ~87% of glyburide to the M5 the placenta play an important role in limiting fetal exposure to glyburide. 5%, 25 ± 5%, and 43 ± 4%, respectively (Hemauer et al., 2010). 5.4 Placental metabolism of glyburide (Hebert et al., 2009). Dose-normalized steady-state plasma concentrations of glyburide were approximately one-half in pregnant women with GDM as compared with those in nonpregnant women with type II diabetes mellitus, consistent with a 2-fold increase in apparent oral clearance of glyburide during pregnancy. Modeling and simulations of this data demonstrate that pregnant women with GDM require much higher dosages of glyburide to achieve comparable concentrations as non-pregnant women. Whether higher dosages will be required during pregnancy to achieve glycemic control still needs further study. #### 5.2 In vivo animal studies In vivo pharmacokinetic studies in pregnant mice have been performed to investigate the mechanism of pregnancy-induced increase in the apparent oral clearance of glyburide (Zhou et al., 2010b). Several groups have shown that CYP3A is a major enzyme responsible for the in vitro metabolism of glyburide (Naritomi et al., 2004; Zharikova et al., 2009; Zhou et al., 2010a). It has also been well established that hepatic CYP3A activity is significantly induced by pregnancy (Hebert et al., 2008; Tracy et al., 2005). Therefore, it has been hypothesized that pregnancy induces the activity of hepatic CYP3A, resulting in an increase in the oral clearance of glyburide (Zhou et al., 2010b). Since it has been shown that the levels of hepatic Cyp3a content in pregnant mice and its activity measured using testosterone as the probe substrate are significantly increased compared with those in non-pregnant mouse controls (Mathias et al., 2006; Zhang et al., 2008), the pregnant mouse was used as the animal model to test this hypothesis (Zhou et al., 2010b). Upon characterization, the pharmacokinetics of glyburide indeed demonstrated a two-fold increase in its hepatic clearance in pregnant mice on gestation day 15 compared to non-pregnant mice, a magnitude of change similar to that observed in the human clinical study, but with no changes in plasma protein binding (Zhou et al., 2010b). To investigate the mechanism of this pharmacokinetic change in pregnant mice, Zhou et al. further determined glyburide depletion in mouse hepatic S-9 fractions and found the halflife of glyburide depletion to be markedly shorter in S-9 fractions from pregnant mice compared to non-pregnant mice. Glyburide depletion was also inhibited to a large extent by the Cyp3a inhibitor, ketoconazole, suggesting that the increase in hepatic clearance of glyburide during pregnancy may be due to an increase in the activity of hepatic Cyp3a (Zhou et al., 2010b). These studies support the notion that CYP3A plays a significant role in the clearance of glyburide in pregnancy. This finding has significant clinical implications. For example, significant drug-drug interactions may occur with glyburide and CYP3A inducers or inhibitors (Lilja et al., 2007). #### 5.3 Placental transport of glyburide Hebert et al. have also shown that glyburide concentrations are measurable in umbilical cord blood at the time of delivery, suggesting that glyburide crosses the placenta and thus may pose adverse effects on the developing fetus (Hebert et al., 2009). The average umbilical cord to maternal plasma concentration ratio of glyburide was 0.7 ± 0.4 (Hebert et al., 2009). This less than unity ratio indicates that glyburide does not cross the placental barrier entirely by passive diffusion, even though it is highly lipophilic (LogP = 4.8). Many ATPbinding cassette (ABC) efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance proteins (MRPs) are highly expressed on the apical membrane of placental syncytiotrophoblasts facing maternal blood, where they (Hebert et al., 2009). Dose-normalized steady-state plasma concentrations of glyburide were approximately one-half in pregnant women with GDM as compared with those in nonpregnant women with type II diabetes mellitus, consistent with a 2-fold increase in apparent oral clearance of glyburide during pregnancy. Modeling and simulations of this data demonstrate that pregnant women with GDM require much higher dosages of glyburide to achieve comparable concentrations as non-pregnant women. Whether higher dosages will In vivo pharmacokinetic studies in pregnant mice have been performed to investigate the mechanism of pregnancy-induced increase in the apparent oral clearance of glyburide (Zhou et al., 2010b). Several groups have shown that CYP3A is a major enzyme responsible for the in vitro metabolism of glyburide (Naritomi et al., 2004; Zharikova et al., 2009; Zhou et al., 2010a). It has also been well established that hepatic CYP3A activity is significantly induced by pregnancy (Hebert et al., 2008; Tracy et al., 2005). Therefore, it has been hypothesized that pregnancy induces the activity of hepatic CYP3A, resulting in an increase in the oral clearance of glyburide (Zhou et al., 2010b). Since it has been shown that the levels of hepatic Cyp3a content in pregnant mice and its activity measured using testosterone as the probe substrate are significantly increased compared with those in non-pregnant mouse controls (Mathias et al., 2006; Zhang et al., 2008), the pregnant mouse was used as the animal model to test this hypothesis (Zhou et al., 2010b). Upon characterization, the pharmacokinetics of glyburide indeed demonstrated a two-fold increase in its hepatic clearance in pregnant mice on gestation day 15 compared to non-pregnant mice, a magnitude of change similar to that observed in the human clinical study, but with no To investigate the mechanism of this pharmacokinetic change in pregnant mice, Zhou et al. further determined glyburide depletion in mouse hepatic S-9 fractions and found the halflife of glyburide depletion to be markedly shorter in S-9 fractions from pregnant mice compared to non-pregnant mice. Glyburide depletion was also inhibited to a large extent by the Cyp3a inhibitor, ketoconazole, suggesting that the increase in hepatic clearance of glyburide during pregnancy may be due to an increase in the activity of hepatic Cyp3a (Zhou et al., 2010b). These studies support the notion that CYP3A plays a significant role in the clearance of glyburide in pregnancy. This finding has significant clinical implications. For example, significant drug-drug interactions may occur with glyburide and CYP3A Hebert et al. have also shown that glyburide concentrations are measurable in umbilical cord blood at the time of delivery, suggesting that glyburide crosses the placenta and thus may pose adverse effects on the developing fetus (Hebert et al., 2009). The average umbilical cord to maternal plasma concentration ratio of glyburide was 0.7 ± 0.4 (Hebert et al., 2009). This less than unity ratio indicates that glyburide does not cross the placental barrier entirely by passive diffusion, even though it is highly lipophilic (LogP = 4.8). Many ATPbinding cassette (ABC) efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance proteins (MRPs) are highly expressed on the apical membrane of placental syncytiotrophoblasts facing maternal blood, where they be required during pregnancy to achieve glycemic control still needs further study. 5.2 In vivo animal studies changes in plasma protein binding (Zhou et al., 2010b). inducers or inhibitors (Lilja et al., 2007). 5.3 Placental transport of glyburide protect the fetus by expelling drugs, xenobiotics, and metabolites from the fetal compartment to the maternal circulation (Behravan & Piquette-Miller, 2007; Ceckova-Novotna et al., 2006; Mao, 2008; Ni & Mao, 2010). Glyburide has been shown to be a substrate of such ABC efflux transporters. Thus, it is likely that ABC efflux transporters in the placenta play an important role in limiting fetal exposure to glyburide. To test this hypothesis, Gedeon et al. examined the role of P-gp, BCRP and MRPs in the efflux of glyburide using stable cell lines expressing these transporters in the presence or absence of selective inhibitors (Gedeon et al., 2006). Their results suggested that glyburide was preferentially transported by BCRP and MRP3, but not by P-gp, MRP1 or MRP2. Likewise, Gedeon et al. demonstrated that inhibition of P-gp or MRP1 did not affect accumulation of [3H]-glyburide in inside-out brush border human placental membrane vesicles; however, inhibition of BCRP did (Gedeon et al., 2008b). Using the dually perfused human placental cotyledon model, Gedeon et al. further showed that the rate of transfer of glyburide across the placenta in the presence of indomethacin (an inhibitor of MRP1, MRP2 or MRP3) was not different from the rate of transfer in the absence of inhibitor (Gedeon et al., 2008a), suggesting that MRP1, 2, or 3 may be only minimally involved in the transport of glyburide across the human placenta. On the other hand, the role of BCRP in the transfer of glyburide across the human placenta was confirmed in a similar human placental perfusion study (Pollex et al., 2008). In contrast, Hemauer et al showed that MRP1 appears to play a greater role in the efflux of glyburide than P-gp or BCRP (Hemauer et al., 2010). Using inside-out brush border membrane vesicles isolated from human term placentas in the presence or absence of selective inhibitors (verapamil for P-gp, Ko143 for BCRP, and indomethacin for MRP1), Hemauer et al. determined the relative contribution of P-gp, BCRP, and MRP1 to the uptake of glyburide into the inside-out membrane vesicles to be 9 ± 5%, 25 ± 5%, and 43 ± 4%, respectively (Hemauer et al., 2010). Zhou et al. confirmed that glyburide is a substrate for human BCRP and mouse Bcrp1 using Madin Darby canine kidney (MDCK) cell transwell transport experiments (Zhou et al., 2008). Zhou et al. also characterized glyburide disposition in wild-type and Bcrp1-/ pregnant mice to elucidate the role of Bcrp1 in limiting glyburide transfer across the placenta to the fetal compartment. The results showed that the maternal plasma concentration-time profiles remained the same between wild-type and knockout mice; however, the fetal area under the concentration-time curve (AUC) of glyburide in Bcrp1-/ pregnant mice was two times greater than that in wild-type mice (Zhou et al., 2008). It is worth noting that the amount of glyburide entering the fetus only accounts for a small fraction of the total amount of glyburide in the body (Zhou et al., 2008). These results confirm that BCRP and Bcrp1 are important determinants of fetal exposure to glyburide (Zhou et al., 2008). All these in vitro, ex vivo, and in vivo studies suggest that ABC efflux transporters, particularly BCRP, are important in protecting the fetus from exposure to glyburide. Thus, if a drug known to be a BCRP inhibitor is co-administered with glyburide, fetal exposure to glyburide may be increased through inhibition of placental BCRP. #### 5.4 Placental metabolism of glyburide The placenta may also protect the fetus by metabolizing drugs or xenobiotics ingested by the mother. Human term placentas have been used in several studies to characterize placental metabolism of glyburide (Jain et al., 2008; Zharikova et al., 2009; Zharikova et al., 2007). Zharikova et al. reported that placental microsomes converted ~87% of glyburide to the M5 Glyburide Disposition During Pregnancy 335 pregnant women with gestational diabetes as compared to non-pregnant women with type II diabetes mellitus (Hebert et al., 2009). This finding implies the need for further evaluation and dosage optimization for glyburide during pregnancy. The mechanism of such a change in glyburide disposition during pregnancy has not been fully understood, but is likely related to increased expression and activity of cytochrome P450 enzymes in the liver, such In summary, glyburide has been increasingly used for the treatment of GDM with similar safety and efficacy to insulin therapy. The mechanistic understanding of pregnancy-induced changes in the disposition of this drug (including fetal exposure) will be important for (2001) ACOG Practice Bulletin. Clinical management guidelines for obstetrician- (2009) Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study: associations with (2010) Hyperglycaemia and Adverse Pregnancy Outcome (HAPO) Study: associations with Anderson GD (2005) Pregnancy-induced changes in pharmacokinetics: a mechanistic-based Anjalakshi C, Balaji V, Balaji MS and Seshiah V (2007) A prospective study comparing Behravan J and Piquette-Miller M (2007) Drug transport across the placenta, role of the ABC drug efflux transporters. Expert Opin Drug Metab Toxicol 3(6):819-830. Bertini AM, Silva JC, Taborda W, Becker F, Lemos Bebber FR, Zucco Viesi JM, Aquim G and Bussing R and Gende A (2002) Severe hypoglycemia from clarithromycin-sulfonylurea drug Cavallari LH and Limdi NA (2009) Warfarin pharmacogenomics. Curr Opin Mol Ther Ceckova-Novotna M, Pavek P and Staud F (2006) P-glycoprotein in the placenta: expression, Coetzee EJ and Jackson WP (1985) The management of non-insulin-dependent diabetes Daskalakis G, Marinopoulos S, Krielesi V, Papapanagiotou A, Papantoniou N, Mesogitis S Dunlop W and Davison JM (1987) Renal haemodynamics and tubular function in human and Antsaklis A (2008) Placental pathology in women with gestational diabetes. localization, regulation and function. Reprod Toxicol 22(3):400-410. during pregnancy. Diabetes Res Clin Pract 1(5):281-287. pregnancy. Baillieres Clin Obstet Gynaeco l1(4):769-787. insulin and glibenclamide in gestational diabetes mellitus in Asian Indian women. Engel Ribeiro T (2005) Perinatal outcomes and the use of oral hypoglycemic agents. 200, December 1994). Gestational diabetes. Obstet Gynecol 98(3):525-538. (2002) The Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study. Int J Gynaecol gynecologists. Number 30, September 2001 (replaces Technical Bulletin Number optimizing dosage guidelines for glyburide during pregnancy. neonatal anthropometrics. Diabetes 58(2):453-459. maternal body mass index. BJOG 117(5):575-584. approach. Clin Pharmacokinet 44(10):989-1008. Diabetes Res Clin Pract 76(3):474-475. interaction. Diabetes Care 25(9):1659-1661. Acta Obstet Gynecol Scand 87(4):403-407. J Perinat Med 33(6):519-523. 11(3):243-251. as CYP2C9 and CYP3A. 7. References Obstet 78(1):69-77. metabolite (ethylene-hydroxylated glyburide), and the rest to other metabolites (Zharikova et al., 2007). When compared to human liver microsomes; however, the total Vmax for all metabolites was much lower for placental microsomes (13 ± 0.8 pmol/min/mg protein) than human liver microsomes (213 ± 37 pmol/min/mg protein). Although the relative contribution of placental drug-metabolizing enzymes to the overall disposition of glyburide may in fact be minimal, the formation of M5 in such close proximity to the fetus could have clinical implications for fetal metabolite exposure. However, at this time, the pharmacological activity of M5 is unknown. Zharikova et al. further identified CYP19 to be the major drug-metabolizing enzyme responsible for the biotransformation of glyburide to M5 in the human placenta (Zharikova et al., 2009). The intrinsic clearance of CYP19 for glyburide was 0.02 µL/min/pmol CYP and only represented 1.8% of the overall intrinsic clearance of human liver microsomes for glyburide. Jain et al. studied glyburide metabolism using placental microsomes isolated from human term placentas from women with uncomplicated pregnancies, women with GDM on diet therapy, or women with GDM on glyburide (Jain et al., 2008). They found that placental microsomes from uncomplicated pregnancies showed higher M1 and M2 metabolite formation rates compared to placentas from women with GDM on diet therapy or on diet therapy plus glyburide. However, there was no difference in glyburide metabolism between placentas from diet therapy and diet therapy plus glyburide (Jain et al., 2008). The differences in placental microsomal metabolite formation may reflect the effects of GDM on the placenta. Histologic abnormalities in the placenta are more common in women with GDM than non-diabetic controls (Daskalakis et al., 2008). Based upon the results obtained so far, the placenta appears to play a very minor role in determining maternal disposition of glyburide, but may play a significant role in controlling fetal exposure to the drug and metabolites. Very limited data is available in this regard, and the role of placental metabolism in controlling fetal drug exposure warrants further investigation. #### 6. Conclusions Although insulin therapy has been the "gold standard" for the treatment of GDM, the increasing use of oral anti-diabetic agents such as glyburide and metformin has begun to change the standard of care. Glyburide is a second generation sulfonylurea. Clinical studies demonstrate that glyburide is a safe alternative to insulin therapy for the treatment of GDM due to its similar efficacy to insulin, relatively low fetal exposure, lower cost and ease of administration. The pharmacokinetic properties of glyburide resulting in low fetal exposure include: high plasma protein binding, a relatively short elimination half-life, and efflux transport by ABC transporters such as BCRP in the placenta. Glyburide is also metabolized in the placenta by CYP19, which may limit fetal exposure to the parent compound but simultaneously expose the fetus to metabolites. However, it is reassuring that the currently used glyburide dosage range for pregnant women with GDM has comparable maternal, fetal and neonatal outcomes as insulin therapy. In addition to the concern of fetal exposure, physiological changes that occur during pregnancy may alter the pharmacokinetics of glyburide, thus affecting the safety and efficacy of the drug for both the mother and the fetus. Indeed, a recent clinical study has demonstrated that the apparent oral clearance of glyburide is increased two-fold in pregnant women with gestational diabetes as compared to non-pregnant women with type II diabetes mellitus (Hebert et al., 2009). This finding implies the need for further evaluation and dosage optimization for glyburide during pregnancy. The mechanism of such a change in glyburide disposition during pregnancy has not been fully understood, but is likely related to increased expression and activity of cytochrome P450 enzymes in the liver, such as CYP2C9 and CYP3A. In summary, glyburide has been increasingly used for the treatment of GDM with similar safety and efficacy to insulin therapy. The mechanistic understanding of pregnancy-induced changes in the disposition of this drug (including fetal exposure) will be important for optimizing dosage guidelines for glyburide during pregnancy. #### 7. References 334 Gestational Diabetes metabolite (ethylene-hydroxylated glyburide), and the rest to other metabolites (Zharikova et al., 2007). When compared to human liver microsomes; however, the total Vmax for all metabolites was much lower for placental microsomes (13 ± 0.8 pmol/min/mg protein) than human liver microsomes (213 ± 37 pmol/min/mg protein). Although the relative contribution of placental drug-metabolizing enzymes to the overall disposition of glyburide may in fact be minimal, the formation of M5 in such close proximity to the fetus could have clinical implications for fetal metabolite exposure. However, at this time, the pharmacological activity of M5 is unknown. Zharikova et al. further identified CYP19 to be the major drug-metabolizing enzyme responsible for the biotransformation of glyburide to M5 in the human placenta (Zharikova et al., 2009). The intrinsic clearance of CYP19 for glyburide was 0.02 µL/min/pmol CYP and only represented 1.8% of the overall intrinsic Jain et al. studied glyburide metabolism using placental microsomes isolated from human term placentas from women with uncomplicated pregnancies, women with GDM on diet therapy, or women with GDM on glyburide (Jain et al., 2008). They found that placental microsomes from uncomplicated pregnancies showed higher M1 and M2 metabolite formation rates compared to placentas from women with GDM on diet therapy or on diet therapy plus glyburide. However, there was no difference in glyburide metabolism between placentas from diet therapy and diet therapy plus glyburide (Jain et al., 2008). The differences in placental microsomal metabolite formation may reflect the effects of GDM on the placenta. Histologic abnormalities in the placenta are more common in women with Based upon the results obtained so far, the placenta appears to play a very minor role in determining maternal disposition of glyburide, but may play a significant role in controlling fetal exposure to the drug and metabolites. Very limited data is available in this regard, and the role of placental metabolism in controlling fetal drug exposure warrants further Although insulin therapy has been the "gold standard" for the treatment of GDM, the increasing use of oral anti-diabetic agents such as glyburide and metformin has begun to change the standard of care. Glyburide is a second generation sulfonylurea. Clinical studies demonstrate that glyburide is a safe alternative to insulin therapy for the treatment of GDM due to its similar efficacy to insulin, relatively low fetal exposure, lower cost and ease of administration. The pharmacokinetic properties of glyburide resulting in low fetal exposure include: high plasma protein binding, a relatively short elimination half-life, and efflux transport by ABC transporters such as BCRP in the placenta. Glyburide is also metabolized in the placenta by CYP19, which may limit fetal exposure to the parent compound but simultaneously expose the fetus to metabolites. However, it is reassuring that the currently used glyburide dosage range for pregnant women with GDM has comparable maternal, In addition to the concern of fetal exposure, physiological changes that occur during pregnancy may alter the pharmacokinetics of glyburide, thus affecting the safety and efficacy of the drug for both the mother and the fetus. Indeed, a recent clinical study has demonstrated that the apparent oral clearance of glyburide is increased two-fold in clearance of human liver microsomes for glyburide. GDM than non-diabetic controls (Daskalakis et al., 2008). fetal and neonatal outcomes as insulin therapy. investigation. 6. Conclusions Glyburide Disposition During Pregnancy 337 Landon MB, Catalano PM, Gabbe SG (2007) Diabetes mellitus complicating pregnancy. Langer O, Conway DL, Berkus MD, Xenakis EM and Gonzales O (2000) A comparison of Leiba A, Leibowitz A and Grossman E (2004) An unusual case of hypoglycemia in a diabetic Lilja JJ, Niemi M, Fredrikson H and Neuvonen PJ (2007) Effects of clarithromycin and Loebstein R, Lalkin A and Koren G (1997) Pharmacokinetic changes during pregnancy and Mao Q (2008) BCRP/ABCG2 in the placenta: expression, function and regulation. 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Curr Pollex E, Lubetsky A and Koren G (2008) The role of placental breast cancer resistance Ramos GA, Jacobson GF, Kirby RS, Ching JY and Field DR (2007) Comparison of glyburide CYP3A and P-glycoprotein. J Pharmacol Exp Ther 316(3):1202-1209. Pregnancies (5), Elsevier Churchill Livingstone, Philadelphia, PA. their clinical relevance. Clin Pharmacokinet 33(5):328-343. maternal serum. Am J Obstet Gynecol 106(3):388-399. disappearance rate. Xenobiotica 34(5):415-427. Pharm Biotechnol 12(4):674-685. Clin Pharmacol Ther 72(3):326-332. Opin Obstet Gynecol 23(2):72-75. outcomes. Am J Obstet Gynecol 195(4):1090-1094. 25(1):51-63. 343(16):1134-1138. 63(6):732-740. 25(6):1244-1255. patient. Ann Emerg Med 44(4):427-428. Chapter 37, In: Gabbe SG, Niebyl JR, Simpson JL ed., Obstetrics - Normal and Problem glyburide and insulin in women with gestational diabetes mellitus. N Engl J Med grapefruit juice on the pharmacokinetics of glibenclamide. Br J Clin Pharmacol pharmacokinetics of anti-HIV protease inhibitors during pregnancy: the role of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate The evidence for effectiveness and safety. Best Pract Res Clin Obstet Gynaecol and glimepiride pharmacokinetics in subjects with different CYP2C9 genotypes. protein in the efflux of glyburide across the human placenta. Placenta 29(8):743-747. and insulin for the management of gestational diabetics with markedly elevated oral glucose challenge test and fasting hyperglycemia. J Perinatol 27(5):262-267. Rochon M, Rand L, Roth L and Gaddipati S (2006) Glyburide for the management of gestational diabetes: risk factors predictive of failure and associated pregnancy Feldman JM (1985) Glyburide: a second-generation sulfonylurea hypoglycemic agent. Gedeon C, Anger G, Lubetsky A, Miller MP and Koren G (2008a) Investigating the potential Gedeon C, Behravan J, Koren G and Piquette-Miller M (2006) Transport of glyburide by Hebert MF, Easterling TR, Kirby B, Carr DB, Buchanan ML, Rutherford T, Thummel KE, Hebert MF, Ma X, Naraharisetti SB, Krudys KM, Umans JG, Hankins GD, Caritis SN, Hemauer SJ, Patrikeeva SL, Nanovskaya TN, Hankins GD and Ahmed MS (2010) Role of Jain S, Zharikova OL, Ravindran S, Nanovskya TN, Mattison DR, Hankins GD and Ahmed Jonsson A, Rydberg T, Ekberg G, Hallengren B and Melander A (1994) Slow elimination of Karakash SD and Einstein FH (2011) Diabetes in pregnancy: glycemia control guidelines and Kirchheiner J, Brockmoller J, Meineke I, Bauer S, Rohde W, Meisel C and Roots I (2002) Klieger C, Pollex E, Kazmin A and Koren G (2009) Hypoglycemics: pharmacokinetic considerations during pregnancy. Ther Drug Monit 31(5):533-541. Jovanovic L and Pettitt DJ (2001) Gestational diabetes mellitus. JAMA 286(20):2516-2518. Kahn BF, Davies JK, Lynch AM, Reynolds RM and Barbour LA (2006) Predictors of rosiglitazone, and metformin. Am J Obstet Gynecol 202(4):383 e381-387. Jacobson GF, Ramos GA, Ching JY, Kirby RS, Ferrara A and Field DR (2005) Comparison of managed care organization. Am J Obstet Gynecol 193(1):118-124. glyburide in NIDDM subjects. Diabetes Care 17(2):142-145. rationale. Curr Opin Endocrinol Diabetes Obes 18:99-103. clinical practice. Clin Pharmacol Ther 85(6):607-614. glyburide efflux in the human placenta. J Obstet Gynaecol 28(5):485-489. Gedeon C, Anger G, Piquette-Miller M and Koren G (2008b) Breast cancer resistance protein: Pharmacotherapy 5(2):43-62. Placenta 29(1):39-43. 12):1096-1102. 84(2):248-253. Am J Perinatol 25(3):169-174. 107(6):1303-1309. 296. History, chemistry, metabolism, pharmacokinetics, clinical use and adverse effects. role of multi-drug resistance protein (MRP) transporters in fetal to maternal mediating the trans-placental transfer of glyburide across the human placenta. placental ABC transporters: implications in fetal drug exposure. Placenta 27(11- Fishbein DP and Unadkat JD (2008) Effects of pregnancy on CYP3A and Pglycoprotein activities as measured by disposition of midazolam and digoxin: a University of Washington specialized center of research study. Clin Pharmacol Ther Miodovnik M, Mattison DR, Unadkat JD, Kelly EJ, Blough D, Cobelli C, Ahmed MS, Snodgrass WR, Carr DB, Easterling TR and Vicini P (2009) Are we optimizing gestational diabetes treatment with glyburide? The pharmacologic basis for better human placental apical membrane transporters in the efflux of glyburide, glyburide and insulin for the management of gestational diabetes in a large MS (2008) Glyburide metabolism by placentas of healthy and gestational diabetics. glyburide failure in the treatment of gestational diabetes. Obstet Gynecol Impact of CYP2C9 amino acid polymorphisms on glyburide kinetics and on the insulin and glucose response in healthy volunteers. Clin Pharmacol Ther 71(4):286- 1. Introduction (Foster-Powell & Cheung, 1998). women bearing age 20 Canada Exercise Guidelines for Women with Gestational Diabetes 1.1 Obesity and type 2 diabetes prevalence are increasing among women of child- 1.2 Women of child-bearing age are inactive, particularly overweight and obese Low levels of physical activity may be contributing to the obesity and type 2 diabetes epidemics in women of child-bearing age. Physical activity levels may be evaluated using different tools but pedometers and accelerometers provide an accurate and objective method of measuring walking and other ambulatory activities. Physical activity levels based on step counts have been defined: <5,000 steps per day = sedentary, 5,000-7,499 steps per day = low active, 7,500-9,999 steps per day = somewhat active, 10,000-12,499 = active and ≥ There has been a significant increase over the past few decades in the the prevalence of women of child-bearing age who are overweight (body mass index, BMI ≥ 25 kg/m2) or obese (BMI ≥ 30 kg/m2). Women of childbearing age are at an increased risk for obesity (Villamor & Cnattingius, 2006) and type 2 diabetes (Lipscombe & Hux, 2007) because of excessive weight gain during pregnancy and weight retention after delivery (Rooney et al., 2005). Data from the 2007-2008 "National Health and Nutrition Examination Survey" (NHANES) showed that 60% and 34% of American women aged 20-39 years were overweight or obese, respectively (Flegal et al., 2010). Abdominal obesity (i.e. waist circumference ≥ 88 cm; (Lean et al., 1995)), a risk factor for many chronic diseases (Després, 2001) also increased and reached 51.3% in 2007-2008 (Ford et al., 2010). Data from the "Pregnancy Risk Assessment Monitoring System" in nine states indicated that prepregnancy obesity increased from 13% to 22% between 1993 and 2002 (Kim et al., 2007). Worldwide population estimates of pre-pregnancy overweight is approximately 34% (Callaway et al., 2006; LaCoursiere et al., 2005) and that of pre-pregnancy obesity is 25% (Chu et al., 2009), which may be an underestimation. This escalating problem may contribute to the obesity and diabetes epidemics, as overweight women who gain 10% or more of their pre-pregnancy body mass are at higher risk for complications such as gestational diabetes mellitus (GDM; (Carducci et al., 1999)) and pregnancy-induced hypertension (Pole & Dodds, 1999). Additionally, higher recurrence of GDM has been associated with greater pre-pregnancy weight, BMI and excessive pregnancy weight gain Michelle F. Mottola and Stephanie-May Ruchat University of Western Ontario, London,Ontario ## Exercise Guidelines for Women with Gestational Diabetes Michelle F. Mottola and Stephanie-May Ruchat University of Western Ontario, London,Ontario Canada #### 1. Introduction 338 Gestational Diabetes Rydberg T, Jonsson A, Roder M and Melander A (1994) Hypoglycemic activity of glyburide (glibenclamide) metabolites in humans. Diabetes Care 17(9):1026-1030. Schelleman H, Bilker WB, Brensinger CM, Wan F and Hennessy S (2010) Anti-infectives and Tracy TS, Venkataramanan R, Glover DD and Caritis SN (2005) Temporal changes in drug Yin OQ, Tomlinson B and Chow MS (2005) CYP2C9, but not CYP2C19, polymorphisms Yogev Y, Ben-Haroush A, Chen R, Rosenn B, Hod M and Langer O (2004) Undiagnosed Zhang H, Wu X, Wang H, Mikheev AM, Mao Q and Unadkat JD (2008) Effect of pregnancy Zharikova OL, Ravindran S, Nanovskaya TN, Hill RA, Hankins GD and Ahmed MS (2007) Zheng HX, Huang Y, Frassetto LA and Benet LZ (2009) Elucidating rifampin's inducing and Zhou L, Naraharisetti SB, Wang H, Unadkat JD, Hebert MF and Mao Q (2008) The breast Zhou L, Zhang Y, Hebert MF, Unadkat JD and Mao Q (2010b) Increased glyburide clearance in the pregnant mouse model. Drug Metab Dispos 38(9):1403-1406. Ther 88(2):214-222. 1490. 73(3):949-959. Obstet Gynecol 192(2):633-639. subjects. Clin Pharmacol Ther 78(4):370-377. pregnancy. Obstet Gynecol 104(1):88-93. and baboon. Biochem Pharmacol 73(12):2012-2019. metabolism. Biopharm Drug Dispos 31(4):228-242. the risk of severe hypoglycemia in users of glipizide or glyburide. Clin Pharmacol metabolism (CYP1A2, CYP2D6 and CYP3A Activity) during pregnancy. Am J affect the pharmacokinetics and pharmacodynamics of glyburide in Chinese asymptomatic hypoglycemia: diet, insulin, and glyburide for gestational diabetic on cytochrome P450 3a and P-glycoprotein expression and activity in the mouse: mechanisms, tissue specificity, and time course. Mol Pharmacol 74(3):714-723. Zharikova OL, Fokina VM, Nanovskaya TN, Hill RA, Mattison DR, Hankins GD and Ahmed MS (2009) Identification of the major human hepatic and placental enzymes responsible for the biotransformation of glyburide. Biochem Pharmacol 78(12):1483- Kinetics of glyburide metabolism by hepatic and placental microsomes of human inhibiting effects on glyburide pharmacokinetics and blood glucose in healthy volunteers: unmasking the differential effects of enzyme induction and transporter inhibition for a drug and its primary metabolite. Clin Pharmacol Ther 85(1):78-85. Zhou L, Naraharisetti SB, Liu L, Wang H, Lin YS, Isoherranen N, Unadkat JD, Hebert MF and Mao Q (2010a) Contributions of human cytochrome P450 enzymes to glyburide cancer resistance protein (Bcrp1/Abcg2) limits fetal distribution of glyburide in the pregnant mouse: an Obstetric-Fetal Pharmacology Research Unit Network and University of Washington Specialized Center of Research Study. Mol Pharmacol #### 1.1 Obesity and type 2 diabetes prevalence are increasing among women of childbearing age There has been a significant increase over the past few decades in the the prevalence of women of child-bearing age who are overweight (body mass index, BMI ≥ 25 kg/m2) or obese (BMI ≥ 30 kg/m2). Women of childbearing age are at an increased risk for obesity (Villamor & Cnattingius, 2006) and type 2 diabetes (Lipscombe & Hux, 2007) because of excessive weight gain during pregnancy and weight retention after delivery (Rooney et al., 2005). Data from the 2007-2008 "National Health and Nutrition Examination Survey" (NHANES) showed that 60% and 34% of American women aged 20-39 years were overweight or obese, respectively (Flegal et al., 2010). Abdominal obesity (i.e. waist circumference ≥ 88 cm; (Lean et al., 1995)), a risk factor for many chronic diseases (Després, 2001) also increased and reached 51.3% in 2007-2008 (Ford et al., 2010). Data from the "Pregnancy Risk Assessment Monitoring System" in nine states indicated that prepregnancy obesity increased from 13% to 22% between 1993 and 2002 (Kim et al., 2007). Worldwide population estimates of pre-pregnancy overweight is approximately 34% (Callaway et al., 2006; LaCoursiere et al., 2005) and that of pre-pregnancy obesity is 25% (Chu et al., 2009), which may be an underestimation. This escalating problem may contribute to the obesity and diabetes epidemics, as overweight women who gain 10% or more of their pre-pregnancy body mass are at higher risk for complications such as gestational diabetes mellitus (GDM; (Carducci et al., 1999)) and pregnancy-induced hypertension (Pole & Dodds, 1999). Additionally, higher recurrence of GDM has been associated with greater pre-pregnancy weight, BMI and excessive pregnancy weight gain (Foster-Powell & Cheung, 1998). #### 1.2 Women of child-bearing age are inactive, particularly overweight and obese women Low levels of physical activity may be contributing to the obesity and type 2 diabetes epidemics in women of child-bearing age. Physical activity levels may be evaluated using different tools but pedometers and accelerometers provide an accurate and objective method of measuring walking and other ambulatory activities. Physical activity levels based on step counts have been defined: <5,000 steps per day = sedentary, 5,000-7,499 steps per day = low active, 7,500-9,999 steps per day = somewhat active, 10,000-12,499 = active and ≥ Exercise Guidelines for Women with Gestational Diabetes 341 29.1 kg) being at increased risk for IGT, compared to those women in the lowest quartile of GWG (i.e -9.4 kg to 7.9 kg), independent of pre-pregnancy BMI (Herring et al., 2009). Finally, Kieffer et al. showed an estimated 2% increase in risk of GDM associated with each pound of maternal weight gained after adjusting for pre-pregnancy BMI (Kieffer et al., 2001). Excessive GWG, especially during the first trimester of pregnancy, has been found to be associated with an increased risk of GDM (Hedderson et al., 2010; Morisset et al., 2011), suggesting that timing of excessive GWG may be important and may be related to different Data from the 2006 "Maternity Experiences Survey", conducted in Canada, showed that 52% of Canadian women gained more than the recommended weight in pregnancy (Lowell & Miller, 2010). Moreover, the survey showed that women with a higher pre-pregnancy BMI were more likely than normal weight and underweight women to gain more weight than recommended. Fifty-five percent of overweight and obese women gained more weight than recommended, compared with 41% of those who were in the normal range and 26% of those who were underweight (Lowell & Miller, 2010). Previous studies reported similar findings, that is, overweight and obese women are more likely to exceed their target weight gain (Caulfield et al., 1996; Tovar et al., 2009; Saldana et al., 2006; Stuebe et al., 2009). Avoidance of excessive GWG may therefore constitute an opportunity for the prevention of impaired 1.5 Insulin resistance, the link between overweight/obesity, excessive gestational The underlying mechanism linking pre-pregnancy overweight/obesity to IGT or GDM is insulin resistance, coupled with an inadequate insulin response due to pancreatic beta-cell dysfunction (Buchanan & Xiang, 2005). Althought overweight and obese women present a similar 50% decrease in insulin sensitivity over the period of gestation, they are more insulin resistant than normal weight women throughout pregnancy (Catalano, 2010). Positive correlations have been reported between maternal body weight, BMI, fat mass and insulin resistance (Ahlsson et al., 2010). Maternal fat mass explained 36% of the variation in insulin resistance, and insulin resistance accounted for 62% of the variation in glucose production (Ahlsson et al., 2010). Adipose tissue is an active organ that secretes numerous factors involved in the development of insulin resistance, such as free fatty acids, leptin, adiponectin, interleukin-6 (IL-6), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-α (TNF-α), resistin, retinol binding protein 4, visfatin, etc. (Rasouli & Kern, 2008). In obese non-pregnant individuals, adipose tissue releases increased amounts of these factors, except adiponectin, which may explain the link between obesity, insulin resistance and type 2 diabetes (Kahn et al., 2006). In pregnant women, it has been reported that mean levels of adiponectin declined, and PAI-1 increased as BMI increments increased (Lowe et al., 2010). Recent studies have focused on the role of adipokines during normal pregnancy and pregnancy complicated by GDM and showed that the pattern of change in maternal circulating adipokine levels may be involved in the pathophysiology of GDM (Briana & Excessive GWG may also further enhance normal pregnancy-induced insulin resistance and be linked to impaired glucose metabolism. Maternal adipose tissue represents 30% of GWG (IOM, 1990) but the proportion of GWG attributed to adipose tissue is more important in early pregnancy (Hedderson et al., 2010). The timing of GWG seems to be important as early patterns of metabolic change affecting glucose metabolism regulation. glucose metabolism during pregnancy. Malamitsi-Puchner, 2009). weight gain and impaired glucose metabolism 12,500= highly active (Tudor-Locke et al., 2008). Pedometer data from the "American On the Move" study showed that women aged 18-39 years took approximately 5500 steps per day (Bassett et al., 2010). Similar results were found by Tudor-Locke et al. using accelerometer data from the 2005-2006 NHANES (Tudor-Locke et al., 2010). They reported that women took approximately 5,800 steps per day (Tudor-Locke et al., 2010). Interestingly, the authors also reported that normal weight women took more steps per day compared to overweight and obese women (6,486, 5,069 and 5,782, respectively). In Canada, accelerometer results from the 2007-2008 "Canadian Health Measures Survey" showed that women aged 20-39 years took nearly 9,000 steps per day. Again, obese women were less active compared to normal weight women. Noteworthy, studies conducted on populations that have a lower prevalence of overweight and obesity, like Japan and Australia, reported higher steps per day (Inoue et al., 2006; McCormack et al., 2003). Achievement of public health recommendation (i.e ≥ 30 minutes of moderate-to-vigorous physical activity per day, accumulated in bouts lasting at least 10 minutes, on at least 5 out of 7 days (Canadian Society for Exercise Physiology & ParticipACTION, 2010; WHO, 2010)) were also examined using accelerometers. Results showed that less than 5% of women of child-bearing age meet these recommendations (Colley et al., 2011; Troiano et al., 2008; Tudor-Locke et al., 2010). Taken together, these findings showed that women of child-bearing age are inactive and suggested that being sedentary, and the prevalence of physical inactivity, may be contributing to the obesity and diabetes epidemics. #### 1.3 Maternal obesity is associated with impaired glucose metabolism Entering pregnancy overweight or obese are closely linked to numerous unfavourable pregnancy outcomes, such as the development of impaired glucose tolerance (IGT) or GDM (Catalano & Ehrenberg, 2006; Chu et al., 2007; Davies et al., 2010; Nelson et al., 2009). Women who are obese prior to pregnancy are more likely to develop IGT as compared to normal weight women (Saldana et al., 2006; Tovar et al., 2009). Similarly, the risk of developing GDM has been shown to increase with increasing BMI: overweight and obese women have 2.14 (95% CI 1.82-2.53) and 3.56 (95% CI 3.05-4.21) times the risk of developing GDM compared to normal weight women (Chu et al., 2007). GDM prevalence rates are 0.7% in normal weight, 2.3% in overweight, 4.8% in obese and 5.5% in extremely obese (BMI ≥ 35 kg/m2) women (Kim et al., 2010). More than 70% of women with GDM have a BMI of 25 kg/m2 or higher (Kim et al., 2010). Similarly, maternal abdominal adiposity in early pregnancy has been associated with a positive glucose challenge test (i.e. glucose levels ≥ 7.8 mmol/L) and an increased risk of GDM (Brisson et al., 2010; Martin et al., 2009). #### 1.4 Excessive gestational weight gain is associated with impaired glucose metabolism Another risk factor for developing IGT and GDM is excessive gestational weight gain (GWG). A cohort study revealed a positive relationship between weight gain in excess of the Institute of Medicine (IOM) guidelines (IOM, 1990) and the development of IGT, although these findings were limited to Hispanic women with a pre-pregnancy BMI ≥ 35 kg/m2 (Tovar et al., 2009). Saldana et al. found in the "Pregnancy, Infection and Nutrition" (PIN) cohort a two-fold increased risk of IGT among overweight women who gained excessive weight up to the end of the second trimester (Saldana et al., 2006). Data from the "Project Viva" showed similar findings, with women in the highest quartile of GWG (i.e. 12.9 kg to 12,500= highly active (Tudor-Locke et al., 2008). Pedometer data from the "American On the Move" study showed that women aged 18-39 years took approximately 5500 steps per day (Bassett et al., 2010). Similar results were found by Tudor-Locke et al. using accelerometer data from the 2005-2006 NHANES (Tudor-Locke et al., 2010). They reported that women took approximately 5,800 steps per day (Tudor-Locke et al., 2010). Interestingly, the authors also reported that normal weight women took more steps per day compared to overweight and obese women (6,486, 5,069 and 5,782, respectively). In Canada, accelerometer results from the 2007-2008 "Canadian Health Measures Survey" showed that women aged 20-39 years took nearly 9,000 steps per day. Again, obese women were less active compared to normal weight women. Noteworthy, studies conducted on populations that have a lower prevalence of overweight and obesity, like Japan and Australia, reported higher steps per day (Inoue et al., 2006; McCormack et al., 2003). Achievement of public health recommendation (i.e ≥ 30 minutes of moderate-to-vigorous physical activity per day, accumulated in bouts lasting at least 10 minutes, on at least 5 out of 7 days (Canadian Society for Exercise Physiology & ParticipACTION, 2010; WHO, 2010)) were also examined using accelerometers. Results showed that less than 5% of women of child-bearing age meet these recommendations (Colley et al., 2011; Troiano et al., 2008; Tudor-Locke et al., 2010). Taken together, these findings showed that women of child-bearing age are inactive and suggested that being sedentary, and the prevalence of physical inactivity, may be contributing to the obesity and diabetes epidemics. metabolism 1.3 Maternal obesity is associated with impaired glucose metabolism mmol/L) and an increased risk of GDM (Brisson et al., 2010; Martin et al., 2009). 1.4 Excessive gestational weight gain is associated with impaired glucose Another risk factor for developing IGT and GDM is excessive gestational weight gain (GWG). A cohort study revealed a positive relationship between weight gain in excess of the Institute of Medicine (IOM) guidelines (IOM, 1990) and the development of IGT, although these findings were limited to Hispanic women with a pre-pregnancy BMI ≥ 35 kg/m2 (Tovar et al., 2009). Saldana et al. found in the "Pregnancy, Infection and Nutrition" (PIN) cohort a two-fold increased risk of IGT among overweight women who gained excessive weight up to the end of the second trimester (Saldana et al., 2006). Data from the "Project Viva" showed similar findings, with women in the highest quartile of GWG (i.e. 12.9 kg to Entering pregnancy overweight or obese are closely linked to numerous unfavourable pregnancy outcomes, such as the development of impaired glucose tolerance (IGT) or GDM (Catalano & Ehrenberg, 2006; Chu et al., 2007; Davies et al., 2010; Nelson et al., 2009). Women who are obese prior to pregnancy are more likely to develop IGT as compared to normal weight women (Saldana et al., 2006; Tovar et al., 2009). Similarly, the risk of developing GDM has been shown to increase with increasing BMI: overweight and obese women have 2.14 (95% CI 1.82-2.53) and 3.56 (95% CI 3.05-4.21) times the risk of developing GDM compared to normal weight women (Chu et al., 2007). GDM prevalence rates are 0.7% in normal weight, 2.3% in overweight, 4.8% in obese and 5.5% in extremely obese (BMI ≥ 35 kg/m2) women (Kim et al., 2010). More than 70% of women with GDM have a BMI of 25 kg/m2 or higher (Kim et al., 2010). Similarly, maternal abdominal adiposity in early pregnancy has been associated with a positive glucose challenge test (i.e. glucose levels ≥ 7.8 29.1 kg) being at increased risk for IGT, compared to those women in the lowest quartile of GWG (i.e -9.4 kg to 7.9 kg), independent of pre-pregnancy BMI (Herring et al., 2009). Finally, Kieffer et al. showed an estimated 2% increase in risk of GDM associated with each pound of maternal weight gained after adjusting for pre-pregnancy BMI (Kieffer et al., 2001). Excessive GWG, especially during the first trimester of pregnancy, has been found to be associated with an increased risk of GDM (Hedderson et al., 2010; Morisset et al., 2011), suggesting that timing of excessive GWG may be important and may be related to different patterns of metabolic change affecting glucose metabolism regulation. Data from the 2006 "Maternity Experiences Survey", conducted in Canada, showed that 52% of Canadian women gained more than the recommended weight in pregnancy (Lowell & Miller, 2010). Moreover, the survey showed that women with a higher pre-pregnancy BMI were more likely than normal weight and underweight women to gain more weight than recommended. Fifty-five percent of overweight and obese women gained more weight than recommended, compared with 41% of those who were in the normal range and 26% of those who were underweight (Lowell & Miller, 2010). Previous studies reported similar findings, that is, overweight and obese women are more likely to exceed their target weight gain (Caulfield et al., 1996; Tovar et al., 2009; Saldana et al., 2006; Stuebe et al., 2009). Avoidance of excessive GWG may therefore constitute an opportunity for the prevention of impaired glucose metabolism during pregnancy. #### 1.5 Insulin resistance, the link between overweight/obesity, excessive gestational weight gain and impaired glucose metabolism The underlying mechanism linking pre-pregnancy overweight/obesity to IGT or GDM is insulin resistance, coupled with an inadequate insulin response due to pancreatic beta-cell dysfunction (Buchanan & Xiang, 2005). Althought overweight and obese women present a similar 50% decrease in insulin sensitivity over the period of gestation, they are more insulin resistant than normal weight women throughout pregnancy (Catalano, 2010). Positive correlations have been reported between maternal body weight, BMI, fat mass and insulin resistance (Ahlsson et al., 2010). Maternal fat mass explained 36% of the variation in insulin resistance, and insulin resistance accounted for 62% of the variation in glucose production (Ahlsson et al., 2010). Adipose tissue is an active organ that secretes numerous factors involved in the development of insulin resistance, such as free fatty acids, leptin, adiponectin, interleukin-6 (IL-6), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-α (TNF-α), resistin, retinol binding protein 4, visfatin, etc. (Rasouli & Kern, 2008). In obese non-pregnant individuals, adipose tissue releases increased amounts of these factors, except adiponectin, which may explain the link between obesity, insulin resistance and type 2 diabetes (Kahn et al., 2006). In pregnant women, it has been reported that mean levels of adiponectin declined, and PAI-1 increased as BMI increments increased (Lowe et al., 2010). Recent studies have focused on the role of adipokines during normal pregnancy and pregnancy complicated by GDM and showed that the pattern of change in maternal circulating adipokine levels may be involved in the pathophysiology of GDM (Briana & Malamitsi-Puchner, 2009). Excessive GWG may also further enhance normal pregnancy-induced insulin resistance and be linked to impaired glucose metabolism. Maternal adipose tissue represents 30% of GWG (IOM, 1990) but the proportion of GWG attributed to adipose tissue is more important in early pregnancy (Hedderson et al., 2010). The timing of GWG seems to be important as early Exercise Guidelines for Women with Gestational Diabetes 343 activity both before and during pregnancy presented a greater risk reduction in both GDM and IGT (51% and 30%, respectively) compared with women reporting no activities in both time periods (Oken et al., 2006). Dempsey et al. reported that women who participated in any physical activity during the year before getting pregnant experienced a 56% risk reduction in GDM compared with inactive women, but those who engaged in physical activity during both time periods experienced a 69% reduced risk (Dempsey et al., 2004b). It has also been shown that pre-gravid physical activity (vigorous activity) was associated with a reduced risk of IGT in pregnancy, an effect likely mediated by enhanced insulin Similar results were found when investigating the effect of physical activity during pregnancy. Dye et al. reported that exercise during pregnancy (i.e. 30 minutes of exercise once or more per week) was associated with reduced rates of GDM, but only among women with a pre-pregnancy BMI>33 kg/m2 (Dye et al., 1997). A study including only Latina women showed that those in the lowest quartile of sports or exercise in mid-pregnancy had a two-fold increased risk of IGT as compared with women in the highest quartile (Gollenberg et al., 2010). Similar association was found with total sedentary behaviours (a composite score of TV watching, sitting at work and sport or exercise reverse scores) (Gollenberg et al., 2010). A case-control study reported that women who participated in any recreational physical activity during the first 20 weeks of pregnancy experienced a 48% reduction in GDM risk, as compared with inactive women (Dempsey et al., 2004a). It has also been shown that women who were previously inactive but became active during pregnancy had a 57% risk reduction in GDM compared to those who remained inactive (Liu et al., 2008). Finally, a prospective study reported that increased physical activity during pregnancy was associated with decreased fasting insulin concentrations (Liu et al., 2010), suggesting that being active during pregnancy may prevent impaired glucose metabolism 2.2 Maternal physical activity is associated with preventing excessive gestational Another mechanism through which physical activity may prevent impaired glucose metabolism is by limiting excessive GWG. Prospective data from the "Project Viva" showed that 30 minutes per day of walking, vigorous physical activity or total physical activity during pregnancy were inversely associated with the risk of excessive GWG (Stuebe et al., 2009). Similar findings were found by Olson et al. who found that decreased physical activity was associated with excessive GWG (Olson & Strawderman, 2003). Lifestyle intervention studies also showed successful results with respect to the prevention of excessive GWG. Two reports using meta-analyses of intervention trials showed that exercise programs, combined with or without nutrition counselling, helps to limit GWG (Streuling et al., 2010a; Streuling et al., 2010b). Interventions that combined exercise and dietary counseling were found to be more successful in limiting GWG, with an average reduction of GWG of 1.2 kg (p=0.01) found in the intervention groups compared to the control groups (Streuling et al., 2010b). Three studies found significant lower GWG in the exercise plus nutrition intervention group compared with the control groups (Asbee et al., 2009; Claesson et al., 2008; Shirazian et al., 2010), 3 studies found a non significant trend in lower GWG in the intervention group compared with the control group (Gray-Donald et al., 2000; Guelinckx et al., 2010; Olson et al., 2004) and three found no significant results (Hui et sensitivity (Retnakaran et al., 2009). weight gain through attenuation of pregnancy-induced insulin resistance. excessive GWG has been associated with GDM risk (Hedderson et al., 2010; Morisset et al., 2011). It is therefore possible that rapid GWG in early pregnancy results in an increased release of "diabetogenic" factors, exacerbating earlier pregnancy-induced insulin resistance and thereby leading to IGT or GDM in women having beta-cell defects. In summary, an increased number of women of child-bearing age are overweight or obese and physically inactive and these women will likely start their pregnancy overweight or obese and sedentary. These factors have been associated with an increased risk for excessive GWG, a deterioration in insulin resistance beyond that induced by pregnancy and impaired glucose metabolism. Therefore, any intervention that helps to limit excessive gestational weight gain and attenuate pregnancy-induced insulin resistance may be successful in preventing impaired glucose metabolism. #### 2. Physical activity is part of a healthy pregnancy A minimal amount of physical activity must be maintained to achieve health benefits during pregnancy. Physical inactivity and a sedentary lifestyle may put the mother and fetus at risk for disease through altered maternal pregnancy adaptations (Mottola 2008). In fact, women who were the most active before pregnancy (Chu et al. 2007) and throughout pregnancy (Dye et al. 1997), had the lowest prevelence of GDM. If GDM and IGT can be prevented, overall rates of obesity and type 2 diabetes, especially in at risk population groups may also be decreased. #### 2.1 Maternal physical activity is associated with reduced risk of impaired glucose metabolism Pregnancy-induced insulin resistance develops at the skeletal muscle level (Buchanan & Xiang, 2005). Investigators have demonstrated that exercise increases the rate of glucose uptake into the skeletal muscle, a process that is regulated by the translocation of the glucose transport protein GLUT-4 (Goodyear & Kahn, 1998; Hayashi et al., 1997; Ryder et al., 2001). Regular exercise in non pregnant individuals results in numerous beneficial adaptations in skeletal muscle, including increases in GLUT-4 expression, which contributes to an increased responsiveness of muscle glucose uptake to insulin (i.e. increased muscle insulin sensitivity) (Goodyear & Kahn, 1998; Hayashi et al., 1997; Ryder et al., 2001). This may explain the observed link between level of physical activity and improvement in glucose homeostasis and insulin sensitivity (Goodyear & Kahn, 1998; Koivisto et al., 1986), and a risk reduction of 50% for type 2 diabetes mellitus (T2DM) in at risk individuals (Madden et al., 2008; Yates et al., 2007). These physiological and molecular mechanisms underlying the beneficial effects of exercise may also be present in pregnant women since successful reduction in the risk of developing IGT and GDM have been reported in women who were active either before and/or during pregnancy (Tobias et al., 2010). In the "Nurse Health Study II", women in the highest quintile of physical activity before pregnancy, specifically vigorous activity, had a 20% risk reduction for the development of GDM compared with inactive women (Zhang et al., 2006). In women not performing vigorous activity, brisk walking was also significantly associated with a reduced risk. Similarly, physical activity before pregnancy, particularly vigorous activity, was associated with a 44% risk reduction of GDM and a 24% risk reduction of any antepartum IGT in women participating in "Project Viva" (Oken et al., 2006). Women who reported physical excessive GWG has been associated with GDM risk (Hedderson et al., 2010; Morisset et al., 2011). It is therefore possible that rapid GWG in early pregnancy results in an increased release of "diabetogenic" factors, exacerbating earlier pregnancy-induced insulin resistance In summary, an increased number of women of child-bearing age are overweight or obese and physically inactive and these women will likely start their pregnancy overweight or obese and sedentary. These factors have been associated with an increased risk for excessive GWG, a deterioration in insulin resistance beyond that induced by pregnancy and impaired glucose metabolism. Therefore, any intervention that helps to limit excessive gestational weight gain and attenuate pregnancy-induced insulin resistance may be successful in A minimal amount of physical activity must be maintained to achieve health benefits during pregnancy. Physical inactivity and a sedentary lifestyle may put the mother and fetus at risk for disease through altered maternal pregnancy adaptations (Mottola 2008). In fact, women who were the most active before pregnancy (Chu et al. 2007) and throughout pregnancy (Dye et al. 1997), had the lowest prevelence of GDM. If GDM and IGT can be prevented, overall rates of obesity and type 2 diabetes, especially in at risk population groups may also 2.1 Maternal physical activity is associated with reduced risk of impaired glucose who were active either before and/or during pregnancy (Tobias et al., 2010). In the "Nurse Health Study II", women in the highest quintile of physical activity before pregnancy, specifically vigorous activity, had a 20% risk reduction for the development of GDM compared with inactive women (Zhang et al., 2006). In women not performing vigorous activity, brisk walking was also significantly associated with a reduced risk. Similarly, physical activity before pregnancy, particularly vigorous activity, was associated with a 44% risk reduction of GDM and a 24% risk reduction of any antepartum IGT in women participating in "Project Viva" (Oken et al., 2006). Women who reported physical Pregnancy-induced insulin resistance develops at the skeletal muscle level (Buchanan & Xiang, 2005). Investigators have demonstrated that exercise increases the rate of glucose uptake into the skeletal muscle, a process that is regulated by the translocation of the glucose transport protein GLUT-4 (Goodyear & Kahn, 1998; Hayashi et al., 1997; Ryder et al., 2001). Regular exercise in non pregnant individuals results in numerous beneficial adaptations in skeletal muscle, including increases in GLUT-4 expression, which contributes to an increased responsiveness of muscle glucose uptake to insulin (i.e. increased muscle insulin sensitivity) (Goodyear & Kahn, 1998; Hayashi et al., 1997; Ryder et al., 2001). This may explain the observed link between level of physical activity and improvement in glucose homeostasis and insulin sensitivity (Goodyear & Kahn, 1998; Koivisto et al., 1986), and a risk reduction of 50% for type 2 diabetes mellitus (T2DM) in at risk individuals (Madden et al., 2008; Yates et al., 2007). These physiological and molecular mechanisms underlying the beneficial effects of exercise may also be present in pregnant women since successful reduction in the risk of developing IGT and GDM have been reported in women and thereby leading to IGT or GDM in women having beta-cell defects. preventing impaired glucose metabolism. be decreased. metabolism 2. Physical activity is part of a healthy pregnancy activity both before and during pregnancy presented a greater risk reduction in both GDM and IGT (51% and 30%, respectively) compared with women reporting no activities in both time periods (Oken et al., 2006). Dempsey et al. reported that women who participated in any physical activity during the year before getting pregnant experienced a 56% risk reduction in GDM compared with inactive women, but those who engaged in physical activity during both time periods experienced a 69% reduced risk (Dempsey et al., 2004b). It has also been shown that pre-gravid physical activity (vigorous activity) was associated with a reduced risk of IGT in pregnancy, an effect likely mediated by enhanced insulin sensitivity (Retnakaran et al., 2009). Similar results were found when investigating the effect of physical activity during pregnancy. Dye et al. reported that exercise during pregnancy (i.e. 30 minutes of exercise once or more per week) was associated with reduced rates of GDM, but only among women with a pre-pregnancy BMI>33 kg/m2 (Dye et al., 1997). A study including only Latina women showed that those in the lowest quartile of sports or exercise in mid-pregnancy had a two-fold increased risk of IGT as compared with women in the highest quartile (Gollenberg et al., 2010). Similar association was found with total sedentary behaviours (a composite score of TV watching, sitting at work and sport or exercise reverse scores) (Gollenberg et al., 2010). A case-control study reported that women who participated in any recreational physical activity during the first 20 weeks of pregnancy experienced a 48% reduction in GDM risk, as compared with inactive women (Dempsey et al., 2004a). It has also been shown that women who were previously inactive but became active during pregnancy had a 57% risk reduction in GDM compared to those who remained inactive (Liu et al., 2008). Finally, a prospective study reported that increased physical activity during pregnancy was associated with decreased fasting insulin concentrations (Liu et al., 2010), suggesting that being active during pregnancy may prevent impaired glucose metabolism through attenuation of pregnancy-induced insulin resistance. #### 2.2 Maternal physical activity is associated with preventing excessive gestational weight gain Another mechanism through which physical activity may prevent impaired glucose metabolism is by limiting excessive GWG. Prospective data from the "Project Viva" showed that 30 minutes per day of walking, vigorous physical activity or total physical activity during pregnancy were inversely associated with the risk of excessive GWG (Stuebe et al., 2009). Similar findings were found by Olson et al. who found that decreased physical activity was associated with excessive GWG (Olson & Strawderman, 2003). Lifestyle intervention studies also showed successful results with respect to the prevention of excessive GWG. Two reports using meta-analyses of intervention trials showed that exercise programs, combined with or without nutrition counselling, helps to limit GWG (Streuling et al., 2010a; Streuling et al., 2010b). Interventions that combined exercise and dietary counseling were found to be more successful in limiting GWG, with an average reduction of GWG of 1.2 kg (p=0.01) found in the intervention groups compared to the control groups (Streuling et al., 2010b). Three studies found significant lower GWG in the exercise plus nutrition intervention group compared with the control groups (Asbee et al., 2009; Claesson et al., 2008; Shirazian et al., 2010), 3 studies found a non significant trend in lower GWG in the intervention group compared with the control group (Gray-Donald et al., 2000; Guelinckx et al., 2010; Olson et al., 2004) and three found no significant results (Hui et Exercise Guidelines for Women with Gestational Diabetes 345 exercise 3 to 4 times per week, starting with 15 minutes of aerobic activity at a target heart rate intensity and increasing time slowly to a maximum of 30 minutes per exercise session. All aerobic activity should be preceded by 10- to 15-minutes of warm-up and followed by 10- to 15-minutes of cool-down. Appropriate exercise intensity may be monitored by using target heart-rate zones, the Borg-scale (rating of perceived exertion, RPE) or the "talk test" (Davies et al., 2003). Heart-rate zones that are provided in the guidelines correspond to moderate-intensity exercise (i.e. 60-80% of maximal aerobic capacity, VO2 max). Aerobic exercise in which large muscle groups are used, including walking, stationary cycling, aqua exercise, or low-impact aerobics are recommended for low risk pregnant women (Davies et Overweight and obese women can participate in exercise, if they have no contraindications to being physically active. Twenty medically pre-screened obese and 20 normal weight pregnant women participated in a graded treadmill exercise test to volitional fatigue to examine the impact of obesity on the ventilatory response to weight-bearing exercise during pregnancy (Davenport et al., 2009). We concluded that exercise ventilatory response is increased during pregnancy but is not affected further by obesity during graded treadmill exercise (Davenport et al., 2009). This is important in that there is no apparent ventilatory limitation to submaximal weight-bearing exercise representing daily living activities such as walking, in pregnant obese women, which lends support to the feasibility of exercise prescription in this population group (Mottola, 2009). Target heart rate zones developed for normal weight pregnant women may be too difficult for overweight and obese women to obtain, and thus we developed and validated target heart rate zones for medically prescreened overweight and obese women at a much lower intensity but high enough to achieve aerobic benefits (Davenport et al. 2008a). The intensity is approximately 20-39% heart rate reserve (HRR), and at a normal walking pace which may help with compliance in this Currently, there are no step recommendations for pregnant women but those that have been defined for adults (Tudor-Locke et al., 2008) may be used for pregnant women. As walking is the most reported activity during pregnancy (Evenson et al., 2004; Evenson & Wen, 2010; Mottola & Campbell, 2003; Petersen et al., 2005), providing step recommendations to pregnant women may encourage them to be more active. Previous studies conducted in our laboratory showed that pregnant women took more than 10,000 steps per day when a 40-minute walk Although maternal physical activity has clear health benefits on pregnancy outcomes, and professional societies strongly recommend active promotion of physical activity for pregnant women, most women remain inactive during pregnancy. In Canada, only 30% of pregnant women meet the adult step recommendations of 10,000 steps per day (Cohen et al., 2010). Data from large cohort studies, such as the "Behavioral Risk Factor Surveillance System" (BRFSS), NHANES and PIN showed that the majority of women do not meet the recommendations for physical activity during pregnancy, based on information collected by interviews or questionnaires (Borodulin et al., 2008; Evenson et al., 2004; Evenson & Wen, 2010; Petersen et al., 2005). The guidelines from the ACOG and from the Center for Controlled Disease (CDC)/American College of Sports Medicine (ACSM) both suggest 30 minutes or more of moderate-intensity activity on most of the days of the week, but differ was added to their usual daily activities (Davenport et al., 2008b; Mottola et al., 2010). 2.4 Are pregnant women meeting the physical activity guidelines? al. 2003). population group. al., 2006; Kinnunen et al., 2007; Polley et al., 2002). Findings from intervention studies using only exercise were less consistent than findings from intervention studies combining exercise and nutrition. Seven trials reported a trend for less GWG in the exercise group (Barakat et al., 2009; Cavalcante et al., 2009; Clapp et al., 2000; Ong et al., 2009; Santos et al., 2005; Sedaghati et al., 2007; Yeo, 2009) which was significant in only one of these trials (Sedaghati et al., 2007). Five trials reported that women in the exercise group did not gain significantly less weight than women in the control group (Collings et al., 1983; Garshasbi & Faghih Zadeh, 2005; Hopkins et al., 2010; Marquez-Sterling et al., 2000; Prevedel et al., 2003). An average reduction in GWG of 0.6 kg (p=0.03) was found in the exercise intervention groups compared to the control groups (Streuling et al., 2010a). A single arm intervention study, combining exercise and diet, reported that 56% of obese women kept their GWG to ≤ 6 kg (study weight goal) and no cases of reduced glucose tolerance were observed (Lindholm et al., 2010). Finally, a recent study conducted in our laboratory showed that excessive GWG was prevented in 80% of overweight and obese women using a Nutrition and Exercise Lifestyle Intervention Program (NELIP) (Mottola et al., 2010). In summary, the prevailing literature clearly indicates that physical activity before and/or during pregnancy has a protective effect against excessive GWG and impaired glucose metabolism. Results from intervention studies suggest that in order to be the most successful in limiting GWG, and thus in helping to prevent impaired glucose metabolism, lifestyle interventions should promote both regular physical activity and healthy eating habits. Promoting a healthy lifestyle during pregnancy, especially in overweight and obese women, becomes increasingly important in the context of the prevention of impaired glucose metabolism. #### 2.3 Guidelines for physical activity during pregnancy Active promotion of physical activity for pregnant women is strongly recommended by professionnal societies, such as the American College of Obstetricians and Gynecologists (ACOG, 2002), the Royal College of Obstetricians and Gynaecologists (RCOG, 2006), the Society of Obstetricians and Gynaecologists of Canada (SOGC) and the Canadian Society of Exercise Physiologists (CSEP) (Davies et al., 2003). The ACOG suggested that "in the absence of either medical or obstetric contraindications, 30 minutes or more of moderate exercise a day on most, if not all, days of the week is recommended for pregnant women" (ACOG, 2002). The recent opinion statement from the SOGC (Davies et al., 2010) on obesity during pregnancy strongly suggests that regular exercise during pregnancy may help to reduce the risk of medical complications associated with maternal obesity. However, all pregnant women should be medically prescreened and consult their health care provider before engaging in an exercise program. In 2008, the Unites States government released physical activity guidelines for Americans, including recommendations specifically for pregnant women to attain at least 150 minutes of moderate intensity aerobic activity per week if not already highly active or doing vigorous intensity activity. Healthy pregnant women who engaged in vigorous aerobic activity or are highly active prior to pregnancy are encouraged to continue physical activity (U.S Department of Health and Human Services 2008). Finally, the joint SOGC/CSEP Clinical Practice Guidelines encourage women to exercise if they have no contraindications (Davies et al., 2003). The SOGC/CSEP Clinical Practice Guidelines provide detailed recommendations about the frequency, intensity, time and type of exercise, following the FITT principle for exercise prescription. Women should al., 2006; Kinnunen et al., 2007; Polley et al., 2002). Findings from intervention studies using only exercise were less consistent than findings from intervention studies combining exercise and nutrition. Seven trials reported a trend for less GWG in the exercise group (Barakat et al., 2009; Cavalcante et al., 2009; Clapp et al., 2000; Ong et al., 2009; Santos et al., 2005; Sedaghati et al., 2007; Yeo, 2009) which was significant in only one of these trials (Sedaghati et al., 2007). Five trials reported that women in the exercise group did not gain significantly less weight than women in the control group (Collings et al., 1983; Garshasbi & Faghih Zadeh, 2005; Hopkins et al., 2010; Marquez-Sterling et al., 2000; Prevedel et al., 2003). An average reduction in GWG of 0.6 kg (p=0.03) was found in the exercise intervention groups compared to the control groups (Streuling et al., 2010a). A single arm intervention study, combining exercise and diet, reported that 56% of obese women kept their GWG to ≤ 6 kg (study weight goal) and no cases of reduced glucose tolerance were observed (Lindholm et al., 2010). Finally, a recent study conducted in our laboratory showed that excessive GWG was prevented in 80% of overweight and obese women using a Nutrition In summary, the prevailing literature clearly indicates that physical activity before and/or during pregnancy has a protective effect against excessive GWG and impaired glucose metabolism. Results from intervention studies suggest that in order to be the most successful in limiting GWG, and thus in helping to prevent impaired glucose metabolism, lifestyle interventions should promote both regular physical activity and healthy eating habits. Promoting a healthy lifestyle during pregnancy, especially in overweight and obese women, becomes increasingly important in the context of the prevention of impaired Active promotion of physical activity for pregnant women is strongly recommended by professionnal societies, such as the American College of Obstetricians and Gynecologists (ACOG, 2002), the Royal College of Obstetricians and Gynaecologists (RCOG, 2006), the Society of Obstetricians and Gynaecologists of Canada (SOGC) and the Canadian Society of Exercise Physiologists (CSEP) (Davies et al., 2003). The ACOG suggested that "in the absence of either medical or obstetric contraindications, 30 minutes or more of moderate exercise a day on most, if not all, days of the week is recommended for pregnant women" (ACOG, 2002). The recent opinion statement from the SOGC (Davies et al., 2010) on obesity during pregnancy strongly suggests that regular exercise during pregnancy may help to reduce the risk of medical complications associated with maternal obesity. However, all pregnant women should be medically prescreened and consult their health care provider before engaging in an exercise program. In 2008, the Unites States government released physical activity guidelines for Americans, including recommendations specifically for pregnant women to attain at least 150 minutes of moderate intensity aerobic activity per week if not already highly active or doing vigorous intensity activity. Healthy pregnant women who engaged in vigorous aerobic activity or are highly active prior to pregnancy are encouraged to continue physical activity (U.S Department of Health and Human Services 2008). Finally, the joint SOGC/CSEP Clinical Practice Guidelines encourage women to exercise if they have no contraindications (Davies et al., 2003). The SOGC/CSEP Clinical Practice Guidelines provide detailed recommendations about the frequency, intensity, time and type of exercise, following the FITT principle for exercise prescription. Women should and Exercise Lifestyle Intervention Program (NELIP) (Mottola et al., 2010). 2.3 Guidelines for physical activity during pregnancy glucose metabolism. exercise 3 to 4 times per week, starting with 15 minutes of aerobic activity at a target heart rate intensity and increasing time slowly to a maximum of 30 minutes per exercise session. All aerobic activity should be preceded by 10- to 15-minutes of warm-up and followed by 10- to 15-minutes of cool-down. Appropriate exercise intensity may be monitored by using target heart-rate zones, the Borg-scale (rating of perceived exertion, RPE) or the "talk test" (Davies et al., 2003). Heart-rate zones that are provided in the guidelines correspond to moderate-intensity exercise (i.e. 60-80% of maximal aerobic capacity, VO2 max). Aerobic exercise in which large muscle groups are used, including walking, stationary cycling, aqua exercise, or low-impact aerobics are recommended for low risk pregnant women (Davies et al. 2003). Overweight and obese women can participate in exercise, if they have no contraindications to being physically active. Twenty medically pre-screened obese and 20 normal weight pregnant women participated in a graded treadmill exercise test to volitional fatigue to examine the impact of obesity on the ventilatory response to weight-bearing exercise during pregnancy (Davenport et al., 2009). We concluded that exercise ventilatory response is increased during pregnancy but is not affected further by obesity during graded treadmill exercise (Davenport et al., 2009). This is important in that there is no apparent ventilatory limitation to submaximal weight-bearing exercise representing daily living activities such as walking, in pregnant obese women, which lends support to the feasibility of exercise prescription in this population group (Mottola, 2009). Target heart rate zones developed for normal weight pregnant women may be too difficult for overweight and obese women to obtain, and thus we developed and validated target heart rate zones for medically prescreened overweight and obese women at a much lower intensity but high enough to achieve aerobic benefits (Davenport et al. 2008a). The intensity is approximately 20-39% heart rate reserve (HRR), and at a normal walking pace which may help with compliance in this population group. Currently, there are no step recommendations for pregnant women but those that have been defined for adults (Tudor-Locke et al., 2008) may be used for pregnant women. As walking is the most reported activity during pregnancy (Evenson et al., 2004; Evenson & Wen, 2010; Mottola & Campbell, 2003; Petersen et al., 2005), providing step recommendations to pregnant women may encourage them to be more active. Previous studies conducted in our laboratory showed that pregnant women took more than 10,000 steps per day when a 40-minute walk was added to their usual daily activities (Davenport et al., 2008b; Mottola et al., 2010). #### 2.4 Are pregnant women meeting the physical activity guidelines? Although maternal physical activity has clear health benefits on pregnancy outcomes, and professional societies strongly recommend active promotion of physical activity for pregnant women, most women remain inactive during pregnancy. In Canada, only 30% of pregnant women meet the adult step recommendations of 10,000 steps per day (Cohen et al., 2010). Data from large cohort studies, such as the "Behavioral Risk Factor Surveillance System" (BRFSS), NHANES and PIN showed that the majority of women do not meet the recommendations for physical activity during pregnancy, based on information collected by interviews or questionnaires (Borodulin et al., 2008; Evenson et al., 2004; Evenson & Wen, 2010; Petersen et al., 2005). The guidelines from the ACOG and from the Center for Controlled Disease (CDC)/American College of Sports Medicine (ACSM) both suggest 30 minutes or more of moderate-intensity activity on most of the days of the week, but differ Exercise Guidelines for Women with Gestational Diabetes 347 there is still controversy regarding the benefits of exercise in improving glycemic control in GDM women, despite endorsements by professionnal organisations. The ACOG (ACOG, 2001) suggests that "women with GDM who lead an active lifestyle should be encouraged to continue a program of exercise approved for pregnancy." The American Diabetes Association (ADA) (ADA, 2004) suggests that "women without medical or obstetrical contraindications be encouraged to start or continue a program of moderate exercise as part of treatment for GDM." The CDA (CDA, 2008) suggests that "physical activity should be encouraged, with the frequency, type, duration and intensity tailored to individual obstetric risk." The recommendation from the Fifth International Workshop-Conference on GDM suggests "planned physical activity of 30 minutes/day is recommended … Advising GDM patients to walk briskly or do arm exercises while seated in a chair for at least 10 minutes Evidence-based studies determining the frequency, intensity, time, and type of activity are needed to provide the best possible outcomes for women with GDM. When exercise was evaluated for controlling blood glucose concentrations or for delaying or preventing insulin therapy, the results were discordant. In the recent ACSM/ADA joint position statement, level of evidence concerning the effect of physical activity to control GDM was non-existent for the ADA, and weak for the ACSM (Colberg et al., 2010). These mixed results could be due to the non-randomization of the subject pool, the different anthropometric characteristics of the women, small sample sizes, lack of well-controlled or reported exercise intensity, the differences in exercise modalities, or questionable compliance to the exercise program. Consequently, because of lack of consistent evidence regarding the benefits of exercise in improving glycemic control in GDM women, exercise remains an adjunctive therapy. The acute effect of exercise on glucose excursion has been evaluated by several authors (Table 1). Avery and Walker (2001) reported that a single 30-minute bout of exercise on a cycle ergometer at 35% or 55% of maximum oxygen consumption (VO2 max) improved glucose excursion compared with rest in women with GDM (Avery & Walker, 2001). Garcia-Patterson et al. found similar results, showing that light postprandial walking at 2.5 km/h decreased glucose excursion in GDM women (Garcia-Patterson et al., 2001). Lesser et al. (1996) determined the effects of a single bout of stationary cycling for 30 minutes at 60% VO2 max, comparing six GDM women to five normal glycemic pregnant women. The effects of a mixed meal 14 hours after the exercise bout were examined. In contrast to the above studies, no improvement in glucose excursion due to the exercise was found in the GDM women. This could be due to a mixed meal being used in the acute experiment and because The chronic effect of exercise for controlling blood glucose concentrations has also been investigated (Table 2). A 6-week arm ergometry exercise program was successful in normalizing fasted and 1-h plasma glucose concentrations and glycosylated hemoglobin (HbA1c) in GDM women randomized to diet therapy plus exercise compared with diet therapy alone (Jovanovic-Peterson et al., 1991). The exercise program consisted of 20 minutes of arm ergometry, three times per week, at an intensity less than or equal to 50% VO2 max. The results of this study gave rise to the recommendation of arm exercise for GDM women mentioned above. In contrast to the above studies, Bung et al. (Bung et al., 1991) randomized GDM women into a group with diet and insulin therapy or diet and exercise. The exercise program consisted of stationary cycle ergometry (50% VO2 max) for 45 minutes (three 15-minute bouts with two rests), three times per week. Because no differences in. after each meal accomplishes this goal" (Metzger et al., 2007). measurements occurred 14 hours after the exercise bout. on the type of activity, as guidelines for ACOG include only exercise and guidelines from CDC/ACSM include any type of physical activity. If the recommendations for physical activity were based on the ACOG guidelines, only 3% of pregnant women meet the recommendations (Borodulin et al., 2008). If the recommendations for physical activity were based on the CDC/ACSM guidelines, approximately 15% of pregnant women meet the recommendations (Borodulin et al., 2008; Evenson et al., 2004; Evenson & Wen, 2010; Petersen et al., 2005). In Spain, 20% of women comply with ACOG criteria whereas 70% comply with CDC/ACSM criteria (Amezcua-Prieto et al., 2010). In summary, most women are physically inactive during pregnancy. This may be contributing to excessive GWG given that women who are meeting the recommendations for exercise during pregnancy are more likely to achieve appropriate GWG (Cohen et al., 2010). Moreover, given the clear association between physical inactivity during the perinatal period and risk for impaired glucose metabolism, physical inactivity may be contributing to the 10 to 100% increase in GDM prevalence observed in several race/ethnic groups during the past 20 years (Dabelea et al., 2005; Ferrara, 2007; Getahun et al., 2008). #### 3. Exercise as an adjunctive therapy for gestational diabetes mellitus management #### 3.1 Conventional management of gestational diabetes mellitus The primary management for women with GDM is control of energy intake, usually referred to as medical nutrition therapy (MNT) (Metzger, 2006). As a dietary intervention, the goals of MNT are to provide adequate nutrition for the mother and fetus, provide sufficient calories for appropriate maternal weight gain, maintain normoglycemia, and avoid ketosis (Franz et al., 2002). The dietary plan suggested by a registered dietitian usually includes eating smaller meals more often, more choices of complex carbohydrates with a low glycemic index, and the elimination of high glycemic foods, including carbonated beverages, sweets, and cake (CDA, 2008). Self-capillary glucose monitoring using a glucometer may be recommended up to seven times per day. The goal of monitoring is to maintain glucose concentrations in acceptable ranges. The Canadian Diabetes Association (CDA) recommends maintaining the following capillary blood glucose values: pre-prandial glucose < 5.3 mmol/L, 1-hour post-prandial glucose < 7.8 mmol/L, and 2-hour postprandial glucose < 6.7 mmol/L. The Fifth International Workshop-Conference on Gestational Diabetes Mellitus guidelines are the same (Metzger et al., 2007). If after 2 weeks of MNT, failure to control capillary glucose concentrations will progress to management of glycemia by insulin injections. It is imperative that maternal blood glucose be maintained below these values, either with MNT plus insulin injections or MNT and lifestyle changes. The type and amount of insulin injected is beyond the scope of this article but depends on medical intervention and management (Metzger et al., 2007). #### 3.2 Exercise/lifestyle management for women with gestational diabetes mellitus Exercise has long been accepted as an adjunctive intervention in the management of diabetes in non pregnant individuals (ADA, 2011; CDA, 2008; Colberg et al., 2010). In type 2 diabetic individuals, exercise has been reported to improve insulin sensitivity and insulinstimulated muscle glucose uptake (Kennedy et al., 1999), to have a positive effect on glycemic control and to decrease cardiovascular risk (Kavookjian et al., 2007). However, on the type of activity, as guidelines for ACOG include only exercise and guidelines from CDC/ACSM include any type of physical activity. If the recommendations for physical activity were based on the ACOG guidelines, only 3% of pregnant women meet the recommendations (Borodulin et al., 2008). If the recommendations for physical activity were based on the CDC/ACSM guidelines, approximately 15% of pregnant women meet the recommendations (Borodulin et al., 2008; Evenson et al., 2004; Evenson & Wen, 2010; Petersen et al., 2005). In Spain, 20% of women comply with ACOG criteria whereas 70% In summary, most women are physically inactive during pregnancy. This may be contributing to excessive GWG given that women who are meeting the recommendations for exercise during pregnancy are more likely to achieve appropriate GWG (Cohen et al., 2010). Moreover, given the clear association between physical inactivity during the perinatal period and risk for impaired glucose metabolism, physical inactivity may be contributing to the 10 to 100% increase in GDM prevalence observed in several race/ethnic groups during The primary management for women with GDM is control of energy intake, usually referred to as medical nutrition therapy (MNT) (Metzger, 2006). As a dietary intervention, the goals of MNT are to provide adequate nutrition for the mother and fetus, provide sufficient calories for appropriate maternal weight gain, maintain normoglycemia, and avoid ketosis (Franz et al., 2002). The dietary plan suggested by a registered dietitian usually includes eating smaller meals more often, more choices of complex carbohydrates with a low glycemic index, and the elimination of high glycemic foods, including carbonated beverages, sweets, and cake (CDA, 2008). Self-capillary glucose monitoring using a glucometer may be recommended up to seven times per day. The goal of monitoring is to maintain glucose concentrations in acceptable ranges. The Canadian Diabetes Association (CDA) recommends maintaining the following capillary blood glucose values: pre-prandial glucose < 5.3 mmol/L, 1-hour post-prandial glucose < 7.8 mmol/L, and 2-hour postprandial glucose < 6.7 mmol/L. The Fifth International Workshop-Conference on Gestational Diabetes Mellitus guidelines are the same (Metzger et al., 2007). If after 2 weeks of MNT, failure to control capillary glucose concentrations will progress to management of glycemia by insulin injections. It is imperative that maternal blood glucose be maintained below these values, either with MNT plus insulin injections or MNT and lifestyle changes. The type and amount of insulin injected is beyond the scope of this article but depends on comply with CDC/ACSM criteria (Amezcua-Prieto et al., 2010). management the past 20 years (Dabelea et al., 2005; Ferrara, 2007; Getahun et al., 2008). 3.1 Conventional management of gestational diabetes mellitus medical intervention and management (Metzger et al., 2007). 3.2 Exercise/lifestyle management for women with gestational diabetes mellitus Exercise has long been accepted as an adjunctive intervention in the management of diabetes in non pregnant individuals (ADA, 2011; CDA, 2008; Colberg et al., 2010). In type 2 diabetic individuals, exercise has been reported to improve insulin sensitivity and insulinstimulated muscle glucose uptake (Kennedy et al., 1999), to have a positive effect on glycemic control and to decrease cardiovascular risk (Kavookjian et al., 2007). However, 3. Exercise as an adjunctive therapy for gestational diabetes mellitus there is still controversy regarding the benefits of exercise in improving glycemic control in GDM women, despite endorsements by professionnal organisations. The ACOG (ACOG, 2001) suggests that "women with GDM who lead an active lifestyle should be encouraged to continue a program of exercise approved for pregnancy." The American Diabetes Association (ADA) (ADA, 2004) suggests that "women without medical or obstetrical contraindications be encouraged to start or continue a program of moderate exercise as part of treatment for GDM." The CDA (CDA, 2008) suggests that "physical activity should be encouraged, with the frequency, type, duration and intensity tailored to individual obstetric risk." The recommendation from the Fifth International Workshop-Conference on GDM suggests "planned physical activity of 30 minutes/day is recommended … Advising GDM patients to walk briskly or do arm exercises while seated in a chair for at least 10 minutes after each meal accomplishes this goal" (Metzger et al., 2007). Evidence-based studies determining the frequency, intensity, time, and type of activity are needed to provide the best possible outcomes for women with GDM. When exercise was evaluated for controlling blood glucose concentrations or for delaying or preventing insulin therapy, the results were discordant. In the recent ACSM/ADA joint position statement, level of evidence concerning the effect of physical activity to control GDM was non-existent for the ADA, and weak for the ACSM (Colberg et al., 2010). These mixed results could be due to the non-randomization of the subject pool, the different anthropometric characteristics of the women, small sample sizes, lack of well-controlled or reported exercise intensity, the differences in exercise modalities, or questionable compliance to the exercise program. Consequently, because of lack of consistent evidence regarding the benefits of exercise in improving glycemic control in GDM women, exercise remains an adjunctive therapy. The acute effect of exercise on glucose excursion has been evaluated by several authors (Table 1). Avery and Walker (2001) reported that a single 30-minute bout of exercise on a cycle ergometer at 35% or 55% of maximum oxygen consumption (VO2 max) improved glucose excursion compared with rest in women with GDM (Avery & Walker, 2001). Garcia-Patterson et al. found similar results, showing that light postprandial walking at 2.5 km/h decreased glucose excursion in GDM women (Garcia-Patterson et al., 2001). Lesser et al. (1996) determined the effects of a single bout of stationary cycling for 30 minutes at 60% VO2 max, comparing six GDM women to five normal glycemic pregnant women. The effects of a mixed meal 14 hours after the exercise bout were examined. In contrast to the above studies, no improvement in glucose excursion due to the exercise was found in the GDM women. This could be due to a mixed meal being used in the acute experiment and because measurements occurred 14 hours after the exercise bout. The chronic effect of exercise for controlling blood glucose concentrations has also been investigated (Table 2). A 6-week arm ergometry exercise program was successful in normalizing fasted and 1-h plasma glucose concentrations and glycosylated hemoglobin (HbA1c) in GDM women randomized to diet therapy plus exercise compared with diet therapy alone (Jovanovic-Peterson et al., 1991). The exercise program consisted of 20 minutes of arm ergometry, three times per week, at an intensity less than or equal to 50% VO2 max. The results of this study gave rise to the recommendation of arm exercise for GDM women mentioned above. In contrast to the above studies, Bung et al. (Bung et al., 1991) randomized GDM women into a group with diet and insulin therapy or diet and exercise. The exercise program consisted of stationary cycle ergometry (50% VO2 max) for 45 minutes (three 15-minute bouts with two rests), three times per week. Because no differences in. Exercise Guidelines for Women with Gestational Diabetes 349 glycemic control were found between groups, the authors suggested that exercise may provide avoidance of insulin therapy through an increase in insulin sensitivity. In another study, GDM women were randomized to a partial home-based exercise program (70% of estimated maximal heart rate) and compared with GDM women with no structured exercise program (Avery et al., 1997). Although the exercise program improved the cardiorespiratory fitness of the GDM women, glucose excursion was not different compared with the women with no structured exercise program (Avery et al., 1997). More recently, Artal et al. (2007) randomized obese GDM women into MNT plus exercise (60% VO2 max) or MNT alone. Results showed that the MNT plus exercise group limited GWG and had no adverse pregnancy outcomes. The authors concluded that placing obese women with GDM on a lifestyle intervention strategy of weight gain restriction may optimize pregnancy outcomes and impact future weight management behaviors. Using a different exercise modality, de Barros et al. (de Barros et al., 2010) randomized GDM women into a resistance exercise program (elastic band) group or MNT alone group. A reduction in the number of patients who required insulin was obseved in the exercise group compared with the MNT group. Furthermore, the percentage of time spent within the proposed target glucose range was In a 2004 retrospective chart review from London, Canada, assessing conventional management of women diagnosed with GDM, Davenport et al. (2005) showed that by 30 weeks of pregnancy, 62% of these women required insulin therapy (after trying conventional management for 2 weeks after diagnoses). Of this cohort, women with a prepregnancy BMI of 25 kg/m2 or greater were 2.6 times more likely to require insulin therapy than those women with a BMI below 25 kg/m2 . The average pre-pregnancy BMI of women requiring insulin therapy was 30.6 ± 6.4 kg/m2. This high incidence of insulin therapy in women with a BMI of 25 kg/m2 or greater may indicate the need for intensive therapy to delay or prevent insulin usage. In women with an early GDM diagnosis (at 16 to 20 weeks of gestation) who followed a structured walking program (30% HRR), 3–4 times per week in addition to conventional management, only 50% required insulin therapy (Davenport et al., 2005). In another study evaluating 30 GDM women, 10 following conventional management plus a low-intensity walking program (30% HRR, 3–4 times per week) matched by insulin useage to 20 women following conventional management alone, we reported lower mean capillary glucose concentrations at the end of pregnancy (fasting and 1h after meals) in the exercising group (Davenport et al., 2008b). The lower glucose concentrations were achieved while requiring fewer units of insulin per kg per day. Using a different exercise modality, Brankston et al. (Brankston et al., 2004) randomized GDM women to a group with diet alone or a group of diet plus circuit-type resistance training. The number of women requiring insulin was not different between groups. However, they found that within the diet plus exercise group, 30% of the women who exercised 2-3 times per week were prescribed insulin therapy compared to 67% of those who exercised <2 per week. Moreover, a subgroup analysis that examined only overweight and obese women showed a lower incidence of insulin use, a lower prescription of insulin and a longer delay from diagnosis to the Taken together, the above results are very encouraging. However, future lifestyle intervention programs are required to confirm these promising results and to determine the frequency, intensity, time, and type of activity that are needed to provide the best possible higher in the exercise group compared with the MNT group. initiation of insulin therapy in the diet plus exercise group. outcomes for women with GDM. NGT – normal glycemia; BMI – body mass index. Table 1. Summary of studies using acute exercise to change blood glucose concentrations in women with gestational diabetes mellitus (GDM). 28-38 w of gestation 30-34 w of gestation 30.7±5.5 w of gestation Table 1. Summary of studies using acute exercise to change blood glucose concentrations in Similar mean values for fasting glucose, peak glucose, area under the glycemic curve with vs without exercise. Similarly, plasma insulin levels did not differ between protocols for either group of subjects. Not successful Blood glucose levels were significantly different after 30 min of rest, low- and moderate-intensity exercise: During control situation, higher 1-h postprandial blood glucose (p=0.001) and 1-h blood glucose excursion (p=0.001) compared to exercise situaton. Successful Rest: 5.2 mmol/L Low: 4.3mmol/L Mod: 3.9 mmol/L. Successful References Population Intervention program Gestational age Main findings breakfast, followed by blood samples. Exercise situation Intensity: 60% VO2 max Duration: 30 min Type: Control situation Standardized stationary cycle. Exercise performed 14 h before standardized breakfast, followed by blood samples. Control situation Remained seated for Exercise situation Intensity: 35% and 55% VO2 max Duration: 30 min Type: stationary cycle. The women exercised at the two intensities for 30 min and rested for 2 h after each Control situation remained seated for 2h after a standard breakfast. Exercise situation walked self-paced (2.5 km/h) in the 1st hour after breakfast and remained seated during the 2nd hour. session. 2h30 (Lesser et al., 1996) (Avery & Walker, 2001) (Garcia-Patterson et al., 2001) N=11: 6 GDM, 5 NGT Ethnicity: na Pre-pregnancy BMI (kg/m2): NGT=24.3±0.9 GDM=25.9±1.8 Age (years): NGT=23.7±20 GDM=27.6±2.8 N=13, GDM Ethnicity: na Pre-pregnancy BMI (kg/m2): 29.0±7.4 Age (years): 31.9±3.6 N=20, GDM Ethnicity: na Pre-pregnancy BMI: na Age (years): 33.5±4.6 NGT – normal glycemia; BMI – body mass index. women with gestational diabetes mellitus (GDM). glycemic control were found between groups, the authors suggested that exercise may provide avoidance of insulin therapy through an increase in insulin sensitivity. In another study, GDM women were randomized to a partial home-based exercise program (70% of estimated maximal heart rate) and compared with GDM women with no structured exercise program (Avery et al., 1997). Although the exercise program improved the cardiorespiratory fitness of the GDM women, glucose excursion was not different compared with the women with no structured exercise program (Avery et al., 1997). More recently, Artal et al. (2007) randomized obese GDM women into MNT plus exercise (60% VO2 max) or MNT alone. Results showed that the MNT plus exercise group limited GWG and had no adverse pregnancy outcomes. The authors concluded that placing obese women with GDM on a lifestyle intervention strategy of weight gain restriction may optimize pregnancy outcomes and impact future weight management behaviors. Using a different exercise modality, de Barros et al. (de Barros et al., 2010) randomized GDM women into a resistance exercise program (elastic band) group or MNT alone group. A reduction in the number of patients who required insulin was obseved in the exercise group compared with the MNT group. Furthermore, the percentage of time spent within the proposed target glucose range was higher in the exercise group compared with the MNT group. In a 2004 retrospective chart review from London, Canada, assessing conventional management of women diagnosed with GDM, Davenport et al. (2005) showed that by 30 weeks of pregnancy, 62% of these women required insulin therapy (after trying conventional management for 2 weeks after diagnoses). Of this cohort, women with a prepregnancy BMI of 25 kg/m2 or greater were 2.6 times more likely to require insulin therapy than those women with a BMI below 25 kg/m2 . The average pre-pregnancy BMI of women requiring insulin therapy was 30.6 ± 6.4 kg/m2. This high incidence of insulin therapy in women with a BMI of 25 kg/m2 or greater may indicate the need for intensive therapy to delay or prevent insulin usage. In women with an early GDM diagnosis (at 16 to 20 weeks of gestation) who followed a structured walking program (30% HRR), 3–4 times per week in addition to conventional management, only 50% required insulin therapy (Davenport et al., 2005). In another study evaluating 30 GDM women, 10 following conventional management plus a low-intensity walking program (30% HRR, 3–4 times per week) matched by insulin useage to 20 women following conventional management alone, we reported lower mean capillary glucose concentrations at the end of pregnancy (fasting and 1h after meals) in the exercising group (Davenport et al., 2008b). The lower glucose concentrations were achieved while requiring fewer units of insulin per kg per day. Using a different exercise modality, Brankston et al. (Brankston et al., 2004) randomized GDM women to a group with diet alone or a group of diet plus circuit-type resistance training. The number of women requiring insulin was not different between groups. However, they found that within the diet plus exercise group, 30% of the women who exercised 2-3 times per week were prescribed insulin therapy compared to 67% of those who exercised <2 per week. Moreover, a subgroup analysis that examined only overweight and obese women showed a lower incidence of insulin use, a lower prescription of insulin and a longer delay from diagnosis to the initiation of insulin therapy in the diet plus exercise group. Taken together, the above results are very encouraging. However, future lifestyle intervention programs are required to confirm these promising results and to determine the frequency, intensity, time, and type of activity that are needed to provide the best possible outcomes for women with GDM. Exercise Guidelines for Women with Gestational Diabetes 351 program CHO 40%–45% Exercise Freq: 1/week in lab, unsupervised ex. session at home Intensity: 60% VO2 peak Duration: 20 min Type: treadmill or stationary cycle Exercise Freq: 3-4/week, Intensity: 30% HRR mild Duration: 25-40 min Type: treadmill Exercise Freq: 3/week (2 at home) Intensity: exercise perception. Duration: 30-40 min Type: resistance training circuit (elastic band) BMI – body mass index; Freq – frequency; CHO – carbohydrate; P – protein; F – fat; na – not given; In 2003, Artal proposed guidelines to develop exercise programs for pregnant women with GDM (Artal, 2003). He suggested 3 to 4 exercise sessions per week, at 50% VO2 max for three Table 2. Summary of studies using the chronic effect of exercise to control blood glucose concentration, to delay or prevent insulin usage in women with gestational diabetes mellitus 3.3 Exercise guidelines for women with gestational diabetes mellitus "somewhat heavy" Diet Length of program <33 w of gestation until delivery Minimum 6 weeks (from diagnosis to delivery) 24-34 w of gestation to delivery Main findings Lower GWG per week in D+EX group than in D group (0.1±0.4 kg vs 0.3±0.4 kg, p <0.05). Similar pregnancy outcomes between the groups. Successful Lower mean capillary glucose levels at the end of pregnancy (fasting and 1h after meals) in EX group but not in CM group (p <0.05). Ex group needed less insulin than CM group. Successful Reduction in the number of patients who required insulin in the EX (7/32) compared with the CON group (18/32) (p=0.005). The % of time spent within the proposed target glucose range was higher in EX group compared with CON group (p=0.006). Successful References Study type Population Intervention N=96: D=57, D+EX=39 Ethnicity: 55-60% caucasian from the US Pre-pregnancy BMI (kg/m2): ≥ 30 Age (years): D=30.6±5.5 D+EX=32.4±5.3 N=30: EX=10, CM=20 Ethnicity: na, from Canada Pre-pregnancy BMI (kg/m2): ≥ 25 Age (years): EX=33.4±3.3 CON=33.3±5.3 N=64: EX=32, CON=32 Ethnicity: na, from Brasil Pre-pregnancy BMI (kg/m2): EX=25.34±4.16 CON=25.39±3.81 Age (years): EX=31.8±4.87 CON=32.40±5.40 (Artal et al., 2007) (Davenport et al., 2008b) (de Barros et al., 2010) (GDM). HRR – heart rate reserve. Self- enrollement in diet (D) vs diet+exercise (D+EX) groups Exercise (EX) vs conventional management (CM) group -matched by BMI, insulin use -2 CM/EX Randomization: exercise (EX) vs control (CON) group program 40% CHO, 20% P, 40% F Exercise Freq: 3/week Intensity: 50% VO2max Duration: 20 min Type: arm ergometry 24 to 30 kcal/kg/24 h; Diet Diet cycle Diet 40% F. 3 meals and 3 snacks. Exercise Freq: 3/week Type: Circuit-type resistance training. Exercise Freq: 3-4/week (2 supervised) Intensity: 70% (220-age) Duration: 30 kcal/kg/day Exercise Freq: 3/week Intensity: 50% VO2max Duration: 45 min (3x15 min) Type: stationary 30 min (including 5 min warm-up and cool-down) Type: stationary cycle or walking 40% CHO, 20% P, Intensity: <140bpm Length of program 6 weeks From diagnosis (30±2 w of gestation) to delivery From <34 w of gestation to delivery From 26- 32 w of gestation to delivery Main findings Lower HbA1C, fasting and 1-hour plasma glucose concentrations in D+EX group compared to D group (p<0.001 for No differences in glycemic control between D+EX and D+I groups. Similar maternal and neonatal outcomes between groups. Successful No difference in HbA1C and insulin usage among EX and CON groups. Similar infant birth weight and incidence of hypoglycemia between groups. Not successful Within D+EX group, 30% of the women who exercised 2-3 per week were prescribed insulin therapy compared to 67% of those who exercised <2 per week. In overweight women only, lower incidence of insulin use, lower prescription of insulin and longer delay from diagnosis to the initiation of insulin therapy in D+EX vs D group (p<0.05). Successful all). Successful References Study type Population Intervention N=19: D=9, D+EX=10 Ethnicity: na Pre-pregnancy BMI (kg/m2): na Age (years): D=31.1±2.8 D+EX=29.5±2.5 N=34: D+EX=17, D+I=17 Ethnicity: Hispanic Pre-pregnancy BMI (kg/m2): na Age (years): D+EX=31.0±4.5 D+I=32.0±5.7 N=29: EX=15, CON=14 Ethnicity: Caucasian Pre-pregnancy BMI (kg/m2): EX=28.4±7.6 CON=25.5±5.5 Age (years): EX=32.2±4.9 CON=30.4±5.1 N=32: D=16, D+EX=16 Ethnicity: na, from Canada Pre-pregnancy BMI (kg/m2): D=28.0±5.7 D+EX=25.9±3.4 Age (years): D=31.3±5.0 D+EX=30.5±4.4 Randomization: diet (D) vs diet+exercise (D+EX) group Randomization: diet+exercise (D+EX) vs diet+insulin (D+I) group Randomization: exercise (EX) vs control (CON) groups Randomization: diet (D) vs diet+exercise (D+EX) group (Jovanovic-Peterson et al., 1991) (Bung et al., 1991) (Avery et al., 1997) (Brankston et al., 2004) BMI – body mass index; Freq – frequency; CHO – carbohydrate; P – protein; F – fat; na – not given; HRR – heart rate reserve. Table 2. Summary of studies using the chronic effect of exercise to control blood glucose concentration, to delay or prevent insulin usage in women with gestational diabetes mellitus (GDM). #### 3.3 Exercise guidelines for women with gestational diabetes mellitus In 2003, Artal proposed guidelines to develop exercise programs for pregnant women with GDM (Artal, 2003). He suggested 3 to 4 exercise sessions per week, at 50% VO2 max for three Exercise Guidelines for Women with Gestational Diabetes 353 factor—in combination with nutritional control—in helping women at risk for GDM and those women diagnosed with GDM, regulate blood glucose concentrations and prevent Obesity and type 2 diabetes are reaching epidemic proportions in society today and women of childbearing age are at risk for developing these diseases because of excessive weight gain during pregnancy and weight retention after birth. If modifiable risk factors for developing diabetes during pregnancy, such as preventing excessive weight gain and preserving glucose tolerance, can be reduced by incorporating physical activity, then exercise can be used as a powerful tool to reduce the diabetes and obesity epidemics in successive generations. Unfortuately, researchers have not be able to suggest an evidencebased program with guidelines for frequency, intensity, time and type of activity (FITT principal for exercise presciption) that would produce the best possible outcomes for women with GDM. Although preliminary results are encouraging, exercise is still considered an adjunctive therapy, and the true effectiveness of a specific exercise program in controlling glucose excursion and reducing the incidence of insulin therapy remains Based on the literature reviewed, it is suggested that in using the FITT principal of exercise prescription, women who are at risk for or who have been diagnosed with GDM, should engage in activity at a frequency of 3-4 times per week, for at least 25 minutes each session, at a mild intensity (walking pace), building to 40 minutes, would be sufficient to provide health benefits. In addition, it is suggested that if pedometers are available, 10,000 steps per day may also regulate glucose metabolism. If women with GDM are overweight or obese, a target heart rate of 102-124 beats per minute (20 to 29 years of age) and 101 to 120 beats per minute (30 to 39 years of age) may also be used to monitor intensity. Continuing research is necessary in this important field especially if new stringent cut-offs for diagnoses of GDM are adopted as guidelines, as they will cause a higher prevalance of GDM, increasing the cost of medical care. Prevention of GDM by adoption of a healthy lifestyle and active living The authors acknowledge funding sources: Canadian Institute of Health Research and the Rx & D Health Research Foundation of Canada, the Lawson Foundation, and the MollyTowell Research Foundation. Dr. Ruchat is funded by a Canadian Diabetes American College of Obstetricians and Gynecologists (ACOG). 2001. Practice Bulletin. September. Gestational diabetes. Obstet Gynecol. 98:525-38. Clinical management guidelines for obstetrician-gynecologists. Number 30, excessive weight gain during pregnancy. 4. Summary and recommendations untapped. may be key. 6. References 5. Acknowledgements Association Postdoctoral Fellowship Award. 15-minute bouts with 5-minute rests between each, for a total of 45 minutes. The joint SOGC/CSEP Clinical Practice Guidelines (Davies et al., 2003) provides detailed recommendations regarding frequency, intensity, time, and type of activity for healthy pregnant women. The same recommendations may be used for pregnant women with GDM, except that the intensity of exercise might be adapted and that precaution should be taken, especially for women using insulin. The SOGC/CSEP Clinical Practice Guidelines provides heart-rate zones corresponding to exercise of moderate intensity (i.e. 60-80% of VO2 max). However, this intensity may be too high for pregnant women with GDM who are overweight or obese and possibly sedentary. The ACSM suggested that previously sedentary overweight and obese pregnant women should initiate an aerobic exercise program at an intensity equivalent to 20% to 39% of reserve aerobic capacity (VO2 reserve) (ACSM, 2005). These developed and validated target heart-rate zones based on age, equivalent to 20% to 39% VO2 reserve are 102 to 124 beats per minute (bpm) for overweight and obese women 20 to 29 years of age and 101 to 120 bpm for those aged 30 to 39 years (Davenport et al., 2008a). Interestingly, lower-intensity aerobic exercise seems to be more efficient in term of glycemic control than moderate-intensity exercise for pregnant women. Indeed, all intervention studies that used lower-intensity aerobic exercise (i.e. ≤60% VO2 max) were successful in controlling blood glucose concentrations and/or limiting/preventing insulin therapy (Artal et al., 2007; Bung et al., 1991; Davenport et al., 2008b; Jovanovic-Peterson et al., 1991) whereas the only study that used moderate-intensity aerobic exercise (i.e. 70% VO2 max) was not succesfull (Avery et al., 1997). Mottola et al. (1998b) investigated low-risk pregnant women and showed that mild exercise (30% HRR) on a stationary bike was better at promoting glucose tolerance in response to an oral glucose load after exercise than moderate intensity exercise (70% HRR) in late gestational women. Biopsies of the vastus lateralis muscle in these late pregnant women showed that total GLUT4 (glucose transporters sensitive to insulin) was elevated in the mild exercise–trained women (starting at 16–20 weeks gestation until delivery) compared with moderately trained women (Mottola et al., 1998a). Subsequently, when nutritional intake was controlled during pregnancy (to ~ 8350 kJ/day, with 200 g of carbohydrate/day), the combination of nutritional control and mild exercise (30% HRR on a stair climber) was better than mild exercise alone in controlling blood glucose concentrations and preventing excessive weight gain during pregnancy. This effect remained at 2 months postpartum (Mottola et al., 1999). The above studies provided groundwork for development of a Nutrition and Exercise Lifestyle Intervention Program (NELIP), in which a mild walking program (30% HRR) was combined with nutritional control (8350 kJ/day; 200 g of carbohydrate/day) for women at risk for GDM (Sopper et al., 2004). Preliminary results are encouraging, in that women at risk for GDM did not develop this disease while on NELIP (N = 23), excessive weight gain was prevented, and normal glucose tolerance remained at 2 months postpartum (Batada et al., 2003). In addition, pregnant women at risk for GDM on NELIP maintained an insulin sensitivity index similar to those at low risk for GDM, and none developed GDM (Mottola et al., 2005b). It is suggested that overweight women at risk for GDM can be given a NELIP at 16 weeks of pregnancy to maintain insulin sensitivity and glucose excursion and to prevent excessive weight gain and GDM. Assessment of HbA 1c in these women also showed values well below the diabetic range (Mottola et al., 2005a). Studies conducted by our lab suggest that mild exercise, regardless of modality (bike, stair climber, or walking), may be a key factor—in combination with nutritional control—in helping women at risk for GDM and those women diagnosed with GDM, regulate blood glucose concentrations and prevent excessive weight gain during pregnancy. #### 4. Summary and recommendations 352 Gestational Diabetes 15-minute bouts with 5-minute rests between each, for a total of 45 minutes. The joint SOGC/CSEP Clinical Practice Guidelines (Davies et al., 2003) provides detailed recommendations regarding frequency, intensity, time, and type of activity for healthy pregnant women. The same recommendations may be used for pregnant women with GDM, except that the intensity of exercise might be adapted and that precaution should be taken, especially for women using insulin. The SOGC/CSEP Clinical Practice Guidelines provides heart-rate zones corresponding to exercise of moderate intensity (i.e. 60-80% of VO2 max). However, this intensity may be too high for pregnant women with GDM who are overweight or obese and possibly sedentary. The ACSM suggested that previously sedentary overweight and obese pregnant women should initiate an aerobic exercise program at an intensity equivalent to 20% to 39% of reserve aerobic capacity (VO2 reserve) (ACSM, 2005). These developed and validated target heart-rate zones based on age, equivalent to 20% to 39% VO2 reserve are 102 to 124 beats per minute (bpm) for overweight and obese women 20 to 29 years of age and 101 to 120 bpm for those aged 30 to 39 years Interestingly, lower-intensity aerobic exercise seems to be more efficient in term of glycemic control than moderate-intensity exercise for pregnant women. Indeed, all intervention studies that used lower-intensity aerobic exercise (i.e. ≤60% VO2 max) were successful in controlling blood glucose concentrations and/or limiting/preventing insulin therapy (Artal et al., 2007; Bung et al., 1991; Davenport et al., 2008b; Jovanovic-Peterson et al., 1991) whereas the only study that used moderate-intensity aerobic exercise (i.e. 70% VO2 max) was not succesfull (Avery et al., 1997). Mottola et al. (1998b) investigated low-risk pregnant women and showed that mild exercise (30% HRR) on a stationary bike was better at promoting glucose tolerance in response to an oral glucose load after exercise than moderate intensity exercise (70% HRR) in late gestational women. Biopsies of the vastus lateralis muscle in these late pregnant women showed that total GLUT4 (glucose transporters sensitive to insulin) was elevated in the mild exercise–trained women (starting at 16–20 weeks gestation until delivery) compared with moderately trained women (Mottola et al., 1998a). Subsequently, when nutritional intake was controlled during pregnancy (to ~ 8350 kJ/day, with 200 g of carbohydrate/day), the combination of nutritional control and mild exercise (30% HRR on a stair climber) was better than mild exercise alone in controlling blood glucose concentrations and preventing excessive weight gain during pregnancy. This The above studies provided groundwork for development of a Nutrition and Exercise Lifestyle Intervention Program (NELIP), in which a mild walking program (30% HRR) was combined with nutritional control (8350 kJ/day; 200 g of carbohydrate/day) for women at risk for GDM (Sopper et al., 2004). Preliminary results are encouraging, in that women at risk for GDM did not develop this disease while on NELIP (N = 23), excessive weight gain was prevented, and normal glucose tolerance remained at 2 months postpartum (Batada et al., 2003). In addition, pregnant women at risk for GDM on NELIP maintained an insulin sensitivity index similar to those at low risk for GDM, and none developed GDM (Mottola et al., 2005b). It is suggested that overweight women at risk for GDM can be given a NELIP at 16 weeks of pregnancy to maintain insulin sensitivity and glucose excursion and to prevent excessive weight gain and GDM. Assessment of HbA 1c in these women also showed values well below the diabetic range (Mottola et al., 2005a). Studies conducted by our lab suggest that mild exercise, regardless of modality (bike, stair climber, or walking), may be a key effect remained at 2 months postpartum (Mottola et al., 1999). (Davenport et al., 2008a). Obesity and type 2 diabetes are reaching epidemic proportions in society today and women of childbearing age are at risk for developing these diseases because of excessive weight gain during pregnancy and weight retention after birth. If modifiable risk factors for developing diabetes during pregnancy, such as preventing excessive weight gain and preserving glucose tolerance, can be reduced by incorporating physical activity, then exercise can be used as a powerful tool to reduce the diabetes and obesity epidemics in successive generations. Unfortuately, researchers have not be able to suggest an evidencebased program with guidelines for frequency, intensity, time and type of activity (FITT principal for exercise presciption) that would produce the best possible outcomes for women with GDM. Although preliminary results are encouraging, exercise is still considered an adjunctive therapy, and the true effectiveness of a specific exercise program in controlling glucose excursion and reducing the incidence of insulin therapy remains untapped. Based on the literature reviewed, it is suggested that in using the FITT principal of exercise prescription, women who are at risk for or who have been diagnosed with GDM, should engage in activity at a frequency of 3-4 times per week, for at least 25 minutes each session, at a mild intensity (walking pace), building to 40 minutes, would be sufficient to provide health benefits. In addition, it is suggested that if pedometers are available, 10,000 steps per day may also regulate glucose metabolism. If women with GDM are overweight or obese, a target heart rate of 102-124 beats per minute (20 to 29 years of age) and 101 to 120 beats per minute (30 to 39 years of age) may also be used to monitor intensity. Continuing research is necessary in this important field especially if new stringent cut-offs for diagnoses of GDM are adopted as guidelines, as they will cause a higher prevalance of GDM, increasing the cost of medical care. Prevention of GDM by adoption of a healthy lifestyle and active living may be key. #### 5. Acknowledgements The authors acknowledge funding sources: Canadian Institute of Health Research and the Rx & D Health Research Foundation of Canada, the Lawson Foundation, and the MollyTowell Research Foundation. Dr. Ruchat is funded by a Canadian Diabetes Association Postdoctoral Fellowship Award. #### 6. References American College of Obstetricians and Gynecologists (ACOG). 2001. Practice Bulletin. Clinical management guidelines for obstetrician-gynecologists. Number 30, September. Gestational diabetes. Obstet Gynecol. 98:525-38. Exercise Guidelines for Women with Gestational Diabetes 355 Buchanan, T.A., and Xiang, A.H. 2005. Gestational diabetes mellitus. 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Rivas Venezuela Diabetes and Pregnancy Unit, Gestational Diabetes Mellitus After Delivery Gestational Diabetes Mellitus (GDM) bring repercussions not only during pregnancy and delivery, but also in the future, with implications for Public Health. Nonetheless, after delivery, there is a tendency of the health team and medical institutions to relax the care given to women with GDM, probably due to the fact that their blood glucose values usually return rapidly to their normal level, wasting in this way the opportunity of preserving the health status of these relatively young group of women with a high risk of cardiovascular events, greatly due to the subsequent development of type 2 diabetes (Bentley-Lewis, 2009; Shah et al., 2008). In recent years an increase in the prevalence of GDM in different populations and ethnic groups has been observed (Dabelea et al., 2005; Hunt & Schuller, 2007). Being GDM, very frequently, a diabetes precursor, a large number of fertile women will be increasingly subjected to a higher risk of developing it in a variable time, generally during middle age. This implies a long period of potential risk for chronic micro and macroangiophatic complications, with implied high social and economical costs. oral health measures (Friedlander et al., 2007; Novak et al., 2006). Women with GDM constitute an excellent target for applying diabetes preventive measures and its comorbidities. In this sense, a continuous and prolonged post-partum follow-up is recommended with two main objectives: to implement non-pharmacological and pharmacological preventive measures, whose efficiency has been shown in some studies (Buchanan et al., 2002; Ratner et al., 2008), and also to carry out early detection of diabetes and other cardiovascular risk factors by guidelines that unfortunately, vary from one organization to another (Asociación Latinoamericana de Diabetes (ALAD), 2008: Metzger et al., 2007: National Institute for Health and Clinical Excellence (NICE), 2008), partly explaining low compliance rates and short duration of follow-ups, even in developed countries (Blatt et al., 2011; Dinh et al., 2003; Ferrara et al., 2009; Kim et al., 2007a). Moreover, maternal breastfeeding stimulus and the most adequate contraceptive method are included (Kim, 2009, 2010; Kitzmiller et al., 2007). More recently, the association between GDM with periodontal disease has been demonstrated, therefore it was considered pertinent to include During pregnancy, women with GDM require education under the premise that glucose intolerance is not always transitory, but that at any time after delivery it can become permanent, in order to increase awareness of the importance of postpartum follow-up, during which educational strategies aimed at correcting knowledge deficits on healthy lifestyles are studied in-depth (Rivas et al., 2010a); moreover, to overcome difficulties found in substituting inadequate habits in practice (Smith et al., 2005; Stage et al., 2004). To do so, it 1. Introduction University of Carabobo - Dr. Enrique Tejera Hospital, Valencia ### Gestational Diabetes Mellitus After Delivery #### Aleida M. Rivas Diabetes and Pregnancy Unit, University of Carabobo - Dr. Enrique Tejera Hospital, Valencia Venezuela #### 1. Introduction 362 Gestational Diabetes Yates, T., Khunti, K., Bull, F., Gorely, T., and Davies, M.J. 2007. The role of physical activity Yeo, S. 2009. Adherence to walking or stretching, and risk of preeclampsia in sedentary Zhang, C., Solomon, C.G., Manson, J.E., and Hu, F.B. 2006. A prospective study of pregravid pregnant women. Res Nurs Health. 32:379-90. diabetes mellitus. Arch Intern Med. 166:543-8. 50:1116-26. in the management of impaired glucose tolerance: a systematic review. Diabetologia. physical activity and sedentary behaviors in relation to the risk for gestational Gestational Diabetes Mellitus (GDM) bring repercussions not only during pregnancy and delivery, but also in the future, with implications for Public Health. Nonetheless, after delivery, there is a tendency of the health team and medical institutions to relax the care given to women with GDM, probably due to the fact that their blood glucose values usually return rapidly to their normal level, wasting in this way the opportunity of preserving the health status of these relatively young group of women with a high risk of cardiovascular events, greatly due to the subsequent development of type 2 diabetes (Bentley-Lewis, 2009; Shah et al., 2008). In recent years an increase in the prevalence of GDM in different populations and ethnic groups has been observed (Dabelea et al., 2005; Hunt & Schuller, 2007). Being GDM, very frequently, a diabetes precursor, a large number of fertile women will be increasingly subjected to a higher risk of developing it in a variable time, generally during middle age. This implies a long period of potential risk for chronic micro and macroangiophatic complications, with implied high social and economical costs. Women with GDM constitute an excellent target for applying diabetes preventive measures and its comorbidities. In this sense, a continuous and prolonged post-partum follow-up is recommended with two main objectives: to implement non-pharmacological and pharmacological preventive measures, whose efficiency has been shown in some studies (Buchanan et al., 2002; Ratner et al., 2008), and also to carry out early detection of diabetes and other cardiovascular risk factors by guidelines that unfortunately, vary from one organization to another (Asociación Latinoamericana de Diabetes (ALAD), 2008: Metzger et al., 2007: National Institute for Health and Clinical Excellence (NICE), 2008), partly explaining low compliance rates and short duration of follow-ups, even in developed countries (Blatt et al., 2011; Dinh et al., 2003; Ferrara et al., 2009; Kim et al., 2007a). Moreover, maternal breastfeeding stimulus and the most adequate contraceptive method are included (Kim, 2009, 2010; Kitzmiller et al., 2007). More recently, the association between GDM with periodontal disease has been demonstrated, therefore it was considered pertinent to include oral health measures (Friedlander et al., 2007; Novak et al., 2006). During pregnancy, women with GDM require education under the premise that glucose intolerance is not always transitory, but that at any time after delivery it can become permanent, in order to increase awareness of the importance of postpartum follow-up, during which educational strategies aimed at correcting knowledge deficits on healthy lifestyles are studied in-depth (Rivas et al., 2010a); moreover, to overcome difficulties found in substituting inadequate habits in practice (Smith et al., 2005; Stage et al., 2004). To do so, it Gestational Diabetes Mellitus After Delivery 365 maternal weight (Macneill et al., 2001) and the presence of impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) in two-month postpartum screenings (Kwak et al., 2008). GDM risk in a second pregnancy has been found nearly ten-folds higher among women with previous GDM than in women without this family history, 41.3% and 4.2%, respectively. The risk increases with the number of previous GDM episodes and thus, with the third pregnancy, GDM recurrence was more marked when GDM had been diagnosed A new GDM pregnancy is not associated itself to a risk increase of type 2 diabetes in women with GDM history (Russel et al., 2008). Nonetheless, the absence of recurrent GDM in a subsequent pregnancy may show a decrease in the risk of developing type 2 diabetes There is strong evidence proving that women with GDM show a high risk of developing diabetes through their lives (Damm et al., 2003). Even though the highest frequency corresponds to type 2 diabetes, type 1 diabetes may also occur, whose proportion will vary For many decades GDM has been acknowledged as a heterogeneous alteration, where autoimmunity against beta cells constitutes the pathogenic basis in a small group of patients, who show a higher risk of developing type 1 diabetes during pregnancy or after In women with GDM, the determination of different specific antibodies against pancreatic beta cells, like antibodies to islet cells (ICA), glutamic acid decarboxilase antibodies (GADA), tyrosine phosphatase tyrosine (IA-2A), and most recently, GAD 65, proven preclinical markers of type 1 diabetes (Mitchell et al., 2000; Murgia et al., 2008), has allowed to know that autoimmune GDM corresponds to 10% of GDM cases in Caucasian women and contributes to a peculiar and complex pre-diabetic state, with a high risk for progressing to type 1 diabetes and to a latent autoimmune diabetes of adulthood (LADA) (de Leiva et al., 2007; Lapolla et al., 2009). The risk increases with the number of antibodies present (Füchtenbusch et al., 1997), and is higher during the first postpartum years (Nilsson et al., 2007). In studies carried out on women with GDM that show positive antibodies, it was found that they were younger, showed lower body mass index (BMI), less proportion of family members with diabetes, less abdominal circumference, and lower levels of plasmatic insulin than in those women without antibodies. Moreover, they had gained less weight during pregnancy and had required insulin treatment in a higher proportion (Bo et al., 2003). Non-Caucasian women with GDM have been less studied looking for autoimmune markers, being found in some cases, similar GADA incidence than in Caucasian women (Kousta et al., 2001), while in other studies, lower incidences have been obtained, but resulting likewise, the presence of antibodies against beta cells, an indicator of future type 1 The presence of antibodies in women with GDM shows immune-mediated pancreas destruction that causes a deficit in insulin secretion and development of type 1 diabetes. Therefore, the importance of making a type 1 diabetes diagnose as soon as possible, to apply therapeutic measures that allow preserving the endogenous insulin secretion and to reach during the two previous pregnancies (Getahun et al., 2010). depending on the population studied (Järvelä et al., 2006). diabetes, even at early stages after delivery (Yu et al, 2009). (Retnakaran et al., 2011). 2.2.1 Type 1 diabetes delivery (Mauricio et al., 1996). 2.2 Diabetes seems essential to incorporate profound changes in the quality of life of the population that favor individual life-style changes. However, for a postpartum follow-up program to be successful, it is also important to amend existing weaknesses regarding knowledge and motivation of the interdisciplinary team responsible for health care in this area (Almario et al., 2008; Clark et al., 2003), and also, to provide health care services with easy access for all women with previous GDM (Kim et al., 2007a). This chapter will expound on focusing the future risks of women with GDM, as well as the basic aspects regarding early detection and prevention of diabetes and other cardiovascular risk factors. ### 2. Maternal risks Maternal GDM repercussions in the future include an increased risk of developing GDM in subsequent pregnancies (Moses, 1997a), type 2 diabetes (Kim et al., 2002) and other cardiovascular risk factors such as obesity (Yun et al., 2007), dyslipidemia (Meyer-Seifer, 1996) and hypertension (Gonçalves et al., 2005), which show up isolated or grouped in the Metabolic Syndrome (Madarász et al., 2008). Generally, they are accompanied by some degree of vascular, fibrinolytic and inflammatory dysfunction (Farhan et al., 2006¸ Heitritter, 2005). Furthermore, the risk of other clinical conditions like polycystic ovary (PCO) (Kousta et al., 2000), periodontal disease (Xiong et al., 2006) and depression (34 Kozhimannil et al., 2009) is increased (Table 1). Table 1. Maternal Risks in women with Previous GDM #### 2.1 GDM in subsequent pregnancies GDM recurrence occurs due to an abnormal glucose tolerance state that aggravates primarily due to physiological demands and hormonal changes of pregnancy itself, but may also show the presence of type 2 diabetes, that was not diagnosed between pregnancies, since a postpartum diabetes screening was not carried out. GDM recurrence rates in subsequent pregnancies show contradictory results, varying in different studies according to the population studied, GDM diagnostic criteria used, diabetes postpartum screenings and the exclusion or not of the preexisting diabetes proportion. In a review, it was found that they ranged from 30 and 84%, being White women rates <40% and in other ethnic groups that include African-American, Latin-American an Asian women >50%, constituting ethnic group different from Caucasian, the most consistent predictor of GDM recurrence (Kim et al., 2007b).Other associations have been reported with GDM recurrence such as fat intake (Moses et al., 1997b), pre-pregnancy maternal weight (Macneill et al., 2001) and the presence of impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) in two-month postpartum screenings (Kwak et al., 2008). GDM risk in a second pregnancy has been found nearly ten-folds higher among women with previous GDM than in women without this family history, 41.3% and 4.2%, respectively. The risk increases with the number of previous GDM episodes and thus, with the third pregnancy, GDM recurrence was more marked when GDM had been diagnosed during the two previous pregnancies (Getahun et al., 2010). A new GDM pregnancy is not associated itself to a risk increase of type 2 diabetes in women with GDM history (Russel et al., 2008). Nonetheless, the absence of recurrent GDM in a subsequent pregnancy may show a decrease in the risk of developing type 2 diabetes (Retnakaran et al., 2011). #### 2.2 Diabetes 364 Gestational Diabetes seems essential to incorporate profound changes in the quality of life of the population that favor individual life-style changes. However, for a postpartum follow-up program to be successful, it is also important to amend existing weaknesses regarding knowledge and motivation of the interdisciplinary team responsible for health care in this area (Almario et al., 2008; Clark et al., 2003), and also, to provide health care services with easy access for all This chapter will expound on focusing the future risks of women with GDM, as well as the basic aspects regarding early detection and prevention of diabetes and other cardiovascular Maternal GDM repercussions in the future include an increased risk of developing GDM in subsequent pregnancies (Moses, 1997a), type 2 diabetes (Kim et al., 2002) and other cardiovascular risk factors such as obesity (Yun et al., 2007), dyslipidemia (Meyer-Seifer, 1996) and hypertension (Gonçalves et al., 2005), which show up isolated or grouped in the Metabolic Syndrome (Madarász et al., 2008). Generally, they are accompanied by some degree of vascular, fibrinolytic and inflammatory dysfunction (Farhan et al., 2006¸ Heitritter, 2005). Furthermore, the risk of other clinical conditions like polycystic ovary (PCO) (Kousta et al., 2000), periodontal disease (Xiong et al., 2006) and depression (34 Kozhimannil et al., GDM recurrence occurs due to an abnormal glucose tolerance state that aggravates primarily due to physiological demands and hormonal changes of pregnancy itself, but may also show the presence of type 2 diabetes, that was not diagnosed between pregnancies, GDM recurrence rates in subsequent pregnancies show contradictory results, varying in different studies according to the population studied, GDM diagnostic criteria used, diabetes postpartum screenings and the exclusion or not of the preexisting diabetes proportion. In a review, it was found that they ranged from 30 and 84%, being White women rates <40% and in other ethnic groups that include African-American, Latin-American an Asian women >50%, constituting ethnic group different from Caucasian, the most consistent predictor of GDM recurrence (Kim et al., 2007b).Other associations have been reported with GDM recurrence such as fat intake (Moses et al., 1997b), pre-pregnancy • GDM in subsequent pregnancies • Type 1 diabetes • Type 2 diabetes • Metabolic Syndrome • Vascular abnormalities • Cardiovascular Disease • Polycystic ovaries • Periodontal Disease • Depression Table 1. Maternal Risks in women with Previous GDM since a postpartum diabetes screening was not carried out. 2.1 GDM in subsequent pregnancies women with previous GDM (Kim et al., 2007a). risk factors. 2. Maternal risks 2009) is increased (Table 1). There is strong evidence proving that women with GDM show a high risk of developing diabetes through their lives (Damm et al., 2003). Even though the highest frequency corresponds to type 2 diabetes, type 1 diabetes may also occur, whose proportion will vary depending on the population studied (Järvelä et al., 2006). #### 2.2.1 Type 1 diabetes For many decades GDM has been acknowledged as a heterogeneous alteration, where autoimmunity against beta cells constitutes the pathogenic basis in a small group of patients, who show a higher risk of developing type 1 diabetes during pregnancy or after delivery (Mauricio et al., 1996). In women with GDM, the determination of different specific antibodies against pancreatic beta cells, like antibodies to islet cells (ICA), glutamic acid decarboxilase antibodies (GADA), tyrosine phosphatase tyrosine (IA-2A), and most recently, GAD 65, proven preclinical markers of type 1 diabetes (Mitchell et al., 2000; Murgia et al., 2008), has allowed to know that autoimmune GDM corresponds to 10% of GDM cases in Caucasian women and contributes to a peculiar and complex pre-diabetic state, with a high risk for progressing to type 1 diabetes and to a latent autoimmune diabetes of adulthood (LADA) (de Leiva et al., 2007; Lapolla et al., 2009). The risk increases with the number of antibodies present (Füchtenbusch et al., 1997), and is higher during the first postpartum years (Nilsson et al., 2007). In studies carried out on women with GDM that show positive antibodies, it was found that they were younger, showed lower body mass index (BMI), less proportion of family members with diabetes, less abdominal circumference, and lower levels of plasmatic insulin than in those women without antibodies. Moreover, they had gained less weight during pregnancy and had required insulin treatment in a higher proportion (Bo et al., 2003). Non-Caucasian women with GDM have been less studied looking for autoimmune markers, being found in some cases, similar GADA incidence than in Caucasian women (Kousta et al., 2001), while in other studies, lower incidences have been obtained, but resulting likewise, the presence of antibodies against beta cells, an indicator of future type 1 diabetes, even at early stages after delivery (Yu et al, 2009). The presence of antibodies in women with GDM shows immune-mediated pancreas destruction that causes a deficit in insulin secretion and development of type 1 diabetes. Therefore, the importance of making a type 1 diabetes diagnose as soon as possible, to apply therapeutic measures that allow preserving the endogenous insulin secretion and to reach Gestational Diabetes Mellitus After Delivery 367 values during pregnancy, mainly, fasting plasma glucose (FPG) (Kim et al., 2002; Golden et al., 2009) and in lower proportion, 2-hour glucose values (Golden et al., 2009; Åberg et al., 2002), area under OGTT curve (Golden et al., 2009; Weijers et al., 2006) and the number of OGTT abnormal values (Chodick et al., 2010). It has also been found that insulin therapy during pregnancy predicts future maternal diabetes (Chodick et al., 2010; Catalano et al., 1991) and it is known that weight gain before, during and after pregnancy increases and accelerates the development of type 2 diabetes in women with previous GDM (Metzger et Other risk factors of type 2 diabetes in women with DGM have been reported, among them, age, non-Caucasian ethnicity, early GDM onset (Sinha et al,. 2003), length of postpartum period, deterioration of beta cell function and use of progestin-only contraception (Xiang et al 2010, Xiang et al 2006a). A series of modifiable factors like low physical activity, low fruit and vegetable consumption, tobacco habits, low educational instruction, and low family income have also been found associated to the risk of type 2 diabetes in women with GDM history (Yun S et al., 2007). There is less knowledge on other risk factors of diabetes described in recent years in women with GDM, such as alteration of some inflammatory and fibrinolytic dysfunction markers, among them adiponectin decrease (Retnakaran et al. 2010), increase in Creactive protein (CRP) (Di Benedetto et al., 2005), homocysteine (Cho et al., 2005) and plasminogen activator inhibitor type 1 (PAI-1) (Morimitsu et al., 2007), among others. GDM is a marker for future development of type 2 diabetes and Metabolic Syndrome in the mother, currently acknowledging pregnancy as a window that reveals future metabolic and Glucose tolerance abnormalities in postpartum have been associated to other components of the Metabolic Syndrome. General obesity (Vohr & Boney, 2008), visceral obesity (Albareda et al,. 2005) and increases in body fat content, particularly visceral fat (Lim et al., 2007), are more frequent in women with GDM history compared to those in control groups. Also there is a higher risk of high blood pressure (Gonçalves et al., 2005; Wender-Ozegowska et al., 2007) and impairment of lipid profile like high triglycerides and LDL-cholesterol values and low HDL-cholesterol values, even though some results are not always consistent (Wender-Ozegowska et al., 2007; Meyers-Seifer & Vohr, 1996; Rivas et al., 2010b). Frequently, varying abnormalities are found both in insulin sensitivity as well as in the secretion function of The prevalence of the Metabolic Syndrome, regardless of the diagnostic criteria used, is three-fold higher in women with previous GDM than in women without this history and increases seven-fold in obese women (Lauenborg et al., 2005). Pre-pregnancy obesity, OGTT fasting plasma glucose during index pregnancy and postpartum weight gain are predictors As in other high risk type 2 diabetes groups, in women with previous GDM, apart from abdominal obesity and insulin resistance, vascular abnormalities including impaired vascular reactivity, increased levels of some markers of endothelial activation, fibrinolysis/coagulation and low grade subclinical inflammation have been found al,. 1993; Baptiste-Roberts et al., 2009; Xiang et al 2010). 2.3 Metabolic syndrome and its components pancreatic beta cells (Tura et al., 2008). 2.4 Vascular abnormalities (Caballero, 2005). cardiovascular risks for the mother (Sattar & Greer 2002). of developing Metabolic Syndrome (Akinci et al., 2010). an adequate metabolic control that lowers the risk of poor pregnancy results (Wucher et al., 2011) and of micro-vascular maternal complications in the future. Thus, in high risk populations of women with GDM, type 1 diabetes screenings must be carried out, looking for antibodies against beta cells. #### 2.2.2 Type 2 diabetes Numerous epidemiological data suggest an association between GDM and type 2 diabetes, showing correspondence in both prevalences in a given population. These two disorders share metabolic aspects, risk factors and genetic susceptibility (Ben-Haroush et al., 2004). Physiopathological changes that occur in GDM, insulin resistance and the relative insulin deficiency due to the pancreatic beta cells deterioration, are similar to processes that occur in other pre-diabetic states of type 2 diabetes (Harlev & Wiznitzer, 2010) and they persist after pregnancy (Kousta et al., 2003). GDM and type 2 diabetes also have in common risk factors like BMI increase, family history of diabetes, increased age, Asian and African ethnic origin (Kim et al., 2002). Likewise, evidence has been gathered regarding that GDM susceptibility, as well as type 2 diabetes, has a genetic component where pregnancy could act as an environmental stressor that catalyses progression to a diabetic state in women with genetic predisposition. It is possible that both conditions are multigenic diseases in whose etiology interact variations of multiple genes with environmental factors, but no definite conclusions can yet be established, since the study on GDM genetics is on its initial stages and also because most researches have been carried out on a small group of White ethnic women (Robitaille & Grant, 2008), finding in some of them, that alleles associated to an increase of developing type 2 diabetes, are elevated in women with previous GDM (Lauenborg et al., 2009). Cumulative incidence of type 2 diabetes varies widely in different reports, with a range of 2.6 to 70% in studies that examined women between 6 weeks postpartum to 28 years postpartum (Kim et al., 2002. During the first months, glucose tolerance abnormalities in women with previous GDM were already found, showing diabetes prevalence rates < 10% in White ethnic women (Pallardo et al., 1999; Feig et al., 2008) and ~10%, or higher in other ethnic groups (Kjos et al., 1990; Lin et al., 2005; Rivas et al., 2007). These rates most probably include women with type 2 diabetes, whose diagnosis had been unnoticed before pregnancy, being impossible to exclude them, due to the GDM definition used worldwide. Progress to diabetes is strong during the first years after delivery, with an annual rate of 5- 10%, reaching ~50% in five years, followed by a slower progression, and appearing a plateau after ten years (Kim et al., 2002; Wollitzer & Jovanovic, 2007). However, more recent studies show that postpartum risk of diabetes in women with DGM increases linearly through the follow-up period, without indication of decrease after five years or plateau evidence of the incidence at ten years postpartum (Feig et al., 2008; Chodick et al.,2010). It has been estimated that in women with GDM, the risk of developing type 2 diabetes along their lives is almost eight-fold higher than in those women who have not developed it (Chodick et al., 2010; Bellamy et al., 2009). In the well-known study Diabetes Prevention Program (DPP), women with GDM history showed a 74% increased hazard for developing diabetes than women without this history (17.1%/year compared to 9.8%/year, respectively) (Ratner et al., 2008) and, in Australia, it was found that 20-30% of women with type 2 diabetes refer previous GDM (Cheung & Byth, 2003). Several maternal risk factors have been associated to the conversion rate to diabetes, resulting highly predictive at short and long-term the oral glucose tolerance test (OGTT) an adequate metabolic control that lowers the risk of poor pregnancy results (Wucher et al., 2011) and of micro-vascular maternal complications in the future. Thus, in high risk populations of women with GDM, type 1 diabetes screenings must be carried out, looking Numerous epidemiological data suggest an association between GDM and type 2 diabetes, showing correspondence in both prevalences in a given population. These two disorders share metabolic aspects, risk factors and genetic susceptibility (Ben-Haroush et al., 2004). Physiopathological changes that occur in GDM, insulin resistance and the relative insulin deficiency due to the pancreatic beta cells deterioration, are similar to processes that occur in other pre-diabetic states of type 2 diabetes (Harlev & Wiznitzer, 2010) and they persist after pregnancy (Kousta et al., 2003). GDM and type 2 diabetes also have in common risk factors like BMI increase, family history of diabetes, increased age, Asian and African ethnic origin (Kim et al., 2002). Likewise, evidence has been gathered regarding that GDM susceptibility, as well as type 2 diabetes, has a genetic component where pregnancy could act as an environmental stressor that catalyses progression to a diabetic state in women with genetic predisposition. It is possible that both conditions are multigenic diseases in whose etiology interact variations of multiple genes with environmental factors, but no definite conclusions can yet be established, since the study on GDM genetics is on its initial stages and also because most researches have been carried out on a small group of White ethnic women (Robitaille & Grant, 2008), finding in some of them, that alleles associated to an increase of developing type 2 diabetes, are elevated in women with previous GDM (Lauenborg et al., 2009). type 2 diabetes refer previous GDM (Cheung & Byth, 2003). Cumulative incidence of type 2 diabetes varies widely in different reports, with a range of 2.6 to 70% in studies that examined women between 6 weeks postpartum to 28 years postpartum (Kim et al., 2002. During the first months, glucose tolerance abnormalities in women with previous GDM were already found, showing diabetes prevalence rates < 10% in White ethnic women (Pallardo et al., 1999; Feig et al., 2008) and ~10%, or higher in other ethnic groups (Kjos et al., 1990; Lin et al., 2005; Rivas et al., 2007). These rates most probably include women with type 2 diabetes, whose diagnosis had been unnoticed before pregnancy, being impossible to exclude them, due to the GDM definition used worldwide. Progress to diabetes is strong during the first years after delivery, with an annual rate of 5- 10%, reaching ~50% in five years, followed by a slower progression, and appearing a plateau after ten years (Kim et al., 2002; Wollitzer & Jovanovic, 2007). However, more recent studies show that postpartum risk of diabetes in women with DGM increases linearly through the follow-up period, without indication of decrease after five years or plateau evidence of the incidence at ten years postpartum (Feig et al., 2008; Chodick et al.,2010). It has been estimated that in women with GDM, the risk of developing type 2 diabetes along their lives is almost eight-fold higher than in those women who have not developed it (Chodick et al., 2010; Bellamy et al., 2009). In the well-known study Diabetes Prevention Program (DPP), women with GDM history showed a 74% increased hazard for developing diabetes than women without this history (17.1%/year compared to 9.8%/year, respectively) (Ratner et al., 2008) and, in Australia, it was found that 20-30% of women with Several maternal risk factors have been associated to the conversion rate to diabetes, resulting highly predictive at short and long-term the oral glucose tolerance test (OGTT) for antibodies against beta cells. 2.2.2 Type 2 diabetes values during pregnancy, mainly, fasting plasma glucose (FPG) (Kim et al., 2002; Golden et al., 2009) and in lower proportion, 2-hour glucose values (Golden et al., 2009; Åberg et al., 2002), area under OGTT curve (Golden et al., 2009; Weijers et al., 2006) and the number of OGTT abnormal values (Chodick et al., 2010). It has also been found that insulin therapy during pregnancy predicts future maternal diabetes (Chodick et al., 2010; Catalano et al., 1991) and it is known that weight gain before, during and after pregnancy increases and accelerates the development of type 2 diabetes in women with previous GDM (Metzger et al,. 1993; Baptiste-Roberts et al., 2009; Xiang et al 2010). Other risk factors of type 2 diabetes in women with DGM have been reported, among them, age, non-Caucasian ethnicity, early GDM onset (Sinha et al,. 2003), length of postpartum period, deterioration of beta cell function and use of progestin-only contraception (Xiang et al 2010, Xiang et al 2006a). A series of modifiable factors like low physical activity, low fruit and vegetable consumption, tobacco habits, low educational instruction, and low family income have also been found associated to the risk of type 2 diabetes in women with GDM history (Yun S et al., 2007). There is less knowledge on other risk factors of diabetes described in recent years in women with GDM, such as alteration of some inflammatory and fibrinolytic dysfunction markers, among them adiponectin decrease (Retnakaran et al. 2010), increase in Creactive protein (CRP) (Di Benedetto et al., 2005), homocysteine (Cho et al., 2005) and plasminogen activator inhibitor type 1 (PAI-1) (Morimitsu et al., 2007), among others. #### 2.3 Metabolic syndrome and its components GDM is a marker for future development of type 2 diabetes and Metabolic Syndrome in the mother, currently acknowledging pregnancy as a window that reveals future metabolic and cardiovascular risks for the mother (Sattar & Greer 2002). Glucose tolerance abnormalities in postpartum have been associated to other components of the Metabolic Syndrome. General obesity (Vohr & Boney, 2008), visceral obesity (Albareda et al,. 2005) and increases in body fat content, particularly visceral fat (Lim et al., 2007), are more frequent in women with GDM history compared to those in control groups. Also there is a higher risk of high blood pressure (Gonçalves et al., 2005; Wender-Ozegowska et al., 2007) and impairment of lipid profile like high triglycerides and LDL-cholesterol values and low HDL-cholesterol values, even though some results are not always consistent (Wender-Ozegowska et al., 2007; Meyers-Seifer & Vohr, 1996; Rivas et al., 2010b). Frequently, varying abnormalities are found both in insulin sensitivity as well as in the secretion function of pancreatic beta cells (Tura et al., 2008). The prevalence of the Metabolic Syndrome, regardless of the diagnostic criteria used, is three-fold higher in women with previous GDM than in women without this history and increases seven-fold in obese women (Lauenborg et al., 2005). Pre-pregnancy obesity, OGTT fasting plasma glucose during index pregnancy and postpartum weight gain are predictors of developing Metabolic Syndrome (Akinci et al., 2010). #### 2.4 Vascular abnormalities As in other high risk type 2 diabetes groups, in women with previous GDM, apart from abdominal obesity and insulin resistance, vascular abnormalities including impaired vascular reactivity, increased levels of some markers of endothelial activation, fibrinolysis/coagulation and low grade subclinical inflammation have been found (Caballero, 2005). Gestational Diabetes Mellitus After Delivery 369 hand, an association between diabetes during pregnancy and prenatal depression in lowincome women has been found (Kozhimannil et al., 2009), showing the importance of 3. Detection and prevention of diabetes and other cardiovascular risk factors Care for women with GDM is extended after delivery in order to assess at an early stage their new metabolic status, and more importantly, to take individual and collective measures that contribute to prevent or at least, retard the progression to diabetes and other cardiovascular risk factors. Thus, it is necessary that health services provide postpartum follow-up programs and motivate women with GDM to attend the activities programmed. Moreover, it is fundamental that the environment surrounding such women promotes lifestyle changes that not only favor them, but also their family and the rest of the population. For many years the importance of postpartum follow-up of women with GDM has been emphasized (Beischer et al., 1997). More recent findings suggest that it should be extended to women with any abnormal glucose homeostasis level during pregnancy (Retnakaran et During the immediate post-partum period of patients with GDM, glucose tests are run to detect those few cases where hyperglycemia persists, which mainly correspond to women whose type 2 diabetes had not been detected before pregnancy. The diabetes diagnose is confirmed if FPG is > 126 mg/dl (7.0 mmol/l) or if hyperglycemia symptoms and a random plasma glucose > 200 mg/dl (11.1 mmol/l) are present (American Diabetes Association All those women not diagnosed with diabetes are programmed for a diabetes screening between weeks six and twelve of postpartum, due to a high incidence of glucose tolerance abnormalities already detected at that time and also because these results allow identifying women with high risks of developing diabetes during the next fifteen years (Kjos et al., 1995). If diabetes screening is not possible at this time, it must be emphasized in the following weeks. Diabetes screening is carried out with a 75g- 2h- OGTT, of higher sensitivity and less expensive per diabetes case detected than FPG (Ferrara et al., 2009; Kim psycho-social aspects. 3.1 Postpartum follow-up of women with GDM • Diabetes screening • Oral health measures • Life-style changes Table 2. Postpartum Follow-up of women with GDM • Pharmacologic intervention • Education 3.1.1 Diabetes screening (ADA), 2011). al., 2008). Such a follow-up includes the aspects shown in Table 2. • Detection of cardiovascular risk factors • Incentive and facilities for breastfeeding • Appropriate family planning and contraception Among the reported vascular abnormalities are increase of peripheral vascular resistance (Heitritter et al., 2005) and impaired endothelium-dependent vasodilation, assessed at the brachial artery by high resolution ultrasound (Anastasiou etal., 1998), but this finding was not found in another study (Hannemann et al., 2002). On the other hand, serum adiponectin levels, an adipokine with known vascular effects, have been found to be lower in women with GDM during pregnancy and postpartum (Heitritter et al., 2005; Vitoratos et al., 2008a; Costacou et al., 2008). On the contrary, PAI-1 has been found elevated, considered as an expression of fibrinolytic dysfunction (Farhan et al., 2006), and of markers of low-grade subclinical inflammation, Interleukin-1beta (IL-1β) has also been found elevated (Vitoratos et al., 2008b), whereas CRP shows contradictory results, since in some studies higher values have been obtained in women with previous GDM than in women with normoglycemic pregnancies (Heitritter et al., 2005; Di Cianni et al., 2007), no differences have been shown in other studies (Thomann et al., 2008) and in some, it is only significant the increase of CRP when diabetes has already been developed (Kim et al., 2008). #### 2.5 Cardiovascular disease (CVD) As it has been described previously, women with previous GDM show in higher proportion, compared to women in control groups, numerous cardiovascular risk factors such as abdominal obesity, insulin resistance, abnormal glucose tolerance, dyslipidemia, high blood pressure values, Metabolic Syndrome, impairments of endothelial dysfunction and inflammation markers. For this reason, it has been proposed that these women have higher long-term probabilities of developing CVD (Bentley-Lewis, 2009), showing in an incidence research review that GDM history increases the risk of CVD about 1.7 folds (Verier-Mine, 2010). This topic is of vital importance since CVD, and particularly, coronary artery disease, constitute one of the main causes of mortality and disability in different countries, thus its prevention and early identification in this group of young women may contribute to improve health indicators. #### 2.6 Polycystic ovaries The prevalence of PCO has been found elevated in women with previous GDM, showing that both pathologies have common associations like obesity and insulin resistance (Kousta et al., 2000; Koivunen et al., 2001). #### 2.7 Periodontal disease Women with GDM have shown higher frequency and severity of periodontal disease during pregnancy and postpartum than women without GDM, after controlling for age, income, smoking, dental calculus (Novak et al., 2006). The prevalence of periodontal disease in women with previous GDM is lower than in non-pregnant women with type 1 and 2 diabetes, but higher than in non-diabetic women and without GDM family history (Xiong et al., 2006). A higher risk of dental caries also seems to exist (Friedlander et al., 2007). #### 2.8 Stress, anxiety and depression Pregnancy generally represents an increase in stress and anxiety in all women, therefore, when GDM develops, considered as a high risk pregnancy demanding comprehensive treatment measures and monitoring of metabolic control, stress levels may increase even more (York et al., 1996). However, there are very few studies on this topic. On the other Among the reported vascular abnormalities are increase of peripheral vascular resistance (Heitritter et al., 2005) and impaired endothelium-dependent vasodilation, assessed at the brachial artery by high resolution ultrasound (Anastasiou etal., 1998), but this finding was not found in another study (Hannemann et al., 2002). On the other hand, serum adiponectin levels, an adipokine with known vascular effects, have been found to be lower in women with GDM during pregnancy and postpartum (Heitritter et al., 2005; Vitoratos et al., 2008a; Costacou et al., 2008). On the contrary, PAI-1 has been found elevated, considered as an expression of fibrinolytic dysfunction (Farhan et al., 2006), and of markers of low-grade subclinical inflammation, Interleukin-1beta (IL-1β) has also been found elevated (Vitoratos et al., 2008b), whereas CRP shows contradictory results, since in some studies higher values have been obtained in women with previous GDM than in women with normoglycemic pregnancies (Heitritter et al., 2005; Di Cianni et al., 2007), no differences have been shown in other studies (Thomann et al., 2008) and in some, it is only significant the increase of CRP As it has been described previously, women with previous GDM show in higher proportion, compared to women in control groups, numerous cardiovascular risk factors such as abdominal obesity, insulin resistance, abnormal glucose tolerance, dyslipidemia, high blood pressure values, Metabolic Syndrome, impairments of endothelial dysfunction and inflammation markers. For this reason, it has been proposed that these women have higher long-term probabilities of developing CVD (Bentley-Lewis, 2009), showing in an incidence research review that GDM history increases the risk of CVD about 1.7 folds (Verier-Mine, 2010). This topic is of vital importance since CVD, and particularly, coronary artery disease, constitute one of the main causes of mortality and disability in different countries, thus its prevention and early identification in this group of young women may contribute to The prevalence of PCO has been found elevated in women with previous GDM, showing that both pathologies have common associations like obesity and insulin resistance (Kousta Women with GDM have shown higher frequency and severity of periodontal disease during pregnancy and postpartum than women without GDM, after controlling for age, income, smoking, dental calculus (Novak et al., 2006). The prevalence of periodontal disease in women with previous GDM is lower than in non-pregnant women with type 1 and 2 diabetes, but higher than in non-diabetic women and without GDM family history (Xiong et Pregnancy generally represents an increase in stress and anxiety in all women, therefore, when GDM develops, considered as a high risk pregnancy demanding comprehensive treatment measures and monitoring of metabolic control, stress levels may increase even more (York et al., 1996). However, there are very few studies on this topic. On the other al., 2006). A higher risk of dental caries also seems to exist (Friedlander et al., 2007). when diabetes has already been developed (Kim et al., 2008). 2.5 Cardiovascular disease (CVD) improve health indicators. et al., 2000; Koivunen et al., 2001). 2.8 Stress, anxiety and depression 2.6 Polycystic ovaries 2.7 Periodontal disease hand, an association between diabetes during pregnancy and prenatal depression in lowincome women has been found (Kozhimannil et al., 2009), showing the importance of psycho-social aspects. ### 3. Detection and prevention of diabetes and other cardiovascular risk factors Care for women with GDM is extended after delivery in order to assess at an early stage their new metabolic status, and more importantly, to take individual and collective measures that contribute to prevent or at least, retard the progression to diabetes and other cardiovascular risk factors. Thus, it is necessary that health services provide postpartum follow-up programs and motivate women with GDM to attend the activities programmed. Moreover, it is fundamental that the environment surrounding such women promotes lifestyle changes that not only favor them, but also their family and the rest of the population. #### 3.1 Postpartum follow-up of women with GDM For many years the importance of postpartum follow-up of women with GDM has been emphasized (Beischer et al., 1997). More recent findings suggest that it should be extended to women with any abnormal glucose homeostasis level during pregnancy (Retnakaran et al., 2008). Such a follow-up includes the aspects shown in Table 2. Table 2. Postpartum Follow-up of women with GDM #### 3.1.1 Diabetes screening During the immediate post-partum period of patients with GDM, glucose tests are run to detect those few cases where hyperglycemia persists, which mainly correspond to women whose type 2 diabetes had not been detected before pregnancy. The diabetes diagnose is confirmed if FPG is > 126 mg/dl (7.0 mmol/l) or if hyperglycemia symptoms and a random plasma glucose > 200 mg/dl (11.1 mmol/l) are present (American Diabetes Association (ADA), 2011). All those women not diagnosed with diabetes are programmed for a diabetes screening between weeks six and twelve of postpartum, due to a high incidence of glucose tolerance abnormalities already detected at that time and also because these results allow identifying women with high risks of developing diabetes during the next fifteen years (Kjos et al., 1995). If diabetes screening is not possible at this time, it must be emphasized in the following weeks. Diabetes screening is carried out with a 75g- 2h- OGTT, of higher sensitivity and less expensive per diabetes case detected than FPG (Ferrara et al., 2009; Kim Gestational Diabetes Mellitus After Delivery 371 levonorgestrel-releasing IUD, which possess known advantages and limitations. Low-dose combination oral contraceptives, with ethinyl estradiol and a progestin, may also be used, but they are not recommended for women that have other cardiovascular risk factors like hypertension. Even though family history of GDM does not mean contraindication of any method, progestin-only pills should be avoided in women who are breastfeeding, since in a study carried out on Latin-women it was found that it was associated to an increase of diabetes risk, assuming that breastfeeding may be a relatively progestagenic state (Kjos et al., 1998). Neither long-acting progestin methods like depot medroxyprogesterone acetate nor medroxyprogesterone, may be suggested as a first option since they may have major effects on the hydrocarbonated metabolism, as it was proven in Navajo women (Kim et al., 2001). In parous women refraining from another pregnancy, surgical sterilization is a good option, particularly in those delivering by cesarean section, since sterilization may be It is important that oral health measures proposed to the population in general for prevention of caries and periodontal disease, are rigorously adopted by women with previous GDM, who moreover, should visit at least annually the dentist in order to carry out an early diagnose of these complications when they arise and to apply the required therapeutic measures. The dentist will be on the alert to detect if progression to diabetes has Childbearing years constitute one of the stages in the life of a woman more prone to weight gain. Thus, life-style changes make up the building blocks for the prevention of diabetes and other cardiovascular risk factors in women with GDM. Providing preventive care leading to reach and maintain an adequate weight, that include strategies on the aspects shown on Table 3, would result extremely beneficial for the health of this vulnerable population group and also cost-effective (Kapustin, 2008). Even though specific studies on this aspect are scarce and show limitations, it has been found that intensive intervention on life-style changes in women with previous GDM, reduced to a 50% diabetes incidence (Ratner et al., In general, nutritional recommendations for women with a high risk of developing type 2 diabetes tend to reduce the consumption of processed foods with high content of sugars, salt and trans fats, favoring the consumption of fresh foods like whole grains, legumes, vegetables, fruits, nuts, seeds, low-fat dairy, skinless poultry, and fish to provide omega-3 occurred, contributing with this pathology screening (Friedlander et al., 2007). 2008). Undoubtedly, there is a need to study in depth this topic. • Low to Moderate alcohol consume • Adequate stress management • Healthy nutrition • Physical activity • Non-smoking Table 3. Life-style changes in women with previous GDM practiced during surgical procedures. 3.1.6 Oral health measures 3.1.7 Life-style changes 3.1.7.1 Healthy nutrition et al., 2007b). If the results are not compatible with diabetes diagnose, whether they are normal or indicators of IGT, or IFG, the test is repeated the following year and then, annually or every three years, according to the different international scientific associations since regarding periodicity of screening, no current criteria uniformity exists (Metzger et al., 2007, American College of Obstetricians and Gynecologists (ACOG), 2009, Asociación Latinoamericana de Diabetes (ALAD), 2008). It is possible that HbA1c will be recommended in the future for postpartum diabetes screening in women with GDM, but studies on this test have not yet been published. #### 3.1.2 Detection and treatment of cardiovascular risk factors Every time diabetes screenings are carried out, for detecting hypertension, obesity and dyslipidemias, women with previous GDM are determined for blood pressure, abdominal circumference, body mass index and triglyceride plasma levels, cholesterol, HDL-c and LDL-c. Determination of insulin levels, insulin-sensitivity indices, markers of endothelial activation, fibrinolysis/coagulation and low grade subclinical inflammation, and other specialized tests are optional in the clinical practice and are reserved in most cases, for research purposes. If the presence of any cardiovascular risk factor is confirmed, the corresponding therapeutic measures are prescribed. #### 3.1.3 Education Education, initiated during pregnancy, constitutes the basis for GDM management. It is imparted in theoretical-practical sessions individually or in groups, whose content and strategies take into account the socio-economical and cultural characteristics of the enrolled women. At the postpartum stage, it is directed basically to imparting knowledge on future maternal risks and on their off-spring, as well as to the life-style changes that contribute to prevent or retard diabetes development and its co-morbidities. It has been found that knowledge on these topics is limited in women with GDM, but increases after participating in an educational program imparted by specialized health personnel (Rivas et al., 2010a). #### 3.1.4 Breastfeeding incentive and facilities As other women, women with GDM must be actively stimulated for exclusively breastfeeding for the longest period during the first year of their child (Metzger et al., 2007). But in this case, it is paramount to contribute in reducing subsequent risks of obesity and glucose tolerance abnormalities. Even though there is currently no definite conclusion on this (Gouveri et al., 2011), many studies show beneficial results of breastfeeding in women with GDM (Kjos et al., 1993; Gunderson et al., 2010). #### 3.1.5 Appropriate family planning and contraception In women with GDM postpartum contraception is recommended (Metzger et al., 2007) in order to prevent a future unplanned pregnancy, with a high risk of developing once more GDM and where teratogenic effects of non-diagnosed diabetes may also be present. There is a wide option of contraceptive methods (Kim, 2009; Kim, 2010; Damm et al., 2007) that in general differ little from the ones used by other women. However, it is important to choose a contraceptive method that does not increase maternal risk of glucose intolerance, metabolic syndrome and CVD, as it occurs with barrier methods, the lactation amenorrhea method during the first six months and intrauterine devices (IUD), both copper and et al., 2007b). If the results are not compatible with diabetes diagnose, whether they are normal or indicators of IGT, or IFG, the test is repeated the following year and then, annually or every three years, according to the different international scientific associations since regarding periodicity of screening, no current criteria uniformity exists (Metzger et al., 2007, American College of Obstetricians and Gynecologists (ACOG), 2009, Asociación Latinoamericana de Diabetes (ALAD), 2008). It is possible that HbA1c will be recommended in the future for postpartum diabetes screening in women with GDM, but studies on this Every time diabetes screenings are carried out, for detecting hypertension, obesity and dyslipidemias, women with previous GDM are determined for blood pressure, abdominal circumference, body mass index and triglyceride plasma levels, cholesterol, HDL-c and LDL-c. Determination of insulin levels, insulin-sensitivity indices, markers of endothelial activation, fibrinolysis/coagulation and low grade subclinical inflammation, and other specialized tests are optional in the clinical practice and are reserved in most cases, for research purposes. If the presence of any cardiovascular risk factor is confirmed, the Education, initiated during pregnancy, constitutes the basis for GDM management. It is imparted in theoretical-practical sessions individually or in groups, whose content and strategies take into account the socio-economical and cultural characteristics of the enrolled women. At the postpartum stage, it is directed basically to imparting knowledge on future maternal risks and on their off-spring, as well as to the life-style changes that contribute to prevent or retard diabetes development and its co-morbidities. It has been found that knowledge on these topics is limited in women with GDM, but increases after participating in an educational program imparted by specialized health personnel (Rivas et al., 2010a). As other women, women with GDM must be actively stimulated for exclusively breastfeeding for the longest period during the first year of their child (Metzger et al., 2007). But in this case, it is paramount to contribute in reducing subsequent risks of obesity and glucose tolerance abnormalities. Even though there is currently no definite conclusion on this (Gouveri et al., 2011), many studies show beneficial results of breastfeeding in women In women with GDM postpartum contraception is recommended (Metzger et al., 2007) in order to prevent a future unplanned pregnancy, with a high risk of developing once more GDM and where teratogenic effects of non-diagnosed diabetes may also be present. There is a wide option of contraceptive methods (Kim, 2009; Kim, 2010; Damm et al., 2007) that in general differ little from the ones used by other women. However, it is important to choose a contraceptive method that does not increase maternal risk of glucose intolerance, metabolic syndrome and CVD, as it occurs with barrier methods, the lactation amenorrhea method during the first six months and intrauterine devices (IUD), both copper and test have not yet been published. 3.1.3 Education 3.1.2 Detection and treatment of cardiovascular risk factors corresponding therapeutic measures are prescribed. 3.1.4 Breastfeeding incentive and facilities with GDM (Kjos et al., 1993; Gunderson et al., 2010). 3.1.5 Appropriate family planning and contraception levonorgestrel-releasing IUD, which possess known advantages and limitations. Low-dose combination oral contraceptives, with ethinyl estradiol and a progestin, may also be used, but they are not recommended for women that have other cardiovascular risk factors like hypertension. Even though family history of GDM does not mean contraindication of any method, progestin-only pills should be avoided in women who are breastfeeding, since in a study carried out on Latin-women it was found that it was associated to an increase of diabetes risk, assuming that breastfeeding may be a relatively progestagenic state (Kjos et al., 1998). Neither long-acting progestin methods like depot medroxyprogesterone acetate nor medroxyprogesterone, may be suggested as a first option since they may have major effects on the hydrocarbonated metabolism, as it was proven in Navajo women (Kim et al., 2001). In parous women refraining from another pregnancy, surgical sterilization is a good option, particularly in those delivering by cesarean section, since sterilization may be practiced during surgical procedures. #### 3.1.6 Oral health measures It is important that oral health measures proposed to the population in general for prevention of caries and periodontal disease, are rigorously adopted by women with previous GDM, who moreover, should visit at least annually the dentist in order to carry out an early diagnose of these complications when they arise and to apply the required therapeutic measures. The dentist will be on the alert to detect if progression to diabetes has occurred, contributing with this pathology screening (Friedlander et al., 2007). #### 3.1.7 Life-style changes Childbearing years constitute one of the stages in the life of a woman more prone to weight gain. Thus, life-style changes make up the building blocks for the prevention of diabetes and other cardiovascular risk factors in women with GDM. Providing preventive care leading to reach and maintain an adequate weight, that include strategies on the aspects shown on Table 3, would result extremely beneficial for the health of this vulnerable population group and also cost-effective (Kapustin, 2008). Even though specific studies on this aspect are scarce and show limitations, it has been found that intensive intervention on life-style changes in women with previous GDM, reduced to a 50% diabetes incidence (Ratner et al., 2008). Undoubtedly, there is a need to study in depth this topic. Table 3. Life-style changes in women with previous GDM #### 3.1.7.1 Healthy nutrition In general, nutritional recommendations for women with a high risk of developing type 2 diabetes tend to reduce the consumption of processed foods with high content of sugars, salt and trans fats, favoring the consumption of fresh foods like whole grains, legumes, vegetables, fruits, nuts, seeds, low-fat dairy, skinless poultry, and fish to provide omega-3 Gestational Diabetes Mellitus After Delivery 373 disease, and its cost (Verier-Mine, 2010; Kim, 2009: Kim, 2010). When metphormine was used in a study, the risk of type 2 diabetes was half reduced in women with overweight and obesity (Ratner et al., 2008). Therefore, further research will provide more insight for its prescription in clinical practice in combination with a healthy life-style, particularly in obese As shown, an adequate postpartum follow-up program for women with GDM comprises a wide range of health-care, clinical-metabolic, gynecological, nutritional, educational, psycho-social, physical training, and odontological activities, among others, carried out by an interdisciplinary team, in order to prevent or retard progression to diabetes and other cardiovascular risk factors, and if this is not attained, to confirm its diagnose as soon as Unfortunately worldwide, postpartum follow-ups of women with GDM are low. In the United States during the first postpartum months, FPG measurements were ordered on 60.5% women and only completed by 34% (Dinh et al., 2003). In a long cohort of women with GDM, 42% were not tested for FPG, the test was not ordered in 21% of them and none were tested for OGTT (Dietz et al., 2008). Only 37% were tested for FPG or OGTT with a mean of ~ 14 months of postpartum (Smirmakis et al., 2005). In Canada, it has been shown that physicians do not order postpartum OGTT, in spite of counting on a publication with guidelines based on expert opinions on the subject (Clark et al., 2003). At a Venezuelan hospital, in a follow-up program, a 66.19% OGTT adherence after a postpartum period of 4.04 years + 2.68 was met, with only 17.98% attendance to all basic education sessions (Rivas et al., 2010c). Achieving favorable changes in life-styles of women with GDM has resulted even more difficult to attain (Stage et al., 2004; Smith et al., 2005), and progression to Individual approach directed to inform women with GDM on the need of reclassifying their metabolic status after delivery, results insufficient for making possible the prevention of diabetes and other cardiovascular risk factors. The need to reinforce knowledge and motivation in the health care team is evident, as well as the access to health care in this area. Moreover, there is a need for stronger research and confrontation on the social determinants that make difficult for GDM women, adherence to postpartum follow-up and the adoption of healthy life-styles (Hjaltested & Conroy, 2010). Structural measures targeted to the population in general, like the ones shown in Table 4, would undoubtedly result in greater benefit. • Promoting the creation and use of public transportation, bicycle lanes, walking lanes • Regulating production, distribution and marketing of processed foods rich in trans • Increasing production, distribution and marketing of fresh foods • Promoting the creation and use of public sport courts and parks • Regulating publicity through communication media of this kind of foods women with glucose intolerance after a GDM pregnancy. possible. diabetes continues increasing. 3.2 Changes in quality of life of the population • Promoting and protecting breastfeeding • Promoting smoke-free environments fats, saturated fats, salt and refined sugars Table 4. Changes in quality of life of the population fatty acids; in other words, foods with low levels of cholesterol and saturated fats but rich in fiber, micronutrients and antioxidants (Melanson, 2008). Daily calorie intake is tailored by nutritionists, according to the characteristics of each woman, so they guarantee weight loss if recommended and at the same time, an adequate nutrition. It is fundamental to provide education on calorie count, food portions, food selection and preparation, reading and interpreting labels, since self-monitoring of intake may help them to incorporate the food plan in their life-styles and adopt these new behavior patterns (Case et al., 2006). Uniform nutritional recommendations in women with a GDM history are needed. #### 3.1.7.2 Physical activity Increasing physical activity is paramount in the daily routine of women with previous GDM, if there is no contraindication for this after a thorough medical evaluation. The goal is to reach a program of aerobic exercises like walking, swimming, dancing, bike riding for 30 minutes five or more days a week, beginning gradually from 5-10 minutes daily in sedentary women. Before and after exercising, stretching exercises must be done for 5-10 minutes. If this is complemented with strength training using light weights or elastic bands, weight loss is increased and muscle tone is improved, having a favorable effect on the glucose metabolism (Case et al., 2006). #### 3.1.7.3 Non-smoking Quitting smoking is recommended in women with previous GDM (Verier-Mine, 2010), in spite of the lack of studies assessing the effects of tobacco in this group with a high risk of diabetes and cardiovascular disease. Nonetheless, it has been found that smoking increases the risk of diabetes in women, without discerning if they have this family history or not (Rimm et al., 1993). #### 3.1.7.4 Light to moderate alcohol consumption Light to moderate alcohol consumption has been found associated with a minor risk of developing type 2 diabetes in middle-aged women and this benefit does not seem to persist when alcohol consumption increases (Wannamethee et al., 2003). It is not known if this result may be generalized to women with previous GDM, but it results sensible to avoid a high level of alcohol consumption due to the possible weight gain and the potential increase of type 2 diabetes risk. #### 3.1.7.5 Adequate stress management Learning and the use of periodic tools to allow managing stress adequately may contribute to the goal of keeping healthy women with GDM history. The presence of symptoms of depression or excessive anxiety is an indication for assessment and treatment by specialized Mental Health professionals, according to each case. #### 3.1.8 Pharmacologic intervention The use of pharmacologic agents has been explored for preventing or retarding diabetes in women with previous GDM. Studies with troglitazone (Buchanan et al., 2002) and pioglitazone (Xiang et al., 2006b) have been carried out, demonstrating effectiveness in both cases in overweight women. Nonetheless, the former has been discontinued for its hepatotoxic effects, and the use of the latter is limited since its prescription is not authorized for prevention, due to its safety profile regarding future cardiovascular and osteoporosis fatty acids; in other words, foods with low levels of cholesterol and saturated fats but rich in fiber, micronutrients and antioxidants (Melanson, 2008). Daily calorie intake is tailored by nutritionists, according to the characteristics of each woman, so they guarantee weight loss if recommended and at the same time, an adequate nutrition. It is fundamental to provide education on calorie count, food portions, food selection and preparation, reading and interpreting labels, since self-monitoring of intake may help them to incorporate the food plan in their life-styles and adopt these new behavior patterns (Case et al., 2006). Uniform Increasing physical activity is paramount in the daily routine of women with previous GDM, if there is no contraindication for this after a thorough medical evaluation. The goal is to reach a program of aerobic exercises like walking, swimming, dancing, bike riding for 30 minutes five or more days a week, beginning gradually from 5-10 minutes daily in sedentary women. Before and after exercising, stretching exercises must be done for 5-10 minutes. If this is complemented with strength training using light weights or elastic bands, weight loss is increased and muscle tone is improved, having a favorable effect on the Quitting smoking is recommended in women with previous GDM (Verier-Mine, 2010), in spite of the lack of studies assessing the effects of tobacco in this group with a high risk of diabetes and cardiovascular disease. Nonetheless, it has been found that smoking increases the risk of diabetes in women, without discerning if they have this family history or not Light to moderate alcohol consumption has been found associated with a minor risk of developing type 2 diabetes in middle-aged women and this benefit does not seem to persist when alcohol consumption increases (Wannamethee et al., 2003). It is not known if this result may be generalized to women with previous GDM, but it results sensible to avoid a high level of alcohol consumption due to the possible weight gain and the potential increase Learning and the use of periodic tools to allow managing stress adequately may contribute to the goal of keeping healthy women with GDM history. The presence of symptoms of depression or excessive anxiety is an indication for assessment and treatment by specialized The use of pharmacologic agents has been explored for preventing or retarding diabetes in women with previous GDM. Studies with troglitazone (Buchanan et al., 2002) and pioglitazone (Xiang et al., 2006b) have been carried out, demonstrating effectiveness in both cases in overweight women. Nonetheless, the former has been discontinued for its hepatotoxic effects, and the use of the latter is limited since its prescription is not authorized for prevention, due to its safety profile regarding future cardiovascular and osteoporosis nutritional recommendations in women with a GDM history are needed. 3.1.7.2 Physical activity 3.1.7.3 Non-smoking (Rimm et al., 1993). of type 2 diabetes risk. 3.1.7.5 Adequate stress management 3.1.8 Pharmacologic intervention glucose metabolism (Case et al., 2006). 3.1.7.4 Light to moderate alcohol consumption Mental Health professionals, according to each case. disease, and its cost (Verier-Mine, 2010; Kim, 2009: Kim, 2010). When metphormine was used in a study, the risk of type 2 diabetes was half reduced in women with overweight and obesity (Ratner et al., 2008). Therefore, further research will provide more insight for its prescription in clinical practice in combination with a healthy life-style, particularly in obese women with glucose intolerance after a GDM pregnancy. As shown, an adequate postpartum follow-up program for women with GDM comprises a wide range of health-care, clinical-metabolic, gynecological, nutritional, educational, psycho-social, physical training, and odontological activities, among others, carried out by an interdisciplinary team, in order to prevent or retard progression to diabetes and other cardiovascular risk factors, and if this is not attained, to confirm its diagnose as soon as possible. Unfortunately worldwide, postpartum follow-ups of women with GDM are low. In the United States during the first postpartum months, FPG measurements were ordered on 60.5% women and only completed by 34% (Dinh et al., 2003). In a long cohort of women with GDM, 42% were not tested for FPG, the test was not ordered in 21% of them and none were tested for OGTT (Dietz et al., 2008). Only 37% were tested for FPG or OGTT with a mean of ~ 14 months of postpartum (Smirmakis et al., 2005). In Canada, it has been shown that physicians do not order postpartum OGTT, in spite of counting on a publication with guidelines based on expert opinions on the subject (Clark et al., 2003). At a Venezuelan hospital, in a follow-up program, a 66.19% OGTT adherence after a postpartum period of 4.04 years + 2.68 was met, with only 17.98% attendance to all basic education sessions (Rivas et al., 2010c). Achieving favorable changes in life-styles of women with GDM has resulted even more difficult to attain (Stage et al., 2004; Smith et al., 2005), and progression to diabetes continues increasing. #### 3.2 Changes in quality of life of the population Individual approach directed to inform women with GDM on the need of reclassifying their metabolic status after delivery, results insufficient for making possible the prevention of diabetes and other cardiovascular risk factors. The need to reinforce knowledge and motivation in the health care team is evident, as well as the access to health care in this area. Moreover, there is a need for stronger research and confrontation on the social determinants that make difficult for GDM women, adherence to postpartum follow-up and the adoption of healthy life-styles (Hjaltested & Conroy, 2010). Structural measures targeted to the population in general, like the ones shown in Table 4, would undoubtedly result in greater benefit. Table 4. 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For those women who do not show this, it is very important to apply strategies directed towards primary prevention like healthy lifestyle changes and possible pharmacological intervention, even though further research on these results is needed. To meet both objectives, it is paramount to carry out, on one hand, life-long postpartum follow-up in women with GDM, and also, to research social determinants that affect compliance to these preventive programs and to put into practice collective measures that create favorable conditions for its adherence. #### 5. References After delivery, women with GDM have a high risk of developing diabetes, metabolic syndrome, CVD and other clinical disorders that imply a decrease in the quality of life and high health-care costs. Thus, over several decades early detection of diabetes and other cardiovascular risk factors has been emphasized. 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The prevalence of GAD antibodies in Korean women with Vol. 55, N° 2, (Feb 2006), pp. 517–522 (Jan 2007), pp. 1-6 and Diabetes Risk in Hispanic Women With Prior Gestational Diabetes. Diabetes, Development of Type 2 Diabetes in Hispanic Women With Prior Gestational gestational diabetes mellitus and their clinical characteristics during and after Among Women With Previous Gestational Diabetes. Prev Chronic Dis, Vol. 4, N° 1, pregnancy. Diabetes Metab Res Rev, Vol. 25, N° 4, (May 2009), pp. 329–334 Yun, S.; Kabeer, N. & Zhu, B. (2007). Modifiable Risk Factors for Developing Diabetes ## Edited by Miroslav Radenković Gestational diabetes mellitus is defined as hyperglycemia with onset or first recognition during pregnancy. The incidence of gestational diabetes is still increasing and this pathological condition has strong association with adverse pregnancy outcomes. Since gestational diabetes can have long-term pathological consequences for both mother and the child, it is important that it is promptly recognized and adequately managed. Treatment of gestational diabetes is aimed to maintain euglycemia and it should involve regular glucose monitoring, dietary modifications, life style changes, appropriate physical activity, and when necessary, pharmacotherapy. Adequate glycemic control throughout the pregnancy can notably reduce the occurrence of specific adverse perinatal and maternal outcomes. In a longterm prospect, in order to prevent development of diabetes later in life, as well to avoid associated complications, an adequate education on lifestyle modifications should start in pregnancy and continue postpartum. Gestational Diabetes Gestational Diabetes Edited by Miroslav Radenković Photo by 10174593\_258 / iStock	doab	2025-04-07T03:56:57.843738	20-4-2021 17:12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/3.0/", "book_id": "0008a8ec-01ff-4139-b26e-22862621b722", "url": "https://mts.intechopen.com/storage/books/337/authors_book/authors_book.pdf", "author": "", "title": "Gestational Diabetes", "publisher": "IntechOpen", "isbn": "9789533075815", "section_idx": 4 }
000f0faf-bfb7-4c4d-9180-ec3d905bbc1f	Albert Fleischmann Stefan Oppl · Werner Schmidt Christian Stary # Contextual Process Digitalization Changing Perspectives – Design Thinking – Value-Led Design Contextual Process Digitalization Albert Fleischmann • Stefan Oppl • Werner Schmidt • Christian Stary ## Contextual Process Digitalization Changing Perspectives – Design Thinking – Value-Led Design Albert Fleischmann Dr. Albert Fleischmann & Partner InterAktiv Unternehmensberatung Pfaffenhofen a.d.Ilm, Germany Werner Schmidt Technische Hochschule Ingolstadt Business School Ingolstadt, Germany Stefan Oppl Department for Continuing Education Research and Educational Technologies Danube University Krems Krems, Austria Christian Stary Department of Business Informatics-Communications Engineering Johannes Kepler University Linz Linz, Austria ISBN 978-3-030-38299-5 ISBN 978-3-030-38300-8 (eBook) https://doi.org/10.1007/978-3-030-38300-8 This book is an open access publication. # The Editor(s) (if applicable) and the Author(s) 2020 Open Access This book is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this book are included in the book's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the book's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland ### Preface "The most important innovations are those that change our thinking." We are pursuing this claim with this book, following the aphorism of Hans-Jürgen Quadbeck-Seeger, a German chemist. Our ingredients: "Comprehensibility is the skill of an expert." Again, we have tried to be faithful to this aphorism. Our work should inspire everyone interested in novel forms of process design in the course of digitalization. Therefore, we attempted to keep explanations as simple as possible. "Luxury ¼ Cultivating the Unnecessary." We want to address all those who want to grasp the essence of processes and their utilization in practical action, without intensive modeling language and application studies, but rather underpinned with graspable concepts. Students may appreciate the textbook character of the book, practitioners the examples, and researchers and developers the conceptual representations and theoretical achievements. "The bigger the project, the quieter it will be buried." For more than a decade, the concept and missionary goal of renewing process management has existed. The result has led to the idea of appreciating simplicity and clarity, without neglecting complexity. As novel drivers of process management have evolved, it is time to look at the digitalization of processes from the perspective of subject orientation. "Adventure tourists are attracted to new and exciting places." The experience is worthwhile, because it opens a view of the world that comes close to our perception of reality, conclusively extending the existing, and thus providing new space for adaptation. Our followers are also adventurers—welcome all! Special thanks go to: • Richard Wright for his essential support in increasing the quality of the book. He transformed the German English of the authors into proper English. With his professional expertise and in-depth understanding of the subject matter he also contributed significantly to the readability of the book. We would like to express our sincere thanks to Richard for his achievement in improving the book. "Innovations are not natural events, we have to seriously want and enforce them"—Ad multos multiplicatores, not only in Ingolstadt, Pfaffenhofen, Steyr, and Vienna. Pfaffenhofen a.d.Ilm, Germany Albert Fleischmann Krems, Austria Stefan Oppl Ingolstadt, Germany Werner Schmidt Linz, Austria Christian Stary ### Contents # Motivation 1 #### 1.1 Business Processes and Business Process Management There is no organization without processes. When people want to collaborate, they use the necessary tools and coordinate their activities to reach the desired result. Since such activities can not only be carried out by humans, but also by machines and computers, their activities must also be included when aligning human requirements and technical capabilities. In particular, different types of actors are involved in at least partially automated processes. A process is triggered by an event that may originate inside or outside the organization, such as a travel request or customer order. Coordinated and targeted action in response to such an event is called a process. In case the organization is a company, this is referred to as business process. There is no company without business processes. There are only differences in their level of maturity. The reactions of an organization to certain business events can always be coordinated anew when these events occur, or a procedure is defined that is then executed in such cases. Events of the same type, such as purchase orders, are referred to as event classes. A predefined procedure for an event class is called a process model. The execution of the activity sequences defined in the model as a reaction to an identified concrete event, e.g., the book order of customer Huber from May 20, is termed a process instance. Every company, irrespective of its type of business, has certain standardized processes that can be designed and tailored to the individual company. For instance, every company has an order-to-cash process designed to react to business events, ranging from the customer order to the receipt of payment, and to document these through booking. Conversely, a procurement process will exist with purchase orders to satisfy individual requirements, the concrete reference (for example, receipt of goods and storage), and the payment of vendors. Other examples are processes for recruitment or logistics. A common classification categorizes processes according to their character into management, core and support processes. The classification is company-specific and depends, among other things, on the industry sector. The more clearly a company defines its business processes and the more consistently it implements them in its daily operations, the more efficient it will be. For many companies, their competitiveness is not (or no longer) based solely on the uniqueness of their products, but on the quality of their business processes. For example, while a publisher's business is primarily determined by its books, at Amazon the customer experience in searching, selecting, purchasing, paying, delivering and returning products, i.e., the smooth, customer-centric process, is the key to success. The models for such processes must be continuously adapted or completely redesigned because the reactions to an event class can change, or additional reactions to new event classes can become necessary. The resulting specifications must also be implemented in the organization and IT infrastructure so that employees can work through instances of the processes in day-to-day business. In doing so, underlying conditions, such as effectiveness, efficiency and compliance, i.e., the requirements to deliver the desired result with the lowest possible expenditure of resources and in compliance with valid external and internal regulations (e.g., laws), must be taken into account. Business Process Management (BPM) has established itself to handle these tasks. It describes an integrated management approach for analysis, design, optimization, implementation, control, monitoring, and further development of the management, core and support processes in a company. From a technical point of view, it also includes IT support for these subtasks through corresponding tools, e.g., for modeling or execution (such as process engines) or more comprehensive Business Process Management Systems (BPMS). In Business Process Management, a company and its immediate environment are regarded as a selected part of reality for modeling and executing. In this dedicated part of the world, one party wants a deliverable from another party in the form of a physical product, a service, or a combination of both. The deliverable should be provided in accordance with associated requirements; the desire for it is the business event to which the company should react as perceived in the defined process model. In Business Process Management, it is therefore necessary to define a model for the provision of services and apply it to the processing of business cases. This means adapting reality according to the model, i.e., analyzing affected sections of reality and changing this reality. Since this reality and the desired changes are very complex, several modeling concepts from the social sciences, business administration and computer science are brought together and combined in BPM. In the following sections we outline an overall view of process management and then explain it in detail in the succeeding chapters. From the perspective of the participants on the world, the various facets of Business Process Management are presented, and a selection of models are introduced which have turned out useful in our practice. The design of such models supports the transition from a more or less unstructured or unsatisfactory way of working to a structured process handling that corresponds to the ideas of a company and its customers. We develop the overall view step-by-step, starting from the individual perspectives of the participants on their work in a process, its structuring and harmonization, then moving forward with the specification in a model and its embedding in the organizational and IT environment of the company and finally culminating in the joint processing of process instances in the resulting sociotechnical systems. A corresponding illustration which grows with this overall view ultimately shows our comprehensive understanding of Business Process Management. #### 1.2 View of the World, Structuring and Modeling As already mentioned, it is important for a company to identify the business events of interest and to define the activities triggered by them. For this purpose, the corresponding extract of reality must be identified and examined more closely. This extract is determined by the customers who demand a service and, for the group of employees of the company involved in the provision of the service, it represents the reality which directly affects and surrounds them. In order to provide the desired service, the parties involved must cooperate directly or indirectly. Everyone makes their contribution in coordination with the others. Based on their personal background in terms of education, knowledge, motivation, experiences and preferences, each group member has his own perception of the process and its context. He develops his individual idea of what his contribution should be, how it is provided, which events with which activities need to be considered and by whom, in which order partial steps take place, which preliminary services are expected by whom and for whom preliminary services are provided. As a result, all affected people possess their own mental "world model" of the extract of reality under consideration (cf., Fig. 1.1). For a successful reaction to business events, it is necessary to structure the different realities of the participants and to transform them into a consistent process model for joint, goal-oriented action. This means that the business process is "agreed upon" by harmonizing the individual, to a greater or lesser extent matching, mental models of the people involved. This joining of the individual ideas of those affected by a business process and the mutual coordination of the different aspects of a business process (cf., Section 1.3) is itself a complex process and the central aspect of BPM. #### 1.3 Components of a Process Description We split a business process description conceptually into three parts (see Fig. 1.2). The first part, called process strategy, makes statements about the purpose, triggers, inputs, end and outputs of the process. The trigger is the event that sets the service provision in motion on the basis of the initiator's expectations, i.e., generates a process instance. This impulse is accompanied by the fact that the initiator provides information or objects which are to be processed according to his expectations. These inputs must be transformed into the expected results and made available to the defined recipient. In this way, the business process creates a value for which a customer pays. This external view of a business process is supplemented by the process logic. This inner perspective describes the actors involved and their coordinated interaction. The actors carry out activities in a logical and timely meaningful order. They transfer the results of their actions to other actors for further processing, or to the intended recipient at the end. Process implementation involves the provision of resources for the processing of process instances. These can be humans, machines and software systems, which take over the activities assigned to them as concrete realizations of the involved persons. In the age of digitalization, software systems (process or workflow engines) synchronize the actions of the actors by controlling the temporal and logically necessary sequence of the sub-steps according to the process model. For the handling of their individual tasks, the actors can use aids such as information, application programs or tools where required. Throughout process realization, it must be ensured that several process instances can be executed in parallel and independently of each other on the basis of the defined exemplary model through appropriate resource allocation. #### 1.4 Determining Factors for Process Models and Process Instances The business model essentially describes how a company affects the world and how it thereby generates revenues and profits. The customer promise as well as the resources and partners with whom this promise is fulfilled are essential. The enterprise architecture describes a machinery with which the business model is to be brought to life. As a typical layer concept, it defines business and IT structures and links them together. The concept of Business Engineering, for [1] example, envisages the business architecture on a strategic level with the definition of goals and services that are interwoven with the business model. At the level of the processes, as implementation tools of the strategy, the process architecture follows with its organizational and operational structure. The transition to the IT structures for supporting the processes leads to the level of information systems with the application architecture and the IT architecture. As a central component of an enterprise architecture, business processes are therefore in a kind of sandwich position that illustrates how they are influenced by other architectural elements. For example, a given organizational structure that is difficult to change can influence the procedures in processes and the way in which a company works together with external partners. The same applies to the availability of resources. But horizontal dependencies within the process organization must also be taken into account, e.g., whether a certain way of working in the ordering process has an effect on the design of payment processing. The underlying technological infrastructure not only affects the content design of the process models, but also the level of detail and accuracy. For the development of IT solutions for process digitalization, rigorous requirements apply to the model definition. Process parts that are to be executed with IT support must be specified precisely. In addition to the internal determining factors explained above by way of example and supplemented in Figure 1.3, external factors also have an impact on process design. Here one can see as an example test steps which have to be included in a process due to compliance regulations. #### 1.5 Process Metrics The processes to be developed or changed have the general goal of supporting the implementation of the business model and the associated strategy. The relationship between the Key Performance Indicators (KPIs) from the business model and the processes is established using Process Performance Indicators (PPIs). These Process Performance Indicators are refinements of objectives from the business model (cf., Fig. 1.4). Typical business Key Performance Indicators are derived from business models and strategies and measure business success at higher aggregation levels, e.g., revenues and costs at the overall company, division, product group level, etc. The focus here is on effectiveness ("Doing the right things"). The business processes are used to implement the strategy and bring together the elements of the enterprise architecture. The associated Process Performance Indicators aim at efficiency ("Doing things right"). They are therefore closely related to the Key Performance Indicators and are partly derived from them. When deriving the performance indicators, it must already be checked whether they can be measured with sufficient precision and justifiable effort. Under certain circumstances, this may also place demands on the process to be developed in order to be able to measure the performance indicators directly or indirectly. If direct measurement is not possible, targets for alternative performance indicators can also be defined and values for the performance indicator actually desired can be derived from them. Target values are defined for the Process Performance Indicators, which are to be achieved by a changed or redesigned process. Throughout the entire process, from the identification of the problem to the implementation of a modified or new process, it is important to constantly check whether the desired goals can be achieved with the resulting process. #### 1.6 Support Concepts The path from individual knowledge and willingness, i.e., from the mental models of the participants, to a process model that can at least in part be digitalized, is complex and costly. In order to reduce complexity and effort, support concepts such as frameworks, process models and description languages were developed. The following overview comprises a thematically grouped selection of such tools, which according to our experience are widely used in practice. They are inserted in Figure 1.5 and are discussed in more detail in the chapters on models (Chapter 2) and modeling languages (Chapter 3). Frameworks for Quality Management: Frameworks for Enterprise Architecture Management (EAM): Frameworks for IT management and IT governance: Description languages for process logic: ### 1.7 Digitalization Today, digitalization is the key word in the transformation of value creation. Digitalization in the economy or in organizations in general means digitalization of business models, products and services as well as of whole processes or parts thereof. For processes, however, this does not necessarily mean full automation without any human intervention. For example, a program that controls a process may, if necessary, include actions executed by humans or by Cyber-Physical Systems. The latter consist of communicating devices with software as well as mechanical and electronic components. In the Industry 4.0 Initiative, the aim is to achieve this comprehensive consideration of processes, i.e., the communication between people, machines and workpieces. On the one hand, these aspects must be expressed in the process models, and on the other hand, the transfer of a business process model into digital execution must be supported as far as possible. Particularly when aspects of quality management, i.e., the continuous improvement of processes, are taken into consideration, it must be possible to implement process changes that entail a change in digitalization quickly and with as little effort as possible. The aspects described in the previous sections must already be included in the creation of the models in order to facilitate the technical implementation of processes, but without already anticipating implementation details (cf., Fig. 1.6). The more precisely the processes are described, the easier this task becomes. Process segments whose flow logic cannot yet be precisely described at the time of modeling must be marked accordingly. However, these parts of a process can be modeled with other suitable methods according to the desired or necessary candor. Such process segments can either be described with Adaptive Case Management methods or, if a communication-oriented description language is used, as a communication loop. The latter is terminated by one of the partners involved after a corresponding result has been achieved, before continuing the process. Important in this context is the granularity, i.e., the level of detail of the process description. Activities should be broken down in such detail that one can clearly determine whether they can be digitalized, partially digitalized (human IT, physical IT), or are performed manually by humans. The tailoring should be based on the business requirements and not on the functionality of a potentially already existing IT system. If necessary, such a system must be adapted to meet the needs of the desired business specification during process implementation. #### 1.8 Process for Creating Processes The definition of the business processes cannot be done schematically or algorithmically, i.e., there is no software that when fed with the business model, the enterprise architecture, and the Key Performance Indicators with associated target values and support concepts, delivers a suitable process description directly. The definition of business processes is an intuitive and creative process. Therefore, creativity techniques and knowledge management methods such as Storytelling, World Café or Value Networks are also used, especially at the beginning of Business Process Management activities. For example, one can use the Design Thinking approach. This is a concept in which interdisciplinary teams work together in an iterative process in an environment which fosters creativity to develop innovative solutions to a problem (see section 5.3). A key point thereby is to develop and consider an in depth understanding of the needs and motivations of people in the target group. Design Thinking offers a comprehensive collection of methods for use in the individual steps of the approach. With these characteristics, it can also be used for the revision or redefinition of a business process. Under certain circumstances, extraordinary solutions can be found that would not have been possible with the usual BPM approach. However, a creative, innovative process concept must also be devised and implemented in detail. Creative design is therefore embedded in a bundle of activities that ultimately makes the process part of the real world. As such activity bundles, we identify analysis and modeling, validation, optimization, organizational implementation, IT implementation as well as operation and monitoring. These activity bundles are a further development or refinement of the Plan-Do-Check-Act cycle. They are usually arranged in a circle, which implies a corresponding flow. This does not always correspond to reality, which is why we present the activity bundles in Figure 1.7 as loosely networked honeycombs (cf., Fig. 1.7). There, the phases of the Design Thinking process and the activity bundles are supplemented. Both concepts are presented in more detail in Chapter 4 and put into relation with each other. Extensive and complex process changes usually require activities from several activity bundles and are carried out as a project. Such a project can thus be regarded as an iteration (process instance) of the process for creating business processes. For this, a detailed project plan must be created with the activities to be carried out, responsibilities and deadlines (cf., Fig. 1.7). The project plan should then be executed according to the methods of project management. #### 1.9 Organizational and Technical Implementation Once the process model has been created, the model must be embedded in the organizational structure of a company. This determines which activity is performed by which person or organizational unit. This mapping does not have to be static, but can vary from instance to instance. For example, the purchasing process can have the same flow logic for parts A and B, but a different purchasing department is responsible for purchasing parts A than for parts B. Process instances for parts A thus affect other organizational units (and persons assigned to them) than for parts B. These rules must be mapped in such a way that a process is correctly linked to the organizational structure. In addition to activities performed by people, there may also be activities in the process that execute application programs or IT services. For this purpose, such actions must be mapped in the process model to functions of software modules, which then execute them at runtime. If during the process modeling attention was already paid to the possible digitalization, this mapping is more or less unproblematic. Software can also control the processing of process steps and assign the tasks specified in the model to the respective persons or IT services as actors. Software systems that support this are also referred to as workflow systems (process engine, workflow engine). Ideally, process descriptions can be transferred directly into workflow systems. After being embedded into the organization and the IT environment, a process can be used for the handling of instances, i.e., real business cases - the goal is achieved. Figure 1.8 shows the now completed path from the individual mental models, including knowledge and intentions of participants, to the joint handling of process instances. #### 1.10 Success Measurement with Performance Indicators When instances of a process are executed, one can check whether the target values defined for the Process Performance Indicators are reached. For this purpose, actual values for the defined Key and Process Performance Indicators (KPIs and PPIs) are measured, calculated, stored and compared with the target values. This comparison can be made in real time or over longer time intervals. Any real-time evaluation leads to the immediate initiation of suitable countermeasures in the event of a deviation from the target. An evaluation of measured values over a longer period of time, on the other hand, shows medium to long-term trends in performance indicators and can trigger corresponding changes. Evaluation results are visualized in process cockpits, among other things (cf., Figure 1.9). #### 1.11 Continuous Improvement Processes are not static but are rather subject to changes in the internal and external determining factors described in Section 1.4. Developments such as business model modifications, new competitors, technical progress or deteriorations in measured PPIs, such as lead time, may require adjustments to a process. To this end, appropriate measures should be taken within the framework of the bundles of activities and procedures presented in Section 1.8. The feedback arrow in Figure 1.10 indicates that the participants may again have diverging views of the selected part of reality. By harmonizing them in the way described, a new instance of the process for creating processes is started. Continuous improvement is a very important aspect of process management. Ongoing adaptations bring one closer to the desired process. However, changes in the environment can influence this convergence. The state being pursued is therefore a 'moving target'. #### 1.12 Corporate Governance and Business Process Management Corporate governance as an institution shapes a company. It has a decisive influence on the business model, corporate strategy, and organization. The business model and strategy are designed to open up future potential for success and thus, secure the sustainable existence of the company. The enterprise architecture creates the infrastructure to exploit the potential for success. The business processes and the business objects (data) processed by them link the business and technical levels of the enterprise architecture. The business processes are the subject of digitalization, i.e., the IT support of process execution by people and machines. In recent years, the associated requirements have increased significantly. In business processes, not only people and IT systems, but also "smart" machines and devices should be able to interact. This refers to highly integrated business processes in the context of Industry 4.0 and the Internet of Things, which integrate human actors as well as individual devices and machines into a common whole. The technical players are often referred to as "smart" or "intelligent". Corporate governance as a process describes the management activities involved in creating and exploiting the potential for success. In the context of BPM, this means the management of socio-technical systems with people that are involved in processes, and machines that support people in their activities or autonomously carry out a chain of activities. Despite the increasing importance of digitalization, the human being, as designer of socio-technical systems and user of supporting technology, is at the center of process management. Not least due to increasing agility requirements, the goal today is for employees to be able to design (model) the operative processes autonomously and independently as far as possible, and for these to then be directly supported by Information Technology without significant delays and additional effort. With a clear commitment to process orientation, management must create the conditions for this as such ("Tone from the top"). These include both the necessary infrastructure and an environment that encourages people to become actively involved in process management activities. The degree of employee involvement is determined by the image of humanity and the associated management philosophy of the company management ("Tone at the top"). In a classical, more hierarchical approach, people and their skills are seen as a resource that is the subject of managerial action, and these people ultimately execute instructions. Such a management philosophy is characterized by direct intervention of the company management and follows Theory X. According to this theory, any lack of motivation is countered by the threat of sanctioning by the company management. In a more systemic, i.e., holistic approach, such as the one that the St. Gallen Management Model is based on, a system is to be created that works itself largely independently on the design of a Business Process Management System. All employees should be able to actively contribute. This management style follows the image of humanity according to Theory Y. According to this theory, the essential characteristics of humans are pleasure in demanding work, self-discipline, responsibility and intellectual power. The image of humanity is supplemented by corresponding organizational theories. The purpose of these is to explain the creation, existence and functioning of organizations. Organizational theories implicitly assume a corresponding view of humanity. Thus, Taylorism is more based on an image of man that corresponds to Theory X. Luhmann's Systemic Organizational Theory, on the other hand, makes no ethical assumptions about the people in an organization; it only assumes that they communicate. Although the Theory of Communicative Action also focuses on communication, the world is to be changed through theory and rationality. It is assumed that man is by nature insightful and open to argumentation. There are management philosophies and organizational theories to match the various concepts of humanity. The nature and use of methods, techniques and tools must be consistent with them. For example, it is not appropriate to propagate the involvement of employees if the company management then does not take their suggestions seriously or does not take notice of these suggestions at all. Before it starts designing processes, a company should therefore be aware of the image of humanity that shapes its leadership and corporate culture. We think that especially for the challenges associated with digitalization, an orientation to Theory Y is necessary, which will often (and must) lead to cultural change in practice. #### Reference 1. Österle, H., & Winter, R. (Eds.). (2003). Business Engineering (2nd ed.). Berlin: Springer. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Models 2 In the previous chapter we outlined the various aspects of Business Process Management. Since models are often used in practice to describe these aspects, the following sections will first look in more detail at the tasks and properties of models and modeling. We then present examples of models from various areas of expertise that in our experience have proven helpful in numerous Business Process Management projects. There is no claim to completeness for this exposition, but it can serve as an orientation for the reader when needing to choose description models for individual project needs. ### 2.1 Model and Reality "You don't have to understand the world, you just have to find your way around in it." According to internet sources, this sentence is attributed to Albert Einstein. Who understands what is going on in the world? Who knows how it works? Therefore, we should take care of our world, namely the part of the world that is important to us at the moment. We should recognize that we create or construct our world on a daily basis. Any excerpt of reality is naturally determined by our subjective interests. We decide which part of the world we want to consider and which aspects seem important to us. In doing so, we identify the artifacts and the relationships between them that are essential for us. Such an abstraction of a part of reality is called a model. It is also possible that the segment of reality considered is already a model itself. This allows parts of an already existing model to be examined more closely. This would then be a model of a model. This process can be repeated as many times as desired. Every person has his or her own subjective view of the world or a part of it. Different people may consider the same part of reality and arrive at different models because they set different priorities (cf., Fig. 2.1). Since models do not cover all aspects of the assigned reality, there are indeed incidents in reality that are not covered by a model and are incomprehensible therein. Figure 2.1: Modeling These phenomena, which cannot be explained in a model itself, form its limits. The creator of a model may decide to adapt it accordingly when intending to include phenomena not yet covered or to omit these if they should no longer be taken into consideration. In any case, a model remains a simplification of reality, which is why there will always be phenomena that are not covered by a model. Each person needs to decide whether the model in use is sufficient for his purposes. If one wants to cover all aspects of reality with a single model, it reaches the complexity of reality. Then one doesn't understand the model to the same extent one didn't understand the reality before mapping it to a model. The intention of modeling, namely, to make reality more comprehensible and manageable, is then no longer achieved. The creation of a model and its use are subjective activities, i.e., the creator chooses the characteristics of the representation of reality according to his ideas. However, it is common for groups of acting participants, subsequently referred to as subjects or actors, to agree on a model for observing reality. Many scientific schools of thought are based on such common models of the involved researchers. Modeling is an essential activity in all sciences, be it philosophy, sociology, physics, chemistry, all engineering sciences, economics, etc. The respective models have different tasks: Either they depict the specific part of reality under consideration, as is traditionally done in the natural sciences, or they serve to try out certain necessary changes to said part (simulation model). This is particularly important to avoid endangering human life. Before going into series production, the properties of cars are checked in safety models in appropriate tests. This does not happen with people, but instead with models of people, so-called dummies. In doing so, two types of models are combined with each other: The car model, which implements the corresponding safety concepts, and the human model (dummy) to investigate the risks of injury. The state of affairs captured in a model can be adapted as often as required until a desired result is achieved for the phenomena considered in the model. For instance, further safety concepts are added to the safety model of a car until the desired reduction of the risk of injury is achieved. Such models are then no longer images of reality, but rather represent a desired reality. The desired properties are then transferred back into reality, e.g., by incorporating the safety concepts tested in the model into the series production of cars. The aim of modeling is always to find our way around in the world, or to try out safely how a corresponding change in reality would affect us. We will not likely succeed in understanding what holds the world together at its core in the foreseeable future. The corresponding model would then be the world that does not exist [1]. #### 2.2 Properties of Models Models serve both as the abstract representation of the observed reality in the sense of a cognitive function and as the design of the observed reality in the sense of a conclusion. As already mentioned, once a model is modified until it corresponds to a desired reality, it can be used as a blueprint for a corresponding transformation of reality. In natural sciences such as physics, models predominantly have a cognitive function, while in engineering sciences and business administration they are intended to support the shaping of reality [2]. In clarifying the model concept used thus far we follow the studies of Herbert Stachowiak. In his model theory he examined the characteristics of models and the properties derived from these more closely [3]. Accordingly, models are identified by at least three characteristics ([3] page 131): #### 1. Mapping A model is always a representation of something. It can be a depiction or representation of a natural or artificial original, whereby this original itself can again be a model. The originals can be created in a natural way, technically produced, or simply exist in some other way. Models can be described or represented in very different ways: A model and the original form a class of attributes. Attributes are characteristics and properties of individuals, relations between individuals, properties of properties, properties of relations, etc. Stachowiak leaves it up to the modeling subject to determine how to conceptualize individuals. He considers individuals as attributes on level 0. Sets of attributes can be combined to form classes, which then form attributes on level 1. These classes can be combined again to form a next attribute level, and so on. #### 2. Reduction In general, a model does not capture all attributes of the original, but only those that appear relevant to the creator or user of the model. Since not all attributes of the original are captured by a model, a pragmatic dimension has been introduced in the broader sense. In the "broader sense" here means that not yet specific pragmatic-operational aspects are considered, according to which the attribute classes that are to be included in a model are selected. This initial selection of attributes is intuitive and arbitrary. In the narrower sense, the reduction is pragmatic only when the intentions and operational objectives of the model creator or user influence the selection of modelrelevant attributes. These adjustments to the intended practical use are made in the next step. #### 3. Pragmatism After the intuitive selection of the attributes, it is checked whether the intended purpose has been achieved. Models are not clearly assigned to their originals. They serve as a replacement function • for certain discerning or acting model-using subjects (for whom?). Models are not only models of something, they are also models for someone. This someone can be a human being or an artificial model user such as a computer program. For a modeler, models can serve as a possibility to find one's way around in the world, i.e., the modeler is also a user of the model. Modelers and model users can also be two different subjects. • within certain time intervals (when?) Models also perform a function over time, i.e., their use is related to a specific point in time or to a defined time interval. During this time, the observed reality or the ideas of the modeler or model user may have changed in such a way that additional attributes should become part of the model. • with restriction to certain mental or actual operations (why?). Models are created for a certain purpose, be it for better understanding a certain part of reality or for creating a blueprint for the transformation of reality. When creating a model, modelers are always in a certain dilemma. On the one hand, the model should sufficiently reflect the desired aspects of reality, whereby it is not clearly defined what is sufficient. On the other hand, the model should not be too complex in order to remain manageable. This conflict of objectives leads to the fact that most models are developed iteratively until they reach the end of their life cycle due to increasing complexity - they are no longer manageable. In the following sections, we present examples of models that consider aspects that are explicitly or implicitly incorporated into business process models. We have grouped the examples into models from the social sciences, business administration, business informatics, and computer science. The classification into these groups is not entirely free of overlaps, since especially business informatics as a cross-sectoral discipline considers issues from various perspectives. #### 2.3 Models of the Social Sciences Business Process Management has to do with people and machines. It aims at organizing their interaction while taking into account additional requirements with regards to technical, economic and ecological feasibility. In particular, the interaction and coexistence of people has been a topic of philosophy for thousands of years. Philosophy, as the doctrine of the basic rules and structures of life, the world and knowledge, seeks to fathom, interpret and understand the world and human existence. The original meaning of philosophy was the teaching of good life. In this sense philosophy is the attempt to create the comprehensive model of our world, but this has not yet been successful. Social philosophers reduce the view and try to create a model of society as some part of reality and thus better understand its meaning and essence. In particular, social philosophies illuminate the relationship between the individual and the community as well as the structures of living together. They are therefore also regarded as variants of philosophy that touch on sociology. They should help sociologists to analyze social processes and support organizational developers in their work and help people to find their way around in the world. There are numerous organizational theories that focus on different aspects in their models (see [4–7]). The question of which organizational theory fits best cannot be answered. Organizational theories are models and thus, according to Stachowiak, the justified but subjective view of the modeler. Organizational theories emphasize the analysis of organizations in highly different ways and pursue different objectives. There are empirical studies on organizational theories that provide results in favor of, or against, a theory. However, the analysis methods used are controversial (see [8] page 68). Organizational theories are based on certain perspectives on people, and the design of Business Process Management is strongly influenced by the prevailing image of people in an organization. Hence, in the following sections we discuss Taylorism, Habermas's Theory of Communicative Action and Luhmann's Social Systems as distinct organizational theories to exemplify different perspectives on human and organizational behavior. #### Taylorism and Fordism Taylorism introduced the 'experiment' into management theory and practice and is one of the classics of organizational theory. With the so-called Scientific Management, organizations received an instrument to design themselves efficiently. One important characteristic is the separation between planning mental work and performing manual work. According to Taylor's view of man, workers are dumb and lazy and must therefore be subject to strict rules. Even today, this view often implicitly influences leadership behavior. For Frederik Winslow Taylor, the most important goals of running a business were the perfection of the means of production and work processes, tighter organization and temporal structuring of workflows in the company, as well as a reorganization of the remuneration system. A core element of Taylorism is the design of work processes on the basis of time and movement studies. A breakdown of the production process into the smallest work steps, an exoneration of workers from mental activities, as well as a change of the wage system should lead to an optimal use of existing performance potentials. The ultimate goal is to increase the productivity of human labor. This is done by dividing the work into its smallest units, which require only little or no cognitive effort to accomplish, and which can be repeated quickly and repetitively due to their small size or the content of the work. Taylorism is based on the following core principles: • Work scheduling The planning of the work is done by other persons than those who carry it out (separation of manual and mental work). In this way Taylor wanted to avoid the shirking that he accused the workers of. Through time and movement studies carried out by the mental workers, the least amount of movement and time required for a work step was to be determined. • Incentivized wages These time and movement studies also revealed what the workers had to achieve in a certain period of time. A bonus ensured that the workers who were classified as dumb and lazy actually tried to achieve the specified performance. • Selection of the most suitable workers One aim was to build up a first-class workforce by means of an appropriate selection mechanism. Tests were developed and used to identify particularly agile and nimble-fingered workers. • Reconciliation between workers and management Taylor believed that the system he had developed could increase productivity to such an extent that the dispute over the distribution of profits would become a minor issue. This achievement should resolve the conflict between employers and employees. Taylorism essentially considers the structuring of work steps but does not focus on their sequence. This aspect was addressed by Henry Ford, who coordinated the individual activities by introducing the assembly line. This step created the basis for the mass production that characterized the 20th century. The assembly line principle was also transferred to administration and strongly influenced Business Process Management. Flowcharts are the assembly line specifications for the execution of administrative tasks or the "production" of services. #### Communicative Action According to Habermas In contrast to Taylorism, Habermas's Theory of Communicative Action is based on insightful people who, through communication among themselves, come to a common rational action [9, 10]. Using his social model Habermas explains the processes in a society, such as the search for truth, for justice, etc. Consequently, it is a model that concerns everyone, because the issues of truth and justice affect all members of societies. The central aspect in Habermas's model of society is the so-called Communicative Action. "Finally, the concept of Communicative Action refers to the interaction of at least two subjects capable of speaking and acting that enter into an interpersonal relationship (whether by verbal or nonverbal means). The actors seek an understanding of the action situation in order to coordinate their action plans, and thus their actions, amicably". (see [11], Volume 1, page 128). According to Habermas, communication enables an individual human being, who is not gifted with rationality on his own initiative, to overcome this deficiency. Communication between people becomes intersubjective action and a possible source of rationality. Communicative Action means acting on the basis of mutual understanding between people. Habermas wants to offer sociologists and politicians a model that they can use to analyze and shape society. The individual can use it to find his way around in today's societies, despite their complexity. #### Social Systems According to Luhmann Similar to Habermas's, Luhmann's Social Model is based on communication. The differences lie in the extent to which communication and action are combined. Luhmann only allows communication as a constituent aspect for organizations. Communication does not occur between people, but between at least two information-processing processors. Luhmann thus sees communication more abstractly. According to him, society does not consist of people or parts of people. Otherwise one would cut off something from society, when one cuts off something from a person. The body of a human being (i.e., as a biological system) with a conscious mind (a psychological system) is in many cases a prerequisite for the functioning of a social system, i.e., communication. However, a human being is not the social system itself. Luhmann makes no assertions about the nature of man in his organizational theory, thus leaving the perspective on humans open. People are only part of the organization insofar as they communicate with each other. The communication between the information-processing processors consists of the so-called selections of information, message and understanding (see Fig. 2.2). The first two selections are for the sender and the third for the recipient. Communication as a piece with at least two actors in three acts is an indivisible unit, namely the smallest unit of a social system and the elementary operation of society Figure 2.2: Luhmann's understanding of communication [12, 13]. This view can serve as a pattern for the definition of communication in business processes, completely independent of a specific perspective on humans. Both Luhmann and Habermas put communication at the center of their organizational theory. The fact that these two important organizational theorists so strongly emphasize the communication aspect of the organization, and that their theories are widely accepted, can be seen as an indication for considering business processes as primarily communication oriented. #### Organizations Complex social systems can be divided into smaller social systems. This structure of a complex social system is called the organizational structure. The criteria according to which the division into smaller social systems takes place are subjective and depend on the respective intentions. According to Luhmann's definition of a social system, the individual social systems communicate within a more complex social system. Organizational structures are thus a model of a more complex social system. In contrast to Luhmann's or Habermas's broad understanding of the term organization, a narrower understanding of organizations has also developed. In business administration, organization is the formal set of rules of a system based on the division of labor. In organizational sociology it refers to a special form of social entity that can be distinguished from other social entities such as families, groups, movements or networks. Essential characteristics of organizations are that people can join them or leave them. In addition, they have a purpose that they are geared to. Organizations have regulations on the division of labor, such as specialization according to performance, function, objects or space, or corresponding hybrid forms. This division of labor requires the coordination of individual activities. Hierarchy is the central instrument of coordination in organizational theory. The hierarchical coordination is supplemented by cadres, commissions, task forces, etc. For one-time problems or problems to be solved for the first time, the hierarchy is supplemented by a project organization. #### 2.4 Models of Business Administration Business administration is the study of economic, organizational, technical and financial processes and structures in companies. Business Process Management is therefore also a part of business administration. Business processes serve to improve the economic efficiency of a company with all of the associated aspects such as customer satisfaction, employee motivation, the integration of partners, etc. For the structuring of all these aspects and for the analysis of their interaction, business administration has developed models which, when applied, have an effect on Business Process Management. #### Business Model A business model refers to the overall concept of a company. It represents the interrelationships as a model, how a company can generate added value for its Figure 2.3: Schema of the Business Model Canvas customers and thus achieve sustainable returns. In addition to the products and services offered, the focus is on the structure of the company, the definition of the target groups (customers) and how they are addressed, as well as the design of the business processes. Beyond this understanding, there are a number of other definitions of the term business model (cf., e.g., [14, 15]). A business model therefore serves to understand the relationship between the company as a system of action and the creation of value. It reflects how a company works and what values it generates for specific target groups. Business models are created within the scope of a company foundation or a reorientation. They consist of several sub-models that describe which resources (materials, information, etc.) are (must be) available to a company as input variables, and how these resources are processed and transformed into marketable products or services, which are then transferred to the customer in order to generate corresponding revenues [16]. Business models can serve multiple stakeholders. The company management can thus better understand its own business and recognize existing strengths and weaknesses as well as opportunities for further development, transformation and improvement of its competitive position. For investors, the business model is often an important aspect of investment decisions. A number of instruments have been developed to create business models. The best known is the Business Model Canvas by Alexander Osterwalder [17], which has found high acceptance in recent years. As the name suggests, the Business Model Canvas approach is based on a poster on which various aspects of the business model are visualized. The canvas provides a grid for nine business model aspects, which is filled with the concrete characteristics for a company at hand (see Fig. 2.3). The focus is on the value proposition (product or service). When completing the form, a series of questions on each of the nine aspects must be answered. The following explanations briefly describe the aspects and provide a selection of associated questions. 1. Customer segments, target groups: All persons or organizations for whom the company in question wants to create values. Questions to be answered include: Each customer segment has its own value proposition, the customer benefit. This is a combination of a product and service tailored to the needs of the respective segment. Questions to be answered include: This factor represents the specific channels through which customers are addressed and promised values are communicated to them. Sales channels determine how interaction with customers takes place. Communication, distribution and points of sale form the interfaces between a company and its customers. The perception of the customer at these points of contact is central and determines the impression a customer has of a company. Questions to be answered include: This section describes how dealings with customers are fostered. Every company should think about what types of customer relationships it wants to establish with different target groups. The design of customer relationships depends not only on the respective target group, but also on the associated objectives of the company (new customer acquisition, existing customer care, etc.). Questions to be answered are among others: The company creates added value with its products and services. The central question is how much the customer is willing to pay for this. The company needs to decide on pricing models and pricing strategy (one-time payment, subscription, etc.). Questions to be answered include: Every company requires certain resources to prepare offers. These can be owned by the company itself, or also leased or provided by strategic partners. #### 7. Key activities: Key activities are the activities necessary for the creation and utilization of services, such as production, sales, and so on. Questions to be answered include: #### 8. Key partners: Key partners are business partners who provide important resources for the realization of the business model. Companies often enter into strategic alliances with these partners. Examples are suppliers, service providers, etc. Questions to be answered include: The cost structure provides information on the most important cost factors of a business model. Questions to be answered include: Key Performance Indicators and associated target values for business processes can be derived from the individual parts of a business model. Conversely, the Key Performance Indicators and target values can influence the design of the processes. If the focus is on low prices, processes will look different compared to a business model focused on high quality. #### Balanced Scorecard The Balanced Scorecard (BSC) was introduced in the early 1990s by Kaplan and Norton [18]. It is a link between the business model, the development of a strategy, and its implementation. In the business world, strategy is classically understood as the (usually long-term) planned behavior of companies in order to achieve their goals. A BSC starts with the vision and strategy of a company and defines the Critical Success Factors (CSF) on this basis with the help of Key Performance Indicators and associated target values. The vision of a company describes the long-term ambitious goal that an organization or company strives for. Typical visions are formulations such as "we want to become the market leader in our market segment," or "we want to become the most profitable company in our market segment". The Key Performance Indicators promote goal setting and performance in critical areas of the strategy to achieve the vision. The BSC is therefore a management system that is derived from the vision as part of the business model and the strategy to implement said model. It reflects the key aspects of the company. The BSC concept supports strategic planning and implementation by bundling the measures taken by all entities of a company on the basis of a common understanding of its goals and by facilitating access to the evaluation and updating of the strategy. Since traditional management based purely on financial indicators no longer meets the requirements of companies for effective planning tools in the information age, Kaplan and Norton have introduced four perspectives for the BSC which allow the activities of a company to be assessed comprehensively. For each perspective, objectives, performance indicators, targets, and measures to be taken are defined (cf., Fig. 2.4). #### Total Quality Management and EFQM The term Total Quality Management (TQM) denotes the optimization of the quality of a company's products and services in all functional areas and at all levels through the participation of all employees. Optimization of quality means neither reaching the highest quality level with the given effort, nor increasing the quality without consideration of costs. Rather, it is a matter of focusing on the interests of the customer and determining quality in terms of the fulfillment of customer requirements. The management of a company decides which requirements the company places on itself and which positioning toward the customer promises the most sustainable business success. This positioning is not static. Knowledge about customer needs and about the procedures to meet these needs require a continuous adaptation of the company. In order to establish TQM, the European Foundation for Quality Management (EFQM) offers organizations assistance in setting up and continuously developing a comprehensive management system. Figure 2.5 shows the structure of the EFQM approach. On the one hand, this structure serves as a tool to build up a TQM and, on the other hand, to identify improvement potentials through a comprehensive evaluation system as well as to increase business success. Enablers in the EFQM model are the methods and concepts used to achieve the results shown in the right half of the figure. The percentages in the presentation indicate the extent to which the individual aspects are included in the overall evaluation of the company. Figure 2.5: EFQM structure The EFQM assumes that the enabling methods and concepts have the largest influence on the results (right side of Figure 2.5). The model thus provides good starting points for identifying Key Performance Indicators and their target values. In addition to the possibility of setting up a management system, EFQM also offers a very sophisticated concept for evaluating its development status. A comprehensive catalogue of questions can be used to carry out an all-round evaluation of an organization. The evaluation can be carried out by employees of the organization itself or by external consultants. The best organizations in Europe score around 750 out of a maximum of 1,000 points in such evaluations. #### EN ISO 9001 Compared to TQM, the EN ISO 9001 standard represents a weakened form of quality management. It describes minimum requirements for a quality management system. Figure 2.6 illustrates the basics of the standard. Management's responsibility means that it defines which customer requirements are met and which quality policy is pursued. The implementation of the quality policy is planned, and the corresponding responsibilities and authorities are defined in the organization. Management is also responsible for evaluating the QM system at planned intervals and, in particular, taking customer feedback into account while doing so. Corporate management must also provide the necessary resources such as personnel, infrastructure, and an adequate work environment. Figure 2.6: EN ISO 9001 The core of an EN ISO 9001-compliant QM system are the processes for realizing the products and the associated customer-related services. Tasks include the planning and definition of suitable processes for the development and manufacture of products, the procurement of inputs, etc. The tools used to monitor product manufacturing and quality must be regularly checked for their suitability. The execution of the processes must be continuously monitored through measurements and analyses of Key Performance Indicators, in order to be able to initiate appropriate improvement measures in the event of deviations. EN ISO 9001 thus provides a framework for Business Process Management. The explanations show that, strictly speaking, there is no difference between Business Process Management and quality management. Without Business Process Management there is no quality management and vice versa. The comparatively lower requirements of EN ISO 9001 are expressed in the fact that a company with a (merely) EN ISO 9001 compliant quality management system can only achieve about 300 points in an EFQM assessment. #### Value Networks The Value Networks concept was introduced by Verna Allee [19]. A Value Network is understood as roles and persons who exchange so-called tangibles and intangibles with each other. Tangible value flows are material value flows between roles and persons, and correspond to the exchange of goods, services, revenues etc. Tangible value flows represent transactions based on contracts. Intangible value flows are an additional benefit through the flow of knowledge; they are not contractually fixed or subject to a charge. Intangible value flows are, e.g., strategic information, planning knowledge, as well as existing emotional components such as mutual trust, common interests, need for knowledge, security, etc. Value Networks should enable participants and organizational developers to actively shape social and professional relationships of interaction in organizational systems by visualizing and creatively handling mutual tangible and intangible performance flows (transactions). Figure 2.7 shows a simple Value Network. The customer sends a value purchase order as a tangible value flow to the supplier. This tangible value flow is accompanied by the intangible value flow of trust. The customer and the logistics company are connected with the tangible value flow of delivery, etc. The numbers on the individual transactions express the sequence in which they are executed. Organizations provide services as a result of their activities that ultimately contribute to the added value of a company or institution. In order to record these services and make them visible, an exchange-oriented view of organizations is recommended. This usually results in a network-like structure in which the roles within an organization and their interaction and communication channels are in the foreground. This perspective enables the transition to a communication-oriented Business Process Management. #### 2.5 Models of Business Informatics Models in business informatics combine aspects from the economic and social fields with computer science to derive requirements for information systems. The models are mainly used to describe socio-technical human-machine systems. The social component covers the aspects around employees and partners. The technical dimension concerns the circumstances of Information Technology. It is important that corresponding models consider the interaction between the two domains, especially the human-machine interaction. In contrast to purely technical systems, which are regarded as deterministic, socio-technical systems can also be non-deterministic, i.e., complex, due to the involvement of social components. The state of development and research on the subject of modeling in business informatics can be found in [20]. We limit ourselves here to the handling of frameworks for enterprise architectures and IT service management. In order to simplify the creation of very similar process models, reference models have been defined over the years by consulting firms or standardization bodies. A wellknown example of such a reference model is ITIL (IT Infrastructure Library, [21]). Due to its wide distribution, we describe it in more detail as an example of a reference model. A reference model that includes ITIL while being more focused on governance and compliance, is COBIT [22]. Due to space limitations we do not provide a representation here but refer to the extensive literature and the official website [23]. #### Enterprise Architectures The understanding of architecture in the context of companies coincides with the original meaning of the term architecture. In many areas of expertise it describes the basic organization of a system with its components and their relationships to one another and to their environment. As already mentioned in Chapter 1, an enterprise architecture specifically describes and links the business and technical elements of an enterprise. The latter include in particular the IT landscape. Both the overall architecture and its parts are described through models. The range of model types used for this purpose extends from a business model through organigrams, data models and process models at the business level, to database models, algorithms and programs in the technical layer. As for business models, there are also numerous frameworks for the modeling of enterprise architectures that provide orientation for those responsible and are intended to facilitate work processes. Dirk Matthes [24] has identified more than 40 frameworks with varying foci, levels of detail and degrees of familiarity. Only four of the most relevant will be handled in the following sections. The Zachman Framework and The Open Group Architecture Framework (TOGAF) with its extension Architecture-Animate (ArchiMate) were named as essential frameworks in surveys (see also [24] page 5). The Architecture of Integrated Information Systems (ARIS) is widely used in practice in German-speaking countries and is of significant importance in the context of process management. #### Zachman Framework The framework presented by John A. Zachman in 1992 [25] in its extended form represents a structure grid similar to the Business Model Canvas, which the user has to complete with the facts for the enterprise at hand. It consists of a matrix with different perspectives in the rows and abstractions to each perspective in the columns. Figure 2.8 shows a condensed representation, a detailed picture can be found on the Zachman International website (www.zachman.com). The perspectives in the rows have the following meaning: Figure 2.8: Zachman framework The columns contain the questions that the company needs to answer: Zachman envisages that a suitable model will be developed for each cell in the table. From this perspective, his framework is a model for a set of models that allow a closer look at different aspects of a company. The users can deviate from the original structure in the rows and columns by changing the emphasis. This flexibility is a strength of the model frame. However, it does not contain any procedure or methodology for defining a concrete enterprise architecture. Processes for their development or transformation have to be exploited elsewhere by the users or have to be designed entirely by themselves. #### The Open Group Architecture Framework (TOGAF) TOGAF is the Open Group's framework for the development of enterprise architectures, including business processes [26]. While the Zachman framework emphasizes the object perspective and offers little support for the architecture development process, TOGAF focuses on the procedure for model creation. It provides methods and tools that help with the introduction, creation, use and further development of enterprise architectures. TOGAF distinguishes four sub-architectures: Figure 2.9: Architecture Development Method from TOGAF The TOGAF framework consists of the following components: Structural model to define, structure and display the results generated with ADM in a uniform and consistent way. • Enterprise Continuum Model for structuring a possible repository that can contain the respective architectures and the possible solutions such as models, patterns, architecture descriptions, etc. • Reference Models Basic models that can be used as a basis for specific models for a company. These are the Technical Reference Model (TRM) and the Integrated Information Infrastructure Model. • Architecture Capability Framework Various reference materials for the development of specific architectural models. The Architecture Development Method (ADM) forms the core of TOGAF as an iterative process model (cf., Fig. 2.9). This creates all of the architecture artifacts. ADM can be applied at multiple levels, allowing architects to define different levels of detail of the enterprise architecture. With the help of the other components, the results are then described, structured and stored. The phases of the ADM are: #### Architecture-Animates [27] (ArchiMate) Architecture-Animate (ArchiMate) is the name of an open and independent modeling language for enterprise architectures published by the Open Group. It provides tools that enable enterprise architects to describe, analyze, and visualize the relationships between business units and their development. The ArchiMate language enables the description of the structure and flow of business processes, organizational structures, information flows, IT systems, and technical infrastructure. The descriptions help the participants to design changes in architectural elements and their relationships, to evaluate the consequences and to communicate them. Figure 2.10 shows the ArchiMate framework. The first three columns correspond to the basic concepts of ArchiMate: • Passive structure elements Passive structure elements are the objects on which the actions from the behavior (behavior elements) are executed. In general, these are information objects, but physical objects can also be modeled as passive structural elements. Figure 2.10: Elements of the ArchiMate framework • Active structure elements Active structure elements are elements that can perform actions. Examples are people, applications, computer nodes, etc. The actions can be triggered via interfaces, which also provide the results. • Behavior elements Behavior elements represent the dynamic aspects of a company. A service is the externally visible behavior of the system that provides the service. The services are used via the corresponding interfaces. Interface events trigger the active structure elements, which then execute the corresponding service function. These three model fragments correspond to the basic elements of natural languages: subject, predicate or verb and object. They are considered on a total of six layers: • Strategy layer Motivation describes what a company wants to achieve. The strategy concepts describe at a high level of abstraction how a company wants to achieve its goals. • Business layer The elements of the business layer can be used to describe products and services that a company makes available externally. The business layer shows how the company realizes these products and services and is intended to help with the analysis of the corporate structure. • Application layer In the application layer, the support of the business layer is represented by applications and data. • Technology layer In the technology layer, the infrastructure needed to implement applications is described. These are essentially the required hardware and software components. • Physical layer This layer focuses on the interaction of IT and physical components such as machines, sensors and actors. Figure 2.11: Relationship between ArchiMate layers and TOGAF ADM • Implementation and migration layer The implementation and migration concept describes how a defined architecture is to be implemented. In particular, it describes the work packages for implementation. The cells of the table therefore contain the core elements for the active and passive structures as well as for the behavior in the respective layer. The motivation column describes the reasons for designing or changing an enterprise model. These influence the modeling and give it the appropriate direction. ArchiMate is seen as a supplement and concretization of TOGAF. TOGAF describes the process for the definition and description of an enterprise architecture (enterprise model), but it does not contain any description languages for the respective sub-models. ArchiMate aims to fill that gap. In addition to the framework shown for the architecture to be developed, it also offers languages for expressing the aspects in the individual layers. Figure 2.11 shows how the individual steps in TOGAF's architecture development method are related to the ArchiMate layers. #### Architecture of integrated information systems (ARIS) The Architecture of Integrated Information Systems (ARIS) is a framework for the definition of enterprise models. It includes the data, function, organization, control, and performance views. For each view, ARIS provides a number of model types for documentation. • Data view: The data view comprises the business-relevant business or information objects and their relationships to each other, that is, all data that is related to the activities of a company. Information objects include states such as article or customer status as well as events such as "Sales order has arrived" or "Production order has been triggered". The relevant model type is the Entity-Relationship Model (ERM). • Function view: The function view describes the business-relevant activities (functions, activities) and their hierarchical relationships. Subordinate functions are sub-functions of the higher-level function. The functions perform operations on the objects described in the data view. In practice, functions are modeled with function trees. • Organizational view: In the organizational view, the organizational structure, that is, the personnel resources of a company and their hierarchical relationships are modeled in an organizational plan. The organigram is the usual model type to represent the organizational view. • Control view: The control view establishes the chronological and factual connection between the individual operational activities. It merges the data, functional and organizational views and thus plays a central integrating role. The control layer is therefore also called the process view. The main model type is the Event-driven Process Chain (EPC). • Performance view: In the performance view, the entries and results of the business process at hand are usually described using product trees. The views, typical model types and their context are shown in Figure 2.12. The picture also makes clear that ARIS, with its integrated control view, places business processes, especially the sequence of activities, at the center of the consideration. Orthogonal to the structuring carried out by the views, ARIS differentiates the abstraction levels functional concept, data processing concept and implementation on the basis of software engineering. This shows the close relationship between the models developed with ARIS and Information Technology. To solve an operational problem, a functional model is created and transferred to a corresponding data processing concept (data processing model). This ultimately serves as the basis for the concrete technical implementation. • Functional concept: The functional concept describes the facts of the operational problem. At this level, data models, functional models, organizational charts, value chains or Event-driven Process Chains (EPCs) and product models are used. • Data processing concept: The data processing concept specifies how the functional concept is to be implemented in IT terms. At this level, database models (data view), structure charts (functional view), network topologies (organizational view) and trigger mechanisms (control view) are considered. The purpose of the data processing concept is to adapt the functional concept to the requirements of Information Technology. • Implementation: At this level, the data processing concept is converted into an executable software system. At this abstraction level, data description languages (data view), programs (function view), network protocols (organizational view), and program control (control view) are considered. #### Framework for IT Service Management: ITIL The IT Infrastructure Library (ITIL) is a collection of predefined processes, functions and roles that typically occur in every IT infrastructure of medium-sized and large companies [21]. The practical assignment of activities is based on roles and functions. These are best practice proposals that have to be adapted to the needs of the company. The collection has since been supplemented by ISO 20000:2005, an ITIL-based certification model for organizations. The IT Infrastructure Library comprises five core volumes with a current total of 37 core processes. Figure 2.13 shows the structure of ITIL. The five core volumes are based on the service life cycle. Based on the service strategy containing the processes strategy development, financial management, service portfolio management and demand management, the service is finally provided via the process groups of service development and service commissioning with the processes of the service provision group. The procedures are subject to continuous improvement. Further books such as "Software Asset Management", "Small-Scale Implementation" or "Building an ITIL based Service Management Department" supplement the core publications. ITIL thus offers comprehensive support in the development of a process system for the IT department of an organization. #### 2.6 Models in Computer Science Models in computer science relate to data structure models and processes in computer systems as well as to various essential accompanying aspects such as security, robustness, etc. On the one hand, these models serve to illustrate a considered part of reality in order to solve a task with the help of information processing. They refer to a defined problem area or certain application areas of computer systems. This includes models that focus on data and the operations running on it, as well as models that look more at the overall structure of complex programs, i.e., their architecture. These two model categories are also called models for programming on a small scale or models for programming on a large scale respectively. In addition to these central model categories of computer science, there are other models that consider flanking aspects such as access models, security models, etc. These are not the subject of further discussion here. #### Figure 2.13: ITIL process groups Figure 2.14: Information is "model - from what - for what - for whom" [11] Instead, in the following sections we explain several model concepts considered essential for Business Process Management. #### Information Information is the more central aspect of data processing, which is why it is also called Information Technology or IT. Information and information systems are models that represent an object of the real world according to the ideas of one or more subjects. The subject's ideas are oriented toward the intended purpose. For example, items in a warehouse are described by their properties such as part number, dimensions, weight, etc. Different users use different parts of the model: The purchasing department looks at information such as purchase price and order limit, while the warehouse worker is more interested in dimensions and storage location. The modeling of the object reality can therefore be understood as an interpretation by the user agents (subjects) such as purchasing or warehouse clerk. Information is then the result and the reason for an interpretation, but it can also itself be an object and thus an object of interpretation and modeling. This relationship between subject, information (model) and original is shown in Figure 2.14. Information receives its value through the interpretation of the overall event by the observing subjects. This observation is partly conscious, but mostly unconscious. The amount of information is reduced and filtered according to the respective need for knowledge, or linked with other information. Data is different from information. A data element is initially only a sequence of characters whose meaning is not unique. The characters can be numbers, letters or symbols. In the marketing department of an online shop, for example, the sequence of numbers 0815 may be found. Although this sequence of characters represents a date, its meaning is not known. The string itself has no meaning except for its individual elements. From this data, however, information can arise, if it is known in which context it is to be interpreted. By combining it with other data, a relationship is created that can be interpreted, and information can be generated. If the data 0815 is in the context "Customer Max Sample, Article 0815", the marketing department can interpret that the customer Max Sample ordered an article with the number 0815. The supplementation of data with other data depends on a subject's interest in knowledge or his Figure 2.15: Example of an Entity-Relationship Diagram intentions to use it. With the information produced in each case, a subject usually wants to influence an addressee, e.g., to perform an action. Information is thus to be understood as "statements that improve the degree of knowledge of a subject (information subject/user) about an object (information object) in a given situation and environment (information environment) in order to fulfil a task (information purpose)" [28] (cf., Fig. 2.14). Modeling is therefore a part of information management. Information is important for politicians and business leaders, but also for every citizen of the world. It reflects a particular situation that applied at a particular point in time and usually allows an update into the future. It serves to make political, economic or personal decisions. When using information, the question always arises of who created it and what intentions that person has. The subjectivity of models thus plays an essential role here. #### Entity-Relationship Model In order to turn data into information, it must be combined with other data and the relevant relationships described. This creates a data model. The best-known method for describing data models is the Entity-Relationship Model (ERM). An Entity-Relationship Model consists of three main elements: • Entities Entities are the object classes that are considered in the part of the real world that is of interest. - Relations describe the relationships between entities. Attributes are properties within the context of an entity. Figure 2.15 shows an example of an ERM. It also indicates which symbols are generally used to express the main elements. However, one can also find ERM representations with notation elements of the Unified Modeling Language (UML). Figure 2.15 describes the following situation: An employee has a name. A project has a name, a start, an end and a budget. The so-called cardinality expresses the fact that an employee can lead several projects, but a project can only be led by exactly one employee. In the modeling concept of classification, objects are combined to form object types (entity sets) and relationships are combined to form relationship types (relationship sets). These types are differentiated according to: #### Flowcharts Flowcharts illustrate an execution sequence of activities or actions and are used in numerous application areas. They can describe the order in which actions are to be performed by people or other actors. Algorithms or computer programs are often documented in the form of flowcharts (e.g., program flowcharts). Due to the broad application of flowcharts, numerous variants have developed which take into account special circumstances of the respective field of application. For data processing, the symbolism for flowcharts was defined in the standards DIN 66001:1983-12 and ISO 5807:1985. Figure 2.16 shows an example of a flowchart. In the explanation of ARIS in section 1.5, we addressed Event-driven Process Chains (EPC) as a model type for the control view. These EPCs are flowcharts of sequences of event nodes, function nodes (operations), and connectors. Arrows as edges represent control flows between the symbols. Functions and events (with the exception of start and end events) each have exactly one incoming and one outgoing edge. If functions are to create several events or if several events should trigger a function, connectors such as an exclusive or (XOR), must be used. The modeler can also express who executes a function with which IT support and what data is manipulated in doing so. For this purpose he assigns symbols for organizational units (for example, departments, jobs, roles), information objects (data), or application systems to the function nodes. These elements must be specified in the corresponding model types. This is referred to as extended Event-driven Process Chains (eEPC). Figure 2.16: Flowchart for account transaction We have already briefly indicated in section 1.5 that an eEPC, as an instrument to describe the control or process view; correlates the interaction between the elements of the other views and model types. Specifically, it expresses itself in the following way: - An event is a state that occurs before or after a function. The symbol for an event is hexagonal. - A function (process) is an action or task that follows an event. Functions are symbolized by rectangles with rounded corners. - Connectors are used to split or join the control flow. The three connectors AND, OR and XOR are available, each represented in a small circle with the corresponding symbol. The decision as to which path is followed after a connector is made by the function preceding the connector. - The function nodes in the control view are linked with nodes from the function tree of the function view and thus specify the activity to be executed. - Information objects are entities from the data model that are bound to carriers such as documents or other data stores. They represent inputs or outputs of the function to which they are connected by a directed edge. The symbol for an information object is a rectangle, the character as input or output is determined by the arrow direction of the connection edge. - Organizational units show which elements from the organizational chart, modeled according to the organizational view, execute the activities (functions) in the process. Organizational units are connected to functions by undirected edges. Figure 2.17: Extended Event-driven Process Chain for an account transaction Figure 2.17 shows an example for an extended Event-driven Process Chain (eEPC). #### Petri Nets [18] One of the first and most widespread theoretical approaches to the description of parallelism are Petri nets. Petri nets are used for the logical modeling of behavior. They consider the behavior of systems, usually information systems, under the following aspects: A Petri net is a structure that is formally and mathematically precisely described as a directed graph with nodes consisting of two disjoint subsets marked with tokens. In the representation as quadruple the following applies: A Petri net is a quadruple: PN ¼ (S, T, K, M) with Figure 2.18: Petri net I According to the above definition, arcs only go from place to transition or from transition to place. Input places of a transition t are places from which arcs run to the transition t. Output places of a transition t are places to which arcs of the transition t lead. A transition can switch if there is at least one token in each input place. Switching means that a token is removed from each input place of the switching transition and a token is added to each output place. The following figure 2.19 show a Petri net with its respective states after performing the switching operations. In the start state in Figure 2.18, only the place s1 has a token, the initial token or the start token. The place s1 is the only input place of transition t1, which can therefore switch. The token is removed from s1 and a token is added to the single exit place s2. The right half of the following figure 2.19 shows the state after switching t1. The place s2 is the input place of the two transitions t2 and t3 (see left side of Fig. 2.19). So it could switch the transition t2 or t3, but not both. Which transition switches is random. This gives us a so-called non-deterministic state. If t3 switches, a token is added to the place s5 and a final state is reached, since no other arc leads out of s5. If the transition t2 switches, a token is added to the places s3 and s4, which are starting points of t2. The corresponding state is shown in the right half of Figure 2.19. Now either the transition t4 or t5 can switch. Here one could conclude that these two transitions switch simultaneously. But this is not allowed in Petri nets. Only one Figure 2.19: Petri net II Figure 2.20: Petri net III transition may switch at a time. Consequently, parallelism as observed in reality cannot be represented. However, it is arbitrary whether t4 or t5 switches first. In our example, t4 switches first and then t5. When both transitions have switched, a final state is reached again. Figure 2.20 shows the corresponding network states. Petri net models allow the analysis and simulation of dynamic systems with concurrent and non-deterministic processes. The type of Petri nets presented here is the basic version. It cannot be used to describe certain situations. For example, it is not possible to assign priorities for transitions, which is why, for example, the right-before-left rule at traffic junctions cannot be modeled. In order to remedy such deficits, extensions have been introduced to map further aspects of reality to the model or to describe certain situations more compactly. Examples are multi-valued, colored or prioritized Petri nets. Details of such extensions can be found in the available literature, e.g., [29]. #### Calculus of Communicating Systems The Calculus of Communicating Systems (CCS) was published by Robin Milner in 1980 (see [30]). This calculus enables the formal modeling of parallel communicating systems. This permits networked systems with a static topology to be described. CCS can be used to formally investigate the properties of programs such as deadlocks, bisimilarity, etc. CCS enables the description of the following aspects: Figure 2.21: Process interaction with CCS The course of a process is described as a tree, i.e., there is a root that represents the initial state and from where the individual branches originate. Each of the branches is marked. These markers represent the actions performed to move from one state to the next. A distinction is made between observable and unobservable actions. Unobservable actions can be executed at any time within a process without affecting other processes. Processes have no common variables. Recursive expressions are used to describe the behavior of a process. Within behavior expressions, variables can be used to reference other behavior expressions. The behavior expressions are described according to the following syntax, in which uppercase letters denote process names and lowercase letters denote actions. • Empty process: Ø • action Process a.P1 executes action a and then behaves like P1. • process name With the expression A :¼ P1 the process P1 gets the name A. Since recursive definitions are allowed, the expression P1 can contain the name A again. • choice Process P1+P2 can be continued with either process P1 or process P2. • parallel composition P1\|P2 means that processes P1 and P2 are executed in parallel. • renaming P1[b/a] describes the process P1, in which all actions with the denotation a are renamed to b. • restriction P1\a denotes the process P1 without the action a. Matching input and output actions in two different processes can synchronize and become an internal action τ. In general, the coaction for an action is marked with a line above the action name. These complementary actions are the send and receive actions. Figure 2.21 shows an example of the interaction between two processes. The example in Figure 2.22 shows how a simple holiday request process can be described with CCS. In the employee process, the vacation request send action is executed (action name with overline). The system then waits for the messages 'rejected' or 'approved'. The manager receives the vacation request message, which he replies to either with Figure 2.22 Example process in CCS the message 'approved' or 'rejected'. If he sends the message 'approved', the message 'approved vacation request' is also sent afterwards. This message is received by the travel department. Messages are exchanged synchronously in CCS, that is, a sender waits until the receiver executes the corresponding receive action. There are also formal rules of derivation for the informal interpretation. This makes process definitions accessible for formal evaluations. #### π Calculus CCS only allows static process structures. Communication relationships cannot be changed dynamically. The π Calculus (see [31]), also developed by Robin Milner, allows the representation of processes with changing structures. Any connections between components can be displayed and these connections can also change, or new ones can be created. Thus, the π Calculus is an extension of CCS to include concurrency. The notation in the π Calculus is largely based on the CCS notation. The following example explains the modeling possibilities of the π Calculus (see Figure 2.23). The agent (process) P wants to send the value 7 to Q via a link a. However, the value is to be transmitted indirectly via another agent R. In Figure 2.24 the individual steps for the execution of system O are shown. The processes P, R and Q are executed in parallel. Process P sends via channel b the name a and then the name 7. Process P receives via channel b the two names. This means that each x is replaced by a and each z by 7. In Figure 2.24 this is the result after step 2. Now the name 7 can be sent via channel a, which is then accepted by process Q. Thus, the value 7 was sent to process Q via process R. The following graphic shows how the structure of System O is changed by its process flow. Since the channel name a is transmitted from P to R, the processes R and Q are linked via channel a after the message has been accepted. This property shows that the π Calculus, in contrast to CCS, permits the modeling of structural changes (cf., Figure 2.25). #### Communicating Sequential Processes Communicating Sequential Processes (CSP) is a methodology for describing interaction between communicating processes. The idea was first introduced by Tony Hoare in 1978 as an imperative language (see [32]). It was then developed into a formal algebra and made famous in 1985 with the publication of the book Communicating Sequential Processes (see [33]). #### Figure 2.24: Execution steps of system O Figure 2.25: Structural change In CSP, as in CCS, the number of processes is static. It cannot be changed during runtime and there are no common variables between processes. Instead, the processes 'know' each other and communicate with each other by sending and receiving messages. For sending, the send process P executes the output command Q! (expr) and the receiver process Q receives the input command P? (vars). Output and input commands are called corresponding if the sequence of expressions (expr) and the sequence of variables (vars) are of the same type in relation to their numbers and components. Analogous to CCS and the π Calculus, CSP is based on an unbuffered message exchange in which the send and receive processes must be explicitly named. With Q!() and P?() a message without content is sent. Such messages are called signals and only serve to synchronize processes. If different signals are required, the distinction is made by means of type designators of the form Q!(Signal1) and P? (Signal1). In addition to the unconditional message exchange described above, there is the receive instruction within a so-called guarded command. A guarded command is only executed if the preceding Boolean condition is true. The formula set $$\mathbf{x} > \mathbf{y}; \mathbf{P}?(\mathbf{z}) - > \mathbf{x} \mathbf{=} \mathbf{x} + \mathbf{y}; \mathbf{y} \mathbf{=} \mathbf{z}$$ is only executed if x is greater than y. Then the message is received by P if P is ready to send the message. To be able to wait for messages from different senders, several guarded commands are combined to form an alternative instruction. $$\left[ \left( \mathbf{x} > \mathbf{y}; \mathbf{P}?(\mathbf{z}) - > \mathbf{x} \coloneqq \mathbf{x} + \mathbf{y}; \mathbf{y} \coloneqq \mathbf{z} \,\right) \right] \mathbf{x} < \mathbf{y}; \mathbf{Q}?(\mathbf{z}) - > \mathbf{y} \coloneqq \mathbf{x} + \mathbf{y}; \mathbf{y} \coloneqq \mathbf{z} \,\big\|\_{\mathbf{x}}$$ In case x > y the message P?(z) is expected, in case x < y the message Q?(z). Alternative instructions can also be executed repeatedly. Syntactically, this is expressed by a in front of the alternative instruction. $$\sim[\mathbf{x} > \mathbf{y}; \mathbf{P}?(\mathbf{z}) - > \mathbf{x} \coloneqq \mathbf{x} + \mathbf{y}; \mathbf{y} \coloneqq \mathbf{z} \parallel \mathbf{x} < \mathbf{y}; \mathbf{Q}?(\mathbf{z}) - > \mathbf{y} \coloneqq \mathbf{x} + \mathbf{y}; \mathbf{y} \coloneqq \mathbf{z} \parallel \mathbf{x}$$ The instruction is executed until none of the conditions are true, then the repetition is terminated. The concepts regarding the concurrency of CSP serve as a design basis for the programming language Go. #### Abstract State Machines In computer science, an Abstract State Machine (ASM) is a model for the formal, operational description of algorithms. The states of an Abstract State Machine are general mathematical structures. The inventor of the model is Yuri Gurevich. Egon Börger has further developed the ASM for practical application [34]. Abstract State Machines (ASM) are finite sets of transition rules of the form #### If condition then action with which the states of an ASM are changed. Condition is any logical expression and action any action. As a rule, action is a value assignment of the form f(t1,. . .. . . . tn) :<sup>¼</sup> s. The meaning of the rule is to execute the specified rule in the current state if the specified condition is met in that state. ASM states are generally defined as arbitrary sets of arbitrary elements with arbitrary functions (operations) and predicates defined on them. In the case of business objects, the elements are placeholders for values of any type and operations such as creating, duplicating, deleting, or algebraically manipulating objects. A calculation step of an ASM in a certain state means that all actions for which the condition is true are executed simultaneously. Simultaneous execution can abstract from irrelevant sequences. Several ASMs can run simultaneously and be linked via so-called controlled or monitored functions. A given ASM M can update controlled functions, but it cannot be modified by other ASMs in its environment. Monitored functions of a given ASM M can only be updated by its environment. By using controlled and monitored functions in pairs, a network of parallel coordinating ASMs can be set up. #### Object-Oriented Models The computer science models considered so far focus either on data or functions. The representation of data in an ERM and functions in a flowchart complement each other only in decoupled representations. The distinction between data and function views in ARIS makes this clear. Object-oriented modeling no longer reduces these individual entities into their separate parts but considers them as an integrated whole in which the individual components are interconnected and interdependent. An object-oriented model is a view of a complex system in which the system is described by the interaction of objects. This type of modeling is intended to reduce the complexity of the description of situations to be mapped in software. The object orientation considers the entities occurring in the real world as objects. A telephone is just as much an object as a bicycle, a person or an employee. Such objects in turn consist of other objects such as screws, rods, arms, feet, head, etc. As is usual in modeling, the objects are reduced to their properties that are significant in the respective situation. For example, an employee in a payroll accounting system is reduced to name, address, employee number, agreed income, tax class, and so on. The objects considered in a model are not designed individually. A rough blueprint with similar properties is created for similar objects. For example, one models the properties of books for a library application that are identical for all books. Such general descriptions of objects are called classes in object orientation. These classes are then used to create the required concrete objects (instances) within the model. The representation of reality in a model is therefore a two-stage process. First, similar objects of reality are identified and described as classes. The individual objects are then created as instances of a class. A class thus describes the structure of a set of similar objects. Figure 2.26 shows a class-object relationship according to which the book "Subject-oriented Process Management" is an instance of the class Book. The notation used comes from the Unified Modeling Language (UML). UML is a language standardized by the Object Management Group for the description of object-oriented models. The properties of a class are Figure 2.26: Relationship class-object Figure 2.27: Description of the Book class Figure 2.27 shows an example of the description of the Book class. This class has five attributes. The Page Number attribute has a constraint that the page number must be positive. The class allows the access and manipulation of its data with six methods. Thus, the title, the authors, the page number, the publisher and the content can be set (Set). The operation (method) DisplayPage(Page) can be used to "read" the contents of the specified page from the book. Objects can now be instantiated from such a class definition. The operations can then be used to set the corresponding attributes for each of these instances. An object-oriented model describes not only the definitions of the classes and the associated objects, but also the relationships between the classes. The types of relationships are: • Inheritance Properties can be passed from one class to the next. This is called inheritance. The class that is used as the basis for inheritance is called the superclass, and the class that inherits is called the subclass. Thus, a class Notebook is a subclass of the class Book. The method "PageEntry(page, content)" is added to the Notebook class. This allows a text to be entered on the specified page; otherwise, the Notebook subclass inherits all attributes and methods from the Book class. Figure 2.28 shows the inheritance relationship between Book and Notebook. The triangular arrow points from the subclass to the superclass. • Associations An association is a relationship between different objects of one or more classes. Associations are represented as a simple line between two classes. The line can be provided with a name (identifier) and number specification. The Figure 2.28: Inheritance of properties associated classes can also receive names regarding the relationship. Figure 2.29 illustrates an example of an association. A person (employee) can be employed by no company or by just one company (employer) and a company can employ one or more persons. Associations are very similar to entity-relationship diagrams. • Aggregations Aggregations are a variant of associations. This is also a relationship between two classes, but with the peculiarity that the two classes are related to each other like one part of a whole. An aggregation is made up of a quantity of individual parts. Figure 2.30 shows an aggregation example in which a company consists of departments and a department of employees. • Composition A special form of aggregation is composition. Here the whole depends on the existence of its individual parts. Figure 2.31 shows a change in the above aggregation. A department does not exist anymore if no one belongs to it. • Objects Communicate with each other, i.e., one object sends messages to another object. The messages then trigger the associated operations. An object therefore only understands the messages for which it contains the corresponding operations. Figure 2.32 shows a person's communication with the notebook to enter a new note. Figure 2.29: Associations between objects Figure 2.32: Message between objects The described constructs form the nucleus of the object-oriented modeling approach. In addition, there are numerous enhancements for depicting certain circumstances, such as the so-called container classes. #### 2.7 Agent/Actor-Oriented Models So far, there is no standardized and generally accepted definition of the agent concept. The term is defined in detail somewhat differently depending on the application domain [34, 35]. What all these definitions have in common, however, is that an agent is a scoped unit that is able to pursue the tasks assigned to it flexibly, interactively and autonomously. The term actor is often used as a synonym for the term agent. Thus [36] define a business actor as "... an entity that is capable of performing behavior". Multi-agent systems consist of several agents that exchange messages synchronously or asynchronously. Multi-agent systems can map the structures of software systems or serve as models for social systems. Depending on the application situation, a distinction can then be made between software agents and human agents. In a way, the processes in CCS, the π Calculus and CSP can be seen as multiagent systems. The term process is used there analogously to the terms agent and actor. An agent-oriented model therefore contains the agents, the communication paths and the messages that are exchanged. A collection of agent-oriented modeling languages and the corresponding procedures can be found in [37, 38]. #### 2.8 Conclusion: Models for Business Processes Business process models depict the part of reality of business processes under consideration. The subjective understanding of the business process concept influences which process aspects are considered essential and therefore placed in the foreground in the models. The intentions and interests of the person creating the model are reflected here. Consequences are numerous interpretations of the business process concept, each of which is neither right nor wrong, but merely sets different focal points. The following definitions of the term business process are examples of this: Both definitions focus on the necessary activities and their consequences. The first example additionally mentions the input and the output with the customer benefit, while in the second definition the processing of the business objects is included instead. On the other hand, neither definition includes the actors and necessary resources. They do not consider by whom and with what the activities are carried out. There is no relation to the organization in which a business process is embedded, or to which IT applications or other resources are required to execute it. We therefore follow an understanding of the term based on that of Gerhard Schewe [41] that also takes these missing aspects into account [41]: As already explained in Chapter 1, we will reorganize the components somewhat and group them as follows: - a. a defined start and input (start event) - b. and a defined end with a result - c. that contributes to the satisfaction of a customer's needs (and thus to the creation of value) 2. Process logic: A process - a. with people and/or machines, that take over the tasks of the respective actor - b. and carry these tasks out with tools (equipment, information, application programs, etc.). With this understanding of business processes, the relationship between the various models from different domains described in this chapter and Business Process Management becomes clear. Figure 2.33 shows the associated integrative character of business process models. Habermas's and Luhmann's models deal with aspects of social systems and organizations. They describe which components and relationships make up an organization and how people are positioned in it. Complex organizations can, for example, be structured into sub-organizations on the basis of operational functions, range of services, geographical aspects or combinations of these. The result is the organization chart. Among other things, business models consider the aspects of customers, suppliers, partners and added value and thus look at the external service relationships on the one hand. On the other hand, they also establish the connection to the more Figure 2.33: Integration of different models through business process models inwardly oriented enterprise architecture, especially with value promises (products and services), activities and resources. At the business level, the organizational structure is modeled within this enterprise architecture with the personnel resources, the processes, and the logical business objects. The linkage with the technical layer of the enterprise architecture leads to the models from computer science. These describe, for example, data structures, control flows and algorithms for programs as well as the design and interaction of other information and communication technology components necessary for the execution of desired actions within the framework of process support and automation. Models of business informatics generally try to unite computer science with models of social systems. These converge in business process models. #### References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. #### # The Author(s) 2020 A. Fleischmann et al., Contextual Process Digitalization, https://doi.org/10.1007/978-3-030-38300-8\_3 # Modeling Languages 3 Modeling languages determine the concepts that can be used to describe an extract from perceived reality and how these concepts can be put into mutual relationship. Modeling languages thus provide the vocabulary and the grammar needed to represent real-life situations in models. They will subsequently be considered in a structured way. The selection of a specific modeling language creates a well-defined point of reference for modeling, to which all actors involved - whether they are actively involved in the creation or passively affected by the model as consumers - can refer [1]. The term "actors" here is not necessarily restricted to humans, who are provided with a common, defined vocabulary by the modeling language in order to form a common understanding of the situation being modeled. It also refers to computer systems that are given the opportunity to further process models with automation support (e.g., in order to use them as a basis for workflow support), as the semantics of model elements are exactly specified in the modeling language (ibid.). The choice of the modeling language therefore determines what can be represented in a model, and what cannot be made visible because the modeling language does not offer any appropriate concepts. The choice of modeling language also influences the usability of the model for different target groups [2, 3]. Some modeling languages are designed for supporting communication among human actors and therefore may allow a "vague" representation of model semantics [4]. Other modeling languages require a more exact specification of semantics. They are preferably used to prepare models for use in IT systems. The choice of a modeling language therefore is dependent on the respective objective of the modeling process. It thus is a major step toward successful support of the activities the modeling process is embedded in. In this chapter we provide the foundation for the appropriate selection of a suitable modeling language to fulfill the requirements of both human actors as well as machines and present different languages with their respective objectives and language elements. The focus here is placed on the mapping of business processes. All selected languages therefore allow the behavior of actors in organizations to be mapped in a broader sense. The languages, however, fundamentally differ in what is considered to be an "actor" and how the behavior of such can be described. The reasons for these differences can be found in the historical context of the languages and their respective objectives. The arrangement of the following sections therefore follows a historical perspective in order to clearly highlight the relationships between the languages and their origins. Finally, we discuss for each of the languages to which extent they allow the mapping of processes according to the definition used in this book, and where they have their respective focal points or gaps in representation capabilities. #### 3.1 Overview Flowcharts, which are still used today, are one of the oldest modeling languages to describe processes. They were originally designed to represent the control flow in a computer program. Due to the generality of their language elements, they are also used for modeling processes in organizations and are therefore presented here as an introduction to graphical process modeling. They introduce the concept of branching in the control flow to represent alternative paths. For a long time eEPCs ("extended Event-driven Process Chains") [5] were a de facto industry standard for Business Process Modeling in Europe. In addition to their capability to map process flows, they also include elements to include responsibilities, data or services in models. This enables the modeling of business processes in their organizational context. In addition, they allow explicit modeling of parallel activities and thus go beyond the expressiveness of flowcharts in the representation of the workflow. Historically, Unified Modeling Language (UML) activity diagrams can be seen as a further development of flowcharts to model software processes [6]. As part of the UML, they today represent the de facto standard for representing control flows in software. Like eEPCs, they also allow the mapping of parallel process flows. Using activity diagrams, we introduce the structuring of models through partitioning and nesting and show how models can be structured based on responsibilities and not just on the workflow itself. BPMN (Business Process Modeling Notation) is the predominant standard for representing business processes today [7]. Originally derived from several different modeling languages, including activity diagrams, BPMN was explicitly designed for representing business processes. In this context, the idea behind BPMN was that it should enable modeling with different objectives - from communication support to execution in Workflow Management Systems. From a conceptual language point of view, BPMN is particularly interesting with regard to its possibilities for compact representation of complex process flows (such as exception handling). S-BPM (Subject-oriented Business Process Modeling) is a modeling approach that puts the actors involved in a business process and their interactions at the center of the modeling process [8]. The resulting modeling language is characterized by a small number of language elements and comprehensive expressiveness for mapping business processes. On the one hand, it is presented here as an example of a not primarily flow-oriented approach to Business Process Modeling, and on the other hand, it represents, in terms of language conception, an alternative to BPMN with its very extensive set of modeling elements. #### 3.2 Flowcharts Flowcharts allow the depiction of simple, sequential processes. A sequential process is characterized by the fact that no more than one activity is performed at any one time – thus parallel processes cannot be represented. Flowcharts were first described in the context of industrial production planning in the 1920s. At the end of the 1940s, they were adapted for the description of processes in the emerging Information Technology sector. Since the mid-1960s, they have been used as a standardized form of representation for computer program sequences. To this day, they are used to represent flows in computer programs or processes in organizations, as long as their complexity does not exceed the expressiveness of a flowchart. The limitation of flowcharts' expressiveness is due to their historical development. In both industrial production planning and in early computer systems, it was not necessary to be able to represent parallel processes. Due to the limited computing resources available (only one CPU or only one processor core), there was no need to provide language elements to design parallel software processes. #### 3.2.1 Notation Elements Flowcharts today exist in many different variants, which mainly differ in the notation used (i.e., the graphical representation). The semantics of the language elements, however, is common to all (cf., Figure 3.1). Basically, any number of operations (i.e., activities, represented as rectangles) is defined. These operations are put into an execution sequence by means of directed connections (represented as arrows). Rounded rectangles indicate the start and end of a process. An essential means of describing processes, both in a computer program and in organizations, is the representation of alternative operations. The selection of an alternative usually depends on a condition that can be checked - in a computer program, this could be exceeding a limit for the value of a variable, in an organization, the existence of a particular document or the decision of a person responsible for executing the process could constitute the criterion for selecting an alternative. Alternatives are represented in flowcharts by branches (represented as diamonds), Figure 3.1: Notation of flowcharts which are connected to the previous operation via an incoming arrow and to the alternatively executed subsequent operations by two outgoing arrows. The restriction to exactly two (and not several) subsequent operations is also due to the origin of flowcharts from the representation of computer programs, since these usually are only capable of evaluating a condition in a binary way (i.e., as true or false). If a condition is to be checked for more than two values, several branches must be cascaded in flowcharts. The outgoing connections are labeled with the respective characteristic value at which the program is continued after the condition has been checked. A repeated execution of operations (for example, as long as there are still documents that need to be processed) is represented by jumping back to an earlier operation via an outgoing connection from a branch. The other outgoing connection then progresses to the operation that is executed when the repeated execution is complete. In addition to these basic elements, flowcharts in some variants also offer special language elements that are used mainly to represent specific operations in the area of application (such as input or output operations in computer systems). However, these are not relevant for the conceptual understanding of flowcharts and especially not for their application in Business Process Modeling. In the following, we present the use of the notation elements using examples from Business Process Modeling. The notation used is based on the symbols defined by ANSI and DIN in the 1960s. #### 3.2.2 Examples The example in Figure 3.2 shows a process with a single operation in which an application (for which we have no detailed information here) is processed. The process ends after the processing of the application has been completed. In the example in Figure 3.3, the process is extended by a decision. The application is checked, and the result of this check allows a decision to be taken on its confirmation or rejection. The confirmation or rejection itself are, in turn, operations. The outgoing connections are merged after the alternative branches are completed. Figure 3.2: Simple flowchart Decisions with more than two possible outputs must be represented in flowcharts using cascaded decision elements. This can be seen in the example in Figure 3.4. Obviously, our applications are investment requests. The first decision now examines whether the sum of investment costs is less than EUR 1,000. If this is the case, the request is confirmed directly. If this is not the case, a second decision is taken. It checks whether the application sum is less than EUR 10,000. If this is the case, the application sum is between EUR 1,000 and EUR 9,999, which leads to an examination of the attachments to the application. If the application amount is not less than EUR 10,000.-, i.e., EUR 10,000.- or more, the application will be forwarded. We don't learn anything about the destination of the forwarding here, because flowcharts don't offer notation elements for depicting this information. As already mentioned, branches can also be used to repeat parts of a process (cf., Figure 3.5). To do this, the decision is inserted at the end of the part to be repeated and an outgoing branch leads back to the point prior to the first operation to be repeated. The other outgoing branch continues the process after the repetition is completed. In the above example, applications are processed as long as there are more applications available. It is important to understand that at least one application must be processed before a decision can be reviewed. If the process should be able to finish if there are no applications at all, an additional decision would have to be inserted at the beginning of the process ("Applications present?"), from which an outgoing connection ("no") leads directly to the end of the process. The other outgoing connection ("yes") would lead to the process flow previously described. Figure 3.4: Flowchart with multiple-outcome decision Figure 3.5: Flowchart with repeated execution of process parts #### 3.2.3 Classification Flowcharts are a simple way to represent business processes in terms of the logical sequence of activities they contain. Other aspects of a business process, such as data or responsibilities, are not accounted for in the language and cannot therefore be represented. It is also not possible to represent parallel process flows. This is a major reason why flowcharts are partly being replaced by more recent languages such as UML activity diagrams or BPMN, which offer constructs for executing operations in parallel. Since flowcharts do not offer any element to depict responsibilities, it is not possible to represent communication processes - this restriction also is addressed in more modern languages, which we introduce in the following sections. #### 3.3 Event-Driven Process Chains For a long time Event-driven Process Chains (EPCs) were the de-facto industry standard in Europe for representing business process models. They were developed as part of the ARIS concept already presented in Section 1.5 and there serve as a means of representing models of the control view of an organization - i.e., the view that deals with the processes in an organization and the associated links between its resources. Resources here can be both acting members and/or departments of an organization from an active perspective, as well as the required and/or manipulated data or goods from a passive perspective. EPCs links the functions that an organization is capable of performing based on upcoming events during business operation. The basic principle of representation is that a function is always triggered by an event - that is, a function must always be preceded by an event in an EPC model in order to determine whether the execution of a function can be started. In the more comprehensive variant "extended EPC" (eEPC), the functions are linked to the elements of the other ARIS views relevant for their execution. In particular, the responsible actors, roles, or organizational units can be assigned from the organizational view, and the relevant documents or data objects from the data view. If a function leads to a billable service, this can be represented by elements taken from the service view. In addition to the representation of decisions, EPCs also enable the mapping of parallel process flows of a business process. For this purpose, it provides additional notation elements. By way of AND connectors process branches run in parallel can be displayed. The XOR connector is used to represent decisions for which exactly one alternative has to be selected (this corresponds to the decision element in flowcharts). The OR connector can be used to represent processes in which one or more alternatives can be chosen. #### 3.3.1 Notation Elements of EPCs The basic element for describing business processes in EPCs is the function (similar to the operation in flowcharts, cf., Figure 3.6). However, the sequence of functions in a process is not determined exclusively by connection arrows. By using events the sequence is specified here in more detail. Every function is triggered by an event and subsequently creates one or more events itself. A process is thus represented by a sequence of events and functions, whereby events and functions always alternate. When naming these, functions should always describe a performable activity (e.g., "Check request"), while events should always describe a state (e.g., "Confirmed request" or "Rejected request"). An EPC always starts and ends with an event. Events that trigger a process are referred to as start events. Events that describe the completion of a process are referred to as end events. Subsequent processes can be triggered by end events of a previous process, that is, an end event can be a triggering start event in another process. By using different connectors in combination with events, it is possible to represent different control flow variants of a process within a model. This includes the possibility of executing functions in parallel (if these are not dependent on each other). The AND, OR and XOR connectors can be used for this purpose: If an AND connector with several outgoing connections is used, all outgoing paths are traversed in parallel. These paths are then usually joined at a later point in time with another AND connector. The function after the joining AND connector is not executed until all the incoming paths have been completed. An OR connector with multiple outgoing connections indicates that one or more of the following paths are traversed in parallel. These paths are usually joined again at a later point with another OR connector, whereby the subsequent function is only carried out when exactly those paths chosen at the original OR branch have been completed. It is important here that the paths to be activated must be selected at the time of reaching the OR connector – it cannot be used to trigger further paths at a later point in time, should this become necessary during execution. Each OR connector must be followed by an event in each of the paths that could be triggered by the function preceding the OR connector. The paths whose first events actually occur are activated during runtime. Finally, an XOR connector is used for representing "exclusive OR" or "either, or" decisions. Using an XOR connector with multiple outgoing connections means that exactly one of the following paths is selected when the process is executed. It is therefore suitable for representing mutually exclusive alternatives in processes. The Figure 3.6: Notation of EPCs paths are joined again with an XOR connector, and the subsequent function is performed when the selected path has been completed. As for OR connectors, XORs must also be followed by an event on each path that could be triggered by the preceding function. These events must be mutually exclusive. The path with the first event that actually occurs is activated during runtime. In contrast to flowcharts, it is also possible to describe more than two alternatives here, as long as the events used are mutually exclusive. #### 3.3.2 Examples of EPCs We here use the same examples as shown for flowcharts to visualize the differences in the notation. The example in Figure 3.7 shows a process with a single function in which an application (for which we have no further information here) is processed. The process ends after the application has been checked. In the example in Figure 3.8, the process is extended by a decision. The application is checked, and the result of this check allows making a decision on its positive or negative assessment. The confirmation or rejection itself are, in turn, functions. The outgoing connections are joined with an XOR connector after the alternate branches are completed. The representation of decisions that have more than two possible options is easier here than with flowcharts. In Figure 3.9, the XOR connector is followed by three events that all refer to the investment amount and are mutually exclusive. The XOR connector can also be used to repeat parts of a process (cf., Figure 3.10). To do this, the connector is inserted at the end of the part to be repeated and an outgoing connection (line) links back to the event that triggers the part to be repeated. The other outgoing branch continues the part of the process to be executed after the repetition has been completed. It must lead to an event that triggers the termination of the repetition. In the above example, applications are processed as long as further applications are available. Figure 3.7: Simple EPC The AND connector can be used if two functions can be executed independently of each other (cf., Figure 3.11). The process modeled in the above example is therefore only correct if the attachments can actually be checked independently of the application. If this is not the case, the two functions would have to be arranged sequentially. From the point of view of modeling, the AND connector, unlike the other connectors, does not have to be immediately followed by events, since no decision is made. All outgoing branches are activated in any case. Figure 3.12 shows an example of the use of the OR connector. Here we assume that an application may contain offers or comments, or both (but must at least contain offers or comments). If offers and comments were completely optional (i.e., if both could be missing), their fundamental necessity would have to be examined with an upstream XOR connector and corresponding events. Alternatively, an additional branch could be added to the OR connector with an event "Application alone is sufficient". In both cases, the condition that events and functions must alternate on all paths through the process must not be violated. If not otherwise possible, this must be ensured by a "dummy" function that does not lead to any actual activity. Figure 3.9: EPC with multi-outcome decision #### 3.3.3 Supplementary Notation Elements in eEPCs The eEPC supplements the business process depicted in an EPC with information about its execution context. In particular, responsibilities and resource requirements are assigned to the functions here. The basic rules of an EPC remain unchanged. The additional elements can only be assigned to functions - events are not affected. At this point we do not provide a complete description of the possible elements of EPCs but focus on the most common ones (cf., Figure 3.13). Responsibilities are represented by organizational units. Such units do not usually represent concrete persons but abstractly identify the name of a role (e.g., managing director) or department (e.g., financial accounting). This ensures that the specification of a process is independent of the availability of concrete staff resources. Concrete persons do not have to be assigned until the time of execution. Undirected connections (lines) are used to show the assignment to a function. In this way, an organizational unit can be assigned to several functions. It is also possible to list organizational units more than once, if the model layout can be made clearer in this way. IT systems are modeled in a similar way. They indicate the need to use a particular IT system (for example, an ERP system or a database) when executing a function. They are also assigned to functions with undirected connections (lines). Figure 3.12: EPC with optional parallel execution of process parts Figure 3.13: Additional notation elements for eEPCs Information objects are used to represent data processing in a business process. An information object can be arbitrarily comprehensive (i.e., a single value, as well as a complete document) and is always assigned to a function by means of a directional connection (arrow) that describes the data flow. If the arrow ends at the function, this means that the information object is required to execute the function. If the arrow ends at the information object, this means that it is created or changed by the function. An information object can have several inbound and outbound connections, which can describe both its origin and its use in a business process. #### 3.3.4 Example of an eEPC To illustrate eEPCs, we use one of the examples described above and add the data flow and the human resources required (cf., Figure 3.14). Application systems would be modeled analogously to the use in organizational units. With regard to the data flow, we can now see that the actual application must be present in order to check the application. This check not only leads to a positive or negative assessment of the application in the process flow, but also to an information object in which the assessment is stored. In the case of a negative assessment, this information object is required to create the rejection (we can therefore assume that the rejection contains a substantive reason). If the application is confirmed, the "Assessment" data object is no longer required - so we can assume that no further justification will be given in this case. With regard to responsibilities, we now recognize that several organizational units are involved in the process. While the application is checked by a clerk, the head of department is responsible for the final confirmation or rejection. It is important here that the events on which the decision is based are triggered by the "Check application" function, for which the clerk is responsible. In the present process model, the head of department has no possibility to revise the decision once made. Figure 3.14: Example of an eEPC #### 3.3.5 Discussion The EPC offers more comprehensive possibilities for representing business processes than the flowchart. What they have in common is their orientation toward using the tasks and activities within an organization as the primary structuring characteristic of the business process (i.e., all information depicted in the model is anchored in the description of the process flow). This is an obvious choice when describing organizational processes, but - as we will see later - not necessarily the only possibility. Other modeling languages use actors or data as their primary structuring elements on which all other information is anchored. This makes aspects of the process visible that can only be implicitly represented in (e)EPCs (such as the transition between responsibilities in the process and the necessary communication between the actors). The requirement to alternate functions and events in the process flow in EPCs often leads to very extensive models that are sometimes difficult to understand. It also bears the risk of tempting modelers to formulate trivial events that do not add any information to the model (e.g., function: "Execute task", event: "Task executed"). When used correctly, however, the EPC approach offers advantages: On the one hand, processes can be described and delimited more precisely than with flowcharts; on the other hand, EPCs explicitly allow the view on the capabilities of an organization (its functions) to be linked to the view on how it uses these capabilities to react to external stimuli or events within the organization itself. Thus, organizational capabilities can be described generically and used multiple times in processes, avoiding inefficiency through replication. From a pragmatic point of view, however, practical experience has shown that the specification of generic functions, as well as process-specific events, in the necessary detail is not always feasible. More modern approaches, such as the activity diagrams discussed in the following or BPMN, therefore still use the concept of events, but only deploy these if an external stimulus (such as an incoming message, an error, or a deadline) actually needs to be addressed. In contrast to the modeling languages with a technically oriented history (such as flowcharts or the activity diagrams described in the next section), the eEPC and the surrounding ARIS framework are concepts originally derived from business administration. They thus pursue a more comprehensive approach to the description of business processes than the technology-centric approaches. The consideration of data, responsibilities, but also goals or services (which were not discussed here), enables a comprehensive modeling of business processes, which still influences the design of contemporary modeling languages for the representation of organizational phenomena (such as business processes or enterprise architectures). #### 3.4 UML Activity Diagrams The activity diagram is defined as part of UML (Unified Modeling Language), which contains a collection of modeling diagrams suitable for specifying software systems. The activity diagram there takes a role similar to that of the original flowchart and is used to illustrate the behavior of a software system. Because of its more recent historical context, it also provides elements for the illustration of distributed and parallel process flows. Like the flowchart, the activity diagram is also suitable for representing organizational processes (i.e., business processes). While it is still used for this purpose today, the focus in the area of Business Process Modeling has shifted strongly toward BPMN (Business Process Modeling Notation). BPMN was specified by the same standardization body as UML and has adopted many elements of the activity diagram. However, BPMN focuses more explicitly on the requirements of Business Process Modeling and the organizational aspects to be represented there, which we have already discussed for EPCs. #### 3.4.1 Notation Elements By definition, an activity diagram always describes an activity that consists of individual actions ("activity" is thus used here analogously to "process"). An action corresponds to an operation for flowcharts, or a function for EPCs, cf., Figure 3.15). An activity usually begins with a start node and finishes with an end node (similar to the associated elements for flowcharts). Between these nodes, the actions contained are specified and brought into sequence by control flow arrows. To influence the process, it is possible to insert decision elements. Decisions can have any number of outgoing branches, for which the activation conditions must be mutually exclusive. The conditions are listed at the outgoing connections. The semantics of the decision symbol corresponds to the XOR in the EPC - there is no equivalent for the OR connector in activity diagrams. The activity diagram provides the split/join element to represent process parts that can be executed independently of each other and in parallel. When used to split the process, it can have any number of outgoing connections that are all activated at the same time. The branches created in this way should be merged again by the join element. The process is not continued until all branches have been finished. Signals are used for communication between process parts in different activities (i.e., in different diagrams), or within an activity when information is to be provided for subsequent process parts. They are incorporated into the control flow like actions. Models do not always have to contain complete signal pairs (i.e., send and receive signals). They can also send signals for processes that are not shown in the diagram (i.e., contain only a send signal) or receive signals from a process that is not shown in the diagram (i.e., contain only a receive signal). Received signals can also trigger an activity and thus replace the start node in a diagram. The activity diagram also provides elements to represent responsibilities and data flows (cf., Figure 3.16). Responsibilities are represented using partitions. Partitions are elements that enclose parts of an activity diagram and thus determine that all elements, in particular the actions they enclose, fall under the responsibility of the stated organizational unit (or, in the case of software systems, system component). If partitions are used (which is not mandatory), all elements of the activity diagram should be enclosed by exactly one of the partitions shown. Overlaps are not allowed, actions outside a partition should be avoided. Data objects in activity diagrams are directly incorporated into the control flow between actions. They can therefore (only) be used to represent the flow of information between two consecutive actions. If a data object is only needed again later in the process flow, it has to be forwarded in the control flow via all intermediate actions, or passed on by means of a signal. #### 3.4.2 Examples In order to work out the differences and similarities to the previously discussed modeling languages, we again make use of the examples already used above. The example in Figure 3.17 shows an activity with a single action in which an application (for which we have no further information here) is processed. The activity ends after the processing of the application has been completed. In the example in Figure 3.18, the process is extended by a decision with three possible outcomes that are mutually exclusive. The application is checked, and the result of this check allows the decision to be taken with regards to the further processing of the application. As with the other modeling languages, we can also use branches here to repeat parts of a process (cf., Figure 3.19). For this purpose, the decision is inserted at the end of the part to be repeated and an outgoing branch is linked back to a closing decision before the first operation of the part to be repeated (a closing decision is used for joining alternative branches and has several incoming and only one outgoing connection). The other outgoing branch of the opening decision element continues the process after the repetition is completed. The split/join element can be used if two action sequences can be executed independently of each other (cf., Figure 3.20). The process modeled in the above example is therefore only correct if the attachments can actually be checked independently of the application. If this is not the case, the two actions would have to be Figure 3.19: Activity diagram with repeated execution of process parts (loop) Figure 3.20: Activity diagram with parallel execution of process parts arranged sequentially. The merging join element waits for both incoming branches to be completed before continuing the process. The example in Figure 3.21 demonstrates the use of partitions, data objects and signals. Partitions are used to represent the responsibilities in the process. The process is triggered by a received signal, which explicitly shows that the execution Figure 3.21: Example of an acitivity diagram with partitions, data objects and signals only starts when an application is received (we had previously always implicitly assumed this for the flowchart and the other examples given for the activity diagram, but, unlike EPCs, were never able to represent it in the model). Send signals are used to transmit the confirmation or rejection to the recipient (who is not shown here). The assessment of the application is only transferred in the case of a negative assessment as a data object for the action "Reject application" (we can therefore assume that the rejection contains a substantive reason). If the application is confirmed, the "Assessment" data object is not required, so we can assume that no further justification will be given in this case. #### 3.4.3 Classification Activity diagrams largely combine the simplicity of flowchart notation with the expressiveness of EPCs (with certain limitations). They allow the handling of data in the process to be represented and introduce a means of clearly representing responsibilities with the partitions. In contrast to the previously discussed languages, the availability of signals allows the depiction of communication processes between participants or with the surrounding environment of the displayed process. The absence of an element corresponding to the OR connector in the EPC does indeed represent a limitation, however this is rarely relevant in practice, since we are usually confronted with mutually exclusive alternatives or completely independent branches of execution in the real world. Activity diagrams are therefore a suitable tool for representing business processes on the whole, especially if the target group using the model has an Information Technology background and is already familiar with the notation. For other target groups, BPMN, which we will discuss in the next section, is preferable due to its more flexible applicability and higher expressiveness for Business Process Modeling. #### 3.5 BPMN BPMN – the modeling language referred to as Business Process Modeling (and) Notation - was developed by IBM in 2002 and subsequently published by the BPMI (Business Process Management Initiative). The aim was to create a universally applicable standard to counter the multitude of process modeling languages used in academia and industry. This language should adopt the essential characteristics of the most common languages and make it possible, in addition to the documentation of business processes, to create models that allow for immediate IT-supported execution. BPMI in turn was merged with the OMG (Object Management Group) in 2005. Thus, BPMN became an OMG standard and complements the already mentioned UML (Unified Modeling Language), which is maintained by the same group. The BPMN 2.0 standard was published in 2010. This standard incorporates several new diagram types: the choreography diagram, the conversation diagram, and the collaboration diagram. In the following, we consider the basic elements of BPMN 2.0 that enable business processes to be represented at the business level. BPMN focusses on business processes, which it presents as a temporally logical sequence of activities (tasks) which are structured in accordance with organizational responsibilities. The representation of data is not as comprehensive as in other modeling languages and only regarded in the context of process flows. #### 3.5.1 Notation Elements for Modeling Process Flows Process diagrams created with BPMN are called Business Process Diagrams (BPD). In its core BPD follows the principles of activity diagrams, which are subsequently supplemented by elements that allow the representation of the potentially more complex control flow in business processes (cf., Figure 3.22). As a basic principle of representation, certain things have to be done in a process (tasks), but possibly only under certain conditions (gateways), and things can happen Figure 3.22: Core notation elements of BPMN (events). These three objects are connected to each other via sequence flows, however only within the confinements of pools or lanes. Pools and lanes are constructs used to represent responsibilities in distributed business processes. They are discussed in more detail below. If a connection is made across pool boundaries, it is modeled using message flows, which we will expand on later. A process consists of tasks. After starting a process (by means of an event), one task follows the other until the process ends (with an event). Tasks can be atomic (i.e., not refined further) or can contain sub-processes. In such cases, tasks are refined by an additional embedded BPD, which represents its detailed sequence of sub-tasks. This detailed sequence can be "hidden" and is represented by a "+" symbol at the bottom of the task. A process begins with a start event and ends with an end event. BPMN offers a multitude of possibilities to define events that can trigger, complete or influence the course of a process. These will be discussed later. At this point it is important to emphasize that a process can start with one or more start events and can end on any path through the process (see sequence flow and gateways below) with one or more end events. There must be a continuous sequence flow from each start event to at least one end event. Tasks, gateways or intermediate events must not be endpoints in the process and therefore always require at least one outgoing sequence flow. A gateway represents a branch in the control flow. The exclusive (XOR) gateway requires a condition for each outgoing control flow, which according to the standard must always refer to the result of an immediately preceding task. The parallel (AND) gateway tracks all outgoing control flows independently and in parallel. The branched control flows can be terminated separately with end events or explicitly merged again with another parallel gateway. After this merger, the control flow only continues once all incoming control flows have been completed (as with the split/join concept for activity diagrams). The inclusive (OR) gateway can follow one or more paths, whereby a condition must be specified for path selection (as with the exclusive gateway). This condition must already be testable at the time of the decision, so the necessary data must have been generated in one of the previous tasks. Decisions, which cannot be made on the basis of previously existing data, can be represented using the event-based gateway. This requires an event in each outgoing branch immediately after the gateway (e.g., an incoming message event or a timer event). When one of these events occurs, its respective branch (and only this branch) is activated. We will discuss this in more detail when discussing the use of events. #### 3.5.2 Examples for Modeling Process Flows In order to work out the differences and similarities to the previously discussed modeling languages, we again make use of the examples already used above. The example in Figure 3.23 shows a process with a single task in which an application (for which we have no detailed information here) is checked. The process ends after the checking of the application has been completed. In the example in Figure 3.24, the process is extended by a decision with three possible results that are mutually exclusive. The application is checked, and the result of this check allows the making of a decision on how to further process the application. In BPMN, it is important that the data used as the basis for a decision is explicitly generated or received before the gateway. Here, too, we can use branches to repeat parts of a process (cf., Figure 3.25). For this purpose, a gateway is inserted at the end of the part to be repeated and an outgoing branch is returned to a closing gateway before the first task of the part to be repeated (a closing gateway is used for merging branches and has several incoming sequence flows and only one outgoing). The other outgoing branch of the opening gateway continues the process after the repetition is completed. The requirement to explicitly create the basis for decision-making prior to the gateway is illustrated here by the additional task of checking the existence of further applications. The parallel gateway is used when two task sequences can be executed independently of each other (cf., Figure 3.26). The process modeled in the example therefore is only correct if the attachments can actually be checked independently of the application. If this is not the case, the two functions would have to be arranged sequentially. The merging gateway waits for both incoming branches to complete before continuing the process. The example in Figure 3.27 shows the use of pools, lanes and data objects. The lanes are used to represent responsibilities in the process. With the BPMN elements introduced so far, we cannot map communication processes. The signals available in activity diagrams can therefore not be represented for the time being, which is why the example here is less specific than the version represented as an activity diagram. The use of message events, which we will introduce in a later chapter, will, however, remedy this deficiency. The assessment of the application is only transferred to the task "Reject application" in the case of a negative assessment as a data object (we can therefore assume that the rejection contains a substantive reason). In case the application is confirmed, the data object "Assessment" is no longer needed, so we can assume that in this case there will be no further justification. Figure 3.26: BPMN diagram with parallel execution of process parts Figure 3.27: Example of BPMN diagram with pools, lanes and data objects #### 3.5.3 Notation Elements for Controlling Sequence Flow with Events A distinguishing feature of BPMN is the very detailed and comprehensive set of event constructs, which enables exact control of the process flow. Events indicate that something has happened and therefore represent points in times, as opposed to tasks that take a certain amount of time and effort to be completed. So far, we have only introduced start and end events. In the following we will describe start, intermediate and end events in detail once again (cf., Figure 3.28). Events are always represented by a circle and usually an enclosed symbol. Simple circles indicate start events, double circle borders indicate intermediate events and thick circle outlines indicate end events. If no enclosed symbol is specified, the event is of no particular type (blank) and can usually only be found at the start or end of a process or as a triggering intermediate event. #### Start Events Start events are used to trigger a process or subprocess. Generic (blank) events are often used if the trigger is either clear from the context, or when it is not yet known. If a process is triggered on the basis of a particular point in time or a period of time or by a periodic event, the timer symbol (clock) is additionally used. A message symbol (letter) is used when an incoming message triggers the process (cf., Figure 3.29). A process can also be triggered by a condition start event, if specific conditions need to be met in the process context in order to start it. Signal start events are used if some observable event from outside or inside the process should lead to the execution of a particular process. The pentagon as a symbol embedded in a start event indicates a combination of several potential start events, whereby only one of the events must occur to trigger the process (cf., Figure 3.30). A process does not have to be limited to a single start event; processes can also have several alternative start events. #### End Events Processes always finish with end events, whereas the same symbols are used here as for the start events, with the exception of the timer symbol, the condition and the combination of parallel triggers. More specific end events include sending a message or issuing a signal to notify other processes about the end event at hand (for details, cf., section "Event Types"). One notable end event type is the termination end event (rightmost symbol in Figure 3.31), which terminates the entire process immediately, regardless of whether Figure 3.31: Examples of BPMN end events Figure 3.32: Example BPMN diagram showing the use of the event-based gateway other sequence flows within the process are still running or not; that is, it terminates the entire process instance. A standard end event on the other hand always only terminates the process branch it concludes. Any further process branches that are still running will continue to be executed. Processes can have several end events. A process without an end event is incomplete. #### Intermediate Events & the Event-Based Gateway Intermediate events can be used anywhere in a process and are represented by a circle with a double border. They are used to represent intermediate results relevant for other processes, or if certain (external) events have an impact on the execution of the process at hand (for example, an incoming message or the expiration of a certain time period). Gateways can also be event-based if the execution of a process is dependent on the occurrence of different events, which require different reactions and therefore follow different paths. Such dependencies can be modeled with an event-based gateway. Figure 3.32 shows a process of applying for a job and waiting for different potential reactions. Depending on whether an invitation or a rejection is received, or whether a deadline of 2 weeks expires, different paths are taken in the process. The event-based gateway is the only gateway where the necessary information required to make a decision does not need to be available at the time the gateway is checked. An event-based gateway blocks the sequence flow until one of the downstream events is triggered, and then continues execution exclusively along the respective branch. Figure 3.33: BPMN notation elements for modeling communication #### 3.5.4 Notation Elements for Modeling Communication BPMN also enables the modeling of distributed business processes. Although BPMN clearly focuses on the process flow during modeling (similar to flowcharts, EPCs or activity diagrams), it also enables the structuring of the process in accordance with the participants involved and their associated responsibilities. The modeling elements available for this purpose are described in this section (cf., Figure 3.33). A pool represents a company or an organizational unit in a company, such as a department. Each (swim) lane in a pool represents a person or role involved in the process that is assigned to this pool. BPMN allows the representation of the interaction of two or more processes. The aforementioned pools and lanes are necessary for the representation of collaborations. Separate lanes are required for all persons or groups involved in a process, and separate pools are necessary for each process or organizational unit that is responsible for this process. Each pool thus contains its distinct processes with separate start and end events. Nevertheless, these individual processes can strongly influence each other, in which case they are coupled via message flows. Message flows indicate that data is exchanged between different processes. Therefore, no message flow can take place within a process (pool). Consequently, there are no message flows within a lane or between different lanes of a single pool. Sequence flows show which activities are executed in which order and do not explicitly constitute an exchange of data. In contrast to message flows, they may only be used within a pool and not between different processes (pools). Message flows can be augmented with message elements, which are used to explicitly represent the exchanged data and contain a more precise specification of the transmitted information. Message flows can be used in different ways: they may either originate from pools and activities, and also end there, or they can be explicitly sent by send message events and received by receive message events. The first case is useful for the descriptive modeling of business processes in which a communication process is to be represented that does not necessarily have to be described exactly. A message originating from an activity or pool is sent at some point during task or process execution - the exact time remains unclear. A message ending at a pool only states that the represented organization receives this message, but not which activity Figure 3.34: Example BPMN diagram showing communication-oriented processes it triggers or how it is handled within a process. This can be useful when modeling external organizational units, whose detailed behavior is unknown. An exact specification of communication processes, however, is only possible by using explicit send and receive events. #### 3.5.5 Examples for Modeling Communication-Oriented Processes In the following, we extend the example that we used to demonstrate the use of events with the communication partner not depicted originally, i.e., we now also model the process of the company to which an application is addressed. The example in Figure 3.34 shows two processes (one per pool) that are linked by message flows. The company's process is triggered by an incoming application, which is represented here in the first message flow. After checking the application, the decision can be made whether to send an invitation or reject the application. In the upper pool, the applicant waits for an answer for a maximum of 2 weeks (as represented by the intermediate timer event). The event-based gateway activates the process branch whose event occurs first. The related send and receive events are linked via message flows. It is important to note here that message flows always represent 1:1 relationships – that is, a sent message can be received exactly once and a receive event can react to exactly one message. Figure 3.35: Example BPMN diagram showing the exclusive use of communication interactions The example in Figure 3.35 shows how BPMN can be used to only represent communication acts between the actors in a distributed process. The pools are used here as black boxes, i.e., the behavior contained in them is not shown and remains unknown. All we see is that messages are exchanged, but the order of the messages is not defined. Since the specified events for sending and receiving are missing here, we augment the model with message elements attached to the message flows in order to be able to comprehensibly describe the nature of communication. Another extension of the original process model is constituted by the modifier in the upper pool, which indicates parallel multiple execution of the process contained in the upper pool. This means that the process in the lower pool could or even must be able to handle several applications arriving in parallel and independently of each other. Empty and filled pools can also be combined as required. If, for example, we wanted to represent the process of handling an incoming application, we could leave the pool "Applicant" unspecific, since we do not know the behavior of applicants (nor is it relevant), but need to know that we can receive an application from them and that we will direct our responses to them again eventually. #### 3.5.6 Notation Elements for Modeling Complex Business Situations The notation elements of BPMN introduced so far enable the representation of business processes from the point of view of the participating organizational units. BPMN allows keeping process models vague or leaving parts of them unspecified if they do not seem relevant for the objective of modeling. In some cases, however, a process needs to be defined as precisely as possible and represented in all its variants, covering all possible exceptions. This is necessary, for example, if the model is intended to serve as the basis for IT-support of the work processes depicted. If aspects are omitted or abridged, the result is a discrepancy between the real work process and the support measures developed based on the model, which ultimately Figure 3.36: Parallel and ad-hoc subprocesses would lead to unsatisfactory tools and work-arounds. This section describes the BPMN notation elements that enable more complex and comprehensive process descriptions. Due to the variety of scenarios that can be represented, examples are given here directly with the descriptions of the respective elements. #### Variants of Activity Modeling In the following section, special features for the general modeling of activities, as well as for the modeling of activities as subprocesses are explained in more detail. #### Subprocesses Processes can include detailed specification of tasks via subprocesses. This method is mostly used to maintain a comprehensive overview of a process when creating large, extensive models, while still being able to specify detailed task descriptions. Subprocesses can be collapsed to tasks, which are then shown in the overall process with a small plus sign. If appropriate tool support is available, collapsed tasks can be dynamically extended again to view the detailed subprocess specifying the exact execution of the task. Subprocesses also can be used to combine several tasks in a single execution context without specifying their exact sequence. The model on the right (an ad hoc subprocess) in Figure 3.36 only indicates that any number of the embedded activities can be performed but makes no statement about their relationships. The left model specifies that all four embedded tasks must be executed before the subprocess is completed. It makes no assertion about their order or other relationships - the activities can be carried out in parallel or in any sequence. #### Types of Tasks Task types describe the character of a task in more detail, indicating for example whether it requires human involvement or can be executed automatically in an IT system. Modifiers as shown in Figure 3.37 are used to distinguish between service tasks, receive tasks, send tasks, user tasks, business rule tasks, script tasks, and manual tasks (illustration from top left to bottom right). These modifiers do not Figure 3.38: Task modifiers for diverse behavior specification necessarily have to be used, but they do specify the semantics of a process model in more detail. #### Execution Behavior of Tasks Tasks can have different markers that describe their execution behavior. Single tasks, entire processes or subprocesses can be executed several times in loops, in parallel, sequentially, or can be marked as ad hoc tasks or as compensation tasks (cf., Figure 3.38). For a looped task or process, a termination condition can be specified in addition to the symbol. If the termination condition is reached, the task or process in question is no longer executed and the superordinate process is continued. If a task can be executed several times in parallel, this is indicated by three vertical lines. For example, the "check application" task could be carried out by several agents for several received application documents. If parallel execution is not possible, but the individual cases are still independent of each other, the sequential multiple instance marker is used, which is indicated by three horizontal lines. With ad hoc tasks, the exact sequence of the sub-tasks contained in the task is unknown a priori and is selected during the execution of the process. It is also Figure 3.39: Different BPMN event types possible to omit some of the sub-tasks and only execute those that are required in the specific situation. Such processes are indicated by a tilde (as shown above). Compensation activities are used in transaction modeling and are described below. #### Event Types BPMN offers a large number of different events targeting different areas of application for detailed process control. While we will deal with these in detail in the following, an initial overview is provided in Figure 3.39 to show the underlying structure of event types. In general, events can occur in three different variants, which we have already introduced above: – Start events trigger new process instances, that is, they start the execution of a process. They are always "receiving" in nature, i.e., they react to stimuli from outside (e.g., incoming messages, time sequences, etc.). Start events can also be differentiated according to their surrounding modeling element. They can be used to either trigger an entire process or to trigger subprocesses. The second case is called an "event subprocess" and can be specified as an "interrupting" or a "non-interrupting" form. An "interrupting" start event indicates that the control flow is completely transferred to the subprocess, i.e., all other task within the respective pool are interrupted and cannot be continued. "Noninterrupting" event subprocesses are started when the respective event occurs without interrupting the execution of the task currently running within the pool the subprocess is placed in. This can be used, for example, to react to events that should not or cannot be handled in the main process of a pool, but whose occurrence should result in a reaction without affecting the main process (such as customer inquiries about the status of an order processing while the order is being processed in the main process). Intermediate events basically exist in "receiving" and "sending" variants. The "receiving" variants (occurring event) block the sequence flow until the specified event arrives. The process therefore cannot be continued until the event has occurred. The "sending" variant (triggered event) indicates the occurrence of certain events in the course of executing a process (or also an occurrence between processes from different pools). Events often are used reciprocally in comprehensively modeled processes, i.e., a receive event exists for each send event. Receiving intermediate events also exist in a "boundary" form. These events are "pinned" to tasks (i.e., are graphically attached to the (lower) boundary of the task) and indicate that it is possible to react on the respective event during the execution of the task. The reaction is specified by a sequence flow originating from the attached event, which leads to the respective tasks to be performed. The boundary intermediate events in general (with some exceptions) again exist in an interrupting and a non-interrupting form. The interrupting form stops the execution of the task marked in this way and continues the sequence flow exclusively via the attached event. The non-interrupting form allows the further execution of the task marked in this way, and the sequence flow originating from the event is triggered in parallel. The triggers that lead to the occurrence of boundary events can come from outside the tasks (for example, incoming messages from other pools) or also from within the task, provided that these triggers are detailed by a subprocess. For example, an error in the execution of a subprocess can lead to activities in the main process via an interrupting error boundary event (such as documenting the error and escalating it to superiors). We have already used message and timer events in basic BPMN modeling. We will now consider the other event types in the context of their respective application areas. Figure 3.40: Link event #### The Link Event For more complex or extensive processes, tracking sequence flows through the diagram can sometimes be difficult. Sequence flows crossing each other or sequence flows with many changes of direction are difficult to read and are detrimental to comprehensibility and clarity. In such cases, the link event can be used (cf., Figure 3.40). In contrast to the other events, it semantically does not represent a real event, but merely serves as a connector between two sequence flows that are far apart. The coupling is carried out via the designation of the sending and the receiving event. There must always be a 1:1 assignment (implicit parallelization by one triggering and several receiving link events of the same name is therefore not permitted). Link events should only be used in cases that cannot be resolved in any other way in order to increase clarity, since the effort involved in searching for related link events can even exceed the tracking of complex sequence flows (as long as there is no tool support for jumping to or visually marking related events). Choosing an alternative arrangement of activities or lanes is usually the better choice. #### Use of Signals In BPMN, messages can only be used for communication between pools. In addition, message-based communication always has exactly two endpoints, so it can only connect exactly one sender to exactly one receiver at a time. If information is to be made available globally within a collaborative process and this is to happen independently of pool boundaries, signals can be used. Signals can be triggered in a process (as intermediate or end events) and are then available both within the pool and in all other pools of the same collaboration. Signals can be used, for instance, to inform all pools of a collaboration about the termination of one of the represented processes. This means that all other processes that are still running can complete their processes cleanly and there are no dangling processes left that can no longer be completed, e.g., because an expected incoming message no longer arrives due to an aborted process of a communication partner. #### Handling of Exceptions and Interruptions Activities, i.e., tasks and subprocesses, can be aborted or interrupted by certain events. This is indicated by event symbols attached to the respective task. Two solid outer circular lines in the event element indicate that the task is interrupted by the event; two dashed circular lines indicate that the task is not interrupted but can be continued while simultaneously reacting on the exception that occurred. In the example in Figure 3.41, we react on a deadline, i.e., we model that the execution Figure 3.41: Non-interrupting and interrupting boundary events Figure 3.42: Example for use of a non-interrupting timer event of the task must not take longer than a certain time span (on the left) or requires some reaction if it lasts longer than a certain time span (on the right). In either case, the model must contain information on what has to happen when task execution takes longer than anticipated. This information is modeled as a sequence flow emanating from the attached boundary event. Reactions to unforeseen triggers such as errors or escalations can also be modeled and displayed in the same way using the respective event type. #### Example: Non-interrupting Timer Events Figure 3.42 shows a subprocess that includes the activities of processing an order in a fast food restaurant, which should not take longer than 5 minutes. If the processing of the order takes longer than 5 minutes, the customer should receive their money back. The order should still be completely processed. If we had used an interrupting boundary event here, the customer would only get their money back, but not their order. #### Different Ways of Terminating Processes Sequence flows in a process are usually concluded with an end event. Such end events, however, only terminate the execution of the respective sequence flow. If other sequence flows are active in parallel (for example, because they were opened events), their execution will continue to be carried out. There are several ways to terminate a (sub)process completely and immediately (i.e., terminate execution in all branches). #### The Terminate Event The terminate event aborts all active branches of a process within a pool immediately. Processes in other pools are not affected and should therefore be informed of the termination by sending a signal before the termination, if necessary (e.g., if there is the risk of waiting for further input from an already terminated process instance). #### The Error Event and the Escalation Event The error event semantically indicates the occurrence of an unforeseen error in process execution and is usually used for subprocesses. It immediately terminates the execution of the whole subprocess. The reason for the error can be given to the event as a parameter. Receiving error events can be attached to the subprocess as a boundary event for the enclosing task element to react to these errors in the superordinate process and trigger corresponding activities. Attached error events are always interrupting, i.e., they terminate the execution of the subprocess (including all active sequence flows in branches in which no error occurred). As a "weaker" variant, the "escalation event" can also be used in an identical way. The escalation event also is available in a non-interrupting form and thus allows the continuation of the execution of the subprocess in which the problem occurred. If the effects of activities already performed have to be reversed when subprocesses are terminated, the transaction handling mechanism and constructs provided in BPMN can be used. They are described in the next section. #### Transactions BPMN also offers the option of representing transactions in a process. A transaction is a set of tasks that is to be executed as a whole, either completely or not at all. In particular, if a task fails, the effects of other already completed tasks need to be reversed. BPMN introduces the concept of transactional subprocesses in combination with compensation events and tasks. Compensation tasks roll back the effects of process steps that have already been executed by means of countermeasures which are initiated in a further process step. In a transaction subprocess (characterized in BPMN by a double border of the enclosing task element), each task is assigned a compensation task via a boundary compensation intermediate event (indicated by a "rewind" symbol). If the transaction is aborted or should explicitly be undone retroactively, the respective compensation task is executed for each task that has already been successfully completed. The abort end event (marked by an "X") can be used to abort a transaction while it is still being carried out. As an end event in a transaction subprocess, it causes its immediate termination and triggers the compensation tasks. When attached to the transaction task as a receiving intermediate boundary event, it determines the further course of the process after the transaction is terminated. As a result of the concept of Figure 3.43: Exampe for transaction subprocesses and compensation events compensation, transactions can also be rolled back after they have been successfully completed. Outside the transaction, a sending intermediate compensation event can be used to retrospectively trigger the compensation tasks contained in the referenced transaction. Figure 3.43 illustrates these concepts using a travel booking process. A travel booking consists of a flight booking and a hotel booking that can potentially be done in parallel. If one of the bookings is not possible, the other booking must be canceled, if it has already been done. An error in one of the bookings leads to the transaction being canceled (triggering the termination event) and leads to sending an error message to the customer. Outside the actual transaction, an error in charging the credit card leads to cancellation of the entire booking by triggering the compensation tasks retrospectively. #### Event-Triggered Subprocesses Event-triggered subprocesses are an alternative to boundary events when handling non-standard incidents that might occur in (sub)processes. While boundary events attached to subprocesses lead to the reaction to such incidents in the superordinate process, strictly local reactions (i.e., reactions that do not have any implications for the overall process) can be kept in the context of the subprocess by using eventdriven subprocesses. Figure 3.44 shows an example of a timer-controlled non-interrupting subprocess. It picks up on the scenario already used above to demonstrate non-interrupting boundary events and shows the process of preparing an order in a fast food restaurant. Event-triggered subprocesses can be started with the same types of events that are available as boundary events, both in their interrupting and non-interrupting versions. Semantically, as already mentioned above, they differ only in the way the incident triggering the event is handled - locally within the subprocess or externally within the superordinate process. Depending on the process, one or the Figure 3.44: Example for non-interrupting event-triggered subprocesses other variant can lead to a more meaningful and/or comprehensible form of representation. #### 3.5.7 Choreography Diagrams The ability to explicitly model the interplay of actors in a collaborative process has been introduced in BPMN 2.0 in the form of choreography diagrams. A choreography depicts the process of exchanging messages between different actors. It thus provides a different view on a collaboration, focusing on the sequence of the transmitted messages independently of the processes of the individual actors. Although a representation of communication is possible in BPMN process diagrams by means of collapsed pools and the messages exchanged, the exact sequence, conditional message flows or loops cannot be represented in this way. For instance, the example of an application process used to demonstrate the use of collapsed pools as shown in Figure 3.35 does not include information on whether the invitation message and the rejection message are mutually exclusive or can occur in parallel. This can be visualized with a choreography diagram. Figure 3.45 shows the choreography representation of the application process shown above as a collaboration process. Here, the process of message exchange is in the focus of representation. Choreography tasks represent the exchange of one or more messages between two or more partners. In their simplest case they correspond to sending a single message from one partner to another. Each choreography task is triggered by one of the partners involved by sending the first message. This triggering partner is entered in a box with a light-colored background at the upper or lower edge of the choreography activity. The names of the other party or parties involved are entered into boxes with darker backgrounds on the other border of the task. Which partner is entered at the top and at the bottom is at the discretion of the modeler. Usually, if there are several choreography tasks Figure 3.45: Example of a choreography diagram between the same partners, the arrangement will remain identical to allow for better comprehensibility. If corresponding collaboration diagrams are modeled, it is recommendable to use the vertical arrangement of the pools as a basis for labeling the partners in the choreography tasks. Choreography activities with more than two partners do not occur in the example shown above. In case more than two partners would be involved, several partner fields can be added at the top or bottom. However, only one field can have a lightcolored background, since only one of the partners initiates message exchange with an initial message. A sequence flow is defined in the choreography diagram between the choreography tasks. Modeling the sequence flow in choreography diagrams essentially corresponds to the sequence flow modeling of ordinary BPMN processes. However, certain elements of process modeling do not make sense in connection with choreography modeling and are therefore not permitted. For example, there are no message events within a sequence flow, since the message exchange is, by definition, part of the choreography tasks. Accordingly, in the diagram above, event-based gateways are not followed by events, but rather by choreography tasks. The path is selected for which the associated choreography task is first started by the respective triggering message. If one wants to know which messages are exchanged in each choreography task, these can be added to the diagram in the form of letter symbols which are linked to the respective partner sending the message. The letters are color coded in the same way as partner fields. A letter symbol with a light-colored background represents the message with which a choreography task is triggered. The letter symbols of the other messages are displayed with darker backgrounds. #### 3.5.8 Classification In recent years, BPMN has advanced to be the standard choice for modeling business processes in industrial practice. Its comprehensive set of language elements makes it suitable for many application areas, from documentation to the automationsupported execution of business processes in organizations. The extensive vocabulary can at the same time be seen as a shortcoming of BPMN due to the increased complexity of the notation. In particular, the large number of event types with semantics that are sometimes hard to distinguish leads to increased effort when learning the language. Potential issues of comprehensibility of the models when using the full set of notation elements is usually countered by using a reduced set of elements in suitable cases. For the descriptive documentation of business processes, it is usually not necessary to use the complete set of events and more complex task types. Only when a process model is to be validated or executed, for example by simulation, is it necessary to enrich the models with information on non-standard cases or exceptions. In such cases, the simpler models can be used as a basis for supplementation. BPMN is one of few Business Process Modeling languages that explicitly deals with communication processes between participating actors, and enables the modeling of the same. During the development of the language, however, the starting point for specifying communication flows was the coupling of technically distributed information systems. BPMN implicitly assumes that within a pool (i.e., between lanes) it is not necessary to explicitly represent communication between actors, because they all have access to the same information infrastructure. Message flows are only modeled between pools. They are used during execution to describe the mapping of the data structures used in the source pool to those of the target pool. A message flow therefore essentially corresponds to a data transfer from one information system to another and therefore always represents a communication process with exactly one sender and exactly one recipient - several recipients cannot be addressed with a single message. While this mechanism can also be used to represent non-technical communication, its expressiveness is limited. In particular, communication between two or more actors without clearly definable messages can only be modeled in non-standard-compliant and ambiguous ways. This limitation is owed to the claim of the executability of the created processes and also exists in other communication-oriented approaches. In addition, BPMN focuses on processes with a fully specifiable control flow. It reaches its limits when process parts are strongly case-specific and cannot be described in detail in advance. In recent years, different approaches have emerged for such processes, which either adopt a declarative modeling approach for representing the execution conditions of process parts or focus on the communication processes between the actors involved. As an example for the latter category, we introduce Subject-oriented Business Process Modeling (S-BPM) in the next section. #### 3.6 S-BPM In contrast to other modeling approaches, subject-oriented process modeling describes business processes primarily from the point of view of communicating actors or systems. When modeling according to the subject-oriented approach, the subjects as representatives for those involved in a process are the focus and starting point of representation. It essentially describes who communicates with whom in which form and how the individual actors react to received messages. Communication is described by defining the messages that are exchanged between the subjects. The behavior of the subjects is described separately by state diagrams, whereby three different state types are used. A subject can wait for a message, send a message, or do something without communicating with other subjects. The latter state type is called the function state and is used to describe the actual behavior, that is, the activities of a subject. #### 3.6.1 Notation Elements When modeling according to the subject-oriented approach, the subjects as representatives for those involved in a process are at the center of attention for modeling. The modeling of a process essentially takes place in two stages with an increasing level of detail. First the interaction diagram is created, in which the subjects and their message exchange are modeled. In a further stage, the behavior of each subject is described in a separate behavior diagram. For the interaction diagram (cf., Figure 3.46), the subjects involved in a process are defined first. A subject is an active entity but does not necessarily have to be a human actor. Technical systems can also be subjects, as long as they play an active role in the process. Subjects must always be described abstractly, i.e., not for specific persons or machines, but on the basis of the necessary tasks to be fulfilled in the process (e.g., "application examiner" and not "Mr. Miller"). Messages are exchanged between the subjects. The interaction diagram only defines which messages exist and who sends and receives them. The order of the messages is not defined here. For each subject, a behavior diagram describes the order in which it sends and receives messages or executes functions (cf., Figure 3.47). The individual states are Figure 3.46: Notation of S-BPM interaction diagrams Figure 3.47: Notation of S-BPM behavior diagrams related to each other by connections which describe the conditions of the transition from one state to the next. Their use depends on the type of condition used: For each function state, what is to be done in the respective behavior step is described. The end conditions of a function state correspond to the outgoing connections that emanate from the respective function state. If the function can lead to different results, different subsequent states can be activated via different transition conditions. This enables the representation of alternative behavior patterns. In a send state, a message is transmitted to a recipient. The subject remains in the state until the recipient is able to receive the message. Who the recipient of the message is and which message is transmitted is described at the outgoing connection of the send state. The respective subject remains in a receive state until one of the messages that the receive state can accept has arrived. Since different messages can be accepted in any particular receive state, different subsequent states can be activated depending on the type of message received. For this purpose, several outgoing connections can be used to describe which message from which sender leads to the corresponding state transition. In this way, it is also possible to react differently to the same message from different senders. #### 3.6.2 Examples In order to work out the differences and similarities to the previously discussed modeling languages, we again make use of the examples already used above. In the first example there is no communication, therefore we focus on the behavior diagram of the only subject involved. The example in Figure 3.48 shows a process with a single task in which an application (for which we have no further information here) is processed. The process ends after the processing of the application has been completed. A behavior diagram must always have a start state, which is marked by a triangle in the upper left corner. There must also be an end state, marked by a triangle in the lower right corner. To fully describe the behavior of the subject, we need a state transition that identifies under what conditions the state "Check application" can be left. Therefore, we insert a state "Done" here, which we mark as the final state and which does not contain any expected activities. In Figure 3.49, the process is extended by a decision with three possible results that are mutually exclusive. The application is checked, and the result of this Figure 3.48: Simple S-BPM behavior diagram Figure 3.49: S-BPM behavior diagram with a multiple-outcome decision examination allows a decision to be taken on further processing. In the case of an investment sum of EUR 10,000 or more, the application will be forwarded. We indicate this by a send state and specify at the outgoing connection who is to receive the request. For the process to be fully specified, a behavior diagram for the head of department would also have to be created at this point. S-BPM also allows the execution of parts of a process repeatedly. For this purpose, a connection is inserted at the end of the part to be repeated, provided with a repetition condition and returned to the first state of the part to be repeated Figure 3.50: S-BPM behavior diagram with repeated execution of process parts Figure 3.51: Example of S-BPM interaction diagram (cf., Figure 3.50). The other outgoing connection continues the process after repetition completion. The example in Figure 3.51 shows the interaction diagram of an application process with two subjects, an clerk and a head of department. Figure 3.52 shows the behavior diagrams of the clerk and head of department respectively. The positive or negative assessment of an application is transmitted as a message. The summary of reasons giving details of the assessment is only sent in the case of a negative assessment. If the application is confirmed, the "Summary of reasons" message is not transmitted, so we can assume that no further justification will be given in this case. #### 3.6.3 Advanced Forms of Communication Modeling and Exception Handling The focus of S-BPM on representing communication processes is reflected in a more comprehensive and flexible description of communication than in all the modeling languages considered above. In particular, S-BPM allows the representation of more complex communication scenarios through the use of input pools and the detailed description of the data exchanged in messages by means of business objects, as well as reacting on unforeseen messages by means of exception handling via message guards. #### Input pools An input pool provides a subject with a mailbox in which incoming messages are stored until they are required in the behavior diagram. In contrast to a simple mailbox, an input pool is configurable. For each subject, the number of storable messages can be specified per type. If the input pool is not able to accept a message according to its configuration, the sender must remain in the send state until the message can be delivered. This allows different communication scenarios to be represented. If the capacity of the input pool for a particular message type is reduced to 0, the sender must always wait until the recipient is ready to receive the message. This is referred to as synchronous communication. If the input pool is configured to accept an arbitrary number of messages, the sender never has to wait until the receiver is in the state in which it can accept the message. This is called asynchronous communication (BPMN only allows the representation of this type of communication). Input pools also enable messages to be received in any order. The messages do not have to be processed in the order they arrive but can be processed according to the recipient's requirements. Input pools have no graphical equivalent in S-BPM but are a concept of execution semantics. They are described for each subject textually or in a configuration tool. If no input pools are defined, the default configuration allows for an unlimited number of messages of any type to be stored. The communication behavior therefore corresponds to the message flows of BPMN (asynchronous communication). #### Business Objects Business objects are used to specify the data that is required for executing the tasks in a business process. Business objects are passive, i.e., they do not initiate any interactions or trigger any actions. Business objects are processed and modified by subjects and can be assigned to messages in order to specify their content in more detail. As for input pools, there is no graphical equivalent for business objects in the notation of the modeling language. Business objects are concepts of execution semantics and therefore are dependent on the technical execution environment. They are usually described in tabular form. The basic structure of business objects consists of an identifier, data structures and data elements. The identifier of a business object is derived from the business environment in which it is used. Examples are business trip request, order, delivery note, invoice, etc. Business objects consist of data structures whose components can be simple data elements of a certain type (for example, character string or number) or nested complex data structures. To allow for better comprehensibility, it is recommended to describe the meaning of the data elements in more detail, especially if the meaning cannot be derived from the identifiers. Figure 3.53 shows an example of a business trip request. This consists, among other things, of the data structure 'Data on requester (employee)' with the data elements for last name, first name and personnel number, and the structure 'Data on business trip' with the data elements for start, end and purpose of the trip. In many cases, the semantics of a business object can change during process execution, for example when a delivery note is transformed to an invoice. Several different statuses can therefore be defined for a business object. When the status is changed, only the data structures or data elements of the previous status that are Figure 3.53: Example of S-BPM business object (business trip request) required in the new status are transferred, and new components are added if required. This ensures that a subject only receives the data it really needs for its work. This also facilitates compliance with data protection regulations. In the business trip request example, the status "Business trip booking" can be derived from the original status "Travel request" of the business object (cf., Figure 3.54). In particular, data elements with internal specifications such as personnel number, compensation group or reason for trip are removed, and thus do not to leave the company if the business object is sent to a travel agency for booking the travel arrangements. As shown in the following Figure 3.54, a new data structure "Booking data" is inserted for this purpose. It contains data elements with which a deadline can be specified for the travel agency to return booking confirmations or to specify certain hotel chains which are preferred by the company. #### Message Guards Handling of an exception (also termed message guard, message control, message monitoring, message observer) is a behavioral description of a subject that becomes relevant when a specific, exceptional situation occurs while executing a subject behavior specification. It is activated when a corresponding message is received, and the subject is in a state in which it is able to respond to the exception handling. In such a case, the transition to exception handling has the highest priority and will be enforced. Exception handling is characterized by the fact that it can occur in a process in many behavior states of subjects. The receipt of certain messages, e.g., to abort the process, always results in the same processing pattern. This pattern would have to be modeled for each state in which it is relevant. Exception handlings cause high modeling effort and lead to complex process models, since from each affected state a corresponding transition has to be specified. To illustrate the compact description of exception handlings, we use a service management process with the subject "service desk" (cf., Figure 3.55). This subject identifies a need for a business trip in the context of processing a customer order - an employee needs to visit the customer to provide a service locally. The subject "service desk" passes on a service order to an employee. Hence, the employee issues a business trip request. In principle, the service order may be canceled at any stage during processing up to its completion. Consequently, this also applies to the business trip application and its subsequent activities. This relatively simple example already shows that taking such exception messages into account can quickly make behavior descriptions difficult to understand because additional receive states have to be added. In these additional receive states it is checked whether a corresponding cancel message has arrived. The concept of exception handling enables supplementing exceptions to the default behavior of subjects in a structured and compact form. Figure 3.55 below shows how such a concept affects the behavior of the employee. Instead of incorporating additional receive states with a timeout zero in order to check whether a message has arrived which interrupts the standard control flow, the behavior description is enriched with an exception handling for the message "service Figure 3.54: Example of business object in different state (Business trip booking) Figure 3.55: Behavior of subject "employee" with exception handling cancellation". Its initial state is labeled with the states from which it is branched to, once the message 'service cancellation' is received. In the example, these are the states 'fill out Bt-request' and 'receive answer from manager'. Each of them is marked by a triangle on the right edge of the state symbol. The exception behavior leads to an exit of the subject, after the message 'service cancellation' has been sent to the subject 'manager'. A subject behavior does not necessarily have to be brought to an end by an exception handling; it can also return from there to the specified default behavior. Exception handling behavior in a subject may vary, depending on from which state or by what type of message (cancellation, temporary stopping of the process, etc.) it is triggered. The initial state of exception handling can be a receive state or a function state. Messages, like 'service cancellation', that trigger exception handling always have higher priority than other messages. This is how modelers express that specific messages are read in a preferred way. For instance, when the approval message from the manager is received in the input pool of the employee, and shortly thereafter the cancellation message, the latter is read first. This leads to the corresponding abort consequences. Since now additional messages can be exchanged between subjects, it may be necessary to adjust the corresponding conditions for the input pool structure. In particular, the input pool conditions should allow storing an interrupt message in the input pool. #### Behavior Extensions When exceptions occur, currently running operations are interrupted. This can lead to inconsistencies in the processing of business objects. For example, the completion of the business trip form is interrupted once a cancellation message is received, and the business trip application is only partially completed. Such consequences are considered acceptable, due to the urgency of cancellation messages. In less urgent cases, the modeler would like to extend the behavior of subjects in a similar way, however, without causing inconsistencies. This can be achieved by using a notation analogous to exception handling. Instead of denoting the corresponding diagram with 'exception', it is labeled with 'extension'. Behavior extensions enrich a subject's behavior with behavior sequences that can be reached from several states equivocally. For example, the employee may be able to decide on his own that the business trip is no longer required and withdraw his trip request. Figure 3.56 shows that the employee is able to cancel a business trip request in the states 'send business trip request to manager' and 'receive answer from manager'. If the transition 'withdraw business trip request' is executed in the state 'send business trip request to manager', then the extension 'F1' is activated. It leads merely to canceling of the application. Since the manager has not yet received a request, he does not need to be informed. In case the employee decides to withdraw the business trip request in the state 'receive answer from manager', then extension 'F2' is activated. Here, first the supervisor is informed, and then the business trip is canceled. #### Choice Segments So far, the behavior of subjects has been regarded as a distinct sequence of internal functions, send, and receive activities. In many cases, however, the sequence of internal execution is not important. Certain sequences of actions can be executed in arbitrary order, this is called freedom of choice. In this case, the modeler does not specify a strict sequence of activities. Rather, a subject (or concrete entity assigned to a subject) will organize to a certain extent its own behavior at runtime. Figure 3.56: Example for S-BPM behavior extensions The freedom of choice with respect to behavior is described as a set of alternative clauses which outline a number of parallel paths. At the beginning and end of each alternative, switches are used: A switch set at the beginning means that this alternative path is mandatory to get started, a switch set at the end means that this alternative path must be completely traversed. This leads to the following constellations: The execution of an alternative clause is considered complete when all alternative sequences, which were begun and had to be completed, have actually been entirely processed and have reached the end operator of the alternative clause. Transitions between the alternative paths of an alternative clause are not allowed. An alternate sequence starts in its start point and ends entirely within its end point. Figure 3.57: Example of process alternatives Thus, the core property of a state chart, namely that a state chart always only contains a single active state is not violated Figure 3.57 shows an example for modeling alternative clauses. After receiving an order from the customer, three alternative behavioral sequences can be started, whereby the leftmost sequence, with the internal function 'update order' and sending the message 'deliver order' to the subject 'warehouse', must be started in any case. This is determined by the 'X' in the symbol for the start of the alternative sequences (gray bar is the starting point for alternatives). This sequence must be processed through to the end of the alternative because it is also marked in the end symbol of this alternative with an 'X' (gray bar as the end point of the alternative). The other two sequences may, but do not have to be, started. However, in case the middle sequence is started, i.e., the message 'order arrived' is sent to the sales department, it must be processed to the end. This is defined by an appropriate marking in the end symbol of the alternative ('X' in the lower gray bar as the endpoint of the alternative). The rightmost path can be started but does not need to be completed. This kind of visualization radically simplifies the representation of the state transitions that would be necessary in case a traditional state chart visualization would be used (using a single connection for each potential state transition). The individual actions in the alternative paths of an alternative clause may be arbitrarily executed in parallel and overlapping, or in other words: A step can be executed in an alternative sequence, and then be followed by an action in any other sequence. This gives the performer of a subject the appropriate freedom of choice while executing his actions. #### 3.6.4 Classification In contrast to the other modeling languages discussed so far, there is no single diagram in S-BPM that fully describes a business process. Rather, separate behavior diagrams are created for all subjects, which are linked by an interaction diagram describing the message exchange. S-BPM thus enables a loose coupling of process parts and an easier adaptation of the behavior of a subject, as long as its communication interface, i.e., the set of received and sent messages and their sequence, remains unchanged. The use of state diagrams to describe the behavior of a subject also constitutes a fundamental difference to the other languages discussed so far. A state diagram – as already indicated by its name - describes the state of a system (here: a subject - this can be a human as well as a machine) and the events that lead to a state transition. A subject can only be in exactly one state at any one time - it is therefore by definition not able to execute process steps in parallel. Rather, all subjects work in parallel and independently of each other. This requires a different approach to modeling, since constructs such as AND connectors (in EPCs), split/joins (in activity diagrams) or parallel gateways (in BPMN) are not available. At the same time, this modeling approach leads to simpler, more compact models and, in contrast to BPMN, a significantly reduced range of language constructs, which contributes to the comprehensibility of the models. #### 3.7 Comparison The modeling languages considered here have different expressive power and, due to their historical development, have different focal points in their approach to represent business process [9]. The following section attempts to summarize these differences again systematically using the process definition presented in the former chapter and thus to compare the languages with respect to their expressiveness. We use the semantics of the presented modeling elements as a starting point. The point of reference for the following considerations is the process definition from the last chapter, which we will reiterate here for simplicity's sake: Table 3.1: Concepts included in process definition - a defined start and input (start event), - and has a defined end with a result, - that contributes to the satisfaction of a customer's needs (and thus to the creation of value) - is the sum of linked activities (tasks), - which, after the start event, are used by actors - in logical and chronological order - for processing a business object in order to - generate the desired result. - with people and/or machines, that take over the tasks of the respective actor, and carry these tasks out - with tools (equipment, information, application programs, etc.). On the basis of this definition, the concepts can be identified which should be representable in a process model in order to be able to model processes comprehensively (according to this definition). Table 3.1 shows these concepts. Concepts that occur more than once are only mentioned when they occur for the first time - such as "result". In the case of concepts which are described in different degrees of detail, only the more concrete concepts are considered - e.g., "linked activities" as a more general formulation of "logical and chronological order". If one now assigns the modeling elements of the languages considered to these concepts, Table 3.2 results. 3.2:Conceptallocationtothenotationelementsindifferentmodeling (continued) Table 3.2: (continued) The conceptual coverage obviously varies across languages. The table also shows that not all languages address all concepts to the same extent or with the same level of expressiveness. The allocation of the modeling elements to the concepts provides a first starting point for estimating the expressiveness of the respective languages. In this overview, the different approaches in the illustration of the logical and chronological connections are only partially recognizable. Here, the languages differ considerably: flowcharts do not offer the possibility of representing parallel processes, EPCs only allow for strong coupling of parallel activity branches by linking them within a process by means of AND or OR operators. UML activity diagrams and BPMN offer the same mechanisms (under different names), but also allow for loose coupling of processes or process parts by means of signals (for activity diagrams) or message flows (for BPMN). Especially the latter mechanism allows a detailed description of communication processes of basically independent process parts. Flexibility, however, is restricted by the necessary unique assignment of sender and receiver for each single message. S-BPM offers a similar communication mechanism, but is more flexible here (especially when using input pools that are not shown in the graphical representation of the language). In S-BPM, a description of process parts running in parallel is only possible by distributing them to different subjects - within a subject, only one functional state can be active at a time, i.e., only alternative branches in the behavior of a subject can be represented. In general, BPMN offers the greatest flexibility in the choice of how to represent a process. Due to the large number of modeling elements, even complex real-world phenomena can be represented in a compact way. This, however, leads to higher demands on language comprehension for the model users. Activity diagrams or S-BPM, which are based on a compact set of modeling elements, follow a different approach here. Their approach leads to larger models in complex contexts, which in turn places higher demands on the model users with regard to their understanding of the model. S-BPM reduces the immediately visible complexity of models by distributing a process over different subjects. While this leads to partial models which can be more easily grasped, it in turn places higher demands on model users when it comes to grasping the overall context within the process. When selecting a modeling language that is suitable for a given task and target group, not only the object of the model (i.e., the business process under consideration) and the target of modeling should be considered. The known or assumed competencies of the modelers and model users also need to be taken into consideration [3, 10, 11]. A fundamental distinction can be made between languages that focus on the flow of activity (such as flowcharts and EPCs) and those that focus on the actors in a process and their communication (such as S-BPM). BPMN and activity diagrams are basically suitable for both types of representation, whereby BPMN offers more expressive means for representing communication processes. The final selection of a modeling language after determining the fundamentally pursued representation approach (activity flow vs. communication flow) is ultimately dependent on the preferences of the modelers or model users. #### References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ### Contemporary Challenges in Business Process Modeling / Management 4 In the previous chapters we have considered the means for describing the sequence of activities executed in a process. In the course of digitalization, processes in an organization and, in particular across organizations, are becoming more complex. In order to describe them nonetheless in a transparent way, it is necessary to organize them as networks of processes, or as a hierarchy of subprocesses. When processes are increasingly supported by IT, two major aspects of IT support seem to be crucial. One aspect concerns the support of the activities specific for a process, e.g., creating a purchase order. The corresponding software applications are specific for each process. Normally, activities in a process are implemented by functions of such applications or, the other way around, activities of a process represent functions of application systems. The other aspect concerns the control of the allowed sequences of activities, which is managed by software solutions in the form of so-called workflow systems. This software for controlling the execution sequence of a process can be derived directly from the process model if the syntax and semantics of the modeling language are precisely defined. This is a very important aspect for the digitalization of processes. In workflow systems functions of software applications which implement certain actions of a process are incorporated into the associated functions of the model. A more detailed discussion on the structure of business process implementations can found in Chapter 7. In the following sections we describe how the different Business Process Modeling languages support the structuring of complex process systems and to what extent the derivation of digital workflow is supported. #### 4.1 Handling of Complex Processes In an organization, business processes are connected with each other either directly or indirectly. The sales process is connected with the order handling process, the order handling process initiates the delivery and invoice processes, and so on. A language for specifying process behaviors should also offer possibilities for structuring complex interconnected process systems. It should allow the representation of the environment of a considered process, which means it should be possible to illustrate the relationships to other processes. If we want to define the activity sequences of the order handling process, we must be able to include the relationships to the sales and shipment processes. The interfaces to these neighboring processes should include the methods for exchanging the data and switching the control flow between the processes. In this chapter we describe the various possibilities to structure complex processes in the modeling languages defined in the previous chapter. #### 4.1.1 Structuring Complex Processes in Flowcharts The only way to structure complex processes in flowcharts is by means of predefined processes. Predefined processes are named processes which are defined elsewhere. The following figure shows an example for using predefined processes. In Figure 4.1 the process on the left side uses the predefined process "do shipment". This process is shown on the right side of the figure. In flowcharts the control flow switches from the process which initiates a predefined process to the predefined process itself. There is no standardized way to describe a process architecture providing an overview of which predefined processes exist and in which other processes they are used. In flowcharts it is assumed that all processes share all data. Hence, it is not necessary to specify the data required by a predefined process explicitly. Due to this tight coupling of processes to predefined processes, flowcharts are not the ideal Figure 4.2: Structuring complex processes using EPCs choice for loosely coupled organizations. In particular, in the case of cross-company processes, the ability to define where the process data is stored is essential. #### 4.1.2 Structuring Complex Processes in Event-Driven Process Chains EPCs use subprocesses for structuring complex processes. The incorporation of subprocesses in EPCs is similar to predefined processes in flowcharts. Figure 4.2 shows the use of the interface symbol for integrating subprocesses. The subprocess approach is not sufficiently expressive to describe complex process systems. Especially if it is necessary to describe the relations between various processes, the problems are the same as with flowcharts. Therefore, Value Chain Diagrams (VCD) have been introduced as an additional model type. VCDs allow describing which processes belong to a complex process system and how they are related to each other. Figure 4.3 shows that the process "order handling" starts the process "shipment". In addition to this successor relationship, there is also the possibility to describe hierarchical relations. Figure 4.4 shows that the process system "order management" consists of the process sequence "order handling" and "shipment", and the process "invoice handling". #### 4.1.3 Structuring Complex Processes as UML Activity Diagrams UML activity diagrams do not contain a specific notational support for structuring complex processes. It is not allowed to use an activity diagram within another activity diagram (recursive use). The only possibility for structuring complex processes is to connect different activity diagrams by means of messages exchanged between them. However, there is no diagram type to specify a communication relationship between activity diagrams. #### 4.1.4 Structuring Complex Processes in BPMN In BPMN the highest modeling level is represented by collaboration diagrams. If there are many involved parties in a process, and thus many pools with several swim lanes, then the 'big picture' of a process system may become quite difficult to understand. In order to overcome this problem, conversation diagrams have been introduced. Conversation diagrams represent an overview of a network of partners and how they communicate with each other. Figure 4.5 shows an example of a conversation diagram. In this diagram a process system with the pools "order handling" and "shipment" is depicted. These pools communicate with each other via the message "start shipment". Conversation diagrams represent a top-level view of a BPMN collaboration diagram. Since nesting conversation diagrams is not allowed, the rectangle must be a pool and cannot consist of a conversation diagram on a lower level. Analogously to the predefined process concept in flowcharts or the subprocess concept in EPCs, there is also a subprocess concept in BPMN. This concept has already been described in the previous chapter. #### 4.1.5 Structuring of Complex Processes in S-BPM In Chapter 3 we introduced the modeling language S-BPM for representing subjectoriented business processes. S-BPM is based on the Parallel Activity Specification Scheme (PASS) [1]. PASS offers useful features for structuring complex process systems in a hierarchical way. Arbitrary levels of descriptions are allowed. We exemplify how levels of communication networks can be used to describe complex process systems with an example of a process for service provision in the case of a car accident service. This service (of an actual company) consists of several connected processes. It encompasses the main process for handling the car accident as well as supporting processes, e.g., for organizing towing and repair shop services, for handling insurance claims, for receiving and paying invoices, etc. These processes are executed by various organizations, such as help desk service companies, towing service companies, car repair workshops, banks, etc. In most business process projects overall processes are not described completely in detail, but rather only in parts of a process. Which part of the process is represented in detail at a specific point in time depends on the perspective taken by the participants or departments involved in that part of the process. For instance, from the perspective of the help desk, only the help desk process needs to be considered in detail. However, we indeed have to take into account the environment in which a considered process is embedded. We must know which relations exist to other processes. It is necessary to know which inputs are required by neighboring processes and which Figure 4.6: Highest process structure level of car accident service results they deliver. A help desk process which organizes the towing services has to know how the towing service is requested and which further interactions are required. For example, it needs to be agreed whether the towing service, or rather the help desk, informs the client with respect to the arrival time of the tow truck. #### Process Architecture In the following we specify the required process architecture. In the diagrams, rectangles represent processes. Each process has a name. Processes consist of other processes and/or subjects. The lines between the rectangles represent the communication channels between processes. Each communication channel has a name and can contain other communication channels and/or messages. Figure 4.6 shows the highest process level of the car accident service. In the "car usage" process the event "car accident" happens. In order to organize support an interaction is initiated with the process "car accident service". These processes exchange messages which are elements of the communication channel "car accident handling". Figure 4.7 shows the next process structure level of the process "car accident service". In this level the process "car accident service" is decomposed into 10 processes. Eight of these processes, namely, the processes "incident management", "mobility service", "towing service", "insurance service", "car repair workshop", "banking", "payment handling" and "payment services" have a communication channel to the process "car usage". This means the communication channel "car accident handling" is separated into eight communication channels. Each of them covers the communication with the related process, e.g., the communication channel "accident notification" is the communication channel between the processes "car usage" and "incident management". A process can also encompass other processes. This means that different levels of processes can be built. Figure 4.8 shows the next deeper level of our process hierarchy. The process "car repair workshop" is broken down into 6 processes. According to this separation the communication sets are also split, e.g., the communication set "handling repair service" is split into three parts, one part is handled by the process "service scheduling" the other by the process "car dropping" and the third by the process "customer satisfaction". As already mentioned, processes cannot communicate directly with each other, but rather the active entities of a process, the subjects, communicate with each other. This means messages which are sent from one process to another process are received by a subject in that process. Messages belonging to a channel are assigned to respective sending or receiving subjects at the lowest level of a process architecture. This lowest level of a process description is the Subject Interaction Diagram Figure 4.9: Interface (border) subjects of the "incident management" process (SID) which shows the involved subjects of a process and the messages they exchange. In the following we consider the process "incident management". This process does not contain other processes as in the case of the process "car repair workshop". The process "incident management" contains a SID. Some of the subjects of a process communicate with subjects of other processes. These subjects are called border subjects because they are at the border of a process to other processes. Figure 4.9 shows the process "incident management" with its border subjects. There is a border subject "help agent" which communicates with the processes "towing service", "mobility service", and "car repair workshop", or more precisely, it communicates with a respective subject in each one of these processes. Another border subject of the process "incident management" which is called "help desk" communicates with a subject in the process "car usage". The border subjects of the process "incident management" must have corresponding border subjects in those processes with which it communicates. The border subject "help desk" communicates with the associated border subject of the process "car usage" and the border subject "help agent" communicates with the respective border subjects of the processes "car repair workshop", "towing service" and "mobility service". The process "incident management" with all of its border subjects is shown in Figure 4.10. The border subjects of the processes "mobility Service", "towing Service", and "car repair workshop" have the same name "service agent" but are different subjects Figure 4.10: "Incident management" process with all border subjects because they belong to different processes. Since the process "car repair workshop" consists of several layers, the corresponding border subject can belong to a process which is part of the process "car repair workshop" on a lower level. From the perspective of the subjects inside the process "incident management", the border subjects of the processes "mobility service", towing service", and "car repair workshop" are interfaces to these processes, therefore they are called interface subjects in the (SID) Subject Interaction Diagram of a process. Figure 4.11 shows the SID of the process "incident management". #### Behavioral Interface Processes to which a considered process has communication relationships are called process neighbors, or just neighbors. Now we want to consider the details of the communication relationships between two neighbors. The interface between two processes is defined by the related border subjects and the allowed sequences in which the messages in a communication channel are exchanged between them [2]. As already described above, each message is defined by a name, and the data which are transported is the so-called payload. A border subject observes the behavior of the border subject of the neighbor process and vice versa. Figure 4.12 shows the border subject "help desk" of the process "incident management" which communicates with the border subject "caller" of the process "car usage". Since we are considering the process "incident Figure 4.11: Subject Interaction Diagram of the "incident management" process management", the border subject "caller" of the process "car usage" becomes an interface subject in the SID of the process "incident management". Figure 4.13 shows the SID of the subject "help desk". Rather than specifying all of the channels, only the messages required for a towing service request are shown. A message "request towing service" stems from the interface subject "caller". This message is accepted by the subject "help desk". The subject "help desk" checks the customer data received with this message by sending a corresponding message "get customer data" to the subject "customer data management". This subject sends the complete customer data back to the subject "help desk" via the message "customer data". The subject "help desk" then checks the customer data. If the data are invalid a message "invalid customer data" is sent to the subject "caller" and the process is finished. If the customer data are valid, the subject "help desk" creates a trouble ticket with this data which is sent to the subject "ticket management" via the message "store ticket". After that, the message "towing service requested" is sent to the "help agent" that organizes the towing service. The part of the communication structure of the subject "help agent" for organizing the towing service is not shown in Figure 4.13. We only see that the subject "help agent" sends the message "towing service ordered" to the subject "help desk". This message contains all the data about the Figure 4.12: Subject interactions of the subject "help desk" service, e.g., name of the towing company and arrival time. The subject "help desk" forwards this data to the interface subject "caller". The behavior described in Figure 4.13 contains the communication of the subject "help desk" with all neighbor subjects, including the communication with the interface subject "caller". From the perspective of this subject, the communication of the subject "help desk" with its other neighbor subjects is not relevant. For the subject "caller" only the communication sequence between itself and the subject "help desk" is relevant. These allowed communication sequences are called the behavioral interface. The behavioral interface between two subjects can be derived from the complete behavior of one of these subjects by deleting the interactions with all the other Figure 4.13: Part of the behavior diagram of the subject "help desk" Figure 4.14. Sample behavioral interface subjects [3]. Figure 4.14 shows how the communication sequence relevant for the communication between the subjects "help desk" and "caller" is derived from the complete behavior of the subject "help desk". #### 4.2 Readiness for Digitalization In this section we investigate to what extent process models are specified in a precise syntactic and semantic notation in the different modeling languages to enable generation of digital workflow support automatically. Readiness for digitalization denotes the capability to support semantically rich process specifications in an accurate way, allowing for a corresponding automated execution of process models. We shed light from this perspective on some notational schemes before demonstrating for subject-oriented representations how behavior models could be handled from a well-defined semantic representation and processing perspective. #### 4.2.1 Readiness for Digitalization of Flowcharts The standard for flowcharts does not contain precise syntax and semantic descriptions. Due to their long history and widespread use, the importance of flowcharts is more or less common knowledge. Flowcharts have no underlying data model to share data between various diagrammatic editing tools for flowcharts, databases or other programs, such as project management systems or spreadsheets. There exist many applications and visual programming languages that use flowcharts to represent and execute programs [4]. However, these tools focus on programming, and not on business processes. #### 4.2.2 Readiness for Digitalization of Event-Driven Process Chains There are many tools for creating EPC-based process models. Up to now there is no standardized way for storing these models. This means models created, for example, with tool A cannot be used in tool B. The tool most commonly used for creating EPC-based process models is ARIS. ARIS uses its own data model. Hence, process descriptions produced with ARIS cannot be processed with another tool. There have been some research activities to define a general data model [5] and also some research projects with respect to the direct execution of EPC models [6]. In practice, however, only the ARIS tool suite is used for creating EPC specifications and the authors are not aware of any projects in which EPCs are executed directly. #### 4.2.3 Readiness for Digitalization of UML Activity Diagrams There are many UML-based tools - a list of them can be found in Wikipedia [7] which support the creation of activity diagrams. The OMG XMI standard specifies a structure that uses XML for interchanging models between various tools. Although in principle, this standard allows the handling of process models with different tools, in practice the transformation of model descriptions between various tools can be cumbersome: Many tool manufacturers create variations of the standard in different ways, e.g., they do not support some notational elements or they add other notational elements. Most UML tools support the generation of code for several target languages. A special code generation for activity diagrams has been proposed in existing research [8]. This code generation targets real time systems and it still needs to be investigated as to what extent it can be used for business processes. The authors could currently not find any UML-based tool suite which is recommended for implementing business processes. #### 4.2.4 Readiness for Digitalization of BPMN The BPMN standard contains an XML data model which allows the processing of a process model by different tools. Since in practice each tool vendor places its focus on different aspects of BPMN and interprets the standard in a special way, the transfer of a process model from one tool to another can be tedious [9]. In the standard's documentation the execution semantics is described in natural language. At the Software Competence Center in Hagenberg, Austria a formal semantic for the process diagrams of BPMN has been defined [10]. For the formal description of the BPMN semantic, the Abstract State Machine formalism has been used [11, 12]. In this project, several ambiguities, inconsistencies and gaps in BPMN have been identified [10]. There are many tools which also support the execution of BPMN processes. However, the majority of these tools do not fully implement the BPMN standard. Most tools support a limited set of execution elements and do not interpret them in fully compatible ways, leading to partially differing execution outcomes for identical process models [13]. #### 4.2.5 Readiness for Digitalization in Subject-Oriented Process Specifications For the S-BPM language, which is based on PASS [1], a standard ontology in OWL [14] has been defined [15]. This ontology allows a PASS specification to be processed with different tools, if these tools follow the standard ontology. Each subject has a base behavior and may have additional subject behaviors for macros and guards. All these behaviors are subclasses of the class SubjectBehavior. The details of these behaviors are defined as state transition diagrams (PASS behavior diagrams). These behavior diagrams are represented in the ontology with the class BehaviorDescribingComponent (see Figure 4.15). The behavior diagrams have the relation "BelongsTo" to the class SubjectBehavior. The other classes are needed for embedding subjects into the Subject Interaction Diagram (SID) of a S-BPM model (see section 3.6). The following figure shows the details of the class BehaviorDescribingComponent. This class has the subclasses State, Transition and TransitionCondition. The subclasses of the state represent the various types of states (class relations 025, 014 und 024 in the Figure 4.16: Main ASM functions of the interpreter figure above). The standard states "DoState", "SendState" and "ReceiveState" are subclasses of the class "StandardPASSState" (subclass relations 114, 115 und 116). The subclass relations 104 and 020 allow a start state (class "InitialStatOfBehavior") and none or several end states (see subclass relation 020). The fact that there must be at least one start state and none or several end states is defined by so-called axioms which are not shown in the figure above. States can be start and/or end points of transitions (see properties 228 and 230). This means a state may have outgoing and/or incoming transitions (see properties 224 and 217). Each transition is controlled by a transition condition which must be true before a behavior follows a transition from the source state to the target state. The ontology defines only the structure of a process description. The dynamic aspect is not covered yet. The execution semantic of S-BPM models is described with Abstract State Machines (ASM) [16]. The ASM defines the algorithm of an interpreter that will "crawl" through a Subject Behavior Diagram (SBD) of a process model defined in the, not explicitly named, Subject-oriented Process Modeling language PASS as defined in the previous section 3. Figure 4.16 shows the ASM-code for the interpretation of the behavior specification [17]. The behavior of a single subject subj is specified by the Behaviorsubj (D) rule, which takes the Subject Behavior Diagram D as a parameter. From there on the Behavior(subj, node) rule defines how a single node behaves. As long as the service of that node is not completed the Perform rule will be called, which is refined for all given services X. Once the node is completed the outgoing transition will be determined by selectEdge, the Proceed rule updates the current SID\_state and initializes the new node with the Start rule, which also is refined for all services X. The following table shows the relationship between the ASM interpreter specification and the classes and properties of the ontology. The meaning of the colors is as following: The interpreter ASM Spec has the following main function or rules that are being executed while interpreted. The following table shows the relationships between the ASM main functions and the classes of the OWL model elements. There are some prototypes of modeling tools which follow the standard ontology of PASS and a prototype of a workflow engine which interprets the standard ontology [18]. #### References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # From Modeling To Digitalization 5 ### 5.1 Overall Context In the previous chapters, we have shown that what happens in organizations (companies, administrations, etc.) is based on models from various disciplines. Business models, which represent enterprise architectures with models for products and services, organizational structure, processes, data and IT infrastructure, describe in which area a company does business, how it does this, which exchange relationships it has with partners, which technical infrastructure it is supported by, etc. In chapter 3, we have focused on approaches and notations for the specification of business processes and thus the design and representation of operational processes. In the course of digitalization, these processes must be augmented, as far as possible and economically sensible, with information and communication technology. Both the appropriate incremental improvements of existing processes, as well as fundamental process innovations, are based on creative design accomplishments, which should lead from process models to executable systems. In this chapter we will therefore first deal with the concept and typical activities of Business Process Management. With the approach of Design Thinking, we then illuminate a methodical approach to creatively produce something new and solve complex problems. Subsequently, we put the two concepts into relationship with one another. #### 5.2 Activity Bundles in Business Process Management #### 5.2.1 Overview In chapter 1 we have already mentioned that the design of business processes up to their execution as instances in the processing of concrete business transactions ("operational business") itself represents a process. This is often understood as a Business Process Management cycle with phases such as strategy, design, implementation and controlling [1]. In practice, however, the sub-activities are often not clearly distinguishable from each other. We therefore see them less as a circle with a sequential sequence, but rather as networked and interwoven, as the honeycomb structure in Figure 5.1 suggests. The diagram also shows that we differentiate the tasks somewhat more and identify them as bundles of activities: analysis and modeling, validation, optimization, embedding into organization, IT implementation and execution and monitoring. Although the usual representation as a cycle suggests that in process management projects all activity bundles are run through as a sequence, their selection and sequence depend on the concrete situation, e.g., the maturity level of a process. The sections 5.2.2 to 5.2.7 explain the activities using an example process. Here, the steps are first run through completely and also in the specified sequence. Such a scenario is realistic, for example, when a process is designed for the first time or completely reorganized. In section 5.2.8, we then discuss several scenarios for improvements that can be derived from the experience gained during operation of the originally designed process environment. They illustrate the situationally different paths through the activity bundles in the further development of the process. #### 5.2.2 Analysis and Modeling The analysis serves to gather information about why a process exists or should be implemented, which goals an organization pursues with it within the framework of its strategy, and how it is currently working. The objectives are the documentation and the acquisition of indications for improvements. The modeling uses, among other things, the results of the analysis and deals with the design of future working methods, i.e., process changes and innovations. If further information is required, the participants switch back to the analysis mode to collect it and then act again in a creative manner. Therefore, analysis and modeling cannot be clearly distinguished from each other. Validation and optimization also usually take place here, when the participants develop the model iteratively to the best of their knowledge and belief, taking into account the weak points identified in the analysis and trying out and discussing possible solutions. In addition to considering determining factors such as strategic significance, objectives and risks, analysis and modeling are essentially concerned with analyzing or specifying (see also section 1.3) For the development of process models based on these findings, the modeling languages presented in chapter 3 with the corresponding graphical notations are used. During analysis and modeling, usually also the ground is laid for operational process controlling in the operating phase. In addition to the process attributes already mentioned, performance parameters (indicators), in particular Process Performance Indicators (PPIs), are defined, systematized in a measurement system and provided with target values [2, p. 265].Typical examples of PPIs are lead time, output per time unit, error rate, customer satisfaction, etc. The PPIs and the target values planned for them form the basis for business process monitoring, that is, operational process control during execution (see sections 5.2.7 and 7.3.3). #### Analysis and Modeling in a Case Study As a case study, we use the strongly simplified process of credit application processing in a bank. There, applications are received from interested parties for the granting of a real estate loan. Before preparing an offer, clerks check the creditworthiness of the respective customer and the value of the property to be loaned on. If the result of both checks is positive, the clerk prepares an offer with data such as loan amount, interest rate, repayment rate and term. If the loan amount is less than €200,000, he signs the offer and sends it to the customer. Otherwise, he must first obtain the approval of his department head and, in the case of more than €500,000, that of the executive board. If the creditworthiness check or the object check reveal any risks, a clerk contacts the interested party to agree on further procedures, such as reducing the loan amount. As part of an analysis of the process, this information was collected and structured (see Table 5.1). Together with additional information, this collected information serves as a basis for modeling the process. Table 5.1: Characteristics of the example process and information obtained for it In the case at hand we use the modeling language S-BPM described in Chapter 3 for the representation of the process, since it exists in a strongly interaction-oriented description. Generally speaking, the representation would also be possible in any other modeling language which allows the depiction of responsibilities (e.g., eEPC or BPMN) The following figures show an excerpt of the associated process model using S-BPM. Figure 5.2 shows the subjects (agents) involved and the messages they exchange during process flow. The communication structure (Subject Interaction Diagram, SID) does not yet contain a sequence in which the respective messages are exchanged. The sequences are described in the behavior of the subjects. Figures 5.3 and 5.4 show the behavior of the subject "interested party" and a behavior excerpt of the "real estate loan processing" subject. The behavior of the subjects "Head of real estate loan department" and "Executive board" is described analogously. They would be involved in the process if the desired loan amount exceeded 200,000 or 500,000 euros. These cases are not modeled in Figure 5.4 – they would be represented by additional branches after the state "approval required". Figure 5.2: Communication structure of the actors in the credit request process Figure 5.3: Behavior of the subject "interested party" #### 5.2.3 Validation In the BPM context, validation means checking whether the designed process generates the output expected by the (external) customer and process owner, for Figure 5.4: Behavior of the subject "real estate loan processing" example in the form of a service or product. This question with regards to effectiveness already refers to the results of partial steps, i.e., process participants of the own organization as customers. For example, it is necessary to evaluate whether an upstream process step provides all the information that a processor requires for a decision (for instance, approval) in his or her subtask. We have already mentioned that parts of a model are repeatedly subjected to validation, even during its step-bystep development. In addition, the object of validation is the completely finished process model, the effectiveness of which should be ensured before it is implemented in terms of Information Technology (cf., section 5.2.6). Otherwise, errors are discovered too late and lead to correspondingly high costs for their elimination. #### Validation in the Case Study The process model for the example process was validated during its development as well as at the end. In doing so, it was initially discovered that the original loan application did not include a field for the applicant's employment status and thus lacked an important risk assessment factor. This led to the extension of the business object and to a positive validation result in the corresponding iteration. #### 5.2.4 Optimization While validation aims at ensuring the effectiveness of business processes, optimization is about efficiency. Process efficiency can be expressed by process attributes for resource consumption such as duration and costs. Optimization means finding the optimal design of a process with regard to such process parameters. Essential starting points are improvements in operational and structural organizational design as well as IT support. Strictly speaking, optimization is not an independent activity bundle, but makes use of modeling, organizational implementation, and IT implementation (see sections 5.2.2, 5.2.5, and 5.2.6). Simulation is a well-known method for comparing alternatives in process execution or resource allocation. It can be used to obtain quantitative information on the development of process parameters for a large number of process instances (orders, production pieces, etc.). The simulation enables the evaluation of a process model with a certain combination of parameters. These can be deterministic or stochastic quantities described by probability distributions. Through the use of parameter changes and alternative process designs, different design options can be analyzed with respect to their behavior. This allows insights to be gained into bottlenecks or inefficiencies and the sensitivity of parameters. The extension of a process model and the gathering of necessary information for conducting simulations can cause considerable effort. Attributes relevant for optimization are often interdependent and contradictory, which makes optimization difficult and requires balancing efforts. A process alternative can, for instance, have a shorter lead time relative to another, but cause higher costs. The decision for an alternative therefore also depends on the priority of the process objectives. #### Optimization in the Case Study The model could already be optimized during its design. The steps for checking customer creditworthiness and value of the property, which were initially sequentially planned, were redesigned to be executed in parallel. #### 5.2.5 Embedding into an Organizational Context For productive operation, validated processes need to be embedded into the existing, redesigned or newly created organizational environment. This is also referred to as the organizational implementation of a process. It quite often requires an adaptation of the surrounding operational and organizational structure. A single process is usually part of an entire value creation environment (value chain, value creation network) into which it must be seamlessly assimilated. Therefore, with regard to the operational integration into the process map, particularly the interfaces with other processes must be considered. This can lead to changes to be carried out at the interfaces of an upstream or downstream process. Such circumstances usually are already taken into account in the upstream activity bundles. Therefore, the implementation should be limited to the chronological coordination of the go-live procedure. This means that processes that are connected via interfaces must go live again at the same time if a change has occurred at an interface that has also made modifications necessary in the partner process. The organizational embedding comprises the assignment of concrete actors, i.e., people as job or role holders, to the actors abstractly specified in the model. One of the challenges is the consideration of the organizational context when using workflow engines. These must be able to dissolve, for instance, dynamic surrogate regulations at runtime, as well as the fact that persons can assume different roles in the same process. For example, a superior in a vacation request process can be the approver of vacation requests for his own employees, but can also be the applicant for his own vacation, which in turn must be approved by his own superior. Therefore, the software must have organizational knowledge, facilitating the correct routing of a process instance through the processing units and steps. Further qualitative and quantitative aspects have to be taken into account during organizational embedding. Care must be taken to ensure that the employees have the necessary qualifications (skills) to carry out the modeled behavior or can gain them through training. Adequate qualification is not only a prerequisite for successful work in the currently valid version of the process; it can also foster improvement proposals by process participants. The number of people assigned to the abstract actors in the model influences the capacity for processing process instances and thus affects parameters such as lead time. #### Embedding into an Organizational Context in the Case Study Column 2 in table 5.2 shows the number of employees who in general are qualified to be assigned to the identified and modeled roles. The third column contains the actual capacity used (short-term absences, e.g., due to illness, are not taken into account). The heads of real estate and consumer credit departments stand in for each other, both in disciplinary and domain-specific matters. This also applies to the members of the executive board. Table 5.2: Potential and concrete assignment of roles #### 5.2.6 IT Implementation Most processes cannot be carried out economically without IT support. Especially when a high degree of automation is strived for, the quality of the mapping of the process in IT becomes very important. But also, and in particular, for steps where human actors are involved (e.g., entering data, making decisions), the user-centered/ friendly design of the IT systems is of high importance. IT-related implementation of a process means mapping it as an IT-supported workflow with integration of a suitable user interface, the execution logic and the IT systems involved. For this purpose, it is necessary first of all to transfer the more or less formal model description (see chapter 3) into a language interpretable by a workflow engine, i.e., into an executable program. This enables the engine to control the execution of a process instance at runtime according to the model. For the completion of individual subtasks during processing, a whole series of software applications and services usually have to be integrated into the process. Typical examples are ERP transactions and document and content management systems. A relatively new approach to automation of standardized, repetitive work procedures is Robotic Process Automation (RPA). This term stands for tools that "perform [if, then, else] statements on structured data, typically using a combination of user interface interactions, or by connecting to APIs to drive client servers, mainframes or HTML code" [13]. Thus, software robots for example imitate the behavior of humans when using the graphical user interface of information systems [14]. This allows, for instance, the quick linking of heterogeneous IT systems and automated data transfer between, or data input in, various existing applications without the need for these to be modified. Artificial Intelligence and machine learning functionality promises to facilitate learning and automated adaptation of RPA tools to changes in the underlying IT systems [15]. Extensive testing of the implemented overall solutions must ensure the quality of the process support provided by IT. #### IT Implementation in the Case Study Table 5.3 shows the essential elements of the IT environment, which was designed to support the process and its sub-steps, together with their most important processrelevant functions. #### 5.2.7 Operation and Monitoring Implemented processes go live after their approval by the responsible authorities. This means that those involved in a process execute it in the form of instances in the organizational and IT environment set up for day-to-day business. In order to obtain information for the deliberate management of processes, it is necessary to observe their behavior during everyday operations. This monitoring records measurement data and calculates actual values for the Key Performance Indicators defined during analysis and modeling. An immediate comparison with Table 5.3: Realized IT environment of the process defined target values leads to escalations along the management hierarchy and, if necessary, to short-term measures in the event of deviations. Medium- and longerterm evaluations reveal structural opportunities for improvement. The analysis of the process behavior and possible deviations allow conclusions to be drawn about causes and triggers feedback into other activity bundles. #### Operation and Monitoring in the Case Study Since the release of the process, the bank has been processing credit applications from interested parties in the described form and environment. Monitoring for the past quarter revealed the following average figures: Since competitors advertise with very short processing times, the bank assumes that the 4 days until applicants receive an offer, all other conditions being equal, is one of the reasons why customers do not sign a contract. This duration also deviates significantly from the previously formulated target of 3 days. #### 5.2.8 Optimization Scenarios in the Case Study The following scenarios show how further analyses of the monitoring results can be used to investigate the causes of the long lead time and to branch out into suitable activity bundles for improvement measures (optimization). In each case, the target point of the branch-out determines the further path through the activity bundles, i.e., which subsequent activities are necessary before the redesigned process can be put into day-to-day operation. In the interests of simplification, we limit our consideration to one measure per scenario. In reality, several optimization possibilities will usually be pursued in parallel. #### Optimization Scenario 1 The frequency distribution for credit application occurrence has shown that on Mondays 25, Tuesdays 15, Wednesdays 6, Thursdays 2 and Fridays also 2 applications are submitted. This could be due to the fact that interested parties view real estate, take purchasing decisions and think about financing mainly on weekends. The analysis of the dormancy period until the real estate loan department processes a request reveals a bottleneck at the beginning of the week, due to the high number of parallel applications. With the currently available capacity of 5 full-time clerks, the average dormancy period is 2 days. In order to reduce the latter and thus also the overall lead time, additional processing capacity, such as available part-time staff, could be employed on Mondays and Tuesdays. In this case, only organizational implementation in terms of staffing is concerned; the process does not change and no further activities are necessary. #### Optimization Scenario 2 A more detailed analysis has shown that the high average lead time is caused by the applications with amounts between €200,000 and €500,000, because the dormancy period until the department management approves the application is very high relative to the other proportions of the total duration. This is due to the fact that the availability of department heads and surrogates for approvals is limited, e.g., due to frequent business trips. The bank's internal process analyst proposes a change of the business rule for approval. In the future, clerks should be allowed to sign and send offers for amounts up to €500,000 themselves. This reorganization affects several bundles of activities. First of all, it requires a change in the model, as the approval loop via the department management is no longer required. The model change requires subsequent validation to ensure that the changed process (still) leads to the desired result. As part of the IT implementation, the modification of the model must also be transferred to the workflow software and tested. The omission of the approval changes the task structure of the department management. For the clerks the tasks remain the same, but competence and responsibility increase. These changes have to be taken into account in the organizational implementation, for example through updated task descriptions and possibly through the qualification of the clerks through training, e.g., for a more comprehensive risk assessment. Compared to case 1, this scenario intervenes massively in the way the process is conducted and therefore requires much more extensive activities. #### Optimization Scenario 3 The bank obtains creditworthiness data on the applicants from the associated credit bureau. For this purpose, the real estate loan processing clerks transfer the necessary customer data from the loan application to the credit bureau's web form. They then enter the results of this query into the banking system for further processing in the bank's own scoring system. The clerks give an account of the time-consuming copying of the data using copy & paste, the errors that occur in doing so, and the resulting reworking. In order to push forward digitalization, the bank decides to use the credit bureau's web service instead of their webform-based internet information service. The web service can be integrated into the workflow in such a way that the process engine triggers it when the clerk pushes a button and transfers the customer data as parameters. The service automatically returns the result to the process engine, which then transfers it to the banking system. In this case, the only activity bundle to be dealt with is the IT implementation, including corresponding software adaptations and subsequent tests, before going live with the modified solution. The work procedures of the participants change only slightly; qualification measures are not necessary. The elimination of manual data transfer relieves them of mindless, time-consuming, and thus cost-intensive and error-prone routine tasks. The more intensive use of IT saves processing and lead time as well as costs, while increasing customer satisfaction. #### Optimization Scenario 4 In section 5.2.4, we described that the customer creditworthiness check and the property value check were deliberately parallelized during modeling in order to save lead time. The analysis showed that the bank rejects five applications per week for creditworthiness reasons. In these cases, however, clerks had already spent effort doing the parallel value check of the real estate in question. Saving on this could initially speak in favor of first checking the creditworthiness and only carrying out the value check if the result is positive. A model change with validation and adaptation of the workflow application would be necessary. However, the sequential order would again increase the lead time and lead to a conflict of objectives. Therefore, it is important to further consider whether the effort for value checking could be reduced through automation so that unnecessary value checks no longer play a role. It is conceivable, for example, that the banking system could be enhanced with valuation functions. It could then calculate a value index after automatic transfer of parameters from the loan application (type, size, year of construction, address, etc.) and being enriched with comparative information (values from the bank's own experience and reference value tables) and geo information (infrastructure with schools, shopping facilities, transportation connections, etc.). This index would accelerate the final value estimation carried out by the clerk. With this option, the parallel execution of the steps could remain. Instead of a model change, the additional functionality in the IT implementation would have to be realized and tested, and the clerks would have to be trained in how to use the software extension. #### 5.3 Introduction to Design Thinking Design Thinking (DT) is a methodical approach to be creative and constructive in order to develop something new and to solve complex problems. It is characterized by innovative approaches to solution-oriented design. Problems can be better solved by focusing on the needs of the (potential) users during continuous iterations and "making comprehensible and graspable" through prototypes. This basic understanding of Design Thinking is shared by practitioners and scientists alike (for an overview of examples of definitions see [3]). The spectrum covered by the approach, on the other hand, is not seen uniformly. There are, for example, interpretations which see it as a mind-set, as a process or as a toolbox [4]. An empirical investigation proves the perception in the continuum between the two poles of toolbox and mind-set (cf., Figure 5.5) [5]. On the one hand, this is due to the different roots, but on the other hand it is also a consequence of the inherent, constant, experience-led further development and adaptation of the concept in different contexts. Larry Leifer, one of the protagonists of the approach at the d.school in Stanford states, that its permanent enhancements are an important part of Design Thinking, and that it would make itself unrecognizable if it were to publish a fixed manifesto one day [6]. The approach traces back to David Kelley from the design agency IDEO and professors Larry Leifer and Terry Winograd from Stanford University. In particular, the latter two recognized, when training engineering students, that the development of marketable products should focus much more on user-related aspects and less on purely technical aspects. This insight led to the development of the DT concept from the 1980s onwards and is still manifested today in the Stanford course on Mechanical Engineering 310 - Design Innovation (me310.stanford.edu). Hasso Plattner made a significant contribution to the further dissemination of this knowledge in research, academic teaching and business practice with his support of the institutions named after him at the d.school Institute of Design at Stanford University and the School of Design Thinking at Potsdam University (HPI D-School). Due to its origin, DT was originally primarily concerned with the development of physical products. However, it is now used in a wide variety of areas, such as the development of services or entire business models, and is increasingly gaining in importance in organizational design and Business Process Management. #### 5.3.1 Core Elements Core elements are a mixture of mindset, procedures and concrete facilities such as work areas. This is reflected in the rough division into the three "Ps", namely into the areas people, process and place. Design Thinking begins with the creation of deep empathy for those affected by a (problem) situation. It identifies the optimal solution in the overlapping area of human desires (human-psychological aspect), feasibility (technological aspect) and Figure 5.5: Understanding Design Thinking profitability (business aspect) [7]. The innovation to be developed should be something, To achieve this, an interdisciplinary team (people) on variable, creativitypromoting premises (place) goes through a procedure (process) with many iterations, whereas a variety of methods can be used. #### People Focusing on the human being takes place in two respects: On the one hand, representatives of the target group of the innovation, i.e., customers or users, with their needs are in the center of interest. Developing empathy for them, putting oneself in their position in the context under consideration, and thus gaining a deep understanding of the problem is the cornerstone for successful innovation and encompasses a large part of the process described in the "Process" subsection below. On the other hand, Design Thinking strongly focuses on the people involved in the project as individuals and as a team. It aims for increasing the quality of results by using the diversity in interdisciplinary teams. Team members should be "T-Shaped", i.e., both experts and generalists. As experts, they are deeply rooted in their specialized field and bring in the appropriate expertise (vertical line of the T). This can also involve the professional representation of a stakeholder group (e.g., sales, production, IT). Looking at a problem from different perspectives and synthesizing know-how and experience from different domains often helps to develop new approaches to solutions. The quality as generalists is a prerequisite for changing from one's own perspective to that of other participants and for being open to cooperation at the (functional) interfaces (horizontal line of the T, "We" thinking) [8, p. 122]. Lewrick et al. plead for interdisciplinary versus multidisciplinary teams because the former truly and collectively generate ideas and stand behind them, whereas the members of multidisciplinary teams often overestimate their own perspective when finding solutions. The latter rather leads to compromise solutions, which are not fully supported by all [8]. Ideally, an interdisciplinary team is made up heterogeneously of representatives not only from as many areas of expertise as possible, but also from different age groups, nationalities, sexes etc. The success of a Design Thinking project is largely determined by the individual characteristics of the members of the associated team and the resulting collaborative working culture and way of thinking. The focus here is on showing high esteem and empathy for people as the starting point for all activities, both for colleagues in the team as well as for users or customers. In addition, the team members should have qualities such as the ability to cooperate, curiosity, joy of experimentation, integrative thinking and optimism. A further success factor is the guidance of the team by a Figure 5.6: Design Thinking process according to Stanford d.school facilitator who is experienced in the process and the use of the method. This person provides orientation for the respective phase of the process and for which instruments can best be used there without, however, intervening in terms of content. #### Process Design Thinking follows a process model with a series of steps. Although slightly different variants of the so-called microcycle with alternative names of the phases have developed over time, their content only differs marginally. We follow the model of the d.school in Stanford, which is based on five phases of the Design Thinking process, also known as working modes (cf., Fig. 5.6). All models are characterized by the alternation between divergent and convergent viewing, thinking and acting, both in understanding the problem space and shaping the solution space. The transition between the expansion of the creative space with an everincreasing amount of information and focusing through containment is called a groan zone, i.e., a "creaking" hinge [8, p. 28f.]. Deliberate iteration is also a fundamental element in the DT process, which is expressed in the motto "fail early, fail often" or "fail forward" in an open culture allowing errors. It describes the idea that ideal solutions can only be found through multiple and early experimentation, testing and consideration of the feedback of the target group, which can lead to more or less extensive new runs of previous modes. The multiple iterations as a so-called macrocycle should lead from the understanding of the problem to the concretization of a vision for a solution and finally to an implementation plan [8, p., 37]. In all process phases the principle "Be visual & show" applies, which means that thoughts, ideas and results are to be visually and vividly documented and presented, for example through post-its with keywords and drawings, mind maps, process maps, tangible prototypes, etc. Because of this principle it is sometimes suggested to speak of Design Doing rather than Design Thinking. For the successful work of a team along the process, a number of rules and tips (rules of engagement) have proven to be helpful, some of which should also be followed in group work in general. They should be communicated at the beginning of a project and called to mind later by the facilitator if necessary: The following sections briefly describe each phase together with tables including a selection of methods and tools that are used in each phase respectively. More detailed explanations can be found for example in the Bootcamp Bootleg of the d. school [9] and in Lewrick et al. [8, p., 36]. #### Empathize (Building Empathy) Empathy is the heart of a human-centered design process. To develop it means to build a deep understanding for the members of the target group with regard to the problem and its context. The aim is to understand why and how people do things, how they think about the world and what is important and useful to them, as well as to learn about their physical and emotional needs. This means observing users, listening to them, interacting with them, imagining and empathizing with their situation and thus immersing into their conscious and unconscious world of feelings, values and needs (engage, observe, immerse). This is the basic prerequisite to steer innovations in the right direction. So-called personas are an essential instrument for documenting the learnings about the target group. Personas represent fictional customers or users together with their objectives, behavior, needs and attributes relevant for the solution to be developed. In the model of the Hasso Plattner Institute, the "Empathize" phase encompasses the stages "Understanding" and "Observing". Table 5.4 shows common instruments for the activities included in this phase. #### Define (Defining Problem) This mode is about building on the findings from the "Empathize" mode, sharing and bringing them together, structuring, weighing and interpreting them. This synthesis serves to test and further develop the personas for ideal-type users and, if necessary, to adopt the perspectives of various stakeholders. The results are a deeper understanding of the users and the problem space as well as a more concrete, meaningful Table 5.4: Methods and tools for the "Empathize" phase problem definition (design challenge). The latter is reflected in a single sentence which, as a so-called Point of View (POV), forms the question for the subsequent phase of idea generation [8, p. 73]. In practice, different POV questions are used. A typical formulation is the "How might we?"-question, for example, "How might we help [user, customer] to reach [a certain goal]?" [8, p. 74]. In the Hasso Plattner Institute model, "Define" corresponds to the "Define point of view" phase. Table 5.5 lists common tools for the activities included in this phase. #### Ideate (Finding Ideas) The aim of idea generation is to develop a wide range of solutions, i.e., to develop and visualize as many ideas, and as many different ideas, as possible. The starting point is the point-of-view question, however all the insights gained so far are incorporated into this phase, including user profile canvas, empathy map and customer/user experience journey. The basic instrument is brainstorming, which can be further and repeatedly stimulated by creativity techniques and specific tasks (e.g., generating ideas for certain functions). The design and testing of initial "lowfidelity" prototypes can also provide further food for thought for solutions and trigger iterations. The use of methods in this mode should enable going beyond obvious solutions, and thus increase the innovation potential by using the collective perspectives and strengths of the team. Unexpected solution directions should be able to emerge and contribute to the quantity and diversity of ideas. This results in a multitude of ideas, which are sorted, condensed and evaluated. The entire process should be strictly separated between the generation and evaluation of ideas, so as not to restrict the creative flow at an early phase. In the Hasso Plattner Institute model, "Ideate" corresponds to the "Finding Ideas" phase. Common instruments for the activities contained in this mode are shown in Table 5.6. Table 5.6: Methods and tools for the "Ideate" phase #### Prototype (Creating prototypes) Prototyping picks up the most highly rated ideas from idea development and continues to develop them further. In doing so, the principle of Design Thinking is implemented: to visualize issues, products and results as early as possible and to test, discuss and further develop them with potential users, incorporating their feedback into tangible models. Prototypes are thus created in order to learn to clarify open questions and discrepancies, to start a conversation or a discourse and to recognize dead ends quickly and at an early stage, which in turn saves costs. In addition to the change requests, the feedback can also lead to complete rejection and thus to a fundamental iteration over more distant previous phases. This procedure is also described by the slogan "Love it, change it or leave it". Prototyping transfers ideas from the mind into the physical world. A prototype can therefore be anything that takes on a physical form and follows the maxim "don't tell me, show me!": a wall with post-it notes, a role play, a room, an object, a storyboard or any combination of different means of expression. The granularity of the prototype should correspond to the progress of the project. In the early stages of a project, prototypes should be created that can be made quickly Table 5.7: Methods and tools for the "Prototype" phase and cost-effectively (low fidelity, quick & dirty), but already generate useful feedback from users and colleagues. In later stages, the prototypes should be refined and allow careful investigating of specific issues. They serve to deepen empathy, to test and to gain further ideas and inspiration. In the Hasso Plattner Institute model, "Prototype" corresponds to the "Develop prototype" phase. Table 5.7 shows common tools for the activities included in this phase. #### Test (Testing prototypes) As discussed in the previous section, testing is closely linked to prototyping. The recommendation "Prototype as if you know you're right, but test as if you know you're wrong" describes the way of thinking that illustrates this relation. Testing offers the opportunity to receive qualitative feedback on the prototypical solutions, to make them better, to learn more about the users, and thus to deepen the empathy for them. The test mode is an iterative learning mode in which the prototypes are placed in the context of the potential user, then used and evaluated by him. Important principles are: "Don't talk, show!", create experiences, and enable the user to make comparisons. The feedback during the tests can lead not only to changes, but also to complete rejection and thus again to a fundamental iteration over more distant previous phases. This procedure is also described by [8, p. 34] the slogan "Love it, change it or leave it". In principle, each iteration loop, regardless of its scope, must reflect which previous results (e.g., personas, user/customer experience journey) have to be adapted as a result of the feedback. In the Hasso Plattner Institute model, "Test" corresponds to the "testing" phase. Table 5.8 contains instruments for the activities carried out during the phase. #### Place For the work of the interdisciplinary teams in the described modes, it is necessary to create a creativity-fostering environment, so-called make or creative spaces. This applies in particular to the availability, size and furnishing of premises as well as to visualization and prototype design tools and materials. The main aim is to provide the teams with freely and permanently available work, interaction, relaxation and storage areas. Flexible furniture with castors, describable and erasable surfaces (walls, tables, boards), as well as good and fast access to information (Internet, libraries, etc.), tools, working materials and catering add to a suitable environment [7, p., 216]. Lighting, ventilation and air conditioning are also important factors to be considered. In practice, teams are sometimes given the opportunity to design the environment themselves (e.g., build their own furniture), especially in the case of long-term projects. Doorley and Witthoft have published instructions and experiences, among other things, in the design of creative environments for the d.school [10]. #### 5.4 Connecting the Concepts #### 5.4.1 Overview As shown, Design Thinking aims at the innovation of products and services, business models and business processes. The focus is on user centricity, creativity and agility in an experimental, iterative process that interdisciplinary teams traverse. Process management pursues a comparable objective with agile and creative process design of new, or redesign of existing, processes under consideration of customer needs. In the following, we put the concepts into relation to one another and discuss the promising use of Design Thinking elements for process management. We pay particular attention to the digitalization of processes, i.e., the reasonable use of information and communication technology for process improvement and innovation. Prototypes and final solutions are therefore always workflow applications with different degrees of automation. #### 5.4.2 User Centricity During process analysis traditional BPM approaches usually involve those participating with interviews and workshops. However, the 'hard' facts of the work in the process with the characteristics listed in Chapter 5.2.2 are in the foreground of the activity-related and process-related interview questions, card techniques or observations. Aspects such as understanding users' motivation, ways of thinking, and values, which are expressly emphasized for the development of empathy in Design Thinking, are largely ignored here. Newer concepts such as Social BPM have changed little in this regard. The Subject-oriented Business Process Modeling (S-BPM) approach, which was already used in the case study at the beginning of this chapter, can build an interesting bridge. It focuses on the subjects as actors in the process. With the associated methodology and language (see Section 3.6) as well as suitable tool environments, representatives of subjects involved in the process can participate in iterative solution development not only as respondents or observers, but also as active designers. They not only explicitly specify the behavior of the subject they represent and its interactions with other participants, but can also immediately test and change the result of their design by executing the resulting model. In doing so, they can implicitly bring in the 'soft' factors mentioned above. #### 5.4.3 Agile Process with Iterations In practice, more extensive process management projects are often still carried out using traditional project management methods in clearly defined phases with milestones, comparable to the waterfall model in software development. This means that the path from analysis of the design of the business model and its organizational and IT implementation to an executable workflow application takes an extensive amount of time. It also increases the likelihood that the resulting IT solution will deviate from the evolving needs and desires of users. For process digitalization in particular, it is therefore advantageous to adapt the agile, iterative process of Design Thinking. This opens up the possibility of meeting the increasing dynamics with regard to the emergence of new processes and changes to existing processes, for example due to new or changed business models such as servitization. For further considerations, we compare the modes of Design Thinking with the activity bundles in process management (cf., Fig. 5.7). Together with the explanations in sections 5.2.2. and 5.3.2 the illustration shows that DT makes a stronger distinction between problem understanding and solution design. The latter only begins with "Ideate". Before this, the actual situation is extensively illuminated and, for example, documented and visualized along the way via personas in the customer/user experience journey, before the point-ofview question is formulated as the starting point for generating ideas. In process management, on the other hand, the problem is usually clearly formulated at the outset. When renewing existing processes, it is usually derived from the desire for improved process performance (e.g., shorter lead times). In practice, therefore, only weak points in the current state are documented and analysis information is used to develop and visualize a new target model, just like in a new process. The creative, design-related part begins earlier than with Design Thinking and tends to be underpinned by less information when being started. It is driven more analytically (e.g., by performance indicators) than by the "soft" factors identified in the course of empathy development in Design Thinking. Regardless of the somewhat different concrete design, the activity bundle Analysis & Modeling can be assigned to the DT modes Empathize, Define and Ideate. The use of proven DT instruments is ideal for a more comprehensive capturing of the problem context and the resulting expansion of the spectrum of solutions for process innovation. Especially when developing a new process, the team members Figure 5.7: Assignment of Design Thinking modes and BPM activity bundles can broaden their horizon and develop a common understanding with a brain dump concerning the problem environment and the discussion of the results. With the help of personas for the process participants in their respective roles as well as interviews and observations, customer/user experience journeys can be described. If participants in the process are team members themselves, they can also visualize their own experiences as journeys. This extends the information base beyond the classic, objective process characteristics to include the user's perspective. This broader foundation for the development of solution ideas should justify the higher effort. In section 5.2.2 we had explained that effectiveness and efficiency, at least of model excerpts, are already taken into account during the analysis and modeling of processes. This is especially true if the future users do this themselves, as in subject orientation. Therefore, the activity bundles Validation and Optimization are assigned to the DT-mode Test, but also cover Empathize, Define and Ideate. With the focus on process digitalization, the DT Prototype mode corresponds to the activity bundle IT implementation in process management. Prototyping in Design Thinking makes the claim to producing a prototype quickly and with simple means, i.e., cost-effectively, in order to quickly obtain feedback from the user (Test mode) and to utilize it. By applying this "fail early, fail often" principle to process management, the team must be able to create an executable model with minimal effort. The focus is therefore on creating a functional prototype in the form of software that allows users to experience what their work with the IT solution would look like. However, assigning the prototype to the IT implementation should not mean that programming is necessary. Rather, it must be possible in the interests of rapid iterations to generate a prototype automatically from the model and have it tested by the users in the activity bundle validation. In the same way, subsequent model changes based on feedback again lead to a new prototype, until a version is found that satisfies the users. Such low-cost and early prototyping possibly prevents more complex reworking during the later realization of the real runtime environment. Using a comparable approach, the user interface can be designed according to the principles of user experience design. Since the respective process model is not only the basis for prototypes, but in its ultimately adopted version also for the workflow application strived for, the activity bundle IT implementation includes also their realization in the way described in section 5.2.6. If software which goes beyond the model-based workflow control has to be developed for this purpose, SCRUM as a user-centered, agile software development method serves as a good choice. In the context of IT implementation, it is also important to decide to what extent the strategy of minimum viable products, often used by software start-ups, should be pursued. This would mean making software with minimal functionality available to customers or users not only prototypically, but also productively in order to obtain their feedback for further development. This could be risky with IT solutions for business-critical processes; on the other hand, it could possibly give an edge over competitors by familiarizing customers with features at an early stage. As explained in sections 5.2.5 and 5.2.7, the model must also be embedded into the organization (Organizational Embedding) before the process can go live (Operation & Monitoring). In Design Thinking, comparable steps follow for the implementation, for example, of a product on the basis of an accepted prototype as well as for its use. However, these steps no longer belong to the modes in the narrower sense (dotted forms in Fig. 5.7). #### Conclusion In order to meet the requirements of digitalizing processes, a process management approach should combine Design Thinking and process management concepts. It must be suitable for quickly mapping processes and their changes both (business) domain-related and in IT, while at the same time adequately involving the users in short iteration cycles in order to approximate the resulting solution to their ideas. In addition to the instruments that can be used for the Design Thinking modes Empathize, Define and Ideate, easy-to-handle methods and tools are particularly necessary for this purpose, with which the team and/or the process participants themselves are able to: An example of a concept to support 1) and 2) is Compare/WP (see chapter 6.1.2). Requirements 3), 4) and 5) are, for example, covered by the S-BPM approach and BPM tools based on it [11, 12]. #### 5.4.4 Interdisciplinary Team The importance of the interdisciplinarity of the facilitator-led team in Design Thinking was explained in section 5.3.2. BPM projects are also usually carried out by teams. We distinguish thereby between four roles: With the traditional phase-oriented approach in BPM, usually a project leader as a facilitator coordinates the collaboration of domain and method specialists as actors and experts during analysis, modeling, validation and optimization. The implementation of the approved business process models is then carried out by IT experts. In section 5.4.3., we have already identified long duration and the resulting deviation from the stakeholders' needs as probable disadvantages of this approach. For some time now, the BizDevOps approach, which is intended to take into account the increasing agility requirements in the course of digitalization, has been becoming more widespread. It strives for a comparatively closer integration of the business departments (business, biz), IT development units (development) and IT operation units (operations)<sup>1</sup> . Right from the start, the agile team includes representatives from all areas in the roles described, who jointly design the process solution. The concept can thus both improve business and IT alignment and also foster enabling through IT. The former means that the degree of coverage of the business departments' needs increases through appropriate IT services. In enabling, IT gives impetus to the use of information and communication technology for business model and business process innovations. Like Design Thinking, the BizDevOps approach therefore involves an interdisciplinary team. The challenge in such teams is to establish the "We" thinking among "T-shaped" individuals. This is due to the fact that line units from which the participants originate (various business departments involved in the process, IT development, IT operations) pursue different goals and often tend to give them a higher priority than a goal to be achieved jointly (innovative or improved process solution). In addition, creativity may be hampered by the involvement of domain experts. On the one hand, process participants are often aware of weak points in existing processes and ways for improvement. On the other hand, they may be 'operationally blind' and too restricted in their consideration of the problem space and, in particular, the solution space. Recruitment should therefore not only take into account diversity aspects such as gender, age and cultural background. Rather, a meaningful balance needs to be found between domain experts and team members who have a background in other fields and have no strong self-interest in the appearance of a solution. The shift in emphasis can be made dependent on whether the goal is more a process improvement in which the experiences and inputs of those familiar with the existing process can be helpful. If, on the other hand, the focus is on a more radical process innovation, this could possibly have a limiting effect. Of course, even with the original objective of improvement, ideas for a fundamental innovation of the process under consideration should not be ignored. #### References <sup>1</sup> When looking beyond company boundaries, one could add partners in the value creation network such as suppliers, customers or logistics service providers (Network Partners) and speak of NetBizDevOps. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Preparation of Process Implementation 6 The aim of the process preparation in the sense of a subsequent implementation is a precise description of the process with a description of the process strategy and process logic. The preparation includes the activity bundles on the left side of the open cycle, i.e., analysis, modeling, validation and optimization (see figure below). The result of these activity bundles is a process description that is sufficiently precise for implementation. The preparation is split into the activities analysis combined with modeling, validation, and optimization. These activities are not carried out in a strict order, but rather the respective priorities can change frequently between activities. Figure 6.1 shows these various activities and their relationships. The following sections present selected methods for these activity bundles. #### 6.1 Analysis and Modeling Analysis and modeling cannot be sharply separated. The analysis focuses on the strategic aspects of processes, while modeling focuses on the process logic. In the analysis the starting point with its associated input, the end state with its generated output, and the therewith satisfied customer needs are clarified. In the analysis, the framework and the essential aspects of the process logic are also defined. In practice, however, the process logic of the actual state is hardly explicitly described when revising processes in this phase. The analysis of the current process logic is accomplished within the framework of the definition of the desired target process. An exclusive reference to the current situation usually makes little sense and is also as a rule unpleasant for all participants to document - What has been "done wrong" lately? As long as one is using the tool 'natural language', the focus is more on the activity of analysis than on that of modeling. The transition from natural language to a more formal process modeling language corresponds to the transition to modeling activities. The modeling can be preceded by a more or less intense analysis method. In extreme cases, a process model is immediately created without prior natural Fig. 6.1 Integration of the preparation in the process management model linguistic analysis. However, it is recommended that at least the strategic aspects of the process under consideration are known and defined. In the following, guidelines for the articulation and coordination of processrelevant knowledge, which can be supported methodically and tool-wise, are presented. An essential element is the understanding of roles which the participants consider relevant for the handling of processes. In addition, it is advisable to consider the exchange relationships between actors, to evaluate their quality for the further design of processes and, if necessary, to derive potential for change from this information. #### 6.1.1 General Information on Articulation and Coordination In most cases, knowledge about workflows and organizational processes rests in the minds of the actors. A context-sensitive, structured survey and analysis is therefore of crucial importance. The survey serves to articulate experiential knowledge and in most cases is carried out within the framework of modeling. However, if it is already handled in advance, the variety of approaches to solving tasks or problems in BPM projects can be dealt with in a more structured way. Nevertheless, in the context, the coordination and alignment of different approaches plays an essential role. Supporting these contributes significantly to the development of solutions that are capable of integration, despite a high degree of diversity and individual approaches to the fulfillment of tasks. This section therefore deals with the survey and negotiation methods and instruments that enable individuals to articulate within the framework of collective reflection and negotiation processes. How people carry out their work, how they react to perceived specifications or deviations and how they cooperate with others is essentially determined by their perception of organizational reality. The interpretation of the perceived determining factors as well as the derivation of the reaction considered adequate of the acting workers can be explained by the cognitive theory of mental models. This theory can also be used as a basis for explaining learning and change processes in organizations that are initiated by operatively active persons. In this section, it therefore forms the basis for the derivation of measures which should enable workers to become aware of their work processes and the organizational interrelationships and determining factors characterizing them. The concept of "mental models" is used to explain how people understand the world - more precisely: how they use their knowledge to make certain phenomena of the world subjectively plausible [1]. Mental models are explanatory models of the world that are formed by people on the basis of everyday experience, previous knowledge, and conclusions based on these. A mental model is used by each individual as a basis to understand the world and, if necessary, to make predictions about its behavior [1]. The knowledge that shapes mental models can be based on everyday experience or can be founded on conveyance or instruction. Seel [1] describes the modification and expansion of one's own knowledge bases and the (further) development of the cognitive abilities necessary for drawing conclusions as "learning". Learning is linked to the processing of individual experiences with, and information about, the world, its structure and evidence, and can be understood as a process of permanent conceptual change [1]. Learning thus presupposes the ability and willingness to understand and accept conveyed world views and then to base one's own mental constructions on them [1]. There are two basic difficulties in changing mental models about work processes. In the case of mental models that have already been recognized as inadequate, there is a fundamental willingness to change (in the sense of adapting the mental model to the environmental conditions perceived as changed), but the challenge is to obtain the necessary information and have it adequately presented. A further difficulty arises in situations in which not all individuals involved perceive the situation as 'problematic' and therefore show no fundamental willingness to change their underlying assumptions with regard to their way of working (i.e., their mental models). This occurs especially in situations where collaborative reflection is not carried out from a generally perceived problem situation, but is either initiated with a purely planning character, or in situations which are perceived as 'problematic' only by certain individuals involved. These problems can be countered with explicit support for the reflection process. Such support must ensure that artifacts are created to represent the individual mental models, which can then serve as the basis for mutual understanding of the respective views on the work process. Such artifacts can serve to coordinate aspects of a work process and to ensure that the ostensive view of a work process encoded in artifacts can be implemented in work practice through performative subjective action knowhow based on it. From a methodological point of view, it must be ensured that all persons involved in the real work process are organizationally and methodologically capable of participating in the collaborative learning process. This requires above all that they can understand and actively use the forms of expression utilized. This, in turn, is a learning challenge that must be explicitly addressed. A widely accepted option for externalizing and harmonizing mental models in the educational sciences is the formation of conceptual models. At the same time, such models can form the basis for the specification of work processes and the configuration of work support systems, as long as they make use of a formally specified semantics (such as BPMN or S-BPM). In accordance with the objective of this section, conceptual models thus represent a means of enabling workers to reflect on their work, to coordinate it, to make the results of these coordination processes accessible to third parties, and to make them usable within the framework of existing system boundaries to support their own work processes. Models are representations of reality that are provided for a particular purpose. Models never represent the real phenomenon as a whole, but contain only those aspects of reality that the modeler considers relevant for the achievement of the respective goal. For modeling, this raises the question of the defining power of these models and the social reality they represent. If a model does not only fulfill an objective of the modeling individual, but is used by other persons, the model influences the mental models of these persons, and thus also their behavior. The active involvement of operative workers in the specification of work processes is therefore an opportunity for their self-empowered development of organizing their work. To this end, however, it is necessary to enable workers to understand such models, to design them by themselves, and to assess their impact on their work processes. Current approaches, on the other hand, continue to assume the need for a process analyst who translates the views of workers into a process model. This can lead to deviations between the real work process and its model representation. In addition, this approach deprives the operatively active persons of the opportunity to sharpen their mental models in the sense of model-based learning and to coordinate them with those of the other participants. In order to enable workers to understand such models, the learning of basic approaches to the creation and interpretation of conceptual models must be the subject of education or training. Workers must be able to identify the models underlying the systems in which they are embedded. In addition, they should be able to assess the implications of external or self-implemented changes to these models and to plan interventions accordingly. For this purpose, the following points need to be methodically supported: about the work in question and to prevent the same real phenomenon from being described by different terms - or vice versa the same term from being used for different real phenomena The following sections show some of the methods used to implement these requirements. #### 6.1.2 CoMPArE/WP The requirements described above are implemented exemplarily in the "CoMPArE/ WP" method. CoMPArE/WP stands for "Collaborative Multi-perspective Articulation and Elicitation of Work Processes". In the application of CoMPArE/WP, the reflection on a real collaborative work process creates awareness of the cooperation in a concrete individual case. Due to its anchoring in concrete work processes, the method is also suitable as a means of organizational development. The form of cooperation of the method is basically determined by its implementation with a card laying technique. The participating operative workers are the essential actors and carry out the components autonomously, whereby articulation and inquiry roles can change. The concrete form of the cooperation differs in the components and is therefore described in the procedure mentioned there. A facilitator is available to support the implementation, but he does not intervene in regard to content. The method should support the articulation and coordination of mental models with respect to work, and at the same time impart basic skills for their expression in conceptual models. The combination of these two sub-objectives has an impact on the framework of the method. From the point of view of teaching modeling competence, it makes sense to introduce the necessary skills step by step with increasing complexity. From the point of view of articulation support, an approach can be contended in three components. Figure 6.2 gives an overview of these three components. Component 1 is used to find a common understanding of where and how the work process to be coordinated begins and ends and to find a common vocabulary. Component 2 is used for articulation and reflection of the respective individual work contribution. Each participant creates here a structured model of their point of view on their respective work contribution, individually and without interaction with others. Due to the uniformly structured presentation of the individual contributions, a collaborative alignment of these is possible in component 3. This alignment is intended to uncover conflicting points of view and to create a common view of the overall work process. The objectives of skill development in modeling are anchored in these components. Component 1 aims to convey the verbalization of mental models and the concept formation based on them. In component 2, the description of the verbalized contents must be represented by means of a predefined category scheme and a notation. In doing so, it needs to be determined which elements of the category scheme remain in the responsibility of the articulating individual and which need to be validated and possibly abstracted or become the subject of negotiation during alignment in component 3. Concrete Implementation of Component 1. It cannot be assumed that all participants have a common understanding of the concepts they use when describing their work. Collaborative concept mapping can be used to align the existing mental models to such an extent that a common vocabulary enables collaboration. In addition, it cannot be assumed that there is a common understanding concerning the borders of the work process to be coordinated. Concept mapping can also help to clarify this issue. In addition to the content dimension, concept maps provide a low-threshold entry into the world of conceptual modeling, since they do not predetermine the meaning of model elements, but rather allow the persons involved to define them during modeling. This facilitates the mapping of the individual mental models into the explicit representation and avoids the necessity of having to carry out a translation to a model with formally defined semantics in addition to the coordination with the other persons involved. Within the scope of this component, the participants are asked to describe all relevant aspects of the environment in which the work process to be reflected is embedded. This is done by individually writing each aspect on a separate card. When the collected individual aspects are brought together, the cards are arranged in turn on a common work surface. The aspects can be put into relation to each other. Cards with different terms for the same aspect are arranged overlapping. Hierarchical or causal relationships between aspects can be represented by drawing explicit connections, but also by the spatial arrangement of the cards. The example in Figure 6.3, which is used throughout this section to explain the method, shows a concept map with relevant aspects for applying for a vacation in a company. The aspects were related to each other by spatial arrangement. The overlapping elements show aspects that are mentioned by several participants and are described using different terms. Concrete Implementation of Components 2 and 3. Components 2 and 3 focus on the articulation and alignment of the perceived course of a work process and the associated interaction within this process. The modeling in component 2 is carried out individually by all persons involved, without interacting with others. This avoids overlapping effects and explicitly reveals different perspectives for the next component. The persons involved describe which of their work steps they see as contributing to the achievement of the work goal, with whom they interact and in what form this interaction takes place. The negotiation of a common point of view in component 3 and the associated creation of a common model is again carried out by means of a structured procedure, which is to introduce more complex modeling tasks and guarantees a uniformly prepared model representation. In doing so, the previously created models are used further. The structuring scheme separates those model aspects that remain in the responsibility of the individual worker from those that are the subject of negotiation. In this step, a structured form of representation with pre-specified semantics is used to represent the work processes. It is based on the current category schemes for the description of collaborative work processes. The categories WHO, WHAT and EXCHANGE (M3) are used. WHO (blue in Figure 6.4) refers to the actors in the work process. WHAT (red in Figure 6.4) is used to describe active contributions in the scope of the work process. EXCHANGE (yellow in Figure 6.4) is used in the context of collaborative work processes to characterize the sharing or exchange of information or material between actors in the context of their own activities. For the sake of usability, these categories are not specified exactly and deliberately leave room for interpretation in concrete use, for example, a WHO element can represent a concrete person, a role, a department or an entire organization. WHAT elements remain the responsibility of the individual participants. WHO and EXCHANGE elements are the subject of coordination in component 3 and must be developed toward a common understanding. Individual Articulation. In component 2, the persons involved individually describe with the help of the elements, what they contribute in the work process, who interacts with them, and in what form this exchange takes place. In order to support the articulation process, a structuring scheme was developed that prepares the models in a coherent form and allows them to be combined in the next step. As shown in Figure 6.4, the structuring scheme defines the spatial arrangement of the model elements. Operative workers represent themselves through a WHO element, under which the perceived contributions to the work process are placed as sequentially arranged WHAT elements. For all other workers with whom an interaction is perceived, another WHO element is placed, under which the interaction is specified in more detail by EXCHANGE elements. Their vertical positioning determines whether an incoming resource is expected (placement above the dependent WHAT element) or provided (placement below the generating WHAT element). Figure 6.4 shows three individual models for the example process described above, which were created according to this structuring scheme. In the example, it can be seen that at this point there may be divergent representations with regard to content, especially in the area of exchange elements (cf., "Application" vs. "Completed application" in the figure above). These divergences become explicitly visible in component 3 and are then subject of the negotiation of a common perspective. Collaborative Alignment. Collaborative alignment is based on the individual conceptional models created in component 2. Figure 6.5 shows an exemplary alignment process for two of the actors represented in the example. The common modeling again takes place on a common work surface (see Figure 6.5 in the middle). The participant, who triggers the real work process, begins by describing his own contributions to the process and adding the corresponding model elements to the surface (steps 1-2 in the following figure). The other participants intervene here only inquiringly to avoid misunderstandings or to disclose ambiguities. An active participation of the others takes place as soon as the first EXCHANGE element is used (steps 3-4). If a fundamentally common view of the work process exists, one of the participants should be able to introduce a correspondingly assigned EXCHANGE element at this point (steps 5-7). If this is the case, the description process is continued by this person (from step 8). In the case of a basic fit, which differs, however, in the designation of the element, e.g., by different abstraction levels, this conflicting designation must be resolved, or the semantic equivalence of the two elements must be represented by overlapping arrangement (e.g., step 7). If there is no element to be assigned, fundamental differences in the representation become visible. This may be due to a lack of awareness of the relevance of an exchange, which means that the addressed participant was aware of the interaction, but did not consider it relevant in the context of the work process. However, if a participant's perceived need for interaction is not reciprocated, this must lead to more in-depth alignment processes. The initial alignment process ends as soon as all of the participants have explained their individual models and added them to the common model. This externalization phase is followed by a collaborative reflection phase, during which the work process is examined on the basis of the common model and discussed with regard to its adaptation to the individual views of the participants. Any necessary modifications are carried out at this point, after consensus has been reached among those affected. The result of the application of the method now represents a consensual representation of the collaborative work process. Due to the limited expressiveness of the modeling language used, it is not possible at this point to map work variants or decisions that are otherwise common in process descriptions. The decision for a semantically limited modeling language was again made from a didactic point of view, since empirical evidence shows that inexperienced modelers can initially describe their views on a work process more simply narratively on the basis of a concrete case. Decisions regarding the concrete implementation of the work process have already been made in the case-based description. Thus, an explicit representation of the same is not necessary in the scope of the modeling. A complete description of the work process therefore requires a multiple execution of the method or its extension by further refinement steps, which however, will not be considered here in detail. In the sense of the formation of modeling competence, the focus in component 1 is on the introduction to the abstraction and conceptualization of perceptions of the real world necessary for modeling. In component 2, the representation and reflection of one's own perception of work, guided by structural aids, is captured in conceptual models and their description by means of given structural elements. Component 3 subsequently focuses on model understanding (of the other individual models), interpretation (with regard to their effects on one's own model), and negotiation (of the jointly justifiable view) of model content, which ultimately conveys the competence to influence work processes in a self-empowering way. #### 6.1.3 Raising Awareness of Process-Relevant Change Potential In the following, the Value Network Analysis [2] is first discussed, as it was introduced in Knowledge Management for the processing of performance relationships between networked actors, before its potential for process design (analysis and modeling) is detailed. #### Value Network Analysis If we consider, as previously mentioned, the added value of organizations and thus the level of performance-related exchange relationships between actors, tangible exchange relationships can be distinguished from intangible ones in the network of actors within the framework of work processes. Tangible exchange is determined by energy and material flows. Intangible exchange, such as knowledge, refers to cognitive processes and action-guiding information. If now participants and exchange relations are described, the structure of an organization or a network can be captured. Exchange relationships represent the molecular level of economic activities. Thus, value creation does not only consist of tangible transactions, but also of intangible transactions. These relate above all to cognitive exchange, since the sustainable success of an organization is based on the exchange of information, knowledge sharing and open cognitive paths that enable appropriate decisions to be made (and thus the successful existence of an organization). However, knowledge and intangible elements behave differently than physical resources in business life, so they cannot be considered tangibles. Due to their proximity to living systems, they constitute a separate category of exchanges and differ from tangibles related to goods, services and revenues. Tangible exchange of knowledge is defined as transactions involving goods, services or revenues, e.g., physical goods, contracts, invoices, delivery and receipt confirmations, inquiries, requests, invitations to bid, and payments. It is essential here that knowledge-intensive products and services that generate income and for which payments are made as part of a service or product or on the basis of a contractual obligation are also regarded as tangible transactions. Intangible exchange of knowledge and performance: Intangible exchange of knowledge and information supports core processes and thus the classic value chain but is not subject to any contractual obligation. Intangibles are 'extras' or (small) courtesies that people exchange in order to build relationships and allow processes to proceed pleasantly or without disturbances. Intangible transactions include the exchange of strategic information, planning knowledge, business-operational knowledge, joint planning activities, collaborative design, and policy development. Intangible transactions are therefore not contractually agreed services for the benefit or support of organizations or their members. They can be extended from one person or group to another, for example, when an organizational unit requests an expert to work temporarily for them in a prestigious position. Recognition often helps in relationship work, so that intangible benefits constitute genuine motivation factors for active participation and engagement in group activities. Intangibles represent the core of all human action, and thus also determine socioeconomic action. Intangible transactions are deliberately seeded. They can be brought about and recognized. If one wants to understand how intangibles generate value, one must first understand how they become visible and work as negotiables in economic exchange relationships. They are often not immediately visible, but rather 'packaged' in services or products. A typical example is to build an understanding for a customer situation (intangible) before offering a service (tangible). For a joint practice, the smooth running of processes is of immediate importance. Thus, those transactions are essential which (also) guarantee by means of intangibles that a common purpose of action is ensured. This must now be methodically taken into account. In a Value Network, tangible and intangible values are generated by means of complex dynamic exchange processes between two or more individuals, groups or organizations, which represent the object of reflective design. #### Holomapping The view of organizational value generation based on networking brings with it a new form of organizational modeling; every exchange requires a mechanism or medium as enabler for transactions. These can be tools such as e-mail or face-to-face interactions in communities of practice. As already mentioned above, typical intangibles pertain to knowledge to gain information from customers and feedback on (product) developments. The representation of tangible and intangible exchange processes in a diagram with flow elements allows the mapping of the dynamics of living systems. First the participants or roles (also groups, teams or organizational units, but not technical aids) are documented - they form the nodes of the network and are visualized through ovals. The participants send or supplement so-called deliverables to other participants. Arrows, which are labeled as the respective deliverable, indicate the direction deliverables take in the course of a particular transaction. Transactions or activities are displayed as directed edges (arrows), which must originate with one participant and end with another. The arrow indicates movement and the direction in which something is happening between two participants. In contrast to participants, who are time-stable, transactions are time-limited and volatile. They have a starting point, a duration, and a conclusion. Deliverables, on the other hand, are real 'things' that move from one participant to another. A deliverable can be material (tangible), like a document or a table, or immaterial (not tied to matter), like a message or a request that is only verbally delivered. Deliverables can also be intangible, for example, when referring to knowledge about a certain fact (cognitive) or in the case of a favor (social/emotional). Arrows are only allowed in one direction - they cover a single transaction between participants. Bilaterally directed arrows are meaningless, in fact, they make it impossible to analyze the processes and exchange relationships. An exchange occurs as soon as a transaction results in a deliverable that is returned. It does not necessarily have to be present in the practical world of action in organizations. However, if it occurs, a Value Network can establish itself, with transactions as molecular elements of value generation. In the context of change processes, it is essential to empower those involved and thus to have the affected role holders create the communication map with tangibles and intangibles (holomap), as well as to process the data collected by them within the framework of the Value Network Analyses. Within the scope of knowledge generation or knowledge collection at the beginning of the work on the network structure, each individual participant considers his/her role, which he/she then communicates to the other participants. In this way, relationships and interdependencies between the individual roles, which are often unknown, become more explicit and clearer. The roles are symbolized as nodes, the exchange of material or immaterial values is represented in the form of lines connecting the roles. The modeling forms the basis for the subsequent analyses for knowledge evaluation and processing. #### Exchange Analysis The holomap shows how people use their work as a starting point for exchange analysis. Tangibles (material value flows) in the network refer to the material exchange between persons (typically goods, services and sales revenue). They represent transactions based on contracts. Intangibles (immaterial, ideational value flows), in contrast to tangibles, are based on knowledge or a certain additional benefit. They are not contractually fixed or subject to a charge. Intangibles often collected are strategic information, process or planning knowledge, as well as existing emotional components such as mutual trust, common interest, need for knowledge, security, etc. - see also Figure 6.6. Fig. 6.6 Extract of a holomap for customer service The exchange analysis examines a Value Network for its conclusiveness, robustness and sustainability. It provides insight into the current structure and dynamics of the network. The following questions should support the exchange analysis: How do the values flow through the organization? Does a certain logic emerge? Is the relationship between the exchange of material and immaterial values balanced or does a certain type of exchange predominate? Does the pattern in the Value Network show reciprocal value flows or are there participants who receive more value flows than they provide? Are there ineffective connections in the network that do not pass on value flows? These questions are intended to check whether the network fulfills its purpose, whether missing end nodes or links can be detected, and how the structure of the network can be optimized. They ensure a general overview of added value and loss of value. The exchange analysis should serve as a stimulus for dialogue, understanding complex systems, and promoting systemic thinking (cf., [3]). The exchange analysis on customer service, under the assumption that the organization is facebook, shows several findings: Customer service is tangible from the point of view of product development as a sink - it only receives feature list. Sales receives lifestyle information, but no trust-related information. The transmission of uncertainty encumbers the relationship between customer service and sales as well as between customer service and product development. This first evaluation may be an indication of the information management shown in Figure 6.6, where features indeed allow for a certain form of feedback, but where these may be acknowledged by users with requests reflecting uncertainty. #### Impact Analysis Impact analysis examines the impact of each individual value input on the participants and thus focuses on the recipients of value inputs. This analysis thus shows which input triggers which reactions and activities and how this affects the material and immaterial assets of the recipients concerned. The costs and benefits of value inputs are then assessed as low, medium or high. In order to gain a better overview of these questions, the answers for each individual recipient of value inputs are entered into a table and the current situation analyzed. The table in Figure 6.7 shows the impact analysis based on the insights gained from the exchange analysis of a customer service employee. The table shows who provides input for which activities and what effects are perceived in the form of material or immaterial value flows. The column entries for the general costs and risks as well as for the benefit of the input addressed are essential for the estimation of change potential. The data from the initial evaluation (exchange analysis) thus form the basis for the two further analyses, whereby value-based detailed evaluations of transactions are carried out from the point of view of input received (impact analysis) and output transmitted (value creation analysis), and in this way provide insights for change processes. For example, as can be seen in the table, it is explained when features enable a successful form of feedback, for example, to avoid requests that reflect uncertainty on the part of users. This is the case even if the current benefit of the presentation of features by customer service is estimated to be low due to a lack of comprehensibility. The entries in the feature list table also show the reference point relevant to value creation, i.e., the quality of information provided to customers by customer service employees. Based on the as-is analysis, strategic perspectives can then be derived and the table can be filled in again and serve as a comparison with respect to its planned, strategic activities (target analysis). In the present case, for example, a customeroriented information service is of increased importance. #### Value Creation Analysis Value creation analysis analyses how values can best be created, increased and used. Like the impact analysis, the value creation analysis also considers the individual role in relation to the entire system. The difference to impact analysis is that, unlike with input, this time the sender or producer of output is considered in his role and with his related activities. Each individual sender of value outputs is analyzed to determine how added value and value accumulation are realized in relation to the existing value output. A costbenefit analysis will also be carried out for this purpose. In the value creation analysis shown in Table 6.1, the results of the work were used to analyze how values can best be created, increased and used. The table shows the analysis based on data from a customer consultant's exchange analysis. Fig. 6.7 Extract from an impact analysis for customer service Table 6.1 Part of the case-related (customer service) value creation analysis For costs / risks and benefits: L¼low M¼medium H¼high Performance indicators can be used to filter out the requirements placed on sales and product development from the analyses. The strategy developed from this can be outlined with the statement 'availability or transparency of information'. As a change measure, it could be decided to increase the identified value creation potential in the Value Network by expanding the tangibles of information flows between all functional units. After the presentation of the actual situation, the value creation analysis also makes it possible to derive strategic perspectives. Here the question should be asked 'Which possibilities should still be used in the future to generate value optimization in value output?' With regard to the concrete question 'What should be done to increase, expand or optimize the value of output?', the table is filled out in a comparative manner to analyze the target situation. The entries in the 'costs/risks' and 'benefits' columns are essential for decision support. Here, the assessment can be put into perspective, especially with regard to costs and risks in the target situation, if, for example, content should be included in training courses, since there is know-how for the creation of corresponding training materials and for the provision of effective mediation formats in the network (e.g., in the context 'Report' - third table entry in the table). A target list usually takes into account knowledge about the feasibility or availability of resources and the associated costs for implementing measures, without anticipating a decision in this regard (see evaluation). #### Evaluation For the evaluation, tangibles and intangibles are evaluated in tabular form (impact analysis, exchange analysis, value creation analysis) according to their significance for the respective role(s). These evaluations reveal the effects on relationships and allow targeted measures to be taken. So far, the exchange analysis has provided insight into the current structure and dynamics of the network. The implementation of the impact analysis allows all participants to deduce their own roles in the network in a context-sensitive way. The impact analysis also provides an overview of the effects each individual value transaction has on the participants. The value creation analysis allows decisions to be made on how values can best be created, increased and used, and how they possibly affect other roles or should include them in the consideration. Based on the derived performance indicators, a strategy can now be developed to increase the identified value creation potential in the Value Network. Typical results of an evaluation to increase added value include the consideration of missing exchange relationships, such as the ones related to the case in question: Both measures show the necessity of networked representation of exchange relations. It is not necessarily the immediacy of exchange relationships, but rather the concatenation of exchange relationships that can bring about added value. If, for example, product development (e.g., software designers) were to start communicating directly with customers, a certain basis for discussion would first have to be established. Such measures could even be counterproductive to the strategic goal to be achieved and increase the existing uncertainty on the part of customers, thus adversely affecting the desired objective. The additions to the network thus allow a context-sensitive system view of value creation through material and immaterial services that should flow between the actors involved. The completed Value Network map, as shown in Figure 6.8, forms the basis for further development planning. For the first time, it shows the inquiry to customers as well as the feedback for the purpose of comprehensibility as tangible deliverables, which are intended to increase the knowledge of those involved through the transparency and availability of information. In the long term, as an intangible exchange between customers and customer service, it is important to strive for a mutually secure relationship that can be expressed through customer loyalty to the organization. Only the open exchange of information enables sustainable customer knowledge management. Value Networks view systems in their entirety and consider their complexity. They enable the holistic identification of both material and immaterial values. In any case, the latter indirectly determine the quality of material exchange relationships and must therefore be taken into account in the development of socio-technical systems. By means of Value Networks the focus on the individuals involved can be captured, which in turn gives them a sense of purpose and fosters their motivation to participate in reflection and actively take part in codesign. The surveys and analyses Fig. 6.8 Adapted network presented here allow the explication of individual roles and their directly or indirectly perceived contribution to the value creation of an organizational system by actors. This facilitates the clarification of roles and the understanding of correlations, since these are visualized graphically in holomaps by means of value exchange relationships, and are thus fed back role-specifically. As such, they represent a viable starting point for the participatory design of socio-technical systems. ### Potentials for Process Analysis and Modeling For the design of processes and their modeling, multiple potentials can be drawn from the Value Network Analysis: which are considered relevant within the framework of the fulfillment of tasks and the perceived organizational events. Thus, the focus is placed on the way of working and the interaction level. Overall, Value Network Analysis is a diagrammatic / tabular technique for organizational development with the goal of process definitions or executable processes, which directly supports the mapping of interaction structures to communication-oriented approaches such as S-BPM ([4]). All information is generated from the point of view of the involved or responsible role holders (see also [5–7]). #### 6.1.4 Structured Asset Records In the following a procedure is described, which leads from a natural linguistic description to a formal behavior model. This approach is based on active sentences of natural languages. The process will be introduced on the basis of the Poly Energy Net (NET) project, an approach funded by the German Federal Ministry for Economic Affairs and Energy. The aim of this project was to develop a solution for a self-organizing distributed energy supply system. The following sections show how the associated software was developed, from a general description to a precise model, which was then converted in a first stage into a program based on process specifications. #### General Information The following steps, which lead from an informal description of a process to a formal model of the process flow, do not have to be performed in the order given. The steps rather serve the goal of a precise process description. If it turns out in one step that something had been forgotten in a previous step, or if it turns out that it is more advantageous to design subprocesses differently, this change is included in the step currently being worked on. The change is not reflected in the previous descriptions. To create a first draft of a process description, the procedures from Design Thinking described in the previous chapter can be used. All documents of the previous descriptions are obsolete by default and no longer valid after completion of a more detailed description, except when a description explicitly states that a previous description is still valid (e.g., as an overview document). Experience has shown that it is not possible to keep several documents consistent. There should therefore only be one valid document, so that a formal behavioral model is available after the preparation has been completed. As a rule, changes should only be made to this model. When creating a process model, one can also start with any step. For example, only active sentences can be used for a natural language description. Thus, the steps which transform a description in arbitrary form into an active description form are omitted. It can also begin with the identification of the actors and continue with the detailing of each actor, including communication with other actors. The concrete procedure depends on the circumstances and preferences of the parties involved. #### Natural Linguistic Description of Processes A process is described in a more or less structured way in natural language. There is no specification as to the structure of the document to be created or the vocabulary to be used. The creators of a process description can follow their preferences. Figure 6.9 shows an excerpt from the rough description of requirements for the energy management system. The initial free use of natural language does not require any special methodological knowledge on the part of the participants. The need for such knowledge could be a major obstacle to the involvement of stakeholders from different departments. Textual descriptions can be supplemented by suitable images. Figure 6.10 shows a functional structure of the system to be created. Such a functional structure is a first approximation to the specification of a process system. Based on these more structural descriptions, a first process-oriented specification can be created. Figure 6.11 shows an excerpt of a process description. This process description is supplemented by an illustration of the effects on a holonic energy network. Figure 6.11 refers to some elements (switches) in Figure 6.12. The tools used at the beginning for an initial requirement definition and process specification are not structured. Basically, texts and supplementary drawings of any Fig. 6.11 Dynamics of a holonic management system kind can be used. In the following steps, this non-technical description of a process is transformed step by step into a precise description of the process flow. #### Process Descriptions in Active Form Informal process descriptions in natural language very often contain passive clauses (see also the above description). However, passive sentences do not contain a direct assertion about the performer of an action. Passive sentences are used when it is not important who the performer of an action is. However, this is not the case with processes. Process descriptions must include the performer of an action. All passive sentences must now be converted into active sentences. To do this, the active elements must first be identified. Active elements can be humans, software systems that run automatically and perform certain activities, physical systems, or any combination of these basic elements. Therefore, in our example, the control center can be a combination of software, people and electrical systems. The software prompts an operator to read a specific measured value and enter it. Depending on the entered measured value, the software initiates the closing of a switch. In order to avoid a process description being too dependent on the organizational and technical environment, abstract actors are introduced. Such abstract actors are entities that send messages, receive messages, or perform internal tasks. Figure 6.13 shows the function diagram with assigned active elements. These abstract active elements are called subjects, following the subjects in active sentences. Subjects are the roles in processes. The tasks of the identified subjects can be briefly described for a better understanding as shown in Table 6.2. With the introduction of actors in the form of subjects, all passive sentences can now be replaced by corresponding active sentences. This makes the process description more complete. Table 6.3 shows a process description with active sentences in tabular form. The numbering of the entries already reflects the control flow. The number of the follow-up action is specified in the associated column. If the follow-up action depends on certain results of the action, this condition is described in the column follow-up action. Depending on the valid condition, there may be another follow-up action. A table with the control flow of a process can be the starting point for a control flow-oriented process model. The individual actors are the swim lanes in BPMN or in a swim-lane-oriented EPK or in UML state diagrams. However, these modeling methods should only be used if no asynchronous events, such as the possibility of changing purchase orders, occur in a process and the parallelism of the agents is not to be modeled. In addition, the number of actors should not be too high. Swim lane representations are usually flat, i.e., there are no hierarchies of swim lanes. More than five swim lanes lead to confusing representations. Thus, a service process contains as a minimum the swim lanes customer, call center, first level support, billing and, where applicable, customer feedback. Experience shows that in real processes there are usually about 10 actors or more involved. Swim lane diagrams even become confusing if the control flow just has to cross several swim lanes in order to switch to another swim lane. Control flows are not a manageable representation for cross-organizational or cross-company processes in a distributed environment. In a distributed environment, messages are the more vivid way to model the collaboration of individual actors. #### Tabular Role-Oriented Description In the last step before the actual process modeling, the process description is structured according to the actors. All sentences with the same actor as subject are summarized in a table. For this purpose, it may be necessary to supplement the process description with interactions between subjects. Phrases such as "informs subject xy" or "engages with" etc. are replaced by send and receive actions (see Table 6.4). Sentence no. 2 "Network monitor reports the situation to the control center" in Table 6.3 is converted into a send and a receive action. In Table 6.4 this corresponds to sentence no. 2 "Network monitor sends status black to control center" in the table section for the network monitor. The counterpart to this is sentence no. 1 in the table section for the control center. After creating the behavior tables, a formal model can be derived in a suitable modeling language. A language should be used in which the aspects deemed important for the process under consideration can be clearly and precisely expressed. In our example we have selected S-BPM. Figure 6.14 shows in the left half the #### Table 6.2 Tasks of identified subjects (continued) Table 6.2 (continued) network structure of the considered process. The rectangles with rounded corners represent the actors involved. The arrows between the actors are labeled with the exchanged messages. The numbers on the message names correspond to the sentence numbers in the table above. The diagram to the right of the process structure shows the behavior of the holon coordinator subject. The circles with the letters E and S are states of communication. Transitions from circles with an E are labeled with the messages expected in this state. The transitions to S states are labeled with the messages sent in this state. All other transitions define local operations on local data. #### 6.1.5 Process Modeling #### Selection of the Modeling Language In the following, some factors which should facilitate the selection of a suitable modeling notation or language are listed as examples. These include the determining factors of a BPM project, the manageability of the language and the support of downstream activities such as validation (see also [8–13]). With regard to determining factors, the question 'What are the properties of the subject matter to be modeled?' is of particular interest. The following properties should be decisive for the selection of a modeling language: Table 6.3 Sequence in the Holon System Table 6.4 Sequence in the holon system (more precise) – Number of actors: At first glance, this parameter does not seem to be necessarily relevant for the selection of a modeling language. However, it becomes more important when it comes to the complexity of processes on the one hand, and the comprehensibility of the models on the other. After all, the instance-model relationship also plays into this factor. If there is a large number of actors involved, a notation should also have modeling mechanisms or language constructs that make them visible, as well as accessible aggregately or through other perspectives (e.g., functional view). With regard to manageability, the question 'How should the subject matter be described?' is of particular importance. The following properties or quality of a modeling language should be decisive for its selection: – Number of symbols: On the one hand, a limited number of symbols can make powerful models easily manageable, but on the other hand it can lead to an undesirable abridgement of facts. Fig. 6.14 Behavioral description of the holon coordinator subject Regarding the support of further activities, the question 'How is the support of the model for next steps?' is of particular interest. When selecting a modeling language, the following features of a modeling language should be considered in this context: i.e., after it has been embedded in the organizational and technical infrastructure and has been transferred into the operational environment or has become operationally effective. In this context, a tool should help to support the introduction phase of a modeled process, i.e., to determine which process steps are transferred to operations and in which order. An appropriate tool should also support monitoring of implemented processes and thus help to ensure the operations of an organization and effectively support its further development. The latter can be done by means of annotations in process models, which mark obstacles to execution. #### Modeling by Construction When constructing a process model, modeling begins with a "blank sheet of paper". The information from the process analysis is used to describe the process step by step. The required activities include several different tasks, depending on the approach chosen: These activities are set in a certain order according to the selected language and lead to differently detailed representations of subject matter. #### Modeling by Restriction In addition to modeling by construction, there is also the possibility of modeling by restriction. This is based on general process models. A typical example is the communication-oriented approach as pursued with S-BPM. In the universal process model, each actor or system involved in a process can send a message to, or receive a message from, any other actor or system involved at any time. This message has the general name 'message' and can transmit any media as a business object. The result is a universal process that is characterized by the number of its subjects. These are marked as boxes with subject 1... n. Their mutual interaction possibilities are marked by arrows between the subject boxes. This results in a similar initial behavior for each subject. Within the framework of modeling by restriction, the following steps are taken to a detailed factual situation: (i) determine number of subjects and subject identifiers, (ii) reduce communication paths, (iii) specify message types, (iv) customize subjects' behavior, (iv) specify and refine business objects. If other, for instance, function-oriented approaches are chosen, then basic structures can be generalized, for example in the form of reference models. In doing so, behavioral patterns will also be used, such as previous events for functions or conditions that determine the further course of business processes after executing a function. #### Combining Approaches While in the construction of process models the modeling begins with a "blank sheet of paper" and is extended step by step with information from the process analysis, in the modeling by restriction a generalized structure of process models and their components is assumed. A typical example of a combination of both approaches is the case corresponding to the middle-out approach. One instance of this case is to start with a construction and, as soon as a (recurring) pattern occurs, to use a reference model and reduce or concretize it. Further application instances would be a pattern comparison and the start with recurring (routine) processes. The latter reverses the above-mentioned case and allows the embedding of special characteristics of process flows into generalized process architectures. The pattern comparison, on the other hand, represents a kind of control process in which the completeness or correctness of a model can be checked by means of a generalized pattern. #### 6.2 Quality Control: Validation and Optimization Validation is closely related to modeling. In modeling the process flow is described according to the objective. This means that during the modeling activity the following question resonates: 'Does the model correspond to the set qualitative and quantitative objectives?' The examination, whether a process model corresponds to the set goals, is called validation and/or optimization. This means that validation and optimization are constantly performed in the course of modeling. After the decision to complete the modeling process, a final check is made as to whether the overall model meets the set objectives. Validation shows whether the process meets all requirements and achieves the intended results. It is also essential for a process whether the desired results are achieved with the least possible effort. Quality control in business processes therefore has two main tasks. It is intended to test the effectiveness and efficiency of processes. Effectiveness means that the process meets the requirements placed on it, i.e., delivers the desired result (output). The process is efficient if it can be carried out with as little financial and time resources as possible in order to deliver the desired result. This is to be achieved through optimization. Both quality controls must be implemented as early as possible, before IT systems are developed at great expense and later users are trained. Figure 6.15 summarizes the individual aspects of validation and optimization. The verification of a process Fig. 6.15 Structure of the quality assurance of a process model model is supported by appropriate tools and reference models. For the validation there are manual tools such as checklists or role-plays, while for the optimization simulation software must be used. The results of the check are prepared and entered into 'ToDo' lists for processing, i.e., modeling activities are started again. This cycle is repeated until the verification leads to a result considered to be good enough. #### 6.2.1 Validation The prerequisite for validation is a model that reflects the subject matter to be represented. It is checked whether the model delivers the expected result according to the specified quality characteristics and whether the process contributes to the goals of the company. This aspect is called semantic correctness. This results from the consensus of the managers as well as the technical and methodological experts who consider the model to be appropriate. The semantic validity is to be distinguished from the syntactic correctness, which concerns the adherence to the fixed description rules, i.e., the description means are used according to the defaults of the modeling language. #### Manual Process Validation Figure 6.16 shows a general procedure for manual validation. Here the process documentation is checked with the help of checklists. The process documentation includes the description of the goals, inputs, results, triggering events, and of course, the model of the process. The process documentation should be checked by all parties involved in a process on the basis of the checklists. The findings of the individual parties are consolidated and clarified in a joint workshop and the necessary revisions are jointly determined. This cycle is repeated until it is jointly decided that no further revisions are necessary. Fig. 6.16 Sequence of a manual process check To prepare a review, the process description and a checklist, according to which the process description is to be verified, are distributed. This checklist contains questions to be answered by the evaluators regarding the process. Examples of such questions are: The above list of questions is only exemplary and not complete. Companies often use lists with up to 100 questions. Reading extensive process documentation and comparing it with long checklists is very tedious. Experience shows that the intensity of the check decreases with an increasing number of pages. The first pages are still read in detail. Then the accuracy decreases continually. In order to compensate for the weaknesses of a visual assessment, a more formalized version of the review, the so-called "walk-through", was developed, whereby the walk-through refers predominantly to the process model. #### Walk-throughs Similar to code inspection in programming, in a walk-through a process is discussed step by step with selected process participants. In order to make the step-by-step process more engaging, a formal process description can be run through with the help of a practical example. A process participant goes through the business process description step by step using a concrete example. For each process step, an expert asks specific questions in order to question the effectiveness of the process description. For example, the understanding of technical terms, the technical necessity as well as the completeness of the process description are questioned. In this way, the process description is evaluated. A walk-through is performed with about two to four process participants representing different user groups. The 'authors' of the process description (e.g., process managers) should remain in the background so that criticism can be formulated openly. All points of criticism and suggestions are collected, documented, and then evaluated with the process participants. This evaluation leads to a revision of the process. The step-by-step analysis of a process can be supported by appropriate tools. The tool used shows the process model on the screen and the current process step is highlighted in color. #### Role-Plays The next level for a tangible review of process models are role-plays. These are particularly useful when communication-oriented modeling languages are used. The actors are then already identified and the roles are subsequently assigned to suitable persons. A game leader triggers the process and provides the necessary input. These process instances are then executed by the individual role holders according to the process descriptions. These 'process flows' are observed by other affected parties and the anomalies identified are noted. After a number of process instances has been executed, the findings are evaluated and necessary adjustments identified. The execution of role-plays can be supported by suitable IT tools. The role owners of a role-play do not receive their role descriptions on paper, they are guided through the process by software that, in particular, implements the flow logic. This software is generated directly and automatically from the process model. The prerequisite for such an approach is that the semantics of the process modeling language used are clearly defined. This prerequisite is, for example, only partly met by BPMN and not at all in the case of EPCs. However, S-BPM entirely fulfills this requirement due to its clearly defined formal semantics. Figure 6.12 shows what an IT-supported validation can look like. The advantage of an IT-supported role-play is that the preparation time is very limited and the process experience is very close to the subsequent productive process execution (Figure 6.17). #### 6.2.2 Optimization After checking the effectiveness (do the processes deliver the desired result at all?), it must be checked whether the result is achieved with the least possible use of resources. Optimization through manual testing, walk-throughs or role-plays is only possible to a very limited extent. The knowledge gained in this way only provides an approximation for determining the resource requirements for an assumed number of process runs. A systematic determination of the resource and time requirements is only possible through simulation. However, the prerequisite for a simulation is that the process model can be executed. When business processes are simulated, the business cases handled by a process are generated randomly according to an assumed probability distribution. As a rule, this is the exponential distribution with a presumed or observed expected value. The individual work steps are assigned the corresponding resources with the required working time. The required working time usually follows a normal distribution with expected values and standard deviations determined from observations. Within the framework of simulation instances, information is provided on the execution capability of processes, on process weak points, and on resource bottlenecks. On the basis of the simulated Process Performance Indicators, various alternatives can be evaluated and a realistic benchmarking carried out in advance of cost-intensive process changes within a company. Modern tools and simulation methods enable the analysis and optimization of processes with regard to costs, lead times, capacity utilization or bottlenecks. In addition, the simulation of business processes forms a starting point for introducing activity-based accounting instead of the relatively inaccurate cost-plus pricing. The profits and losses of the individual departments thus become transparent at an early stage. As already mentioned, conducting a simulation study requires a precise description of the process under consideration. This means that a formal method must be used to define the process flow. In addition, the most precise knowledge possible of the probability distributions, their parameters and the examined performance indicators is required. In practice, simulations are not often used due to the high effort involved, although the findings can be compelling. #### References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Realization 7 With the specification of effective and efficient processes, the foundation for their implementation has been laid. Following these preparatory activities, we now deal with the implementation of process specifications in an execution environment and the handling of process instances in live operation. The implementation of a process specification as an executable process comprises the activity bundles organizational implementation, IT implementation and operation including monitoring. Figure 7.1 shows the classification of these activities in the process management model. Based on reflections on the documentation of elaborated process specifications, the following sections present selected methods for the activity bundles organizational and IT implementation, as well as for operating and monitoring of processes. The activities of the process management model provide the conceptual framework for the implementation of business processes in a working system. As already discussed in previous chapters, the activity bundles represent a certain standard classification criterion, but they can be performed in any order. Each of the phases contains a bundle of activities that are typical for accomplishing the respective tasks. Every business process is in a certain phase at any particular point in time - or in other words, in a certain state: either being modeled, put into effect, executed, or analyzed. The life cycle model thus defines a phase or state space for business processes. #### 7.1 Process Documentation Since an organization's or company's business processes define how products and services are developed, manufactured and delivered, it is useful to document these processes in a structured way. Documentation should be made centrally available to all employees. It must be available at the beginning of the implementation activities at the latest. Business process documentation is the result of activity bundles during preparation measures. Figure 7.1: Classification of implementation activities in the process management model Nowadays, digital documents serve as the standard for documentation. Ideally, the provision of these documents takes place by means of a generally available intranet, with which a basic access control can also be designed. The documents themselves can be, for example, PDF files that cannot be easily changed (possibly also digitally signed), or HTML files that can be viewed via a browser. For smaller organizations and companies, it is usually sufficient to use existing office software for creating and maintaining process documentation. For more extensive collections, the use of specific software, so-called Business Process Management Systems (BPMS), appears to be worth considering. In any case, the chosen form needs to support its users and the management systems based on it in the best possible way. The documents should also contain a unique identifier, a version number and a date. In case a word processor is used, a style sheet is recommended in order to achieve a uniform appearance and uniformly defined structures. In addition, a list with all documents including their history is recommended to provide a general overview. The process owner or process coordinator is responsible for ensuring that process documentation is up-to-date and complete. The process office specifies what the process documentation should contain and provides appropriate templates and explanations. In case a dedicated IT system (BPMS) is to be used, respective training of all employees - depending on their role - is strongly recommended. In the sense of change management, the employees should also be involved in the selection or development procedure in an adequate form. Roughly speaking, the following content for a process documentation has proved to be useful, although not all items are relevant for every business process and additional items might be added where needed: #### 7.2 Linking Elements of the Enterprise Architecture #### 7.2.1 Overview As mentioned in chapters 1 and 2, business processes are part of the enterprise architecture. Enterprise architectures address the internal aspects and structures of a company. They are essentially models of the internal structure of a company and cover not only organizational but also technical aspects, in particular the deployed IT infrastructure. When implementing the process models, it is now necessary to establish the relationship between the process model and the available resources. Figure 7.2 shows the individual steps from a process model to the executable process instance. In a process model, the actors, the actions, their sequences and the objects manipulated by the actions are described. Actions (activities) can be performed by humans, software systems, physical systems or a combination of these basic types of actors. We call them the task holders. For example, a software system can automatically perform the "tax rate calculation" action, while a person uses a software program to perform the "order entry" activity. The person enters the order data via a screen mask. The software checks the entered data for plausibility and saves it. However, activities can also be carried out purely manually, for example when a warehouse worker receives a picking order on paper, executes it, marks it as executed on the order form and returns it to the warehouse manager. When creating a process model, it is often not yet known which types of actors execute which actions. Therefore, it can be useful to abstract from said model when starting to describe processes by introducing abstract actors. A modeling language should allow the use of such abstractions. This means that when defining the process logic, no assertion should have to be made about what type of actor is realized. In S-BPM, the subjects represent abstract actors. In BPMN, pools or swim lanes can be interpreted as abstract actors, while in EPCs roles can be used for this purpose. In the description of the control logic of a process, the individual activities are also described independently of their implementation. For example, for the action "create a picking order" it is not specified whether a human actor fills in a paper form or a screen mask, or whether a software system generates this form automatically. Thus, with activities the means by which something happens is not described, but rather only what happens. The means are of course related to the implementation type of the actor. As soon as it has been defined which types of actors are assigned to the individual actions, the manner of realization of an activity has also been defined. In addition, the logical or physical object on which an action is executed also needs to be determined. Logical objects are data structures whose data is manipulated by activities. Paper forms represent a mixture between logical and physical objects, while a workpiece on which the 'deburring' action takes place is a purely physical object. Therefore, there is a close relationship between the type of task holder, the actions and the associated objects actors manipulate or use when performing actions. A process model can be used in different areas of an organization. The process logic is applied unchanged in the respective areas. However, it may be necessary to implement the individual actors and actions differently. Thus, in one environment certain actions could be performed by humans and in another the same actions could be performed by software systems. In the following, we refer to such different environments of use for a process model as context. Hence, for a process model, varying contexts can exist, in which there are different realization types for actors and actions. In BPMN, the modeler can define for each task separately whether it is a so-called human task, service task or user task. User tasks are performed by humans together with software systems, such as filling out a picking order on the screen. This means that the description of the implementation type in BPMN is part of the process logic. Since in BPMN pools and swim lanes can be interpreted as actors, care must be taken that no contradictions arise with the implementation type of tasks within, for example, a pool. For instance, in case the designer specifies that a pool is only executed by humans, this pool cannot contain any service or user tasks. Since the definition of the implementation type is part of the process logic in BPMN, it may be necessary to create a separate process model for each context. In S-BPM, actors are not assigned to individual activities, but rather the actor type is assigned to an entire subject. In S-BPM, this assignment is not part of the process logic, but is done instead for each process in a separate two-column table. The left column contains the subject name and the right column the implementation type. If there are several contexts for a process model, a separate assignment table is created for each of them. The assignment of the implementation type forms the transition between the process logic and its implementation. Subsequently, it has to be defined which persons, software systems and physical systems represent the actors and how the individual actions are concretely realized. These aspects are described in detail in the following subsections. #### 7.2.2 People and Organizations For each context in which people are involved it is necessary to determine the respective action (activity) holders, and thus the concrete persons or organizational units that carry out the actions. In companies and administrations there are people with different educations, qualifications, preferences, and interests. There are merchants, developers, craftsmen, etc. who will take care of the arising tasks. Organizations can therefore also be described as structured resource pools. Depending on the type and scope of the tasks, an organization forms units in which the respective specialists are combined. There are purchasing departments with procurement experts, or development departments that are made up of several development engineers and other specialists. The relationship between the persons and organizations in the organizational structure and the abstract actors in the processes can be established statically or dynamically. #### Static Assignment In the simplest case, the two-column table already mentioned provides information on which actor defined in the process (column 1) is assigned to which person (column 2). The second column can also contain an organization or organizational unit. Then, all its members are assigned to the abstract actor. Such a specification ensures that in case of illness or vacation any person from the organization can take over the arising tasks of the process. In addition, the workload can also be distributed dynamically if actions from several process flows (process instances) have to be handled. With BPMN, pools, swim lanes and individual actions can be assigned separately to an actor. It is important to ensure that there are no inconsistencies when using all assignment options. If a software system executes a swim lane as an actor and there is a human task in this swim lane, it is not clear what this means. Actors were therefore introduced in Bonitasoft's BPMN-based tool (see [1]). They are placeholders for task holders, to whom, similar to the subjects in subject-orientation, concrete actors are assigned. In S-BPM, a complete subject is embedded into the organization, i.e., the assignment then applies to all actions of this subject. #### Dynamic Assignment In many processes, the assignment of the actors of a process to persons and organizational units cannot be determined statically. For a business trip application, the person handling the request can depend on whether the trip is domestic or international. While the process logic may be the same in both cases, the executor may differ, for example because an employee has special expertise in international travel with visa issues. In such cases, it makes sense to first determine and assign the persons or organizational units involved during execution of the process logic, for example, depending on data values in business objects (in this case, the travel request). For a flexible dynamic assignment of physical actors, tables are usually no longer sufficient, but instead programming language constructs are necessary. [2] shows how such a language can be used to embed subject-oriented Process Models into organizations. Since in BPMN the assignment of actors is possible via pools, lanes and tasks, the description of the assignment is very complex here. Various BPMNbased tools such as Bonita [1] or Activiti [3, 4] integrate processes into the organization by programming this in Java or XML. #### 7.2.3 Physical Infrastructure Particularly in manufacturing processes, physical systems are involved as actors. In this way, blanks can be delivered to a machine via a transport system, the machine processes the blanks, and the processed parts are then transported to the next processing step. If such a machine is regarded as a subject or pool, the delivered parts are modeled as messages to be received and the dispatched processed parts as messages to be sent. The modeler represents the processing step as a single task in a subject or pool. #### 7.2.4 IT Infrastructure Digitalization in an economic system means implementing processes with the most comprehensive support possible through software systems. In corresponding scenarios, computer systems or machines carry out the activities for the most part; human intervention is reduced as far as possible and sensible. Essential aspects thereby are: • The control of the process flow: The sequence of actions described in the process (control flow) are automatically controlled by computer programs. • The execution of actions Actions on data objects can often be performed fully automatically by appropriate computer programs. Software systems are the means to merge the different types of actors. For example, during the control of the process flow, a process engine integrates human and machine operators at runtime in the processing of instances, according to the individual situation. The following explanations deal with the control of the process flow and the manipulation of the associated business objects by Information Technology, although without going into detail about the integration of humans or physical systems as task holders. This will be covered in section 7.2.5. #### Control Flow The process logic corresponds to the control flow logic of a computer program. The exclusive automated execution of the process logic by a computer program is only possible for highly structured processes. All conceivable process possibilities must be covered. No human intervention is planned. To avoid that the computer program's execution logic differs from the described process logic, it is useful if the computer program can be automatically derived from the description of the process logic. The following prerequisites must be met for such cases: The importance of distributed systems, which are already widely used today, continues to grow with the development of cloud and edge computing. This can mean that parts of a fully automated process are executed on different computer nodes of a distributed system. Since these can be based on different technologies, appropriate variants may have to be available for the automatic conversion of a process description into a computer program. Thus, individual subjects of an S-BPM description of a process can run on different computer nodes of a distributed system. This can mean that the program code might need to be generated separately for each subject. This can, however, be avoided if the generated target code is available on preferably all the node types of the distributed system, and the target system has a framework through which the program parts running on different nodes can work together. The software components usually synchronize their collaboration by exchanging messages. Such an enabling programming language is, e.g., Java with the AKKA framework. It allows messages to be exchanged across computer boundaries. [5] has investigated this possibility on the basis of S-BPM models. In BPMN, individual pools can be executed theoretically on different computer nodes. This requires that communication between the pools is possible across computer boundaries. A search for possibilities of a corresponding code generation tool for BPMN models in spring 2018 brought no results. #### Activities and Data In fully digitalized processes, computer systems also perform the activities contained in the process flow. This can be done using functions or services that are already available in existing application programs and can be inserted or called at the appropriate places in the program that implements the process control flow. For the integration of newly developed software for activities, technologies such as web services, REST interfaces, or simple APIs are often used. Database access for direct manipulation of data can be integrated analogously depending on the interfaces of the database systems used. When using S-BPM, data belongs to a subject. Multiple subjects are not allowed to access shared data. If several subjects want to use the same data, e.g., stored in a database, it has to be 'packed' into a subject. This subject receives the corresponding requests from other subjects, performs the desired action, and sends the result back to the requesting subject. In software technology, activities together with their data are called classes. Several such classes can be combined to form services. In IT, this is then called a Service-oriented Architecture. The services are invoked according to the control flow of the process. Tasks in BPMN can be realized through services. Similarly, functions can be implemented within a subject in this way. Microservices [6] are a current concept for structuring software systems. Microservices describe an architecture in which individual small functional building blocks are independently programmed, tested, approved and made available on different technical platforms on demand. Through their interaction by means of interfaces which are independent of the programming language, complex application software can be realized. In doing so, microservices usually communicate by exchanging asynchronous messages. This also corresponds to the concept of Reactive Programming [7], whose main characteristic is the coupling of program blocks by asynchronous messages. In BPMN, pools can be implemented as microservices if all tasks in a pool are executed by the same actor. In S-BPM, subjects are always assigned to a task holder and communicate via the asynchronous exchange of messages. Therefore, a process described in a subjectoriented format corresponds, in view of IT architecture, to a microservice-oriented structure that meets the requirements of Reactive Programming. #### 7.2.5 Combinations of Task Holders Tasks are often not performed by a single type of task holder. With the increase of digitalization, the number of tasks performed exclusively by humans is decreasing continuously. Today, a warehouse worker often receives his picking orders via a tablet connected to an IT system. After he has assembled the goods, he also confirms task completion via the tablet. This action closes the commissioning task. The IT system updates the data and automatically triggers the subsequent task, such as preparing the shipping documents. The picking task is therefore carried out by humans and IT as types of task holders, a combination that is very common in business processes. The IT controls at least the process logic and manages the data while people enter data or operate machines. Machines controlled by small computers with corresponding software are increasingly replacing machines as purely mechanical actors. These embedded systems control the mechanics and handle communication with other machines or higher-level business applications. Communication with other intelligent machines is referred to as horizontal communication, and with business applications as vertical communication. The latter links production systems with business processes and is an integral part of the Industry 4.0 initiative. The following sections describe in more detail how the various task holders are integrated when implementing a process logic. #### Combination of Humans with IT The combination of people and IT in the execution of business processes is the core of their digitalization. The IT at least takes over the control of the process logic, i.e., it arranges the handling of the intended tasks by the respective task holders according to the process logic. In addition, tasks can be carried out directly by IT as task holders without the need to involve people. If a task can only be completed with human support, the software responsible for the control flow prompts the responsible human task holder via an appropriate user interface. This may concern not only the input of data, but also the confirmation that the user has executed a manual action. IT systems as control software and as actors for activities usually also store the data generated during process execution, both the content of business objects, as well as metadata, such as time stamps for the start and end of instances and execution steps. A comprehensive platform for implementing business processes executed by people and IT solutions is called a Workflow Management System (WFMS). The Workflow Management Coalition (WfMC) has developed a reference model for this purpose. Figure 7.3 shows the components and interfaces of the reference model [8]. The interfaces between the individual components are defined as follows: Figure 7.3: Reference model for Workflow Management Systems (WFMS) [8] The essential components in the reference system of the WfMC are the workflow enactment service and the workflow engine. The actions and their sequence are defined in the process definition and controlled by the workflow engine. In a company usually several instances of a business process run simultaneously. A process instance is created when the execution of a business process is triggered by an event defined for this purpose. Process instances follow uniform process descriptions but are executed independently of each other. For example, two different customers can place an order. Each of these individual customer orders creates an independent instance of the business process "order processing". The workflow enactment system uses workflow engines to manage the individual instances. A workflow engine provides the execution environment for a workflow instance. Its main tasks are: A workflow enactment service is a service that starts, manages and executes one or more workflow engines. Workflow systems use application interfaces to access the functions of application systems that perform tasks defined in a process. A worklist handler is a component that involves users in a process. It can be part of a Workflow Management System or defined and programmed by a workflow expert. Through the worklist handler the involved actors know which tasks they have to perform in which process instance. Workflow functions can be embedded in other common applications, such as an e-mail program, so that users have a uniform user interface when handling process instances. For such an integration, there must be a communication mechanism between the workflow enactment service and the other applications. The reference architecture also provides an interface for administration and monitoring. The software connected there serves to monitor the simultaneous execution of different instances of several business processes. On the one hand, this monitoring refers to the functional level of the processing of business cases, for example with the recording and evaluation of response and processing times (see section 7.3.3). On the other hand, the IT systems used must be monitored at the technical level, for example, with regard to load and malfunctioning behavior. Most workflow systems currently available on the market only support orchestration, i.e., a single control flow of tasks. For BPMN, this means a workflow system can only execute the functions in a single pool. If several pools are used in a BPMNbased process description, a separate workflow engine is required for each pool. These workflow engines must then as a rule be connected by programming via Interface 4. Especially if a process should be executed on a distributed infrastructure, such structures can become complex and cost-intensive, as many software vendors charge license fees for each installed instance of the workflow engine. This increases not only technical complexity but also the costs. Subject-oriented process descriptions are process choreographies, i.e., the subjects are independently executed in parallel, without central control for all subjects. To coordinate their individual activities, the subjects exchange messages asynchronously. An execution environment for subject-oriented defined processes is therefore, strictly speaking, a so-called multi-workflow system. Each subject corresponds to an orchestration performed by its own workflow system. The asynchronous exchange of messages between several workflow systems is realized in Business Subject-oriented Process Management using the input pool concept. A number of multi-workflow systems have been developed for S-BPM (see [9]). #### Combination of Physical Devices and IT: Cyber Physical Systems In a process, actors can also be machines that perform tasks in a purely mechanically or electrically controlled way, and fully automated without human intervention. Machine control is carried out mechanically, electromechanically or, in the case of the embedded systems already mentioned, increasingly by computer and software systems. Sensors monitor the physical condition of the machine and ambient conditions, and actuators such as servomotors, switches, controllers, etc. intervene with the operation of the machine. The control software largely determines what happens on and at intelligent machines and defines their vertical and horizontal communication with other systems. Machines communicate with each other not only by exchanging data, but also by forwarding and receiving physical artifacts. The message "workpiece to be deburred" can be realized by automatically transporting a workpiece from a metalcutting production machine to a machine that deburrs edges. At the same time, the deburring machine can receive additional messages that contain a more detailed description of the workpiece and specify the type of deburring. Combinations of physical and Information Technology components are referred to as Cyber Physical Systems (CPS). Since they bring together production and business processes, they are of particular importance for Industry 4.0 applications. In BPMN, the modeler can model a task executed by a combination of humans and IT as a service task and describe an intelligent machine as a separate process in a pool. If there is a corresponding number of machines working together in a complex manufacturing situation, the use of numerous pools in BPMN can lead to representation problems. The reason lies in the horizontal arrangement of the pools: In case of a larger number, the message edges inevitably have to cross pools, which can be confusing. In S-BPM, on the other hand, a machine is always modeled as a subject, since the decision as to which type of task holder will execute the subject is first made in the implementation stage. If a physical task holder is used in a process, there cannot be an arbitrary number of parallel instances of this process. A physical unit cannot be split logically, as is possible with IT or human actors. #### Combination of Humans, Physical Devices, and IT There are also scenarios in which people carry out certain tasks when intelligent machines are used. For example, a machine receives the workpiece via a transport system together with the associated information as a message, e.g., contained in a RFID device. Together the machine and an operator carry out the intended tasks in accordance with the information contained in the RFID device. The operator confirms the completion of his work via a user interface. The workpiece is then dispatched and the updated accompanying information is transmitted to the other parties involved. In such situations, the aim is to reduce the share of human labor and replace it with more sophisticated mechanics or improved embedded systems. This does not necessarily mean that changes to the process logic are required. #### 7.3 Execution and Monitoring #### 7.3.1 Putting the Process Into Operation After the definition of task holders and the implementation of the activities, the process has to be put into operation. To this end, the task holders must be prepared by setting up the physical and IT infrastructure and by training the human task holders. IT-based task holders are loaded with the respective programs and the mechanics of machines are configured accordingly. During the go-live phase, it is important to establish the link with other processes. In a company, processes are integrated into a coherent network of processes. There should be no isolated processes. If, for example, a new process for shipping preparation is introduced, it needs to be linked to the order acceptance process. With communication-oriented process descriptions, this is done simply by exchanging messages. Another possibility is shared data, i.e., processes write data required by other processes into a shared database. After the preparatory work, the process should be tested as an overall construct. An advantage would be a test environment that represents a realistic image of the operational environment. However, for cost reasons this is often not possible. Irrespective of that, it is advisable to introduce the process step by step. It is an advantage if a process is described and implemented as a loosely coupled system of communicating task holders. The individual strands of action for the actors can be put into operation step by step. Since they are only loosely coupled to each other through asynchronous message exchange, other task holders can be initially simulated easily. With BPMN, pool by pool is thus put into operation. However, if the pools are very complex and include several swim lanes, the go-live phase is also complex. When using S-BPM, a system can be built by gradually adding the individual subjects and their behavior. If modelers do not use several pools in BPMN, i.e., they define and implement a process exclusively in a control flow-oriented way, it can only be put into operation as a whole. In the case of choreographies, i.e., when using subjects or several pools, the process can be executed, even though, for example, one pool contains software which is still faulty. The messages sent to this pool can alternatively be received and worked on by people, and the result sent back by them as a message. The other pools recognize the described logic and flow (i.e., the observable behavior), although it is not yet implemented in its final form. #### 7.3.2 Process Instances The actual execution of a process is referred to as a process instance. A process instance is created when the start event occurs. This can be a call from a customer who wants to order a product. In this case, a telephone salesperson explicitly creates an instance by starting the digital ordering process and entering the necessary data. When ordering in an online shop, the customer enters the order data himself and creates an instance as soon as he confirms the purchase. In both cases, an instance then runs through the processing steps and positions defined in the process logic. Since a company normally has several purchase orders at any particular point in time, several independent process instances, which are in different processing stages, exist at the same time. Employees of a company are usually involved in several process instances and carry out the tasks assigned to them in these instances. They allocate, so to speak, a time slot of their working time to each process instance. This happens analogously with IT systems. The capacity of both the human resources and the IT systems can thus be divided into time slots in order to process several instances of the same process, or different processes. This cannot be readily done with physical or cyber-physical systems. Such a system can only be involved in one instance at a time. A machine cannot be instantiated several times, it is only available once. Only after a machine has completely finished a task or task sequence in a process instance, can it work for another process instance. A physical system is therefore assigned to exactly one process instance at a particular point in time. This fact is important when processes that can be easily instantiated because they do not contain physical task holders are linked to processes that contain physical components. A manufacturing system almost entirely consists of physical or cyber-physical task holders. A process running in such a system is instantiated just once and exists as an instance until the whole production environment is switched off. A physical task holder cannot work in a time slot for a process instance 1, then do something for a process 2, and then continue working on process instance 1. The actions of a physical task holder for different process instances would not be independent of each other. If, for example, a valve for process instance 1 is to be half opened and then fully opened for process instance 2, the valve is of course also fully opened for process instance 1, which should not happen. If a machine is assigned to a task in BPMN and the assigned pool can be instantiated several times, the machine must always be assigned to a single instance at runtime. The machine must always know which instance it is working for, in order to retrieve the correct data from the memory common to all instances, and then take the work piece to be processed out of the work piece container. When using S-BPM, the machine is assigned to the corresponding subject. The latter receives the work piece and the accompanying data as a message. The data also contains an identification of the associated higher-level process instance, usually the order number. With this, the machine knows for which process instance it is now working. The message by which a machine declares its work finished is transmitted to a subject instance of the triggering process instance. #### 7.3.3 Monitoring In day-to-day business, process participants execute business processes according to the modeled design in the environment created during organizational and IT implementation, and consisting of personnel and technical resources. Each business transaction runs in this execution environment as an instance. Transaction systems such as Enterprise Resource Planning (ERP) applications or workflow engines record the behavior of the instances in the form of entries in a log file (event log, audit log). A log data record contains, among other things, a unique instance identifier, a partial step identifier and time stamps for the start and end. This results in raw data for the calculation of Process Performance Indicators (PPIs). Reliable management information based on such key indicators is the prerequisite for continuous adaptation of process design with respect to increasing the degree of target achievement. The periodic ex post facto evaluation of a large number of instances over longer periods of time such as weeks, months, quarters, etc. primarily serves to identify structural improvement potential, for example with regard to scheduled personnel deployment, process logic, or the degree of IT coverage. This traditional monitoring and reporting follows the concept of Business Intelligence with the principle 'store and analyze' and the methods of data and process mining. The resulting changes are primarily of a medium- and long-term nature. In order to meet the increasing real-time requirements, traditional process monitoring is supplemented by Business Activity Monitoring (BAM), which evaluates event-driven data almost in real time, reports results promptly, and thus enables short-term, instance-related measures [10]. An example is the prioritized processing of an ordering instance of an "A" customer once the system recognizes and reports that it lags behind the usual processing progress at a measuring point, and therefore the promised delivery date cannot be met (predictive analysis). BAM uses the concept of Complex Event Processing (CEP) with the principle 'stream and analyze' and stream mining methods. This means that the system constantly searches for patterns of complex events in the stream of recorded individual events (e.g., set time stamps, passed measuring points), which only become relevant for certain purposes by linking the individual events. A typical example is the recording of two transactions with the same credit card in Hamburg and New York. These simple single events (low level events) are registered in the event stream as normal events. The CEP system only combines them into a complex event if both transactions take place within a short period of time, in this example about 3 hours. In this case, an event pattern of geographical distance and time frame would lead to classification as a complex event of "assumed credit card fraud". Business activity monitoring is intended to monitor a large number of data sources permanently and simultaneously. Event data generators include applications that execute process instances (e.g., ERP, CRM, workflow engines), and which provide other information from inside or outside the enterprise, such as surrounding conditions, weather and traffic data. Increasingly, this also includes (sensor) data produced by smart phones and devices that are part of the Internet of Things. BAM analyzes and aggregates the flood of data using defined rules and transmits the results to entitled and interested recipients. Figure 7.4 juxtaposes traditional and Business Activity Monitoring. The activities listed in the left column serve as attributes for comparison [11]. The timing and type of exploitation of the recorded/registered data depend on the design of the monitoring and reporting according to the user requirements. Utilizing the pull principle, the user can retrieve the desired evaluation at any time. According to the push principle the system generates evaluations on a time-controlled basis, e.g., daily, weekly, monthly, quarterly at specified times, and informs the predefined recipient group accordingly. If limits or tolerance thresholds of Process Performance Indicators of individual instances are exceeded at runtime, alarm messages can also be transmitted by push to those responsible, or other processes can be started, e.g., an extensive escalation procedure with corrective measures for the case in question. Figure 7.4: Properties of Traditional and Business Activity Monitoring Management cockpits containing dashboards dominate for the presentation of evaluation results. These usually include representations in the form of tachometers, traffic lights and bar or pie charts. For space-saving displays with high information density, word graphics such as spark lines or bullet graphs are also used. Examples of evaluations and their presentation are: Figure 7.5: Instance report (overview) Figure 7.7: Instance report (current processing status) #### 7.3.4 Process Mining Process mining is a special form of evaluating process data. Log files are thereby used to extract process-related information from the transaction data of central IT systems (e.g., ERP, CRM and SCM), and thus visually reconstruct and analyze the actual process flow. This allows insights to be gained into the actual behavior of executed process instances [12]. Hence, process mining concepts and tools deliver valuable information for analysis and continuous improvement of processes (see section 1.3.5). The most common and frequently analyzed business processes are purchasing, sales, accounts payable and accounts receivable, as well as ticketing systems (e.g., in IT service management). An event log contains sequentially recorded events (trace) with attributes such as case ID, activity, timestamp, resource, and business objects (data elements). Depending on the process, additional information can be added, such as the name of a customer or vendor, order quantity and value, delivery quantity, etc. Figure 7.9 shows an extract of an example of an event log. Combining such protocol data for process execution with process models allows identifying three essential types of process mining approaches [12]: • Discovery: Algorithms reconstruct the actual process flow and its manifold variants from the log data (without additional information). This allows the Figure 7.9: Extract from event log (Image taken with permission of Celonis SE (www.celonis. com)) formal description of processes that are already in day-to-day operation ('lived') but not yet documented. In particular, the conformance check should be carried out as part of Business Activity Monitoring during the runtime of instances, in order to promptly detect violations and mitigate their consequences, e.g., stopping a bank transfer in the event of irregularities in the release of payments. The following example is taken from the process mining tool provided by Celonis SE (www.celonis.com). It shows selected mining results for an ordering process (purchase to pay) that is handled by an ERP system. The analysis of the associated event log for a given period has produced the following information (see Figure 7.10): Figure If the scope is expanded to include the seven most frequently occurring process variants, 83% of all instances are covered. The process graph now also shows the seven paths with the corresponding frequencies (see Fig. 7.11). Alternatively, the average duration of paths and state transitions can be displayed. With regard to the process flow, it can be seen that a considerable number of instances (18,938) begins with the scan of the invoice and only then is an order created in the system (see CaseID 10003 in Fig. 7.9). This is an indication of so-called maverick buying, i.e., departments procure something without prior involvement of the purchasing department. In a next step, this usually undesirable process behavior can be specifically investigated in order to eliminate its root causes. With the display of complementing information, processes can be examined in greater depth. Figure 7.12, for example, provides information on the number of order instances distributed over months with the respective values as well as their distribution among suppliers (Fig. 7.12). The presented analyses cover only a small part of the extensive capabilities of the Celonis environment. In addition to the default evaluations provided by the system, users can define their own evaluation scheme on the basis of their individual data models by using a large number of existing analysis components, such as various diagram types, Process Performance Indicators, and OLAP tables. Furthermore, the software allows executing individualized process evaluations with the help of the Process Query Language (PQL). Additional functions allow, for example, the comparison of the actual process flow with the one intended in the model (conformance check), or the parallel visualization and thus the comparison of different behaviors of the same process, e.g., in two branch offices (benchmarking). The future of process mining lies in the combination of process analysis with intelligent algorithms. For example, the Celonis Proactive Insights Engine (Pi) integrates machine learning and Artificial Intelligence techniques, enabling automated identification of process weaknesses and their causes for users [13]. Building on this information, the user can retrieve intelligent recommendations for process improvements. Given the described features, process mining plays an important role in the context of Robotic Process Automation (RPA), in particular when it comes to assessing RPA potential of processes and developing respective applications [14]. #### 7.3.5 Continuous Improvement The aim of monitoring, evaluating and reporting process instances is to generate and provide management information on process behavior. The addressees can analyze it, investigate the causes of deviations from target values and derive short-, mediumand long-term needs for action and restructuring measures. In the following we discuss typical changes in process design, the goal of which is to have positive effects on process behavior with respect to optimization. For illustration we refer to the loan application example in chapter 5. Parallelism and overlapping execution. Logically and technically independent activities can be executed completely or partially simultaneously and by different task holders. Although the number of different activities can increase as a result of the splitting, executing these in parallel often accelerates the process. For example, a review step in loan application processing could be broken down into the creditworthiness check for the customer and the value check for the object to be financed, and these could be run in parallel. Aggregating activities. Aggregating as the opposite of splitting means that activities that were originally carried out separately and by different task holders are now carried out by a single task holder. This reintegration of tasks reduces the division of labor and thus, for example, reduces interfaces. The number of activities in a business process and its model decreases as a result of the grouping and the sequence of steps in the relationships between the activities change. In the credit application process, the (re-) combination of the two checking steps could again be reasonable, in case the automation of the customer creditworthiness check previously described outweighs the time gained through parallelism. Changing the sequence. The sequence of events in relationships between activities, or between groups or bundles of activities, can possibly be reversed, which may have advantages in terms of time, cost or capacity. In the loan application example, the creditworthiness check should be accomplished before the value check of the object to be financed, because the latter is not necessary, or the entire process ends, if a potential customer is not creditworthy. The sequencing of checks contrasts to running them in parallel. Therefore, in order to choose one of these variants, it would be helpful to know how often an application is rejected due to a lack of creditworthiness, and thus how many resources are wasted through a parallel object value check in these cases. Elimination of activities. Verbal discussions, process mining, path analyses, and simulation experiments can reveal activities that are not needed (dead paths), activities that are very rarely carried out, and activities in which hardly any value is added or that are inefficient. The number of activities of a process decreases due to elimination and the structure of the relationships change. An example of eliminating low-value-adding activities could be the omission of an additional credit application approval by the department management for amounts over €200,000 as mentioned in section 5.2. Elimination or reduction of cycles. When business transactions are iterated along cycles of activities, the lead times of processes are generally increased, which often leads to waiting times during execution. With path analysis, such cycles can be identified and localized, and possibly eliminated by changing the process design. During loan application processing, for example, the loan officers may repeatedly have to ask the prospective customer for information because there is a lack of sufficient details to assess the financing project. If the electronic application form requires input (mandatory form fields, annexes, etc.) for such information, the workflow system can prevent the submission as long as information is missing. Although this does not ensure that the applicant will provide the correct information in a complete and valid manner, the probability that he will do so increases, and the number of enquiry loops necessary due to missing details will most likely decrease. Insourcing and outsourcing. Under certain circumstances, it may make sense to have entire business processes, or parts of them, carried out by specialized external service providers rather than by the own organization. External partners might accomplish tasks in a more cost-effective way and/or much faster, e.g., due to economies of scale. In this way, fixed costs can often be replaced with variable costs through the use of partner services. For example, instead of employing its own experts for property valuation, the bank could, on a case-by-case basis, engage an architectural firm. If the right conditions are in place, the reverse route can also bring advantages, for example in case previously outsourced activities can be organized more economically internally, e.g., because interfaces and transaction costs are no longer necessary. Automating. Technical progress opens up possibilities to have manual work steps supported by IT applications and machines, e.g., robots, or to have them executed completely automatically. This is particularly relevant for time-consuming, less motivating and error-prone activities. In order to implement automation options, the development and market for corresponding technologies needs to be continuously monitored. In doing so, suitable solution modules can be identified and included in process adaptations. As an example, the credit bureau's web service can be used for obtaining customer information. Its integration not only fosters automation but is also an example of partial outsourcing. Reduction of interfaces. Naturally, process execution based on the division of labor has interfaces on the organizational and technical level. It involves various organizational units and external partners, who often use heterogeneous IT systems and tools to generate and exchange intermediate results which are sometimes linked to different media. The consequences are media disruption and associated duplication of work effort, transmission errors, loss of time, costs, etc. Reducing the number of interfaces counteracts these deficiencies. It can be achieved through organizational changes such as reintegration or elimination of activities and changes in the allocation of tasks to task holders. On the technical level, integrated IT systems such as Enterprise Resource Planning software and workflow applications are helpful. In the loan application example, the partial transfer of signature authority to the processing level has changed the tasks assigned to the clerks and the department management. As a consequence, one branch of the process flow could be eliminated, and the corresponding set of interfaces reduced. Since many of the mentioned optimization approaches are mutually interdependent, the effects of a measure on other design factors must always be taken into account. For example, outsourcing of process parts can increase the number of interfaces and increase efforts for service management. These effects counteract the advantages of outsourcing and always require an assessment of associated advantages and disadvantages. #### References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Industrial Use Case 8 The following case study shows how some of the previously described BPM concepts, methods and languages were used to improve a core process of a company. On the basis of the individual aspects of the open BPM cycle their instantiation and intertwining will be demonstrated as occurring in a real industrial development project. In the following, the course of the project is described in a structured way. The respective activities of the bundle of activities in Figure 8.1 are noted in the margin of the use case description. The activities of each activity bundle are not only carried out with a high degree of overlap, there are also frequent shifts with respect to the considered focus throughout the project. ### 8.1 Background and Setting ENGEL is a traditional Austrian manufacturer of injection moulding machines and was founded in 1945 by Ludwig Engel. Following the introduction of the first injection moulding machine in 1952, ENGEL had developed into the world market leader by 2016 with a total sales of 1.36 billion euros. The fully owner-managed company employs around 5,900 people worldwide in 9 production plants and over 85 branches [1]. ENGEL is a strongly customer-oriented company with a focus on flexibility and innovation. Analysis: Determining the Key Performance Indicators based on the business model and strategy The strong orientation toward customer needs and the ongoing development toward shorter delivery times has led to the definition of a company-wide goal: Figure 8.1: Shift between activities in the sample use case reduction of the total process lead time for all variants of a standard component for injection moulding machines by 30%. A project team was set up to survey and analyze the existing process and to implement the necessary improvements. The first step in any optimization process is to capture the actual status to gain more detailed knowledge of the material and information flows, and of any factors that may affect the process. At the start of the project there was hardly any explicit information about the overall process, the detailed process steps, or the process actors involved. Initially, it was only known that the process involved two production sites in two different countries (Factory A and Factory B) and three relevant product groups: Analysis: A look at the "world" as it is. Analysis&Modeling: Outline the current process Figure 8.2 Supply chain between factories Figure 8.2 shows a rough diagram of the supply chain. The triggering event is the order submitted to Factory A from the production department (as internal customer). Factory A saws the blanks and then delivers the necessary parts (Product 3) for Product 2 to Factory B for assembly. The intermediate Product 2 is delivered to Factory A for assembly and then subsequently delivered to the internal customer (production line for the injection moulding machine). This process is controlled by orders that are exchanged between the factories. When an order arrives at a factory, it is entered into the factory's ERP system, and a production order is created with a corresponding delivery date. An order is not productively effective until this entry has been made. If the process up to the entry takes longer than 2 working days, the production order does not reach production on time because the order lead time is set to two working days. In the initial situation, approximately 95% of all orders between the factories were entered too late, i.e., more than two working days passed between the arrival of the order in the factory and the actual entry. These orders then had to be processed manually with enormous additional effort, which led to an internal delivery reliability for Product 2 of only 39%. This in turn resulted in problems with production planning in Factory A and further delays in the production of Product 1. Analysis: First rough planning and diagnosis of enterprise architecture: No fundamental changes to the organization and the IT In order to achieve the specified goal of reducing the lead time by 30%, a time frame of only 10 weeks was specified. The two factories were located in two different countries with different languages. The tight timeframe led to further restrictions for the project; new software solutions or technologies could not be introduced on-the-fly. The introduction of such far-reaching changes is a strategic decision and requires a lot of manpower, budget, risk management, and time. This meant that changes to the existing processes had to be implemented within the existing organization and IT environment. Only after achieving the original project's objective it would be possible to implement further measures for additional improvements. #### 8.2 Implemented Measures #### Analysis: Process analysis and selection of a modeling language Apart from a superficial description of the material flow between the factories, no explicit process information was available to the project team. Since proper process documentation is essential for understanding the overall process and identifying optimization potential, the next step was to document and analyze the already established production process using Value Stream Analysis (VSA) [2, 3], i.e., the standard tool for documenting production processes in the company. For an initial illustration of the process, it was necessary to select a suitable representative product which covered the basic production steps and most of the material flow. The project team decided to use a variant of Product 2 for a first as-is analysis. This selection was based on an ABC analysis of all product variants and the corresponding work plans. The variant selected for Product 2 accounts for 30% of total production. It had the most complex work schedules and the highest total lead time of the three defined product groups. #### Analysis&Modeling: Modeling of a part of the as-is process By tracking the material flow at both production sites, collecting relevant KPIs (inventory levels, production lead times, customer cycles, etc.), and interviewing the responsible employees, the project team was able to create a Value Stream Model (VSM) for Product 2. Figure 8.3 shows the VSM of the production process and its hierarchical structure. #### Analysis&Modeling: Identification of optimization potential The results of the Value Stream Analysis were as follows: Figure 8.3 Value Stream Analysis of the production of "Product 2" #### Analysis&Modeling: Detailing the process model In addition, the project team could obtain more detailed information on the ordering process between the factories and extend the process description accordingly: The Value Stream Analysis enabled the project team to identify two potentials: The lack of production synchronization and the non-optimized order processing. However, production synchronization is directly linked to the respective production planning process. During a short analysis of the planning process the project team came to the conclusion that long-term improvements would only be possible if the procedures in the planning process were completely reorganized and restructured, and the mindset in itself was changed. Although this would have been a necessary modification, the project team could not implement it within the timeframe of the project. The team members therefore focused on the ordering process and its optimization potential, as they identified it as a possible 'quick win' for their project. #### Analysis&Modeling: Modeling still missing aspects This was the starting point for a comprehensive process survey. However, the VSM still lacked relevant process information to describe the overall process and the corresponding information flow in detail, such as, Analysis&Modeling: selection of an additional modeling language Since the VSA focuses on the production processes and especially describes the material flow between the process steps, the project team quickly realized that it needed a different approach. They had to use an additional method to describe the flow of information in a level of detail that allowed an accurate analysis to be conducted. The next step was to create a process model of the people and SAP systems involved in the process and their respective interactions and information flows. To supplement the company's existing VSM, the project team introduced Subject-oriented Business Process Modeling (S-BPM) [4]. The project team chose S-BPM because of past experience and the problems they had had with flowcharts and swim lane diagrams, which are occasionally used alongside the VSA. Analysis&Modeling: Rationale for the addition of Subject-oriented Modeling In previous use of swim lanes, the process models either provided an overview of the process (and were not detailed enough for a thorough process analysis), or the process models were so detailed that it became very difficult to keep track of them. Furthermore, in the team's experience, swim lane models are not suitable for visualizing the individuals involved and their interdependence in a transparent way, especially if there are more than five or six participants. Experience has also shown that the people involved in the processes, their individual approaches, their knowledge and their experience are a decisive driving force for the processes of a company and are indispensable for successful processes [5, 6]. Since the way in which the flow of information between process actors is organized has a significant impact on business process performance ([7], the project team focused on the S-BPM representation of processes. It concentrates specifically on the point of view of the involved process actors, so-called "subjects", and their interaction in the process environment. A subject can be a machine, an IT system or a person. Subjects are abstract agents without any indication of how they are finally implemented. Analysis&Modeling: Description of S-BPM and tools used The S-BPM modeling method uses two levels to describe processes, the Subject Interaction Diagram (SID) and the Subject Behavior Diagram (SBD). The interaction between the subjects is visualized in the SID, which describes the exchanged messages between the involved parties (subjects). The SID provides a process overview and helps to identify the role of a subject in the overall process. The Subject Behavior Diagram describes the individual process steps of a participating subject's role in the process. The behavior of a subject is described by three different actions (the so-called "states" in S-BPM) it performs: "send", "receive", and execute an internal task ("function"). Since the relevant part of a process for a process actor is encapsulated within the respective subject, each SBD represents an independent actor within the process. It is not necessary to model the whole process at once or in a strict sequence, e.g., if information is missing or not available, or if part of the process is not relevant for the task at hand. The S-BPM notation consists of five symbols which are defined by their meaning and not by their form, although recommendations exist in associated literature. Two symbols are used in the SID to represent a subject (e.g., a rectangular shape) and the interaction (e.g., an arrow), and three symbols (most often rectangular shapes in different colors) in the SBD to represent each state type (send, receive, function). One advantage of the two levels and such a simple modeling notation is the possibility to model processes simultaneously in a top-down and/or bottom-up approach, whereby the traditionally separate areas of Business Process Management and Lean Production [8] are combined dependent on the various demands and requirements, and on the available depth of detail of the information. Although there are dedicated software solutions for modeling S-BPM, the project team used their own individual MS Visio template. This allowed them to immediately focus on process modeling and analysis without investing resources and time in the application of an external technical solution - a common mistake in many companies when process modeling is implemented [9]. Based on more detailed personal interviews, the project team created a model of the first level of the process, the SID. The resulting process model of the current situation showed, as expected, that the logistics and production process were very complex. Approximately 40 subjects were involved in the production, production control and logistics of all three products in both factories. #### Analysis&Modeling: Detailed modeling using S-BPM The final version of the resulting as-is SID and the notation used are shown in Figure 8.4. The SID shows the general communication structure of the process and which subjects exchange which messages with each other. The concrete names of the subjects or the content of the exchanged messages are not relevant for the further understanding of the implemented measures. The rectangular shapes represent the various subjects involved in the process, and the arrows represent the messages exchanged between the subjects. In order to distinguish between the two factories, the project team marked the corresponding subjects in green for Factory A and in orange for Factory B. In addition, they marked the subjects that represent SAP systems with hatching to highlight the already digital parts of the existing process. Although both plants use the same ERP system, the project team dealt with it separately according to the respective departments for a more structured visualization (SAP System A, SAP System B, and SAP System A Dispatching). Figure 8.4: Communication structure (Subject Interaction Diagram) of production and the ordering process Optimisation: Identification of further optimization aspects Due to the number of subjects involved and the complexity of the entire process, it would not have been practicable to collect and model all subject behaviors for the next steps without a defined framework. To define such a framework, the project team used the now existing SID to identify and analyze the main nodes and bottlenecks of the process with respect to the corresponding products (Figure 8.5). The most striking part of the process was the order processing itself. The processing of the order for Product 1 by Factory A, the order processing and the procurement of Product 2 by Factory B, and the production of Product 3 in Factory A employed up to 12 subjects (3 SAP systems and 9 persons) and took up to 15 working days. In addition, only 65% of Product 2 was completed on time because order processing took too long and orders were received too late at the production center (approximately 95% of all orders). This had a direct impact on the production of Product 1 and on process stability. The delivery times could only be met with a lot of effort in production. Figure 8.5: Relationship between the communication structure and the corresponding products The project team decided to focus on this material procurement process because the process itself is very complex and time-consuming relative to the complexity of the components provided (Product 3). The project team defined the scope of the process survey as follows: Validation: Validation of the process model The project team examined the relevant process steps by interviewing the involved employees in individual interviews, accompanying the employees during the process, and at the same time modeling the Subject Behavior Diagrams (SBD) in the presence of the interview partners. This enabled the project team to describe the process flow for Figure 8.6: Communication structure (SID) of the process for the production of Product 3 Product 3 in detail (see arrows in Figure 8.6), document detailed information about the SAP ERP system and the transactions used, and enable the interview partners to directly accompany the process modeling procedure while verifying the model. Analysis: Further process modeling and as-is analysis of process implementation After the SAP transactions were clearly described in the SID and SBD and a dummy request was tracked through the system, the project team specified the different steps of the SAP system. This allowed the team to distinguish between automated and manual steps, validate the verified process model, and document actual process lead times. For the SBD rectangular shapes and different colors were used to represent the three states: red for the "send" state, green for the "receive" state, and yellow for the "function" state (see also Figure 8.7). Figure 8.7 visualizes the process behavior of an employee who processes production orders in Factory B. This employee checks whether production plans exist for planned production orders. Subsequently, all planned production orders with available production plans are combined according to a defined set of rules and Figure 8.7: Example for a behavioral description of an employee released for production. The employees do this manually for each production order, with several thousand orders per day. Product 3 alone causes a total workload of approximately 7 hours per day. Validation: Identification of optimization potential on the basis of the as-is model The total hours worked for the entire survey, all interviews, and the time required to complete and review the process models was approximately 200 hours. This is a relatively low effort compared to other process optimization projects in view of the complexity of the process models and the depth of detail investigated. The now available detailed knowledge about the subjects involved enabled the project team to create a time schedule for the process based on the collected data and the data documented in the SAP system (order times, delivery times, etc.). This schedule includes all organizational and production steps and their respective lead times. For example, the lead time for one of the Product 1 variants, from order acceptance in Factory B to delivery of the finished Product 1 to the assembly line in Factory A, was approximately 30 working days (see Table 8.1). #### Organisational and IT-Implementation: In cooperation with the employees, the existing work plans were revised, updated and improved. These changes led to shorter lead times for the individual work steps, as well as to a reduced number of work steps as a result of merging existing steps. In this case, a reduced number of work steps means fewer subjects as well as fewer behavioral states. During the analysis, the project team identified several similar process steps that were carried out differently in Factory A and Factory B. In one factory, necessary process steps were executed manually, while the same steps were executed automatically by the SAP system in the other factory. In addition, existing automated SAP batch jobs were interrupted because the required manual input between jobs was missing. These jobs were scheduled at two defined times during the working day, and if manual input was missing at that time, the entire order would have to wait up to a full working day. This could happen several times with each order for different jobs, which could ultimately lead to a delay of several working days. The subjects described provided precisely defined processes that describe all relevant process steps in the SAP system, all required SAP transactions, who executes these transactions, and the interaction between the system and employees. This detailed process documentation allowed the company's IT department to directly implement relevant subject behavior to create new standardized, digitalized, and automated processes, as well as enabling them to revise process steps and streamline the processing schedule of existing batch jobs for both factories. This included steps such as order acceptance, order entry, order opening, order release in both factories, and delivery of the production documents to production. The automated order processing enabled the company to process Product 3 in Factory A on an order-related and timely basis, which in turn allowed the introduction of KANBAN inventories with defined critical parts, the reduction of the inventory of non-critical parts, and the shipment of externally purchased parts directly to manufacturing. Table 8.1: Time consumption for as-is process #### 8.3 Achieved Results #### Operation and monitoring: Measured Key Performance Indicators One of the achievements was a new warehouse strategy and a reassessment of the inventory, which made it possible to revise the entire inventory and implement a KANBAN system for critical parts of Product 3. The newly created KANBAN inventory and the higher value of the affected parts led to an overall increase in fixed capital of approx. 209%. However, this had only a minimal influence on the existing stock value of approx. €10,000 in total. This new strategy increased availability and reduced delivery times for all parts purchased from external suppliers. The interruptions in the production of Product 2 due to missing parts could originally last up to 15 working days. After the changes, all required components were available within one working day either directly on site or via the supplier's safety stock - an enormous improvement in process stability and a reduction in rotating stock compared to a relatively small increase in inventory. Value Stream Analysis is an established tool in the company for the analysis of production processes. Although associated literature ([3, 10]) and external consultants have often highlighted VSA's ability to describe not only material flows but also information flows, the company's expectations were not met when trying to document and visualize these. When most relevant information is available, the administrative processes and information flow can be described with a Value Stream Model. Based on the project team's experience, however, a VSA is not suitable for a representation of the flow of information with partially abstract information. S-BPM provided the project team with an easy-to-learn modeling notation that can still provide very accurate and detailed process models. The employees involved were able to independently understand, read and correctly interpret the S-BPM notation and began to verify their own process models (subject behavior) without the input of the method specialists. This led to a high acceptance of the process survey and the subsequent changes in the process, as the employees were directly involved in the documentation and the optimization steps. The restructuring and digitalization of previously manual process steps has led to a standardized process and a reduction of the subjects involved from 12 to 8 (see Figure 8.8). Fewer subjects mean fewer interfaces in the process, which in turn reduces process complexity and increases process stability and transparency. In addition, employees were freed from time-consuming and repetitive tasks. The increased degree of digitalization and the newly planned process has led to a new process lead time of 2 days for order processing (originally 5-10 days). Thanks to the detailed and clearly defined process, the company's IT department was able to implement the process changes in the existing system environment within just 3 working days. The production and shipping process for Product 3 was reduced to 3 days, from 5-6 days originally. This means that the project team was able to reduce the total lead time for Product 3 from 11-15 working days by 87% to just Figure 8.8: Communication structure of the updated process for Product 3 2 working days. These changes led to an increase in on-time delivery for Product 3: delivery reliability rose to 89% just four weeks after implementation and to 97% after one year. The relatively long period of time needed to process orders for Product 3 in the initial phase meant that most orders arrived at Factory A too late or at very short notice. The newly created automated SAP processes resulted in faster processing of Factory A orders within the associated Factory B departments. This in turn led to a shorter ordering time for Product 3 and an earlier start of production for other components required for Product 2. The result was a reduction from an initial 95% of orders registered too late to only 12%, which again significantly increased process stability and quality, and reduced the need for troubleshooting in both factories. The total lead time of the production and ordering process for Product 2 could be shortened by 7 working days (approx. 38%), from 19-23 days to only 12-14 days. The conversion of manual work into automated, digitalized processes running in the SAP system has led to a reduced workload of the employees involved from 5-6 hours per day to as little as one hour per day. The employees now only have to manually process the purchase orders for very specific components or special cases that could not be covered by the SAP system. The effects of these changes add up to a calculated process cost reduction of around €65,000 per year. The implemented improvements and corresponding changes at the process level reduced the lead times of Products 2 and 3 and led to a shortened overall lead time for Product 1: from initially 26 to 33 working days to 18 to 20 working days. This is a total reduction of approximately 60% for the entire ordering and production process (see Table 8.2 and Table 8.3). Not only was the project team able to achieve the original target of a 30% reduction in lead time, but rather to more than double this reduction by digitalizing and automating the process steps and the corresponding information flow. In addition, this has led to a reduction in rotating stock with a total value of several hundred thousand euros over the entire process. These results show that it is possible to significantly reduce lead time and manual workload by optimizing and digitalizing the flow of information. The increased degree of digitalization and the associated process transparency can help to achieve further improvements and to better understand the processes in future analyses [11]. Although the implementation of specialized S-BPM tools was deliberately avoided in the case at hand, their introduction, thanks to the S-BPM methodology, could provide the basis for an even more comprehensive digitalization of existing processes. The S-BPM method and supporting modeling tools enable a direct transformation of process models into running processes [4], which could significantly reduce the effort for the future digitalization of processes. #### References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ### Index #### A Abstract actors, 203 Abstract State Machine (ASM), 144 Actions, 84 Active sentences, 203 Activity bundles, 152, 253 Activity diagram, 84 Actors, 175, 225, 226, 228–230, 232, 233, 235–237 Adaptive Case Management, 12 Ad hoc tasks, 100 Agility, 171 Analysis, 152–154 AND connector, 76 ArchiMate, 10 Architecture of integrated information systems (ARIS), 143 Articulation, 180 Automation, 159 #### B Behavior, 119, 138, 140, 141, 143, 144, 146 Behavior diagram, 110 Behavior tables, 206 Boundary intermediate events, 102 BPM project, 208 Branches, 71, 75 Business Activity Monitoring (BAM), 239, 240, 245 Business events, 1, 3 Business Model Canvas, 31, 39 Business models, 7, 10 Business objects, 115 Business Process Diagrams (BPD), 89 Business processes, 1, 3, 12, 26, 46, 65–67, 84, 130, 133, 143, 144 Business Process Management (BPM), 1–3, 10, 12, 16–21, 23, 27, 28, 30, 37, 38, 50, 66, 253, 259, 260, 267, 271 Business Process Management cycle, 151 Business Process Management Systems (BPMS), 224 Business Process Model and Notation (BPMN), 10, 70, 89, 226, 228–232, 235–238 #### C Choice of the modeling language, 69 Choice segments, 120–123 Choreography diagrams, 107 Collaboration diagram, 132 Collaborative alignment, 188 Collaborative concept mapping, 185 Communication, 28–30, 38, 63, 64, 67 "CoMPArE/WP" method, 183 Compensation tasks, 105 Condition, 71 Control flows, 206 Control logic, 226 Control Objectives for Information and Related Technology (COBIT), 10 Control view, 75 Conversation diagrams, 132, 133 Creativity, 171 Cross-company, 131 #### D Data objects, 85 Decision, 71 Decision elements, 84 Define, 167 Design Thinking (DT), 12, 14, 163–171 Design Thinking process, 166 Deviations, 160 Digitalization, 129, 143–146, 171 Diversity, 165 # The Author(s) 2020 A. Fleischmann et al., Contextual Process Digitalization, https://doi.org/10.1007/978-3-030-38300-8 #### E Effectiveness, 157, 218 Efficiency, 157 Embedding, 157 Empathize, 167 Empathy, 163 End events, 90, 94 Enterprise architecture, 6, 7, 12, 16 Escalations, 160 Event-based gateway, 90 Event-driven Process Chain (EPC), 143 Events, 75, 76, 134, 225, 234, 237–239, 243, 245, 250 Event-triggered subprocesses, 106 Event types, 101 Exception handling, 119 Exceptions, 103, 117 Exchange analysis, 193 Exclusive (XOR) gateway, 90 Execution behavior, 100 Execution context, 99 Execution semantics, 115 Execution sequence, 71 Experiential knowledge, 180 Experts, 175 Extended Event-driven Process Chains (eEPCs), 70 #### F Facilitators, 175 Flowcharts, 70, 71, 130–132, 143 Functions, 75, 76 Function state, 111 #### G Gateways, 89 Governors, 175 #### H Holomap, 192 Holonic energy management system, 201 "How might we?"-question, 168 #### I Ideate, 168 Impact analysis, 194 Implementation, 157 Inclusive (OR) gateway, 90 Individual articulation, 186 Information objects, 81 Information Technology (IT), 38, 39, 43, 44, 46, 48, 50, 52, 65 Input pool, 115 Intangible exchange of knowledge, 191 Intangible value, 37 Interaction diagram, 110 Interdisciplinary teams, 165, 175–176 Intermediate events, 95, 102 Interruptions, 103 IT implementation, 159, 223, 238 IT systems, 80 #### K Key Performance Indicators (KPIs), 7, 12, 33, 34, 36, 37 Knowledge management, 12 #### L Lanes, 90, 96 Log, 238, 243, 245 #### M Macrocycle, 166 Mental models, 181 Message flows, 96 Message guards, 117–120 Messages, 110 Microcycle, 166 Middle-out approach, 214 Model, 1–3, 6, 7, 12–14, 16, 20, 23–34, 36, 39, 41, 42, 44–48, 50–53, 56, 60–62, 64, 65, 182 Modeling, 152–154 Modeling by construction, 213 Modeling by restriction, 213 Monitoring, 159 #### O Object, 228, 250 OMG standard, 89 Open cycle, 179 The Open Group Architecture Framework (TOGAF), 10 Operations, 71, 159 Optimization, 153, 157, 218 OR connector, 76 Organization, 1, 2, 6, 14, 16, 17, 20 Organizational, 157 Organizational implementation, 223 Organizational units, 79 OWL, 144, 146, 148 #### P Parallel (AND) gateway, 90 Parallel Activity Specification Scheme (PASS), 133, 144, 146, 149 Parallel process flows, 75 Partitions, 85 People, 163 Philosophy, 24, 27 Place, 163, 170 Plan-Do-Check-Act, 10, 14 Point of view (POV), 168 Poly Energy Net, 199 Pools, 90, 96 Process, 1–3, 5–8, 10–12, 14–16, 20, 129–139, 143, 144, 146, 163, 223–240, 243, 245–248, 250, 251 Process controlling, 153 Process implementation, 6 Process instances, 1, 3, 14, 223, 225, 226, 229, 234, 235, 237–239, 243, 250 Process logic, 6, 10, 66, 226, 228, 229, 231–233, 236, 237, 239 Process model, 225–228, 245 Process Performance Indicators (PPIs), 7, 9, 16, 153 Process Query Language (PQL), 247 Process realization, 66 Process strategy, 3, 65 Project plan, 14 Prototype, 169, 170 #### Q Quality control, 214 #### R Reality, 2, 3, 14, 16 Receive state, 111 Robotic Process Automation (RPA), 159 Role-plays, 217 #### S SAP system, 260 S-BPM, 110–123, 226, 228, 229, 231, 232, 235–238 Semantics, 129, 143, 144, 146 Send state, 111 Service, 133–139, 146, 148 Signals, 85, 103 Simulation, 157, 218 Social BPM, 171 Software robots, 159 Split/join element, 84 Start events, 90, 94, 102 State diagrams, 110 Strategy, 32–34, 44, 48 Subject-oriented Business Process Management (S-BPM), 10 Subject-oriented Business Process Modeling (S-BPM), 70, 154, 171 Subject-oriented process modeling, 110 Subjects, 110 Subprocesses, 99, 129, 131 Supply chain, 255 Swim lane, 259 Syntactic, 143 Syntax, 129, 143 #### T Tangible exchange of knowledge, 191 Tangible value, 37 Tasks, 89 Task types, 99 Test, 170 Textual descriptions, 200 Theory X, 20 Theory Y, 20, 21 Total Quality Management (TQM), 10 Transactions, 105 Transaction subprocess, 105 T-Shaped, 165 #### U Unified Modeling Language (UML), 132, 143, 144 UML activity diagrams, 70 Use of natural language, 200 User centricity, 171 #### V Validation, 153, 155, 215 Value creation analysis, 194 Value network analysis, 190 Value Stream Analysis (VSA), 256–258, 267 Value Stream Model (VSM), 256, 258, 259, 267 #### W Walk-through, 217 #### X XOR connector, 76	doab	2025-04-07T03:56:57.944958	10-2-2021 14:38	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "000f0faf-bfb7-4c4d-9180-ec3d905bbc1f", "url": "https://library.oapen.org/bitstream/20.500.12657/37369/1/978-3-030-38300-8.pdf", "author": "Fleischmann, Albert\|\|Oppl, Stefan\|\|Schmidt, Werner\|\|Stary, Christian", "title": "Contextual Process Digitalization", "publisher": "Springer Nature", "isbn": "", "section_idx": 0 }
00134e2c-4cd5-4041-bb42-ea5f0ee762e6	Magdalena Kolasa # The Scope and Limits of Protection for Distinctive Signs against the Community Design The Application and Implications of Art. 25 (1) (e) of the Community Designs Regulation 17 https://doi.org/10.5771/9783845243856, am 03.04.2025, 22:22:53 Open Access – - https://www.nomos-elibrary.de/agb https://doi.org/10.5771/9783845243856, am 03.04.2025, 22:22:53 Open Access – - https://www.nomos-elibrary.de/agb BUT\_Kolasa\_7910-2.indd 1 27.08.12 13:12 MIPLC Studies Edited by Prof. Dr. Christoph Ann, LL.M. (Duke Univ.) Technische Universität München Prof. Robert Brauneis The George Washington University Law School Prof. Dr. Josef Drexl, LL.M. (Berkeley) Max Planck Institute for Intellectual Property and Competition Law Prof. Dr. Thomas M.J. Möllers University of Augsburg Prof. Dr. Dres. h.c. Joseph Straus, Max Planck Institute for Intellectual Property and Competition Law Volume 17 BUT\_Kolasa\_7910-2.indd 2 27.08.12 13:12 Magdalena Kolasa # The Scope and Limits of Protection for Distinctive Signs against the Community Design The Application and Implications of Art. 25 (1) (e) of the Community Designs Regulation Nomos BUT\_Kolasa\_7910-2.indd 3 27.08.12 13:12 Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über http://dnb.d-nb.de abrufbar. The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at http://dnb.d-nb.de . a.t.: Munich, Intellectual Property Law Center, Master Thesis (LL.M. IP), 2011 ISBN 978-3-8329-7910-2 #### 1. Auflage 2012 © Nomos Verlagsgesellschaft, Baden-Baden 2012. Printed in Germany. Alle Rechte, auch die des Nachdrucks von Auszügen, der fotomechanischen Wiedergabe und der Übersetzung, vorbehalten. Gedruckt auf alterungsbeständigem Papier. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other than private use a fee is payable to »Verwertungsgesellschaft Wort«, Munich. BUT\_Kolasa\_7910-2.indd 4 27.08.12 13:12 ## Abstract The Community design is a relatively new legal instrument, showing a considerable amount of open questions of practical relevance. One of those is the application and the implications of Art. 25(1)(e) CDR – the ground for invalidation of a Community design on the basis of infringement of a prior distinctive sign. This potentially attractive provision, allowing for an invalidation on the basis of a reference to various, Community and national, legal provisions, to date results in a lower number of invalidations than Art. 25(1)(b) CDR – the other ground for invalidation available for the owners of distinctive signs. This paper addresses the scope and limits of protection for the distinctive signs under Art. 25(1)(e) CDR by defining them on the Community and on the national level with reference to the German jurisdiction. An analysis of the scopes of protection of the relevant distinctive signs follows, including trade marks, trade names, company symbols, work titles and names. Additionally the scope of protection provided for the distinctive signs by the unfair competition provisions is described. This analysis is a starting point for addressing some of the controversial issues concerning the application of Art. 25(1)(e) CDR as a ground for invalidation. The issue of admissibility of application of limitations to the right to a distinctive sign in design invalidity proceedings and propositions as to their implementation are presented. Furthermore an analysis of problems arising from the use of Art. 25(1)(e) CDR with regard to the applicable law is provided, presenting the possible solutions of those controversies. The paper also addresses the question of whether an action for infringement of a prior distinctive sign will be successful unless an invalidation of the design right is obtained first. Finally, the relationship between the invalidation on the basis of lack of novelty, lack of individual character and based on infringement of prior rights is addressed. As the discussion of the abovementioned problems shows, Art. 25(1)(e) CDR grants the proprietor of a distinctive sign a broad scope of protection against a Community design. However due to the fact that the Community Desig Regulation has left many questions regarding the implementation of that protection unanswered, a considerable level of legal uncertainty is attached to its application and this ground for invalidation still remains an alternative infrequently used by the owners of distinctive signs. In spite of the conceptual challenges connected to its application, Art. 25(1)(e) CDR provides for a potentially attractive ground for invalidation of Community designs, which might gain in importance, depending on the future development of the case-law. https://doi.org/10.5771/9783845243856, am 03.04.2025, 22:22:53 Open Access – - https://www.nomos-elibrary.de/agb Table of Contents ## V. Summary 73 ## List of Works Cited 75 https://doi.org/10.5771/9783845243856, am 03.04.2025, 22:22:53 Open Access – - https://www.nomos-elibrary.de/agb ## Acronyms and Abbreviations 12 ZPO Zivilprozessordnung [ZPO] [Civil Procedure Statute], Sept. 12, 1950, BGBl. at 455, as amended (Federal Republic of Germany). https://doi.org/10.5771/9783845243856, am 03.04.2025, 22:22:53 Open Access – - https://www.nomos-elibrary.de/agb ## I. Introduction The Community system of design protection,1 proved to be appealing for the Internal Market participants.2 Its power of attraction owes largely to the fact that it grants a fast and cheap protection for a relatively long period of time. Fast, because the registered Community design does not undergo a substantive examination upon registration3 and the unregistered Community design does not require registration at all.<sup>4</sup> Cheap, because due to the lack of examination and the online instruments available in the OHIM the fees covered by the applicant for a registered Community design are minimal.<sup>5</sup> The long-lasting protection is granted for up to 25 years in the case of the registered Community design6 and 3 years in case of the unregistered Community design.7 However, this simplified acquisition of an exclusive right may lead to a conflict with other rights. The grant of a quick and cheap protection is balanced by the possibility of invalidation of a Community design on various grounds enumerated in Art. 25 CDR. Hence the burden of clearing the register is shifted on the market participants,8 which include the owners of signs that identify their persons, entities or their products. Those signs can be described as distinctive since their common feature is that they distinguish goods or undertakings. The existence of a design using such a sign might be a threat for the owner of that sign, leading to confusion, dilution, damage to reputation, or gaining an unfair advantage over the owner of the sign who has made an investment in its development and promotion. The invalidation of a Community design on the basis of its conflict with a prior distinctive sign can be founded on the design's lack of novelty,9 lack of individual character10 or on it falling into the scope of protection of that sign.11 Since <sup>1</sup> Created by the CDR and the DD. <sup>2</sup> To date over 460.000 registered Community designs and an unestimated number of unregistered Community designs, as reported on the OHIM webpage, http://oami.europa.eu/ows/rw/ pages/RCD/index.en.do (last visited June 5, 2012). <sup>3</sup> Art. 45 CDR. <sup>4</sup> The making available being sufficient for grant of an exclusive right , Art. 11 CDR. <sup>5</sup> See: http://oami.europa.eu/ows/rw/pages/RCD/index.en.do (last visited June 5, 2012). <sup>6</sup> Art. 12 CDR. <sup>7</sup> Art. 11(1) CDR. Art. 25(1)(e) CDR refers to the protection for signs granted by both Community and national laws, the owner of such a prior sign may avail himself of various legal provisions from any of the countries of the EU in pursuing the invalidation of the design. This ground for invalidation, even though potentially powerful, still seems to be a less attractive alternative than Art. 25(1)(b) CDR, partially due to a considerable level of legal uncertainty connected to its application. This thesis tries to analyse the scope of Art. 25(1)(e) CDR and to address some of the controversial issues connected to its application. Due to the constraints of this paper, it is not possible to refer to the legal regimes of all EU Member States, i.e. those that due to the geographical scope of a Community design may provide for legal grounds for its invalidation. Therefore the analysis will be limited to the harmonized rules governing trade mark laws of all the Member States.12 With regard to other distinctive signs, reference will be made to German law, Germany being the largest economy13 within the Internal Market. The first part of the thesis delineates the background by defining the notion of a distinctive sign and that of the Community design and by identyfying the area of conflict between them. Subsequently the scopes of protection of the relevant distinctive signs and their limits are described in the context of design invalidation. An attempt to analyze the implications of the construction of the CDR provisions in practice follows, highlighting the challenges for the owners of prior rights in enforcing them on the basis of Art 25(1)(e) CDR. Finally, a summary of the research is presented. <sup>12</sup> Additionally, the constraints of this thesis do not allow for a detailed discussion of all aspects of the trade mark infringement, hence the stress of the analysis will be put on the issues specific for the conflict with a design and common for all EU Member States. <sup>13</sup> according to the International Monetary Fund, World Economic Outlook Database, Apr. 2011, Report for Selected countries and subjects, available at: http://www.imf.org/external/pubs/ft /weo/2011/01/weodata/weorept.aspx?sy=2007&ey=2010&scsm=1&ssd=1&sort=country&ds =.&br=1&c=941%2C946%2C137%2C122%2C181%2C124%2C918%2C138%2C964%2C18 2%2C968%2C423%2C935%2C128%2C936%2C939%2C961%2C172%2C184%2C132%2C 134%2C174%2C144%2C944%2C178%2C136%2C112&s=NGDP\_RPCH%2CNGDPD%2C PPPPC&grp=0&a=&pr1.x=15&pr1.y=15 (last visited June 5, 2012). # II. Distinctive signs, the Community design and the conflict between them ## A. The notion of a distinctive sign Distinctive signs lack a legal definition. In the literature, they have been described as signs used in relation to commercial activities which have a distinctive character, i.e. are capable of distinguishing goods or services of one undertaking from those of other undertakings, thereby allowing the customers for the identification of the source of those goods or services.14 However some authors do not limit the definition only to commercial activities and source–identifying function. Hildebrandt includes those signs which have a distinguishing function regardless of the matter that is being distinguished, thereby including trade marks – as distinguishing between the products, company indicia – as distinguishing between the companies, titles of works – as distinguishing between works, geographical indications – as distinguishing between the regions from which the products originate and names – as distinguishing between different persons.15 A sign as such also lacks a legal definition. The ECJ has only stated that it should be perceivable with one of the five senses.16 In the design context, this must be limited to the eligibility for perception by sight, as a design is the appearance of a product.17 It seems that the most relevant aspect of a sign's distinctiveness is that it allows the consumers to distinguish between goods, services and undertakings, therefore enabling them to differentiate between the source of goods or services or between traders. With respect to distinctive signs other than registered trade marks, the OHIM includes in their characteristics the fact that they are based on use, are trade or business related and not merely personal, serve a distinguishing function in the course of trade and are of exclusive nature, i.e. confer ownership or an ownership-like position.18 In that respect the likely ground for the application of Art. 25(1)(e) CDR will be the conflict with prior trade marks, rights of similar character such as company symbols, trade names, work titles19 other names, and signs which can be protected under unfair competition provisions. While geographical indications and domain names can be seen as distinctive signs in a broad sense, they either do not point to a single commercial source of the goods or services (geographical indications), or do not incorporate a proprietary right to prohibit the use (in the case of domain names)20 and therefore go beyond the scope of this thesis. Designs cannot be qualified as distinctive signs because they are protected as such, not as indicators of origin.21 The Community trade mark is a unitary right governed by a single legal act<sup>22</sup> and national trade mark laws have been harmonized by the TMD. Other distinctive signs remain in the competence of the national legislators. ## 1. Trade Mark ## a) General remarks Trade marks are any signs that are capable of being represented graphically and of distinguishing the goods or services of one undertaking from those of other undertakings.23 This distinctive character, i.e. recognisability among the relevant consumers is defined as "capacity of a trade mark to (…) be retained in the <sup>18</sup> The Manual of Trade Mark Practice, available at: http://oami.europa.eu/ows/rw/resource/doc uments/CTM/legalReferences/partc\_nonregisteredrights.pdf (last visited June 5, 2012), C.4.5.3.1. <sup>19</sup> Special protection for work titles is a German peculiarity. Under §5(3) MarkenG their function is to identify the work as such, rather than its source, Franz Hacker, Markenrecht. Das deutsche Markensystem [2011] Carl Heymanns Verlag 2011, 302 (hereinafter: Hacker). <sup>20</sup> Hildebrandt supra note 15, §1 para. 1, 1. <sup>21</sup> Uma Suthersanen, Design law: European Union and United States of America [2010] Thomson Reuters (Legal) Limited, 166 (hereinafter: Suthersanen). <sup>22</sup> Art. 2(2) CTMR. <sup>23</sup> Art. 4 CTMR, Art. 2 TMD, §3 MarkenG. memory and to be recognized again".24 Therefore a sign constituting a trade mark is not protected as such, but only as an indicator of origin25 being an instrument allowing for a communication on the market between the competitors and consumers.26 This origin – indicating function27 has been recognized28 as the essential of a trade mark. However, as the development of law has shown, it is not the only ground for the protection of trade marks, as further functions have been accepted. They are founded on the trade mark's essential function29 and include the guarantee of quality of the goods or services,30 advertising function,31 communication and investment functions.32 For further remarks on the trade mark functions in the context of infringement – see Chapter III C. 1. b. The trade mark system in the EU consists of the Community trade mark,<sup>33</sup> which requires registration and grants its owner a unitary right valid for the entire EU and the national laws harmonized under the TMD, which provides for normalisation concerning the requirements for the grant of protection34 and scope35 of the exclusive right. Therefore in the EU, there are 28 (Community and 27 national) systems of protection of trade marks, which are to a large extent corresponding. As a result the trade marks that can conflict with a Community design include registered Community trade marks, registered national trade marks and unregistered national trade marks, which is possible due to the fact that TMD foresees a minimum harmonization.36 In particular §4 No 2 MarkenG allows for trade mark protection of signs as long as they have been used as an origin indicator for specific goods or services and as such have acquired recognition among the relevant public,37 registration not being a condition for such protection. Consequently even though the national laws of the Member States of the EU show some divergences as far as the catalogue of signs protectable under the trade mark law is concerned,38 once they come to existence, they are granted the same scope of protection39 and therefore will be described together in Chapter III of this thesis. b) Trade marks that may conflict with a Community design Subject to the limitations regarding certain characteristics of a sign,40 the law does not provide any exceptions to eligibility for trade mark protection as far as the type of sign is concerned. The respective provisions of Art. 4 CTMR and Art. 2 TMD provide mere lists of examples of such signs, including i.a. words, designs and shape of goods or of their packaging. In particular, as the ECJ has stated, the requirements for grant of protection for the three-dimensional marks do not differ from those applicable for other types of marks, although the relevant consumer does not usually perceive the product forms as indicative of source.41 20 The trade marks which are most likely to be conflicting with a design are the three-dimensional signs, in particular product shapes,42 product surfaces or trade dress. A further example are two dimensional signs, which will be in conflict upon the use in a two dimensional design, e.g. a pattern,43 but also upon a reproduction of such mark in a three-dimensional design of a product.44 The case law provides also examples of invalidation based on a prior word mark45 and a figurative mark.46 Due to the characteristics of a position mark,47 which determines a specific use of a sign, it is also likely to be successfully used as ground for invalidation. In some countries (e.g. France and Belgium) the distinctive signs are required to be registered if they are to be granted protection and no additional safeguard is available to protect a trader's reputation. Other regimes, e.g. British and Ger- man, allow for protection without registration, i.a. under unfair competition provisions.48 This safeguard, unlike trade mark law, does not refer to any particular sign, but rather to the efforts of a market participant, his time and investment put into the creation of any subject-matter on the one hand and the behaviour of his competitor influencing that effort in an unfair way – on the other. The considerations here focus on the nature of the behaviour, the underlying achievement is protected only additionally49 and due to the unfair competition rules having a character of general clauses, they are able to fill-in the gaps in protection provided for distinctive signs by IPRs.50 Even though one of the basic rules governing exclusive rights prescribes the freedom of copying51 outside the limits of IP, the unfair competition law provides for its limitations.52 However such restriction, if applied too broadly, might limit the competition and as a result harm both the consumers and the market. Therefore the application of unfair competition provisions is limited to behaviours which are unfair. "Unfairness" of a behaviour is a term which each national legislation needs to define for itself. The harmonized notion of unfair competition codified in Art. 5 of Directive 2005/29/EC of the European Parliament and of the Council of 11 May 2005 concerning unfair business-to consumer commercial practices in the internal market, is a general clause, broad enough for national laws to incorporate their developed legal attitudes. ## b) Signs protected under unfair competition that may conflict with a Community design The object of protection under unfair competition provisions are i.a. products in which the customers are interested for their origin or reputation and able to rec- <sup>48</sup> William Cornish, David Llevelyn and Tanya Aplin, Intellectual Property: Patents, Copyright, Trade Marks and Allied Rights [2010] Sweet&Maxwell, 638-639 (hereinafter: Cornish/Llevelyn/Aplin). <sup>49</sup> Eckhart Gottschalk and Sylvia Gottschalk, Das nicht eingetragene Gemeinchaftsgeschmacksmuster: eine Wunderwaffe des Designschutzes? [2006] GRUR Int 461, 466 with further references (hereinafter: Gottschalk/Gottschalk). <sup>50</sup> Ansgar Ohly, Designschutz im Spannungsfeld von Geschmacksmuster-, Kennzeichen- und Lauterkeitsrecht, [2007] GRUR 2007, 731, 736 (hereinafter: Ohly 2007). <sup>51</sup> Id. 735. <sup>52</sup> Ansgar Ohly, The Freedom of Imitation and its Limits – A European Perspective [2010] IIC 505, 512. ognize them due to their characteristics.53 These have been defined in the caselaw as products having a competitive individuality which as a whole or through their features are able to transfer to the consumers the message as to their origin or characteristics.54 In the case of distinctive signs, most of them can be perceived either as a product or as its feature that possesses the competitive individuality. The protection under unfair competition is in some respects broader than that under trade mark law.55 Still, the underlying notion of both trade mark distinctiveness and competitive individuality is that the more uncommon the sign – the more likely it is to possess both distinctiveness and competitive individuality.<sup>56</sup> The existence of competitive individuality is a question of fact and is judged taking into account all the relevant circumstances, such as novelty, originality, recognisability among the relevant public, level of advertising or fame, and even costs and effort of promotion.57 Therefore it can be inherent to a product due to its characteristics,58 or it can be gained through time, similarly as secondary meaning in trade mark law.59 Company symbols and work titles are protected under the German trade mark law. According to §5(2) MarkenG company symbols are "signs used in the course of trade as names, trade names, or special designations of business establishment or enterprises. Business symbols and other signs intended to distinguish one <sup>53</sup> Dissmann in: Maximiliane Stöckel and Uwe Lüken, Handbuch Marken- und Designrecht [2006] Erich Schmidt Verlag 495 (hereinafter: Stöckel/ Lüken). <sup>54</sup> Ohly in: Henning Piper, Ansgar Ohly, Olaf Sosnitza, Gesetz gegen den unlauteren Wettbewerb [2010] C.H. Beck, §4 No.9, para. 9/32 (hereinafter: Piper/Ohly/Sosnitza); BGH GRUR 1997, 754, 756 - "grau/magenta". <sup>55</sup> Ohly suggests that the unfair competition can protect the "small coins" of distinctive signs due to the lower requirement of competitive individuality, Ohly 2007, supra note 50, 738, though the taking unfair advantage of distinctiveness may be pursued only under trade mark law, BGH GRUR 2007, 795, 799 - Handtaschen. <sup>56</sup> Ansgar Ohly, Die Europäisierung des Designrechts [2004] ZEuP 296, 309 (hereinafter: Ohly 2004). business from another which are regarded within the affected circles as the distinctive signs of a business establishment, shall be equivalent to the special designation of a business establishment".60 The difference between those signs and trade marks is that while marks refer to goods or services and only indirectly to their source, the company symbols convey a direct information on the origin.61 The company symbols come into existence with the begin of their use, regardless of registration, if they are inherently distinctive, or if that is not the case when they acquire secondary meaning.62 Nevertheless the notion of company symbols includes trade names which require registration,63 and which are protected as company symbols additionally to the protection provided for them by the HGB. Under §5(3) MarkenG, work titles cover designations of printed publications and cinematographic, musical, dramatic or other works. Since they refer to the work itself, they may confer an information about origin only indirectly. ## b) Company symbols that may conflict with a Community design Company symbols include names, i.e. words which identify a person (natural or legal) or an object and can only be represented with words. Any other sign, including symbols,64 logos, colours, or even slogans can be protected under §5(2) MarkenG, once they acquire a distinctive character among the relevant consumers as indicating the company.65 Taking into consideration their characteristics, and the characteristics of work titles (§5(3) MarkenG) any of those symbols could potentially be used in a design. 65 Lüken in: Stöckel/ Lüken, supra note 53, 254. <sup>60 §5(2)</sup> MarkenG, English translation taken from http://www.ip-firm.de/markeng\_e.pdf (last visited June 5, 2012). <sup>61</sup> Lüken in: Stöckel/ Lüken, supra note 53, 251. <sup>62</sup> Id. 254. <sup>63 §29</sup> HGB. <sup>64</sup> BGH GRUR 2005, 419, 422 - Räucherkate. Trade name is a registered name of a merchant which he uses in his commercial activities.66 It identifies the trader (a natural or legal person) in his activities on the market, thereby allowing for the recognition of the market participants and their activities. Trade name differs from commercial symbols in that it points to a person or entity rather than to a commercial activity, therefore a trader can have only one trade name, while at the same time owning different commercial symbols, identifying different activities that he exercises.67 b) Trade names that may conflict with a Community design Trade names must be distinctive and capable of identifying their owner and may not include information that might be misleading for the market participants.68 Therefore they can consist only of words and symbols possessing a recognised meaning that can be pronounced (e.g. &).69 As such – they might be used in a Community design, especially of a pattern or logo. §12 BGB regulates the protection of names, i.e. designations which allow for an individualization of natural or legal persons and other entities,70 allowing them to act against unauthorised uses of those names by others, potentially also use in a Community design. Since trade names are seen as names, and names – may be seen as company symbols, while at the same time they all may constitute trade 68 §18 HGB. <sup>66 §17, §29</sup> HGB. <sup>67</sup> Heidinger in: Münchener Kommentar zum HGB [2010] C.H. Beck §17 para. 35. <sup>69</sup> Heidinger in: Münchner Kommentar zum HGB [2010] C.H. Beck §17 para. 12. <sup>70</sup> Thomas Nägele Das Verhältnis des Schutzes geschäftlicher Bezeichnungen nach §15 MarkenG zum Namensschutz nach §12 BGB [2007] GRUR 2007, 1007, 1008 (hereinafter: Nägele). marks - there exists a crossover of protection under the trade mark rules, §15 MarkenG, §37 HGB and §12 BGB.71 ## b) Names that may conflict with a Community design Protection of names under §12 BGB covers any signs that may identify a person or entity as their name, therefore not only words (as under protection of trade names), but also figurative elements, such as emblems, seals or logos.72 ## B. The notion of a Community Design The design is a legal instrument for the protection of creations that form external shapes of products or their parts,73 and result from the features of a product and/or its ornamentation, as long as they are new and have individual character.74 It protects the visual appearance, which includes two-dimensional representations, such as get-up and typefaces. This protection is of an abstract character, not confined to a defined range of products.75 The substantive requirements of novelty76 and individual character77 of a design have an essential bearing on the validity of the Community design as the existence of prior rights may lead to the destruction of the design's novelty or individual character and as a result – form a ground for declaration for its invalidity under Art. 25(1)(b) CDR. Novelty under Art. 5 CDR is judged against an identical design that has been made available78 prior to an unregistered Community design, or – in case of the 77 Art. 6 CDR. <sup>71</sup> This multiple protection is accepted in §2 MarkenG. The relationship between the various provisions is examined more closely in Chapter III C. 2-5. <sup>72</sup> BGH GRUR 1993, 151, 153 - Universitätsemblem. <sup>73</sup> Casado Cerviño and Wahl in: Charles Gielen and Verena von Bomhard (eds.), Concise European Trade Mark and Design Law [2011] Wolters Kluwer, 360 (hereinafter: Gielen/ von Bomhard). <sup>76</sup> Art. 5 CDR. <sup>78</sup> The concept of making available referred to in Art. 5 and Art. 6 of CDR is clarified in Art. 7 CDR and is limited to events that could have reasonably become known to the specialized business circles in the Community. The discussion of this concept goes beyond the scope of this thesis. For more detailed analysis see: Green Lane Products Limited v PMS International registered Community design – prior to the filing of the application for its registration or its claimed priority. Immaterial differences79 between the Community design and the novelty-destroying design should be disregarded, while the assessment is made from an objective perspective, which, unlike copyright, excludes protection for designs that were independently created.80 The design's individual character, similarly as novelty, is assessed against a single piece of prior art.81 However, the requirement it involves is on the one hand less stringent, on the other - more difficult to prove: if the Community design does not produce on the informed user a different overall impression than the prior design, it is lacking individual character and hence is eligible for invalidation under Art. 25(1)(b) CDR. The impression both designs make is judged from the perspective of an informed user, who is defined as a notional user of the designs at issue, who is "particularly observant and has some awareness of the state of the prior art, that is the previous designs relating to the product in question".82 The Community design system includes registered83 and unregistered84 Community designs. The unregistered Community design is granted protection upon the making available85 and the registered Community design - upon registration in OHIM which however does not include a substantive examination, in particular of novelty and individual character.86 Group [2008] EWCA Civ 358 and comments of Johanna Brückner-Hofmann in: Hatrwig, Designschutz in Europa [2009] Vol.3 Carl Heymanns Verlag 234, 251. ## C. The area of conflict between distinctive signs and the Community design Community designs protect the appearance of a product and cover i.a. threedimensional objects, packaging, patterns, logos and typefaces. As has been shown above - all those objects of a design may be protected as distinctive signs. Since the existence and possible conflict with a prior sign are not part of the considerations made upon grant of protection for a Community design, the eligibility for such protection is in fact judged in invalidation proceedings instigated only by the interested market participants.87 The conflict with a prior distinctive sign may lead to the invalidation of a Community design either on the basis of Art. 25(1)(b) CDR – when it can be shown that the sign forms part of the prior art and the Community design is either identical (in the case of asserted lack of novelty) or, though not being identical, does not produce a different overall impression on the informed user. The third ground for invalidation relevant for holders of distinctive signs is Art. 25(1)(e) i.e. situation where the owner of a prior right is able to show that the Community design in fact infringes his prior right, whereas this infringement claim may be based on any, Community or national, legal ground as long as it confers on the owner of the sign a right to prohibit the use of his sign. If a design is not novel it will also not possess individual character.88 However, even if the design is novel and possesses individual character, it might nevertheless infringe a prior distinctive sign (especially in cases where there is no likelihood of confusion between the signs but there exists a likelihood of association or where the prior sign has a reputation). As will be shown in the subsequent chapters, the ground for invalidation of a Community design on the basis of its conflict with a prior distinctive sign, grants its owner a broad selection of weapons against the design. <sup>87</sup> Anyone – in case of Art. 25(1)(b) CDR or the holder or a prior sign in case of Art. 25(1)(e) CDR, as prescribed in Art. 25(3) CDR. <sup>88</sup> Musker in: Gielen/ von Bomhard, supra note 73, 367. III. Art. 25 (1)(e) CDR as ground for invalidation of a Community design A Community design will be declared invalid under Art. 25(1)(e) CDR if an earlier distinctive sign is used in this design and the Community law or the law of the Member States governing that sign confers on the rightholder of the sign the right to prohibit such use. The analysis will therefore encompass the following considerations: ## A. Use of a distinctive sign in a subsequent design As the OHIM Invalidity Division stated with regard to trade marks, "registered Community design is deemed to use a sign which is identical or similar to the sign of the earlier trade mark, where the following two conditions are met: (1) The registered Community design contains a feature which is perceived as a sign. (2) That sign is identical or similar to the sign of the trade mark. A feature of a registered Community design cannot be perceived as a sign where that sign is devoid of distinctive character".90 Hence, the starting point of the analysis is the existence of a feature in the design which has a distinctive character. If that requirement is fulfilled the second step is to establish whether this feature of the design is similar or identical to the sign that is seeking protection. Other ele- 90 ICD 000007030 - AM Denmark A/S v Kuan-Di Huang, OHIM Inv. Div. Sept. 17, 2010, available at: http://oami.europa.eu/ows/rw/pages/RCD/caseLaw/decisionsOffice/invalidity.en.do under the ICD number, para. 22. <sup>89</sup> Oliver Ruhl, Gemeinschaftsgeschmacksmuster. Kommentar [2007] Carl Heymanns Verlag 465 – 468 (hereinafter: Ruhl 2007). ments of the design which do not form part of the allegedly used sign should be disregarded.91 The use of a sign in a subsequent Community design does not require an exact and detailed reproduction of that sign.92 Judging similarity or identity involves a comparison between the feature of the design and the sign as protected, i.e. in case of registered trade marks, the comparison must be between the design at issue and the mark as registered, not as used.93 To assess whether a sign is used, Hager proposes a determination whether the feature corresponding to the sign "is swallowed up in the overall appearance to such an extent that it is only interpreted as part of the product like any other element or design feature".94 If so – then the design at issue will not use any feature that might conflict with a distinctive sign. ## B. Prior distinctive sign and a subsequent design The decision on whether the design is junior in relation to the distinctive sign boils down to comparing the date of begin of the Community design and the date of the commencement of the prohibiting effect of the distinctive sign. Whether the distinctive sign has existed and provided its owner with the right to prohibit an unauthorised use of that sign prior to the design at question will be judged, in case of the registered rights, by the date of the publication of registration or application for registration95 or, in the case of rights that do not require registration - under national laws regulating the protection of the respective distinctive sign.96 The existence of a Community design begins in the case of the registered Community design on date of filing of the application for registration with the OHIM, a central industrial property office of a Member State or with the Bene- <sup>91</sup> Unlike assessment of novelty under Art. 5 CDR and of individual character under Art. 6 CDR where the design as a whole is compared with the prior sign. <sup>92</sup> Neville Cordell and Tim Austen, European GC highlights conflict between trade marks and designs [2010] 5 JIPLP 622, 623, Community Design invalidity Manual, supra note 15, C.7.2. <sup>93</sup> GC Case T-148/08 - Beifa Group Co. Ltd. v OHIM, 2010 ECR II-01681, para. 114; Hager, supra note 24, 413. lux Design Office,97 and of the unregistered Community design – on the date on which the design was first made available to the public in the Community.98 Hence those respective dates will be taken into account when judging whether the design is "subsequent" within the meaning of Art. 25 (1)(e) CDR. In the context of Art. 8(4) CTMR the General Court has expressed the view that the existence or protection of a prior sign invoked under the provisions of the UK law of passing off must be established at the time of the filing of the contested trade mark rather than on the date when the goods or services bearing the contested mark were offered on the market.99 It is submitted that these considerations are adequate also in the Community design context. ## C. Right to prohibit the use of a prior distinctive sign Not every use of a prior distinctive sign in a Community design will lead to its invalidity. It is necessary that the owner of the prior sign has the right to prohibit the use, i.e. that the design falls into the scope of protection of the sign and infringes the owner's rights. The scope of protection of trade marks, company symbols and work titles, trade names, names and unfair competition provisions, with the view on the potential conflict with a design right are described below. ## 1. The scope of protection of trade marks The provisions on scope of protection of the CTMR mirror those of the TMD, and hence should be interpreted in the same way.100 Therefore the protection for trade marks based on the CTMR and harmonized national laws will be described simultaneously, with a reference to the relevant provisions of both texts. Additionally, Art. 8 CTMR and Art. 4 TMD use substantially identical terms as Art. 9 CTMR and Art. 5 TMD respectively, accordingly the interpretation given by the <sup>97</sup> Art. 12 and 34 CDR, however if the documents from the national office reach OHIM later than 2 months after the national filing – the date of receiving the documents by OHIM counts as the filing date. Remarkably, the priority does not influence the term of protection of the registered Community design, Art. 43 CDR, Suthersanen, supra note 21, 147-148. <sup>98</sup> Art. 11 and 110a CDR. <sup>99</sup> GC Case T-303/08 - Tresplain Investments v OHIM, O.J. (C 30) 35, available at: http://curia.europa.eu/jmcs/jmcs/j\_6/ under the case number, para. 98-99. <sup>100</sup> Cornish/Llevelyn/Aplin, supra note 48, 708. Court on Art. 8 CTMR or Art. 4 TMD applies also to infringement, as confirmed by the European Court of Justice.101 The owner of a trade mark is entitled to prevent others from using in the course of trade: ## a) Use in the course of trade The first requirement for the trade mark protection, common for all three types of infringement is that the allegedly infringing sign (the design at issue) is used in the course of trade.105 Use in the course of trade will be found where the "sign is used in the scope of a commercial activity in pursuit of an economic advantage, instead of acts for private purposes or acts that are not directly or indirectly aimed at gaining an economic advantage".106 The registration of a Community design will presuppose its use in the course of trade "since the purpose of registering a design is its use for commercial purposes".107 The requirement of use in the course of trade became a starting point108 for a discussion on whether the infringing sign must furthermore be used "as a trade mark" and if yes what exactly does it mean. 101 ECJ Case C-425/98 - Marca Mode CV and Adidas AG, 2000 ECR I-04861, para. 26-28. <sup>102</sup> Art. 9(1)(a) CTMR, Art. 5(1)(a) TMD. <sup>103</sup> Art. 9(1)(b) CTMR, Art. 5(1)(b) TMD. <sup>104</sup> Art. 9(1)(c) CTMR, Art. 5(2) TMD. The implementation of Art. 5(2) TMD was optional, however all Member States transposed this provision into their national laws (Cornish/Llevelyn/Aplin, supra note 48, footnote 530). b) Trade mark use: use affecting the trade mark function As the ECJ stated in Arsenal, "the exclusive right was conferred in order to enable the trade mark proprietor to protect his specific interests as proprietor, that is, to ensure that the trade mark can fulfil its functions" and therefore that this right may only be exercised where the use by the third party of the sign affects or is liable to affect its functions as trade mark.109 This requirement is no longer an explicit requirement of granting protection,110 it is nevertheless taken into consideration by the courts finding infringement under double identity and likelihood of confusion.111 It is not required for the protection of marks with a reputation.112 There are three113 main functions of trade marks:114 <sup>109</sup> ECJ Case C-206/01 – Arsenal Football Club plc v Matthew Reed, 2002 ECR I-10273, para. 51. certain uses of a trade mark may damage its value "by detracting from the allure and prestigious image of the goods in question and from their aura of luxury".117 After L'Oreal v Bellure, where the ECJ contended that the functions of a trade mark that can be affected by the infringing use include not only the origin function, but also any other function, and giving by way of example the quality, communication, investment and advertising functions, the problem of trade mark infringement by way of affecting its function remains unclear. Recognition of new and undefined functions of a trade mark stirs doubts as to whether this requirement remains part of the infringement test. It also lowers the legal certainty on the market. Some authors suggest that such harmonization is a negative development of the trade mark law and has no solid legal ground, since it conflicts with the protection provided under Art. 5(5) TMD which refers the protection of trade mark functions other than that of distinguishing goods or services to national law.118 Despite the broadening of the trade mark protection by acknowledging new functions, the courts have also recognised certain types of uses as not influencing any of the functions. Merely descriptive use on goods or on their packaging does not influence any trade mark function and the public does not perceive such use as use of the sign for the goods in question, hence such use is not infringing.119 If that is the case, there would be no need for the defendant (Community design owner) to call upon any of the defences to the infringement, because with the lack of trade mark use the infringement is denied already at an earlier stage.120 As an example of a use that does not influence any of the trade mark functions, the ECJ has recognized the use to denote particular characteristics of the goods.121 The use as embellishment was considered to be a use that does not influence any trade mark function by the Advocate General Jacobs in his opinion in the case Adidas v Fitnessworld.122 However the ECJ123 did not share this ap- proach and held rather that if the relevant public considers the element to be a pure embellishment, they will not establish a link between the two marks – and that will be the basis for non-infringement. Various types of infringing use are likely to influence different functions of a trade mark, therefore the assessment regarding the function that is being influenced by a certain use is part of the analysis of the three types of trade mark infringement presented below. ## c) Use for goods or services The protection for trade marks is granted when the allegedly infringing sign is used "in relation to goods or services".124 Since a design is defined through the notion of a product,125 it is imminently connected with the goods (and more loosely – with services). However one of the characteristics of the design protection is that it is not limited as far as products to which it is applied are concerned.126 Trade marks on the other hand are protected with regard to the goods or services for which they have been obtained or similar goods.127 This has raised a question of whether by the fact that a design stretches onto any products, it automatically is used for the goods or services covered by any trade mark, or whether it should be established if the products covered by the design are at least similar to those protected by the trade mark. As the OHIM Board of Appeal has found, "when the registered Community design contains a two-dimensional figurative logo, which may be applied to an infinite range of products and services, including those protected by the prior trade mark, the contested design is liable to jeopardise the guarantee of origin, which constitutes the essential function of the trade mark".128 Thus in such cases it is irrelevant whether the goods or services of the trade mark are similar to those for which the design is or may be used. A stronger opinion was presented by Schlötelburg, who stated that a design comprises all possible goods or ser- 124 Art. 9(1) CTMR, Art. 5(1)–(2) TMD. <sup>125</sup> Art. 3 CDR. vices and therefore it is obsolete to compare the goods or services because the ones for which the infringing design may be applied are always identical to those covered by the trade mark at issue. Consequently, according to him, the invalidation of the design should result already when it is established that the signs used by the prior trade mark and by the design are identical or similar, comparison of goods or services is not necessary.129 Ruhl and the OHIM itself propose a different approach by stating that one should not give up the comparison of goods or services for which the trade mark and the design in question are applied. How far the comparison should go depends on the characteristics of the design, which may be applicable only to certain goods (e.g. shape of a product), to many types of goods (e.g. designs for surfaces) or to any possible good (e.g. logos).130 Furthermore the design needs not be attached to goods – it can be used in relation to them, which is judged by the relevant public.131 Therefore it is possible to apply for the invalidation of a design which has not yet been put into use, the abstract judgement of possible use in relation to goods for which the trade mark is applied is sufficient to establish the infringement of such trade mark. d) Double identity The infringement under Art. 9(1)(a) CTMR / Art. 5(1)(a) TMD will be found where a third party uses an identical sign without the authorisation of the trade mark owner for identical goods or services and this use affects or is liable to affect "the functions of the trade mark, in particular its essential function of guaranteeing to consumers the origin of the goods or services".132 This protection is absolute133 and unconditional. In the case LTJ Diffusion v Sadas, the ECJ clarified the notion of identity of signs by stating that "sign is identical with the trade mark where it reproduces, <sup>129</sup> Martin Schlötelburg, Musterschutz an Zeichen, GRUR 2005 123, 126-127 (hereinafter: Schlötelburg); similarly: Gottschalk/Gottschalk, supra note 49, 467. <sup>130</sup> Ruhl 2007, supra note 89, Art. 25 para. 31; Hartwig and Traub also suggest examination of goods or services for similarity in Comments to ICD 000001477 - Hee Jung Kim v Zellweger Analytics Limited, OHIM Invalidity Division March 1, 2006, in Hatrwig, Designschutz in Europa [2007] Vol.1 Carl Heymanns Verlag 211, 220 (hereinafter: Hartwig 2007), the same approach has been included in Community Design Invalidity Manual, supra note 15, C.7.4. <sup>131</sup> Amanda Michaels, A Practical Guide to Trade Mark Law [2002] Sweet&Maxwell 4.17. <sup>132</sup> L'Oreal v eBay [2009] EWHC 1094 (Ch) para. 283 and the caselaw cited therein. <sup>133</sup> In comparison to protection under Art. 9(1)(b) CTMR and Art. 5(1)(b) TMD, double identity does not require proving likelihood of confusion, Simon 2005, supra note 119, 412; confirmed in Recital 8 to CTMR, Recital 10 to TMD, and in ECJ Case C-245/02 - Anheuser-Busch Inc. v Budĕjovický Budvar, národní podnik, 2004 ECR I-10989 para. 63. without any modification or addition, all the elements constituting the trade mark or where, viewed as a whole, it contains differences so insignificant that they may go unnoticed by an average consumer".134 Whether the signs and goods are identical is judged from the standpoint of the average consumer, that is a consumer of the goods in question who is reasonably observant and circumspect135 and compares the signs and goods globally,136 relying on his imperfect recollection of the signs that he has come across on the market, not side-by side, while the level of his attention will vary according to the category of the goods or services for which the sign is protected under the trade mark.137 Even though the stronger, more distinctive signs are granted more protection,138 the decision-making body may not include the level of distinctiveness of the mark claiming protection and its elements upon comparison of the signs to such an extent as to call into question the validity of the earlier mark.139 Despite the absolute character of the protection, for the protection under double identity to step in it must be established that the allegedly infringing use is a use that affects any of the functions of the trade mark.140 Additionally, where the infringing goods are identical to the ones of the trade mark owner, Art. 5(1)(a) TMD establishes a presumption that those functions are compromised.141 Therefore where there exists an identity of both signs and goods or services, the analysis of the infringement under double identity boils down to answering the question whether the design at issue uses an identical sign to the sign of the mark. The case-law has provided for an example of invalidation of a Community design on the basis of Art. 25(1)(e) CDR in connection with Art. 5(1)(a) TMD in the "pasteboard multi package container" design which in the drawing included bottles bearing a trade mark.142 ## e) Likelihood of confusion Under Art. 9(1)(b) CTMR / Art. 5(1)(b) TMD respectively, the scope of protection of a trade mark covers the use of a sign which is identical or similar to the trade mark and is used for goods which are identical or similar to those covered by the trade mark, when there exists a likelihood of confusion on the part of the public between the sign and the trade mark, which includes the likelihood of association between them. The similarity of signs, of goods and likelihood of confusion are assessed from the point of view of relevant consumer of the goods or services in question143 (for further analysis of the notion of the relevant consumer see supra under d). ## (1) Similarity of signs The assessment of the degree of similarity of signs follows the same considerations as the review under the double identity test (see supra under d). The elements of the mark in question which are devoid of distinctive character may not be taken into account upon comparison, as they do not contribute to the mark's function as origin indicator.144 The comparison is made between the trade mark as registered (or as used – in the case of unregistered trade marks in Germany) and the alleged infringer's actual practice,145 i.e. in the case of the registered Community design – the design as registered and in the case of the unregistered Community design – the design as made available to the public. It has however been suggested by Hager that in the case of shape marks the shape features not directly apparent from the registration should not be ignored, because the public does not perceive the shape marks in their two-dimensional graphic representation but in the form in which they are actually used. Ignorance of the features deriving from use would "falsify the identifying function of shape trade marks". According to him these considerations should however not go as far as to create a different object of comparison than that which was registered.146 This approach has not been shared by the General Court who, judging on invalidation of a Community design, annulled the decision of the OHIM Board of Appeal stating that it was issued on the basis of a comparison with a three-dimensional image, 145 Cornish/Llevelyn/Aplin, supra note 48, 785. <sup>143</sup> ECJ Case C-210/96 - Gut Springenheide and Tusky, 1998 ECR I-04657, para. 31. <sup>144</sup> Just as "if an element of a product is not perceived by the public as an indication of origin, the protected sign as such cannot be impaired", Hager, supra note 24, 410. <sup>146</sup> Hager, supra note 24, 414. while the registered trade mark was two-dimensional. The reason for the annulment being the fact that "a three-dimensional mark (...) is not necessarily perceived by the relevant public in the same way as the figurative mark", the threedimensional sign being perceived from a number of angles, the two-dimensional only as an image.147 However the OHIM Board of Appeal was of the opinion that when a two-dimensional pattern (protected as trade mark) is put on a threedimensional design, the overall impression may be such that the design uses the trade mark.148 The global appreciation of the signs covers the visual, aural and conceptual similarity and must be based on the overall impression given by the marks. However a finding of similarity on all those levels of comparison is not required. It is enough that the existence of at least one of them is found by the court, taking into account the situation in which the consumer encounters the products bearing the mark.149 Therefore the comparison of signs is not made in isolation from the goods which are covered by the trade mark (even though the goods are compared at a subsequent step of the test). The visual similarity is the core of comparison in judging the conflict between a design and a prior mark, as design is defined through the appearance of a product150 and the visual comparison includes the mark's colour, size, shape and position.151 However aural and conceptual elements should not be disregarded when judging infringement of a trade mark by a subsequent design, as the elements of a design may also have sound and meaning (when they include words which need to be pronounced152 or accordingly words or symbols that may be ascribed a certain meaning153). It is submitted that while these should not be disregarded upon the assessment of the overall impression, they should not be given as much weight as the visual elements precisely because the design is the appearance of the product and not its sound or meaning. As far as similarity of signs is concerned, the prior trade mark does not need to be reproduced identically in the Community design at issue. It is sufficient 148 Case R 211/2007-3 - Burberry Ltd. v Jimmy Meykranz, OHIM Third Board of Appeal Mar. 3, 2008, available at: http://oami.europa.eu/search/legaldocs/la/EN\_boa\_index.cfm under the case number, para. 15, as a result invalidating the CD for lack of individual character, not under Art. 25(1)(e) CDR. 151 Philips, supra note 149, 322. 153 E.g. trade mark 007 and a design including a picture of a man in a tuxedo pointing a gun. <sup>147</sup> GC Case T-148/08 - Beifa Group Co. Ltd. v OHIM, 2010 ECR II-01681, para. 121. <sup>149</sup> Jeremy Philips, Trade Mark Law. A Practical Anatomy [2003] Oxford University Press, 320 (hereinafter: Philips). <sup>150</sup> Art. 3(a) CDR. <sup>152</sup> Especially in cases of word marks and designs for logos, e.g. word mark FOR YOU and a logo with a "4U" element. that the mark is incorporated in the design. Therefore additional elements may not change the perception of the design as using the trade mark,154 although their incorporation might lead to the trade mark being "swallowed up" in the design and therefore not being used in it at all.155 Conversely, the OHIM Invalidity Division did not find similarity of signs where the prior trade mark was figurative, even though the phonetic comparison pointed to identity as both signs used the word "flex", however that element was found to be the only similarity and due to the presence of other elements in both signs, was considered to be "not sufficient to constitute similarity between a feature of the registered Community Design and the sign of the Community trade mark".156 ## (2) Similarity of goods or services As indicated above (see supra at c), depending on the characteristics of the design and its capability to be used in relation to different goods or services, the comparison of goods or services for which the allegedly infringing design might be applied will include different scope of goods or services, and in cases where the design (e.g. logo) can be applied to any goods or services it can be assumed that the goods or services are identical. Similarly as establishing the similarity of signs, the decision on similarity of goods is a question of fact.<sup>157</sup> When assessing the similarity of goods, "all relevant factors relating to those goods should be taken into account, such as nature of the goods, (...) intended purpose, method of use and whether they are in competition with each other or are complementary".158 The final question that needs to be asked is however: Would a relevant consumer, taking a global appreciation approach, consider the goods as being similar? Answering this question often requires a balancing exercise between the various factors, as in different circumstances one might outweigh the other. Some authors have also argued that "the Canon factors are sub- <sup>154</sup> Case R 609/2006-3 - Honeywell Analytics Ltd v Hee Jung Kim, OHIM Board of Appeal May 3, 2007, available at: http://oami.europa.eu/search/legaldocs/la/EN\_boa\_index.cfm under the case number, para. 18, where a design was found using a word mark "MIDAS". Confirmed in GC Case T-148/08 - Beifa Group Co. Ltd. v OHIM, 2010 ECR II-01681, para. 50. <sup>155</sup> See supra under A. <sup>156</sup> ICD 000002756 - Flex Equipos de Descanso S.A. v The Procter and Gamble Company, OHIM Invalidity Division Jul. 26, 2007, available at: http://oami.europa.eu/ows/rw/pages /RCD/caseLaw/decisionsOffice/invalidity.en.do under the ICD number, para. 20. <sup>157</sup> Gert Würtenberger, Community Trade Mark Law Astray or Back to the Roots! [2006] E.I.P.R. 549, 550 (hereinafter: Würtenberger). <sup>158</sup> ECJ Case C-39/97 - Canon Kabushiki Kaisha and Metro-Goldwyn-Meyer Inc., 1998 ECR I-05507, para. 23. concepts which should be used to define whether the goods may in the actual marketplace come from the same or linked companies. Mere similarity of goods of themselves is (...) not sufficient to prove that the goods are similar".159 In Canon, the Court has also stated the principle of proportionality, prescribing that the more similar the goods are, the lower is the degree of similarity between the signs which will result in finding the likelihood of confusion, while the less similar the goods the higher degree of similarity of signs will need to be found in order to find likelihood of confusion.160 ## (3) Likelihood of confusion The decision on existence of likelihood of confusion is a question of law.161 It requires the assessment of all circumstances by way of global appreciation from the point of view of the average consumer of the goods or services in question162 judging the "capacity of the mark to identify the goods or services for which it has been registered as coming from a particular undertaking".163 The global appreciation takes into account both the goods or services in question and the strength of the protected mark164 and also the level of consumer attention with regard to different kinds of goods, which means that if "the objective characteristics of a given product mean that the average consumer purchases it only after a particularly careful examination, it is important in law to take into account that such a fact may reduce the likelihood of confusion between the marks relating to such goods at the crucial moment when the choice between those goods and marks is made".165 It follows additionally that the comparison and assessment of the likelihood of confusion are made at the point of sale. However in Arsenal, the Court recognized also post-sale confusion.166 The General Court recognised that the relevant public in the case of instruments for writing should comprise of <sup>159</sup> Jukka Palm, Canon, Waterford… How the Issue of Similarity of Goods Should be Determined in the Field of Trade Mark Law [2007] E.I.P.R. 475, 478. <sup>160</sup> ECJ Case C-39/97 - Canon Kabushiki Kaisha and Metro-Goldwyn-Meyer Inc., 1998 ECR I-05507, para. 17. <sup>161</sup> Würtenberger, supra, note 157, 551. The author submits therefore that the likelihood of confusion should not be judged from the perspective of relevant consumer, but rather should only be a means to help the decision-maker decide the question of law. <sup>162</sup> ECJ Case C-342/97 - Lloyd Schuhfabrik Meyer & Co. GmbH v Klijsen Handel BV, 1999 ECR I-03819, para. 25. <sup>163</sup> Id. para. 22. the public at large, since the goods are everyday products and the level of attention is relatively low.167 On the other hand in a case concerning high-end mixers, the UK High Court defined the relevant public narrowly. "As both mixers were premium priced products, targeted at design-conscious consumers", the expectations and knowledge of those consumers had to be taken into account when judging infringement.168 The basis for the likelihood of confusion must be the level of similarity between the signs and goods or services, "recognition of the mark on the market, the association that can be made with the used or registered sign",169 in fact all factors relevant to the circumstances of the case need to be taken into account,170 i.e. the spectrum of the relevant factors will vary from case to case. In particular, under the "neutralisation doctrine" when there are "confusing similarities in visual, phonetic, conceptual or figurative respects, the significant differences in one of these criteria may neutralise the likelihood of confusion arising from other criteria".<sup>171</sup> When it is established that there exists a similarity between the signs, upon assessing the likelihood that the relevant consumer will be confused by them, the descriptive or only weakly distinctive elements should not be disregarded, but judged as a part of the overall impression that the signs make. Because of their descriptiveness or low level of distinctiveness, similarity between such elements is less likely to create likelihood of confusion, as the relevant consumer will not concentrate on such elements when making his judgement.172 Conversely, the protection of a distinctive element of a mark must be recognized if such a component "maintains an autonomous distinctive position in the composite mark, even without constituting its dominant element".173 Under Art. 9(1)(b) CTMR / Art. 5(1)(b) TMD, the finding of likelihood of confusion includes the likelihood of association between the signs. In Marca Mode the ECJ stated that the likelihood of confusion and the likelihood of asso- <sup>167</sup> GC Case T-148/08 - Beifa Group Co. Ltd. v OHIM, 2010 ECR II-01681, para. 108. <sup>168</sup> Whirlpool Corp v Kenwood Ltd [2008] EWHC 1930 (Ch)., confirmed in Whirlpool Corp v Kenwood Ltd [2009] EWCA Civ 753, E.T.M.R. 7 para. 83. <sup>169</sup> Recital 8 CTMR, Recital 10 TMD. <sup>170</sup> ECJ Case C-251/95 - Sabel BV and Puma AG v Rudolf Dassler Sport, 1997 I-06191, para. 22. <sup>171</sup> Würtenberger, supra, note 157, 549; CFI Case T-6/01 - Matrazen Concord GmbH v OHIM, 2002 ECR II-04335 para. 35; similarly: Paola A. E. Frassi, The ECJ Rules on the Likelihood of Confusion Concerning Composite Trade Marks: Moving Towards an Analytical Approach [2006] IIC 438, 442-443. <sup>172</sup> Philips, supra note 149, 346-347; Hager, supra note 24, 412, who suggests the complete exclusion of non-distinctive elements when comparing marks consisting of both protectable and non-protectable elements (at 413). <sup>173</sup> ECJ Case C-120/04 - Medion AG v Thomson multimedia Sales Germany & Austria GmbH, 2005 ECR I-08551, para. 30. ciation are not separate concepts that should be applied alternatively, but that likelihood of association constitutes part of the likelihood of confusion concept and serves to define its scope.174 Additionally mere association, without the element of confusion is not enough to find infringement.<sup>175</sup> ## (4) Influence on trade mark functions Since the likelihood of confusion must concern the source of the products, it is required that the use of the allegedly infringing sign influences the mark's origin function. As the ECJ stated in Canon, there can be no likelihood of confusion, "where it does not appear that the public could believe that the goods or services come from the same undertaking or, as the case may be, from economicallylinked undertakings".176 Therefore the OHIM Board of Appeal found it conceivable that when the public encounters a logo (incorporating a design) applied to products or their packaging, they might perceive that logo as an indication of commercial origin. That would lead to jeopardising the essential function of a trade mark.177 ## f) Protection for trade marks with reputation The protection under Art. 9(c) CTMR / Art. 5(2) TMD is granted against the use of a sign which is similar or identical to the trade mark which has a reputation in the Community (in the case of a Community trade mark) or nationally (in the case of a national trade mark) and where the use of that mark by the defendant is without due cause and takes unfair advantage of, or is detrimental to, the distinctive character or the repute of the mark. Establishing the similarity or identity of signs and goods follows the same considerations as in the two prior types of infringement (see supra at d and e).178 <sup>174</sup> ECJ Case C-425/98 - Marca Mode CV and Adidas AG, 2000 ECR I-04861, para. 34. <sup>175</sup> ECJ Case C-251/95 - Sabel BV and Puma AG v Rudolf Dassler Sport, 1997 I-06191, para. 26. <sup>176</sup> ECJ Case C-39/97 - Canon Kabushiki Kaisha and Metro-Goldwyn-Meyer Inc., 1998 ECR I-05507, para. 30. Confirmed in the context of invalidation of a Community design in GC Case T-148/08 - Beifa Group Co. Ltd. v OHIM, 2010 ECR II-01681, para. 97. <sup>177</sup> Case R 609/2006-3 - Honeywell Analytics Ltd v Hee Jung Kim, OHIM Board of Appeal May 3, 2007, available at: http://oami.europa.eu/search/legaldocs/la/EN\_boa\_index.cfm under the case number, para. 28. <sup>178</sup> ECJ Case C-252/07 - Intel Corporation Inc. v CPM United Kingdom Ltd, 2008 ECR I-08823, para. 62-63. The infringement can be found irrespective of whether the sign is used for different goods, or for similar or identical goods or services.179 In this context, the ECJ does not require a degree of similarity of signs that would lead to likelihood of confusion.180 It is sufficient that the similarity is such that "the relevant section of the public establishes a link between the sign and the mark"181 i. e. it is enough that the design at issue "brings the mark to mind",182 even though the public does not confuse the two signs. The owner can enforce his rights even in the cases where it is clear that the consumers are not misled as to the relation between the two signs. And, since the detriment to the distinctive character or to the mark's repute is not required either,183 it is enough to show that the advantage taken is unfair, without furnishing further evidence as to the consequences of such an advantage being taken.184 To be granted protection, the mark must have a reputation185 in a substantial part of the territory for which it exists - in the case of a Community trade mark it will be a substantial part of the Community, which can be a country186 or in the case of a national mark - part of a region.187 When establishing the reputation of a mark the Court needs to take all relevant factors into account, i. a. "the market share held by the trade mark, the intensity, geographical extent and duration of its use and the size of investment made by the undertaking in promoting it",188 but "showing a niche reputation189 is sufficient to meet the (...) standard of marks with a reputation".190 The first type of infringement of a reputed trade mark occurs when the accused design takes unfair advantage of the distinctive character or the repute of the mark. Even though the ECJ pointed to the fact that "a trade mark with a reputation necessarily has distinctive character, at the very least acquired through use"191 and usually considers them together, the distinction between such marks can be made "because there are trade marks with limited distinctive character but of good repute and trade marks with considerable distinctive character but of only moderate repute".192 The concept of taking unfair advantage of the distinctive character or repute of the mark was addressed by the ECJ in L'Oreal v Bellure where the court referred to the notions of "parasitism" and "free-riding" known from national unfair competition laws. The Court defined the unfair advantage as "seeking by [the use of the mark] to ride on the coat tails of mark with a reputation in order to benefit from the power of attraction, the reputation and the prestige of that mark and to exploit, without paying any financial compensation, the marketing effort expended by the proprietor of the mark in order to create and maintain the mark's image".193 Such exploitation does not in fact need to cause damage to the reputation, even potentially. The stress of the assessment lies in taking unfair advantage.194 The other type of infringement is the use that is detrimental to the distinctive character of the trade mark (blurring). It has been defined by the ECJ that the "detriment is caused when the mark's ability to identify the goods or services for which it is registered is weakened, since the use of an identical or similar sign by a third party leads to dispersion of the identity and hold upon the public mind of the earlier mark"195 and it "requires the evidence of a change in the economic behaviour of the average consumer (...) consequent to the use of the later mark, or a serious likelihood that such a change will occur in the future".196 The more distinctive and/or known a trade mark is, the larger is the risk of the detriment to its distinctive character.197 Unfortunately this proved to be an insufficient guid- <sup>191</sup> ECJ Case C-252/07 - Intel Corporation Inc. v CPM United Kingdom Ltd, 2008 ECR I-08823, para. 73. <sup>192</sup> Jehoram/van Nispen/Huydecoper supra note 39, 308 -309, giving as examples of the first category the trade mark "Ideal Standard" and of the second – "Lidl". <sup>195</sup> Id. para. 39, ECJ Case C-252/07 - Intel Corporation Inc. v CPM United Kingdom Ltd, 2008 ECR I-08823, para. 29. ance as the rightowners have difficulties in enforcing their rights in cases of blurring.198 The third type of infringement, causing detriment to the reputation, occurs "when the goods or services for which the identical or similar sign is used by the third party may be perceived by the public in such a way that the trade mark's power of attraction is reduced", in particular where the goods on which the sign is used "possess a characteristic or quality which is liable to have a negative impact on the image of the mark".199 The provisions require that the use takes unfair advantage of or causes detriment to the distinctiveness or repute of the mark, without due cause. This notion has not been so far clarified by the Court of Justice. It has however been suggested that due cause could be derived from the limitations provisions of Art. 12 CTMR and Art. 6 TMD respectively. This would mean that the use of the trade mark in a descriptive manner, the necessity to use the mark or having earlier rights could serve as a justification for the use of a mark with a reputation.200 The protection afforded to marks with a reputation is granted irrespectively of the use influencing any of the trade mark functions.201 Establishing a link is enough to find infringement and "this is a link between the two parties' marks and not between the later mark and the earlier user, as is required in the confusion cases".202 Whereas when the sign is viewed purely as an embellishment by the relevant public, no link with the mark is established and therefore the requirement for grant of protection is not fulfilled. However where the sign is seen as an embellishment but nevertheless such a level of similarity exists that a link is established, the infringement is given.203 The use as an embellishment as a concept should be distinguished from the descriptive use which does not influence any of the trade mark functions. Since a trade mark with a reputation is granted protection irrespective of detriment to any of its functions – descriptive use could not be used as a defence in the sense of double identity and likelihood of confusion. The fact that the sign is used as pure embellishment does not constitute an infringement because it does not lead to establishing a link between the signs, not because it does not influence the mark's function.204 <sup>198</sup> Ilanah Simon Fhima, The Court of Justice protection of the advertising function of trade marks: an (almost) sceptical analysis [2011] JIPLP 325, 328. Therefore it must be stated that the protection under Art. 9(1)(c) CTMR/ Art. 5(2) TMD "does establish and was intended to establish a wider form of protection than is laid down in Art. 5(1) [TMD] and that only one of the three types of 'injury' covered by Art. 5(2) need to be proved".205 The "anti-dilution" protection under Art. 9(1)(c) CTMR/ Art. 5(2) TMD and Art. 5(5) TMD is strongly affected by unfair competition considerations. While it can be a reasonable solution in jurisdictions such as Benelux, "where unfair competition laws are generally precluded from the sphere of trade marks",206 they might prove problematic in the countries with elaborate unfair competition protection, resulting in overprotection of trade marks and limiting the freedom of traders to develop products, which includes the freedom of copying. A detailed analysis of Art 5(5) TMD goes beyond the scope of this thesis as it has been implemented only by the Benelux countries. 2. The scope of protection of distinctive signs under unfair competition law The lack of comprehensive harmonization of law in the EU results in large differences between the treatment of distinctive signs under unfair competition rules. Since Art. 25(1)(e) CDR includes application of national laws, it is necessary to consider the national protection of signs. In this part of the thesis German regulations of such protection will be described. According to §1 UWG the statute protects against unfair commercial practices, i.e. such behaviours of the market participants which can to an appreciable extent influence the behaviour of competitors, consumers or other market participants. In this respect the parties interested in protection of their distinctive signs under unfair competition will usually be the competitors of the accused design owner. As signs are basically protected under trade mark law, the German caselaw207 developed a rule that the protection under unfair competition provisions will be available when the rules of trade mark law do not provide for a relevant protection or when there has been a gap left on purpose by the legislator.208 The rationale for such an approach is that the overprotection might hinder the competition when the owners of signs could use both exclusive rights and unfair competition to exclude others from using the same subject – matter and thereby <sup>205</sup> Christopher Morcom, L'Oreal v Bellure – Who Has Won? [2009] E.I.P.R. 627, 634. <sup>206</sup> Cornish/Llevelyn/Aplin, supra note 48, 792-793. <sup>207</sup> BGH GRUR 1999, 161, 162 - MAC Dog. <sup>208</sup> Wirtz in: Horst-Peter Götting and Axel Nordemann, UWG. Handkommentar [2010] Nomos §3, para.83 (hereinafter: Götting/Nordemann); BGH NJW-RR 2003, 1551, 1552 - Tupperwareparty, English translation available in [2004] IIC 459, 461. stretch the boundaries of trade mark law.209 Hence the German courts rightly, it is submitted, try to avoid overlaps of protection under IP and unfair competition laws.210 It has also been proposed in the literature, that due to the fact that the unfair competition protection under §4 Nr 9(a) UWG relies on the same considerations as the trade mark law, it should be available only to subject-matter not eligible for trade mark protection.211 On the other hand, since the interests protected differ, the applicability of general rules of civil law, unlike trade mark law, does not exclude protection under unfair competition.212 Due to the fact that the German trade mark law protects also unregistered trade marks, the unfair competition protection becomes most relevant for signs which, even though distinctive, are precluded from the trade mark protection. Accordingly, in the context of distinctive signs and design rights, the cases actionable under unfair competition provisions are those of product imitation exemplified in §4 No. 9 UWG. This protection is related to the goods or services, rather than to the sign as such213 and is available even for shapes that are excluded from trade mark protection e.g. because of their functionality,214 however the requirement of unfairness of the behaviour of the alleged infringer must not be based on considerations of a purely trade mark nature, because otherwise would lead to bypassing the compulsory requirements of trade mark eligibility.215 The protection under §4 No. 9 UWG will therefore be applicable for goods which due to their distinctiveness can be qualified as sign, bur are excluded from protection by the trade mark law. It is granted where there exists a competition between the products in question, the allegedly infringing design includes a copy of the sign seeking protection, and the behaviour of the design owner is considered unfair towards the owner of the prior sign under a general assessment of all circumstances of the case. 210 BGH GRUR 1996, 581, 583 – Silberdistel, however overlapping protection has been accepted in the case of an unregistered Community design, see: BGH GRUR 2006, 79, 80 - Jeans I. 48 <sup>209</sup> Ohly 2007, supra note 50, 737. <sup>211</sup> Joachim Bornkamm, Markenrecht und wettbewerblicher Kennezeichenschutz. Zur Vorrangthese der Rechtsprechung [2005] GRUR 2005, 97, 102. <sup>214</sup> Art. 7(1)(e)(ii) CTMR, Art. 3(1)(e) TMD. <sup>215</sup> Nordemann in: Götting/Nordemann supra note 208, §4 No.9, para. 9.26. ## a) Competition between the products The rules of law on unfair competition may be applicable only if there exists a competitive relationship between the applicant and the holder of the design in question.216 Therefore the unfair competition will not step in where the products are so far apart that their consumers differ, e.g. fast moving consumer goods and nuclear power plants technology. This requirement might become problematic in the case of luxury goods. Even where there is no confusion as to source of the imitation, the owner of the luxury sign might rely on the protection for taking unfair advantage of the reputation of his sign. But if the products do not compete with each other, especially where it can be clearly established that they are sold via different trade channels and bought by different consumer groups – the protection against unfair competition might nevertheless be unavailable for the proprietor of the sign. Such an approach was taken by the Federal Supreme Court in the Handtaschen case,217 where the protection under unfair competition was denied for undoubtedly famous Hermès "Kelly" and "Birkin" bags, i.a. due to the fact that the sale of the allegedly infringing bags was carried out via different channels of trade. ## b) Copying The German law has acknowledged two types of use of the enforced sign that can be described in the design context as: the exact copying (identity of signs, in terms of trade mark law), which includes changes or additions which are so insignificant that can be considered irrelevant taking into account the sign's overall impression (similar approach as that of the ECJ in LTJ Diffusion218) and incorporating the sign into the later design, with changes or additional elements, that still allow for the recognisability of the underlying sign (similarity, in trade mark terms).219 However, unlike trade mark law, the similarity in unfair competition terms requires that the alleged infringer knows the sign that he is using, which is assumed in the case when the design is subsequent. Consequently, a proof of independent creation would immune the design holder from liability.220 In this respect Nordemann suggests an application of the copyright considerations which distinguish between a derivative work (which leaves the elements of the original 217 BGH GRUR 2007, 795, 799 - Handtaschen. <sup>216</sup> Ohly in: Piper/Ohly/Sosnitza, supra note 54, §4 No.9 para. 9/31. <sup>218</sup> ECJ Case C-291/00 - LTJ Diffusion SA v Sadas Vertbaudet SA, 2003 ECR I-02799. recognizable and therefore infringes the rights of the author of the original) and a result of a mere inspiration (where the characteristics of the original work fade taking onto account the effort provided by the author of the derivative).221 The comparison between the sign and the design is based on the overall impression both of them create in the relevant public. However the conclusion as to the existence of copying can be drawn only on the basis of identity or similarity of those elements of the sign claiming protection that convey the message as to the source of the goods.222 This makes the comparison of signs similar to that made upon the assessment of the trade mark likelihood of confusion. ## c) Additional circumstances (§4 No 9 (a)-(c) UWG) Under the unfair competition rules, imitation as such, even of a product that has a competitive individuality, is not regarded as unallowable. There is a need to establish the existence of additional circumstances that make the copying unfair. The time of judgement as to the unfairness of the behaviour is tied to the nature of the provisions which protect the market participants and their actions. Therefore, unlike trade mark law,223 the German unfair competition does not recognize post-sale confusion or taking unfair advantage. The assessment is to be taken at the time of the allegedly infringing action.224 It is submitted that the assessment for the purposes of Art. 25(1)(e) CDR should be taken at the time of registration of the design, since it is the existence and not the use of the design which is being challenged. The examples provided in §4 No 9 (a) – (c) UWG do not exhaust the possibilities of an infringement. Any action that is unfair and able to influence the behaviour of market participants may result in liability under the general clause of §3 UWG.225 50 <sup>223</sup> ECJ Case C-206/01 – Arsenal Football Club plc v Matthew Reed, 2002 ECR I-10273, para. 57. ## (1) Avoidable confusion as to source The first, and most important226 case of unfair behaviour is causing avoidable confusion as to source of the goods, §4 No 9 (a) UWG. It has been submitted that the assessment of confusion requires similar judgement as that under trade mark law,227 i.e. the comparison should be based on the overall impression made by both signs, taking into account their distinctive elements and not be taken side by side but taking into account how the sign and the design are encountered.228 The BGH however has not recognized such an approach and requires either that the product seeking protection is known on the German market or that the comparison could be made in abstracto, side-by side.229 This has been criticized, as the requirement of certain awareness of the public in Germany discriminates against foreign market participants and the abstract comparison ignores the interests of unfair competition protection which include the regulation of behaviour on the market.230 The judgement on whether the confusion as to source exists is made from the point of view of the consumer of the product in question, similarly as in trade mark infringement. Hence, the characteristics of such a consumer must be taken into consideration.231 Furthermore it is sufficient that the relevant consumer knows the product, it is not required that he is able to ascribe the product to a certain source.232 The additional requirement that the confusion as to source was avoidable, inquires whether the accused design owner did undertake all the necessary steps, according to the circumstances of the case, in order to avoid such confusion that could objectively have been avoided. A confusion as to source that could not have been avoided requires only that the accused acted against it.233 The steps to be taken include i.a. putting information onto the products as to their source,234 or adding a disclaimer. However the use of any elements of the prior sign (product) that are not capable of indicating origin235 or that form part of the public <sup>226</sup> Covering about 90% of the case-law on unfair competition according to Nordemann in: Götting/Nordemann, supra note 208, §4 No. 9, para. 9.52. <sup>227</sup> Art. 9(1)(b) CTMR, Art. 5(1)(b) TMD. <sup>228</sup> Nordemann in: Götting/Nordemann, supra note 208, §4 No. 9, para. 9.58. domain and are freely accessible to everybody may not be seen as infringing and must be accepted even if it might cause confusion.236 Furthermore, only reasonable steps can be required from the alleged infringer, therefore the interests of the owner of the sign and the design proprietor must be balanced and that is done by the courts under consideration of all relevant facts of the case, although the copying of aesthetic features may not generally be excused, while copying of technical features is generally allowed.237 In case of doubts, the rule of freedom of copying should prevail, since it satisfies the public interest in the use of the elements of products designs. ## (2) Unfair advantage or damage to reputation The second type of behaviour covered by §4 No 9 (b) UWG occurs when unfair advantage is taken of or the reputation of the competitor's goods is damaged. The considerations behind this type of infringement are similar as in trade mark "dilution"238 cases. Therefore, even though the existence of the reputation of a sign is not an explicit requirement, it must be shown that the sign seeking protection is to a certain extent known among the consumers.239 The taking of unfair advantage requires a substantial transfer of the market success of the sign. It is not sufficient that the design brings the sign into mind, a stronger association must be created.240 The examples of taking unfair advantage include causing confusion as to source and causing association with the renown sign,241 even when no confusion can be found, which can be relied on only if the protection under Art. 9(1)(c) CTMR / Art. 5(2) TMD is not available. Due to the fact that the German trade mark law protects also unregistered trade marks, company symbols and work titles,242 the unfair competition protection steps in also for <sup>236</sup> BGH GRUR 2007, 339, 344 - Stufenleitern. <sup>237</sup> Nordemann in: Götting/Nordemann, supra note 208, §4 No.9, para.9.64; copying of technical elements is prohibited when the sign claiming protection consists of a multitude of technical and functional elements and the entire combination is copied in the design, Michael Loschelder Der Schutz technischer Entwicklungen und praktischer Gestaltungen durch das Markenund das Lauterkeitsrecht – Versuch einer Bewertung der Rechtsprechung der letzten zwei Jahre [2004] GRUR Int 2004, 767, 770. Furthermore, technical features that need to be copied will not be seen as possessing competitive individuality and if the copied solution is appropriate – the risk of confusion has been recognized as unavoidable in Towel Hooks (I ZR 131/02) BGH [2006] IIC 348, 351. <sup>238</sup> Art. 9(1)(c) CTMR / Art. 5(2) TMD. those signs in cases where the design is not used as source indicator or when the goods or services are not similar to those covered under trade mark law, also in the cases of lack of likelihood of confusion, on the condition that that use leads to a competitive disadvantage on the part of the owner of the sign. The unfair association can also lie in using the sign in such a way that the positive image associated with the sign or its advertising power is negatively influenced243. 244 Causing detriment to the sign's reputation requires that the use of the design results in lowering the opinion and positive image connected to the quality or the luxury image of the sign seeking protection.245 These considerations correspond to those covered by protection of marks with a reputation under Art. 9(c) CTMR / Art. 5(2) TMD. (3) Breach of confidence Breach of confidence is the third type of product imitation situation and covers two types of behaviours: acquiring the know-how in a dishonest way, e.g. by industrial espionage246 and classical breach of confidence which includes public use of legally obtained information (e.g. during employment or negotiations that did not lead to signing of a contract) against a secrecy clause, which can also be implied247. 248 ## d) General assessment and interplay of factors Unfair competition protection requires balancing of interests of the persons involved. Therefore there is a certain interdependence between the "level of competitive individuality, kind, way and intensity of copying and the additional circumstances of the behaviour".249 The higher the level of the competitive individuality or the bigger the similarity of signs, the lower is the required level of unfairness of the behaviour.250 ## 3. Scope of protection of company symbols and work titles The scope of protection of company symbols and work titles under §15 MarkenG, resembles closely that of trade mark. Although §15 MarkenG does not include double identity, the protection against confusion as to source (§15(2) MarkenG) and protection of indicia with reputation (§15(3) MarkenG) cover most cases of infringement.251 These provisions are regarded as lex specialis towards §12 BGB and therefore this general clause cannot be a ground for protection for a distinctive sign whenever there are grounds for the owner to rely on §15 MarkenG.252 On the other hand, HGB provisions can be relied on additionally.<sup>253</sup> Company symbols and work titles are protected against confusion. However, instead of comparison of goods or services for which the sign is used, under protection of company symbols it is rather the comparison of the scope of activities of the owner of the sign seeking protection and the design proprietor. Whereas the complete identity of those fields is not required, it is sufficient that both fields show some crossovers.254 The interdependent factors that need to be taken into account, include an assessment of identity or similarity of the signs, the level of distinctiveness of the prior sign and the fields of activity in which both signs are used.255 Therefore in this case the comparison seems more straightforward than under trade mark law – regardless of goods or services for which the symbol and the design are used, it is the field of activity of their owners that needs to be taken into account and hence, unlike trade mark law, the corresponding factual situations are being compared. Of course, when establishing the field of activities, it is necessary to take into account the goods offered or the services rendered by both entities, however these will not be the only circumstances under assessment. MarkenG in §15(3) provides for protection of company symbols and work titles with reputation. It corresponds to the provision of §14(2) No 3 MarkenG,256 and so it has been submitted in the literature that due to the fact that company symbols usually constitute also the company's trade mark or are at least signs eligible for trade mark protection, the applicability of §15(3) MarkenG should correspond closely to that of §14(2) No 3 MarkenG. In addition, because of the broad understanding of the concept of confusion under §15(2) MarkenG, the practical importance of protection of company symbols with reputation is relatively low.257 For this reason for a detailed analysis of the scope of protection it is referred to the analysis regarding protection of marks with reputation in Chapter III C. 1. f. ## 4. Scope of protection of trade names (Firma) The protection under §37 HGB requires that the Firma is used as a trade name and without the authorisation of the proprietor, §37(2) HGB requires further that the applicant's rights are infringed by that use. This general clause is sufficiently broad to cover double identity, likelihood of confusion and protection of trade names with reputation.258 Use as trade name has been defined as "any action that has a direct relation to the operation of one's business and can be understood as an expression of the user's intention to use the sign as his own trade name".259 Whether this is the case is judged from the point of view of the commercial circles that encounter the sign. It has been recognised that use as a trade name is given in situations in which a trade name is usually utilised and therefore the public expects that such a name will be used.260 Accordingly it seems that in an application for invalidation of a Community design the evidence must be produced that the use of the accused design infringes the rights to a trade name. The registration of a design as such does not lead to use as a trade name, the context of use and possible impression among the public need to be shown. With respect to an infringement of rights to a trade name by a design, a recent decision of the Higher Court in Cologne261 provides for a relevant guidance. According to this decision, since under §18 HGB the trade name must be able to characterise its owner and possess a distinguishing character, it must not include any figurative elements and like other names can be composed only of words. Therefore the use of the trade name with additional elements, for example as part of a logo, might lead to lack of use as a trade name and consequently – not be infringing under §37 HGB. The protection of a trade name under §37(2) HGB requires further that the rights of the applicant for the invalidation of a Community design are infringed. <sup>257</sup> Ingerl/Rohnke, supra note 24, §15 para. 79-80. <sup>258</sup> Lüken in: Stöckel/ Lüken, supra note 53, 255. <sup>259</sup> BGH NJW 1991, 2023, 2024 - Case II ZR 259/90. <sup>260</sup> Ingerl/Rohnke, supra note 24, Nach §15 para. 166. <sup>261 6</sup> U 67/10 [2010] OLG Köln, Nov. 5, 2010 with comments by Fabian Zigenaus, GRUR-Prax 2011, 10. It has been recognised by the case-law that those rights must be of an economic nature.262 ## 5. Scope of protection of names The protection under §12 BGB is pre-empted by the possibility to rely on trade mark law.263 Therefore in the context of design infringement it is not likely to be relied on in many instances and is prone to be called upon in the cases of use in a design of a sign which by its owner is not used commercially and therefore is protected neither as a trade mark nor as a company symbol.264 This general clause allows for sufficient flexibility to cover double identity, likelihood of confusion and protection of names with reputation.265 Finding of an infringement under §12 BGB requires an unauthorised use of a name in such a way that infringes the legitimate interests of the owner of that name. The provision protects the identification function of a name and therefore use that can be prohibited by the owner of the protected name must be such that it influences the association of the name with its owner266 and has been described not as likelihood of confusion as to source but rather as ability to cause such confusion.267 As a result – the protection under §12 BGB requires a lower threshold of proof on confusion as it seems to be judged in more abstract terms than the likelihood of confusion closely connected to the judgement of the relevant public. The requirement of infringement of legitimate interests of the owner of the name goes beyond the protection against the likelihood of confusion, likelihood of association and dilution of his name, whereas, differently as under §15(2) MarkenG, the protection against likelihood of confusion does not require the proximity of the fields of activity between the proprietor of a name and the alleged infringer.268 Furthermore, depending on whether the name for which the protection is claimed is a name of a natural or a legal person, it is required that <sup>262</sup> II ZR 259/90 [1991] BGH Apr. 8, 1991, NJW 1991, 2023. <sup>263</sup> Ingerl/Rohnke, supra note 24, Nach §15 para. 3. <sup>264</sup> Ingerl/Rohnke, supra note 24, Nach §15 para. 7; opposite view presented by Nägele in: Nägele, supra note 70, 1009, himself being of the opinion that the applicability of §15 MarkenG or §12 BGB should depend on the rationale of protection under each of the provisions and giving examples of such factual configurations, id. 1013. <sup>265</sup> Lüken in: Stöckel/ Lüken, supra note 53, 255. <sup>266</sup> BGH GRUR 1993, 151, 153 – Universitätsemblem. <sup>267</sup> Nägele, supra note 70, 1008. <sup>268</sup> Id. 1008-1009. the infringed interests are of personal or of purely economic269 nature respectively.270 The OHIM presents a different approach, including in the scope of distinctive signs only those names that are trade or business-related and not merely personal,271 which consequently would allow for invalidation of a Community design only if the owner of a right to a name invokes his economic, and not personal interests against the design right. ## D. The limits of protection of distinctive signs ## 1. The limits of protection of trade marks In infringement proceedings under the trade mark law, the defendant has a range of defences that, if they prove successful, render his behaviour legal. Since the invalidation of a Community Design under Art. 25 (1)(e) CDR is based on the concept of infringement of the prior distinctive sign, the question can be posed, whether these defences can be called upon in invalidation proceedings by the holder of the design. The most far reaching defence strategy is challenging the validity of the prior mark or accusing it of being subject to revocation (Art. 99(3) CTMR, Art. 11(3) TMD272) and furnishing a proof of lapse of the right (e.g. due to lack of payment of the renewal fees, Art. 46 and 47 CTMR), as a non-existing right is unenforceable. Challenging the validity of the prior trade mark in the design invalidity proceedings has not been accepted. The registered rights are subject to the presumption of validity273 and there is no legal ground that would allow challenging such presumption in the design infringement proceedings. If the validity of a distinctive sign is contested – the Invalidity Division may suspend its proceedings on invalidation,274 however OHIM will not of itself inquire into the question of existence or validity of the sign on which the invalidation application is based and <sup>269</sup> BGH GRUR 1998, 696, 697 - Rolex-Uhr mit Diamanten. <sup>270</sup> Ingerl/Rohnke, supra note 24, Nach §15 para. 19-20. <sup>271</sup> Manual of Trade Mark Practice, available at: http://oami.europa.eu/ows/rw/resource/documents /CTM/legalReferences/partc\_nonregisteredrights.pdf (last visited June 5, 2012), C.4.5.3.1. <sup>272</sup> Art. 11(3) TMD provides for optional harmonisation. <sup>273</sup> Art. 99(1) CTMR. <sup>274</sup> Art. 2.6 OHIM Guidelines on Invalidation of Registered Community Design; Community Design Invalidity Manual, supra note 15, B.1.6.2, providing a list of situations in which the proceedings can be suspended and stressing the OHIM's discretion in the decision on suspension. will treat the prior right as valid.275 Submitting a proof that the right does not exist or has lapsed will result in rejection of the application, due to the nonexistence of the prior right to a distinctive sign,276 although in fact it is on the applicant to establish the existence of his right. A defence that has been recognised by the case-law in design invalidation proceedings is requiring the applicant to provide the proof of genuine use of the trade mark which has been registered for at least five years.277 As the General Court stated in Beifa, since the national law allows the alleged infringer to require in the infringement proceedings that the proprietor of a trade mark invoking his rights provides proof of genuine use of his mark, the proprietor should do it and if he fails – he has no right under the national law to prohibit the use of the Community design, which results in the inapplicability of Art. 25(1)(e) CDR.<sup>278</sup> This decision was issued on the basis of national (German) trade mark,279 however Art. 99 (3) CTMR provides for similar defence with respect to the Community trade mark280 and Art. 11(3) TMD provides for optional implementation of a corresponding provision into national laws of the Member States. In its decision, the General Court referred to the rules of infringement of the trade mark law and defences provided therein. This seems a correct approach since Art. 25(1)(e) CDR refers to "right to prohibit use" which suggests that a regular assessment of infringement under national or Community law should be made on application for invalidation of a Community design. However, Art. 99(3) CTMR allows not only for the request of proof of use but also for raising a defence of invalidity of the trade mark based on earlier rights of the design proprietor (similarly 11(3) TMD, however the TMD does not provide for defence of invalidity of the trade mark, unlike Art.99(3) in fine CTMR), nevertheless a defence stating that the Community trade mark or a national trade mark could be declared invalid should not be allowable. The question of invalidity of the trade mark invoked against the design should be judged in separate proceedings. Under Art 53 CDR the Office is entitled only to examine the invalidity of the Community design, therefore allowing for the examination of a trade mark validity would go beyond the competences of the OHIM in design invalidity <sup>275</sup> Ruhl 2007, supra note 89, Art. 53 para. 38, Community Design Invalidity Manual, supra note 15, C.7.4. <sup>276</sup> José J. Izquierdo Peris, OHIM Practice in the Field of Invalidity of Registered Community Designs [2008] 2 E.I.P.R. 56; Schlötelburg, supra note 129, 126; Ruhl 2007, supra note 89, Art. 25 para. 34. <sup>277</sup> Art. 99(3) CTMR, Art. 11(3) and 12(1) TMD. <sup>278</sup> GC Case T-148/08 - Beifa Group Co. Ltd. v OHIM, 2010 ECR II-01681, para. 65-66; this rule has been incorporated in Community Design Invalidity Manual, supra note 15, B.1.1.2. <sup>279 §25(2)</sup> MarkenG. <sup>280</sup> Ingerl/Rohnke, supra note 24, §25 para.4. proceedings. This however does not exclude the applicability of request of proof of genuine use of the mark, since it has no direct bearing on the mark's validity, only on its enforceability. Hence this approach is consistent with both the caselaw and submissions of the scholars and practitioners. Another provision stating a ground of defence in infringement is the "fair use" provision of Art 12 CTMR / Art. 6(1) TMD. This defence has not been addressed by the case-law as of now. It has been raised by the design holder in Zygmunt Piotrowski v Danone,281 however the Board of Appeal confirming the Invalidity Division's decision on invalidation, did not address this issue. It is submitted that since Art. 25(1)(e) CDR requires that the owner of the prior distinctive sign "has the right to prohibit such use", the "fair use" defence should be accepted, just as it is accepted in infringement proceedings. Although for it to be allowed, the design proprietor will have to fulfil the stringent requirements of proving that the use of the mark in a Community design is in accordance with honest practices in industrial or commercial matters and taking into account all possible circumstances in which the design might be used.282 Similarly as in the assessment under unfair competition rules, the decision on this defence depends very much on the factual pattern and therefore might lead to only some uses of the mark being fair. It is submitted that such an inconsistency might be solved by an application of a disclaimer under Art. 25(6) CDR. A defence of express consent to registration (Art 53(3) CTMR / Art. 4(5) TMD) could be considered to be used by the proprietor of a Community design in invalidation proceedings by way of analogy to the provisions of the CTMR. One argument for it could be the systematic interpretation of the CDR, which has been based on the provisions of the CTMR.283 However a strong argument against such an approach is the fact that such provisions have not been included in the CDR, hence it should be seen as a deliberate decision of the EU legislator and analogical application of the CTMR should not be accepted. Nevertheless such arguments could be enforced in national courts284 – in the case of application for an invalidation of a Community design its owner could apply for the na- <sup>281</sup> Case R 137/2007-3 - Zygmunt Piotrowski v Compagnie Gervais Danone, OHIM Third Board of Appeal Sept. 18, 2007, available at: http://oami.europa.eu/search/legaldocs/la/EN\_boa \_index. cfm under the case number, para. 4. <sup>282</sup> ECJ Case C-533/06 - O2 Holdings Limited and O2 (UK) Limited v Hutchison 3G UK Limited, 2008 ECR I-04231, para. 66. <sup>283</sup> Commission of the European Communities, Green Paper on the Legal Protection of Industrial Design [1991] (hereinafter: Green Paper), states (8.2.1 at 106) that unless there are solid reasons for not doing so, solutions adopted in CTMR should be accepted for Community design. <sup>284</sup> E.g. affirmative action under §256 ZPO. tional court's declaratory judgement and for suspension of proceedings in OHIM until the national judgement becomes final. The application for the invalidation of a design can be submitted as long as the design exists. However, since acquiescence (Art. 54 CTMR/ Art. 9 TMD) precludes the trade mark proprietor from opposing the use of that trade mark, it is arguable that under Art. 25(1)(e) CDR, such a defence could be accepted by way of analogy. However, similarly as with express consent to registration, CDR lacks legal ground for such an application. ## 2. Limits of protection of other distinctive signs and statute of limitations With the exception of protection for company symbols and work titles, which under §23 MarkenG are subject to the fair use limitation in the same way as trade marks are, and names, which cannot be enforced in cases of use of own name and where freedom of speech has priority, there are no special limitations of protection for enforcement of the other types of distinctive signs. The application of the statute of limitations should however be considered with regard to all distinctive signs. The right to prohibit use, as required by Art 25(1)(e) CDR is not limited in time and exists as long as the infringing activity takes place, i.e. in case of a Community design – as long as it is registered or protected as unregistered Community design. This lack of limitation can be questioned, as on the one hand the justification for the invalidation of a Community design is certainly the public interest in clearing the register of rights that do not deserve protection,285 which should not be limited in time, but on the other hand – Art. 25(1)(e) CDR expressly refers to the fact that the invalidation can go only as far as the owner of the prior sign has the right to prohibit the use of the allegedly infringing design under the Community or national law, which may include the national provisions regulating the statute of limitations. As it has been argued by Hacker, the registration of a sign leads to a constant infringement and therefore the right to apply for its invalidation cannot be limited in time.286 On the other hand arguably due to the public character of the Design Register, it must be assumed that the registration has become known to the holders of prior rights and the begin of the term of limitation is easy to establish. It is submitted that since Art. 25(1)(e) CDR refers to right to prohibit use, the assessment should not differ from that of <sup>285</sup> Ruhl 2007, supra note 89, Vor Art. 24-26, para. 3. <sup>286</sup> Hacker, supra note 19, 261. infringement, including the statute of limitations. The commencement starting from the publication in the register seems an appropriate solution. Under unfair competition protection, there is no explicit limitation of protection in time. As long as the competitive individuality exists and is able to indicate the origin or the specific features of the product and the anticompetitive behaviour is still effective (e.g. the design remains registered), the owner of the prior sign may institute an action under unfair competition rules. The right to claim protection under national law for trade marks, company symbols and work titles is precluded under the statute of limitations after three years from the obtaining knowledge of the infringement,287 according to §20 MarkenG. Similar rule applies to trade names and names under the general rule of §195 BGB.288 <sup>287</sup> It is submitted that in the case of registered design it should be from the date of the registration and not actual gaining knowledge, due to the public nature of the registers. 288 Ingerl/Rohnke, supra note 24, Nach §15 paras. 27, 172. https://doi.org/10.5771/9783845243856, am 03.04.2025, 22:22:53 Open Access – - https://www.nomos-elibrary.de/agb ## IV. The application of Art. 25(1)(e) CDR and its implications ## A. The procedure of invalidation of a Community design ## 1. Applicant and forum According to Art. 52 and Art. 25(1)(e) CDR it is solely the holder of a prior distinctive sign that has standing in the invalidation proceedings, which he is obliged to prove by submitting relevant documents, such as a registration certificate.289 In the case of unregistered signs the applicant will have to provide evidence as to the existence of his right to the distinctive sign. He can initiate the invalidation in OHIM – as regards a registered Community design, or in a Community design court290 – by way of counterclaim for invalidation of a registered or unregistered design when he has been sued for infringement of that design, or by a stand-alone action for invalidation of an unregistered Community design.291 ## 2. Applicable law and procedural challenges The substantive law relied upon in the invalidation proceedings depends on the distinctive sign that is being invoked against the Community design. In the cases where the application is based on the right to a Community trade mark it will be the provisions on the scope of protection stipulated in the CTMR.292 When a national right to a distinctive sign is relied upon – the OHIM or the Community design court will need to apply the provisions of the relevant national law.293 In the OHIM it is the duty of the applicant to substantiate both the facts294 and the legal ground in the same way as he would have done in the national court and he is also obliged to establish the applicable rules e.g. by filing the copies of relevant statutes or case-law.295 The OHIM or a Community design court will apply their own procedural rules in the invalidation proceedings: those stated in the CDR – in case of proceedings in the OHIM, or rules applicable for proceedings governing national designs, unless the CDR expressly provides otherwise – in an action in a Community design court.296 The fact that the respective bodies apply their own procedural rules, but depending on the invoked prior right, can apply national substantive laws, may cause tensions influencing the scope and limits of protection of distinctive signs, especially in the instances where the owner of a Community design wants to invoke defences. National laws, in particular in those aspects that have not been harmonized, allow for various defences, requiring diverse evidence and providing for different rules on burden of proof. These are often part of the national procedural rules. Therefore a question can be posed as to how far the application of the national rules should go, especially in the proceedings in the OHIM which are of an administrative and not judicial nature. In Beifa,297 the only case on Art. 25(1)(e) CDR adjudicated by the General Court so far, the court accepted the application of national German provisions allowing the design owner to request proof of use of the trademark serving as ground for invalidation, applying the German substantive rule of Art. 25(1) MarkenG298 limiting the right of the trade mark owner to assert claims under it and the arguably procedural defence under Art. 25(2) MarkenG299 allowing the defendant to request a proof of genuine use of a trademark registered for at least five years. Additionally the Court accepted the analogical application of the procedural rules on opposition to the registration of a Community trade mark, stating that the request of proof of genuine use should be filed in due time and cannot be made for the first time before the Board of Appeal.300 In many instances the CDR itself remains unclear as regards the applicable rules on procedure and in the absence of procedural provisions in OHIM refers to the rules generally recognised in the Member States.301 In particular, it does not include rules on defences that might be applicable in the invalidation proceedings. However, while the rules on defences may be generally seen as substantive provisions and therefore reference to the respective national laws should be acceptable, the rules on evidence are of procedural nature. The general rule of conflict of laws302 prescribes that upon application of foreign law the court may use the foreign substantive provisions, but must apply its national procedural law. Therefore, since the CDR does not foresee any specific procedural rules on application of foreign national laws during invalidation proceedings, the availability of those especially as defences should be limited. On the other hand, the General Court in Beifa seems to have accepted the application of national rules of a procedural character, at least to some extent. Whereas this might be practical in the case of national trade mark laws harmonised under the TMD, it seems problematic in the not harmonised regimes of unfair competition and of other distinctive signs, requiring the OHIM to gain expertise in 27 national legal regimes on both substantive and procedural level. Furthermore, Art. 25(1)(e) CDR uses the phrase "Community law or the law of the Member State governing that sign confers on the rightholder of the sign the right to prohibit such use". It is not clear whether this refers to the procedural or the substantive rules. This was also not explained in the travaux preparatoires.303 Other provisions of the CDR that include a reference to the national laws of the Member States include Art. 89(1)(d)304 and Art. 96(1) – which seem to refer to national substantive rules, but also Art. 84(3)305 and Art. 92(2) – which are more prone to be referring to national procedural rules. It is submitted that the harmonisation goal is more likely to be achieved if the application of certain provisions by both national courts and the Office leads to the same result. However the acceptance of the application of both substantive and procedural national rules by the OHIM finds no support in the rules governing conflict of laws and might lead to overloading the Office with tasks. Certainly guidance from the European Legislator on this matter would be desirable. 305 Ruhl 2007, supra note 89, Art. 83, para. 4. <sup>301</sup> Art. 68 CDR. <sup>304</sup> Which according to Ruhl should include also procedural rules of foreign countries which should be transformed into corresponding national provisions of the forum, Ruhl 2007, supra note 89, Art. 89 para. 75. ## 3. Effect of the invalidation The invalidation of a Community design has an ex tunc effect306 – the design is to be treated as if it had not existed at all, to the extent that it has been declared invalid. This effect is introduced when the decision on declaration of invalidity becomes final307 and is subject to the possibility to maintain the Community design in an amended form in spite of its eligibility for invalidation, as long as that form complies with the requirements for protection and the identity of the design is retained, which may include a disclaimer and which can be done upon a motion of the design's holder or by way of a decision declaring the design's partial invalidity.308 Due to the abstract nature of the design protection309 such an amendment may limit only the content of the design and not the goods or services for which it may be applied – therefore if that is not possible it should be declared invalid in toto.310 Despite the CDR being modelled on the rules of the CTMR, it does not include a provision corresponding to Art. 112 CTMR, which would allow for a conversion of a design challenged for validity into national design applications. Moreover, an amendment may not limit the territorial character of the Community design. Even in the cases where the application or counterclaim for invalidation are based on a national right – due to the unitary character311 of the Community design right, the effect of invalidation stretches onto the entire territory of the European Union,312 and is not limited to the territory where the prior right exists. This is different under German law as regards the unregistered trade marks313 and company symbols314 which may cause invalidation of a national design only when they are nation-wide, while in the cases where they exist locally – they have an effect of a territorial limitation of the design right.315 Therefore it has been suggested by the German authors, that an unregistered German trade 306 Art. 26(1) CDR, Suthersanen, supra note 21, 6-078. 315 Eichmann in: Helmut Eichmann and Roland Vogel von Falckenstein, Geschmacksmustergesetz [2010] C.H. Beck, §34 para. 3. <sup>307</sup> Art.87 CDR. <sup>311</sup> Art. 1(3) CDR. <sup>314 §5</sup> MarkenG. mark or a company symbol can provide a ground for invalidation of a Community design only when this prior right exists for the entire territory of Germany.<sup>316</sup> ## B. Invalidation of the design or action for infringement of the prior sign? As has been shown (supra in Chapter III.) the invalidation of a Community design on the ground provided in Art. 25(1)(e) CDR grants the prior distinctive sign a very broad protection, based on both harmonised and not harmonised legal grounds, requiring different conditions for grant of protection and level of proof and hence giving the holders of prior signs a rich arsenal of weapons against a Community design. Taking into consideration that if the design is novel and possesses an individual character, the owner of a prior sign can still invalidate it arguing that it infringes his distinctive sign, a question can be asked whether this owner could be more interested in invalidation of the entire Community design, or rather in starting a case on infringement of that sign, since the arguments he would be making in both proceedings correspond. After all, the invalidation of a Community design does not result in prohibition of use of the sign – it will only deprive the design owner of a negative right to stop others from using the design. What most owners of distinctive signs are interested in is in fact an injunction against the use of a design which can be obtained only in infringement proceedings and not upon application for invalidation of a Community design. But since a Community design benefits from an assumption of validity,317 a legitimate doubt arises as to whether the owner of a distinctive sign may obtain an injunction against the use of a later Community design on the ground of infringement of his rights to a sign, without first obtaining a decision on invalidation of such a design. This matter, although based on a slightly different factual pattern, has been a subject of a preliminary question to the CJEU by the Community Design Court in Alicante on 11 October 2010.318 The case refers to a conflict between two designs in a situation where the subsequent registration was effected after the receipt of a cease and desist letter from the owner of the prior design, who subsequently filed a lawsuit for infringement of his right. The other party's defence was that as long as the design is not declared invalid, its owner has a positive <sup>316</sup> Eichmann in: Helmut Eichmann and Annette Kur, Designrecht. Praxishandbuch [2009] Nomos, 93. <sup>317</sup> Art., 85 and Art. 94 CDR. <sup>318</sup> Reference for a preliminary ruling from the Juzgado de lo Mercantil No 1 de Alicante (Spain) Case C-488/10 - Celaya Emparanza y Galdos Internacional S.A. v Proyectos Integrales de Belizamientos S.L., available at: http://curia.europa.eu/jmcs/jmcs/j\_6/ under the case number. right to use the design under Art. 19(1) CDR and therefore a claim for infringement by such a design should be rejected for lack of the plaintiff's legal standing.319 The question referred inquired whether in the proceedings for an infringement of a Community design the owner has the right to prohibit the use by a third party of a later design that does not produce a different overall impression or by a party who uses such a design registered in his name as long as the later design is not declared invalid and whether the answer should depend on the intention of the third party in registering the design.320 The infringement actions are regulated by the national procedural laws, since all of them, based both on Community and national rights, are dealt with by national courts. An example of a provision that allows for an infringement action without prior invalidation of the accused registered right is Art. 110 CTMR which allows the owners of prior rights to invoke their claims for infringement of those rights by a later Community trade mark. This is independent from the opposition or invalidation proceedings and leads to a different result: it allows for a national court to prohibit the use of a Community trade mark on the territory of a Member state where the conflicting prior right exists.321 If an analogical application of Art. 110 CTMR to the Community design was accepted, the owner of a prior right would not need to apply for invalidation of a Community design, but would be able to limit the territorial scope of this right. It is submitted, that even though the CDR was modelled on the CTMR,322 it does not include a provision corresponding to Art. 110 CTMR, therefore it should be seen as an intentional decision by the legislator and analogical use of the CTMR should not be accepted. A further argument for a necessity of prior invalidation could be that due to the presumption of validity of a Community design which is binding not only in infringement action based on the design323 but also in other proceedings,324 in actions which are not enumerated in Art. 81 CDR (e.g. proceedings on infringement of a prior trade mark or other rights), it is not possible to challenge the validity of a Community design – neither by way of counterclaim nor as a defence. A separate application for invalidation of the design should be filed, subject to the suspension of the main proceedings.325 Arguably, the presumption of validity implies that the Community design does not collide with other rights, until it is invalidated due to such a collision. Hence an action for infringement of a distinctive sign by a subsequent Community design would be successful only after the invalidation of the design is declared. A different view326 was presented by the Advocate General Mengozzi in his opinion in the case referred to the CJEU for a preliminary ruling and was subsequently adopted by the Court.327 According to the ruling, the decisive consideration should be the "priority principle under which the earlier registered Community design takes precedence over later registered Community designs".328 Furthermore, the lack of substantive examination of the design, allowing for a quick registration of those rights must be taken into account. If prior invalidation of a design allegedly infringing earlier rights was required, it might lead to defendants registering their designs in order to block infringement proceedings instigated by owners of prior rights. This, according to the AG and the Court could result in unacceptable abuses of law. Therefore, an invalidation of a Community design is not a prerequisite for filing for a decision that that design infringes a right to a prior design and consequently that its use is prohibited. Even though the decision of the CJEU refers to a prior design, which does not constitute a distinctive sign, it is submitted that the argumentation presented by the Court can be extended onto cases of infringements of prior signs. The rationales of the ruling, referring to the principles of priority and possibilities of abuse of law, do not so much depend on the type of the allegedly infringed right, but more on the nature of the design right, which despite of the CDR containing pro- visions suggesting otherwise,329 remains an unexamined right, granting its proprietor only a negative right to prohibit others the use of that design, but not granting him an absolute right to use it as long as it remains valid. ## C. Invalidation based on a prior distinctive sign: novelty, individual character or Art 25(1)(e) CDR? The community design, being a relatively novel legal instrument330 still reveals a considerable number of open questions. Some of them are the controversies connected to the application of Art. 25(1)(e) CDR, especially as far as employment of national laws is concerned. Furthermore, due to the evidentiary burden resting on the applicant, covering not only the evidence on facts but also on law, Art. 25(1)(e) does seem less attractive than the other ground for invalidation available for the owners of prior distinctive signs, i.e. Art 25(1)(b) CDR. Even though when applying for invalidation of a Community design, the applicant can avail himself of many legal grounds simultaneously, the OHIM can base its decision on only one of them without referring to the others. As the information on the Invalidity Division decisions shows,331 more often than on the ground of Art. 25(1)(e) the applications are successful on Art. 25(1)(b) CDR. Whether this trend changes will depend on the expansion of the case-law on the Community design. A recent development in that respect was the definition of the "informed user" relevant for the assessment of the design's individual character. In the PepsiCo332 case, it has been suggested by the General Court and accepted by the Advocate General Mengozzi, that "the informed user is particularly observant and has some awareness of the state of the prior art, that is to say, the previous designs relating to the product in question that had been disclosed on the date of filing of the contested design, or, as the case may be, on the date of priority claimed".333 This definition has been accepted by the CJEU who con- firmed that this notion "must be understood as lying somewhere between that of the average consumer, applicable in trade mark matters, who need not have any specific knowledge and who, as a rule, makes no direct comparison between the trade marks in conflict, and the sectoral expert, who is an expert with detailed technical expertise. Thus, the concept of the informed user may be understood as referring, not to a user of average attention, but to a particularly observant one, either because of his personal experience or his extensive knowledge of the sector in question".334 Hence it is submitted that this development will not result in a major change in the attractiveness of Art. 25(1)(e) CDR as ground for invalidation in comparison to the test for the lack of individual character. It must nevertheless be stressed that the infringement test is not just a lower threshold of the individual character requirement. The tests of Art. 6 CDR, Art. 25(1)(b) CDR and of Art. 25(1)(e) CDR differ. The starting point of the assessment of the individual character is the design at issue. The informed user should compare it as a whole with the prior sign335 and if the additional or different elements of the design are such that they result in the design producing a different overall impression – the Community design will be deemed valid because it possesses an individual character. Conversely, the starting point of the assessment under Art. 25(1)(e) CDR is the prior sign and the decision whether it has been used in the design. If it has been used in the same form – the use is confirmed, regardless of any additional elements that the design might have, i.e. the design does not need to "limit itself" to the use of the prior sign to be using the sign and therefore be eligible for invalidation.336 The comparison is not made between the prior sign and the design as in Art. 6 CDR but between the prior sign and the sign constituting an element of the design. It is also made from a perspective of a relevant consumer, which in many cases will involve a lower level of attention than the informed user and make the infringement case easier to argue. 336 Id. 152. in CJEU Case C-281/10P – PepsiCo, Inc. v Grupo Promer Mon Graphic SA, O.J. (C 362) 9, available at: http://curia.europa.eu/jcms/jcms/j\_6/ under the case number, para. 55. <sup>334</sup> CJEU Case C-281/10P – PepsiCo, Inc. v Grupo Promer Mon Graphic SA, O.J. (C 362) 9, available at: http://curia.europa.eu/jcms/jcms/j\_6/ under the case number, para. 53. <sup>335</sup> Suthersanen, supra note 21, 114-115. https://doi.org/10.5771/9783845243856, am 03.04.2025, 22:22:53 Open Access – - https://www.nomos-elibrary.de/agb ## V. Summary The analysis presented above shows some of the advantages and disadvantages of Art. 25(1)(e) CDR as ground for invalidation of a Community design. The basic advantage from the perspective of the owner of a sign seeking protection is that it allows him for recourse to many legal regulations in order to invalidate the design, which may include Community and national, harmonised and independent legal grounds, as depicted on the example of German law. Thereby the scope of protection for distinctive signs against the Community design proves broad and flexible. On the other hand, even though most of those grounds are part of well established national systems, their application in a Community context remains unclear. In spite of Art 25(1)(e) CDR referring to "right to prohibit use" which would suggest the application of all national rules, it is doubtful that national procedural rules could be applied in OHIM. Furthermore, the legal uncertainty associated with the admissibility and scope of defences against the claim for infringement results in a relatively infrequent application of that ground for invalidation. Often the owners of prior distinctive signs are more interested in obtaining injunctions against use of the design than in invalidating it. The decision of the CJEU337 not requiring an invalidation of a Community design before an injunction against its use can be issued will not result in a rise of design invalidations and more frequent application of Art 25(1)(e) CDR. On the whole, the applicability of Art. 25(1)(e) CDR as ground for invalidation of a Community design remains relatively less attractive for the holders of prior signs, who more often avail themselves of the ground for invalidation provided in Art. 25(1)(b) CDR. Nevertheless, a further development of case-law might create incentives for a more frequent application of infringement as ground for invalidation of a Community design. <sup>337</sup> CJEU Case C-488/10 – Celaya Emparanza y Galdos Internacional S.A. v Proyectos Integrales de Belizamientos S.L., Feb. 16, 2012, available at: http://curia.europa.eu/jcms/jcms/j\_6/ under the case number. https://doi.org/10.5771/9783845243856, am 03.04.2025, 22:22:53 Open Access – - https://www.nomos-elibrary.de/agb ## List of Works Cited ## Legal texts ## European Union ## Germany ## Benelux Convention Bénélux en matière de propriété intellectuelle (marques et dessins ou modèles) [Benelux Convention concerning Intellectual Property (Trademarks and Designs)], February 25, 2005 MB 26.04.2006 (Benelux) (Benelux IP Treaty). ## Books ## Journal articles Víctor Sáez, The Unregistered Community Design [2002] E.I.P.R. 585. Martin Schlötelburg, Musterschutz an Zeichen [2005] GRUR 2005 123. Fabian Zigenaus, Comments to OLG Köln, Case 6 U 67/10, GRUR-Prax 2011, 10. Case-law European Court of Justice / Court of Justice of the European Union ECJ Case 102/77 - Hoffmann-La Roche & Co. AG v Centrafarm, 1978 ECR 01139. ECJ Case C-251/95 - Sabel BV and Puma AG v Rudolf Dassler Sport, 1997 I-06191. ECJ Case C-337/95 - Christian Dior BV v Evora BV, 1997 ECR I-06013. ECJ Case C-210/96 - Gut Springenheide and Tusky, 1998 ECR I-04657. ## Opinions of Advocates General ## Court of First Instance / General Court CFI Case T-6/01 - Matrazen Concord GmbH v OHIM, 2002 ECR II-04335. CFI Case T-130/01 - Sykes Enterprises Incorp. v OHIM, 2002 ECR II-05179. GC Case T-9/07 - Grupo Promer Mon Graphic SA v OHIM, 2010 ECR II-00981. GC Case T-303/08 - Tresplain Investments v OHIM, O.J. (C 30) 35, available at: http://curia.europa.eu/jmcs/jmcs/j\_6/ under the case number, appealed to CJEU C-76/11P. ## References for preliminary rulings ## OHIM Invalidity Division ## OHIM Boards of Appeal Case R 211/2007-3 - Burberry Ltd. v Jimmy Meykranz, OHIM Third Board of Appeal March 3, 2008, available at: http://oami.europa.eu/search/legaldocs/la/EN\_boa\_index.cfm under the case number. Germany BGH GRUR 1966, 503 - Apfel-Madonna. BGH GRUR 1983, 377 - Brombeer-Muster. BGH GRUR 1985, 876 - Tchibo/Rolex I. BGH NJW 1991, 2023 - Case II ZR 259/90. BGH GRUR 1993, 151 - Universitätsemblem. BGH GRUR 1995, 57 - Markenverunglimpfung II. BGH GRUR 1996, 210 - Vakuumpumpen. BGH GRUR 1996, 581 - Silberdistel. BGH GRUR 1997, 754 - "grau/magenta". BGH GRUR 1998, 696 - Rolex-Uhr mit Diamanten. BGH NJW-RR 2003, 1551 - Tupperwareparty, English translation available in [2004] IIC 459. BGH GRUR 2005, 349 - Klemmbausteine III. BGH GRUR 2005, 419 - Räucherkate. Towel Hooks (I ZR 131/02) BGH [2006] IIC 348. ## United Kingdom (decisions available at: http://www.bailii.org/databases.html) Green Lane Products Limited v PMS International Group [2008] EWCA Civ 358. Tesco Stores Ltd v Elogicom Ltd [2006] EWHC 403, E.T.M.R. 91. L'Oreal v eBay [2009] EWHC 1094 (Ch). Whirlpool Corp v Kenwood Ltd [2008] EWHC 1930 (Ch). Whirlpool Corp v Kenwood Ltd [2009] EWCA Civ 753, E.T.M.R. 7. ## Reports, Manuals and Guidelines ## Internet sources	doab	2025-04-07T03:56:57.985567	12-2-2021 04:05	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "00134e2c-4cd5-4041-bb42-ea5f0ee762e6", "url": "https://www.nomos-elibrary.de/10.5771/9783845243856", "author": "Kolasa, Magdalena", "title": "The Scope and Limits of Protection for Distinctive Signs against the Community Design", "publisher": "Nomos Verlagsgesellschaft mbH & Co. KG", "isbn": "", "section_idx": 0 }
001c1372-3390-4f92-8b42-7dfe9fe510d8	# Reza Che Daniels # How Data Quality Affects our Understanding of the Earnings Distribution How Data Quality Affects our Understanding of the Earnings Distribution Reza Che Daniels # How Data Quality Affects our Understanding of the Earnings Distribution Reza Che Daniels School of Economics University of Cape Town Rondebosch, South Africa ISBN 978-981-19-3638-8 ISBN 978-981-19-3639-5 (eBook) https://doi.org/10.1007/978-981-19-3639-5 © The Editor(s) (if applicable) and The Author(s) 2022. This book is an open access publication. Open Access This book is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this book are included in the book's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the book's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore ## Preface How data quality affects our understanding of the earnings distribution. by Reza Che Daniels In household surveys, estimating parameters of the earnings distribution is frequently complicated by multiple sources of survey error, often leading to claims of "poor data quality". However, it is not always the case that multiple sources of survey error leads to poor data quality, and knowing the difference between "good data" and "bad data" is important for both research and policy-making purposes. Far too often in my experience, claims of "poor data quality" are based on ignorance at best, intellectual laziness at worst, providing too easy an escape for dealing with the mostly manageable (statistically) repercussions of data production optimisation decisions that must balance cost, timeliness and accuracy. The 'flip-side' to this is the researcher that makes too brazen a claim about point estimates of paramaters from data that is not intended to measure the kind of outcomes reported on. For individual income data, or employee income data that we shall call "earnings", estimates of poverty and inequality are often estimated with statistically unsound methodologies that leave more doubt about those estimates than inspire confidence. Due to the politically sensitive nature of constructs such as poverty and inequality, it is the responsibility of the research community to provide sound guidance in this respect. This book is concerned with developing a statistically sound methodology for estimating parameters of the income distribution in household surveys that often contain multiple sources of survey error—some that are observable and some that are unobservable. The country of interest is South Africa, and we focus on a period in South Africa's history just after the transition to democracy in 1994 when the geopolitical boundaries of the country had stabilized, but the best way to measure income was still being debated not only in South Africa (SA), but internationally. In 1996 the International Expert Group on Household Income Statistics (that became known as the Canberra Group), was formed in response to widespread recognition of data quality concerns in household income distribution statistics. They published the 2001 Canberra Group Handbook, which became a key reference manual for national statistics agencies. The whole theme of household income statistics was given a greater spotlight by the newly formed (at the time) Millenium Development Goals, the first of which was to "eradicate extreme poverty and hunger" by 2015. Researchers and statisticians interested in creating baseline estimates of poverty around the world turned to the 2001 Canberra Group Handbook for guidance. We discuss how Statistics South Africa (SSA) deviated from, but later came closer to, key recommendations in the Canberra Manual. Along the way, however, there were important limitations in the data that need to be addressed by researchers interested in analysing this period in SA's history. Two periods are of interest in this book: (1) the immediate post-1994 period during which the geopolitical boundaries of South Africa were recast; and (2) 1997–2003, which is after both the transition to democracy in 1994 and the 1996 national census. The 1996 census was the first time that Statistics South Africa could enumerate the now contiguous geopolitical entity of the democratic country, after reincorporation of the former Apartheid-era "Bantustans" (which were supposed to be independent tribal homelands set up by the Apartheid government and excluded from the definition of South Africa in the Bantu Homeland Citizenship Act of 1970). This afforded SSA the opportunity to create a new sampling frame from which to draw more representative samples of the SA population for household surveys. Between 1997–2003, SSA continued to run the October Household Survey (OHS), which was started in 1993 to obtain general information (including income) from a representative sample of individuals. However, prior to the 1996 Census the OHS had an outdated sampling frame. By isolating the period 1997–2003, we are able to zoom in on a period of important changes to the way income questions were asked. In 2000, SSA discontinued the OHS and commenced the Labour Force Survey (LFS) a biannual survey that continued until 2008, when it was replaced with the Quarterly Labour Force Survey (QLFS). We restrict the analysis of the LFS to 2000–2003 only, because it captures a period during which questionnaire design changes to the income question stablised. Over the course of the book we develop a framework that researchers can use to investigate data quality, and then apply this framework to SSA household surveys in the post-Apartheid era. Chapter 1 introduces the book and provides some background to debates in the survey methodology and econometrics communities concerning income statistics and the data quality concerns that must be addressed in order to generate estimates of poverty headcounts and inequality indices. Chapter 2 develops a framework for investigating microdata quality that is a guide for researchers working with any public-use dataset, often with poor information about the survey quality control process, about how to identify different components of survey error. It is largely based on integrating the total survey error paradigm with data quality metrics that shed light on the possible factors that influence point estimation of key parameters of interest. The framework is then utilised to investigate the evolution of data quality in SSA's labour market surveys. Preface vii Chapter 3 isolates questionnaire design and item nonresponse for the employee income question in two South African labour market surveys: the October Household Survey (OHS, 1997–1999) and the Labour Force Survey (LFS, 2000–2003). This time period isolates a period of changing questionnaire design for the income question. Between 1997 and 2000, the employee income question gradually included new response options for the respondent to state that they don't know or refuse to answer the question, which turn out to be important distinctions. We use sequential logistic response models to evaluate how improvements to the income question improved the capacity to understand the nonresponse and bounded response options. We then evaluate the empirical stability of predictors of response type between 1997–2003. Chapter 4 is concerned with conducting univariate multiple imputation for employee income with nonresponse and bounded responses. A variable with this mixture of data types is called coarse data. Because the income question consists of two parts—an initial, exact income question and a bounded income follow-up question—the resulting statistical distribution of employee income is both continuous and discrete. An analysis of the interrelationship between the exact income and bounded income variables released in the public-use data also reveals a non-trivial degree of processing error for certain survey years between 1997–2003. We identify two forms of processing error that have to be dealt with before multiple imputation can be performed. We then conduct multiple imputation using four differently specified models to test the sensitivity of imputed draws of income to mis-specification in the imputation algorithm. We also evaluate the point estimates of quantiles and moments of the multiply imputed income distributions as the number of imputations increase. Chapter 5 draws on the lessons learnt in the preceding chapters to identify how data quality will always influence our understanding of the income distribution. We focus on what can be 'fixed', what cannot be, and what might matter for different sorts of analyses. We also generalise the findings in the book so that the methods enumerated can be applied to any household survey concerned with measuring income, anywhere in the world. Chapters three and four taken in combination are key to this. It is my hope that this monograph provides guidance to researchers and data scientists about how to both frame and deal with data quality in microdata, specifically when analysing income and the constructs of poverty and inequality that are so important to policymakers and to measuring socio-economic progress. The methods are reproducible and I'm sure others will improve upon them. This is welcomed. As a research community, let us do what we do, well. Cape Town, South Africa Reza Che Daniels ## Acknowledgements First and foremost, I would like to thank my family for their constant support. I would also like to thank my colleagues Murray Leibbrandt and Martin Wittenberg for their valuable advice in the creation of this monograph. Steve Heeringa at the University of Michigan played a seminal role in stimulating my intellectual curiosity in this field, and I am also grateful for his insights in the development of this book. I cannot stress enough how much being part of the incredible teams at the Southern Africa Labour and Development Research Unit (SALDRU) and DataFirst—both at the University of Cape Town—assisted me by providing a rich intellectual ecosystem for the ideas in this book to be birthed, trialled and tested over multiple years and survey instruments. SALDRU also funded the open access costs of this book, for which I am eternally grateful. # Contents #### Contents xiii # About the Author Reza Che Daniels is Associate Professor in the School of Economics, University of Cape Town. He was one of the Principal Investigators of the National Income Dynamics Study (NIDS), South Africa's first nationally representative longitudinal household survey. He is also one of the Principal Investigators of the NIDS-Coronavirus Rapid Mobile Survey (NIDS-CRAM), which uses a sub-sample of the NIDS to monitor the impact of COVID-19 in South Africa. # List of Figures # List of Tables ## Chapter 1 Introduction This book is concerned with the measurement and quality of employee income from household survey (micro) data. The empirical applications are based on South African household surveys compiled by the national statistics agency (Statistics South Africa). Despite this specificity, the insights are generalisable to any household survey concerned with measuring income. Data quality is a central theme in any data compilation effort. However, it is often very difficult to diagnose where exactly in the data production process data quality falters. Data quality is a concern for both macro- and microdata. For macro-economic data, the International Monetary Fund (IMF) presides over the process of ensuring standards are developed for the production of national economic statistics associated with the System of National Accounts (see IMF, 2003 for the latest such framework). For household survey data, there are data quality frameworks for surveys themselves (see Statistics Canada, 2003, 2009 and Statistics South Africa, 2006a; 2006b), and for specific themes like income. In all household survey data, several forms of error are present in different magnitudes, including coverage error, sampling error, nonresponse error, adjustment error, processing error, measurement error and validity. These components of error form part of the total survey error paradigm (Groves et al., 2004), and can often be exacerbated by poor data quality management within statistical organisations. To understand data quality therefore requires some understanding of the practises inside statistical organisations with respect to data quality control. Examples of such data quality control elements from Statistics Canada (2003) and Statistics South Africa (2006a) include relevance, timeliness, accessibility, interpretability, coherence, integrity, methodological soundness and accuracy. For income data measured in household surveys, the Canberra Group's (2001; 2011) recommendations on household income statistics is the main reference. The Canberra Group was a group of national statistics and other data compilation agencies from over fifteen countries, plus representatives from many international agencies, whose main objective was to "... enhance national household income statistics by developing standards on conceptual and practical issues related to the production of income distribution statistics" (Canberra Group, 2001, xi). The global level of importance accorded to this task was noteworthy, for it coincided with the adoption of the Millennium Development Goals, the first of which was to halve absolute poverty, defined as all those living below US\$1.00 per day in constant purchasing power parity (PPP) adjusted terms, between 1990 and 2015.1 The income distribution has been a central preoccupation of economists since the inception of the discipline due to its positive correlation with individual and societal welfare. An important formalisation of the work on income distributions was made by Vilfredo Pareto in the nineteenth century, who found after analyses of empirical income data on several European countries that the probability distribution of income was right-skewed (Kirman, 2008). More detailed analyses of income distributions since then led to the realisation that several possible statistical distributions have valid application to income over different ranges of the variable (see (Cowell, 2000) for discussion). As long as people have analysed income distributions there have been debates about the data utilised for this purpose. Income is measured both in the national accounts and with household survey data. However, the methodologies used to collect and aggregate this data renders income measured in the national accounts to be quite a different construct to income measured in household surveys (Havinga et al., 2010). This book is concerned with income measured in household surveys only. #### 1.1 The Income Construct in Household Surveys Generally, when income distribution is discussed, the debate concerns the distribution of total income. But total income is comprised of many components. The Canberra Group (2001, 18) distinguish the following types of income that together sum to total income: This book is primarily concerned with employee income. Employee income is considered to be a form of cash income that is easily and accurately measured relative to property income and cash transfers (Canberra Group 2000, 13). However, the employee income question in household surveys is complicated by a feature that is designed to increase the probability that a respondent answers the question. That is, a second, bounded income bracket question is presented to respondents as a followup to the exact income question in the event that they refuse to answer or state that <sup>1</sup> See http://www.un.org/millenniumgoals/poverty.shtml. they don't know. This leads to an income variable with a continuous distribution for exact income responses and a discrete, grouped continuous distribution for bounded income bracket responses. Respondents can also refuse to answer the follow-up question, or once again state that they don't know their income or that of the proxy respondent on whose behalf they are reporting. Consequently, there is also nonresponse to the employee income question. How researchers treat the many issues that confront them with income data in public-use household surveys can often be very different, leading to different estimates of parameters of the income distribution from the same dataset. The advantage of having a follow-up income question with a lower level of information disclosure is that it reduces the social sensitivity of the question, but can also aid respondent recall. Consequently, some form of follow-up question that bounds the range of income is often also asked in household surveys for other components income, including income from self-employment, rentals, property and transfers. Therefore, while the emphasis in this book is on employee income, the insights are generalisable methodologically to any component of income that is measured in a similar way. The overall quality of household surveys also has an important bearing on the accuracy of individual income statistics. In South Africa (SA), nationally representative sample surveys have only been compiled by Statistics South Africa (SSA) since the early 1990s. Before 1994, the geopolitical borders of SA included the Bantustans, considered separate by the Apartheid Government to the state of SA. Consequently, in the national statistics community in the mid 1990s, more emphasis was placed on creating new sampling frames for the democratic SA than refining questionnaire design for constructs like employee income. This necessary trade-off in the data production process led to poorer quality income data initially that gradually improved as other operational aspects of the household surveys themselves improved. #### 1.2 Objectives and Chapter Typology The main objectives of this book are: Chapter Two is directed at understanding the universe of errors that can arise in household surveys and linking these to data quality protocols inside statistical organisations. It identifies specific data quality metrics for each component of survey error that can arise. It then applies this framework to South African labour market household surveys. The chapter provides a general taxonomy for investigating data quality that can be useful to researchers whose aim it is to understand the relationship between survey error and data quality in public-use datasets. In order to demonstrate this, the individual income variable is reviewed for the employed population of South Africa, evaluated over multiple survey instruments and time periods, ranging from 1995–2007. Chapter Three in this book isolates the design of the employee income question in household surveys and the propensity of respondents to provide a particular response type. Employee income is typically measured in a way that allows respondents to provide either an exact income value or an interval into which it falls. It is then the user's responsibility to generate a variable that combines these two response types appropriately. However, missing data is also present when respondents refuse to answer the question or state they don't know. Understanding the different subsets of respondents sheds light on the trade-offs of questionnaire design for employee income, and provides valuable insight into the response process that can inform single and multiple imputation exercises. The final substantive chapter then goes on to investigate public-use employee income data with a mixture of continuously distributed income observations, groupedcontinuous observations and item nonresponse. This mixture of data types is called coarse data in the literature, and has important implications for imputing plausible values for such data. In SSA's household surveys, we also find that there is a nontrivial degree of processing error in the two income variables released in the publicuse dataset that must be treated appropriately before multiple imputation exercises can commence. We then conduct multiple imputation and discuss several aspects of the imputation algorithm – from the estimation method, to constraints on the bounds of the plausible draws, to the specification of prediction equations – all of which have a bearing on the reliability of imputed draws. Once these concerns have been addressed, multiple univariate imputations of employment income from coarse data can be obtained in a manner that allows researchers to account for the greater uncertainty inherent in that data. This then allows for the reliable estimation of univariate parameters of the income distribution. The time-frame for the analysis spans the mid 1990s to the latter part of the 2000s for Chapter Two. However, for Chapters Three and Four, the time-frame is restricted to 1997–2003. This is because this period was associated with important changes in the way household surveys were conducted in Statistics South Africa. Between 1995–1999, the October Household Survey was a repeated cross-sectional survey that collected labour market data as well as more general household information. From 2000 onwards, this survey was split into the Labour Force Survey (LFS) and the General Household Survey. Only the LFS is analysed in this book. This allows us to identify the role of questionnaire design in improving the quality of income data. The LFS was designed as a rotating panel survey whose explicit purpose was to obtain accurate estimates of employment and unemployment. In Chapters Three and Four, only the September Waves of the Labour Force Survey are analysed in conjunction with the OHS 1997–1999. Because the LFS is a rotating panel household survey (see Cantwell, 2008 for a definition), a proportion of the respondents change with each Wave of the survey, ensuring that it is representative of the South African population at the time of going to field. Therefore, it is possible to analyse the crosssectional OHSs in combination with individual waves of the rotating LFS panel. The final chapter in this book concludes the discussion. Since each chapter contributes original insight into different aspects of data production and use, the Conclusion stresses the need to factor all of the issues discussed in this book into an overall set of guidelines for estimating parameters of the income distribution. The discussions in chapters three and four, in combination, provide particularly powerful insights about how to ultimately derive reliable points estimates about poverty and inequality. #### References SSA. (2006b). Data quality policy 001: Policy on informing users of data quality. Pretoria: SSA. Statistics Canada. (2003). Statistics Canada quality guidelines (4th ed.). Ottawa: Statistics Canada. Statistics Canada. (2009). Statistics Canada quality guidelines (5th ed.). Ottawa: Statistics Canada. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Chapter 2 A Framework for Investigating Microdata Quality, with Application to South African Labour Market Household Surveys #### 2.1 Introduction This chapter identifies a framework for investigating microdata quality that is particularly useful to researchers working with public-use micro datasets where limited information about the data quality protocols of the survey organisation are present. It then utilises this framework to investigate South African labour market household surveys from the mid 1990s to 2007. In order to develop the framework, we rely on the total survey error (TSE) framework to articulate the forms of statistical imprecision that exist in any public-use dataset. The magnitudes of statistical imprecision are largely dependent on the efficacy of the survey organisation's data quality control protocols, which are, in turn, affected by human resource and budget constraints. The objective of this chapter is to provide researchers with the tools needed to assessthe quality in public-use datasets, to the extent that components of survey error are identifiable. Researchers will always have imperfect information in this regard, yet in South Africa at least, this has not stopped both the academic community and policymakers from making public statements about data quality that are often ill-informed and frequently incorrect. The choice of time-period to investigate microdata quality in South Africa (SA) coincides with a period of profound change in the country associated with the transition to democracy in 1994. Geopolitical changes included the provincial boundaries within SA and the incorporation of former Bantustans, which were previously "homelands" for Black South Africans (some of which were self-governing) created by the Apartheid government. The national statistics agency (Statistics SA) therefore had to increase the scope of their operations and develop new sampling frames. Over time, new surveys were conducted and gradually more attention was devoted to the quality of the data and sophistication of the survey instruments. The October Household Survey (OHS) was the first household survey conducted in democratic South Africa to include a labour market component, and officially started in 1993. However, both the 1993 and 1994 versions of the survey have magnitudes of survey error that have resulted in very few researchers utilising them (see Wittenberg, 2006 for discussion). We therefore commence with the OHS 1995 to OHS 1999. The Labour Force Survey (LFS) replaced the OHS as the labour market survey for SA in 2000. We analyse the data from the LFS until 2007, whereafter it became the Quarterly LFS and changed in frequency and design. In order to understand what was going on inside the national statistics agency in the mid 1990s, a qualitative interview with a retired sampling statistician (Professor David Stoker) was conducted (see Daniels andWittenberg, 2010). Prof Stoker worked in Statistics SA (SSA) in various capacities from the late 1980s until the early 2000s, and was in a unique position to shed light on the data quality pressures facing SSA over the time period. Information from this interview is supplemented by the survey Metadata and other survey documentation released to the public by SSA in each year of the OHSs and LFSs. In narrating these issues, a valuable historical record has been created of microdata quality in South Africa during one of the most fascinating periods in the country's history. The rest of this chapter proceeds as follows. Firstly, we discuss the importance of framing data quality debates such that they do justice to both data production (the perspective of the survey organisation) and data consumption (the perspective of the researcher). Then we consider the interaction between specific data quality elements and components of survey error. This creates the framework for investigating microdata quality. We then apply this framework to SA labour market household surveys from 1995 to 2007. Lastly, we discuss the generalisability of the framework and its scope for application to other surveys and countries. #### 2.2 Framing the Discourse on Data Quality Microdata quality is an artifact of a data production process controlled by survey organisations with finite budgets. This data production process commences with the conception of a project and concludes with public release of the data. Consumers of data (researchers) become concerned with data quality in the public-use dataset when it becomes apparent that univariate, bivariate and/or multivariate distributions in the data are problematic. This means that both the production and consumption dimensions of microdata need to be considered when attempting to create a framework for investigating microdata quality. In this section we locate the discourse of creating a framework for microdata quality at the nexus of the data production and consumption process, i.e. when considering parameters of interest on variables released in a public-use dataset. Researchers only observe the final product released by the statistical organisation, and so do not have the information to make accurate judgments about where in the data production process data quality falters. However, they can see inconsistencies in the statistical distributions of variables of interest that often hint at poor data quality. Survey organisations, on the other hand, rarely consider bivariate and multivariate relationships before publishing the data, and so often miss the insights researchers glean as users of the data. Below we define data quality elements in the data production process. This helps clarify the context in which survey organisations operate. Then we discuss a taxonomy of statistical errors in the survey process encapsulated by the total survey error (TSE) framework. TSE has proved itself useful to survey organisations to guide an understanding of the relationship between data quality and sources of statistical error. For researchers, the TSE framework is useful as a conceptual map to think more clearly about data quality in public-use datasets. #### 2.2.1 Data Quality Elements in the Data Production Process Data quality management, evaluation and reporting has become an increasingly important issue to statistical organisations and (inter)national agencies tasked with generating or compiling information for third-party users. In turn, for users of the data, understanding data quality necessitates an understanding of the processes leading up to public release. Formal recognition of the need for data quality indicators has been acknowledged in the broader statistical community for some time. Recent efforts by the economics community with respect to microdata quality has also raised the primacy of this debate (see Flinn et al., 2001). Brackstone (1999) identifies six dimensions of data quality: relevance, accuracy, timeliness, accessibility, interpretability, and coherence. Underlying these six dimensions is the idea that the data ought to be 'fit for use'. "Fitness for use encompasses not only the statistical quality concepts of variance and bias, but also other characteristics such as relevance and timeliness that determine how effectively statistical information can be used" (StatCan, 2003, 6). These ideas have become the bases for many national statistical organisations developing data quality manuals, such as Statistics Canada (2003, 2009). Statistics South Africa (2009, 2010) define two additional dimensions of data quality, namely methodological soundness and integrity (SSA, 2010). These two additional qualities hint at resource constraints (particularly human resource constraints) that may be more binding in developing countries. However, they are not necessarily separate from Brackstone's data quality concerns and can in fact be considered to be fully nested within them. Brackstone's (1999: 143) six themes are worth elaborating: "relevance" refers to the degree to which statistical information meets the needs of users or clients; "accuracy" refers to the degree to which the information correctly describes the phenomena it was designed to measure, and includes such concepts as mean square error; "timeliness" refers to the delay between the reference period and the date of public release, and typically involves a trade-off against accuracy; "accessibility" refers to the ease with which users can obtain the information; "interpretability" refers to the availability of the supplementary information and metadata necessary to interpret and use the data correctly; and "coherence" refers to the degree to which it can be successfully brought together with other statistical information within a broad analytical framework and over time. These components of data quality are resource-dependent, and for a well funded statistical organisation like Statistics Canada (which Brackstone, 1999 based his work on), the scope to invest in each of these dimensions of data quality is high. That said, Groves (2004) and Heeringa and Groves (2006) note that regardless of the size of resources available, there is always an optimisation problem when it comes to maximising data quality with a finite budget. But the size of the budget itself is not trivial. In fact, in low-income countries survey operations in national statistical offices can be severely restricted due to very small budgets (compared to their more well funded high-income country counterparts). Glewwe (2005) notes that in developing countries, these constraints imply that more careful planning is needed before a survey goes to field in activities such as drafting budgets and securing financing, developing a work plan for remaining activities, drawing a sample of households to be interviewed, writing training manuals, training field and data entry staff, preparing fieldwork and data entry plans, conducting pilot tests and launching publicity campaigns. Data quality concerns must therefore also be considered within the environment in which statistical organisations function. #### 2.2.2 The Total Survey Error (TSE) Framework The TSE framework can be used as a taxonomy to understand the scope of potential error sources in a micro dataset. The determinants of data quality are principally under the control of the survey organisation, where conscious effort needs to be invested in each step of the survey process in order to manage the quality of the data obtained. When the data finally get to a stage ready for public release, certain forms of survey error may still be present in the data. It is then up to researchers to identify if, how and when any remaining sources of error will affect their analyses. But researchers do not have the necessary auxiliary information to diagnose all forms of survey error precisely. This is exacerbated when survey organisations release poor documentation with public-use datasets. Under these circumstances, researchers can often face grave doubts about whether their analytical results are indeed valid or if they are rather an outcome of an unreliable data generating process. Components of survey error can generally be split into two forms: errors of observation and errors of nonobservation. Errors of nonobservation are those arising because measurements were not taken on part of the population, whereas observational errors are deviations of the answers of respondents from their true values (Groves, 1991, 2). In line with this, the TSE framework disaggregates the components of error into two themes: (1) measurement of the variable of interest, and (2) representation of the population of interest. Figure 2.1 presents a schematic overview of TSE. Under the measurement theme, the possible sources of error include validity of the construct, measurement error and processing error. For the representation theme, the sources of error include coverage error, sampling error, nonresponse error, and adjustment error. Because researchers and survey organisations frame the concept Fig. 2.1 Agency (i.e. Survey Organisation (SO), Researcher (R)) in the total survey error framework. Source Adapted from Groves, Fowler, Couper, Lepkowski, Singer & Tourangeau, 2004, 48 of data quality differently, it is helpful to consider the agency of these two groups in the TSE framework. A few terms in the figure require explanation (taken from Groves, 2004, vi). Coverage error stems from the failure to give some person or group of persons any chance of inclusion in the survey sample. Non response error stems from the failure to collect data on all persons in the sample, while sampling error arises from differences in the survey sample compared to the population it is trying to measure. Measurement error stems from inaccuracies in responses recorded on the survey instruments, and can be attributable to four different components: (a) effects of interviewers on the respondent's answers to survey questions; (b) error due to respondent's inability to answer questions, lack of effort, or other psychological factors; (c) error due to weaknesses in the wording of survey questionnaires; and (d) error due to effects of the mode of data collection (e.g. face-to-face surveys, telephone surveys). Non response error can be split into unit nonresponse (meaning entire sampling unitsrefuse to participate in the survey) and item nonresponse (meaning an individual responds to some questions in the questionnaire, but not to others). End-users of the data are unable to deal with unit nonresponse, but are able to deal with item nonresponse, where single and multiple imputation methods become applicable given a plausible model about the response mechanism. Adjustment error arises out of the need to adjust the survey for coverage error, sampling error and (unit) nonresponse error. Typically this is done by calculating weights. In South Africa, survey organisations usually combine individual weights into a single weight that is included in the public release version of the dataset. When this is the case, researchers are unable to separate out the components of the weight, and so are left without the means to investigate how each weight was calculated. From Fig. 2.1 we can see that on the measurement side of the TSE framework, researchers have insight into processing error and certain forms of measurement error. However, it is unusual that any informed insight can be gleaned about construct validity in public use datasets—certainly insofar as understanding the sensitivity of question wording on outcomes is concerned, which would be part of the question pre-testing phase presided over by the survey organisation. Cases where researchers are able to directly engage with construct validity do exist though, especially when appraising whether a questionnaire accurately captures some externally defined construct, such as (broad or narrow) unemployment or the informal sector. On the representation side of the TSE framework, item nonresponse and adjustment error are the two components that researchers can gain some insight into. Item nonresponse can be imputed by either the researcher or the survey organistion, but adjustment error is usually the domain of the survey organisation. However, there are circumstances when researchers are able to identify whether errors have been made in the adjustment process. In South Africa, Branson and Wittenberg (2007, 2011) and Branson (2009) have analysed the weights in Statistics SA's labour market household surveys and found several inconsistencies. Finally, it is incumbent upon both the survey organisation and the researcher to compute final survey statistics appropriately. It is the former's responsibility to provide all the documentation, weights and survey design features (such as variables used to stratify, cluster and make finite population corrections) necessary for researchers to generate accurate point estimates from public-released data. It is then the researcher's responsibility to account for survey design features in their univariate, bivariate and multivariate analyses (for example, see Daniels and Rospabe, 2005). #### 2.3 The Interaction Between TSE and Data Quality While the TSE framework provides data users with a quick schematic overview of potential error sources, the data quality controls within survey organisations provide insight into the protocols for data production that can have a direct bearing on the overall quality of public-use data. In this section we demonstrate how data quality guidelines interact with the TSE framework. We use two editions of "Statistics Canada Quality Guidelines" (2003, 2009) to inform the discussion, as well as two editions of Statistics South Africa's Statistical Quality Assessment Framework (SSA, 2009, 2010). We can summarise the relationship between the TSE framework and components of data quality as per Fig. 2.2. From Fig. 2.2 we see that the concept of "accuracy" is what brings together both the TSE framework and the functional operational concerns of the Survey Organisation. Indeed, Statistics Canada (2003, 6) note that the very purpose of publishing quality guidelines is to inform the debate on "how to assure quality through effective and appropriate design or redesign of a statistical project or program from inception through to data evaluation, dissemination and documentation." Below we elaborate on how each component of the TSE framework interacts with components of data quality. #### 2.3.1 Validity of the Construct of Interest In the TSE framework, validity is defined as the observational gap between constructs and measurements (Groves et al., 2004, 50). In other words, validity is concerned with how well the survey instrument measures the construct of interest. In statistical terms, the notion of validity acknowledges two sources of variability—one at the level of the individual respondent and another at the level of different trials of the survey (ibid, 50). From a data quality perspective, it is very difficult to know a-priori how valid a particular construct may be over different trials of the survey. It is also very expensive to run multiple trials of a survey simply to obtain sufficient data to be able to estimate this. However, it is possible to assess how respondents' responses may vary given a different phrasing or wording of the survey questions for example. This is the idea behind pre-testing questionnaires, which can span any number of different dimensions from wording a particular question differently and testing whether respondents respond differently, to translating questionnaires into different languages and conducting similar diagnostic exercises. Questionnaire design is thus partly relevant to the idea of validity. Pre-testing questionnaires can aid the understanding of both validity and measurement error. To concretise the discussion, consider the construct validity of income. From a practical point of view, income can refer to many different sources. Thus the validity of income has to do with everything from the component of income being measured to the scope of income (i.e. whether that income is an individual or household measure). Different types of income measurements in a household survey include employee income, income from self-employment, rental income, property income and income from transfers (Canberra Group, 2001, 2011). Household surveys in South Africa that measure all of these types of income include the Income and Expenditure Surveys (SSA, 1995, 2000, 2005) and the National Income Dynamics Survey (SALDRU, 2008, 2010–2011). The main data quality elements associated with validity are relevance. The process of transcribing the constructs of interest to the questionnaire is a very important part of any survey. #### 2.3.2 Measurement Error Measurement error is defined as the observational gap between the ideal measurement and the response obtained (Groves et al., 2004, 51). The "error" component implies a departure from the true value of the measurement as applied to a sample unit and the value provided (ibid, 52). The effects of different sources of measurement error can be very difficult (and sometimes impossible) for researchers to identify in public-use datasets. For example, Wittenberg (2004) notes that in trying to measure the occupational distribution of manufacturing sub-sector employment in South Africa using the Manufacturing Census, the Population Census and the October Household Surveys, one of several possible explanations of divergences in the point estimates could be due to fieldworker errors. The difficulty here though lies in the inability of researchers to precisely determine the potential sources of the problems, for Wittenberg (ibid) also notes that the discrepancies discovered could have been due to a range of other factors, all of which can only be speculated upon when investigating the empirical magnitudes. On the other hand, changes in questionnaire wording are precisely identifiable by researchers given careful analysis. For example, Bhorat (1999) noted that the definition of the informal sector in the October Household Surveys 1995 was problematic. This changed in later years of the survey, but in so doing Yu (2009) made the point that it made time-series analyses of the repeated cross-sections of informal sector workers problematic. Yu (2007) notes that the manner in which broad and narrow unemployment rates were measured also changed across survey years, and that these kinds of changes to questionnaire wording impose important trade-offs. Due to the multidimensional nature of measurement error, data quality guidelines need to be developed for each possible source of error. Groves (2004, 359) notes that when considering the interviewer as a source of measurement error, it is crucial to understand the manner in which they can affect the survey. It is also possible (and necessary) to monitor the results of interviewers as close to real time as possible. When developing indicators to assess interviewer variance in household interview surveys for example, Groves (ibid, 364–5) discusses Kish's (1965) original interviewer intraclass correlation coefficient, which is the ratio of variance between interviewers to the total variance of a measure. This is a very direct way to assess interviewer performance, and can aid the discussion of measurement error when it becomes apparent that certain interviewers behave erratically (e.g. submit completed questionnaires with identical values for many questions). The respondent is also a source of measurement error, and the manner in which errors can be introduced by the respondent are numerous. Groves (2004, 407–408) notes that from models of the interview process and newer cognitive science perspectives, there are five stages of action relevant to survey measurement error, including: (1) how the respondent encodes (processes and stores) the information asked of him/her; (2) how the respondent comprehends the question; (3) how the respondent retrieves the information; (4) how the respondent judges the appropriate answer to provide the interviewer with; and (5) how the respondent communicates the information to the interviewer. Clearly the relationship between the interviewer and the respondent is important here, and this reiterates the need for interviewer training and possible matching of interviewers to respondents on socio-cultural grounds (such as race or language). The importance of designing a sound questionnaire is related to the discussion above in that it has an impact not only on the influence and image of a statistical agency, but also, from a data quality perspective, on respondent behaviour, interviewer performance, collection costs and respondent relations (StatCan, 2009, 28). The principles for designing a questionnaire include that it should collect data that corresponds to the survey's Statement of Objectives while taking into account the statistical requirements of data users, administrative and data processing requirements as well as the nature and characteristics of the respondent population. Furthermore, it should flow smoothly from one question to the next, facilitate respondents' recall, facilitate the coding and capture of data, minimise the amount of editing and imputation that is required, and lead to an overall reduction in the cost and time associated with data collection and processing (ibid, 28). There are consequently several different data quality elements involved for this source of error, including accuracy, methodological soundness, coherence and relevance. All of these must be managed effectively in order to minimise measurement error in public-use data. #### 2.3.3 Processing Error Processing error is defined as the observational gap between the variable used in estimation and that provided by the respondent (Groves et al., 2004, 53). Processing error is about data collection, capture and coding. These operations use a large portion of the survey budget, requiring considerable human and physical resources as well as time (StatCan, 2009, 32). Depending on the degree of automation of these tasks, there can also be a large amount of paradata (e.g. indicators of whether or not a unit is in the sample, history of visits, mode of data collection, administrative information and cost information) generated in this process (ibid, 32). In the evolution of SSA's household surveys, there are many instances of processing error. For example, Yu (2007) identifies inconsistencies with several variables related to earnings, such as work experience and hours worked, which have some values greater than logical upper bounds (though, alternatively, this could be a source of measurement error if the respondent or interviewer was the source of the information). Yu (2007) also identifies coding inconsistencies with race, marital status and education in several October Household Surveys (ibid). Processing error also exists in the component statistical files of the publicly-released OHS 1998, where some observations are repeated in the person file but absent in the worker file (ibid). These examples demonstrate an important feedback loop on data quality from researchers to the survey organisation. It is rare that the survey organisation will be able to pick up errors of this nature in a set of routine checks, but researchers who are concerned with very specific issues relating to the data will. The main data quality element involved in data capture, collection and coding is accuracy (StatCan, 2009, 37). The key principle guiding data collection is to minimise the burden on the respondent while ensuring privacy and security of the information provided in all data gathering and processing operations (ibid, 32). Because these operations have a high impact on data accuracy, quality and performance measurement tools should be used to manage the collection, capture and coding processes within the survey organisation (ibid, 32). While these principles point to explicit guidelines for data capture, collection and coding, the degree of success in minimising processing error is rarely perfect (see StatCan, 2009, 32–36). Newer forms of technology (e.g. computer assisted interviewing software) can aid the degree to which the process is minimised, but whenever there is a human element involved there is the scope for making mistakes. #### 2.3.4 Coverage Error Coverage error is defined as the nonobservational gap between the target population and the sampling frame (Groves et al., 2004, 54). Coverage itself is the completeness of the information for the target population that would be derived if all of the frame units were to be surveyed (StatCan, 2009, 19). Coverage errors include missing in scope units, included out-of-scope units, misclassified units and duplicates. Coverage errors therefore are a function of both frame undercoverage (or overcoverage) and differences in the survey estimate for those actually covered from those for which an estimate is required (ibid, 19). Coverage error is a particularly important source of error in poorer countries or countries in transition, where the geopolitical units may be new or changing. South Africa during the mid 1990s is such an example, where the names and internal geopolitical boundaries of provinces were redefined more than once in the 1990s. Furthermore, in poorer countries national statistical agencies often have more limited budgets, and the capacity to keep sampling frames up to date is more limited (Yansaneh, 2005). There are international organisations that can assist statistical organisations in these countries with optimising resources for improved frame maintenance and sample selection, such as the United Nations Statistics Division (see "Development of National Statistical Systems", UNSD, 2011). For cost minimisation purposes, master sampling frames combined with master samples are frequently advocated for statistical organisations with limited resources (see Pettersson, 2005). These are methods that generate frames and samples to be used in many different surveys by the same statistical organisation over time. The data quality elements that arise for coverage error pertain largely to the degree to which the sampling frame accurately captures the target population; hence, accuracy and relevance are the key elements (StatCan, 2009, 21). For survey organisations, this means that sampling frames need to be well designed and kept up to date. Certain countries have very different conventions on the type of information that can be stored by public statistical agencies. For example, in Sweden there is a population register and an updated list of names and addresses for almost all residents, whereas in the USA the population is so large that telephone numbers are often used as frames (Groves et al., 2004, 55). The specific type of coverage errors that can arise therefore also depend on the country, its population size (or number of firms in the event of enterprise surveys), and the degree to which information can be stored about individuals. An important relationship between coverage and frames is to ensure that the survey population is reasonably consistent with the target population on the one hand, and that the frame then conforms to the survey population on the other (StatCan, 2009, 19). Coverage error can reduce the degree to which the frame and the survey populations match and can result in cost increases, loss of timeliness and a diminished accuracy of the estimates from a bias and variance point of view (ibid, 19). Consequently survey organisations need to implement procedures to minimise this discrepancy. Contemporary ways of doing this include using remote sensing and satellite imagery. #### 2.3.5 Sampling Error Sampling error is defined as the nonobservational gap between the sampling frame and the realised sample (Groves et al., 2004, 57). Sampling error consists of two components, namely sampling variance and sampling bias (Krotki, 2008). Sampling variance is the part that can be controlled by sample design factors such as sample size, clustering strategies, stratification, and estimation procedures (ibid, 2012). Sampling is a means of selecting a subset of units from a target population for the purpose of collecting information that can be used to draw inferences about the population as a whole (StatCan, 2009, 23). The sample design encompasses all aspects of how to group units on the frame, determine the sample size, allocate the sample to the various classifications of frame units, and select the sample (ibid, 23). Sample designs are either probability-based or non-probability based, the latter being generally fast, easy and inexpensive to undertake (ibid, 23). Some of the principles for dealing with probability-based sample designs include that it should be as simple as possible within the context of a design that (1) is based on randomisation, (2) has population units that have a known positive probability of being selected, and (3) has calculable selection probabilities (ibid, 23). When probability-based samples are designed to be used for more than one survey, i.e. when dwelling units or clusters of dwellings on the same sampling frame are reserved for use in future surveys, then that kind of sample is known as a master sample. Master samples are frequently used in developing countries for cost reduction purposes and to ensure that investments in creating probability-based designs can be utilised for more than one survey (Pettersson, 2005). An important data quality element associated with sampling is accuracy (StatCan, 2009, 26). This means that every decision that is made about the survey needs to be thought about in relation to how well the sample represents the population. The size of the sample is also important in reducing sampling error. This point naturally extends to subsample sizes that may be necessary to obtain representivity at geographical levels smaller than the nation state (e.g. provincial and/or urban-rural representation). The variables of interest in the survey are also important. For example, to obtain provincially representative statistics on poverty requires that sufficiently large enough samples are drawn for the population groups that are most likely to live in poverty in those provinces. The design of the sample needs to balance accuracy within the budget constraint. Multi-stage complex samples are therefore the norm when it comes to probabilitybased surveys, and will include careful thought about stratification, primary sampling units, clusters, weights and design effects from previous surveys that may aid sample size considerations for current surveys (StatCan, 2009, 25). If the survey is a rotating panel, then the sample needs to be designed to account for rotation, whereas if it is a periodic survey, then the sampling process can be a simpler process. Attrition in any panel survey further complicates sampling error, and needs to be carefully monitored as the panel progresses over time. The importance of survey documentation that correctly reflects the choices that were made and the problems that were encountered then becomes key, since it records and catalogues the information needed to understand the trade-offs of decisions that affect the accuracy of the outcomes. #### 2.3.6 Nonresponse Error Nonresponse error is defined as the nonobservational gap between the sample and the respondent pool (Groves et al., 2004, 58). "Nonresponse error arises when the values of statistics computed based only on respondent data differ from those based on the entire sample data" (ibid, 59). Nonresponse can be split into two components: unit nonresponse and item nonresponse. Unit nonresponse is when an entire sampling unit (e.g. individual, household or firm) does not participate in the survey because they could not be contacted or refused to participate in the survey for some reason. Item nonresponse is when a particular question in the questionnaire is not answered by the respondent, either because the respondent refused to answer the question or because the interviewer failed to ask the question. The main data quality element involved in nonresponse error is accuracy (Stat-Can, 2009, 49). Nonresponse can have two effects on data: (1) it biases estimates when nonrespondents differ from respondents; and (2) it increases the variance of estimates because the sample size is reduced (ibid, 46). It is therefore important to understand what has become known as the nonresponse mechanism, i.e. the process that leads to nonresponse. For unit nonresponse, the degree of effort expended by the survey organisation on minimising non-contacts and refusals to participate in the survey is key to reducing its incidence. This has budgetary implications, so unless the survey organisation explicitly allocates resources for this process, the degree to which they understand the unit nonresponse mechanism is compromised. Depending on the survey, if no effort is invested in following up unit nonrespondents, then it is frequently addressed by reweighting the data. The basic ideas behind nonresponse were developed by Rubin (1976, 1987), as were a set of solution methods based on imputation strategies of various forms. The key idea behind nonresponse analyses is to establish whether the process that leads to missing data can be ignored. Ignorability refers to a property that permits the survey organisation (in the case of unit nonresponse or item nonresponse) or the researcher (in the case of item nonresponse only) to not take explicit account of the process that leads to missing data when conducting analyses. Ignorability was first developed as a condition for missing data by Rubin (1976, 1987), and helped distinguish the conditions of missing completely at random (MCAR—what Rubin, 1976 originally called Observed at Random), missing at random (MAR), and not missing at random (NMAR). For item nonresponse, understanding the response mechanisms amounts to determining whether the missing data are missing completely at random (MCAR), missing at random (MAR), or not missing at random (NMAR). Statistics Canada (ibid, 46) define these "classic" response mechanisms as follows: uniform nonresponse is an MCAR mechanisms where the response probability is completely independent of the units and the measurement process, and is constant over the entire population; nonresponse depending on an auxiliary variable is a MAR mechanism where response depends on certain auxiliary data or variables available for all units measured; and nonresponse depending on the variable of interest is a NMAR mechanism where the response probability depends on the variable of interest. The principles for dealing with nonresponse in a survey are related to budget, time and staff constraints, the impact on overall quality and the risk of nonresponse bias (ibid, 46). It is also dependent on the mode of the survey (e.g. personal interview, telephonic), auxiliary information for respondents, an effective respondent relations programme, a well designed questionnaire, and the use of active management to ensure regular follow-up on collection operations and adaptive data collection (ibid, 46). For researchers, dealing with item nonresponse often involves reweighting or imputation methods. The latter ought to be based on careful analyses of the response mechanism in a manner analagous to how survey organisations investigate unit nonresponse (this is the focus of the next chapter in this book). This allows the item response process to be understood using the same general methods for understanding unit response. #### 2.3.7 Adjustment Error Adjustment error is defined as the discrepancy between the sample of respondents and the post-survey adjustments necessary to ensure the sample represents the population of interest. These adjustments are efforts to improve the sample estimate in the face of coverage, sampling and nonresponse errors, and use some information about the target or frame population or response rate information on the sample to make adjustments (Groves et al., 2004, 59). Adjustments are usually made by creating appropriate weights, so the data quality concerns associated with adjustment error pertain to weighting and estimation. The key data quality element associated with adjustment error is accuracy (StatCan, 2009, 61). The three reasonably standard weights associated with probability-based surveys are probability of selection weights, unit nonresponse and post stratification weights. The first weights observations in the survey by the inverse of their probability of selection. The second assigns a weight to missing units relative to observed units that match some known characteristics between the two (e.g. cluster, psu location). Post-stratification weights adjust demographic survey population totals in a given survey period to the most recent national demographic population totals on record. These weights can then be multiplied together to obtain a composite weight for each observation in the survey that will be included with the publicly released dataset. The principles associated with creating weights and correct estimation procedures that affect adjustment error depend on the type of weight produced and the method by which the weights get accounted for in the estimation process. Accurate information at the sampling and response stages of the survey help with the creation of sampling and unit nonresponse weights. Sampling weights need to reflect the sample design, so if a multi-stage design has been used (including stratification and clustering for example), then the probability of selection weight needs to correctly reflect the probabilities associated with each stage of selection. For the nonresponse weight, the observed sample is smaller in size than the original sample, so to compensate, reweighting can be performed by adjusting the design weights by factors that account for each unit's probability of response (StatCan, 2009, 59). These factors are usually obtained using response models (ibid, 59). If auxiliary data are available, an improvement to the precision of certain estimates can be achieved by a process known as calibration, which consists of adjusting the weights such that estimates of the auxiliary variables satisfy known totals (ibid, 59). The post-stratification weight is one such example, but more generally, desirable properties of calibration include (1) coherent estimates between different sources of data; (2) potential improvements to the precision of the estimates; and (3) potential reduction of unit nonresponse error and coverage error (ibid, 59). Final estimates of key statistical quantities of interest are then about correctly accounting for these weights in the estimation process. #### 2.4 Data Quality and Survey Errors in Statistics South Africa Household Surveys Evident from the above discussion is that every component of survey error links through to data quality metrics. But it is also important to be aware of the broader efforts within the statistical organisation to produce the dataset from inception of the project to public-release. Therefore, in order to make an accurate assessment of microdata quality, the TSE framework is an important start. We now investigate the quality of South African labour market household surveys from the mid 1990s to the mid 2000s. This was a unique period in the country's history during which many changes were taking place, including inside the national statistics office. The surveys considered are the October Household Surveys (OHS, 1995–1999) and the Labour Force Surveys (LFS 2000 September—2007 September). The variable of interest is employment income (a necessary choice when discussing the measurement side of TSE), and we will be tracking the evolution of the income question over time within the context of changing survey instruments and methodological innovations. An analytically challenging part of this discussion is trying to understand the changing situational environment within Statistics South Africa (SSA) over the period of interest. In order to do this, the results of a personal interview with a retired sampling statistician—Professor David Stoker—will be utilised (see Daniels and Wittenberg, 2010). Prof Stoker worked with SSA in various capacities from 1985 onwards, and his institutional knowledge about what was happening at the time is thought to be unique. As far as the surveys themselves are concerned, the OHS and LFS share the same mode, namely they are face-to-face household interview surveys where an interviewer asks a household member a set of questions from a questionnaire about that member's activities and about other household members' activities. However, the OHS was always a single cross section, while the LFS was a biannual rotating panel commencing in February 2000 and extending until September 2007. In 2008, SSA changed the LFS to a quarterly panel, but stopped releasing questions about income to the public; hence, the QLFS will not be reviewed here. #### 2.4.1 Representation of the Population of Interest In this section we evaluate the errors of nonobservation associated with the TSE framework, including coverage error, sampling error, nonresponse error, and adjustment error. As before, the time period of interest is 1995–2007. At the start of this period the newly formed geopolitical region of democratic South Africa had just been born out of an Apartheid state that excluded what were known as the Bantustans (Transkei, Bophuthatswana, Venda, Ciskei—the TBVC states). The challenge for the national statistics agency was therefore to help everyone understand this new country, and there was much urgency on the part of policymakers to know the socio-economic features of the new South Africa. While surveys like the OHS were conducted during this period to achieve these ends, survey documentation was often very poor, complicating attempts to understand everything that was going on at the time. #### Coverage Error The new geopolitical entity of South Africa required a new sampling frame, which took time to create. In fact, the 1996 Census was the first time that Statistics South Africa (SSA) had the opportunity to send fieldworkers to every part of the country. As such, it served as an opportunity to validate the existence of dwelling units in remote areas that had escaped previous enumeration attempts and only been observed by satellite imagery. The next major effort to understand the limitations with the sampling frame was the 1996 Post Enumeration Survey (PES). A PES is an independent survey that allows comparisons to be made with Census results, permitting estimates to be made of coverage and content errors (Whitford and Banda, 2001). One of the major objectives of a PES is to develop a methodology for the calculation of the undercount or overcount of the Census, which can be differentiated by geographical area or demographic characteristics (e.g. age, race, sex). Since the OHS 1995 was conducted before the 1996 Census, it is likely to suffer from the greatest degree of coverage error compared to all other surveys investigated in this document (OHS 1995—LFS 2007 September). However, SSA did release updated OHS 1995 weights based on the population totals in the 1996 Census (a few years after it was completed) in order to reduce this source of error. The next major effort to update the sampling frame was the 2001 Census and the subsequent 2001 PES. The 2001 Census also experienced problems in the field with interviewers, such as interviewers stopping work because they had not been remunerated (this was reported in the local press at the time). However, between the Census and the PES, the national sampling frame would have been appropriately updated. The final concerted effort to update the sampling frame was the 2007 Community Survey, but that falls outside the scope of this document. It is important to note that despite the discussion above, sampling frames are not just updated at discrete points in time. Because SSA undertake surveys every year, and employ fieldworkers to administer questionnaires, feedback from interviewers concerning the absence of existing dwelling units or the presence of new units takes place on a continuous basis. This information impacts the measure of size of each cluster the fieldworkers visit, and therefore has an important implication for the calculation of the correct selection probability of each dwelling unit or household within the cluster. In summary then, the fact that a new geopolitical unit was created with the democratic South Africa in 1994 meant that the Statistics Agency had their work cut out for them. Coverage error was therefore likely to be largest in the mid to late 1990s, diminishing steadily as the frame became fully enumerated. Since SA is a developing country, we also expect migration patterns and new housing developments to have a significant effect on coverage error over time. This means that the sampling frame is likely to continue to change on an annual basis. The importance of using a combination of technology (e.g. GIS) and skilled interviewers with a virtuous feedback loop to the sampling statisticians then becomes the key to reducing coverage error. #### Sampling Error It is important to understand key developments in the sample design of the various surveys over time. The type of surveys evaluated (the OHS and LFS) also raise different questions with respect to sampling error: the OHSs were all single period cross-sectional surveys with complex probability-based designs, while the LFS was a rotating panel survey. Sampling error for a rotating panel is expected to be slightly different compared to a cross-section (see StatCan, 2009, 23–26). There were important changes made to the sampling design of the OHS 1995 compared to all previous surveys conducted by SSA before that, namely that (1) the focus switched to households rather than dwelling units, (2) the number of households drawn within each EA was reduced while the number of EAs was increased, and (3) race stopped being used as an explicit variable upon which to stratify the sample (Daniels and Wittenberg, 2010). These were changes in the sample design that improved the representivity of the sample relative to the population, and increased the cost of the surveys (specifically in the case of increasing the number of EAs). The OHS 1996 sample was produced in conjunction with the sample for the 1996 Post Enumeration Survey (SSA, 1996, Metadata), while the OHS 1997 was based on the administrative records of the 1996 Census, which are records kept by interviewers for each EA they visit (Daniels and Wittenberg, 2010). The 1998 OHS was based directly on the Census 1996 (SSA, 1998, Metadata), while the OHS 1999 was based on the 1998 Master Sample. However, due to the concurrent implementation of the Census in 1996 and Post Enumeration Survey in 1996, the budget for the 1996 OHS was reduced and the sample size reduced substantially, thereby increasing sampling error. The 1998 Master Sample then came to play a major role for many SSA surveys including the LFS Rotating Panel. SSA developed the first master sample in 1998, and then updated it in 2003 and 2008 (Daniels and Wittenberg, 2010). The master sample reserves certain clusters of households for certain planned surveys in the future as well as ad hoc surveys that may arise. The SSA 1998 master sample was reserved for the last of the OHSs, the LFS, the General Household Survey and the 2000 Income and Expenditure Survey (ibid, 2010). Anecdotally, the budget for the OHS in 1998 was also lower, possibly due to resources diverted to the development of the master sample, and this reduced the sample size of the OHS in 1998 accordingly, increasing sampling error in this year too. The advantage of a master sample is that even though it is expensive to develop initially, it becomes more cost effective in the long-run because more than one survey can be based on it (Pettersson, 2005, 72). However, the disadvantage of a master sample is that because it fixes the households that will be selected in each EA for each survey at the time of development, it can become outdated the longer it is used. The LFS experienced many problems initially with successfully implementing a rotating panel survey design. The first wave of the panel was in February 2000, but subsequent to that two problems arose: (1) the rotating part of the sample was improperly implemented, and (2) fieldworkers were not properly trained to do what they were supposed to in terms of interviewing the same household (Daniels and Wittenberg, 2010). The correct implementation of the rotating panel design only commenced in LFS 2002 February (ibid, 2010). From a sampling point of view, a panel differs from a single cross-section in that while the sample for a rotating panel is nationally representative in the first wave, it can loose that representivity over time. The rotation of the sample is designed to reduce this loss of representation. Attrition can cause bias in panel surveys, but this was never rigorously explored by SSA over the life of the LFS, most likely due to the role that the rotating component of the panel played in frequently refreshing the sample. #### Nonresponse Error There are two components of nonresponse, namely unit and item nonresponse. Our focus here is on unit nonresponse only (Chapter Four of this book will focus on item nonresponse for employee income data). Unit nonresponse occurred in every survey under review. However, SSA's description concerning how they dealt with unit nonresponse is completely absent for every OHS. The LFS is also silent on unit nonresponse until the LFS 2000 September, when Table 2.1 Intended and realised sample sizes it is only mentioned with respect to the weights (SSA, 2000, Metadata). Despite this, it is possible to track the extent of unit nonresponse. We do this in Table 2.1 below by showing the difference between the intended sample size for each survey from OHS 1995—LFS 2007, compared to the realised sample size (computed by evaluating the number of households in the datasets) released for each survey. Table 2.1 shows that there are very high response rates in SSA's household surveys, particularly in the 1990s. Kerr and Wittenberg (2012) provide evidence that this was because SSA substituted for unit nonresponse in the early OHSs, yet there is no indication of this in the Metadata survey documentation that accompanies the surveys (see OHS and LFS Metadata, 1995–2007). #### Adjustment Error There are three principal weights used for adjustment purposes: (1) probability of selection, (2) unit nonresponse, and (3) post-stratification. The survey documentation for the OHS is only ever useful when it comes to understanding the first of these for households and individuals. From a reading of the Metadata files for each OHS, it seems that SSA never corrected for unit nonresponse using weights (see SSA, Metadata: OHS95-99). Unit nonresponse weights are only officially mentioned in the LFS 2000 September survey documentation (see SSA, 2000, Metadata). The post-stratification weight is also never discussed or even hinted at in any OHS survey documentation (see SSA, Metadata: OHS95-99). The LFS 2000 February is the first survey in the series evaluated here to include a discussion of post-stratification and how it was conducted. Adjustment error therefore seems to be possibly one of the largest sources of TSE in the OHSs. For the LFS, the weights seem to be fine. However, neither unit nonresponse weights nor post-stratification weights featured in the official documentation of the OHSs. Researchers have for some time been struggling to understand the apparent jumps in key weighted variable estimates over time using SSA's household surveys (see Branson and Wittenberg, 2007 and Branson, 2009). This goes at least part of the way to explaining why these apparent trend-breaking patters are found over time. #### 2.4.2 Measurement of the Construct of Interest We now turn to the measurement side of the Total Survey Error framework and use the employment income variable to anchor the discussion. The income question is directed to employees only in the OHSs, but to both employees and self-employed in the LFSs. In the discussion below, we evaluate the employee income question only, thereby tracing the evolution of the question over time. The surveys instruments evaluated include the OHS 1995—OHS 1999, and the LFS 2000 February—LFS 2007 September. #### Validity The construct of interest for all surveys reviewed in this section is income earned in the main job for all individuals that were employed in the last seven days, except in the OHS 1995 where the "seven days" is not made explicit in the wording of the question. Throughout the OHSs and LFSs, income is always distinguished into various components in the instrument, including (a) salaries and wages, (b) bonuses and (c) income from overtime. The question thus requires the respondent to provide the sum of the three components of income in a single estimate. This amount is before tax. Key features of the income question in the OHS and LFS are summarised below. Table 2.2 Features of the income instrument The extent to which this income question loses validity is negligible. The focus is on income in the main job, and consequently remuneration in that job would yield the correct distribution of salaries earned by the employed. If individuals have more than one job, then total income earned by the individual would be higher, but total income is a different construct to income earned in the main job. Consequently, results should be interpreted as such. There is no mention in the survey documentation of SSA whether the questionnaire was ever pre-tested or how it fared when translated. This shows the paucity of information relating to data quality for many of these surveys. However, we can observe from the income questions themselves important changes to the wording over time. In 1995, the time period options for reporting income included daily, weekly and monthly, but that changed after 1998 to weekly, monthly and annually. This had a deleterious effect on aggregation and standardisation of income values for the sample. It also renders comparisons over time problematic because researchers have to make very arbitrary decisions about how to treat daily income. #### Measurement Error As noted above, Groves (1991, vi) differentiates measurement error into four components including the interviewers, the respondents, the questionnaire and the mode of data collection. The two components that are most important for the income question are interviewer effects and errors due to the psychological issues impacting respondents (viz. social sensitivity of the income question). The wording and the mode also play a role, though are likely less significant. The wording of the income question is identical in every SSA survey investigated except for the OHS95. Whatever weaknesses are associated with this wording are held constant across the surveys. Similarly so for the mode of data collection, since the OHSs and LFSs are both face-to-face surveys. The impact of interviewers on respondents is multi-dimensional. Because income is such a socially sensitive question, respondents may be influenced by any number of psycho-social and socio-demographic factors, such as the race and gender of the interviewer and even the tone of voice used. As a consequence, interviewer training is very important when trying to solicit income information in face-to-face household interview surveys (Groves and Couper, 1998). Survey organisations consequently often try and match the race of the interviewer with the expected racial majority of the geographical areas of responsibility of the interviewer. Further training of interviewer conduct and behaviour within households is also frequently undertaken. As far as the wording and sequencing of the income question is concerned, there are two parts to the question in all the OHSs and LFSs except 1996. The first is when the interviewer asks the respondent for the actual value of their income. A respondent is then faced with three options: (a) to provide the actual value, (b) to refuse to provide the value, or (c) to state that they don't know the value. Only if the respondent does not provide an actual value, is s/he presented with a list of income brackets. For a respondent to then decide to provide an answer after having failed to do so at the first prompt suggests either that they did not want to reveal the precise value of their income and now have been persuaded to do so by the showcard with income brackets, or that they are unsure of the exact value of their income (or other people in the household's income that they are asked to provide a value for). This latter feature of the question, where the respondent is asked to provide the income of other members who live in the household, potentially induces a considerable source of measurement error. One would expect that cohabiting or married partners would have better information about each others' income, but multiple unrelated employed people in one household may know very little about the income of other household members. The ratio of self-reporters to proxy reporters in the surveys are presented in Table 2.3. An identifier for self-reporting was only included in the questionnaire from 1999 onwards. We can see from the table self-reporters generally constitute no more than sixty percent of the sample in any given year. This implies that the scope for measurement error due to proxy reporting is rather substantial. There is very little that can be done about this, save to be aware of it and control for it where possible. The existence of a bracket reporting option in the income question is designed to reduce item non-response, but in so doing, an additional component of measurement error is introduced. This is the case simply because we now no longer know the exact wage of the respondent, but rather the range into which it falls. However, nonresponse is more expensive to deal with for survey organisations and statistically poses tougher challenges, so this trade-off between components of total survey error is important for the income question. In surveys where point and interval options are presented to the respondent, the sequencing of the prompts and nature of the alternatives are important because they can aid recall and provide information about the response process. Often, the practises of survey organisations differ in important respects on this matter. SSA sequence the income question in the OHSs and LFSs to firstly ask the respondent for an exact value of their income before the interval prompt takes place. In the Health and Retirement Study (HRS) in the USA, however, the sequencing is the same as the Labour Force Survey (proceeding from an exact value to an interval estimate), but the nature of the prompt for the intervals is very different. Instead, the HRS has an unfolding bracket design where the respondent is first asked if they earn greater than \$25,000. If they respond in the affirmative, the interviewer then proceeds to ask whether they earn a higher amount (>\$50,000); if they respond in the negative, a lower value is prompted (>\$5,000). This proceeds logically until a narrower interval is obtained (see Heeringa, 1995 for a discussion of the income variable in the the HRS instrument). The National Income Dynamics Study (2010–2011) in South Africa employs a similar unfolding bracket design to the HRS for all income questions. The analytical implications of the different designs are non-trivial. As Vazquez-Alvarez (2006) and Melenberg et al. (2006) have demonstrated, the unfolding bracket design introduces anchoring bias. Anchor strategies are purposefully introduced into surveys to aid respondent recall (see Blair et al., 1991). However, they also introduce potential biases into the results. While the sequencing and format of the brackets in SSA's design is likely to be free from anchoring bias, it remains an open question Table 2.3 Self and proxy reporting per survey year whether it is an improved method. Casale and Posel (2005) note the non-randomness of the bracket subset of respondents, identifying differences between self- and proxyreporting to be significant. Table 2.4 shows the evolution of the distribution of response types in the Labour Force Survey for the employed, economically active population only. We restrict the analysis to this survey only and this particular subsample in order to demonstrate how the empirical magnitudes change when we hold the instrument constant. From the table we can see that over time, the continuous subset of observations has reduced, but not monotonically. The percentage of bracketed response categories fluctuated around 20 percent in every year except 2000, when a disproportionate number of respondents provided a continuous response. This may have been due to greater training of interviewers by SSA to assure respondents of the confidentiality of the information. "Don't Know" and "Refuse" response options increased to about their steady state after the year 2000, when they were at their lowest. This again suggests that unusual effort was expended by the survey organisation in 2000 to obtain good quality income responses, and better interviewer training may have been the key here. Table 2.4 Distribution of Response Types Per Survey Year #### Processing Error The impact of processing error on the survey is often difficult to detect for the income question specifically, but it has potentially significant implications. Because of the release of three variables into the public-use data for employee income (i.e. continuous income, categorical income and the time unit of reporting), processing error has the potential to exist when more than one response type exists for the same individual (we explore this further in the next chapter in this book). Other examples of processing error in the income question include: It is not always possible to identify all of these forms of processing error in the surveys, but some forms of error are easily identifiable from the variables released in the data. Furthermore, because processing error can impact all variables unevenly in a public-use dataset, it is important to check all variables of interest for processing error before analysis. Sometimes processing error may be suspected when there are other ambiguities in the data. For example, one of the far-reaching implications of the wording of the income question in 1995, where the question prompts the interviewer to clarify from the respondent whether the amount of income reported is daily, weekly or monthly, is that when one multiplies the number of respondents who reported a daily value for their income by their income, the resulting values are extremely high. On the one hand, this is an artifact of poor question wording; on the other hand, it could be interviewer error. Thankfully the income question changed permanently and for the better subsequent to 1995, but it does render comparisons with that year problematic. #### 2.5 Discussion For South Africa during the mid to late 1990s, there were extraordinary demands on SSA. On the one hand it had to define and enumerate a new sampling frame for a revised geopolitical entity. On the other, there were pressing demands by policymakers for information about the new SA, and this pressure likely reduced the time available for thorough documentation and quality control. The mid 1990s was marked by poor operational standards, suggesting that SSA was still very much finding its feet as an institution, itself undergoing internal restructuring as an organisation. For the representation side of the TSE framework then, we saw that researchers could do very little about coverage error, even though it is likely an important source of error in the OHSs. The 1996 Census and 1996 Post Enumeration Survey played a very important role in defining the new sampling frame. However, it reduced the budget available for the OHS in 1996, which resulted in a reduced sample sizes in that year. The 1996 Census and 1996 PES helped statisticians develop the first Master Sample in 1998, which was then used to define the Labour Force Survey sample and many other household survey samples in SA. The switch from the OHS to the rotating panel of the LFS introduced new sampling errors, for rotation was improperly implemented, suggesting once again that SSA was undergoing a process of learning about this new survey instrument. Fieldworkers play a very important role in updating the measure of size of Enumerated Areas (EA) drawn in the master sample as new dwelling units are added or destroyed. As the master sample gradually becomes outdated, improper enumeration or failure to re-enumerate can introduce a form of coverage error. Inbetween updating the master sample, then, fieldworkers also have an impact on this source of error. For the probability of selection, (unit) nonresponse and post-stratification adjustments, survey organisations usually provide weights that must be taken into account when analysing the data. However, the weights in SSA datasets seemed to be problematic and certainly not subject to sufficient methodological documentation until later waves of the LFS. The weights always combined at least the probability of selection weight with a post-stratification weight (in the OHSs), and also with the unit nonresponse weight (in the LFSs), to form one composite weight differentiated by individual and household. Because the process was never described in relevant documentation, researchers were never aware of exactly what SSA did in this regard. The weights that were released to the public generated population totals on key variables of interest that were often unstable and highly variable when the datasets were stacked over time. For item nonresponse on individual variables like income, Stats SA have never provided single or multiple imputations of missing data. It therefore falls to researchers to evaluate the patterns of missing data on variables of interest, and then to develop solutions like single or multiple imputation strategies to deal with this form of potential bias in public-use datasets. This issue is explored later in this book. For the measurement side of the TSE framework, validity of the constructs in the questionnaires are usually established by pre-testing exercises. But there is no record of this in the documentation throughout the period of 1995–2007. For specific variables like income, the design of the question is usually targeted at reducing item non-response (by including the income brackets as a follow-up prompt), but it does so at the cost of introducing measurement error on the value of income reported. From a survey design point of view, this can be interpreted as a trade-off between non-response bias and measurement error attributable to the instrument. In other words, it is preferable to have some measurement error on the income variable than to have non-response on it, which is much more difficult to understand or treat appropriately if it is non-ignorable non-response. Non-ignorable non-response cannot be understood effectively without incorporating and budgeting for a specific study of non-respondents to be undertaken by the survey organisation. However, this was never done with SSA's OHSs and LFSs. The actual wording of the income question did change over time, however, despite no clear documentation of pre-testing questions. In fact, the income question changed with almost every OHS until it stabilised in the LFS. The time units for income reporting eventually moved away from daily, weekly and monthly (up until 1998, though in 1995 an annual option was also available) to weekly, monthly and annually (from 1999 onwards). "Don't know" as a response option was added to the question in 1999, and "Refuse" was added as a further response option from the commencement of the LFS. The ranges of the income brackets changed between 1995 and 1996 and 1997, after which those ranges remained constant all the way through to the 2007 LFS. Finally, the self employed were asked a different income question in the OHSs, while they were asked the same income question in all of the LFSs. Measurement error attributable to the interviewer was anecdotally rife throughout these surveys due to poor fieldworker practises (e.g. recruitment and training). One can only speculate about whether and how interviewers influenced respondents, thereby introducing another form of measurement error, but this is impossible to quantify. Finally, because of the release of three variables in the public-use data for income (i.e. continuous income, categorical income and the time unit of reporting), processing error was introduced into the data when more than one response type existed for the same individual. This gradually reduced over time though, suggesting more careful data cleaning or interviewer training on this question. We discuss the scope processing error further in the next chapter in this book. #### 2.6 Conclusion At the heart of any discourse on scientific method is debate about data quality. For producers of data, modern expectations are that greater disclosure of the limitations of data is required. For consumers of data, judicious analyses of that data mandates a thorough understanding of what the data is intended to measure, versus what it can be stretched to accommodate. Scientific research often shapes policy dialog, and so another interest group begins to weigh in on data quality debates. Unfortunately, debates that are ostensibly about data quality can often hide disingenuous attempts to thwart results based on sound data, particularly in the policy domain. The need for a clear framework for investigating data quality is therefore a cogent one. The main contribution of this chapter has been to (1) adapt the TSE framework into one that recognised the limited agency of researchers to assess data quality; and (2) integrate the TSE paradigm with the data quality paradigm. This helped create a framework for investigating microdata quality that was sensitive to the capacity of agents to diagnose data quality in the first place, while at the same time recognising the pressures that shape data quality within data production organisations. It is important to recognise that improvements to data quality did happen over time with SSA labour market surveys, partly as a natural consequence of the learning process from previous mistakes and partly because of the involvement of researchers and policymakers who communicated their data quality concerns to Stats SA. As researchers focussed specific effort on only a few variables in the surveys, they often uncovered deficiencies in the data that were much harder for the survey organisation to detect. Consequently, improving data quality is an iterative process that should ideally promote a virtuous cycle of interaction between producers and consumers of data. For producers of data, the preparation and publication of detailed data quality frameworks is recommended in much the same way as Statistics Canada and SSA have gone about developing them. These frameworks are also excellent documents to inform users about issues of relevance to survey organisations, such as confidentiality issues. The advantage of using a coherent framework to discuss data quality is that it directs attention to components of the data production process and the likely data quality elements that led to that error. However, for researchers as consumers of data, the TSE framework is insufficient in itself to inform efforts to rigorously interrogate data quality, for it is rarely possible to identify those errors or quantify their magnitude in public-use datasets. In the absence of clear data quality documentation for each survey instrument, considerable thought therefore needs to be given to the likely errors that exist and their impact on analyses. For example, comparing poverty estimates between the mid 2000s and the mid 1990s using the LFS and OHS is likely an exercise riddled by coverage errors that researchers can do very little about. Yet these numbers often dominate the policy discourse. Under such circumstances, it is far better to acknowledge uncertainty more explicitly and to consider the bounds of sensitivity of key estimates to alternative assumptions about the data generating process. #### References Bhorat, H. (1999). The October household survey, unemployment and the informal sector: A note. South African Journal of Economics, 67(2), 320–326. Blair, J., Menon, G. & Bickart, B. (1991). Measurement effects in self vs. proxy responses to survey questions: An information-processing perspective. In Biemer, P. P., Groves, R. M., Lyberg, L. E., Mathiowetz, N. A., & Sudman, S. (Eds.) Measurement error in surveys. New Jersey: Wiley. Rubin, D. B. (1987). Multiple imputation for nonresponse in surveys. New York: Wiley. Rubin, D. B. (1976). Inference and missing data. Biometrica, 63, 581–592. Statistics South Africa (SSA). (1995). October household survey metadata. Pretoria: SSA. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Chapter 3 Questionnaire Design and Response Propensities for Labour Income Microdata #### 3.1 Introduction The income question in household surveys is one of the most socially sensitive constructs. Two problems that arise with social sensitivity concern the probability of obtaining a response and the type of response provided. In survey error terms, this translates into an important relationship between questionnaire design (construct validity) and item non-response. In turn, these affect the statistical distribution of income that has both univariate and multivariate implications. Consequently, the interrelationship between questionnaire design and response type is crucial to understand when conducting analyses of the income variable. This chapter discusses the design of the employee income question and evaluates the characteristics of respondents who report their incomes as exact values, bounded values, and three additional response types that we will initially group into item nonresponse: (a) those who state they don't know their income or that of the proxy individual on whose behalf they are reporting, (2) those who refuse to answer the question, and (3) responses that are coded unspecified responses in the public-use dataset. The focus is therefore on the response process for a particular variable, which is conditional on the respondent having already agreed to participate in the survey. In all of Statistics South Africa's (SSA) Labour Force Surveys (LFS), which began in 2000, the employee income question commences by asking individuals what the exact value of their income is. If they refuse to answer or state that they don't know, respondents are then presented with a showcard that displays ascending bounds of income categories. Here they are required to pick an income category that most likely captures the correct income value. If they refuse a second time or repeat that they don't know the value, the final response is recorded as such. The treatment of nonresponse groups in the income question differed across the October Household Surveys (here we focus on the OHS 1997–1999). In 1997 and 1998, there were no options for don't know and refuse, whereas in 1999 only an option for don't know was included in the questionnaire. This resulted in a large number of unspecified income responses in the publicly released OHSs, which confound the understanding of the nonresponse mechanism. Only in the LFS were options introduced into the employee income question to differentiate nonresponse into both don't know and refuse response types, yet there were also always a positive number of unspecified responses in the LFS 2000–2003. The introduction of new response groups to the income question allows us to examine the impact of these questionnaire design changes on the response propensities of participants in the survey. From this, we can understand the item nonresponse mechanism far more precisely, and this has profound implications for imputation strategies that become the subject of the next chapter in this book. The factors that influence respondents to provide a particular kind of response become important for two main reasons: firstly, it helps shed light on the possible socio-cultural factors that influence social sensitivity or social desirability, and secondly it provides insight into the correlates of bounded responses and nonresponse. An important part of the analytical process required for understanding nonresponse is to attempt to diagnose whether that data is ignorable for the type of analysis envisaged. For applied purposes, ignorability determination amounts to establishing whether the data are missing at random or not. Analysing response propensities therefore also helps to characterise the missingness mechanism. Response propensity models are traditionally employed by survey organisations when investigating the determinants of survey participation and unit nonresponse (see Groves and Couper, 1998). The innovation in this chapter is to investigate item nonresponse process analgously. The chapter proceeds as follows: firstly, different designs of the employee income question in household surveys is discussed. This provides insight into the trade-offs of varying approaches to asking respondents about their incomes, a traditionally very sensitive question and one where evasive behaviour by the respondent is common. Secondly, we discuss the methodology for analysing item response propensities. We draw from the survey participation literature for this purpose, and discuss suitable models to tailor the approach to item nonresponse. Finally, the results are presented and discussed, before the conclusion summarises. #### 3.2 Questionnaire Design and the Income Question #### 3.2.1 The Response Process and the Cognitive Burden of Answering Income Questions Like any survey question, the decision by the respondent to provide an answer to the income question is broadly influenced by (1) whether they can answer, and (2) whether they will answer. Psychological research has demonstrated that respondent knowledge is a matter of degree rather than a dichotomy of knowing and not knowing, where respondent knowledge can be classified in terms of four cognitive states: whether that knowledge is available, accessible, generatable (i.e. able to be cued), or inestimable (Beatty and Herrmann, 2002, 73). Given this, it would be reasonable to assume that an important objective of questionnaire design should be to structure the sections and questions in such a way as to improve respondent recall, which means framing the instrument and using anchoring strategies to be as supportive as possible in assisting recall. The design of the questionnaire, including section and question presentation order, is therefore a non-trivial issue when it comes to the quality of responses to questions (Schwarz and Hippler, 1991). Response propensity is not only affected by respondent attributes such as age, race and gender, but also by factors such as the survey mode, interviewer training, question topics and structure, and institutional dimensions (e.g. public or private statistical agency or marketing company) of the survey (Dillman et al., 2002). For the income question, key goals for the design of the question are not only to reduce item nonresponse, but also to minimise misreporting, under-reporting and measurement error. Hurd et al. (2003) note that questions about incomes are among the most difficult to answer in household surveys for several reasons, including that (1) respondents may be reluctant to reveal information they consider private and sensitive; (2) cognitive issues make it difficult for respondents to accurately report their income, especially when that reporting is done for other household members; (3) the time period for which a source of income is asked in the questionnaire may be quite different to the time period the respondent usually receives that income; and (4) taxes may or may not be included in different sources of income. Hurd et al. (2003) conclude that all of these issues can lead to significant bias (particularly in the case of under-reporting) and measurement error. In the case of the employee income question, many of these negative potential outcomes are mitigated by the introduction of a follow-up prompt that applies if a respondent initially states that they don't know or refuse to provide a value. The follow-up then asks the respondent to identify some range of values into which their (or the other household member on whose behalf they are reporting) income falls. The objective of this follow-up prompt is to provide an anchoring strategy for the respondent in the form of a lower and upper bound to income, but it also reduces the social sensitivity of the question because it reduces the level of information disclosure. The precise type of follow-up prompt differs between surveys, and there is some discussion in the literature about the relative merits of alternative questionnaire designs. Anchoring is an important principle that facilitates respondent recall by triggering indirect cues in the cognitive response process that bear on the target judgement (Frederick et al., 2010). However, Jacowitz and Kahneman (1995) note that the disadvantage of using an anchor to prompt the respondent into some form of indirect answering of quantitative estimation questions (such as income), is that it introduces the possibility of anchoring bias. Anchoring bias is when respondents provide a value for their income that is closer to the value of the anchor itself, which introduces uncertainty surrounding the reliability of the answer. Jacowitz and Kahneman (1995) develop a simple quantitative methodology to measure anchoring bias. They find that anchoring effects are "surprisingly large", sometimes evident in the original evaluation of the anchor as high or low (in the questionnaire design phase), and inversely related to respondents' confidence in their judgements but substantial even in judgements made with high confidence. For the income from employment question, the extent of anchoring bias is partly related to the exact form of the income follow-up prompt, to which we now turn. #### 3.2.2 Different Types of Income Questions In household face-to-face interview surveys the employee income question differs mainly with respect to the nature of the follow-up prompt that follows an initial request for an exact amount (of either gross income or net income). This follow-up prompt can differ in three primary ways: There are several different dimensions to take into account when discussing the merits of alternative designs. However, all three question types share the commonality that they reduce item nonresponse on the question by providing an alternative response option to an exact response. In order to distinguish the relative merits between the question types, we focus on (1) how they affect the response process, and (2) their analytical implications. Schwartz and Paulin (2000) conducted an experiment to assess the merits of these three questions types to respondents. Eligibility to participate in the experiment was based on whether a respondent received any money in wages or salary in the past twelve months. An instrument similar to the Consumer Expenditure Quarterly Interview Survey in the USA was developed, with different types of bracketing techniques used including show cards, unfolding brackets and respondent generated intervals (RGIs). Upon completion of the mock interview, a cognitive interview was conducted to evaluate respondents' subjective experience of the process. It was found that across experimental groups, the show-card conventional bracketing technique received the highest overall preference rating and it was rated the easiest with which to reach an answer, possibly due to the fact that it is the only question with a combination of a visual aid (ibid, 967). This was followed by the RGI technique, with unfolding brackets selected as the least popular technique. Schwartz and Pualin (2000, 969) suggest that while respondent preference may not be an issue for surveys that rely on only one interview, for longitudinal surveys this factor may become more important. Here, conventional brackets and RGIs are considered to be preferable by the authors. An important finding was also that conventional brackets were likely to have been considered preferable by high-income respondents because there was limited disclosure if their income was in the highest, open-ended bracket. With RGIs, however, high income respondents had to disclose a lower and upper bound that lead to the (self-selected) bounds becoming wider as income increased. In the final analysis, Schwartz and Paulin (2000) suggest that RGIs are likely to lead to higher data quality on income questions because, unlike the conventional bracket which is essentially a recognition memory task, the RGI technique is a twostep memory task. Here, the respondent must firstly estimate the actual amount and then decide how to bound that amount. Their experiment suggested that one way respondents chose to limit the complexity of the RGI task was to skip it and instead provide an exact value. It was noted (ibid, 969) that exact values are statistically preferred to range responses for income questions because they are more precise, and consequently RGIs would improve data quality. Analytically, the existence of the bracketed subset raises the issue of anchoring bias. For RGIs and the conventional show-card bracket question, anchoring bias (or entry-point bias) is non-existent, but for the unfolding bracket design it is potentially substantial. For salary income though, Hurd et al. (2003) find that there is little evidence of anchoring bias in the Health and Retirement Study (HRS) in the USA, but Juster et al. (1999) find that there is evidence of anchor bias in measures of saving and income from components of wealth. However, Vasquez-Alvarez (2003) postulates different types of anchoring effects for the HRS's (1996) salary income variable when it is treated as a covariate in a model of differences in smoking prevalence between the sexes, and finds evidence that anchoring biases play a significant role in model inferences. The detection of anchoring bias is a non-trivial issue and much work remains to be done on this topic (see especially Juster et al., 2007). While conventional show-card brackets and RGIs are not subject to anchor biases, they are not without their problems. Show-cards can only be administered in faceto-face interview surveys, whereas unfolding brackets and RGIs can be presented telephonically too. RGIs are the most recent innovation to questionnaire design for financial data. Press and Tanur (2004) find that the interval length between the lower and upper bounds of RGI questions is directly related to the respondent's confidence in their answer, and that sometimes question wording has a direct relationship to the response rate, and to accuracy of the population parameter. Press and Tanur (2005) suggest that to improve the accuracy of RGIs it is helpful to have respondents provide confidence scores about how sure they are of their answers. RGIs also impose specific estimation tasks concerning interval estimation at the individual level, as opposed to show-cards and unfolding brackets where the length of the interval is standardised in questionnaire design. The relevance of this discussion for our purposes is that the choice made by respondents about how to answer the income question matters. The precise nature of the follow-up prompt for income helps overturn initial refusals to the income question and therefore conveys information about the response process. Questions then arise about whether groups of respondents with particular characteristics behave in similar ways and are more likely to disclose their incomes with the follow-up question. This can help shed light on the socio-cultural and ethno-linguistic determinants of social sensitivity or social desirability. Social desirability is when respondents want other people to know what incomes they earn, as a type of demonstration effect. #### 3.2.3 Analysing Response Groups in the Income Question Common to all employee income question types is a three-fold differentiation of response groups into exact responses, bounded (bracketed) responses and nonresponse (don't know and refusals).<sup>1</sup> In this section we discuss how models of survey participation can be used to develop response propensity models for individual questions like employee income. Traditionally, survey methodologists develop response propensity models to understand survey participation (or unit nonresponse), often decomposing nonparticipation into noncontacts and refusals (see De Leeuw and de Heer, 2002). This literature provides an important basis for adapting the models to item nonresponse. Groves and Couper (1998) note that there are four hypotheses about survey participation: (1) the opportunity cost hypothesis; (2) the exchange hypothesis; (3) the social isolation hypothesis; and (4) the concept of authority and survey cooperation. The opportunity cost hypothesis states that people will participate in surveys if they don't have anything better to do. For example, employed people may have less discretionary time than unemployed people. The exchange hypothesis relates to the fact that people generally feel more obligated to participate if they are given an unconditional gift. The social isolation hypothesis suggests that more isolated individuals have a lower probability of survey participation. An example of this is when an individual is a victim of crime and chooses to close their home off to outsiders. Finally, a survey organisation can use its authority to encourage participation. This is possible for a national statistics agency in particular, but may be less so for a marketing company. <sup>1</sup>Note that our treatment of "Don't Know" responses as a form of nonresponse takes its precedence from Rubin et al. (1995). However, this definition imposes no constraints on the analysis, and later in this chapter we consider "Don't Know" as a partial form of response because it reveals at least some information about income, as opposed to refusals. The dependent variable in survey participation models is usually binary, coded zero for conducting the interview and one for not participating (either refusals or non-contacts, but not both). The explanatory variables include variables for environment (e.g. central city urban or suburbia, population density, crime rate, percent under twenty years old); social isolation (including race, mixed ages (e.g. greater than 69 years old), single person household, children less than 5 in the household; residential exchange in last five years); and social exchange (owner occupied house, monthly rental, house value). Models of response behaviour also incorporate more elaborate individual factors. For example, Johnson et al. (2002) describe the impact of culture on nonresponse. They suggest that cultural variability matters for nonresponse for everything from survey question comprehension, to memory retrieval, judgement formation and response editing processes. As a consequence, it is also important to factor these variables into response propensity models, though it is unlikely that every relevant variable in this respect will be available in public-use datasets. #### 3.2.4 Questionnaire Design Changes in SA Labour Market Household Surveys We evaluate employee income in South Africa's two major household interview labour market surveys: the October Household Surveys (OHS; 1997–1999), and the Labour Force Surveys (LFS; 2000–2003 September waves only). The OHS was a repeated cross-sectional survey, while the LFS was a biannual rotating panel survey. Only the September Waves of the LFS are chosen in order to allow the series to be more comparable with the OHS. Since the LFS is a rotating panel survey, it poses no methodological problem to take only one wave in a given year because each wave of a rotating panel is designed to estimate the population of South Africa at the time of going to field. The rotation part of the panel ensures that a portion of the sample changes in every Wave of the survey (Cantwell, 2008). In both of these surveys, the employee income question developed by Statistics South Africa (SSA) had a show-card follow-up for bracketed responses, but evolved over time with respect to its treatment of nonresponse. In the OHS 1997 and 1998, there were no options for don't know and refuse; in the OHS 1999 don't know was added as an option for the first time; only with the commencement of the LFS in 2000 were both don't know and refuse added to the question. We want to exploit these changes in questionnaire design to evaluate how they affected the capacity to understand the response process for employee income. Figure 3.1 displays the employee income question in the LFS 2000 that became the standard after much trial and error in the 1990s. For both the OHS and LFS, the surveys required a single adult respondent to answer the income question for every member in the household. When responses are provided for household members other than the respondent, this is called proxy Fig. 3.1 The income question: labour force survey 2000 September reporting, which has been subject to some attention in the literature due to the anticipated increase in measurement error associated with a proxy reporter (see Blair et al., 1991). The intuition behind this is simple: a proxy reporter is less likely to know the exact value of the income of other members of the household. While this may be less likely in the case of cohabiting partners in an intimate relationship where the intra-household allocation of resources is shared, it is increasingly likely in multiple adult households either in the same extended familial group or unrelated individuals living in the same household. One way to account for this is to include a variable for self or proxy reporting directly into the analysis (see for example, Casale and Posel, 2005). However, the ability to do so was not present in the majority of October Household Surveys and only became part of the questionnaire in 1999. The differences between the questionnaires over time therefore has an important bearing on the degree to which we can understand the response process. The final major difference in the questionnaires between the OHS and the LFS is that in the OHS more general information is provided about the household including their household conditions and exposure to crime for example. In fact, when the OHS ended in 1999, two surveys were designed to replace it: the Labour Force Survey (LFS) and the General Household Survey (GHS, although the GHS was only implemented some years later). The LFS contained all the labour market information from the previous OHS questionnaire with improvements to sections like the income question, while the remainder of the OHS questionnaire was directed to the GHS. Note that despite the differences in the length of the overall questionnaires between the OHS and LFS, the income question appears at roughly the same point in each questionnaire, implying that respondent fatigue by the time they reached the employee income question during the interview was not altered too drastically between the two survey instruments. The evolution of the survey instrument and the income question in these surveys provides us with a valuable opportunity to evaluate how changes to questionnaire design impacted the response process. #### 3.3 Methodology The principle of developing response propensity models for an individual question like income shares its motivation from the analagous requirement to understand the response process for the survey more generally. We begin by describing the evolution of the employee income question and the resulting structure of the data released to the public. Thereafter, the response propensity models are developed before estimation, specification and testing are discussed. #### 3.3.1 Response Propensity Models for the Employee Income Question Models of survey participation propensity, such as those in Groves and Couper (1998), De Leeuw and de Heer (2002) and Johnson et al. (2002), model the process as a function of (1) variables that reflect the possible perceptions of the respondent to the relative burden of participating in the survey, in combination with (2) variables that reflect the capacity of the survey organisation to shift the perception of the respondent about that burden. Unlike survey participation propensities, however, response propensities to particular questions in a survey already have buy-in from the respondent about survey participation. Consequently, modelling the process is dependent on the features of the variable(s) of interest. Another way of saying this is that survey participation and response propensities on individual questions are always related in that item nonresponse is conditional upon unit response. For the income from employment question, we saw from the literature that there are two primary concerns: the cognitive burden of answering the income question, which is partly related to recall and social sensitivity issues; and the expected correlates of income itself, since both bounded response and nonresponse is thought to be related to higher income levels. We therefore also need to incorporate variables that best predict this effect. Here we are limited by the questionnaires themselves. In the OHS and LFS questionnaires, the following variable groups of interest can be identified in some or all of the instruments: Important variables that would help shed light on the response process are interviewer codes and any diagnostic information about the interview itself (often called paradata). However, none of this information is available in any of the public-use versions of the OHSs or LFSs. <sup>2</sup>The number of retirees will be omitted in order to prevent a perfectly collinear relationship between the household composition variables and household size. The above variables are included in all of the response propensity models when they become available in the survey questionnaires. Because the same variables are utilised in every survey year, it is important to note that we invoke the assumption that the response process is stationary over time. This implies that, a-priori, we do not expect changes to the direction of influence of the covariates over time. However, their direction of influence can change depending on the response type under investigation. We discuss each variable's rationale for inclusion in the section on model specification and testing below. #### 3.3.2 Questionnaire Design Changes and the Resulting Structure of Income Data in Publicly Released Datasets An important difference between the OHSs and LFS was that in the OHS, selfemployed individuals answered a different income question to employees, whereas in the LFS both employees and the self-employed were asked the same question. In order to standardise the sample to employees only, we drop all self-employed from all surveys and further restrict the sample to the economically active population (16–64 years old). In the OHS97 and OHS98, the time period for reporting income was daily, weekly and monthly, whereas in 1999 (and, thankfully, every year since then), the periods changed to weekly, monthly and annually. In all of SSA's public datasets, employee income is differentiated into three variables: (1) a continuous variable that reflects the range of exact income responses; (2) a categorical variable that reflects the ascending bounded income ranges of the bracketed subset; and (3) a variable for the time unit of income recorded. These three variables need to be used to derive a single income variable for analysis. The two surveys of interest are the OHS (1997–1999) and LFS (2000 September– 2003 September). During the OHS, the income question changed (the don't know option was added in 1999 and the time period of reporting changed from daily, weekly and monthly in 1997 and 1998 to weekly, monthly, annually in 1999), and new questions were added to the questionnaire that can help explain the response process (e.g. the introduction of self versus proxy reporting in 1999). The OHS also asked more general questions about the neighbourhood the respondent was living in and their experience of crime, whereas the LFS omitted these questions from the questionnaires. While in the OHS, both the employee income question and the questionnaire changed, in the LFS, neither the employee income question nor the questionnaire changed on key variables of interest. Fig. 3.2 The employee income response process in OHS 1997 and 1998 #### 3.3.3 Estimation, Specification and Testing #### Estimation We can think of response propensity models for employee income as modelling a latent variable for the unwillingness to disclose income. This variable is not directly observed, but we do observe the response type for the income question, which gives us information about the level of information disclosure the respondent is willing to provide. An important estimation task is then to adequately account for the sequential nature of the response process that reveals the level of information disclosure. In the income question, the interviewer first asks the respondent for an exact income value; if they refuse or state that the don't know, the interviewer asks a follow-up question where a showcard is presented to the respondent with bounded income ranges. The respondent can then choose a bracket into which their income falls. Only if the respondent states that they don't know or refuses again, is the final response coded as don't know or refuse.3 Because the income question itself evolved over the survey years under investigation (particularly between 1997–2000), the sequential nature of the response process differs over time. Figures 3.2 and 3.3 depict this. From Fig. 3.2, we see that the respondent can first provide an exact income value or state that they don't know or refuse (collectively grouped as "nonresponse" in the figure). The interviewer then prompts the respondent to answer again, this time with a bounded response follow-up question presented with a showcard. If the respondent refuses again or states that they don't know, the OHS 1997 and 1998 data record an unspecified response for that individual, which we know can be either don't know <sup>3</sup>Note that we assume the showcard that the interviewer presents to the respondent only has the bounded income ranges printed, rather than the additional options to state that they "Don't Know" or "Refuse", which is present in the questionnaire as per Fig. 3.1. This would ensure that the interviewer does not inadvertently prompt the respondent for a "Don't Know" or "Refuse" response by presenting it on the showcard. Fig. 3.3 The employee income response process in LFS 2000–2003 or refuse, but which cannot be identified as such from the questionnaire and so is conflated into a grouped "nonresponse" option that concludes the response process for these survey years. In the OHS 1999, don't know was provided in the income question for the first time, and hence the sequential structure of the response process has an additional branch that decomposes the final "nonresponse" option into don't know and unspecified. Here, unspecified responses are confounded with refusals because no option for refuse is present in the OHS99 questionnaire. In the LFS 2000–2003, we have the same sequential structure as the OHS 1999, but this time the final "nonresponse" option is decomposed into its exhaustive subsets of refusals and don't know responses. Figure 3.3 presents this sequential structure. A suitable characterisation of this kind of problem is the sequential response model of Maddala (1983). Adapting this model to the problem of the employee income question as depicted in Fig. 3.3, define the outcome variable Y to have four possible alternatives: The probabilities of each outcome in the sequential response model can be written as: $$\begin{aligned} P\_1 &= F(\beta\_1' \mathbf{x}) \\ P\_2 &= \{1 - F(\beta\_1' \mathbf{x})\} F(\beta\_2' \mathbf{x}) \\ P\_3 &= \{1 - F(\beta\_1' \mathbf{x})\} [1 - F(\beta\_2' \mathbf{x})] F(\beta\_3' \mathbf{x}) \\ P\_4 &= \{1 - F(\beta\_1' \mathbf{x})\} [1 - F(\beta\_2' \mathbf{x})] [1 - F(\beta\_3' \mathbf{x})] \end{aligned} \tag{3.1}$$ where F is the cumulative distribution function and the betas are parameters to be estimated. As Maddala (1983, 49) notes, this kind of model is easy to analyse because the likelihood functions can be maximised by maximising the likelihood functions of dichotomous models repeatedly. By doing this, note that we therefore make the assumption that the probability of choice at each stage of the response model is independent of the choice at the previous stage. In other words, the independence of irrelevant alternatives (IIA) assumption of more general polytomous discrete choice models is applicable here too. Despite the invocation of the IIA assumption, however, note that unlike the multinomial response model, the sequential response model estimates dichotomous models that combine multiple outcomes against a changing base outcome sequentially until the stages of the sequence are exhausted. Therefore, as implied by Fig. 3.3 and Eq. 3.1, the first stage of the sequence is estimated combining bounded responses, don't know responses and refusals, {Y = 2 + Y = 3 + Y = 4}, against the base outcome of a continuous response, {Y = 1}. The second stage of the sequence is estimated combining don't know and refusals, {Y = 3 + Y = 4}, against the base outcome of a bounded response {Y = 2}; and the third stage of the sequence is estimated as {Y = 4} against the base outcome of a don't know response, {Y = 3}. In other words, the parameter β<sup>1</sup> in Eq. 3.1 is estimated from the entire sample by dividing it into two groups, continuous responses and initial nonresponse (to the first exact income question); β<sup>2</sup> is estimated from the subsample of remaining response types divided into bounded responses and final nonresponse (to the follow-up income question); and β<sup>3</sup> is estimated by dividing the subsample of final nonresponse into refusals and don't know responses. In this context, the IIA assumption is entirely reasonable because the respondent has to refuse or state that they don't know twice: once to the initial income question for an exact response, and a second time to the follow-up question that presents a showcard. The third stage simply decomposes nonresponse into refusals and don't know, exhausting the possible response alternatives. Hence the IIA assumption is reasonable to defend. Buis (2011) discusses a modern application (and some limitations) of the sequential response model, and we use the estimator he developed called the sequential logistic model, implemented in Stata version 12 using the package written by Buis (2012, Version 1.1.15). #### Specification In this section we discuss variable selection over the different survey years, possible omitted variables and the possibility of measurement error in the explanatory variables. Recall from Sect. 3.3.1 that we have four broad variable groups: (1) cognitive burden of answering income variables; (2) willingness to disclose variables; (3) personal characteristics of respondent; and (4) correlates of income variables. The rationale for including each variable under these themes is presented in Table 3.1. Across the survey years from 1997–2003, we observe almost all of these variables, but in some years certain variables are not available or they change from categorical to continuous. For example, an identifier for self reporter (versus proxy reporting) only becomes available from 1999 onwards, while the variable for feeling unsafe in the neighbourhood you live is only available in 1997 and 1998. The variable for total household expenditure changes from continuous in 1997 and 1998 to categorical in 1999. It then changes again in 2000, when it was not asked at all in the LFS 2000 (September) because of the concurrent 2000 Income and Expenditure Survey that was administered to the same households. For this survey year, we merge in the continuous variable from the IES 2000. For all LFS after that, expenditure was asked in the same way as the OHS 1999, when a bounded expenditure range was presented to respondents. Note that only in the years when there is a categorical expenditure variable are there options for don't know and refuse to the question. It is important to note that for the variable 'first language of respondent', the rationale for including it in the models is to capture socio-cultural influences of social sensitivity to reporting income. In other words, we are interested in whether it affects the willingness to disclose income. However, it is very difficult to predict a-priori what the direction of the coefficients will be, for very little research has been done into this topic in South Africa. In order to ensure that we do not get spurious results in this respect, we are insulated by the fact that the response propensity models will be run over multiple, independent samples of individuals in the South African population over multiple time periods from 1997–2003. Consequently, we get a chance to observe the stability of the findings for language over time. Note that two different language variables are constructed for the analysis: one that introduces dummies for all eleven official SA languages, and one that keeps Zulu, Xhosa, English and Afrikaans, but aggregates the more regional languages together (including Ndebele, Northern Sotho, Southern Sotho, Tswana, Swazi, Venda, Tsonga and Other language). The rationale for the latter is that the cell sizes for some of these regional languages get very small when included with all of the other covariates. Zulu is SA's most spoken first language, and we consequently use it as the reference category in all regression models. A similar problem exists with the race variable. In contemporary discourse in SA, race is still disaggregated into the main classifications of the Apartheid era, namely African/Black (hereafter referred to only as African), Coloured, Indian/Asian (hereafter referred to only as Indian), and White. An option for the respondent to report their race as "Other" was present in all survey years from 1997–2003. However, Table 3.1 Explaining response type: covariate selection the number of individuals in the employed economically active subpopulation who report their race as "other" is very low, ranging from a minimum of zero in 1997 to a maximum of 49 in 2001. We therefore set "other race" to missing in the regression models due to the small cell sizes associated with it, and rather estimate race as a dummy variable for the four main racial groups only, with African as the reference group. On the question of the construct of race, it should be noted that there is very likely to be some measurement error on this variable. This is because the race question in all survey years (1997–2003) has a reporting option called "African/Black". During and even after Apartheid, the convention among supporters of certain political parties including the African National Congress was to follow the Black Consciousness movement's recommendation to label all historically disadvantaged groups "Black". So, for example, Indian/Asian and Coloured people who were historical supporters of the liberation struggle during Apartheid were (and still are) far more likely to report their race as "Black" compared to the Apartheid classifications given to them (especially among older generations). There is very little we can do about this form of measurement error in the data, other than note it for reference. It should also be noted that important omitted variables in this analysis include information about the interviewer that administered the questionnaire to the respondent, such as their race, age and gender, and information about the behaviour of the respondent in the interview, such as whether they were hostile or not. However, it is rare that this information is released by the survey organisation to the public, so very little can be done to compensate for these omitted variables other than to acknowledge their importance. The response propensity models developed in this chapter are not models that allow for causal inference. However, the stability of the signs and effect sizes of coefficients, over independent samples of the employed economically active population of South Africa from 1997–2003, does provide very useful insight into the stability of the correlates of the response process. #### 3.4 Results In this section we report the main findings. We commence by conducting a descriptive analysis of the distribution of different response types to the income question, before evaluating the probability of a bounded income bracket response as income increases. We then present the response propensity models. All results are not weighted because we are interested in the characteristics of the sample itself, rather than the population. #### 3.4.1 A Descriptive Analysis of Employee Income Response Type Table 3.2 shows the distribution of income subsets when the exact income variable is combined with the bounded income variable to form one derived monthly employee income variable that will henceforth be used for analysis. The percentage of exact responses in each survey year ranges from 87 percent in 2000 to 54% in 1999. This suggests that interviewer effort and training on socially sensitive questions may yield high dividends. Anecdotal evidence of greater effort by Statistics SA to train interviewers in 2000 is given in Daniels and Wittenberg (2010). Bounded responses vary from 9% of the sample in 2000 to 37% of the sample in 1998. However, there is no clear trend in the response propensity of this subset over time, though it does rise consistently after 2000. If we sum the responses for Don't Know, Refuse and Unspecified, we can evaluate the percentage of the sample for each year that represent the group of item nonrespondents for the income question. This number ranges from approximately 3% in 2000 to about 7% in 2003. This suggests that the bracket follow-up prompt is very successful at reducing nonresponse for employee income. The percentage of Don't Know responses doesn't seem to have a discernible trend, but the percentage of Refusals increases steadily from the LFS 2000–2003. For the bounded subset of observations, preliminary insight into the response mechanism can be obtained by evaluating the probability of a bounded response within each income category. Here, all observed income responses (including the Table 3.2 Distribution of response types: OHS97–LFS03 exact subset) are converted into bounded ranges before the probability is calculated. Table 3.3 presents the results. The table shows the percentage of respondents who provide a bounded response when all income observations are grouped into income categories. Don't know, refuse and unspecified responses are omitted from the calculations. A value of 0.98 as the first number for the zero income category in 1997 therefore implies that 98% of respondents who replied that their income was zero did so only when prompted by the interviewer for a bracketed response. There were 46 observations in total for this reporting option in 1997, 98% of which answered inside the bracket bound. The zero income category is somewhat peculiar to the SSA income question and generally has a low number of observations, ranging from 2 in 1998 to 46 in 1997. For income categories above zero, there is a near monotonic increase in the probability of reporting a bounded response as income itself increases, and this finding holds for almost every survey year. In other words, social sensitivity increases as income increases. Two notable exceptions to the monotonicity finding are in 1998 and 1999, where the highest probability of a bracket response is in the R11,001- R16,000 range in both years. Finally, the total probability of a bounded response in each survey year is presented at the bottom of Table 3.3, where we see it is lowest in 2000 at 10% and highest in 1998 at 38%. This considerable fluctuation may be due to interviewer training on the approach to the income question, as 2000 is considered to be the year that a substantial investment in interviewer training by Statistics SA was made (Daniels and Wittenberg, 2010). The overall conclusion from this section is that, in general, the probability of a bounded response increases as income increases. This is most likely due to the social sensitivity of income and the higher cognitive burden of answering the income question as an individual's remuneration increases and possibly becomes more complex (e.g. has benefits added or deductions subtracted). We now turn to multivariate analysis to evaluate the predictors of the various response types. #### 3.4.2 Sequential Response Propensity Models In this section we report results for the sequential response propensity models over two time periods: (1) 1997–1999, and (2) 1999–2003. In the first period, a two-stage sequential logistic response model is estimated for response type as per Fig. 3.2. The inclusion of OHS99 here means we do not decompose nonresponse into don't know and unspecifieds initially. Instead, we do this in the second time period, when we also analyse the LFS. Here, a three-stage sequential logistic response model is estimated as per Fig. 3.3 and Eq. 3.1. For all models, odds ratios are reported for the coefficients. The results are unweighted because we are interested in the sample itself. Standard errors are robust and clustered at the level of the primary sampling unit. Table 3.3 Probability of a bounded response within each monthly income category: OHS97–LFS03 #### Two-Stage Sequential Logistic Response Model We now present the findings for the two-stage sequential response models used for the OHS 1997, 1998 and 1999. For 1999, don't know responses are combined with unspecifieds. The first-stage results are reported in Table 3.4 and the second-stage results are reported in Table 3.5. Recall that the first stage of the sequential logistic model evaluates initial nonresponse to the exact income question, whereas the second stage evaluates final nonresponse compared to bounded responses (see Fig. 3.2). Odds ratios are reported for all model coefficients, and the effects are discussed for each group of explanatory variables (see the "Testing" column in Table 3.1 for a recap of the variable groups). Table 3.4 shows the odds ratios for the first stage of the system of equations that represent the sequential response model of equation refeq:rp1, for survey years 1997– 1999. Subsequent stages of the model are presented in the tables below. Regardless of the stages of the model, however, it is important to note that the specifications differ slightly between 1997 and 1999 due to changes in questionnaire design. Specifically, the variable "felt unsafe in neighbourhood" appears in 1997 and 1998, but is absent from 1999 onwards. Similarly, the variable for self reporter only appears in 1999. While this renders strict comparison of the stability of predictors over time impossible, it does give us insight into how questionnaire design changes impacted the capacity to diagnose the response process. Evident from Table 3.4 is that for the cognitive burden variables, none are repeatedly significant across the survey years except household head, and the direction of influence also changes for the number of kids and the number of economically active individuals (aged 16–64 years old) within the household between the survey years. Individuals in cohabiting relationships have lower odds of reporting initial nonresponse, but this effect in only significant in 1998. A self-reporter is significant, but only appears in 1999 and so its repeated effect cannot be assessed yet. In 1999, a self reporter to the income question reduces the odds of initial nonresponse by approximately 29%. Variables reflecting the personal characteristics of the respondent show a little more stability. Men have higher odds of not reporting an exact response, and this effect is significant in every year. The turning point of age is calculated as the coefficient on age divided by two times the coefficient of age squared, and is presented at the bottom of the table. Note that while the turning point is calculated using the log of the odds, the coefficients in the table itself are odds ratios (this convention will be maintained for the rest of this chapter). Note that while the odds ratios in the table are rounded to the third decimal place, the signs for the log of the odds of the coefficients on age squared are all negative. This implies that the shape of the relationship between age and the probability of initially refusing to answer the income question in all three years increases up to the turning point, after which it decreases. Important to note is that in 1998, the turning point lies outside the upper bound of the sample of economically active individuals (64 years old), suggesting a monotonic relationship between age and response type for this survey year. In 1997 and 1999, however, that relationship is quadratic with a turning point reached at about 52 years of age. Therefore, in 1997 and 1999 individuals are increasingly likely to refuse the initial income question up until 52, whereafter they become more likely to provide an exact income response. The race dummies show changes in direction of influence across the years for Indian and Coloured people, where the odds ratio suggests a negative relationship for these two groups relative to Africans in 1997, but this changes to a positive relationship in 1998, then changes again to negative in 1999 for Indian people. A Table 3.4 First-stage response propensity: initial nonresponse compared to exact responses: OHS 1997-OHS 1999 Reference: Number >65yr; African; no education; Zulu; expen R0-R399; Dwelling = owned formal dwelling. Significance: \* = 10%, \\ = 5%, \\\* = 1% Table 3.5 Second-stage response propensity: final nonresponse compared to bounded response: OHS 1997-OHS 1999 (continued) Table 3.5 (continued) Dwelling = owned formal dwelling. Significance: \* = 10%, \\ = 5%, \\\* = 1% stable effect is observed for White people, where the odds of nonresponse is always greater than Africans. Education shows predictable effects given its correlation with income, with the odds of nonresponse increasing as education increases (relative to those with no education). For the willingness to disclose variables, we see that rarely does any language have the same direction of influence across survey years, and sometimes the same language has statistically significantly negative odds in one year (relative to Zulu speakers), and statistically significantly positive odds in another year (e.g. Xhosa and Venda). This suggests that linguistic differences are ambiguous predictors of the first stage sequential response process. For the neighbourhood safety variable, which is only available in the OHS97 and OHS98,we see that it is associated with about ten percent higher odds for nonresponse reporting, but the coefficient is not statistically significant in either year. On the other hand, an urban location is always statistically significant and always has greater odds for nonresponse reporting compared to exact response reporting. For 1997 and 1998, variables that are thought to be correlated with income show the expected signs and significance, except the dwelling ownership and type variables. For 1999 the dwelling type variables show predicted effects and are significant. The reference category is an owned formal dwelling, a strong signal of wealth, so we would expect respondents who live in unowned formal dwellings, sub-let arrangements or informal areas to have lower odds of initial nonresponse, which is indeed the case. For those who own a vehicle, another stock of wealth variable, the odds of not providing an exact response are always higher than those who do not own a vehicle, and this result is statistically significant across the three years. Living in an urban area is a positive and significant predictor of nonresponse reporting in each year. For household expenditure, when it is measured as a continuous variable, the results suggest that a one percentage point increase in expenditure increases the odds of nonresponse by 0.23% in 1997 and 0.33% in 1998. However, there seems to be a nonlinear effect of expenditure on income reporting type, which is discernible only when expenditure is reported in brackets. Here, we see that while almost every expenditure category has higher odds for nonresponse and bounded response reporting relative to the R0-R399 expenditure category, the highest, open-ended expenditure category (>R10,000) has a lower effect size than the second highest category (R5,000-R9,999), and is not statistically significant (we return to this in the threestage sequential response model below). We now turn to the second stage of the sequential logistic response model. Here we are comparing nonresponse to bounded response, with the same set of explanatory variables as the first stage model. Nonresponse in 1999 conflates don't know responses with unspecified, whereas in 1997 and 1998 there are only unspecified responses for this subset. What we're looking for in this second stage response model is any stable change in direction of the effects previously observed, which will tell us that the response process has changed as the response options evolve into the second income question. Important to note is that because we now exclude the exact subset of responses, the effective subsample size differs from the estimation subsample. The effective subsample includes only the bounded responses and nonresponse subsets of respondents in the second stage of the sequential model.4 Evident from Table 3.5 is that there are far fewer statistically significant coefficients across the entire range of predictors compared to the first stage model, except in 1999. In 1998 only two coefficients are significant, namely cohabiting and other language. At first consideration, the lack of significance doesn't seem to tell us much about this stage of the response process. But it is important to note that a lack of significance for so many covariates in the second stage suggests a very different response process to the follow-up employee income question. This would be equivalent to stating that the observed wealth effect in the first stage has been removed in the second income question, and that now both nonresponse and bounded response groups are indistinguishable on this set of predictors. However, some caution is perhaps prudent here, for the findings in 1999 in particular are quite different to 1998 and 1997. The predictors themselves are also different, for in 1997 and 1998, self-reporter is not available while feeling unsafe in neighbourhood is available. The latter is insignificant in both years, as it was in the first stage response model (see Table 3.4), suggesting perhaps that it is an irrelevant variable in both stages of the employee income response process. On the other hand, self-reporter is highly significant in 1999, and is clearly a more relevant variable in these models. We shall examine this in more detail for the LFS surveys below. In 1999, Table 3.5 shows that the cognitive burden variables are very important predictors of final nonresponse. A household head reduces the odds of nonresponse by about 45%, while a self-reporter reduces the odds of nonresponse ten-fold. Since <sup>4</sup>Note that the effective subsample size is not available using Buis's (2012) algorithm for the sequential logistic response model. Here, and in every other table presented in this chapter, the effective subsample size is estimated by fitting separate logistic regression models to each stage of the sequential response process. The validity of doing so is given by Maddala (1983), and discussed in Sect. 3.3.3 above. household size is held constant, the interpretation of the coefficients on the number of children and adults in the household is relative to them replacing a senior citizen (65 years or older). Thus, if a child was to replace a senior, it would reduce the odds of nonresponse by 42%, while an adult (aged 16–64) would reduce the odds of nonresponse by 32%. The coefficient on household size reflects the addition of one more senior citizen because the number of children and adults are being held constant. Therefore, the addition of one senior citizen increases the odds of final nonresponse by 71%. The presence of senior household members is clearly correlated with greater reluctance to provide an income response, or greater confusion about that income (leading to a higher incidence of don't know responses). Also in 1999, for the personal characteristics variables, cohabiting with a romantic partner reduces the odds of nonresponse by 26%. Men have odds that are 63% higher than women for final nonresponse, but the age, race and education variables are generally insignificant. This is the first indication that the correlates of income variables may no longer be playing the powerful role in explaining the response process that they did in the first-stage model. If we consider the coefficients and significance of housing, vehicle ownership and expenditure variables, this effect would seem to be reinforced. Consequently, it suggests that variables that are correlated with income do not explain final nonresponse (alternatively we may simply not be able to measure this effect accurately). This is a very important finding, but preliminary at this point. We explore this further in the three-stage models below. For the willingness to disclose variables, the effects for language is once again ambiguous, even though many of the coefficients are significant in 1999. Living in an urban area is significant in 1997, but the direction of influence changes across the survey years. In summary, we can see that there are very different factors explaining the first stage of the sequential response model compared to the second stage. The qualifier on these findings, is that nonresponse in the final stage confounds don't know and refuse, providing limited insight into the construct of nonresponse itself. Below we are unconstrained by this, and explore the three-stage models for 1999–2003. #### Three-Stage Sequential Logistic Response Model In this section we present results for the three-stage models for the survey years 1999– 2003. The first stage evaluates the determinants of initial nonresponse compared to exact responses; the second stage evaluates the determinants of final nonresponse against bounded responses, and the third stage decomposes nonresponse into refusals compared to don't know responses. For the OHS 1999, which doesn't have an option for refusals in the questionnaire, we use the response group coded "unspecified" in the public-use dataset as the indicator of interest. This group of unspecified responses presumably conflates refusals with processing error. By analysing the predictors of this response type along with the LFS, we have an opportunity to see if the same relationships hold over time. Note, however, that because of the lack of the refuse option in the OHS 1999, it is not strictly comparable to the LFS in the third-stage of the sequential response model, and we will interpret the results accordingly. For the first two stages of the model, the lack of a refuse option doesn't prejudice the comparability of the output. Table 3.6 shows that for the cognitive burden variables, there are many significant effects, particularly during 2000–2002, but less so in 1999 and 2003. The household head variable is significant in every year until 2003, when its direction of influence changes. A self reporter is always significant and always reduces the odds of nonresponse. The household composition variables are not repeatedly significant across all survey years, but the direction of influence of additional kids or economically active people (16–64 years old) is almost always lower than the reference category of seniors. The household size variable is also not significant in 1999, 2002 and 2003. Cohabiting individuals reduce the probability of nonresponse, but the variable is only significant in 2000 and 2002. The importance of self-reporters in this section is noteworthy relative to the findings in 1997–1999. For personal characteristics, men always have slightly higher odds of nonresponse, but this is not significant in every year. The coefficients on age are significant in every survey year except 2000, and for those years when it is significant, the turning point is approximately 47 years of age. The sign of the coefficients once again suggest an inverted-u shape to the relationship between age and response propensity, with the probability of refusing to answer the first income question increasing until 47, after which it decreases. The race variables are fascinating. Coloured and White people have higher odds of nonresponse compared to Africans (though only the coefficients for Whites are significant in every year), but Indian people have significantly lower odds of nonresponse compared to Africans. This suggests that, all else equal, people of Indian or Asian descent in SA actually have a preference for reporting an exact response. Thus, rather than there being a socially sensitive dimension to the exact income question, for Indian people there seems instead to be a socially desirable dimension to it—a possible demonstration effect. The education category dummies show the expected directional influence given their correlation to income, with effect sizes generally increasing over time. Thus, tertiary education respondents have much higher odds of initial nonresponse compared to those with no education. After primary school, all of the education categories have coefficients that are statistically significant in every year, suggesting stable direction of the effects relative to the base of no education (except in 1999), even though the coefficients are quite different in magnitude. For other variables that are correlated with income—including housing type and ownership, vehicle ownership and total household expenditure—the coefficients are also always in the expected direction and always significant (with one or two exceptions) in every survey year. This is perhaps the most important affirmation that, for initial nonresponse at least, it is strongly related to higher income levels. The exception to this is the finding for Indian people, who are on average the second wealthiest population group in South Africa after Whites, but here demonstrate behaviour that Table 3.6 First-stage response propensity: initial nonresponse compared to exact responses: 1999– 2003 (continued) Table 3.6 (continued) Reference: Number >65yr; African; no education; Zulu; expen R0-R399; dwelling = Owned formal dwelling. Significance: \* = 10%, \\ = 5%, \\\* = 1% suggests a cultural difference in their attitude to social sensitivity. Because we are controlling for the partial effect of language and race in these models (note that in these three-stage sequential logistic models, a more aggregated language variable (see Table 3.1) is used to ensure large enough cell counts for the models to run), the finding for Indian people can be interpreted as a socio-cultural effect, and is highly noteworthy. We now turn to the second stage of the sequential response model, which evaluates final nonresponse (including refusals combined with don't know responses) compared to bounded response. Table 3.7 presents the results. Evident from the table is that the cognitive burden variables are important predictors of final nonresponse compared to bounded response. The household head and self reporters always have lower odds of nonresponse, and these coefficients are statistically significant in every year except in 2003 for the household head. However, for the household composition variables, the effects are not significant in 2000 and 2001, though the coefficients go in the same direction as every other year. Similarly, for household size, in 2000 and 2001 the effects are in different directions and not significant, whereas they are both positive and significant in other years. For cohabiting status, 2000 and 2003 have insignificant results and the effect is in different direction in 2000, while for the remaining years they reduce the odds of nonresponse and are significant. The results for personal characteristics variables, including gender, age, race and education are rarely consistently statistically significant over all years, and the coefficients for language show no consistent direction of influence over time. The failure of age to play a significant role in the second stage of the response process (except in 2001) is identical to the second stage of the response models for OHS97-99 presented in Table 3.5 above, suggesting that it plays a diminished or non-existent role in explaining further nonresponse beyond the first stage of income reporting. Table 3.7 Second-stage response propensity: final nonresponse compared to bounded responses: 1999–2003 (continued) Table 3.7 (continued) Reference: Number >65yr; African; no education; Zulu; expen R0-R399; dwelling = Owned formal dwelling. Significance: \* = 10%, \\ = 5%, \\\* = 1% The housing wealth dummies are also almost never significant, nor the vehicle ownership variable (except in 2001). However, the expenditure variables are frequently significant, especially in the highest income category which is significant in every year. The direction of the effect is surprising though, for it seems that as total household expenditure goes up, the odds of nonresponse go down. The coefficient on the log of expenditure also suggests lower odds for nonresponse reporting as expenditure increases. The take-home message from the second stage of the response model is that the odds of final nonresponse do not seem to increase with income. The most consistent effects over time are for the cognitive burden variables, notably self reporter followed by household head. The lack of explanatory power in the wealth variables suggests that the follow-up employee income question that presents the showcard to the respondent is very successful in persuading higher income individuals to disclose their earnings, albeit as a bounded response. This would suggest that any remaining nonresponse should no longer be unambiguously positively correlated with income. We now turn to exploring this in the third stage of the sequential response model. Table 3.8 shows the results of the third stage response model, where the dependent variable decomposes final nonresponse into refusals compared to don't know responses, except in 1999 where unspecified responses confound refusals with other possible sources of missing data, such as processing error or measurement error. However, there are generally no stable predictors over time in this stage of the response process despite a standardised instrument between 2000–2003. Small sample sizes also suggest weaker power in these models. In this table we also start seeing very large effect sizes for certain variables. The large coefficient sizes are potentially indicative of small cell sizes in this stage of the response model, leading to near perfect prediction of the outcome. To get some idea about whether it is a small sample size that is driving this, the effective sample size at the bottom of the table is useful to consult, as is Table 3.2 above, which provides the counts of each response type that constitute the dependent variables in these models. As far as the effective subsample size is concerned, the results for 2000 demonstrate that it has the smallest sample of nonresponse groups, and is very different to every other survey year. We evaluate further diagnostics of these models in the next section of this chapter below. Among the cognitive burden questions, only self-reporter is repeatedly significant (except in 2000), and it increases the odds of refusing by the largest order of magnitude. The strength of the self-reporter variable is unsurprising though because those respondents who are proxy reporters are much less likely to know the incomes of other household members, whereas self-reporters are much more likely to refuse on social sensitivity grounds. Hence the large coefficients are to be expected here, though a magnitude of 33 times the odds (in 2001) is surprising in light of the relatively large effective sample size (of 864 observations, roughly equally distributed between don't knows and refusals—see Table 3.2). For personal characteristics variables, there is no stable effect for age, sex or race, with odds ratios often below one for a given year and then above one for the next year. For age and age squared, it is not meaningful to discuss the turning points as the results are insignificant for all survey years. Education categories have odds ratios generally greater than one, and in 2002 the results are large and significant. The very large coefficients for education in 2000 suggest small cell sizes in this year in particular. For the willingness to disclose variables, language is again inconsistent over time, while living in an urban location is almost always significant, but the direction of influence on the odds change from negative to positive and back again over time. For the correlates of income, the results for expenditure in 2002 and 2003 suggest an increasing chance of refusing as expenditure increases, but the results are not always significant at the lower expenditure categories. However, owning a vehicle and housing wealth is almost never significant, suggesting an absence of a wealth effect on the odds of refusing. The overall conclusion to this stage of the response model is that self-reporting is the major explanatory factor impacting upon the probability to refuse to answer the income question. The wealth effect seems to be absent, while a positive but nonmonotonic relationship with household expenditure seems to be present, a slightly contradictory set of results. Finally, an important concern that arises in each of the sequential response models, but particularly in the case of the third stage models where the effective sample size is smallest, is the interrelationship between covariate nonresponse on expenditure and nonresponse on income. If these two forms of missingness are correlated, then it is possible for a simultaneity problem to exist that could lead to biased results. We now turn to evaluating this question along with other diagnostic tests of the response models. Table 3.8 Third-stage response propensity: refuse compared to don't know responses: 1999–2003 (continued) Table 3.8 (continued) Reference: Number >65yr; African; no education; Zulu; expen R0-R399; dwelling = Owned formal dwelling. Significance: \* = 10%, \\ = 5%, \\\* = 1% #### 3.4.3 Diagnostics of the Sequential Response Models In this section we evaluate model fit and the sensitivity of the results above to simultaneous income and expenditure missing data. This helps shed light on the limitations of the analysis, and provides some useful insights for further research. #### Model Fit In this section we discuss model fit for the sequential logistic response models estimated in the main text of this chapter by presenting Hosmer-Lemeshow (H-L) statistics. The sequential logistic model fitted to the data is estimated as a system of equations in the algorithm by Buis (2012). Theoretically, however, it is also possible to derive the same results by fitting binary logistic models to each stage of the sequential response process. This is immediately evident from Eq. 3.1 above. The H-L test results in Table 3.9 are calculated as post-estimation statistics after binary logistic models for each stage of the sequential response models are fitted to the data. The pseudo R<sup>2</sup> values from those models are also presented as a further model diagnostic. The table shows the response stage for each year investigated, the number of observations involved in the post-estimation procedure after each binary logistic model is fitted in order to calculate the H-L statistic, the number of groups used, the H-L statistic itself with p-value, and the pseudo R2. Large H-L statistics and small p-values indicate a lack of fit of the model. The results from Table 3.9 suggest that the models do not fit the data well in the first stage of the sequential response process in every survey year except 2002. This is unsurprising because multiple response groups are collapsed into the dependent variables of the first stage models, namely bracketed responses, don't know, refuse and/or unspecifieds, which are all compared against exact responses (the base outcome in the first stage). It is only from the second stage of the response process that the models begin to fit well. For the second and third stages the H-L tests suggest that we fail to reject the null of good model fit in all survey years except in the third stage of 2001 (at the 5% significance level). It should be noted that the small sample size in 2000 indicates weak statistical power of the H-L test in this year, but for every other year this is unlikely to be the case. However, the pseudo R<sup>2</sup> values suggest that the specification of the models best explain the variance of only the third stage of the response process: that is, predictors Table 3.9 Hosmer-Lemeshow (H-L) test for model fit and pseudo r squared in logistic regression of each sequential response stage Response Stage 1: missing + bracket compared to continuous Response Stage 2: missing compared to bracket Response Stage 3: refuse compared to don't know of refusals compared to don't knows. For the first and second stages, the pseudo R<sup>2</sup> is typically very weak. Important to note here is that on statistical grounds, the pseudo R<sup>2</sup> is not a particularly informative statistic for discrete (and particularly binary) dependent variable regression models due to the limited variation in the dependent variable itself. Nevertheless, its magnitude does impart some information on how the response models perform. #### The Sensitivity of Model Estimates and Inferences to Omitted Expenditure It is important to conduct an analysis of simultaneous nonresponse on employee income and expenditure because these two variables are correlated and expenditure is an explanatory variable in every response propensity model. The role of the total household expenditure variable in these models is to provide us with a correlate to individual employee income, but the capacity of this variable to do its job effectively is reduced if nonresponse on it occurs jointly with nonresponse on income. It should be noted that while employee income is measured at the individual level for the employed economically active population, expenditure is measured at the household level. Therefore, the extent to which these two variables are correlated will be higher in smaller households. Table 3.10 presents the percentages of joint nonresponse for each survey year and the denominator subsample size in the percentage calculations. The changing form of the expenditure variable over time provides for different levels of detail in this analysis. Firstly, when total household expenditure is a continuous variable, then the only form of nonresponse that we observe on it is an unspecified response. This is compared against the number of don't know, refuse and unspecifieds on income. The number jointly observed as nonresponse on expenditure and income then enters into the numerator of the percentage calculation, while the total number of don't know, refuse and unspecified responses for employee income enters the denominator. From this we see that for the OHS97, OHS98 and LFS00, simultaneous nonresponse on income and expenditure accounts for between 17 and 26% of all nonresponse. These numbers can be further decomposed when a bounded expenditure bracket is asked for rather than an exact response, because additional response options exist in the expenditure question for don't know and refuse. As with income in the OHS99, the expenditure question also does not have an option for "refuse", which was only introduced in the LFS questionnaires. The most important row of Table 3.10 for the OHS99 and LFS00-03 is the last one, in which all forms of nonresponse on expenditure is compared to all forms of nonresponse on income. Here we see that simultaneous nonresponse is in fact much larger than for the continuous expenditure variable in every year investigated, averaging about 30% of all nonresponse on income in the LFS, but rising to a very high 47% in the OHS99. The first-order impact of nonresponse on expenditure in the regression models is to reduce the estimation sample size by the number of nonrespondents on expenditure. Table 3.10 Jointly observed nonresponse subsets for expenditure and income In the limiting case, if all nonrespondents on household expenditure were the highest income earners, then the loss of covariate information for these cases could introduce biases into the sequential response models. But since the numbers here are quite low, this concern is mitigated to some extent, particularly in the first and second stages of the sequential logistic response models where the subsample sizes are always in the several thousands for each survey year. However, expenditure nonrespone becomes non-trivial in the third stage of the sequential response models when the outcome variable is refusals (for the LFS, unspecifieds in 1999) compared to don't know responses. From Table 3.10, we can see the potential estimation sample sizes for the outcome variable sometimes involves observations counts in the hundreds. Here, nonresponse on household expenditure will play an important role because it reduces the estimation sample size for all other covariates too, and to the extent that these covariates also help predict refusals and don't know responses in the income question, the explanatory power of the models and for refusals compared to don't know responses in particular—is compromised. We therefore re-estimate the three-stage sequential response model of Sect. 3.4.2, omitting the expenditure variables from each year. Table 3.11 presents the results for Table 3.11 Third-stage response propensity: refuse compared to don't know responses omitting expenditure Reference: Number >65yr; African; no education; Zulu; expen R0-R399; dwelling = Owned formal dwelling. Significance: \* = 10%, \\ = 5%, \\\* = 1% the third stage of the response model only.<sup>5</sup> By way of summary, in the first and second stages of the model, almost all coefficients were in a similar direction. More common was that the significance levels changed, and this occurred for about 10% of the coefficients, though never consistently over time. However, for the third stage of the model, there are important changes in the direction of influence of coefficients and in statistical significance. Table 3.11 shows the results of the third stage of the sequential response model when expenditure is omitted from the specification. At the bottom of the table, we introduce a row that shows the gain in estimation sample size attributable to omitting expenditure from the model. This number ranges from 336 in 2000 to 1631 in 1999, the latter clearly more likely to influence results than the former. Comparing the results of this stage of the model with its counterpart in Table 3.8 shows somewhat similar findings, but given that the main finding in Table 3.8 was that there were no stable findings across the years, this is not particularly informative. One identical effect in Table 3.11 is for the self reporter variable, where the coefficient sizes are again very large and significant in the same four years as in Table 3.8 (i.e. 1999, 2001–2003). In the two years when the expenditure category is always significant in Table 3.8, namely 2002 and 2003, the effect of omitting expenditure is to deflect its influence into other variables in the model. In 2002, vehicle ownership and unowned formal dwellings becomes significant when they were not before. On the other hand, the education variables reduce in magnitude and become insignificant when expenditure is omitted. One interesting effect in Table 3.11 is for education in 2003, where the coefficients have now nearly doubled in magnitude and become significant (compared to Table 3.8). To the extent that education is picking up a correlate of income effect, the omitted expenditure variable may be influencing the results for education. However, because this only happens in 2003, it is not possible to generalise the result. Nevertheless, it does suggest that the effect of omitting expenditure in the sequential response models is not trivial, and may cause more problems than it solves in certain survey years. #### 3.5 Conclusion The main objective of this chapter was to carefully establish the interrelationship between questionnaire design and response propensities in order to identify the characteristics of respondents that have the highest probability of not responding to the employee income question. Analytically, an important part of the analysis was to assess the stability of the effects over multiple time points. Two periods were distinguished: (a) 1997–1999, which allowed us to evaluate how improvements to the income question affected our understanding of the response process, and how the <sup>5</sup>For the first and second stages of the sequential response model excluding expenditure, results will not be presented (but are available from the author.). addition of the self-reporter option and omission of unsafe neighbourhood influenced our understanding of income response type; and (b) 2000–2003, which allowed us to evaluate the stability of groups of predictors over time given a fixed instrument. The latter ensured that the findings were not exclusively due to transient empirical fluctuation in any given year. Improvements to the design of the income question unambiguously positively impacted the ability to understand nonresponse on it. This was particularly so for decomposing final nonresponse into both refusals and don't knows. In 1999, when only the don't know option was provided, unspecified responses seemed to mimic the patterns associated with those who refuse to answer the question for the first two stages of the sequential response models, but by the third stage began to differ in the signs and significance of important covariates. The addition of a self-reporter indicator in the questionnaire was equally important for explaining final income nonresponse in all survey years, except 2000 which was clearly an anomaly in the history of Statistics South Africa's surveys. The sequential logistic response model proved to be a suitable estimator for response propensities to employee income when it was measured by an initial exact prompt followed by a showcard bracketed follow-up prompt. The overall results from the first stage of the sequential response models was that initial nonresponse was strongly associated with variables correlated with income. This result was stable over almost every survey year from 1997–2003. There was also an interesting social desirability or demonstration effect discernible for people of Indian/Asian descent in this first stage response process, though this was most apparent in the LFS. However, in the second stage, there seemed to be a reversal of the finding that response propensities were correlated with income. Instead, a rise in the importance of household characteristics and self-reporting was apparent. What this implied was that the follow-up income question actually overturned initial refusals from higher earning respondents, and therefore neutralised the correlate of income effect in the (non)response process. The third-stage response propensities showed that, with or without expenditure included in the specification, the results were unstable across the years except for selfreporting, which was large and significant in every survey year except 2000. A small sample size is the most likely explanation for the anomalous results in 2000. Notable for this stage of the response models was the strength of the Hosmer-Lemeshow tests and pseudo r-squared statistics. But the fact that no subset of predictors remained consistently statistically significant across the years suggests some variation in this part of the missingness mechanism over time. Finally, it should be remembered that a limitation with this analysis is the inability to observe variables related to (1) the characteristics of the interviewer conducting the survey, and (2) the respondent's behaviour during the survey. These (omitted) variables could have helped better explain the final refusal response in particular. #### References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Chapter 4 Univariate Multiple Imputation for Coarse Employee Income Data #### 4.1 Introduction Employment income data are coarsened as a result of questionnaire design. In the previous chapter we saw that Statistics South Africa (SSA) ask two employment income questions: an exact income question with a showcard follow-up. In publicuse datasets, this results in two income variables: a continuously distributed variable for exact income responses and a categorical variable for bounded income responses with separate categories for nonresponse. It is the task of the researcher to then generate a single income variable that effectively deals with this mixture of data types. Following Heitjan and Rubin (1991), we call a variable with this mixture of data types "coarse data". Coarse income data pose non-trivial implications for researchers concerned with analysing that data. The primary problem that arises from an inconsistent treatment of this variable is that parameter estimates may be biased and dependent on the particular researcher's choice of method to overcome the problems posed by the instrument's design and resulting data structure. This leads to potentially erroneous inferences on important univariate parameters of the income distribution, including quantiles and moments. Multiple imputation is potentially an effective solution for coarse data problems (Heitjan and Rubin, 1990; Heitjan, 1994). It involves substituting coarse data values with plausible draws of those values multiple times. Multiple imputation has been applied to coarse wealth data by Heeringa (1995, 2002), and it has been applied to coarse earnings data by Daniels (2008) and Vermaak (2010). Ardington et al. (2006) conducted multiple imputation for total income. However, because multiple imputation is effectively a simulation-based technique (Schafer, 1999), it is very dependent on the setup of the imputation process and can frequently perform suboptimally for reasons that may not be easy to identify. Van Buuren et al. (1999), Royston (2004), White et al. (2007) and Graham et al. (2007) discuss various aspects of the multiple imputation process that can affect the reliability of imputed draws and statistical inference, such as covariate selection, the imputation algorithm itself and the numbers of imputations needed for reliable inference. In this chapter the imputation algorithm is simplified by imputing univariately for coarse income data only, rather than also imputing covariate missing data. This has both advantages and disadvantages. The main disadvantage is that it removes all units with covariate nonresponse from the estimation sample, which is equivalent to treating covariate nonresponse as missing completely at random (MCAR). The cost of doing this is dependent on the application, with Allison (2000) noting that more sophisticated treatments of covariate nonresponse can impose equally stringent (but often more opaque) assumptions on the data. However, a distinct advantage of multiple imputation is that imputed draws can be made for many variables with missing data simultaneously, making it computationally efficient. There is, therefore, a definite trade-off in ignoring covariate nonresponse. The main advantage of imputing multiple times for a single variable is that it allows us to be far more precise about exactly which aspects of the multiple imputation algorithm lead to implausible results. The two primary dimensions of the imputation algorithm that will be explored are specification of the prediction equations and sensitivity of the results to the number of imputations. The reason we need this precision is because, as shown in the previous chapter on questionnaire design and response propensities, we saw that respondents who chose to answer the bounded income question generally were higher income individuals. However, when we accounted for predictors of higher incomes in the sequential response propensity models, it was revealed that the final nonresponse subset had refusals that were largely indistinguishable from don't know responses on observable covariates. It was this finding that led to the suggestion that final nonresponse was likely an ignorable form of nonresponse. In this chapter a key objective is to assess where in the income distribution the bounded, refuse, don't know and unspecified subsets of the employment income question lie when we generate plausible values of their incomes using multiple imputation. The coarse data framework allows us to characterise the nature of the problem in a theoretically sound manner. The simplified univariate multiple imputation algorithm then allows us to test the sensitivity of inferences to covariate selection and the number of imputations. The usefulness of doing this is that we learn how robust imputations are to mis-specification. Lessons learnt from this process can then feed into more complex multivariate missing multiple imputation exercises. In order to examine the performance of the imputation algorithm, we test four differentspecifications of the prediction equations: one that is completely mis-specified to establish a baseline of how wrong the imputed draws can be; one with covariates selected identically to the response propensity models of the previous chapter; one with Mincerian earnings function based covariates; and one with a combination of response propensity and Mincerian earnings function covariates, which we treat as the first-best specification method for reasons discussed below. Data for this exercise is identical to the previous chapter: the October Household Surveys (OHS, 1997–1999) and Labour Force Surveys (LFS, 2000–2003 September Waves only). As with the previous chapter, the sample is restricted to economically active (16–64 year old) employees only. We can therefore also observe how improvements to the income question over time affect the imputation process. #### 4.2 Preliminaries #### 4.2.1 Coarse Income Data A variable with continuous, bounded and missing observations is not simply an example of nonresponse, but in fact a more complicated problem known in the literature as "coarse data". The theory of coarse data stems in part from the theory of missing data, which was principally developed by Rubin (1976, 1987). However, "coarse data" is in fact a generalisation of the various ways that data may not reflect their true values, and includes as special cases rounded, heaped, censored, partially categorised and missing (i.e. completely coarse) data (Heitjan and Rubin, 1991). Two principal papers established the theory of coarse data: Heitjan and Rubin (1991) and Heitjan (1994). To show the direct precedents to missing data theory, it is useful to note that the theory of coarse data generalised Rubin's (1976, 1987) theoretical phraseology–an association partially mandated by the result that missing data was simply one form of coarsening. As a consequence, the concepts of missing completely at random" (MCAR), "missing at random" (MAR), and "not missing at random" (NMAR) were distinguished from "coarsened completely at random" (CCAR) and "coarsened at random" (CAR). Heitjan and Basu (1996) explicitly differentiate between these five concepts, but the epistemological extensions provided by coarse data theory are particularly useful to income in public-use micro datasets. For the purposes of this discussion, coarse data is defined to consist of a combination of continuous data (assumed not to be coarsened at all), bounded data (bracket responses), and item missing data.We formally define what this means for the univariate statistical distribution of income, commencing with the missing data framework and then incorporating the more general coarse data framework. Following Little and Rubin (2002, 12), we define the complete data matrix as Y = (yi j) and the missing data indicator matrix M = (Mi j). Y is differentiated into an observed and unobserved component, Yobs and Ymis. The distribution f (·) of missingness is conditional upon Y and unknown parameters φ, denoted f (M\|Y, φ). If f (M\|Y, φ) = f (M\|φ) ∀ Y, φ, the unobserved data are said to be Missing Completely at Random (MCAR). Here, missing data do not depend on the observed or unobserved components of the complete data matrix. If f (M\|Y, φ) = f (M\|Yobs, φ) ∀ Ymis, φ, the unobserved data are said to be Missing at Random (MAR), a more restrictive condition than MCAR because now the missing data depend on the observed data. If the missing data M depend on the missing values in the data matrix, the mechanism is called not missing at random (NMAR). The missing data mechanism is said to be "ignorable" if the unobserved data are thought to be MCAR or MAR; in this case, a separate model for the mechanism that causes non-response is not needed (i.e. can be ignored). The missing mechanism is said to be "non-ignorable" if the unobserved data are NMAR. The coarse data framework incorporates missing data as a type of coarsening, but is also generalisable to bounded data such as income reported in brackets. To see the extensions, we again rely on Little and Rubin's (2002, 127–129) formulation of the problem. Let Y be the complete data matrix in the absence of coarsening with sample space , and let f (Y \|φ) denote the density of Y for the complete data with unknown parameters φ. The observed data are now thought to consist of a subset of the sample space in which Y is known to fall. This subset is a function of Y and a coarsening variable G that determines the bounds of Yobs, so that Yobs = Yobs(Y, G). To see the extension to bracketed responses such as those present in income microdata, note that the characterisation of Yobs = Yobs(Y, G) assumes that the observed data fall within known upper and lower bounds and not outside these bounds. Since the bounds are assumed known, the coarse data framework is flexible enough to be applied not only to bracketed response types, but also to data that is thought to be imprecisely coarsened, such as rounded data, heaped data, or otherwise partially categorised data (see Heitjan and Rubin, 1991). In each case the coarsening mechanism needs to be precisely modelled. To incorporate missing data into this framework, call the unobserved data completely coarsened, and allow plausible values of that data to lie within the sample space of Y . In this case, G is simply the missing data indicator matrix. Thus: $$\mathbf{y}\_{obs,ij} = \begin{cases} \begin{Bmatrix} \mathbf{y}\_{ij} \end{Bmatrix}, \text{ the set consisting of the single true value, if } G\_{ij} = 0\\ \mathbf{\hat{y}}, \quad \text{the sample space of } \mathbf{Y}, \text{ if } G\_{ij} = 1 \end{Bmatrix} \tag{4.1}$$ From this, the data Yobs are called coarsened at random (CAR) if f (g\|yobs, ymis, φ) = f (g\|yobs, φ) for all ymis. To apply the framework to a mixture of continuous responses, bounded responses and missing data, we follow Heeringa's 1995 example and simply allow G to precisely define whether the data are observed as continuous, bracketed or missing. To make the framework specific to the income question in the OHS and LFS, we will characterise the coarsening process to match what is found in the public-use datasets. $$\mathbf{y}\_{obs,ij} = \begin{cases} \begin{Bmatrix} \mathbf{y}\_{ij} \end{Bmatrix}, & \text{if } G\_{ij} = \{0\} \\ \begin{Bmatrix} \mathbf{y}\_{L} \le \mathbf{y}\_{ij} < \mathbf{y}\_{U} \end{Bmatrix}, & \text{if } G\_{ij} = \{1, 2, \dots, 14\} \\ \mathbf{y}, & \text{if } G\_{ij} = \{15, 16, 17\} \end{Bmatrix} \tag{4.2}$$ Here, Gi j = {0} indicates that yi j is observed as a set consisting of the single true (exact) income value; Gi j = {1, 2,..., 14} indicates that yi j falls within the lower bound yL and upper bound yU of one of the fourteen possible brackets in the OHS and LFS income questions; and Gi j = {15, 16, 17} indicates that yi j is observed as "Don't Know", "Refuse" or "Unspecified", and would then fall within the sample space of Y . A key implication of the coarse data framework is that the variable G itself is measurement error free (Heitjan and Rubin, 1991;Wittenberg, 2008). This effectively implies that if a respondent reports their income to be within a given bracket, it cannot lie outside of those bounds. It also implies that if a respondent provides an exact income response, that response is assumed to be precisely reported. One of the implications of this relates to the imputation process for it implies that plausible draws of income for the bracketed subset of observations have to lie within the lower and upper bounds of those brackets, while draws for the missing data can be made over the sample space of income. #### The Special Case of Unspecified Responses in the Coarse Data Framework In Statistics SA's household surveys between 1997 and 2003, nonresponse to the employee income question was often recorded in the public-use data as an unspecified response. This response type exists even when there are options for don't know and refuse in the questionnaires. In 1999, the don't know option was introduced to the question for the first time, before both don't know and refuse options were added in 2000. Despite this, in each of the LFS, unspecified responses still exist for the subsample of employed economically active individuals. This represents a form of either processing or measurement error because don't know and refuse exhaust the possible nonresponse types in the income instrument. Because of this, the nature of the coarsening mechanism for unspecified responses is opaque. Unspecified responses in the OHS 1997 and 1998 are the only identifiable form of nonresponse because the income question does not present any options to the interviewer for recording a don't know or refuse response. Therefore, we are forced to treat those as nonresponse. In 1999, the unspecified responses are confounded with refuse responses. But in the LFS, unspecified responses are identifiable as a form of processing error. Observations that are deemed to be a result of processing error cannot simply be included in the coarse data framework as applied here, for it represents a mutually exclusive error mechanism in the data. We deal with this below by firstly exploring the extent of processing error in the data and then conducting independent multiple imputations for these observations. #### The Special Case of Zero Income Brackets An idiosyncratic feature of the bounded income question in all of the surveys analysed in this chapter (OHS97-LFS03) is that it has a zero income option in the showcard. The existence of zero income brackets is thought to be related to false income reporting by Vermaak (2010), who imputes a proportion of these responses based on an assessment of the share that seem plausibly zero. The coarse data framework does not allow measurement error in the coarsening process to exist. Therefore, simply imputing the zero responses without a theoretical basis for doing so is arbitrary. Vermaak (2010) seems to include the self-employed in her subsamples of economically active individuals, which increases the number of zero responses substantially. This is easy to do in the LFS because the same question is asked to both the employed and the self-employed, whereas in the OHS the income question was different for self employed individuals. We restrict the sample here to employees only in all survey years. Zero income values can exist as a valid response type for the subsample of economically active employees because respondents can be off work on unpaid leave. We evaluate the prevalence of zero income responses below, but keep all such observations in the data without imputing them. #### 4.2.2 Multiple Imputation Multiple imputation has gained recognition as one of the most effective methods for handling multivariate item nonresponse in public-use datasets. However, its use requires a clear understanding of its limitations. The coarse data framework is very useful for characterising the possible ways in which observed data may differ from their true values, and while it incorporates missing data as a type of coarsening, its extension to other data problems such as measurement error is limited on theoretical grounds. Recent advances in multiple imputation theory do indeed pose solutions to data measured with error (see, particularly, Ghosh-Dastidar and Schafer, 2003),but associated with this is (1) a necessary change in the operation of imputation algorithms and, (2) a modification of the combination rules required for valid statistical inference from multiply imputed datasets (Reiter and Raghunathan, 2007). Multiple imputation has to address the pattern of coarsening present in a dataset. It was traditionally envisaged as a tool for data base constructors whose use of the methods was assumed to be independent from the data analyst's (Rubin, 1996). However, as the algorithms became more widely available and as more researchers became familiar with the methods, its use has burgeoned across the social and life sciences to a vast array of different applications. Indiscriminate use of multiple imputation is clearly discouraged by the major proponents of the method. As Schafer (1999) points out, multiple imputation is neither the only principled method for handling missing values, nor is it necessarily the best. Indeed, "(f)rom a statistical standpoint, …a naive or unprincipled imputation method may create more problems than it solves, distorting estimates, standard errors and hypothesis tests…" (Schafer, 1999, 3). This view echoes Rubin's (1996: 475), who reminds all that the "actual objective (of multiple imputation) is valid statistical inference not optimal point prediction under some loss function, and replacing the former with the latter can lead one badly astray". One of the important implications of the coarse data framework discussed in Sect. 4.2.1, and directly implied by Eq. (4.2), is that the type of coarsening is defined to be precise; in other words, there can be no measurement error in the coarsening variable (G). The use of the coarse data framework thus places particular restrictions on the manner in which multiple imputation can be conducted. Its utility lies in the fact that it provides clear rules for multiple imputation for the data structure resulting from the income question in the surveys considered. There are examples in the literature of multiple imputation being used to deal with other forms of survey error. In particular, Ghosh-Dastidar and Schafer (2003) demonstrate how multiple imputation theory can be extended to the case of nonresponse and measurement error (without a validation study). They call their process multiple edit multiple imputation (MEMI), and note that producing MEMI requires assumptions about the distribution of the ideal data, the nature of nonresponse, and a model for the measurement error mechanism. This approach can also be adapted to suit other uses of multiple imputation, such as anonymising confidential survey information (ibid, 2003). However, in each case both the imputation algorithms and the rules for estimation and inference from the multiply imputed datasets differ, and have to be derived for the intended application. #### 4.3 Setup of the Problem In this section we firstly discuss the data preparation tasks needed before working with the employee income variables. Here, the existence of bounded zero responses and processing error will be evaluated. We then develop an appropriate multiple imputation algorithm for coarse income data and identify the rules for estimation and inference given the nature of the coarse data problem and the imputation process. #### 4.3.1 Data Preparation #### Zero Income Responses Since the subsample of interest is economically active employees, zero income responses ought not to exist in general, unless the person is off work temporarily and on unpaid leave. However, in each survey year, there are a positive number of zero responses in the OHS and LFS. Moreover, the majority of zero responses are reported in the bounded income question in the OHS and LFS questionnaires, rather than the exact income question. Table 4.1 presents the number of observations reported in each response type. Evident from the table is that the number of zero responses is usually very small, ranging from two in 1998 to forty-five in 1997. Most of these are reported in the bounded income question. Of those employees who reported a zero income response (either in the bounded question or the exact question), the percentage that also reported that they have been absent from work in the past week due to illness ranges from zero in 1997–1999 to Table 4.1 Distribution of response types: OHS97—LFS03 29% in 2000, 42% in 2001, 53% in 2002 and 24% in 2003. There is no question for whether individuals are on unpaid leave for other reasons, however, so we cannot investigate this phenomenon. Because there are legitimate reasons for zero income reporting, we keep all zero responses in the subsamples of employees for each survey year and do not impute any of them. #### Processing Error and/or Measurement Error in the Data Two anomalies exist in Statistics SA's OHS and LFS: (1) instances where both an actual and a bracketed value are observed for the same individual; and (2) observations that are coded as "Unspecified" (i.e. missing), when in fact response options already exist in the questionnaire for the respondent to reply that they "Don't Know" or "Refuse" to answer the question. It is impossible to tell from the data or the survey documentation whether these anomalies are by design or whether they constitute a form of processing or measurement error, but they need to be addressed before imputation can take place. To formalise the problem, consider that the universe of potential outcomes for income responses consists of a continuous (exact) income subset, a bounded subset, and a missing (don't know, refuse or unspecified) subset. These three subsets are mutually exclusive because a bracketed outcome is only observed if the respondent chose not to answer the actual income prompt from the interviewer. A missing outcome is only observed if the respondent chose not to answer both the actual and the bracketed response prompt. Let the event that an exact income response is reported by the respondent be denoted P(A), the event that a bounded response is reported be denoted P(B), and the event that a missing response be reported be denoted P(M). For these three events to be mutually exclusive, P(A ∪ B ∪ M) = P(A) + P(B) + P(M) = 1, and P(A ∩ B ∩ M) = 0; P(A ∩ B) = 0; P(A ∩ M) = 0; P(B ∩ M) = 0. A first form of (either processing or measurement) error can then be defined to exist if any of these outcomes are violated. Because the design of the income question evolved between the OHS 1997-LFS 2000, P(M) is not defined by don't know and refuse for every survey year. We therefore need to decompose P(M) into its observable parts: don't know responses (denoted P(D)), refusals (denoted P(R)), and unspecified responses (denoted P(U)). Across the survey years we will then observe missing responses as: A second form of error can be defined to exist only for the LFS if P(M) P(D) P(R) = + + P(U), where P(U) = 0. This is because don't know and refuse responses in the LFS complete the possible forms of nonresponse for the employed, economically active population. In the OHS 1999, unspecified responses cannot be identified as a form of error because those responses confound refusals in the same way that unspecified responses confounded both don't know and refusals in the OHS 1997 and 1998. Table 4.2 presents the extent of these errors in the OHS97-LFS 2003. In order to estimate the subsets correctly, we use the raw data from the surveys of interest before any transformations of the variables are made. In the table, the column for 2000 is repeated for presentation purposes only, simply to show (1) how the transition from the OHS to the LFS proceeded, and (2) how all of the LFSs compare. We can see from the table that the sum of the probabilities do not always add up to one; this is the first clue that something is amiss. The first form of error exists for the OHS97-LFS00, but only for the subset P(A B). That is, we sometimes jointly observe values for exact and bounded income ∩ for the same respondents in these public-use datasets, which should not be happening. The findings for 1997 and 1999 are noteworthy because of the magnitude of the error in the data, at 68 and 53%, respectively (obtained from the "Sum" row in the table). For both years, these numbers match the percentage of actual income observations in the survey. This suggests that for each exact income observation, there is also a bounded observation. It is unclear why this is the case, or what motivation Statistics SA could possibly have had in doing this. One potential reason is that it is not a form of error at all, but rather that the survey organisation intentionally did this for some reason (it was not apparent from a reading of the survey organisation's accompanying literature and metadata whether or why this was done). In order to investigate this further, we checked the consistency between the exact values that were also observed as brackets by transforming actual income into a new Table 4.2 Subsets of interest in the observed income data monthly income variable, and then converting that variable into a bracketed variable with the same bounds as the SSA's bounded variable. The result was that about 85% in 1997 and 99% of actual income observations in 1999 were in the correct monthly income bracket. For 1998, only 16% of actual income observations were in the correct bracket. While it is true that the extent of this error is mitigated to some extent when there is a match between the variables, the existence of two data points on income for the same person should never, as a rule, exist. We do not observe this form of error for the other possible subsets, namely P(A ∩ M) or P(B ∩ M), in any of the datasets. This is unsurprising, for the actual placement of the "Don't Know" and "Refuse" options in the public-use dataset is as an option in the bounded income variable, making it impossible to confuse these subsets (when they enter the data electronically). It is clear from the table, though, that SSA really improved their performance on this dimension of the problem over time, with this form of error dropping to zero by the LFSs. That said, the LFS2000–2003 all have non-zero complements to P(A ∪ B ∪ M), which ought to no longer exist given that the income question had specific response options for don't know and refuse. Consequently, a second form of error exists, and is non-zero in each LFS dataset. It is substantial in the OHS 1999 and LFS 2000, at approximately 2.5 and 2%, respectively, of the sample of employed economically active individuals. The first type of error discussed for these datasets can easily be dealt with by generating a new derived income variable from the combined actual and interval variables in the raw data, and overwriting the bracketed responses with the exact responses. The rationale for doing this is that exact responses are preferred to bounded responses from an information content point of view (see Schwartz and Paulin, 2000). For the second type of error, we deal with it differently across the survey years: the observations are kept in the OHS 1999 because they are confounded with refusals; but they are omitted for imputation purposes from the LFS, where the nonrespondent subset is fully defined by don't know and refusals. However, we will evaluate and impute these response types separately in the analysis below to examine their distribution. #### 4.3.2 The Imputation Algorithm There are several important steps required for the development of appropriate multiple imputation methods. These include: - Variables that are required in the complete data model of interest; - Variables that appear to determine missingness; - Variables that explain a considerable amount of the variance of the target variable, which helps to reduce the uncertainty of the imputations. It is important to note that in this chapter we are concerned with multiply imputing for coarse income data only, which sets the pattern of coarseness as univariate. Consequently, we are not interested in multivariate coarsening or the effect of coarse data on the earnings covariate vector. An important consequence of this is that the multiple imputation algorithms simplify tremendously because the process of drawing plausible values from the conditional distribution of each variable with coarse data is restricted by design to one conditional distribution—income. Practically, this means our task is to develop a univariate multiple imputation algorithm. This has two implications: (1) it is no longer necessary to characterise the coarse data mechanism in a multivariate sense (e.g. to establish whether it is monotonic or a general multivariate coarse data pattern); and (2) it is no longer necessary to use a sequential regression multiple imputation approach to the problem because there is only one variable with coarse data.1 For this purpose we utilise <sup>1</sup> The two most common sequential imputation algorithms are variants of Van Buuren et al. (1999) multiple imputation by chained equations (MICE) algorithm, and Raghunathan et al. (2001) sequential regression multiple imputation (SRMI) algorithm. Royston's (2004, 2005, 2007, 2009) imputation by chained equations (ICE) algorithm is similar in principle to Van Buuren et al. (1999) procedure, while StataCorp (2011) developed a flexible multiple imputation package that can perform monotonic multiple imputation, fully conditional specification procedures (such as MICE, ICE and SRMI), and explicit Bayesian algorithms that allow the user to specify prior and posterior the interval regression-based multiple imputation procedure developed by Royston (2007) and modified by StataCorp (2011). #### 4.3.3 Estimation and Inference from Multiply Imputed Data Multiple imputation was suggested as a potential solution to missing data problems by Rubin (1976), and the rules for inference from multiply imputed datasets came to be known as Rubin's Rules. These essentially state that analyses of multiply imputed datasets should be conducted based on standard complete-data techniques, but parameter estimates must be combined across datasets. Formally, Rubin's Rules are presented as follows (we follow Royston's, 2004 exposition): Let θˆ <sup>m</sup>, Wm, m = 1,..., M be M complete-data estimates and their associated variances for an estimated parameter θ. The mean of θ is then calculated as: $$ \bar{\theta}\_M = \frac{1}{M} \sum\_{m=1}^M \hat{\theta}\_m. \tag{4.3} $$ The variance of θ has both a within component and a between component. The within component of the variance is: $$ \bar{W}\_M = \frac{1}{M} \sum\_{m=1}^M W\_m. \tag{4.4} $$ The between component of variance is: $$B\_M = \frac{1}{M-1} \sum\_{m=1}^{M} (\hat{\theta}\_m - \bar{\theta}\_M)^2. \tag{4.5}$$ Combining the within and between-components then leads to the formula for total variance: $$T\_M = \bar{W}\_M + \frac{M+1}{M} B\_M,\tag{4.6}$$ The reference distribution for confidence intervals and significance tests is a t distribution, $$(\theta - \bar{\theta}\_M) T\_M^{-1/2} \sim t\_v,$$ distributions, amongst others. The algorithm in StataCorp (2011) also has the functionality to be restricted to the type of univariate multiple imputation procedure utilised in this chapter. with degrees of freedom, $$\nu = (M - 1) \left( 1 + \frac{1}{M + 1} \frac{\bar{W}\_M}{B\_M} \right)^2 \dots$$ In the analysis below, we obtain parameter estimates for the marginal distribution of post-multiply imputed income using these rules for a variety of different parameters. #### 4.4 Results: Univariate Multiple Imputations for Coarse Income In this section we conduct univariate multiple imputation for coarse income data. Our objective is to draw plausible values for both the bracketed and missing subsets in each survey year. The multiple imputation algorithm employed for this purpose is based on an interval regression procedure developed by Statacorp in Stata Release 12 (2011). The algorithm allows for imputed draws to be restricted to the income bracket lower and upper bounds, and it simultaneously allows for imputed draws for missing data to be unrestricted. The sensitivity of estimates and inferences to a range of different specifications of the prediction equations of the imputation algorithm is tested. Four models are developed for this purpose: #### 4.4.1 Quantiles and Moments Across Four Imputation Models The results for weighted univariate income parameter estimates for each imputation model are presented in Table 4.3. The table shows parameter estimates of the multiply imputed nominal employment income variables ("Yimp"), for each of the four imputation models discussed above and the estimation sample size ("Est.N") in each survey year. Quantile estimates are calculated post-imputation for each of m imputed income variables using Rubin's Rules (see Eq. (4.3) above). For this section, the variance of the estimates are omitted, but they will be evaluated in detail below in Sect. 4.4.6. 2 Results from the table are discussed thematically. The following issues are of relevance: Evident from Table 4.3 is that up until the median, the differences between the imputations are relatively trivial. This is expected, for we know that the probability of a bounded response increases as income increases, so any difference in imputed draws for this subset will only make its presence felt higher up the income distribution. That said, an important feature of the imputation algorithm is that it limits the range of imputed draws to the bounds of each income category. For the highest income category, however, this is an open ended interval with no upper bound. Therefore, imputations for respondents in this group have no upper limit. At the top of the income distribution, we see substantial differences between the distributions. At the 99th percentile, the OHS 1999 has the widest range between the four imputation models. The mis-specified method of model 1 leads to substantially higher estimates than any other model. The differences between distributions in model 2 (that has response propensity covariates) and model 3 (that has earnings function covariates) is also substantial, but the difference in estimates between model 3 and the first-best imputation model 4 (which combines response propensity and earnings function covariates) is much lower. In fact, in every survey year and for every quantile other than the minimum, the first-best imputation model always generates distributions with the lowest estimates. The importance of this is particularly stark for the maximum values in each distribution. Important to note here is that in survey years where an exact income value is extreme, such as in 1999 and 2000, the imputed values rarely exceed this outlier, except for the mis-specified imputation model one in 1999, where an imputed draw <sup>2</sup> Note that the variance of a quantile has to be computed manually after m multiple imputations using Rubin's Rules (see Eq. (4.4) to (4.6) above). The total variance of a quantile contains only a between-imputation component of variance (see Eq. (4.5) above), but Rubin's total variance formula in Eq. (4.6) still has to be used to calculate the variance of a quantile because of the (m + 1)/m adjustment for finite m. is larger than the maximum in that year. But there is nothing generalisable from this observation, for in 2001 where an exact income value also represents the maximum, the imputation model one does not exceed it. The relationship between outliers in the observed distribution and multiple imputation is therefore important to be aware of. The differences between the four imputation models at the maximum are substantial in 1997, 1998, 2002, and 2003. This suggests that specification of the imputation algorithm is most significant to the upper tail of the income distribution. The fact that the model 4 estimates are the lowest for each parameter across the entire distribution suggests that covariate selection based on explaining both the outcome variable of interest (income) and the response process leading to coarse data (response propensities), is crucial for plausible draws of income, but even more important for the highest income earners. However, it is not clear that a congenial imputation model that only focuses on earnings covariates (model three) is substantially worse than model four. Model two is slightly more volatile across the survey years, suggesting that choosing covariates that explain the response process alone is not an optimal way of specifying multiple imputation algorithms. Finally, the reduction in the estimation sample size for model 4, although relatively modest, is nevertheless an important limitation associated with increasing the number of covariates in the prediction equations. #### 4.4.2 The Distribution of Multiply Imputed Bounded Income Values In this section we compare the subsets of multiply imputed income. We restrict the analysis initially to the first-best imputation model only. The kernel densities of the five multiply imputed bounded income distributions are presented in Fig. 4.1. The density for exact income responses is on the same graph. The solid lines represent the bounded distributions and the dashed line the continuous distribution for exact responses. We can see from Fig. 4.1 that the densities of imputed draws for the bracketed subset are always to the right of the actual income response distribution. This is entirely expected from the analysis in Chapter Three, where we saw that the probability of a bounded income response increases as income increases. The densities for each of the five imputed draws are very similar, and generally have similar skewness and kurtosis. This is to be expected given the bounds of the brackets, which restrict where in the distribution the draws can be made. An important observation concerns the maxima of the imputed draws for the bracketed subset of income respondents. In 1997 and 2003 we see clearly that the maximum monthly income value in the data is generated by the imputed draws for bounded income. It is also apparent that the minimum income values are determined by respondents who answer the bracketed section of each questionnaire. It should be remembered Fig. 4.1 Multiply imputed bracketed income (solid line) compared to observed continuous income (dashed line): 1997–2003 that the lowest bracket in each questionnaire is zero. And in each survey year we observe a non-zero count of such responses. This is highest in 2000, but is also noticeable in 1997, 2001–2003, where it clearly affects the kernel densities. The existence of zero values for employee income is not unreasonable given the fact that the income question asks respondents about their labour market activities in the week preceding the interview, during which respondents could be earning no income. #### 4.4.3 The Distribution of Multiply Imputed Missing Income Values The kernel densities of multiply imputed draws for the nonresponse subset (combining unspecifieds, don't know and refusals as appropriate to the survey year) of observations are compared to the observed responses (bounded and continuous) in Fig. 4.2. As before, each of the five multiply imputed income distributions are plotted on the same graph for each year. The densities for imputed draws of missing income observations are the solid lines while observed income has dashed lines. We can see from this figure that the distribution of imputed missing values changes over time, relative to the distribution of observed responses. In 1997 the densities for the missing income respondents generally overlaps that of the observed respondents. This suggests that respondents who didn't answer the income question had similar predicted values of income compared to respondents who did provide an answer to the question, based on observables in the public-use dataset. That begins to change immediately after 1997, however, where in 1998 it becomes clear that the missing subset of respondents had predicted income values discernibly more to the right than the observed subsets of income respondents. The location of the densities for the missing subset of observations gradually moves further to the right over time. To explain this trend, it is noteworthy to remember that we are observing the nominal distribution of monthly income over time. It is therefore reasonable to expect that the distribution of income in the population itself would shift to the right over the time frame. #### 4.4.4 The Distribution of Multiply Imputed Refusals and Don't Know Income Values In this section we evaluate the distributions of multiply imputed refusals and don't know income values. The time frame is restricted to 2000 and beyond, since these response options only appear in the questionnaires from 2000 onwards. The kernel densities for the multiply imputed draws of refusals are plotted with a solid line while draws for don't know responses are plotted with dashed lines. Because imputed draws for refusals and don't know responses are of particular interest, we Fig. 4.2 Multiply imputed missing income (solid line) compared to observed (multiply imputed bracket and continuous—dashed line) income: 1997–2003 compare the four multiple imputation models against each other. In Fig. 4.3, the misspecified imputation method (model 1) is on the left hand side while the first-best imputation method (model 4) is on the right hand side. It is evident from Fig. 4.3 that there is now a lot more variation between the imputed draws for each response group, and there are very different inferences about the distribution of don't know and refuse responses depending on which multiple imputation method is used. According to model one, the two groups are nearly indistinguishable, whereas in model four they are always very different. The densities of imputed income draws for refusals always lie to the right of the don't know responses. This shows a clear advantage of correctly specifying multiple imputation algorithms. To evaluate the sensitivity of this finding, we now compare the results for multiple imputation models 2 and 3 against each other. Figure 4.4 presents the densities where refuse responses are the solid lines while don't know responses are the dashed lines. We can see from Fig. 4.4 that regardless of whether the multiple imputation algorithm is specified with response propensity covariates only, or whether it is specified with earnings function covariates, the imputed draws for don't know and refuse subsets of the income distribution show very different distributions. The fact that both models predict this difference is unsurprising because some of the response propensity covariates were chosen precisely because they're correlated with income. Consequently, despite the fact that the response process for the income question was explained in the previous chapter, where it was evident that refusals were not discernibly different to don't know responses on observable covariates, when we impute for refusals and don't knows there are discernible differences between these subsets of the income distribution. The former finding reinforces the fact that this was likely due to weak power associated with small sample sizes for the third stage response propensity models. However, when refusals and don't know responses are set to missing and imputed off observed incomes, discernible differences do exist between these groups. #### 4.4.5 Unspecified Responses as a Source of Error In this section we isolate two survey years where unspecified responses represent a significant source of error, namely 1999 and 2000. Unspecified responses in 1999 are confounded with refusals; they consequently enter into the multiple imputations models discussed above. However, in 2000 unspecified responses represent a source of error only because don't know and refuse responses complete the nonresponse possibilities. Therefore, these responses are not imputed in models 1 through 4 above. However, in this section we conduct a new multiple imputation exercise for the LFS 2000 that is identical to model 4 above, but that does multiply impute values for unspecified responses. We then evaluate the densities of these unspecified responses compared to the other nonresponse subsets (Fig. 4.5). Fig. 4.3 Multiply imputed missing income: refusals (solid line) compared to don't know (dashed line): 2000–2003 Fig. 4.4 Refusals (solid line) compared to don't know (dashed line): response propensity (model 2) and earnings function (model 3) imputations: 2000–2003 Fig. 4.5 Unspecified response error imputations: 1999 and 2000 Table 4.1 on page 126 presents the subsample sizes for unspecified responses. We now want to compare the multiply imputed draws for these responses against the imputed draws for don't know responses in 1999, and against both don't knows and refusals in 2000. Figure 4.5 presents the results. In 1999, the densities for unspecified income draws are the dashed lines, while the solid lines represent don't know responses. In 2000, the densities for unspecified income draws are the bold dashed lines, whereas refusals are the solid lines and don't know the narrower dashed lines. From the figure it is clear that unspecified responses are substantially different to identified nonresponse groups in both 1999 and 2000. In 1999, if the unspecified responses were only refusals, then we would expect the distribution of these responses to lie to the right of the imputed don't know densities, as they do for every survey year in Figs. 4.3 and 4.4. However, they are much more widely spread across the income distribution than refusals. The same is true in 2000, when there is no longer confounding with refusals. Here, the densities for the imputed unspecified responses are spread across a much larger range than either the don't know or refuse imputations. This suggests that processing error is a completely different error mechanism to nonresponse on the income question, and should consequently not enter multiple imputation algorithms that do not explicitly account for the very different properties of this component of error. #### 4.4.6 Stability of Parameter Estimates as the Number of Multiple Imputations Increase The final section of this paper evaluates the stability of parameter estimates of imputed income as the number of imputations increase from two to five to twenty. We conduct multiple imputations using the specification of model 4 only. A-priori, we know that there is not much variation in imputed draws below the median of monthly income from previous analysis (see Table 4.3 on page 138). However, above this level there is more scope for variation. In particular, the largest (open-ended) income bracket as well as the distribution for imputed refusals and don't know responses should be considered to be highly variable given the analysis above. We therefore need to establish the bounds of sensitivity due to the number of multiple imputations conducted. Tables 4.4 and 4.5 present the results of this exercise. Parameter estimates in Table 4.4 are calculated as the mean of the two, five and twenty multiply imputed monthly income variables in the each respective datasets, as per Eq. 4.3 of Rubin's Rules. Evident from the table is that quantile estimates are almost identical below the median. For the mean of monthly income, the estimates are also very close across the two, five and twenty imputations for each survey year. In fact, this observation holds for every quantile including the maximum in every survey year. Even when we sum up all of the observations for monthly income to create a population-based estimate of the total monthly income earned by employees in South Africa, we can see that estimates do not differ substantially. The coefficient of variation of these estimates is presented in Table 4.5. Given that the means of parameter estimates are stable over two, five and twenty imputations–as presented in Table 4.4–the coefficient of variation is informative about the magnitude of the inflation in the variance observed as the number of imputations increase. We can see from the table that the ratio of the standard deviation to the mean is very small across every quantile and moment as the number of imputations increase. The largest values for the coefficient of variation are all found in the maximum column, Table 4.5 Coefficient of variation of quantiles and moments as number of imputations increase for the survey years 1997 and 2003. Even here though, the numbers are less than 0.5. Aside from these larger values, every other estimate of the coefficient of variation is always below 0.1. Despite the small magnitude of these coefficients, an important observation is the fact that they do not simply reduce in size as the number of imputations increase. This prevents any strong conclusions about the relationship between the number of imputations and its impact on inference. Two contributing factors to this finding are that (1) the percentage of missing observations is small (at between 3 and 5% for each survey year), and (2) the range of the bounded subset of observations is restricted through the imputation algorithm to lie within the lower and upper bound of each income bracket, thereby formulaically reducing the variance for imputed draws for all but the highest, open-ended income bracket. For the highest, open-ended income bracket, we saw that specification of the prediction equation in the imputation algorithm is important for reducing the right skewness of the upper tail. Since parameter estimates in Tables 4.4 and 4.5 use both response propensity and earnings function covariates in the model, the variance even in the upper tail of the distribution is relatively low. The overall conclusion from this analysis is that stability in the point estimates of parameters of multiply imputed income is achieved with as little as two multiple imputations. #### 4.5 Conclusion This chapter conducted univariate multiple imputation for coarse subsets of the employee income distribution in South African household surveys from 1997 to 2003. During this time, the employee income question itself evolved, shedding greater light on the coarse response mechanism. The coarse data framework was very useful in guiding the approach not only to the imputation algorithm, where an important implication was restricting the range of the imputed draws to lie within each income bracket, but also to the treatment of unspecified responses when they were identified as a source of survey error. This is one of the major advantages of the coarse data framework: it encourages an explicit approach to the characterisation of the response mechanism, which then leads to clear rules about what can and cannot be accommodated in the imputation step. For processing error, the fact that two variables are released in the public-use dataset—one for actual income responses and one for bracketed responses—implies that there is a non-zero chance of error between these variables that needs to be addressed when it exists. We identified two types of survey errors: one where duplicate income responses were identified for the same individual, and another where unspecified responses were present in the data even when response options that complete the missing data subset were present in the questionnaire (i.e. don't know and refusals). The solution to the first type of error was to create a new variable for income that overwrites the duplicate records of bounded income with the actual income values. However, for the second type of error, there was no simple solution because the problem ought not to exist for the subsample of interest (employed economically active individuals). Hence these observations were not imputed in the main analysis and analysed separately instead. An important relationship that repeatedly presented itself in each section of this chapter was that of the relationship between questionnaire design and the resulting data structure. This made the analytical task iterative to an extent more than complex, for it required careful data checks and question wording and sequencing checks that mandated a fastidious and detail-oriented approach to the problems and interpretation of the results. An overall lesson learnt from this chapter is that it is incumbent upon researchers to be absolutely meticulous in their data preparation, imputation, estimation and analysis tasks when working with micro datasets. The univariate approach to multiple imputation utilised here allowed for very specific sensitivity analyses to be performed. Four different specifications of the imputation models provided the basis for sensitivity analysis to mis-specification in the imputation algorithm. We used four different models for this purpose: a misspecified algorithm (model 1), one that explained the response process only (model 2), one that explained income itself (model 3), and a final one that combined covariates from model 2 and 3. It was this fourth model that was chosen as the first-best model, given the recommendations for covariate selection of Van Buuren et al. (1999). The main limitation with this model was a reduction in the estimation sample size due to the greater prevalence of covariate missing data compared to the other models. The advantage of incorporating predictors for the response process in the imputation algorithm as well as earnings covariates was that it evidently reduced the rightskewness of the imputed monthly income values. The plausibility of imputed draws for the highest, open-ended income bracket, the refusals, don't know and unspecified response groups, was clearly affected by covariate selection in the imputation process. The fact that the first-best model reduced these values relative to the other three specifications suggests there is considerable merit to paying close attention to the response process in multiple imputation algorithms and not simply to predictors of the outcome variable. This has important implications for more sophisticated multiple imputation exercises that seek to impute for covariate coarse data too, for it suggests that each variable with coarse observations needs: (1) a model of the coarse data mechanism for that variable (this would include checks for additional forms of survey error); (2) an analysis of the factors explaining the response process for that variable; and (3) appropriate prediction equations for that variable, which include covariates that explain both the response process and the outcome variable of interest. #### References Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. # Chapter 5 Conclusion: How Data Quality Affects Our Understanding of the Earnings Distribution Household survey data are subject to multiple forms of survey error that can have a direct bearing on data quality, influencing end-user estimates of parameters of interest in unpredictable ways. This book has focussed specifically on employee income, but the insights are generalisable to any component of income. Chapter Two developed a framework for investigating microdata quality that was based largely on the total survey error (TSE) paradigm, but that also included specific data quality control elements. The TSE framework decomposes survey error into coverage error, sampling error, nonresponse error, adjustment error, processing error, measurement error and validity. We focussed on adapting the total survey error framework to shed light on which aspects of data quality researchers can observe and do something about. This framework then served as the basis for evaluating the evolution of data quality in Statistics South Africa's labour market household surveys from the early 1990s to 2007. It was argued that efforts to improve data quality should involve a virtuous interaction between producers and consumers of microdata and should be considered an evolving process. For producers of data, the preparation and publication of detailed data quality frameworks was emphasised, and two such examples were reviewed (the Statistics Canada and SSA Data Quality Frameworks). These frameworks are also excellent documents to inform users about issues of relevance to survey organisations and how these may affect the overall quality of the public-release data. For example, the late 1990s would have been an excellent time for the national statistics office (SSA) to inform users to expect variation over the repeated cross-sections of survey data due to non-sampling errors, and to explain that process in some detail. However, data quality frameworks were not in use by SSA at that time. For consumers of data, judicious analyses of the univariate, bivariate and multivariate relationships in public-use versions of the datasets shed light on different components of survey error in variables of interest. Any problems associated therewith should be communicated back to survey organisations. However, this does not make the analysis task any easier, and comparisons of repeated cross-sections of income data are particularly vulnerable to components of survey error directly under the control of the survey organisation. Ultimately, it was noted that improving data quality for income in particular is about improving data quality for household surveys in general. Chapter Three isolated questionnaire design and item nonresponse for the employee income question in two South African labour market surveys: the October Household Survey (1997–1999) and the Labour Force Survey (2000–2003). The choice of time period isolated a period of changing questionnaire design for the employee income question. Between 1997 and 2000, the income question gradually included new response options for the respondent to state that they don't know or refuse to answer the question. We used sequential logistic response models to evaluate how improvements to the income question improved the capacity to understand the nonresponse and bounded response mechanisms. The use of these models represents an important contribution to the literature, for they can be used to evaluate the response process regardless of whether the bounded response question is in the form of a showcard, an unfolding bracket or a respondent generated interval. It was found that the probability of initial nonresponse to the exact income question was correlated with income, but when the second follow-up bounded income question was presented to respondents, final nonresponse was no longer repeatedly associated with predictors of income. This suggested that the bounded income question overturned initial nonresponse to the exact income question and included more high income earners in the observed response subset. The addition of refuse and don't know response options to the employee income question played a very important role in improving the understanding of the nonresponse process, but in the final analysis of this chapter at least, respondents who refused to answer the employee income question were no longer significantly different to those who stated that they didn't know their income, at least as far as predictors of income were concerned. Rather, correlates of the knowledge of income became significant, with self-reporters and those cohabiting with romantic partners having the most consistently higher odds of refusing over time. Chapter Four was concerned with conducting univariate multiple imputations for coarse response subsets of the employee income question. An analysis of the interrelationship between the exact income and bounded income variables released in the public-use data revealed a non-trivial degree of processing and/or measurement error for certain survey years between 1997–2003. We identified two forms of error that had to be dealt with effectively before multiple imputation could be performed. We also noted an idiosyncratic feature of the bounded employee income question in all of SSA's household surveys, namely the existence of a zero bracket. This was left in the data and not imputed because it was deemed to be a reasonable response value to the income question given the fact that employees could state they were not working due to being ill. Once these features of the public-use data were effectively treated, we then conducted multiple imputations for coarse income observations using four differently specified models to test the sensitivity of imputed draws of income to misspecification in the imputation algorithm. It was found that a combination of response propensity and Mincerian earnings function covariates led to imputed draws that were the least likely to be extreme values in the income distribution, relative to alternative specifications. This has very important implications for more complex multiple imputation algorithms that seek to simultaneously impute income and covariate coarse data, an exercise that will require much initial data preparation and analysis before the integrity of the algorithm can be validated. We then also evaluated the point estimates of quantiles and moments of the multiply imputed income distributions as the number of imputations increased, where it was found that stability in the estimates and inferences was achieved after only two imputations. This was likely a product of both the low percentage of item missing data and the restricted ranges of plausible imputed draws for the bounded income respondents. However, despite the low percentage of item missing data, it was found that imputed draws for refusals always had higher values than don't know respondents. This was a very important finding that was not discernible in Chapter Three, where predictors of the refuse subset no longer seemed to be different to the don't know subset on variables correlated with income. The coarse data framework proved to be very useful in Chapter Four in guiding the approach to multiple imputation, not only because it informed the use of an interval censored regression algorithm, but also because it led to the decision rule to exclude unspecified responses in the LFS from being imputed in the primary analysis. When we then conducted a separate imputation process for these unspecified responses in 1999 and 2000, it was found that imputed draws were very differently distributed compared to imputed draws for don't know and refuse responses. This suggested that unspecified responses was an altogether different error process to item nonresponse on the employee income question, and should be treated as such. Taken in combination, Chapters Three and Four show the necessary steps that researchers need to take when preparing the data for final estimates of univariate parameters of the earnings distribution. Post-imputation, poverty and inequality estimates can only then be thought of as accurate to the maximum degree possible given the data. This is true regardless of the country for which data is collected, which makes the methodology generalisable to any context. Limitations could still exist though, to the extent that unobservable components of survey error, such as frame error and sampling error, remain material. In summary then, the presence of multiple sources of survey error in microdata need not impose undue constraints to the reliable estimation of parameters of the income distribution. What is required is that each source of survey error's potential impact on that distribution is known, even though nothing can be done about some of those components of error after public release of the data. For those components of error that can be observed, statistically rigorous methodology has to inform the approach to univariate and multivariate analyses, and researchers need to be explicit about their treatment of each relevant component of error. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.	doab	2025-04-07T03:56:58.000066	14-7-2022 06:01	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001c1372-3390-4f92-8b42-7dfe9fe510d8", "url": "https://library.oapen.org/bitstream/20.500.12657/57371/1/978-981-19-3639-5.pdf", "author": "Daniels, Reza Che", "title": "How Data Quality Affects our Understanding of the Earnings Distribution", "publisher": "Springer Nature", "isbn": "", "section_idx": 0 }
001e4a90-6b26-4650-bf99-81d98b68a57f.0	# Mapping, Monitoring and Assessing Disasters Edited by Spyridon Mavroulis and Efthymios Lekkas Printed Edition of the Special Issue Published in Applied Sciences www.mdpi.com/journal/applsci ## Mapping, Monitoring and Assessing Disasters	doab	2025-04-07T03:56:58.028243	7-3-2023 17:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001e4a90-6b26-4650-bf99-81d98b68a57f", "url": "https://mdpi.com/books/pdfview/book/6898", "author": "", "title": "Mapping, Monitoring and Assessing Disasters", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036565392", "section_idx": 0 }
001e4a90-6b26-4650-bf99-81d98b68a57f.1	Mapping, Monitoring and Assessing Disasters Editors Spyridon Mavroulis Efthymios Lekkas MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors Spyridon Mavroulis National and Kapodistrian University of Athens Greece Efthymios Lekkas National and Kapodistrian University of Athens Greece Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Applied Sciences (ISSN 2076-3417) (available at: https://www.mdpi.com/journal/applsci/special issues/disasters mapping monitoring assessing). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Volume Number, Page Range. ISBN 978-3-0365-6539-2 (Hbk) ISBN 978-3-0365-6540-8 (PDF) © 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. ### Contents Operational Mapping and Post-Disaster Hazard Assessment by the Development of a Multiparametric Web App Using Geospatial Technologies and Data: Attica Region 2021 Wildfires (Greece) Reprinted from: Appl. Sci. 2022, 12, 7256, doi:10.3390/app12147256 ................. 185 Gabriela Az ´ocar de la Cruz, Gabriela Alfaro, Claudia Alonso, Rub´en Calvo and Paz Orellana Modeling the Ignition Risk: Analysis before and after Megafire on Maule Region, Chile Reprinted from: Appl. Sci. 2022, 12, 9353, doi:10.3390/app12189353 ................. 207 #### Sotiris Valkaniotis, George Papathanassiou,Vassilis Marinos, Charalampos Saroglou, Dimitrios Zekkos, Vasileios Kallimogiannis, et al. Landslides Triggered by Medicane Ianos in Greece, September 2020: Rapid Satellite Mapping and Field Survey Reprinted from: Appl. Sci. 2022, 12, 12443, doi:10.3390/app122312443 ................ 225 ### About the Editors #### Spyridon Mavroulis Dr. Spyridon Mavroulis is a Geologist with an MSc in the Prevention and Management of Natural Hazards, MSc in Environmental, Disasters and Crises Management Strategies and a PhD in Geology and Earth Sciences. His research interests focus mainly on the fields of neotectonics, tectonic geomorphology, hazard and risk assessment, disaster prevention and management of natural hazards and their effects on public health and on various elements of the built and the natural environment. He has participated in research projects on the integrated management of the geoenvironmental impact from natural hazards and related disasters. He has participated in over 20 scientific missions in Greece and worldwide in areas affected by earthquakes, landslides, tsunamis, volcano eruptions and megafires, to study the generation mechanism, assessing and mapping the impact on the geoenvironment and the built environment and evaluating actions and measures for disaster risk reduction. He has published articles on mapping, monitoring and assessing disasters in national and international scientific research journals and conferences. #### Efthymios Lekkas Dr. Efthymios Lekkas is a Professor of Dynamic, Tectonic, Applied Geology and Natural Disasters Management in the National and Kapodistrian University of Athens (NKUA) and the President of the Earthquake Planning and Protection Organization of Greece (EPPO). He is also Director of the Postgraduate Studies Program of "Environmental, Disasters and Crises Management Strategies" of the NKUA. He has participated as Principal Investigator and Lead Researcher in more than 300 applied research projects on earth sciences and disaster prevention and management. He has published books on natural and technological hazards and related disasters and scientific research publications in peer-reviewed journals and national and international conferences dealing with dynamic, tectonic, and applied geology, seismotectonics, earthquake engineering and the management of natural and technological disasters. He has participated in and/or coordinated over 70 scientific, operational and humanitarian missions for major disasters during the last 30 years in Greece and worldwide in order to assist local authorities and Civil Protection agencies and to offer scientific and technical assistance not only to the authorities of the affected countries but also to search and rescue teams and volunteers. ### Preface to "Mapping, Monitoring and Assessing Disasters" The detection, mapping and monitoring of disasters and assessing their impact play a key role in disaster management and disaster risk reduction. In the past, the acquisition of disaster data from the disaster-affected area, the rapid extraction of disaster information and the disaster situation reporting was a time consuming process, the results of which were available long after the completion of the immediate response phase. In recent decades, the rapid scientific and technological developments and especially their synergy have contributed to the progression and evolution of the field of disaster prevention and management with significant results in disaster risk reduction. The geospatial technological advances have contributed significantly to the early detection of upcoming disasters, the automatic delineation of affected areas, the accurate mapping of disaster impact and the real-time or near-real-time monitoring of their evolution. Furthermore, they have enabled scientists to acquire related data in real time, to quickly analyze available information and timely disseminate critical information to the scientific community, the disasters and crises management agencies and the general public, aiming at the more effective mitigation of adverse effects on all sectors of human activity and on the majority of elements of the natural and built environment. The purpose of this Special Issue "Mapping, Monitoring and Assessing Disasters" is to collect research advances in this scientific and operational field. It comprises 10 research articles addressing various issues of mapping and monitoring disasters and assessing their impact. The phenomena studied by the authors comprise the seismic activity in the Ionian Islands (Western Greece) from 2014 to 2018, which included four earthquakes with magnitudes ranging from Mw=5.9 to 6.8, the seismic sequence of Thiva (Central Greece) from 2020 to 2021, the destructive 2021 Arkalochori (Crete, southern Greece) Mw=6.0 earthquake, landslides triggered by earthquakes in the Ionian Islands from historical times to the present day, the landslides caused by the Medicane Ianos that occurred in mid-September 2020 in western and central Greece, the fires in the wildland–urban interface (WUI) zones in the Attica Region (central Greece) during the 2021 spring and summer season (May-August), the megafire in the Maule region (Chile) during the 2021 summer season (January–February), which was one of the largest megafires in the history of the south central zone of the country, and the historic 1917 Samoa tsunami in the Pacific Ocean, which constituted the second most deadly recorded tsunami event in this area after the fatal 2009 event. Joint pre-, co- and post-seismic ground deformation and seismological analysis was performed for the analysis of the seismicity in the Ionian Islands from 2014 to 2018. It was based on geodetic data from the commercial and institutional continuous Global Navigation Satellite System (GNSS) networks in the area, as well as seismological data from the Hellenic Unified Seismic Network (HUSN). Double-difference relocation was utilized to assemble a high-resolution earthquake catalogue and to examine, in detail, the distribution of hypocenters and the spatiotemporal evolution of the 2020–2021 Thiva (central Greece) sequence. The local deformation was delineated by applying instrumental and imaging geodesy, and the long-term trends or anomalies that could have contributed to stress loading were identified. Interdisciplinary research was conducted for the analysis of the 27 September 2021, Mw=6.0 Arkalochori (Crete, southern Greece) earthquake. It comprised synergistic geological mapping, tectonic analysis, fault photorealistic model creation by unmanned aerial system (UAS) data processing, as well as post-seismic surface deformation analysis by differential interferometry synthetic aperture radar (DInSAR) image interpretation coupled with accurately relocated epicenters recorded by locally established seismographs. UAS-aided photogrammetry and terrestrial laser scanning (TLS) was applied to high-visit coastal areas in the western part of the Lefkada Island (Ionian Sea, western Greece), often affected by earthquake-triggered landslides (ETL). This application aimed to explore how the capabilities of these cutting-edge methodologies contribute to the improvement of our understanding and monitoring of the structural integrity of slopes. This approach allowed the initial identification of high-risk zones and the subsequent prioritization of measures and strategies for risk-mitigation-driven development. Scientific publications and numerous contemporary sources were reevaluated for the compilation of the inventory of earthquake-triggered landslides (ETL) in the Cephalonia Island (Ionian Sea, western Greece). The related landslide susceptibility was examined by exploiting 10 landslide causal factors in the frame of a geographic information system (GIS)-based analytic hierarchy process (AHP). The comparison of the ETL inventory and the landslide susceptibility index (LSI) map highlight the high to critically high susceptible zones. UAS photogrammetric survey guidance was developed for accurate landslide mapping and monitoring in steep terrains based on identical tests within landslide areas with different characteristics, with high-resolution orthophotos and digital surface models (DSMs) emerging from the UAS imagery processing through structure-from-motion (SfM) photogrammetry. Landslides triggered by the September 2020 Ianos medicane were identified in Greece by using early remote sensing data and by conducting a series of post-event field surveys for verification. The rapid landslide recording was then compared with new methods of automated landslide mapping through the detection of changes in satellite imagery. All applied methods captured large events in mountainous areas and landslides with significant dimensions and/or long outflow distances. The 1917 Samoa tsunamigenic earthquake was modeled from its origin to produce outputs of tsunami inundation extent and depth at a spatially flexible grid resolution, which were validated using available run up observations and tide gauge records. Then, the first detailed 1917 tsunami inundation model was combined with the digital distributions of buildings to produce exposure metrics to evaluate the likely impacts on present-day coastal assets and populations if a similar tsunami were to occur. The effects of the 2021 wildfires in the Attica region (Greece) were examined based on Earth observation and GIS-based techniques for the development of a web app that included the derived knowledge. Sentinel-2 satellite imagery was used for extracting the burned area extent and severity using a normalized burn ratio (NBR)-based method. The erosion risk was modeled on a pre- and post-fire basis with the revised universal soil loss equation (RUSLE). Several variables comprising human activity, geographic, topographic, and land coverage were used to develop a model of ignition risk in Maule (Chile), a large zone with a Mediterranean climate, which was affected by a megafire in 2017. The derived information should be integrated into decision-making processes. The authors who contributed to this Special Issue come from universities, research centers, observatories and ministries in Greece, New Zealand and Chile. They are typical examples of countries that, from ancient times to the present day, have faced a multitude of disasters from geophysical hazards including earthquakes and their induced hazards (landslides, liquefaction, tsunami), to hydro-meteorological hazards (floods and fires among others), and have developed a culture of risk prevention. This Special Issue is specifically addressed to scientists working in relevant scientific fields and to the staff of operational actors involved in each of the stages of the disaster management cycle, from prevention to recovery, and generally to those interested in effective environmental, disaster and crises management strategies. Through its applications and examples, this Special Issue aims to provide the latest high-level knowledge on the above scientific fields and to update knowledge on relevant issues. Furthermore, it aims to inspire the further development of this scientific and operational field and the effective applications for disaster mapping and monitoring, and disaster impact assessment. The Guest Editors of this Special Issue congratulate all the authors for their valuable contributions and all the reviewers for their valuable time and constructive comments that helped to improve the overall merit of this Special Issue. The Editorial Office of "Applied Sciences" is acknowledged for the collaboration and the Section Managing Editor for the excellent cooperation and continuous support throughout the preparation of this Special Issue. ### Spyridon Mavroulis and Efthymios Lekkas Editors ### Editorial Special Issue on Mapping, Monitoring and Assessing Disasters *Spyridon Mavroulis \ and Efthymios Lekkas Section of Dynamic Tectonic Applied Geology, Faculty of Geology and Geoenvironment, School of Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15784 Athens, Greece \* Correspondence: [email protected] Mapping, monitoring, and assessing technologies and related studies and applications play a significant role in disaster management and disaster risk mitigation. In recent years, synergies of modern and innovative methodologies have augmented the efficiency of disaster mapping shortly after their advent and made it possible for involved scientists and researchers to acquire and analyze related data and to disseminate critical information to first responders during emergencies, authorities involved in disaster management and recovery processes, the affected population, and the general public. This present Special Issue comprises 10 research papers addressing various issues of mapping and monitoring disasters and assessing their impact. They highlight recent advances in the field and are valuable for understanding the complexity of the generated phenomena. The phenomena and their effects included in this Special Issue can be divided into: (i) earthquakes and related ground deformation [1–3], (ii) earthquake-triggered landslides (ETL) [4–6], (iii) landslides triggered by hydrometeorological hazards comprising medicanes [6,7], (iv) tsunamis [8], and (v) wildfires [9,10]. Sakkas et al. [1] present the results of monitoring seismicity and ground deformation in the Ionian Islands (western Greece) during a period of intense seismic activity (2014–2018) with destructive earthquakes. Joint pre-, co-, and post-seismic ground deformation and seismological analysis is performed based on geodetic data from the commercial and institutional continuous Global Navigation Satellite System (GNSS) networks in the area, as well as seismological data from the Hellenic Unified Seismic Network (HUSN), respectively. Kaviris et al. [2] monitored the 2020 to 2021 Thiva (Central Greece) earthquake sequence. They utilized double-difference relocation to assemble a high-resolution earthquake catalogue and examine, in detail, the distribution of hypocenters and the spatiotemporal evolution of the sequence. By applying instrumental and imaging geodesy, they delineated the local deformation and identified long-term trends that could have contributed to stress loading. Vassilakis et al. [3] monitored the 27 September 2021, Mw = 6.0 Arkalochori (Crete, southern Greece) earthquake. They conducted interdisciplinary research comprising geological mapping, tectonic analysis, fault photorealistic model creation by unmanned aerial system (UAS) data processing, as well as post-seismic surface deformation analysis by differential interferometry synthetic aperture radar (DInSAR) image interpretation coupled with accurately relocated epicenters recorded by locally established seismographs. Mavroulis et al. [4] applied UAS-aided photogrammetry and terrestrial laser scanning (TLS) to high-visit coastal areas in the western part of Lefkada Island (western Greece), often affected by ETL. This application aims to explore how the capabilities of these cutting-edge methodologies contribute to the improvement of our understanding on and monitoring of the structural integrity of slopes. This approach allows the initial identification of high-risk zones and the subsequent prioritization of measures and strategies for risk-mitigationdriven development. Mavroulis et al. [5] studied ETL from historical times to present in Cephalonia Island (western Greece). Based on scientific publications and numerous contemporary sources, they compiled an inventory of sites affected by ETL, several of which caused human losses Citation:** Mavroulis, S.; Lekkas, E. Special Issue on Mapping, Monitoring and Assessing Disasters. Appl. Sci. 2023, 13, 963. https:// doi.org/10.3390/app13020963 Received: 10 January 2023 Accepted: 10 January 2023 Published: 11 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). and injuries. The study further examines the ETL susceptibility, exploiting 10 landslide causal factors in the frame of a geographic information system (GIS)-based analytic hierarchy process (AHP). The comparison of the ETL inventory and the landslide susceptibility index (LSI) map highlights the high to critically high susceptible zones and reveals that the majority of ETL was generated within the highlighted susceptible zones. Nikolakopoulos et al. [6] focused on developing a UAS photogrammetric survey guidance for accurate landslide mapping and monitoring in steep terrains. They conducted four identical tests within landslide areas with different characteristics. High-resolution orthophotos and digital surface models (DSMs) emerge from the UAS imagery processing through structure-from-motion (SfM) photogrammetry. Accuracy assessment is carried out using quantitative and qualitative comparative approaches, and a strong relation is revealed between UAS acquisition geometry and landslide characteristics. Valkaniotis et al. [7] identified the landslides triggered by the 2020 Ianos medicane by using early remote sensing data and conducting a series of post-event field surveys for verification. The rapid landslide recording is then compared with new methods of automated landslide mapping through the detection of changes in satellite imagery. All applied methods captured large events in mountainous areas and landslides with significant dimensions and/or long outflow distance. In terms of comparing the compiled inventory with past events, they concluded that the Ianos landslides were triggered along roughly the same locations of historical occurrences, revealing a relationship with long-term climatic and lithological/geomorphological conditions. Sischka et al. [8] modeled the 1917 Samoa tsunamigenic earthquake from its origin to produce outputs of tsunami inundation extent and depth at spatially flexible grid resolution, which are validated using available run up observations and tide gauge records. Then, they combined the inundation model with digital distributions of buildings to produce exposure metrics for evaluating the likely impacts on present-day coastal assets and populations if a similar tsunami were to occur. They provided the first detailed 1917 tsunami inundation model, supporting an appreciation of the regional risk to local tsunamis. Falaras et al. [9] examined the effects of the 2021 wildfires in the Attica region (Greece) based on Earth observation and GIS-based techniques for the development of a web app that includes the derived knowledge. The effects of wildfires are estimated with the use of Sentinel-2 satellite imagery concerning burned area extent and burn severity using a normalized burn ratio (NBR)-based method. In addition, the erosion risk is modeled on a pre-fire and post-fire basis with the revised universal soil loss equation (RUSLE). This study highlights the importance of assessing the effects of wildfires with a holistic approach to produce useful knowledge tools in post-fire impact assessment and restoration. Azócar de la Cruz et al. [10] used several factors, such as human activity, geographic, topographic, and land cover variables to develop a model of ignition risk. The study area corresponds to Maule region (Chile), a large zone with a Mediterranean climate, affected by a megafire in 2017. Wildland fire management requires integrating this information into decision-making processes if we consider that the impact of climate change persists. Funding: This research received no external funding. Acknowledgments: We congratulate all authors for their valuable contributions to this Special Issue and to all reviewers for their valuable time and constructive comments that helped improve the overall merit of this Special Issue. We also thank the Editorial Team of "Applied Sciences" for the collaboration, and we express our appreciation to the Section Managing Editor for the excellent cooperation and continuous support throughout the preparation of the Special Issue. We hope that the papers will inspire further development and effective applications for disaster mapping and monitoring and impact assessment. Conflicts of Interest: The author declares no conflict of interest. #### References Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. ### Article The Use of Innovative Techniques for Management of High-Risk Coastal Areas, Mitigation of Earthquake-Triggered Landslide Risk and Responsible Coastal Development *Spyridon Mavroulis 1,\, Emmanuel Vassilakis 2, Michalis Diakakis 1, Aliki Konsolaki 2, George Kaviris 3, Evangelia Kotsi 1, Vasilis Kapetanidis 3, Vassilis Sakkas 3, John D. Alexopoulos 3, Efthymis Lekkas <sup>1</sup> and Nicholas Voulgaris <sup>3</sup> Abstract: Coastal areas constitute a very dynamic environment, balancing between numerous natural and anthropogenic processes liable to sometimes hazardous geomorphic phenomena. Especially in tectonically active coastal regions and areas of high economic value, slope failures can have significant impacts and therefore need careful and detailed examination. This work uses Unmanned Aerial System (UAS)-aided photogrammetry and Terrestrial Laser Scanning (TLS) in tectonically active segments of the coastal zone of the Ionian Islands in Greece, to explore how their capabilities can help to improve our understanding of the structural integrity of the slopes. Results show that the two approaches are able to extract large numbers of discontinuity facets, in a more practical, rapid and safe way than conventional methods of rock slope stability analysis extending to unreachable yet important parts of the slope. Through this holistic record of the structural condition of the slope the two applications allow the identification of segments that are more prone to instability and failure. In this way, they improve our understanding of the prioritization of interventions aiming to enhance the prevention of slope failures, mitigating the associated risk and improving local development in these high-value locations. Keywords: coastal areas; landslides; lidar; UAS; remote sensing; coastal development; TLS; Ionian #### 1. Introduction Globally coastal areas balance in a highly dynamic regime formed by a variety of complex natural processes [1] and human intervention [2]. The evolution of these constantly changing environments is affected by numerous natural geomorphic mechanisms such as erosion, mass wasting, deposition, wave action, as well as tectonic and volcanic activity [3]. Despite these processes and the risks associated with them, coastal areas contain a large part of the world's population [4] and an important portion of socio-economic activities [5], including trade, tourism, transportation and others. This activity heavily influences [6] some segments of the coastline. Coastal zones are home to many interests including economic (ports, fishing, navigation), recreation, water quality and nature conservation. Assets developed in coastal areas can be very valuable [7] and the disruption of some of the activities can be very costly [8]. There are examples where the interests and exploitation Citation:** Mavroulis, S.; Vassilakis, E.; Diakakis, M.; Konsolaki, A.; Kaviris, G.; Kotsi, E.; Kapetanidis, V.; Sakkas, V.; Alexopoulos, J.D.; Lekkas, E.; et al. The Use of Innovative Techniques for Management of High-Risk Coastal Areas, Mitigation of Earthquake-Triggered Landslide Risk and Responsible Coastal Development. Appl. Sci. 2022, 12, 2193. https://doi.org/10.3390/app12042193 Academic Editor: Hyung-Sup Jung Received: 18 January 2022 Accepted: 18 February 2022 Published: 20 February 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). schemes of these areas are conflicting with each other or lead to expansion into areas of high risk [9–11]. Thus, coastal zones are often characterized by both high exposure of valuable assets [8], as well as high hazard levels as a result of the various dynamic processes that take place. Particularly, in seismically active areas, high inclination, deformation of geological formations and strong earthquakes lead to instability phenomena [12–14] that are often associated with significant damage to property and infrastructure [15,16] and loss of human lives [17,18]. On many occasions, these failures can become a very significant threat in coastal areas. The complexity of the processes together with the high risk caused by such mass-movement events create a demand for in-depth knowledge and understanding of the mechanisms (both natural and anthropogenic) that drive them [19]. For example, it is crucial to understand the role of discontinuities in geological formations that affect their integrity and stability, acting as catalysts in various geomorphic processes, including landslides. Monitoring this and other structural properties of rock formations is important to predict slope behavior in coastal areas. Coastal slope failures are a significant source of hazard and a constraint for human activities in rocky coastal areas [20]. The most prevalent process responsible for the high risk of coastal cliffs is slope mass movements [21]. The extent and economic impact of this problem tend to increase over time, due to the general increasing trend in the use of coastal areas, especially steep and rocky coastlines, which correspond to one-third of the coasts worldwide [22,23]. The value of land in these locations is constantly increasing due to the high demand for exploitation, especially beaches of special beauty, which are often formed at the base of cliffs and are a tourism product of high value. There are also issues related to the presence of urban areas and archaeological and historical heritage sites close to the top of the cliffs [24,25]. In this context, coastal evolution and its processes have been examined through a variety of techniques, some of which belong to the field of remote sensing [26]. New techniques, such as photogrammetry and laser scanning, have shown interesting capabilities in studying changing terrain and particularly geomorphic processes, including slope stability and structural integrity [27,28]. For example, unmanned aerial systems (UASs) and UAS-aided photogrammetry have been used before in applications in coastal areas including surveying topographical changes [29], cliff erosion [30], coastline changes [31], and coastal floods [32–34]. In the field of mass movement, previous studies [35,36] have demonstrated that UAS, with the proper processing of data, can provide fairly accurate results [37,38]. Similarly, Terrestrial Laser Scanning (TLS) has demonstrated its capabilities in slope stability [39] including applications in coastal zones [40,41]. TLS has been used in numerous works to study landslides [42–44], cliff erosion [45–47] and rock formation discontinuities [48–50]. Despite the demonstrated capabilities of these technologies and their high-resolution imaging aptitude, there are very limited applications in highly visited areas or areas of high value that combine active tectonics and frequent instability phenomena. In addition, there are few applications that exploit outcomes of these techniques regarding slope stability to improve risk mitigation and enhance development. Given that, in many cases, decisions on the exploitation of coastal zones are made with a poor understanding of the evolutionary processes and functioning of the coastal environments, in the case of high-value and highly active areas, it is considered crucial to gain an improved understanding of dangerous instability phenomena. Any sustainable and responsible effort of development on these types of zones requires a deep understanding of the complexity of geomorphic processes and the roles of influencing and instabilitytriggering factors [51–53], including tectonic and earthquake dynamics, mass wasting, erosion, deposition, land-use changes, human intervention, and others. In this context, the aim of this work is to exploit innovative techniques such as UASaided photogrammetry and TLS to improve our understanding of the processes taking place, segments of the coastal zone of the Ionian Islands in Greece and, in particular, the island of Lefkada. The two study sites are situated in an earthquake-prone area, with frequent strong earthquakes leading to various mass-movement and slope failure phenomena, enhanced by active tectonics, but also subject to high-intensity storms (including Medicanes) that have also contributed to the high landslide risk. The present study aims to apply the two techniques at the Egremnoi and Porto Katsiki beaches, two highly visited tourist attractions both characterized by noteworthy economic and ecological value. The study aims to determine the zones of higher risk through the two applications and discuss the outcomes in relation to local development, considering that Lefkada is a typical example of a Mediterranean busy coastal zone, home to hundreds of thousands of visitors each year. The study is organized as follows. First, we present the broader area and the study site, in terms of its geodynamic characteristics, then we describe the methodology for the UAS and the TLS applications, followed by the results. Then we discuss the practical implications of the findings and describe the most important conclusions in the last section. #### 2. Study Area #### 2.1. Geological and Seismotectonic Setting Lefkada Island is located in the southern Ionian Islands (Western Greece), in the front of the Hellenides orogenic belt, in a region dominated by the presence of the Cephalonia Transform Fault Zone (CTFZ) [54,55]; (Figure 1). The latter is a 140-km long right-lateral fault zone, as evidenced by a large number of focal mechanisms [56–59]. The study area lies in the transition zone between the subduction of Tethys beneath the Aegean microplate to the south and the continental collision to the north (Figure 1). The CTFZ consists of two major segments, i.e., the Cephalonia Segment (CS) to the south and the Lefkada Segment (LS) to the north. The proximity of Lefkada to the CTFZ is the main reason the island is among the most seismogenic areas in Europe. Regarding the historical era, large and destructive earthquakes have occurred during the last six centuries [60,61] (Figure 2a). Most of these events mainly affected the western coast of the island, such as the 1704 (M = 6.3), 1769 (M = 6.7), 1783 (M = 6.7) and 1869 (M = 6.4) earthquakes [62–64], (Figure 2a). Thirteen disastrous events that took place at the western onshore area of Lefkada occurred between 1612 and 1869 [64,65]. On the contrary, the 1723 (M = 6.7) earthquake was the sole historical event that occurred at the southwestern part of Lefkada Island [58]. Intense seismic activity has been recorded in the Lefkada region during the 20th century, characterized by the frequent occurrence of destructive events [66]. More specifically, 87 events with Mw ≥ 4.1 occurred between 1911 and 1998, 36 of which with Mw ≥ 5.0 and 5 with Mw ≥ 6.0 (Figure 2a). Both of the largest events of the 20th century, with Mw = 6.5, occurred in 1948. The first event of 22 April destroyed the southwestern part of the island, whereas the second on 30 June caused damage to the northwestern part [66]. Two large events occurred in the vicinity of Lefkada Island during the 21st century. The first was the Mw = 6.3 on 14 August 2003, located northwest of the island at a depth of 9 km. Its focal mechanism indicated dextral strike-slip faulting (e.g., [58,67,68]). The most recent destructive event occurred on 17 November 2015 (Mw = 6.4) close to Athani, at the southwestern coast of Lefkada, on an almost vertical dextral strike-slip fault [55,69]. Most aftershocks were aligned parallel to the western coast of Lefkada Island, along an NNE-SSW direction, with another group of epicenters located close to the northern part of Cephalonia Island [63]. The seismogenic layer ranged from 3 to 16 km in depth, whereas more than one fault plane with different strike and dip values were activated [64]. Despite its larger magnitude, this event caused less damage than the 2003 earthquake, proving the structural efficiency of the local buildings [69]. Figure 1. Map of the Hellenic Arc showing the location of Lefkada Island at the northwesternmost part of the Hellenic Arc along with the prominent morphological features of the Hellenic Arc and the major morphoneotectonic features based on Mariolakos and Papanikolaou [70,71] and the seismic risk zones of Greece. Lefkada belongs to zone III of the current Greek Building Code with a Peak Ground Acceleration (PGA) value of 0.36 g for a return period of 475 years. The geological structure of the southern Ionian Islands is characterized by the occurrence of alpine formations of the Ionian and Paxoi geotectonic units and post-alpine deposits lying unconformably on the alpine basement [72–77]. Similarly, Lefkada is composed of (a) alpine formations that belong to Ionian and Paxoi geotectonic units, (b) molassic formations and (c) recent deposits that lie unconformably on the previous formations [74,78] (Figure 2a). Ionian formations are observed in the largest part of the island, while Paxoi formations are only observed in the southwestern part of the island and more specifically in the Lefkata peninsula (Figure 2a). As regards the tectonic structure, the faults dissecting the island are mainly normal or strike-slip with a sinistral or dextral sense of shear [77,79,80]. The active structures are major faults detected mainly along the margins of fault blocks [72,77]. In particular, Lefkada is composed of the following 8 fault blocks [78] (Figure 2a): Some of the aforementioned main fault blocks are composed of smaller fault blocks. The most characteristic case is the Dragano-Athani graben within the fault block of the Lefkata peninsula. This graben is bounded to the west and east by NNE-SSW striking faults. Its western margin is defined by the AFZ with a total horizontal displacement of approximately 860 m corresponding to a Quaternary slip rate of about 8 mm/year [81] and similar geometric and kinematic characteristics with the Lefkada segment of the Cephalonia Transform Fault Zone [79]. Figure 2. (a) The neotectonic map of Lefkada Island from Lekkas et al. [77]. The Egremnoi and Porto Katsiki coastal areas are located in the southwestern part of the fault block of Lefkata peninsula and are composed of Jurassic–Miocene limestones of the Paxoi geotectonic unit. (b) Based on the landslide susceptibility map of the Ionian Islands compiled by Mavroulis et al. [82], these slopes are formed in coastal areas highly susceptible to failure. #### 2.2. Study Sites In the frame of susceptibility and hazard assessment in the Ionian Islands for highlighting sites of earthquake-related hazards, Mavroulis et al. [82] studied the landslide susceptibility of the Ionian Islands by applying the Analytical Hierarchical Process (AHP) used along with the Weighted Linear Combination (WLC) method in the context of multicriteria decision analysis for the calculation of the Landslide Susceptibility Index (LSI). Based on the applied methodology and the respective results, it is concluded that the abrupt coastal slopes and scarps in the western part of Lefkada are characterized by high and very high susceptibility to earthquake-triggered landslides (Figure 2b). Indeed, the western coastal part of the island has been heavily affected by landslides triggered by historical and recent earthquakes [64,79,83]. In particular, landslides comprising mainly rockfalls have been triggered by earthquakes on: Among the areas affected by earthquake-triggered landslides are the coastal areas of Egremnoi and Porto Katsiki (Figure 3), located in southwestern coastal Lefkada [64,84,85]. They constitute typical cases of extensive coastal slopes, where the change in morphological slope is so abrupt that almost vertical planes are formed. In addition to faults and joints, the continuous marine and terrestrial erosion processes play an important role in the evolution of these coastal slopes. The geological structure of Egremnoi and Porto Katsiki is similar, as both are composed of Jurassic-Miocene limestone of the Paxoi unit. They are intensively faulted resulting in their disintegration and brecciation, while locally completely pulverized and extremely unstable. They are extremely susceptible to landslides, a fact that has been revealed not only by the landslide susceptibility assessment of Mavroulis et al. [82], but also by the triggering of landslides not only by nearby earthquakes but also by distant earthquakes (Figure 3). Typical examples of landslides induced by nearby earthquakes are the rockfalls and slides caused by the earthquake on 17 November 2015 (Figure 3). In the Egremnoi area, a large mass comprising loose limestone blocks, semi-cohesive scree with limestone breccia and red clay was mobilized during the 2015 earthquake and rolled down towards the adjacent beach, which was almost entirely covered by landslide material [78] (Figure 3a–c). Furthermore, the part of the road network leading to the beach was completely destroyed, and some buildings found on the edge of the slope were at risk of total collapse [86,87]. As regards the presence of residents and tourists in this area, it was fortunate that the earthquake was generated during the winter season and the beach was empty. In Porto Katsiki, loose brecciated limestone blocks were detached from the abrupt limestone slope and fell on the narrow beach [78] (Figure 3f). It is significant to note that both beaches are amongst the most beautiful and visited in the Mediterranean with thousands of locals and tourists attracted during the summer season staying close to the steep limestone slopes or inside coastal caves. Fortunately, the 2015 earthquake was generated during the winter season with very limited tourist flow in Lefkada and both beaches were totally empty. A typical example of landslides induced in these areas by distant earthquakes comprises the rockfalls triggered in the large coastal slope of Porto Katsiki by the 8 June 2008, Mw = 6.8, Andravida (NW Peloponnese, Western Greece) strike-slip earthquake [88] (Figure 3d). The epicenter was reported at a distance of 110 km southeast of the affected slope. Due to this large distance from the epicenter and due to the absence of similar phenomena in the intermediate areas with similar geological and morphological properties, these rockfalls were considered to be far-field earthquake environmental effects, attributed to the combined effect of the rugged morphology, the intensively faulted and eroded limestones and the pre-existing instability conditions along the abrupt coastal slopes in the western part of the Lefkata peninsula [88]. Figure 3. Views of the Egremnoi (a–c) and Porto Katsiki (d–f) slopes in the southwestern part of Lefkada Island. (a) The Egremnoi slope and the adjacent impressive beach were heavily affected by the 2015 Mw = 6.4 Lefkada earthquake. Extended landslides were induced (b) and covered almost the entire longitudinal narrow beach (c) leaving only small parts intact and inaccessible after the earthquake. Due to the fact that the shock occurred in November, the beach was empty, and no effects were reported to people. However, the road leading from the upper parts of the slope to the beach was totally destroyed and houses founded on the slope were found on the edge of the cliff and on the verge of collapse. (d) The Porto Katsiki slope has been affected not only by nearby earthquakes but also by distant earthquakes. The 8 June 2008, Mw = 6.8 earthquake with the epicenter located 110 km southeast of Porto Katsiki triggered rockfalls in the southern segment of the slope, while the mobilized material ended up on the adjacent part of the beach fortunately without fatalities and injuries. (e) Boulders and smaller fragments are concentrated mainly in the southern part of the beach. (f) Rockfalls triggered by the 17 November 2015, Mw = 6.4 Lefkada earthquake. They were mainly generated in the southern segment of the slope, while they were limited or absent in the central and the northern segment respectively. Since 2015, protection marking on the beach with poles and ropes (red dashed line) and rockfall warning signs have been placed for the safety of visitors. #### 3. Methodology In this context, the coastal morphology evolution, including short-term onshore changes and processes, has been examined through several remote sensing techniques based on data acquisition with the use of state-of-the-art equipment. The swift development in sensor technology and geomatics during the last decade has improved the acquisition and processing of remotely sensed information, especially regarding the monitoring of infrastructure as well as medium-sized territorial zones due to geological risks [89]. Rather prominent techniques include the generation of point clouds of the areas of interest, which can be composed either directly by using surveying equipment such as Terrestrial Laser Scanners (TLSs) or indirectly by processing numerous photographs taken by Unmanned Aerial Systems (UASs). Both Light Detection and Range (LiDAR) and Structure from Motion (SfM) processing techniques can provide large amounts of digital data of very high resolution from which point clouds of similar quality can be generated. High-resolution 3D point clouds and 3D meshes are the fundamental types of data for any quantitative structural analysis afterwards. Laser scanning is an optimized technique for obtaining 3D surveying data. Usually, TLS has a spatial resolution ranging from millimeters to centimeters. Regarding this, the widespread use of UASs, together with SfM and Multi-View Stereo (MVS) technologies, has improved the flexibility of topographic surveys and structural analysis. In fact, nowadays, due to swift technological development, it can be used rather easily under adverse conditions, as nadiral, oblique, or frontal images can be acquired with minimal effort. Therefore, cliffs, steep slopes, hanging rocks, and morphological discontinuities in general, are either being scanned or photographed for the generation of a basement dataset on which further processing will be applied. The mapping of structural failures and rock discontinuity density at coastal cliffs proves to be very critical concerning the safety regime in the beaches beneath them, especially the popular ones [90]. During the last few years, there has been an ongoing debate as to whether the direct TLS point clouds are more accurate and precise than the indirect data constructed after photogrammetric processing [91–93]. In this context, more attention has been given to the latter, which can also generate high-resolution 3D models of slopes, focusing on the advantage of low costs, flexible oblique view sensing and photo-realistic vision information when compared with terrestrial LiDAR point clouds [94], which are produced by rather expensive equipment. On the other hand, the accuracy of these is much more adequate, not to mention the data density, which is also difficult to be obtained with UAS flights at a reasonable elevation (e.g., 75 m). Furthermore, the use of UASs is a practical way of mapping areas larger than TLS can provide, and it has been proved to be optimal for landslides and rockfalls that often cover areas that range from less than one square kilometer up to a few square kilometers. Additionally, the limitations of beach width where in situ observations or the use of other techniques such as TLS are unattainable can be overcome via UAS and restrictions associated with LiDAR instrumentation (e.g., high weight and occlusion areas), since the final product is very similar, as mentioned above. In this context, we chose to apply the two different approaches at the two case studies of Egremnoi and Porto Katsiki, taking into consideration the most appropriate technique for each case. A point cloud derived from SfM processing was utilized for creating the basement datasets (Digital Surface Model and ortho-photograph) for the Egremnoi coastal zone, whilst LiDAR scanning was used for direct point cloud acquisition at the popular Porto Katsiki beach. The flowchart below illustrates the main steps of the methodology along with the software solutions used for each one of them (Figure 4). Figure 4. Basic steps of the applied methodology. #### 3.1. UAS-Aided Survey at Egremnoi The raw image data acquisition was conducted with a rotor-wing UAS (DJI Phantom 4 RTK) equipped with a stabilized, built-in 20M camera (of 8.8 mm focal length) bundled on a two-axis gimbal. This unmanned platform has been chosen due to its relatively reasonable cost and easy on-site operation in combination with the equipped miniaturized Geodetic Navigation Satellite System (GNSS) antenna, which provides sufficient precision, particularly for the horizontal positioning required to facilitate proper alignment of the images captured during the survey [95]. Although ground control points (GCPs) are a vital tool for ensuring geolocation accuracy, in this case study, in which the morphology of the area consists of very steep slopes with limited accessibility, the use of the RTK antenna with which the drone is equipped is efficient enough for highly accurate results. Nevertheless, we used the GNSS receiver in the Network Real-Time Kinematic (NRTK) mode, connected to the SmartNET provider accuracy service [96] for obtaining subjective geolocation, which is also necessary for the comparison between independent surveys [49]. The UAV flight survey was designed to cover an area of 0.3 km2, of a 160 m steep slope consisting of fragmented limestones that fall on the coastal zone, especially after the occurrence of earthquakes [78]. The take-off point was placed at an elevation of 190 m, and the UAV was programmed to fly along four transects almost parallel to the coastline, at the absolute height of 230 m. The camera was set pointing 30 degrees off nadir, with an image overlap value of 75%. This resulted in the acquisition of 592 images that were processed within Agisoft Metashape photogrammetry software, to produce (a) DSM (0.12 m resolution), (b) a vertical ortho-mosaic image of the coastal zone (0.06 m resolution) and (c) an ortho-mosaic image normal to the steep morphological discontinuity (0.06 m resolution) (Figure 5), based on a dense point cloud that consisted of 31 million points. Regarding accuracy, the processing resulted in a root mean square error (RMSE) of 0.08 m on the xy-axes and 0.18 m on the z-axis, which are rather acceptable values. Figure 5. The SfM technique led to the construction of several high-resolution products such as (a) DSM, (b) ortho-image and (c) vertical photo-mosaic of the Egremnoi steep slope. #### 3.2. TLS-Aided Survey at Porto Katsiki A series of reasons led us to use a TLS instead of a UAV for the point cloud generation at the Porto Katsiki white limestone steep slope, which bounds the beach zone and increases the rockfall risk, especially during the tourist season. The crowd presence during the data acquisition and the unstable NRTK signal due to the bad reception of the cellular network prevented the surveying team from using a UAV at this site. Thankfully, the morphology of the surrounding area provided us with a rather ideal place for establishing the LiDAR equipment and using it as a base for the tripod on which the TLS was placed (Figure 6). Figure 6. (a) Spatial location of the TLS point clouds acquired from two base stations (in red and blue colors). The black dots delineate the common scanned area of the steep slope with the minimal shadowing effect. (b) Panoramic view of Porto Katsiki beach during the fieldwork. Note the scanning equipment established at the bottom right (TLS base 2). The state-of-the-art, long-range Terrestrial Laser Scanner, Leica P50, was used, with the ability to acquire high-precision point data at distances up to 1 km. According to its specifications, the point accuracy may reach the order of 3 mm over the range that it was used at Porto Katsiki, since the TLS base was established at a distance between 120 and 430 m opposite the steep slope. This resulted in a rather dense point cloud as the laser beam adds a point almost every 0.9 cm at a distance of 120 m or every 3.6 cm at the furthest slope areas, producing a rather reliable and valuable dataset for further processing. For the latter, in addition to the xyz coordinates, RGB values (using the internal 4MP camera) are included along with intensity laser signal values as well. We acquired two point clouds from two different TLS bases, with known coordinates, 25 m away from each other (Figure 6) in order to change the acquisition angle towards the slope as much as possible and therefore reduce the shadowing during the scanning. Both point clouds were processed together and merged, after co-registration and geolocation. The final dataset consisted of 90 million points, and after the cleaning procedure, a point cloud of 65.5 million was generated for further interpretation (Figure 7). The latter includes several types of point classification and meshing, leading to detailed morphotectonic analysis of the rock slope and calculation of structural discontinuities' orientation (Figure 7). Figure 7. (a) The point cloud of the cliff uphill from the Egremnoi beach, cleaned from the noise caused by the vegetation, which might alter its steep topography. Classification of the extracted facets according to their calculated dip in degrees (b) and each one's dip direction that is color-coded regarding the statistical analysis (c). The cliff was segmented into three segments (North, Middle and South) and below each one, the wedge-sliding kinematic analysis is presented. #### 4. Results The point clouds that were generated at both areas were used for further analysis, which was rather common for both datasets, with no restrictions concerning their origin (UAS and TLS, respectively). The structural analysis was carried out using the facets/fracture detection and more specifically following the Kd-tree method [97] of the freeware CloudCompare [98]. The Kd-tree method is a kind of high-dimension binary tree, and by using it, one can search the nearest neighbor points in a relatively quick way [99] based on splitting the space of the mesh into two parts. The top node splits the space in one dimension, and the next nodes spilt the space in another dimension. The splitting causes approximately half of the points to be stored in the left subtree, and the other half is stored in the right subtree. It stops splitting when the points in one node achieve the given maximum count [99]. In this case study, the Kd-tree method has been used for the extraction of the orientation at each discontinuity located on the slopes under investigation [100,101]. Since the original data are points, the Kd-tree was used to organize them in k-dimensional space, searching for the neighboring points. Afterward, this point organization is used for dividing the cloud into small planar patches, which are then grouped into facets. The Egremnoi Beach dataset consisted of the final point-cloud, which was generated after the application of the SfM method, followed by the construction of a 3D model using Agisoft Metashape software in mesh format (i.e., Triangular irregular network—TIN) with almost 25 million facets, after excluding large parts of the slope that were covered by vegetation, which altered the slope morphology (Figure 7a). The vegetation removal succeeded after classifying the points of the cloud, due to relatively high values of green in the Red–Green– Blue color model. The resulting dataset was used at the next stage for the structural analysis of the steep slopes along the coastline, considering that a large accumulation of debris occurred at certain locations next to the slope's prone base. The dip of every facet was calculated by using CloudCompare [98] algorithms (Figure 7b), and the patterns that were identified at the dip profile map proved to be more than helpful for the prone segmentation, as it was quite large for further processing. Therefore, it was divided into three segments and the discontinuity planes were statistically interpreted individually (Figure 7c). The average geometric characteristics of the topography of the northern segment are 55/275, and the orientation of 4141 discontinuity planes that were extracted from the pointcloud processing was concentrated in two main groups at 24/289 and 43/237. The kinematic analysis of the discontinuity poles using Dips software v.7.0 [102], in the context of the wedgesliding risk, indicated that 29% of the facet pairings are under serious rockfall potential, considering that the friction angle is set at 30◦ [87,103]. The 3259 facets identified at the southern segment are concentrated more or less in the same orientations (42/302 and 43/246) and the morphology is approximately identical (55/273). The kinematic analysis for wedge sliding showed that 25% of the discontinuity pairings could lead to failure, especially after an earthquake occurrence. The respective percentage of potential rockfall at the median segment of the Egremnoi cliff is lower (23%), even though the statistical significance of one of the main facet groups was dramatically decreased and substituted from a new, almost vertical one, with E-W trending (87/358). The latter is the result of the kinematic analysis of 4058 discontinuity planes and the second pole concentration is at 39/301, which is very similar to those concentrations identified at the other two segments' interpretations. A similar processing methodology was followed with the Porto Katsiki cliff TLS data, even though it was based on more objective management of the mesh product, due to higher point density and a lack of any kind of vegetation altering the slope morphology, which led to impressive and much more clear results (Figure 8a). A large number of facets (23,380 records) were identified, each one characterized by dip and dip-direction measurements, among other location and precision information, which was extracted following the above-mentioned methodology. Several groups of oriented facets were recognized and classified according to their dip (shown with color coding in Figure 8b). The facets were grouped based on their dip direction in six categories shown with different colors in Figure 8c. Each classification contained classes that were assigned different colors, providing images where sectors with similar rock mass characteristics regarding the discontinuity orientation were defined (Figure 8d). At the next stage, the prone area was segmented into each one of those sectors, which seem to behave homogenously in the context of safety due to rockfalls. The average slope orientation characteristics were calculated using tools provided by CloudCompare freeware [98], and the facet list was filtered by excluding the morphology facets and keeping the facets that coincide with rock fracture discontinuities (6175 records). The latter was used at the next step of the kinematic analysis related to the wedge-sliding risk along the prone. Figure 8. The geometrically corrected point cloud of the Porto Katsiki cliff, above the beach, in real color (a). The generated mesh file was classified according to each facet's dip in degrees (b) and each one's dip direction (c). The latter led to (d) a cliff segmentation into three generic morphological planes; Blue (81/244), Green (68/287) and Red (63/301). Specifically, at the northern morphological segment (Blue plane at Figure 8d) where the plane of the average slope is calculated at 81/244, no more than 21.5% of the discontinuity plane combinations would lead to wedge sliding (Figure 9), which is the most common in this prone area, considering that the limestone friction angle is set at 30◦. The percentage rises to 27% for the median segment (Green plane at Figure 8d), where the average slope is oriented at 68/287 and almost reaches 32% for the southern segment (Red plane at Figure 8d). Therefore, it is apparent that the factor of safety in the context of visiting this beach is rather critical, but it is significantly increased while moving to the southernmost segments of the beachfront. Additionally, a back analysis indicates that the rock mass sensitivity to failure increases at the intersection of the three segments since several fallen blocks are found adjacent to these locations. Taking into account the results from the UAV and TLS surveys in Egremnoi and Porto Katsiki coastal areas, respectively, as well as the data obtained from the statistical analysis of the slope discontinuities, we can draw important conclusions about the condition of the slopes and their response to a possible strong earthquake in the future. As emerged from the statistical analysis of the discontinuities and the kinematic analysis of the wedge-sliding risk for the Egremnoi slope, the percentages of facet pairings, which are under serious rockfall potential in the case of an earthquake occurrence, vary from 23 to 29%. Based on this small difference from section to section of the slope, as well as its overall response during the Lefkada earthquake in November 2015, as shown in Figure 3b,c, it is concluded that the slope is characterized by a uniformity in the discontinuities' orientation and its response to strong shocks. Figure 9. Wedge-sliding risk analysis for the three individual segments of Porto Katsiki beach (B for blue morphological plane, G for green morphological plane, R for red morphological plane of Figure 8d). However, nothing similar emerges for the coastal slope in Porto Katsiki. Although particularly susceptible to earthquake-triggered rockfalls, effects that have already occurred during both nearby and distant earthquakes, the Porto Katsiki slope has not been shown in the past and is not expected to show a uniform response to a future earthquake, regardless of the distance from the epicenter. From the statistical analysis of the discontinuities and the kinematic analysis of the wedge-sliding risk for the slope in Porto Katsiki, it is found that the parts more susceptible to falls are located along the intersection of the three generic morphological planes (blue plane—northern segment, green plane—middle segment and red plane—southern segment in Figure 10). This fact, in combination with the generation of failures and the increased presence of boulders and smaller fragments from previous earthquakes (2003 and 2015 Lefkada and 2008 Andravida events) in the southern part of the slope and the beach, respectively, indicates that this part presents the highest susceptibility to slope failures. On the contrary, this does not apply to the middle and northern parts of the slope. In the middle part, large rock fragments resulting from failures are very limited, while in the northern part, they are entirely absent. This indicates the best response of these segments during the applied earthquake loads. Therefore, it is concluded that the rockfall risk during earthquakes in the southern part of Porto Katsiki is high. The effects on people staying in the adjacent narrow beach are inevitable when rockfalls occur unless special care and preventive measures are taken for their mitigation by the bodies and municipal authorities involved in the prevention and management of disasters caused by earthquakes and earthquake-related hazards. Even before the 2015 Lefkada earthquake, the beach had been divided into two zones of safe and unsafe, the distinction of which was made by placing protection poles and ropes, while there are also visible rockfall warning signs for visitors. However, the monitoring and updating of measures must be continuous, always taking into account all the factors that can affect the slope and increase the potential for slope failures and impact on the adjacent beach. Figure 10. The three morphological planes detected from the TLS survey in the Porto Katsiki coastal area. The blue plane corresponds to the northern segment, the green to the middle and the red to the southern. The discontinuities detected along the planes' intersection create conditions favorable to failure. Taking into account the presence of boulders and other smaller fragments along the beach, it is concluded that the southern segment of the slope (red plane) is characterized by the highest susceptibility to large rockfalls with impact on the adjacent beach. Please note the colors used correspond to the three generic morphological planes detected through the TLS application and are shown in Figure 8d. #### 5. Discussion This work used UAS-aided photogrammetry and TLS in tectonically active segments of the coastal zone as a tool to improve our understanding of the mass-movement processes and the spatial dimension of landslide risk through a highly localized lens, as an approach that improves our capabilities in coastal management in slope-failure-prone areas. The outcome of the two applications at Egremnoi and Porto Katsiki beaches is a strong indication these cutting-edge technologies have the capabilities to produce valuable results in the field of slope failures, allowing the identification of higher-risk zones and relative prioritization of any interventions. Given the significance of slope failures as a hazard in coastal cliffs and the extent of its impact acknowledged in the introduction of the present study, rock slope stability analysis is considered very important for reducing the effects of instabilities and increasing the safety of adjacent or neighboring areas, while preserving the value of the sites in question. However, the classical survey methods comprising conventional data collection, which have been proposed and applied in various environments and settings (e.g., [104,105], present significant problems. These problems have to do with the time and cost needed for implementing fieldwork, the safety of researchers during data acquisition and the coverage of the data collection [43,106–108]. The visual inspections close to steep slopes, scarps and cliffs and direct measurement conducted by the researchers, who act as rock climbers, affect their safety during the field survey by exposing them to potential failure impacts. Furthermore, some discontinuities cannot be directly observed during visual inspections and are partially recorded and mapped, unless suitable aerial or satellite imagery is used. Consequently, a limited number of manual compass data measurements of dip and dip direction can be collected during the implementation of the classical survey methods in steep slopes, affecting the results as discontinuities measured in small areas are not representative of the whole rock masses. If an effort is made to fully cover the slopes, then field data acquisition may be a timeconsuming and expensive procedure. In the case of rock slope stability analysis on the coastal slopes of Egremnoi and Porto Katsiki, the classical methods, which include surveys with a geological compass measuring dip and dip direction directly on the discontinuity, face many problems, which are strongly related and attributed to the geological and geomorphological structure. These issues are strongly related to the current geological and geomorphological setting of western Lefkada. Its tectonic structure is characterized by active and seismic faults, which, in combination with other morphological discontinuities, form a geotechnically unstable area with high and steep slopes composed of highly fractured, brecciated, unsupported and almost powdered geological formations, mainly limestones of the Paxoi geotectonic unit with secondary welding and extensive semi-cohesive scree with limestone breccia and red-clay-filled fractures as well as similar geometries of beds and discontinuities. These intense tectonic processes have a great impact on the geomorphology of the study area. They have increased the height and inclination of slopes resulting in almost vertical slopes with maximum elevation larger than 100m in several sites. In addition, the combined action of (i) endogenous processes, including deformation, which has caused lithological heterogeneity and mechanical anisotropy of the alpine formations and post-alpine deposits, and (ii) surficial processes including repeated cycles of mechanical, chemical and organic weathering and marine and aeolian erosion, have contributed to decreased cohesion and formation loosening along the steep coastal slopes. All the aforementioned factors make it excessively difficult or even impossible to fully apply conventional methods for rock slope stability analysis in sites such as Egremnoi and Porto Katsiki. They would be time-consuming and expensive due to the equipment that must be used for the researchers' safety during field survey, while the results will only refer to accessible parts of the slopes as most of them will be excluded due to inaccessibility. On the contrary, the application of UAS-aided photogrammetry and TLS surveying offer examination of the Egremnoi and Porto Katsiki slopes, through a highly localized lens across all parts of the surfaces under study. Furthermore, the large number of planes identified in the studied slopes (14,849 discontinuity planes from UAS in Egremnoi and 23,380 facets from TLS in Porto Katsiki) is at first essential for the thorough study of the deformation and then for the determination of the potential for failures along the slopes, either by one structure or by the synergy of two or more structures, which in turn reveals individual parts of the slopes susceptible to failures in the case of important external loads applied during an earthquake. The large number of data recorded during the application of these methods as well as the speed with which these data are processed is a significant advantage over conventional methods [109]. In addition, it should be noted that the UAS- and TLS-derived datasets are available for capturing on-demand data in the sense that the surveyor chooses the exact desired time of recording the study area and all of its features. This is an advantage compared to other image-exploitation approaches (e.g., satellite imagery analysis), especially when it comes to disaster management, in which capturing the natural processes is time-sensitive. In addition, UAS and TLS data capturing do not have the limitations of cloud cover. Furthermore, the capabilities of the UAS and TLS surveys fit the opportunistic nature of surveying a geomorphic phenomenon in the sense that the required equipment can be rapidly deployed in the field to collect information (e.g., after morphological changes), within the short time window that it is available (i.e., slopes affected by cleanup works). With respect to the field data gathering process, the suggested approaches have important benefits. Firstly, the segment of the coast under examination can be revisited virtually (back at the lab) at a later time, as the geometrical data captured are stored and can be reused, which is also essential for multi-temporal processing. This is particularly useful in the case of remote sites or unsafe areas. This availability of terrain data for a part of the coastal cliffs/zone allows the processing of multiple parts, permitting a more holistic study of the conditions. In this way, the captured scarp information in the form of a DSM offers increased flexibility and provides the opportunity to perform different trials on the study site, depending on the prevalent risk (e.g., a housing complex on top of the hill or an area filled with visitors). The very high resolution provides a description of the slope in very high detail, with nodes every few centimeters. This is a strong advantage of this approach, as it increases practicality, reduces time and personnel needs and minimizes safety concerns, especially considering steep slopes and inaccessible areas that would otherwise be surveyed using handheld instruments, putting surveyors at risk. In highly touristic areas of special natural beauty, such as the coastal areas in the western part of Lefkada, the interpretation of data originating from either UAS-aided photogrammetry or LiDAR scanning is not only an important scientific innovation for assessing the landslide susceptibility of slopes, but also a significant tool in the direction of responsible and effective management of coastal areas through the cooperation of authorities involved in the prevention and management of disasters from natural hazards. This effectiveness is achieved through the adoption of invasive measures to reduce and eliminate the adverse effects of the occurrence of such phenomena on infrastructure and visitors. These measures may require prohibiting access to the adjacent beaches until their completion, thus imposing enormous constraints on the socio-economic development of the highly touristic areas. However, landslide risk mitigation measures could be of low cost and without major interventions in the landscape. They do not presuppose a total ban on access to susceptible areas and therefore they do not cause any disruption to tourism and socio-economic activities of the areas where they are applied, in addition to the enhanced safety that they provide. The most suitable approach for the beaches adjacent to the Egremnoi and Porto Katsiki slopes is their division into access zones. This distinction mainly takes into account the results of the UAS and TLS survey and especially the landslide-susceptible parts of the slopes, which are adjacent to the beaches. Additional elements that are evaluated are the presence of unstable fragments on slopes with a high potential to detach and fall on or slide to the beaches in the case of a strong earthquake, as well as the location of the fragments, which have ended up on the beach from previous earthquakes or other episodes that also triggered landslides, such as heavy and rapid rainfall. After evaluating these data, the beaches could be divided into three zones: (1) The free-access zone of low landslide risk, (2) the restricted-access zone of moderate risk and (3) the prohibited-access zone of high risk. The boundaries of the zones can be modified at any time depending on whether the conditions that favor the occurrence of landslides are aggravated or not. Thus, prohibitedand restricted-access zones can be merged into a single access zone of high risk after the occurrence of a major earthquake, during a prolonged aftershock sequence, as well as during or after a prolonged period of heavy rainfall. This high-spatial-resolution mapping of slope deformations can be used as a landslide precursor, assisting prevention measures. This zonation enables stakeholders to reduce the adverse effects of potential landslides without, however, barring access to beaches for long periods of time, which are required by the implementation of large-scale projects and interventions. Additionally, in the context of the multi-temporal approach, repeated data acquisitions could be scheduled at regular time periods or after triggering episodes, which might potentially disrupt the slope stability. A comparative process could quite easily be merged into an automated procedure, and an alert might be generated for the re-establishment of the safety zones. Even though the point-cloud differences are not crucial, regardless of their origin, a matter of point density arises. TLS scanning provides denser datasets, with higher accuracies (depending on the specifications of the LiDAR) provided that there are suitable steady spots for establishing the equipment, at small distances, opposite the monitored cliff behind the beachfront. The latter adds credit to continuous multi-temporal observation and the quantification of any displacement, which might spur strong objections when it comes to the exact positioning of UAS imagery and therefore the overall model accuracy. Moreover, restrictions for UAS flying admittance due to safety may apply in no-fly zones or in areas with a human presence. A strong argument for preferring UAS photogrammetry products instead of directly acquired data from TLS scanning would be the presence of vegetation cover on the cliff slopes. Even though its existence is an objective parameter that might affect both point-cloud datasets by—sometimes—hiding important fractures, its removal during the photogrammetric processing seems to be rather more harmless. That is due to multiple angles of imagery acquisition, which allows for information of point generation to be available even after the removal of points that are classified as vegetation. On the contrary, the canopy at the vegetated areas that are scanned with TLS does not permit laser penetration, and rather large areas of "no-data" appear, with no participation in further processing. #### 6. Conclusions This study applies two cutting-edge methodologies that enhance our capabilities to understand slope stability and monitoring in tectonically active coastal areas. The two sites studied in the present work are a very common setting in the scenic coastline of the highly touristic Ionian Islands and other parts of the Mediterranean, where slope failures and instabilities threaten high-value locations and their visitors. The application demonstrates that the UAS- and TLS-aided surveys are able to provide a very detailed description of the structural condition of the slopes, in terms of discontinuities, using large numbers of measurements in a more practical, reliable and safe way in comparison with conventional surveys. Evaluation of the outcome of the surveys allows the identification of specific segments more prone to slope failures, improving the understanding of risk in the study sites and creating circumstances for the prioritization of interventions. This improved understanding is feasible because of the applied technologies. Regarding future research in the field, it will be interesting to further explore the combined use of UAS and TLS applications and the evaluation of the respective outcomes, as these first results show that it has the potential to maximize the information content provided to stakeholders and decisionmakers. In this way, the synergy of the two applications has the potential to provide related knowledge in order to adopt site-specific mitigation measures and strategies and can be an effective and responsible method for risk-mitigation-driven development of high-value coastal areas. Further research could also explore how these applications would compare in terms of performance against conventional rock slope stability analysis and ground-based slope-failure monitoring techniques. The approaches presented can be a useful, efficient and transferable tool that would benefit risk-mitigation efforts and the design of protection measures by expanding applications to other sites with similar geological and geomorphological properties worldwide. Author Contributions: Conceptualization, E.V. and S.M.; methodology E.V., A.K., E.K., S.M. and M.D.; software, E.V., A.K., S.M. and M.D.; validation, E.V.; formal analysis, E.V., S.M., M.D. and G.K.; investigation, E.V., S.M., M.D. and G.K.; resources, N.V. and E.L.; data curation, E.V., A.K., E.K., S.M. and M.D.; writing—original draft preparation, E.V., S.M., M.D. and G.K.; writing—review and editing, E.V., S.M., M.D., G.K., V.K., V.S., J.D.A., E.L. and N.V.; visualization, E.V. and S.M.; supervision, S.M. and M.D.; project administration, N.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the "Telemachus–Innovative Operational Seismic Risk Management System of the Ionian Islands" project, included in the Priority Axis "Environmental Protection and Sustainable Development" of the Regional Operational Programme "Ionian Islands 2014–2020", grant number MIS 5007986, which is funded by the European Regional Development Fund (ERDF) and National Resources under the National Strategic Reference Framework NSRF 2014–2020. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data available on request from the corresponding author. Acknowledgments: Special thanks to Metrica S.A. for providing support during the field data acquisition and processing in order to achieve maximum precision and accuracy of the TLS data. Conflicts of Interest: The authors declare no conflict of interest. #### References ### Article Seismological and Ground Deformation Study of the Ionian Islands (W. Greece) during 2014–2018, a Period of Intense Seismic Activity *Vassilis Sakkas 1,\, Vasilis Kapetanidis 1, George Kaviris 1, Ioannis Spingos 1, Spyridon Mavroulis 2, Michalis Diakakis 2, John D. Alexopoulos 1, Danai Kazantzidou-Firtinidou 1, Ioannis Kassaras 1, Spyridon Dilalos 1, Emmanuel Vassilakis 3, Evelina Kotsi 2, Gerasimos Tselentis 1,4, Efthymis Lekkas <sup>2</sup> and Nicholas Voulgaris <sup>1</sup> Abstract:** Seismicity in the Ionian Sea (W. Greece) is mainly generated along the Cephalonia–Lefkada Transform Fault Zone (CLTFZ) in the central Ionian, and on the northwestern termination of the Hellenic subduction margin in the south. Joint pre-, co- and post-seismic ground deformation and seismological analysis is performed at the broad Ionian area, aiming to homogeneously study the spatiotemporal evolution of the activity prior to and after the occurrence of strong (M > 6) earthquakes during the period of 2014–2018. The 2014 Cephalonia earthquakes (Mw6.1 and Mw5.9) were generated on a faulting system adjacent to CLTFZ, causing local ground deformation. The post-seismic sequence is coupled in space and time with the 2015 Lefkada earthquake (Mw6.4), which occurred on the Lefkada segment of the CLTFZ. Co-seismic displacement was recorded in the broader area. Seismicity was concentrated along the CLTFZ, while its temporal evolution lasted for several months. The 2018 Zakynthos earthquake (Mw6.7) caused regional deformation and alterations on the near-velocity field, with the seismicity rate remaining above background levels until the end of 2021. In the northern Ionian, convergence between the Apulian platform and the Hellenic foreland occurs, exhibiting low seismicity. Seismic hazard assessment revealed high PGA and PGV expected values in the central Ionian. Keywords: Ionian Islands; Cephalonia–Lefkada Transform Fault Zone; seismicity; ground deformation; GNSS; seismic hazard #### 1. Introduction The area of the Ionian Islands in western Greece plays an important role in the kinematic processes of the eastern Mediterranean. This tectonically complex area is by far the most seismically active region in Greece and among the most seismogenic regions in Europe. It is characterized by the frequent occurrence of destructive large earthquakes and undergoes intense ground deformation. The central Ionian Islands constitute part of the Eastern Mediterranean lithosphere that is subducted beneath the Aegean lithosphere along the Hellenic Arc. Citation: Sakkas, V.; Kapetanidis, V.; Kaviris, G.; Spingos, I.; Mavroulis, S.; Diakakis, M.; Alexopoulos, J.D.; Kazantzidou-Firtinidou, D.; Kassaras, I.; Dilalos, S.; et al. Seismological and Ground Deformation Study of the Ionian Islands (W. Greece) during 2014–2018, a Period of Intense Seismic Activity. Appl. Sci. 2022, 12, 2331. https://doi.org/10.3390/ app12052331 Academic Editor: José A. Peláez Received: 19 January 2022 Accepted: 17 February 2022 Published: 23 February 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The Ionian Sea (Figure 1) hosts areas with different fault geometries and kinematics. The prevailing tectonic structure is the NNE–SSW-trending, right-lateral Cephalonia– Lefkada Transform Fault Zone (CLTFZ), which is the most seismically active structure not only in the Ionian Sea, but also in Greece. This zone is a major boundary in the kinematic field of the region, as it separates the Ionian Margin into two different areas. The North Ionian Islands, comprising the Diapontia Islands, Corfu, Paxoi and Antipaxoi Islands, move slowly northward and northwestward at rates lower than 5 mm/year with respect to Eurasia. The South Ionian Islands, comprising Lefkada, Cephalonia, Ithaca and Zakynthos, move rapidly southwestward with velocities of 6–30 mm/year [1–3]. The CLTFZ is composed of two segments: the 40 km-long, NE–SW-striking, ESEdipping, right-lateral Lefkada segment, extending from the northwestern offshore part of Lefkada Island to the northern offshore part of Cephalonia Island [4,5], and the 90-kmlong Cephalonia segment, close to the western offshore part of Cephalonia, with similar geometry and kinematic properties to the other segment [6,7]. Figure 1. Seismotectonic background of the Ionian Islands region. Focal mechanisms of stronger events (M ≥ 5.5) are presented (see Data Availability Statement for references). Historic seismicity is presented by gray squares (SHEEC database, [8,9]) Principal stress axes S<sup>1</sup> (blue) and S<sup>3</sup> (red) after [10]. Fault lines after [11–19]. CLTFZ: Cephalonia–Lefkada Transform Fault Zone, WAFZ: Western Achaia Fault Zone. The South Ionian Islands (Zakynthos and Strofades) are located close to the northwesternmost tip of the Hellenic Arc, a few kilometers east of the Hellenic Trench in the southern Ionian Sea. The trench represents the active plate boundary where the eastern Mediterranean lithosphere is being subducted beneath the Aegean one. This subduction zone terminates against the Cephalonia segment of the CLTFZ. The CLTFZ is not the only major right-lateral fault zone in the Ionian Sea. A few kilometers south of Zakynthos, another structure occurs. It constitutes the southward extension of the seismic NE–SW-striking, right-lateral Western Achaia Fault Zone (WAFZ), which extends from the northwestern part of the Peloponnese to offshore southern Zakynthos [20]. The epicenter of the 8 June 2008, Mw6.4 Andravida earthquake [21,22] and its aftershock sequence were distributed along the onshore part of the WAFZ, at the northwestern part of the Peloponnese. It is worth noting that the earthquake did not induce direct expression of primary surface faulting [23]. Furthermore, the onshore part of the WAFZ has no direct surficial morphotectonic or geological evidence onshore of the Western Peloponnese. On the contrary, its offshore extension is linked with an offshore pull-apart basin NE of the Strofades Islets [24,25]. The part of the Ionian Sea located south of Zakynthos constitutes a downthrown block of the External Hellenides at the northern end of the Hellenic Trench. Flat thrusts, strike-slip and normal faults are detected [25], with thrusting prevailing over strike-slip or normal faulting [7,26]. The prominent feature of this area is a 46 km-long, NW–SE-trending thrust system [27], responsible for the generation of the 1997 Mw6.6 [28] and the 26 October 2018 Mw6.7 [29] Zakynthos earthquakes. The intense seismicity on the southern part of the Ionian Sea is attributed to the proximity of this area to the CLTFZ, the Hellenic Trench and many onshore faults in the South Ionian Islands. These islands have been formed either on the margins or within fault blocks. At Lefkada Island, typical cases are the fault zones in the western coastal part [15], along which extensive environmental effects were generated from the two strong Lefkada earthquakes on 14 August 2003 (Mw6.3) [30,31] and on 17 November 2015 (Mw6.4) [32], triggering many rockfalls and landslides in the western coastal part of the island [17,33]. In Cephalonia, several active faulting zones at the northern, western and southwestern parts of the island are susceptible to triggering of earthquake environmental effects, with the most typical example being the numerous rockfalls and slides due to the August 1953 earthquakes [34]. In Zakynthos, typical cases of active faults are the Volimes fault zone in the north part of the island, which has been formed in the transition from the Northern Zakynthos fault block to the Central Zakynthos one. Similar active faults are also located at the eastern and southern parts of Zakynthos Island [16]. Regarding the North Ionian Islands, a NNW–SSE-trending system, with a relatively extended shelf width represents the convergence (continental collision) between the Apulian Platform and the Hellenic foreland, with Corfu lying on the northwestern edge of the Hellenic Fold and Thrust Belt. The E–W-striking Southern Salerno–North Corfu fault zone is a major right-lateral structure that crosses Corfu from coast to coast [35] and which has resulted in the displacement of N–S-trending fold axes and thrusts [36]. This part of the Ionian Sea is characterized by lower seismicity than the southern one. The North Ionian Islands have suffered damage from earthquakes generated in adjacent areas, such as the 20 February 1743 Salento peninsula (Apulia, southern Italy) earthquake with Mw7.1 and I0 = IX, which triggered landslides in Corfu [37] and severe structural damage, including building collapses in Corfu town [38], attributed to very efficient strong propagation with NW–SE preferential directivity [39]. The complex geotectonic status of the area resulted in the high seismic activity that occurs in the broad Ionian area. However, during the last decade, and mainly during the period between 2014 and 2018, increased seismicity was observed, and strong events (M > 6.0) shocked the central Ionian Islands. Early 2014, two earthquakes (Mw5.9 and Mw6.1) occurred on Cephalonia Island, while in November 2015 and in October 2018 two earthquakes of Mw6.4 and Mw6.7 took place at South Lefkada and offshore south of Zakynthos, respectively. The regional crustal motion along the entire Ionian Sea and western Greece, as well as the local deformation on the central Ionian Islands, have been studied and monitored with local dense Global Positioning System (GPS) networks [13,40,41] and continuous Global Navigation Satellite System (GNSS) stations [42,43]. Previous work focused on the seismological analysis and interpretation of each of these strong earthquakes [15,29,32,44–49], on the study and modeling of the co-seismic deformation during every event [50–58] and on the geological impact close to the epicentral areas [14,15,17,59,60]. The purpose of the present work is to study and present in a unified and homogeneous approach an overview of the seismicity and the ground deformation in the broad area of Ionian Sea, extending from Corfu Island to the north to Strofades Islet in the south. The time span of the data covers the period before, during and after the occurrence of the strong earthquakes of 2014–2018. The spatiotemporal evolution of the seismicity involving these significant events is analyzed. Pre-, co- and post-seismic deformation is quantitatively described, aiming to understand the pattern of the ground motion associated with the recorded seismicity. The regional implications of joint seismological and geodetic analysis are also considered and discussed with respect to the geotectonic status of the area. Seismological data from the Hellenic Unified Seismic Network (HUSN) and geodetic data from the commercial and institutional continuous GNSS networks in the area were used in the framework of this study. #### 2. Seismological Data and Results In the Ionian Islands there are historical reports of 94 earthquakes from the years 358 to 1898 [9,61]. A significant number of historical earthquakes are located onshore, most possibly due to the limitations that arise from the use of historical sources. Instrumental seismicity, since 1900, is concentrated around the islands of Lefkada, Cephalonia and Zakynthos [28]. As described above, epicenters in Lefkada and Cephalonia are linked to local faults and mainly to the CLTFZ, whereas seismicity offshore and close to Zakynthos is related to the border between the Eurasian and African plates. Focal depths are limited to the upper 30–40 km of the crust, while foci at greater depths are related to the submerged African plate. The high seismicity rate is reflected in the high seismic hazard of the central Ionian Islands that belong to the highest category (Zone III) of the current Greek Building Code, with a Peak Ground Acceleration (PGA) value of 0.36 g for a return period of 475 years [62]. Since the implementation of the HUSN in 2007 [63], the monitoring of seismic activity in Greece has been enhanced, with increased detectability and improved location accuracy. This was particularly important for the region of the Ionian Islands, as it is situated at the western margins of the network and a significant part of its intense seismicity is located offshore. Herein, a catalogue of ~62,000 earthquakes was compiled. These events have occurred in the broad Ionian Islands area, including part of mainland western Greece. The seismological data were collected from the database of the Geodynamics Institute of the National Observatory of Athens (GI-NOA) for the period from February 2011 to May 2018. For the consecutive period from June 2018 to November 2021, the seismological data were extracted from the database of the Seismological Laboratory of the National and Kapodistrian University of Athens (SL-NKUA). Furthermore, in the present analysis, relocated seismicity catalogues were incorporated for the 2014 Cephalonia, 2015 Lefkada and 2018 Zakynthos aftershock sequences [29,32,64–67]. #### 2.1. Seismicity Results After visual inspection, the seismicity of the study area during the last decade was divided into 12 spatial groups (Figure 2), to enable the description of its spatiotemporal evolution. Seismicity marginal to the area of interest was placed in the miscellaneous group #13 (white). The magnitude of completeness is estimated at M<sup>c</sup> = 2.0 ± 0.2, with variations depending on the method used for its determination or the time period of a selected subset. At times, it reaches lower values, e.g., 1.5, whereas during the occurrence of significant earthquakes that produce series of large aftershocks, the magnitude of completeness may temporarily surpass 2.5. The cumulative number of events per spatial group (Figure 3a) and the spatiotemporal projection (Figure 3b) along the A–B profile of Figure 2a are drawn after the application of the magnitude threshold (M ≥ Mc = 2.0). The herein referred magnitudes are ML, unless otherwise stated. Figure 2. (a) Seismicity in the region of the Ionian Islands during the period February 2011–November 2021 from the databases of GI-NOA and SL-NKUA, including relocated catalogues for the three main aftershock sequences [29,32,64,65]. Colors and numbers correspond to different spatial groups. The north–south-oriented profile A–B is used for the spatiotemporal projection of Figure 3b. (b) Close-up of the seismicity in the region of Cephalonia–Ithaca–Lefkada Islands. (c) Close-up of the seismicity in the region of Zakynthos Island. Starting in early 2011, scattered seismicity was observed throughout the Ionian Islands region. An M4.5 earthquake occurred near the northern tip of Cephalonia Island on 16 March 2011, an M4.1 event occurred just offshore south of the island on 5 December 2011 and an M4.2 event near the northwestern part of the Paliki peninsula (Atheras area) on 23 April 2013. The background seismicity rate with M ≥ 2.0 was ~0.4 events/day, occurring mainly onshore of the island (group #1, red) and in Myrtos Gulf (group #2, orange). Overall, very few events are located near to or onshore of Ithaca Island during the study period. Background seismicity was also recorded along the SW extension of the CLTFZ, at a rate of 0.1–0.2 events/day. Further south, near Zakynthos Island, clustered seismicity was recorded following events of magnitude around 4 at group #4 (indigo; ~0.3 events/day) and group #8 (yellow; ~0.5 events/day), which extended from the area between Zakynthos and Cephalonia to the NW Peloponnese. An M5.0 event occurred at the westernmost end of group #4 on 19 July 2011, triggering seismicity along SW–NE-trending streaks. It should be noted that such delineations of epicenters in that area have been argued to be affected by the large azimuthal gap, causing location biases along this direction [29], so they should not be overinterpreted. The southernmost group #6 (cyan) near the Strofades Islets has a background rate of ~0.2 events/day. On Lefkada Island (group #3, green; with a background rate of 0.1–0.2 events/day), seismicity often occurs in spatiotemporal clusters, whereas the background seismicity on Cephalonia Island tends to be more randomly scattered. Significant earthquakes that took place during the study period also include an M4.5 event on 23 October 2012 offshore north of Lefkada and west of Preveza, and an M4.7 event on 23 May 2013 at the southern tip of the island. Figure 3. (a) Cumulative number of events per spatial group of Figure 2 (different colors), for events with M ≥ M<sup>c</sup> = 2.0. The larger events (M ≥ 4.0) are marked by circles. (b) Spatiotemporal projection along the north–south-oriented profile A–B of Figure 2a, for events within a range of ±50 km from the profile and M ≥ M<sup>c</sup> = 2.0. Symbol size is proportional to magnitude. Events with M ≥ 5.0 are marked with stars. Close-ups for the periods January 2014 to September 2017 and August 2016 to November 2021 are presented in Figures A1 and A2 of Appendix A, respectively. Group #9 (black), near the eastern shore of Amvrakikos Gulf, with a background rate of ~0.1 events/day, contains an M5.2 event that occurred on 24 October 2014, producing a small cluster. Further north, Corfu Island is characterized by very low seismicity onshore throughout the study period. Sparse seismic activity was recorded offshore to the south and a bit denser distribution of epicenters is identified to the north, near the shores of Albania (group #5, purple; with a background rate of 0.1–0.2 events/day), related with events of magnitude 4.0 ≤ M < 5.0. West of Corfu Island, at NW Epirus, group #10 (blue; background rate of 0.2–0.4 events/day) included an M = 5.6 thrust event that occurred on 21 March 2020 near Kanallaki [68,69]. Group #11 (beige; background rate of 0.1 events/day) contains an M5.3 event that occurred on 15 October 2016 near Ioannina. At the northern edge of the study area, group #12 (gray; background rate of 0.1 events/day) near the Greco-Albanian border, contains an M5.3 event that occurred on 1 June 2019. The most significant bursts of earthquake activity at the Ionian Islands region during the study period comprise the 2014 Cephalonia (groups #1 and #2), the 2015 Lefkada (groups #3 and #2) and the 2018–2019 Zakynthos mainshock–aftershock sequences (group #4), which are described in more detail in the following subsections. #### 2.1.1. The 2014 Cephalonia Sequence The 2014 Cephalonia sequence was initiated by an Mw6.1 mainshock that occurred on 26 January 2014, ~2 km NE of the city of Lixouri, on the Paliki peninsula [44–46,60,64]. About one week later, on 3 February 2014, another significant earthquake of magnitude Mw5.9 occurred at the NW part of Paliki. The seismicity on Cephalonia Island during 2014 was relocated using the double difference method (HypoDD; [70]) and a minimum 1D velocity model [64,65]. The aftershock distribution extended ~32 km in a N20◦ E direction, covering the entire Paliki peninsula (group #1), but seismic activity was also triggered in a spatially separated cluster inside Myrtos Gulf (group #2). The southern half of the onshore seismicity, related mostly to the first major earthquake of 26 January 2014 at a depth of 16 km, appeared more complex, being distributed between 5 and 17 km and extending to a width of 15 km in a N110◦ E direction, whereas the northern half, mostly related to the second major earthquake, was more linearly distributed (width ~5 km) and shallower, at focal depths between 5 and 12 km [65]. Seismicity after the first earthquake was mainly concentrated near its hypocenter during the first hours, but quickly spread throughout the aftershock zone. Some clusters further south, offshore Cephalonia, were triggered at a later stage. A significant cluster occurred south of group #1 after an M5.0 event on 8 November 2014, soon followed by clustered activity in Myrtos Gulf (group #2). The focal mechanisms of the two major events and most of the major aftershocks indicate dextral strike-slip faulting along SSW–NNE-trending, subvertical faults. However, seismicity inside the Myrtos Gulf is characterized by several smaller clusters trending E–W, i.e., transverse to the axis of the CLTFZ, which suggests antithetic sinistral faulting. The 2014 sequence temporarily increased the seismicity rate of the neighboring groups #7 and #8. The temporal evolution of the post-seismic activity in Cephalonia (group #1) shows a long relaxation period that lasted up to July 2017, considering a relatively high background seismicity level (0.3 events/day) for M ≥ M<sup>c</sup> = 2.0 (Figure 4). In the northern area of Cephalonia (group #2), with a smaller background seismicity rate (0.1 events/day prior to 2014), increased activity was observed up to September 2017, due to its reactivation after the 2015 Lefkada mainshock, which is presented in the following Section 2.1.2. Figure 4. Temporal evolution of the seismic sequence on Cephalonia Island (group #1), for events with M ≥ M<sup>c</sup> = 2.0. The upper panel shows the cumulative number of events, with the occurrence of M ≥ 4.0 events depicted by stars, while the lower panel describes the seismicity rate on a semilogarithmic scale, measured for sliding windows of various lengths (5 days to 100 days). #### 2.1.2. The 2015 Lefkada Sequence A few months after the Cephalonia events, on 17 November 2015, an Mw6.4 earthquake struck Lefkada Island, with its epicenter located near the mid-southern part of the western coast. The seismicity during the period from 17 November to 3 December 2015, relocated with the double-difference method [65], revealed a very different distribution than that of the 2014 Cephalonia sequence. The aftershocks were divided in distinct clusters, distributed at focal depths between 5 and 15 km. The aftershock zone extended to ~60 km in a N16◦E direction, mainly onshore of the island. Very few events occurred south of the mainshock, suggesting that a major fault patch was ruptured, leaving only few small unbroken asperities. The largest aftershock was an M5.0 event that occurred on the same day as the mainshock and close to its vicinity. The densest cluster was located just north of the mainshock, in the same region that was activated during an M5.1 event that had occurred on 29 November 1994 [71], and also triggered after an Mw6.3 earthquake that struck the northern part of the island on 14 August 2003 [30,31]. Notably, the mainshock of 2015 at Lefkada Island, which mainly involved group #3, also triggered seismicity in group #2, between Cephalonia and Lefkada, and even inside the Myrtos Gulf. Furthermore, these offshore southern clusters presented similar characteristics as those in the gulf of Myrtos, i.e., east–west alignment, likely related to antithetic sinistral faulting, transverse to the CLTFZ. The earthquake rate of group #3 that increased since the occurrence of the 17 November 2015 earthquake, remained higher than the background level (0.1 events/day; for M ≥ M<sup>c</sup> = 2.0) as late as September–October 2017. To exclude possible biases due to the increase of event detectability, setting a higher magnitude threshold of 2.3 (Figure 5) indicated a return of the seismicity rate to its background level around October 2016, dropping to very low levels in May–July 2017. Figure 5. Temporal evolution of the seismic sequence on Lefkada Island (spatial group #3) for events with M ≥ 2.3. Panels are similar to Figure 4. #### 2.1.3. The 2018–2019 Zakynthos Sequence The most recent significant seismic occurrence in the region of the Ionian Islands during the study period is the 2018–2019 Zakynthos mainshock-aftershock sequence [29,49,72]. An Mw6.7 offshore earthquake occurred on 25 October 2018, 45 km SW of Zakynthos Island, at an estimated depth of ~12 km. The focal mechanism of the mainshock indicates dextral strike-slip on a low-angle (25◦), east-dipping plane. The relocated catalogue of over 4000 aftershocks in the period between 25 October 2018 and 31 March 2019 reveals an extensive aftershock zone, ~80-km-long in a N110◦ E direction and ~55 km wide [29]. The largest aftershock was an Mw5.5 event that occurred on 30 October 2018, 15 km NNW of the mainshock, with a similar focal mechanism, albeit with a steeper dip angle. The distribution of epicenters permits the distinction of some sub-clusters within the seismicity cloud. A large group in the vicinity of the mainshock presented a smoothly diminishing seismicity rate. On the other hand, other clusters, particularly at the northern part of the aftershock zone, but also at the southern end, presented outbreaks, some related with the occurrence of significant aftershocks. The most persistent activity was observed at the northern subclusters of group #4, with intense activity that lasted until October 2019. A last burst of seismic activity occurred between March and July 2020 and was triggered at the southern end of the 2018 aftershock zone, after an M4.3 event on 26 March 2020, followed by a few more M > 4 events. A final significant M5.2 event occurred on 21 October 2020 in the same region, after a period of quiescence. The 2018 Zakynthos mainshock–aftershock sequence was preceded by several foreshocks since the beginning of the year in the vicinity of the mainshock. An M4.8 event had occurred at the northern end of group #4 on 21 February 2018 and another Mw4.8 event was recorded half an hour before the mainshock. Earlier activity was also documented near the mainshock's region between September 2016 and March 2017, in at least three outbreaks, while a smaller one occurred in August 2017. Focal mechanisms of the 2018 Zakynthos aftershock sequence involved both low-angle and steeper strike-slip faulting [29]. The latter is mainly related with the northern subclusters, which also present a form of SW–NEtrending streaks, similar to the cluster at the southern end of group #4. The kinematics of the mainshock and many of the major aftershocks are consistent with SW–NE-trending compression occurring in this region that is found in a transition zone between strike-slip faulting in the north (CLTFZ) and to the east (WAFZ in the NW Peloponnese, e.g., the 2008 Andravida earthquake [21,22]) and the northwestern end of the Hellenic Arc subduction zone. The temporal evolution of the post-seismic activity indicates that even towards the end of 2021 the seismicity rate had still not returned to its background level, despite the magnitude threshold being set as high as 2.5, quite above the average Mc = 2.0 (Figure 6). Figure 6. Same as Figure 4, but for the temporal evolution of seismicity south of Zakynthos Island (group #4), for events with M ≥ 2.5. #### 2.2. Seismic Hazard The intense and large magnitude seismic activity in the broad area of the Ionian Islands makes the assessment of seismic hazard crucial in the urban planning, as well as in the construction of critical infrastructure. Additionally, the tourism industry in the Ionian Islands is a major concern for the local and national economy that requires enhancing its earthquake resilience. The strong earthquakes that occurred in the area have yielded high values of Peak Ground Acceleration (PGA). At Cephalonia, the two events of 2014 induced shakings reaching ~560 cm/s<sup>2</sup> and 735 cm/s2, respectively [73]. At Lefkada, PGAs of ~412 cm/s2 were recorded in the 2003 event [74] and 363 cm/s2 during the 2015 earthquake [67]. Finally, the Mw6.7 earthquake of Zakynthos led to a recorded PGA of ~382 cm/s2 [75], on the island. On the framework of this study, the seismic hazard of the Ionian Islands was evaluated, aiming to estimate the maximum anticipated ground motion values in this highly active seismogenic area. A probabilistic seismic hazard assessment (PSHA) method has been applied. Based on the regional character of this study, a source-zone approach was selected. Zone boundaries were obtained from the SHARE project [76,77]. However, the b-value, earthquake rate and maximum magnitude for each area were re-estimated, from the catalogue compiled in the context of the current study. To account for time periods of incomplete data, the modified b-value (β) was determined using a maximum likelihood method [78]. The ground motion prediction equations (GMPEs) of [79], which have been successfully applied in seismic hazard studies in Greece [80–82], were used to obtain PGA values in the final PSHA model. A reverse/strike-slip focal mechanism type and a rock basement were considered in the GMPE, for all areas. Finally, the seismic hazard was assessed using the R-CRISIS software [83]. The software can calculate the expected hazard value (in this case, PGA and the Peak Ground Velocity; PGV) for estimated earthquake occurrence probabilities at specific timeframes, for the given distribution of sources and the event magnitudes within the limits of each zone. The PGA and PGV values were computed for a 10% exceedance probability level in 50 years (return period of 475 years) (Figure 7). Figure 7. Maps showcasing the spatial distribution of PGA (a) and PGV (b) for a return period of 475 years. Major cities in the Ionian complex are marked by gray boxes. The two hazard quantities follow similar distributions. Highest values are observed at Cephalonia and the nearby Ithaca Island. The high seismic activity on these islands fully complied with the obtained hazard values. Similarly, increased values are observed at the NW edge of Zakynthos. The hazard quantities decrease towards the central part of Zakynthos, in accordance with the lack of strong events in this area. The high hazard zone is terminated at the southern part of Lefkada Island. The lowest hazard values in the area are observed in the north Ionian Islands (Paxoi and Corfu); as expected, since no large-magnitude events (M ≥ 6.0) have occurred in this area during the past 120 years [28]. The Greek Building Code [62] classifies the Ionian islands into two zones: Zone II (with PGA of 235 cm/s2) and Zone III (with a value of 353 cm/s2). Lefkada, Cephalonia, Ithaca and Zakynthos Islands are in Zone III, with Corfu and Paxoi classified in Zone II. While the code provides wide ranges, the current analysis indicates that there may be a need for a more detailed approach. The expected PGA at Corfu and Paxoi was found to be the lowest in the Ionian Islands, being less than 353 cm/s2. However, it was observed that for Zone III islands, a much finer distribution could be extracted (Figure 7a). Moreover, Cephalonia and Ithaca seemed to feature PGA values much higher than that of the Seismic Code. Making this distinction by introducing a new Zone with stricter building guidelines could prove to be a useful approach to reduce seismic vulnerability—and therefore, risk—in a financially significant area of the Ionian prefecture. Revising the national code would also offer the opportunity to rethink local actions for improving the seismic resilience of older and historical buildings in cities and towns (e.g., Argostoli in Cephalonia) that teem with them. Other seismic hazard studies, albeit Greece-wide, also find PGA values that well exceed those proposed by the building code [80,84]. #### 3. GNSS Data and Results Daily GNSS data from stations located in the Ionian Islands and western mainland Greece and the Peloponnese were processed for the period from 2009 to 2021. The analysis intended to determine the crustal velocity field of the broad area, detect possible preseismic displacements and study co- and post-seismic deformation. The continuous GNSS stations extend from the northern Corfu Island to the Strofades Islets, south of Zakynthos. In western mainland Greece and the Peloponnese, the stations are located in the broad area of Patras Gulf and in the city of Pyrgos in the western Peloponnese (Figure 8). Sites KASI, SPAN, PONT, KIPO, VLSM, ZNTE, STRF, KTCH and RLSO belong to the National Observatory of Athens (NOA network) [42,43]. The commercial network of METRICA SA (HexagonSmartNet) [85] provided data for stations KERK, PAXO, ARGO, ZAKY, AGRI and PYRG. In Cephalonia Island, the sites SISS, KARA and SKAL are part of the National and Kapodistrian University of Athens network (NKUA net) [54]. Meanwhile stations EXAN and DRAN in Lefkada Island and FISK and ASSO in Cephalonia are part of the GNSS network of the Corinth Rift Laboratory (CRL GNSS net) [86], which is the only international Near-Fault Observatory (NFO) of the European Plate Observing System. Finally, PAT0 station in the city of Patras is a European Reference Frame (EUREF) station [87]. Figure 8. Horizontal velocity vectors (black arrows) for the continuous GNSS sites (blue triangles) of the broad Ionian Islands area. Red arrows indicate the horizontal velocity component estimated for the period after the strong (M > 6) earthquakes in the area (ITRF 2014). The raw GNSS data were processed using the Bernese v5.2 software [88]. Several GNSS stations of the EUREF and IGS were included in the processing together with the local GNSS sites, while auxiliary files were introduced in the procedure. The absolute antenna phase center corrections were used, together with precise orbital solutions from the Center for Orbit Determination in Europe (CODE) and the Vienna Mapping Functions for the tropospheric modeling. The FES2004 model (http://holt.oso.chalmers.se/loading; accessed on 11 January 2022) was used for the tide-loading corrections. The precise double-difference method was used for static-mode solutions. Several ambiguity-resolution strategies were applied, based on the length of the formed baselines between the GNSS stations. The daily calculated coordinates for the GNSS stations were evaluated for the repeatability error on a weekly basis, and solutions were excluded in cases of large deviations from the weekly solution. The processing resulted in the estimation of high-precision station coordinates. Time series were formed, annual velocities were calculated (Figure 8; Tables 1–5) and co-seismic displacements were determined (Appendix B). The daily coordinates of the Ionian GNSS stations were estimated on the global ITRF2014 reference frame. Table 1. Velocity components for the GNSS stations on Corfu and Paxoi Islands (ITRF 2014). Table 2. Velocity components for the Lefkada GNSS stations (ITRF 2014). Table 3. Velocity components for the Cephalonia GNSS stations (ITRF 2014). Table 4. Velocity components for the Zakynthos—Strofades GNSS stations (ITRF 2014). #### 3.1. Corfu–Paxoi Islands At the North Ionian Islands, three GNSS stations were processed (Table 1): two in Corfu, KASI and KERK; and the PAXO station located at Paxoi Island. The linear type of motion in all three components for the whole-time span is evident in these stations. There was no static deformation due to the seismic activity that occurred in the south, i.e., related to the 2015 Lefkada or the 2014 Cephalonia events (Figure S1). The calculated velocity vectors are similar to the ones that are presented in [68,89]. For the two Corfu stations (KASI and KERK), the horizontal velocity components are almost parallel, with the southern site exhibiting a slightly increased eastward motion. The most noticeable aspect is the significant subsidence that occurs at the KERK and PAXO stations. Table 5. Velocities of GNSS stations in western Greece and the NW Peloponnese (ITRF 2014). #### 3.2. Lefkada Island On central Lefkada Island, data from two GNSS stations (SPAN in the north and PONT in the south) were processed for the period from 2009 to 2021 (Figure S2). Partial data from two other sites, EXAN and DRAN, were available after the strong (Mw6.4) 2015 earthquake (Table 2). For the two stations—SPAN and PONT—which operated prior, during and after the November 2015 event, a linear type of motion was observed for the pre-seismic period. There was no clear evidence of any kinematic changes or any alterations on the pattern of motion, in any of the three components, prior to the earthquake. Baseline changes between the two sites calculated for the pre-seismic period (2009 to 2015) indicated shortening of the distance, with a rate of V = (−2.50 ± 0.03) mm/year. The latter is indicative of the compressional status that occurred along the island during the pre-earthquake period. The co-seismic displacements at the two sites (SPAN and PONT) were quite significant, mainly in the southern PONT station, which is located in the near vicinity of the epicenter (Appendix B). Static co-seismic displacements, which were smaller but not negligible, were also observed at stations located in Cephalonia (VLSM, KARA and SKAL), in Zakynthos (ZAKY) and in western Greece (AGRI) (Appendix B). During the post-seismic period, the kinematic status of the area was characterized by a long relaxation process, expressed as a non-linear motion on the stations. This post-seismic kinematic behavior was observed at the two Lefkada stations, but mainly in PONT [90], as well as at sites of northern Cephalonia (FISK, ASSO). The slow relaxation was also evident at the two new sites that were established on the island, EXAN and DRAN (CRL GNSS net). The relaxation period on the PONT station was estimated to last up to August 2016, exhibiting a non-linear type of motion during that period. The estimation was performed with an average moving window of 5–60 days in length. The relaxation process was characterized by a significant southward motional component and a strong uplifted tension, following the significant co-seismic subsidence. For the subsequent period (from 2016 to 2021), the time series of the two stations showed an analogous motion to the pre-seismic period, but with slightly smaller horizontal amplitudes (Figure 8). Similar velocity vectors for the post-seismic period were also estimated for the EXAN and DRAN GNSS sites. Calculation of the baseline changes between SPAN and PONT sites after August 2016 shows that the shortening of the distance between these two sites remains, as was the case for the pre-seismic period, but with a slightly decreased amplitude (−1.81 ± 0.05 mm/year). #### 3.3. Cephalonia Island Regarding the island of Cephalonia, most GNSS stations were established after the 2014 intense seismic activity. For the period prior to 2014, data were available only from the VLSM station. The time series of this station (Table 3) showed an ENE linear type of motion with small seasonal fluctuations, mainly in the north component, and a subsiding pattern (Figure 9). Figure 9. Time Series of VLSM Cephalonia GNSS station. Red lines mark the dates of the strong earthquakes in the Ionian Islands. The two strong events of Mw6.1 in late January and Mw5.9 in early February 2014, recorded on the VLSM and KIPO stations, caused measurable static displacement (Appendix B). KIPO station, located on the western part of the Paliki peninsula, closer to the earthquakes' epicenters, revealed the most significant displacement. VLSM station, to the southeast of the epicentral area, situated on the limestone unit of central Aenos Mountain, exhibited smaller co-seismic motion. The co-seismic displacement vectors in these two GNSS sites indicated a right-lateral activated faulting zone [50]. Nevertheless, these events have not caused any displacement to GNSS stations located at the neighboring islands or in western Greece (Appendix B). The GNSS velocity vectors for the sites located at Cephalonia Island after the seismic activity of 2014 exhibited a gradual rotation on the horizontal component from ENE in the north to ESE motion to the southern part of the island (SKAL station) (Figure 8). Most stations showed linear type of motion since 2014, with a subsiding vertical component (Table 3). The motional behavior of the VLSM station is, however, also worth noting. The time series of VLSM (Figure 9) show irregular motion, in both the north and vertical components, for the period from February 2014 to September 2016. During that period, the station shows a non-linear ESE horizontal motion and uplifting behavior. The station acquired the pre-seismic ENE and subsiding motional character in the subsequent period (from September 2016 to present). This long-lasting irregular behavior—which may be described as a post-seismic relaxation period and also encompassed the 2015 Lefkada earthquake—has not been observed at any other site in the Ionian Islands. Calculating for the post-seismic period (from 2017 to 2020) the baseline changes between the stations in Cephalonia, extension occurred between the northernmost (FISK) and the southernmost (SKAL) stations (V = 4.66 ± 0.10 mm/year). Meanwhile, in the central part of the island the distance between the ARGO and VLSM stations remained almost stable (V = −0.44 ± 0.04 mm/year), for a similar period (from September 2016 to October 2021). #### 3.4. Zakynthos–Strofades Islands In the southern part of the Ionian Islands, GNSS data from three stations were processed (Table 4). For the sites of ZAKY and STRF, located in the city of Zakynthos and at the islet of Strofades, respectively, data were available prior to the strong 2018 earthquake (Mw6.7). Both stations exhibited a linear type of ground motion. ZAKY station revealed an ESE horizontal motion, with a negligible subsiding pattern, while STRF station showed an SE motion with lower amplitude (Figure 8). Baseline change calculation between these two sites for the pre-earthquake period showed a lengthening of the distance with an estimated velocity of V = 3.50 ± 0.10 mm/year. ZAKY station's pre-seismic kinematic behavior indicated shortening relative to the GNSS station PYRG, located in the western Peloponnese (V = −5.12 ± 0.09 mm/year). Likewise, the baseline distance between the STRF and PYRG stations decreased with a similar velocity, V = −4.52 ± 0.08 mm/year. These observations revealed a compressional regime between the Peloponnese and the South Ionian Islands, while extension occurred between Zakynthos and Strofades. The 2018 earthquake, southwest of Zakynthos, caused strong ground displacement in both Zakynthos and Strofades. Regarding GNSS stations at the island of Cephalonia, significant ground displacement was recorded at the central ARGO and the southern SISS and SKAL stations (Appendix B), but not at the VLSM site. Moreover, static ground displacement was observed at stations located in the western (PYRG) and northwestern (PAT0) Peloponnese, even in the southwestern part of mainland Greece (KTCH). After the occurrence of the mainshock, a new continuous GNSS station was established in the island (station ZNTE). The two sites that operated in Zakynthos after the Mw6.7 event, together with the PYRG station, revealed an almost eight-month-long post-seismic relaxation period. During this period, the kinematic behavior of ZAKY and PYRG stations showed a significant alteration of motion with respect to the pre-seismic one (Figure 10). A distinctive alteration of the east motional component was observed at PYRG station, where the eastward pre-seismic pattern reversed to a westward post-seismic motion. The post-seismic relaxation period was estimated to last up to early July 2019. During the following period (from July 2019 to the end of 2021), both ZAKY and PYRG stations again presented a pre-seismic motion pattern, with small alterations in the horizontal components (Figure 10). Similarly, the ZNTE station indicated a velocity vector significantly different compared to that of the post-seismic period, with a pattern of motion consistent to that of ZAKY. #### 3.5. Neigboring Stations–Overall Image Together with the continuous GNSS stations in the Ionian Islands, stations located in western mainland Greece (AGRI, KTCH) and western and northwestern Peloponnese (PAT0, RLSO, PYRG) were also processed (Table 5). The goal was to study the velocity field, ground deformation and differential motions in the broad region of the Ionian Sea, focusing on the area close to the highly activated central Ionian Islands. Figure 10. Time series for the two GNSS stations in Zakynthos (ZAKY) and the western Peloponnese (PYRG). Red lines are the same as Figure 9. The deduced velocity field of the area shows the differentiation of the motion between western Greece and the Peloponnese, resulting in the opening of the Patras Gulf with an estimated rate of about 8.0 ± 0.5 mm/year. As previously described, the seismic activity on Cephalonia has not caused any displacement to the broad area, as co-seismic deformation was observed only at local sites. The Lefkada 2015 event affected only the northern AGRI station, located in western Greece. It was only the strong 2018 Zakynthos event that caused noticeable static displacement in both western Greece and the Peloponnese (Appendix B). The most distinct co- and post-seismic deformation was recorded at PYRG station. A small displacement was also observed at KTCH station (Figure S3). The strong events in the Ionian Islands did not appear to affect the velocity field of these stations, which exhibited a linear type of motion throughout the processed period. The only noticeable alterations were observed in the vertical component of PAT0 station (NW Peloponnese) (Figure S3). In this station, the vertical component shows a non-linear motion. Initially, up to the end of 2015, an uplift pattern was observed, which since the beginning of 2016 has reversed to a subsiding motion. A similar pattern also was observed at the neighboring RLSO site. The overall image of the velocity field in the broad Ionian Islands area shows a gradual rotation of the horizontal velocity vectors (Figure 8). GNSS stations located to the north exhibited a NW horizontal motion, while towards the south, the horizontal vector component shifted to a SW motional direction. The latter has been combined with a decrease in the amplitude of the horizontal velocity from ~26 mm/year in the northern Ionian Islands, to ~13 mm/year in Zakynthos Island and ~7 mm/year in Strofades Islet, respectively. The transition from a northward to southward motion pattern occurs in Cephalonia Island. A similar rotation pattern is also observed at the stations located in western Greece and the NW Peloponnese, with the transition occurring between AGRI and KTCH stations, roughly at the same latitude as Cephalonia. Analyzing the baseline-length changes for GNSS stations located on different Ionian Islands, as well as between stations in Ionian and western Greece, information about the tectonic status of the area can be extracted (Appendix C). In the northern Ionian Sea, the distance between Corfu and Paxoi has remained almost unchanged (VKERK-PAXO = −0.60 ± 0.11 mm/year). Intense lengthening was calculated between the northern islands (Corfu, Paxoi) and Lefkada (i.e., VPAXO-SPAN = 7.55 ± 0.11 mm/year). The latter indicates an extensional regime between the northern and the central Ionian Islands. Extension with a lower rate is also observed by the baseline changes between the stations located in the central and southern Ionian Islands. Fluctuations in the baseline velocity rates were detected before and after the strong seismic events that occurred in the area during the years 2014–2018. According to the baseline velocity between stations on the islands of Lefkada and Cephalonia, lengthening was taking place during the period before the 2014 seismic events at Cephalonia (i.e., VPONT-VLSM = 2.91 ± 0.04 mm/year). For the period following the 2015 Lefkada earthquake, up to late 2016, the observed intense shortening (VPONT-VLSM = −13.61 ± 0.73 mm/year) could be attributed to the post-seismic process. Re-establishment of the pre- 2014 lengthening character then gradually took place. Negative baseline velocity changes between the central Ionian Islands and stations located in western Greece and the Peloponnese imply compressional forces in this area which were not affected by the intense seismic activity. The latter is supported by the small variations on the velocity values before and after the strong earthquakes (Appendix C). #### 4. Discussion Three major tectonic features in the Ionian Islands define the regional kinematic field and control the seismic activity: the convergence of the Apulian Platform and the Hellenic foreland in the north; the long NNE–SSW Cephalonia–Lefkada Transform Fault Zone offshore and west of the respective islands in the central part; and the northwestern tip of the Hellenic Arc in the southern Ionian Sea. These major structures, together with smaller local faults, create a complex tectonic environment generating intense seismic activity and strong ground deformation. In the present study, seismological and geodetic data from the area of the Ionian Islands were combined for the periods before, during and after the occurrence of strong earthquakes in 2014–2018. In the following, the results of the joint analysis are discussed in terms of the overall tectonic status of the study area. #### 4.1. North Ionian Sea The northern Corfu and Paxoi Islands located at the Hellenic foreland present the largest amplitude regarding the horizontal velocity vectors (with respect to ITRF), but the recorded seismicity is by far the lowest in the region. The seismicity in this area is located mainly west of Corfu and Paxoi Islands, in mainland Greece and north of Corfu, close to the shores of Albania. The estimated horizontal velocities for the local GNSS stations match with the ones calculated by [68], although the datasets cover slightly different time periods. Considering that the horizontal velocity amplitude of the Apulian Platform is ~31.1 mm/year (VEast = 25.1 mm/year, VNorth = 18.4 mm/year) [68], the Paxoi Island shows a similar horizontal amplitude of ~29.7 mm/year, while Corfu stations have smaller average velocity ~25.8 mm/year. The latter may indicate that, kinematically, Paxoi is controlled by the Apulian motion, as [68] has suggested, although the baseline change between stations KERK and PAXO is estimated to be rather small (about −0.6 ± 0.1 mm/year). The subsiding pattern observed in the southern Corfu and Paxoi stations (KERK, PAXO), though they could be attributed to local geological setting, may also reflect the continental collision that occurs in the area. The absence of significant seismic activity along the NNW–SSE-trending boundary of the Apulian Platform and Hellenic foreland—not just in the limited period of the present study but also during the past (Figure A3 in Appendix A)—indicates a nearly aseismic convergence process, mainly owed to the relatively low motion rate (~5.5 ± 0.7 mm/year; that is, the average converge velocity of the two Corfu stations towards Apulian Platform, similar to the velocity estimated by [89]). #### 4.2. Central Ionian Sea The two strong earthquakes in early 2014 on Cephalonia Island triggered a long-lasting aftershock sequence, partially overlapping with that of the 2015 Lefkada mainshock. Prior to the 2014 events, the background seismicity occurred along the regional CLTFZ and onshore at the northern part of the island, likely associated with the local major faulting features. The baseline change between the VLSM station and the southern PONT site in Lefkada indicated that extension occurred between the two islands. The 2014 events occurred on a faulting system adjacent and parallel to the CLTFZ on the Paliki peninsula. Strong co-seismic deformation occurred mainly at the western part of the island, as has been detected from local GPS network measurements [50]. However, ground displacement was observed only on Cephalonia; none of the continuous GNSS stations in the near vicinity of the island, either on Lefkada or western Greece, showed any deformation, proving the local character of the activated seismogenic faulting zone. The post-seismic activity expanded mainly along Paliki, towards the north, but seismic clusters were also observed at the southern part of the activated zone. Seismicity in the area of the Myrtos Gulf indicated activation of structures transverse to the axis of the CLTFZ, oriented with local faults, e.g., the Agia Efimia fault (Figure 1), as the major thrust fault that separates the northern Erissos peninsula from the main island is called. It is important to notice that a similar cluster, on the northern part of Cephalonia, was also formed after the Mw6.3 2003 northern Lefkada earthquake [13,30]. The seismicity rate remained at a high level (above the background level) for the whole period until the occurrence of the November 2015 event on Lefkada. This long post-seismic period is correlated with the ground deformation observed on the GNSS VLSM station, where different pattern of motion was exhibited with respect to the pre-seismic era. Analytically, a southward motion with uplift tension occurred, and increased lengthening of the baseline distance between PONT and VLSM was observed. The late 2015 Mw6.4 Lefkada earthquake was the second strong event to occur in less than two years in the central Ionian Islands. However, this earthquake occurred on the Lefkada segment of the major CLTFZ or a sub-parallel structure. The regional character of the event reflected on the ground deformation observed in the broad area, extending to Zakynthos Island to the south and to AGRI station in western Greece. The post-seismic activity expanded onshore of Lefkada Island, but also triggered clustered activity on the northern part of Cephalonia, in an area previously activated after the 2003 Lefkada event and, more recently, after the 2014 Cephalonia events. The increased seismicity rate (above the background level) after the strong earthquakes, marks a long relaxation period (up to October 2017), indicative of the activated area (Figures 4 and 5). The recorded motion at the PONT GNSS site, near the epicentral area, as well as to other neighboring GNSS sites at Lefkada and Cephalonia, confirmed the long relaxation period after the earthquake [90]. Based on the geodetic data, the kinematic field of the area returned to its pre-seismic pattern during August–September 2016, for both Lefkada and Cephalonia. However, the amplitude of the velocity vector at the sites on Cephalonia and Lefkada show small but noticeable differentiation (~15%) with respect to the pre-seismic period from 2009 to 2014. Regarding the possibility that the 2014 seismic sequence caused or triggered the 2015 earthquake, previous studies [48,91] claimed that the southern Lefkada event was a continuation of the seismic process of the 2003 earthquake in the northern part of CLTFZ. The seismological data in the present study reveal that after the occurrence of the 2014 earthquakes, seismicity propagated northwards, while it has been shown [48] that the 2014 main events caused Coulomb stress changes in the epicentral area of 2015, where the 2003 earthquake also induced stress transfer. The GNSS data, for the inter-seismic period from February 2014 to November 2015, showed motional changes on the VLSM station (southward pattern), while the PONT Lefkada site maintained its kinematic status. The latter resulted in increased velocity (lengthening character) of the baseline change between these two stations, doubled during that period (Appendix C). Assessing the spatiotemporal expansion of the 2014 Cephalonia seismic sequence, together with the geodetic evidence presented in this study, it may be argued that the occurrence of the anticipated event after the 2003 earthquake in southern Lefkada Island may have been accelerated—and even triggered—by the 2014 Cephalonia events. It is likely that the cause of the activity, in both the Cephalonia and Lefkada epicentral areas, was the 2003 event on the major CLTFZ structure [48], expressed as a local-scale activity in Cephalonia, and as a regional one in Lefkada. #### 4.3. South Ionian Sea The last strong earthquake (Mw6.7) in the Ionian Sea occurred south of Zakynthos Island, three years after the major event at Lefkada. The source of this earthquake was located on the northwestern end of the active plate boundary between the eastern Mediterranean lithosphere and the Aegean one. Geodetic data from the broad area prior to the earthquake show extensional status between Cephalonia and Zakynthos, as well as between Zakynthos and Strofades. Compression occurred between Zakynthos–Strofades and the Peloponnese, as depicted by the baseline change between the GNSS sites (Appendix C). The earthquake was preceded by a number of intermediate events (~M5.0) in the near vicinity of the epicentral area. Detailed seismological and geodetic work by [58] has suggested slow-slip events in the area, with transient signals on the surface deformation. This earthquake resulted in the longest and most intense seismic sequence of the study period (Figure 6), accompanied by significant ground deformation and motional alterations in the affected area. The Mw6.7 event caused strong ground deformation on a broad area, extending to Cephalonia Island, to Patras city (NW Peloponnese) and to western mainland Greece (KTCH GNSS station). The extent of the co-seismically deformed area highlights the regional character of this event. The post-seismic activity is concentrated offshore, north and east of the epicenter. It is worth noting the limited activity on the northern onshore part of the island. The latter observation, which may indicate a differentiation between the northern and southern parts of the island, has also been observed on the kinematic field detected by local GPS measurements [41]. Post-seismic evolution of the ground deformation suggests a significantly shorter period until the area returns to the pre-seismic kinematic pattern. This period extended up to July 2019, as revealed by the horizontal component of the three closer sites to the epicenter. The orientation of the seismic clusters on the northern extent of the Zakynthos sequence (group #4; Figure 2c) in a SW–NE-trending direction, coincide with the direction of the 2008 activated zone in NW Peloponnese [21]. This is an area where the geodetic data show a differential motion between the PAT0, RLSO and the PYRG GNSS stations. #### 4.4. The Broad Ionian Sea Area The overall image of the seismic activity in the broad area of Ionian Islands is dominated by the three seismic sequences generated after the large-magnitude events (M > 6) in Cephalonia, Lefkada and Zakynthos Islands (Figures 2 and 3). The Lefkada sequence is mainly aligned and concentrated along the north Lefkada segment of the regional CLTFZ structure, expanding offshore and west of the northern part of Cephalonia. The 2014 Cephalonia sequence occurred on a local faulting zone, associated with the Cephalonia segment of the CLTFZ but not comprising part of it. It is distributed spatially on a wider zone, along the western Paliki peninsula, and extends offshore, west of the northern Erissos peninsula. These two sequences appear to be coupled to each other in both time and space and linked with the earlier 2003 Lefkada earthquake. The southern Zakynthos sequence also has regional characteristics and is spatially distributed in a region much larger than anticipated for the magnitude of the mainshock [29]. This is an area affected by the transition of the northwestern end of the Hellenic Arc subduction zone to the CLTFZ major faulting structure. The geodetic data clearly depicted the kinematic pattern of the broad area, with respect to major tectonic formations and seismic activity (Figure 11). Comparing the regional velocity field on the northern Ionian Islands with the one of the central islands, a clear kinematic boundary emerges between these two areas. The northern islands show a uniform motional pattern, while the velocity field at the central and southern Ionian Sea shows a clockwise rotation around an axis located on Cephalonia Island (calculating the horizontal velocities with respect to VLSM station). Based on the results of this study, a rotation rate of 6.9 ± 2.1 degrees/Myr was estimated. This agrees with previous regional studies [92,93], as well as with local geodetic studies on Cephalonia [13]. Baseline changes between several GNSS sites reveal the extensional regime along the Ionian Islands, which is gradually reduced from north to south, while compression occurs between the central Ionian Islands, and western Greece and the Peloponnese. Extension is also observed between western Greece and the Peloponnese, as well as across the NW Peloponnese. The strain regime alternates the periods after the strong earthquakes, marking the postseismic relaxation process. The spatial extension of the co-seismic displacements reflects the regional or local character of the activated area. The Lefkada and Zakynthos earthquakes caused static displacement in the broad area, while the Cephalonia events affected only the local GNSS sites. Figure 11. Overview of the Ionian Islands area showing the main tectonic structures together with seismic activity (M > 4) (black circles), and strong (M > 6) events (yellow stars). Black arrows show the relative Eurasia–Africa motion in the SW [94], and the motion of the Hellenic foreland with respect to the Apulian Platform (estimated herein) in the north. The extensional (red arrows) and compressional (blue arrows) pattern calculated based on baseline changes between GNSS stations (green triangles) is qualitatively presented. The temporal and spatial joint analysis of the seismological and geodetic data revealed that the 2014 and 2015 seismic sequences are connected, while the Zakynthos one is part of the tectonic process in the western termination of the Hellenic subduction margin. The expansion of the seismic activity after the 2014 and 2015 events offshore of northern Cephalonia is the spatial link. In this area, previous work [52] identified a seismic gap, indicating that a segment of the CLTFZ has not ruptured yet and may generate a strong event in the future. The present analysis shows that seismicity in this region is clustered in a large number of small segments oriented almost in an E–W direction, transverse to the axis of the CLTFZ. The latter may indicate the transition between the Cephalonia and Lefkada segments of CLTFZ, and the increased seismicity may relieve the accumulated stress. Thus, the occurrence of a strong event in this region seems less likely, but cannot be excluded. Meanwhile, the area between Cephalonia and Zakynthos is characterized by low seismic activity. It is a region whose kinematic status is affected by the CLTFZ in the north and the subduction process in the south. The last major earthquake in the area dates to 1983, with an Mw6.8 earthquake [52] occurring offshore SW of Cephalonia and NW of Zakynthos. It may be argued that this is an area that has the potential to generate another strong event. However, the geodetic data show small changes in the baseline length between the SKAL GNSS site in southern Cephalonia and Zakynthos station, ZAKY (Appendix C), and a similar pattern of motion, in both direction and amplitude. Another area where low seismic activity was observed during the study period is Ithaca Island. Although the general seismic activity in this area is relatively low (e.g., Figure 2b), a few strong earthquakes have occurred in the past (Figure A3). Previous GPS campaign results [54] showed a differential motion between the northern and southern parts of the island, and compression between Cephalonia and Ithaca, compatible with the regional field [93]. The area is considered to have the potential to generate a significant earthquake in the future, taking into account the strong events in its vicinity, but more data are required to assess such a scenario. The strong earthquakes and large co-seismic ground deformation recorded in the study area clearly exhibit the necessity for seismic hazard analysis in such a seismically active area. As expected, large earthquakes yield violent shaking that coincides well with the expected PGA from the probabilistic analysis. The latter confirms the definition of the SHARE seismic source zones. However, future studies in the area should focus on much smaller scales and identify potential hazards from specific active fault zones in an area as tectonically complex as the broad Ionian Sea. The process of seismogenesis, as described by statistical laws in any PSHA approach, should also be revised according to fault-specific knowledge. Finally, a detailed survey of local site conditions would be crucial in better refining PSHA results and offer greater accuracy of the estimations. #### 5. Conclusions Seismological analysis and geodetic results from continuous GNSS stations were combined to study the spatiotemporal evolution of seismic activity and ground deformation in the Ionian Islands during the period from 2014 to 2018, characterized by the occurrence of strong earthquakes in Cephalonia, Lefkada and Zakynthos Islands. Low seismicity was observed in the north Ionian Sea, where the collision between the Apulian platform and the Hellenic foreland is taking place. The velocity field of the area, deduced by stations on Corfu and Paxoi Islands, shows horizontal NW motion, transverse to the collision front. The vertical component reveals a subsiding pattern, compatible with the convergence process in the area. The CLTFZ feature offshore of the central Ionian Islands of Cephalonia and Lefkada is the prevailing structure, generating the strong and intense seismic activity observed in the region. The 2014 Mw6.1 and Mw5.9 earthquakes occurred on an adjacent local faulting zone, causing strong ground displacement on the island but not in the broader area. The seismic sequence following these two events expanded in the vicinity of the activated area along the Paliki peninsula, and offshore northwards. Few months later, on 17 November 2015, another strong earthquake (Mw6.4) occurred on or parallel to the Lefkada segment of the CLTFZ. Co-seismic displacement was recorded in almost all the southern Ionian Islands, highlighting the regional character of the seismogenic source. The post-seismic activity expanded along the CLTFZ and southwards in the same area as the 2014 sequence. The temporal evolution of the post-seismic activity and the ground deformation show a long relaxation period. However, the motional pattern obtains its pre-seismic character earlier (August 2016) compared to the respective seismic excitation. The seismic activity reaches its background levels (0.1–0.2 events/day) after a longer period, between late 2016 and mid-2017. The two seismic sequences are linked in space and time, with the 2014 activity likely accelerating the 2015 event. The October 2018 Mw6.7 Zakynthos earthquake occurred close to the northwestern tip of the Hellenic Arc. Strong ground displacement took place on the near-field GNSS stations, as well as on Cephalonia Island and the Peloponnese. The seismic sequence expanded in the vicinity of the epicentral area, but mainly offshore south and west of Zakynthos. The motion field of the stations in Zakynthos and the western Peloponnese showed significant alterations with respect to the pre-seismic period, on both north and east motional components, which regained its anticipated pattern about eight months after the mainshock. However, the seismicity rate has remained at higher values, compared to the background activity (0.2 events/day), for more than three years. Probabilistic seismic hazard assessment was performed in the Ionian Islands to estimate PGA and PGV for a return period of 475 years. The smallest values were obtained, as anticipated, for the northern Ionian Islands, corelating with the low seismicity of the area. However, the calculated PGA values in Cephalonia and Ithaca are significantly higher and well exceed the ones proposed by the current Greek Building Code. Based on the hereinperformed PSHA analysis, a finer, more detailed zonation of the area and re-evaluation of the PGA values provided by the Building Code must be considered in the future. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/app12052331/s1, Figure S1: Time series for station KERK on Corfu Island, and PAXO on Paxoi Island; Figure S2: Time series for station SPAN and PONT on the northern and southern part of Lefkada, respectively; Figure S3: Time series for station KTCH in Western Greece and PAT0 on the NW Peloponnese, in the city of Patras. Author Contributions: Conceptualization, V.S. and G.K.; methodology, V.S., V.K., G.K. and I.S.; software, V.S., V.K. and I.S.; validation, V.S., V.K., G.K. and S.M.; formal analysis, V.S., V.K., G.K. and I.S.; investigation, M.D., J.D.A., D.K.-F., I.K., S.D., E.V. and E.K.; resources, G.T., E.L. and N.V.; data curation, V.S., V.K. and G.K.; writing—original draft preparation, V.S., V.K., G.K., I.S. and S.M.; writing—review and editing, V.K., G.K., I.S., S.M., J.D.A., I.K., S.D., E.V. and E.K.; visualization, V.S., V.K., I.S. and S.M.; supervision, G.K., J.D.A., I.K., E.V., G.T., E.L. and N.V.; project administration, N.V.; funding acquisition, N.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the project "Telemachus Innovative Seismic Risk Management Operational System of the Ionian Islands" (MIS 5007986) which is part of the Regional Operational Programme «Ionian Islands 2014 2020» and is co-financed by the European Regional Development Fund (ERDF) (National Strategic Reference Framework NSRF 2014 20). Informed Consent Statement: Not applicable. Data Availability Statement: Focal mechanisms presented in Figure 1a are compiled from various sources, including [5,95–106]. Seismological data from GI-NOA and SL-NKUA are available at https://bbnet.gein.noa.gr (accessed on 18 January 2022) and http://www.geophysics.geol.uoa.gr/ stations/gmapv3\_db/ (accessed on 18 January 2022), respectively. GNSS data from NOA-network and for station SISS (NKUA-network) are available at http://geodesy.gein.noa.gr:8000/nginfo/ (accessed on 18 January 2022). Acknowledgments: The authors would like to thank the personnel of the Hellenic Unified Seismological Network (http://eida.gein.noa.gr/; accessed on 18 January 2022) for the installation and operation of the seismological stations used in the current study. Continuous GNSS data were also provided by METRICA SA (HexagonSmartNet). Some of the figures were made using the Generic Mapping Tools software [107]. Conflicts of Interest: The authors declare no conflict of interest. #### Appendix A Close-up diagrams of the spatiotemporal distribution in the areas of Cephalonia– Lefkada Islands and Zakynthos Island. Figure A1. Close-up of Figure 3b in the region of Cephalonia–Lefkada between January 2014 and 12 September 2017. Figure A2. Close-up of Figure 3b in the region of Zakynthos between August 2016 and November 2021. Figure A3. (Left) Historic seismicity [8,9] and (right) significant earthquakes (M<sup>w</sup> ≥ 5.9) during the instrumental era (1900–present) (from [28] and the databases of GI-NOA and SL-NKUA) in the broad region of the Ionian Islands. #### Appendix B The co-seismic displacements on the continuous GNSS stations processed in this study are presented in Table A1. The co-seismic displacement and its error were estimated based on the average value of each coordinate seven days prior to the earthquake and one to seven days after its occurrence, depending on the proximity of each site to the epicentral area. Results are presented only for the stations where the co-seismic displacement was larger than its error, otherwise it is considered that no displacement occurred. Table A1. Co-seismic displacements on the continuous GNSS stations. #### Appendix C The baseline change between two stations was estimated based on the daily coordinates of the selected sites. Baselines were formed only when data were available for both sites and for a period exceeding 24 h. The following Table A2 lists some characteristic formed baselines between the Ionian Islands, as well as between GNSS stations in Ionian Sea and western Greece and the Peloponnese. Table A2. Baseline velocity between continuous GNSS stations. #### References ### Article Investigation of the Thiva 2020–2021 Earthquake Sequence Using Seismological Data and Space Techniques *George Kaviris 1,\, Vasilis Kapetanidis 1, Ioannis Spingos 1, Nikolaos Sakellariou 1, Andreas Karakonstantis 1, Vasiliki Kouskouna 1, Panagiotis Elias 2, Andreas Karavias 3, Vassilis Sakkas 1, Theodoros Gatsios 1,3, Ioannis Kassaras 1, John D. Alexopoulos 1, Panayotis Papadimitriou 1, Nicholas Voulgaris <sup>1</sup> and Issaak Parcharidis <sup>3</sup> Abstract:** We investigate an earthquake sequence involving an Mw = 4.6 mainshock on 2 December 2020, followed by a seismic swarm in July–October 2021 near Thiva, Central Greece, to identify the activated structures and understand its triggering mechanisms. For this purpose, we employ doubledifference relocation to construct a high-resolution earthquake catalogue and examine in detail the distribution of hypocenters and the spatiotemporal evolution of the sequence. Furthermore, we apply instrumental and imaging geodesy to map the local deformation and identify long-term trends or anomalies that could have contributed to stress loading. The 2021 seismic swarm was hosted on a system of conjugate normal faults, including the eastward extension of the Yliki fault, with the main activated structures trending WNW–ESE and dipping south. No pre- or coseismic deformation could be associated with the 2021 swarm, while Coulomb stress transfer due to the Mw = 4.6 mainshock of December 2020 was found to be insufficient to trigger its nucleation. However, the evolution of the swarm is related to stress triggering by its major events and facilitated by pore-fluid pressure diffusion. The re-evaluated seismic history of the area reveals its potential to generate destructive Mw = 6.0 earthquakes; therefore, the continued monitoring of its microseismicity is considered important. Keywords: seismic swarm; seismology; geodesy; double-difference relocation; Coulomb stress transfer; Thiva; Greece #### 1. Introduction Greece is located at the southeastern margin of Europe, a region with rich localized seismotectonic phenomena, a result of the convergence between the Eurasian and African tectonic plates. These processes are expressed through intense deformation and high seismicity along thrust, strike-slip, and normal faults [1,2]. Geodetic, geological, and seismological surveys have revealed that the N–S to NE–SW extensional tectonic regime of Central Greece in the Quaternary is a result of the back-arc extension between the Hellenic arc and the transtensional tectonic regime of the North Aegean [3–5]. Thiva (Central Greece) is located at the transition zone between two major WNW–ESEand NW–SE-striking rifts: the Corinth Gulf in the south and the Euboekos Gulf in the east (Figure 1). The Gulf of Corinth is an area characterized by high seismicity [6,7] expressed through the frequent occurrence of seismic swarms [8–12]. Citation: Kaviris, G.; Kapetanidis, V.; Spingos, I.; Sakellariou, N.; Karakonstantis, A.; Kouskouna, V.; Elias, P.; Karavias, A.; Sakkas, V.; Gatsios, T.; et al. Investigation of the Thiva 2020–2021 Earthquake Sequence Using Seismological Data and Space Techniques. Appl. Sci. 2022, 12, 2630. https://doi.org/ 10.3390/app12052630 Academic Editor: Valerio Comerci Received: 31 January 2022 Accepted: 28 February 2022 Published: 3 March 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Figure 1. Seismotectonic map of part of Central Greece showcasing Thiva (Boeotia) and the Corinth and Euboekos gulfs. The study area is marked by the blue rectangle. Focal mechanisms of significant earthquakes obtained from the catalogue of the Seismological Laboratory of the National and Kapodistrian University of Athens (SL-NKUA) and the compilation in [13] are shown, along with the locations of seismological stations of the Hellenic Unified Seismic Network (HUSN; red triangles) and GNSS stations (inverse yellow triangles). Green stars indicate epicenters whose parameters were estimated through macroseismic data in the present study. Yellow stars are epicenters of events adopted by the SHEEC catalogue [14]. Faults, after [15,16], are also shown (black lines). Inset: Greece and its main geotectonic features. The area of the larger map is marked by a red rectangle. NAT: North Aegean Trough; NAF: North Anatolian Fault. The broad Thiva area is characterized by normal faulting (Figure 1). It is worth noting that recent strong and destructive earthquakes in Greece have occurred on normal faults, such as the Lesvos 2017 [5,17,18], Kos 2017 [5,19,20], Samos 2020 [21–24], Thessaly 2021 [25–30], and Arkalochori (Crete) 2021 [31,32] earthquakes. Fault segmentation is common near Thiva, with the presence of a multitude of smaller normal faults all around the area. One of these segments is the 6 km long Kallithea fault, striking between N 80◦ E and N 115◦ E, with a probable eastward extension [33]. North of Thiva, a denser fault network with smaller south-dipping ruptures dominates the area around Yliki Lake. Due to the small length of the mapped faults, the occurrence of an earthquake with a magnitude (M) of at least 6.0 seems unlikely [34,35]. On 2 December 2020, 10:54:56 UTC, an Mw = 4.6 earthquake occurred east of Thiva [36]. Moment tensor inversion yielded a normal focal mechanism with strike N106◦ E, dip 31◦, and rake −81◦ [36]. The analysis of both geodetic and seismological data indicated an uncommonly shallow depth of approximately 2 km for the main event. The mainshock and its aftershock sequence, which lasted until 3 January 2021, were triggered by ruptures on or adjacent to the Kallithea fault [36]. A few kilometers to the west and 7 months later, a new earthquake sequence was initiated, characterized by high productivity, lasting for over 6 months. The whole activity led to the recording of over 2500 earthquakes. In this study, we present a detailed analysis of the seismic crises in the Thiva area between late November 2020 and mid-October 2021 through high-resolution doubledifference relocation. The spatiotemporal evolution of the various outbreaks composing the 2021 seismic swarm is presented, along with the activated structures at depth and their relation to surface traces of mapped faults. The triggering mechanisms of the 2021 Thiva seismic swarm are investigated, such as stress redistribution due to the 2 December 2020 mainshock or possible aseismic factors, including fluid-induced pore-pressure diffusion, through the migration of seismicity, as well as deformation transients. The deformation study resulted from the joint analysis of local GNSS data and satellite aperture radar interferometry (InSAR) products. #### 2. Past Earthquakes The study area has had a mild-to-moderate seismic footprint since antiquity. There are ancient reports of various incidents in the broad area of Thiva that could be interpreted as geotectonic in nature [37]. Limited archaeological evidence suggests earthquake activity, which has had an impact on Thiva between 1350 and 1230 BCE without any substantial proof regarding the epicentral area [38]. Many centuries later, in 1321–1323 CE, the eyewitness Jordanus reports collapse and damage of many houses in Thiva [14,39]. In [40] it is noted that, in general, the areas of Boeotia, Locris, Fokis, and Fthiotis have suffered from catastrophic earthquakes, such as the 427 BCE Orchomenos earthquake sequence, which shook Boeotia, Euboea, and Athens and was perhaps a forerunner of the large Maliakos Gulf 426 BCE earthquake [39]. After another long period of seismic quiescence, significant activity was observed in the 19th century. On 13 December 1833, [41] reports a local earthquake was felt in Thiva. On 18 August and 29 September 1853, two destructive earthquakes struck Thiva, affecting the area between it and Yliki Lake, a few kilometers to the north. The first shock destroyed the northern outskirts and caused considerable damage to the rest of the urban environment, leading to 11 fatalities and 60 injuries. The second one caused the collapse of the already-damaged houses and ruined the rest, causing one casualty. At least 3 felt aftershocks occurred up to 1854 [14,39]. Thiva, with a population of 4000, was rebuilt by 1860, with wider spaces between houses. On 26 September 1872, a rather strong event occurred, preceded by a few foreshocks, causing nonstructural damage [39]. The earthquake of 23 May 1893 was also preceded by damaging foreshocks, ruining Thiva and nearby places [39]. The earthquake of 17 October 1914 occurred without a foreshock and caused heavy damage in Thiva and nearby villages. A strong aftershock, a few hours later, extended the damage area with a westward migration of seismicity [14,39,42,43]. This earthquake was recorded by the two seismographs of the National Observatory of Athens, of Mainka and Agamemnone type, followed by a series of aftershocks. All recorded events were located at a distance of 45 km from Athens, assuming their epicenters approximately at 6 km SE of Thiva, in agreement with the epicenter of the mainshock derived from the isoseismals drawn from a significant number of direct macroseismic observations [40,44]. The Annales de l'Observatoire National d'Athènes reported another damaging earthquake near Thiva on 23 September 1917, preceded by two foreshocks and followed by at least 37 recorded aftershocks lasting up to the end of the year. This information is passed on to the catalogues of Galanopoulos [45], Kárnik [46], and Shebalin et al. [47]. However, the event is not reported in the catalogue of Makropoulos et al. [6]. For the three events with I > 8, information on coseismic effects is also reported. More specifically, regarding the 18 August 1853 event, in the S. Euboekos gulf successive waves were observed and the sea flooded the coast without any damage. In Kopais Lake (artificially drained in 1880, approximately 25 km NW of Thiva) the banks were flooded. Rockfalls fell from a mountain near Thiva and from Mt. Ptoon (18 km to the north). Liquefaction near Atalanti was also reported. No ground cracks or surface ruptures were observed [39]. For the 23 May 1893 earthquake, ground cracks were reported near Thiva and Mulki, a settlement near Aliartos (Figure 1). Rockfalls and ground cracks, triggered by landslides, were reported from localities in the vicinity of Thiva. No information about ground deformations was reported [39]. For the 17 October 1914 earthquake, no ground cracks or any kind of ground ruptures was found [40]. For the pre-1900 events, macroseismic intensities were collected from [48], whereas for those in the 20th century, intensities were assigned using EMS98 (European macroseismic scale) [49] from the reports of the Annales de l'Observatoire National d'Athènes [40,44], as well as from Ambraseys's unpublished material. The latter was donated to SL-NKUA and consists of notes, earthquake lists, slides, maps, and books. Having this information, we compiled the seismic history of Thiva, that is, a distribution of EMS98 intensities, I, over time, for events in the study area between 1893 and 1934 (Figure 2a). The apparent clustering of damaging (I ≥ 6) and felt earthquakes is associated with two periods (1914–1917 and 1923–1927) of the 20th century, the first related to the two most damaging shocks in this period (1914 and 1917). The activity in 1914–1917 is almost continuous, including foreand aftershocks of the major events. However, at least five subclusters can be identified in the year 1915 (Figure 2b). This activity resembles the recent 2020–2021 activity in the vicinity of Thiva regarding its duration. It is noted, however, that the 1915 events represent the long aftershock activity of the 1914 event, a fact also mentioned in [40,44]. Figure 2. Seismic histories of the area (a) between 1893–1934 and (b) 1915 in terms of EMS98 (macroseismic scale). The red rectangle in panel (a) marks the window presented in panel (b). The significant number of available intensity data points allowed for the macroseismic estimation of earthquake parameters (MEEP) using the MEEP software [50], described in detail in [51]. The software calculates four types of epicenters (Centroid, MEEP, Bakun– Wentworth, and Pairwise). The first is based on the [52] "boxer" method, while the second is the MEEP procedure with iterations adopting the point of lowest RMS [50]. The other two procedures are the methods in [53,54], respectively. The uncertainties of the parameters are calculated via a bootstrap resampling routine of the intensity data points (IDP) data set [55], repeating the procedure 1000 times, from which a standard deviation is calculated. However, the final uncertainties concerning both the epicenter coordinates and the magnitude are obviously significantly larger compared with the respective ones obtained through analysis of recent instrumental data, depending on the number and distribution of the available IDPs. Table 1 and Figure 1 (green stars) present the results of MEEP software applied to the four most damaging earthquakes in the study area, with updated, in comparison with previous studies, parameters due to the increased number of IDPs (Table 1). It is observed that, within a period of 65 years, Thiva has experienced three events of equivalent moment magnitude, Mw, ranging from 6.0 to 6.2. Figure A1 presents the spatial distribution of macroseismic intensities assigned in this study and the calculated macroseismic epicenter of the 1914 earthquake derived from 62 IDPs. Our analysis yielded an epicenter between Thiva and Kallithea (site of the December 2020 activity). For the 1917 event, the epicenter is located in Thiva, where the sole maximum intensity is observed. Table 1. Estimated macroseismic parameters in comparison with previous studies for the four damaging earthquakes in Thiva (Figure 1, green stars). I0: epicentral intensity; ϕ: latitude of estimated epicenter; λ: longitude of estimated epicenter; Uepi: epicentral uncertainty; Mw: equivalent moment magnitude (macroseismic); D: focal depth; IDPs: number of intensity data points. #### 3. Seismological Data and Methods Here, we study the seismicity in the area of Thiva between 25 November 2020 and 16 October 2021. During that time, P- and S-phase arrivals for over 2000 earthquakes were manually determined at the Seismological Laboratory of the National and Kapodistrian University of Athens (SL-NKUA). The catalogue data were enriched with additional events and manually picked arrival times from the database of the Geodynamics Institute of the National Observatory of Athens (GI-NOA). The compiled catalogue contains a total of ~2800 events for the study period. The sequence was recorded by the regional Hellenic Unified Seismic Network [56], complemented by the deployment of temporary local stations, which play a significant role in constraining the focal depths and increasing the detection threshold. Specifically, two stations (AMPE and THIV; Figure 3) were installed by SL-NKUA, and three stations (THVA, THI1, and THI2; Figures 1 and 3) were installed by GI-NOA in the immediate area of Thiva. Figure 3. Seismicity in the area of Thiva between 25 November 2020 and 16 October 2021. (a) Initial locations with HypoInverse and (b) after double-difference relocation with HypoDD. Fault lines are from [35] and the NOAfaults database [15,16]. Local seismological and GNSS stations are marked with red triangles and a yellow inverted triangle, respectively. #### 3.1. Location and Relocation of Seismicity Initial locations for the earthquake sequence were acquired using the HypoInverse code [57], employing the P-wave velocity model of [58] for the neighboring Eastern Gulf of Corinth, as well as for Boeotia, obtained from recordings of the local CORNET network [59–61]. Other velocity models that were tried out (e.g., regional models for Central Euboea or Atalanti; [62]) provided similar results and comparable uncertainties; however, the [58] model was preferred for consistency with previous results for the 2020 Kallithea sequence, where the same model was used [36]. The importance of local velocity models for earthquake location has recently been demonstrated in the case of the 2019 Constance Lake (Central Europe) seismic sequence [63]. Regarding the present study, the Vp/Vs ratio was confirmed to be ~1.79 with the Chatelain [64] method (Figure A2), as in the original model of [58]. The initial locations are presented in Figure 3a. The average root mean square (RMS) travel-time residual for the dataset is 0.19 s, with average horizontal and vertical location uncertainties, as reported by HypoInverse, at 0.38 and 0.80 km, respectively (Figure A3). The average focal depth is 9.4 km, although much shallower hypocenters (a median focal depth of 4.2 km) are resolved for the eastern group, related to the 2020 Kallithea sequence, previously presented by [36]. The magnitude of completeness for the dataset is M<sup>c</sup> = 1.6, determined by the entire magnitude range (EMR) method [65]. As this sequence involves a large number of events that occurred in a series of outbreaks clustered in space and time within a limited area, it is important to reduce the relative location uncertainties in order to better distinguish the activated structures. For this purpose, we employed the double-difference relocation code, HypoDD [66]. This algorithm minimizes the double difference between calculated and observed P- and S-wave travel times for pairs of neighboring events, resulting in the reduction of location uncertainties due to unmodeled velocity structure. Waveform cross-correlation data are also incorporated in the relocation scheme to enhance the hypocentral distribution of strongly correlated events by reducing relative location uncertainties due to arrival-time picking inconsistencies. To facilitate the relocation procedure, the dataset was divided into subsets, each one with a smaller number of events. This was performed after examining the initial locations and separating the sequence into groups, roughly based on their spatiotemporal distribution. Several outbreaks were distinguished in time related to both temporal and spatial clustering of the occurring earthquakes. The Thiva earthquake sequence was divided into 9 temporal groups, each covering a different period and beginning with an abrupt increase in the seismicity rate. The event waveforms were initially examined in terms of waveform similarity using reference stations located in the vicinity of the epicentral area and operational during the whole study period. For this purpose, waveforms of the station VILL (Figure 1) at a distance of ~16 km from Thiva were mainly employed, while additional data were also used from the temporary station THVA (Figure 3), deployed by GI-NOA at Thiva, 1 day after the 2 December 2020 M<sup>w</sup> = 4.6 Kallithea event, and the station STFN at an epicentral distance of ~16 km from the 2020 Kallithea sequence (Figure 1). The full event waveforms (both P and S waves) were cropped and band-pass-filtered between 2 and 8 Hz for the stations VILL and STFN and in the range of 2–15 Hz for the station THVA. The waveforms of all combinations of event pairs were cross-correlated, and the RMS values of cross-correlation maxima from each component were registered in a correlation matrix. Nearest neighbor linkage was applied, and multiplets were formed with an optimal threshold value of Cth = 0.88, which maximized the difference between the size of the largest cluster and the sum of clustered events [67]. A total of 1948 events were classified in 163 multiplets. Next, for each of the temporal groups, the P- and S-wave windows of events belonging to the same multiplets were cross-correlated at each station with available arrival-time data, and their cross-correlation maxima with their respective time lags were registered as input for HypoDD. The double-difference relocation procedure was performed separately for each one of the 9 temporal groups. The parameterization of the procedure for each group varies slightly due to the different numbers of events, travel-time data, and clustering properties. However, a similar scheme was employed for all cases, favoring stronger a priori weights for catalogue data during the first steps, followed by stronger weights for cross-correlation data during the last stage. A total of 2700 events were successfully relocated, representing 96.4% of the initial catalogue (Figure 3b). #### 3.2. Spatiotemporal Evolution of the Sequence To examine the spatiotemporal distribution of the 2020–2021 Thiva sequence in detail, the dataset was divided into spatial groups, permitted by the achieved reduction of relative location uncertainties with the application of the double-difference relocation. Ward's linkage was applied to the matrix of 3D hypocentral distances between all combinations of relocated event-pairs, and a large number of spatial clusters were formed. After visual examination, some smaller clusters were merged, and a final subdivision into 12 spatial groups was adopted (Figure 4). An exception to this procedure is group #1, which is related to the 2 December 2020 event, but is also covering all seismicity that occurred during the first period of the seismic crisis, that is, before 10 July 2021, which mainly took place at the eastern part of the study area. In the following, we examine the spatiotemporal evolution of the sequence with reference to the 12 spatial groups, distinguished by different colors and numerical labels (Figure 4). Figure 4. (a) Map of relocated epicenters with different colors and numbers representing the 12 spatial groups to which the sequence was divided. Events with M ≥ 3.5 are depicted as stars. The profile line A–B is used for the spatiotemporal projection of Figure 5. (b) Cumulative number of events with M ≥ M<sup>c</sup> = 1.6 per spatial group represented by different colors and numbers (at the right end of each curve) for the period between July and 16 October 2021. The occurrence of events with M ≥ 3.0 is marked with circles with size proportional to magnitude. Major events related to outbreaks at certain groups are labeled with their date and largest event magnitude. Group #1 is presented in an alternate version of this plot in Figure A4, covering the entire study period, starting 25 November 2020. Figure 5. Spatiotemporal projection of relocated epicenters along the N 110◦ E-oriented profile A–B in Figure 4a for the period between 9 July and 16 October 2021, with colors and numbers corresponding to different spatial groups. Events with M ≥ 3.5 are depicted as stars. The red lines indicate possible fluid-driven seismicity triggering fronts with a hydraulic diffusivity of 0.20 m2/s (dashed line) or 0.25 m2/s (solid line). The dashed blue line is drawn for reference, representing a seismicity migration speed of 70 m/day towards ESE. The sequence commenced in late November 2020, with a small cluster of earthquakes occurring near Kallithea (eastern group #1, blue), including an M<sup>L</sup> = 3.0 on 25 November. Then, on 2 December 2020, 10:54:56 UTC, an Mw = 4.6 earthquake struck near Kallithea. This earthquake apparently had a very shallow focus at a focal depth of 1.5 km after relocation in agreement with the results in [36]. The shallow focal depth of this event is evidenced by the fact that, despite its small magnitude, the deformation field was clearly identified by InSAR, exploiting Copernicus Sentinel-1 data in both ascending and descending orbital directions [36]. This sequence was short-lived, as it practically ceased after 17 December 2020, with a few earthquakes happening until January 2021, and was followed by a long period of quiescence (Figure A4 in Appendix B). The seismic activity at the western part of the sequence started on 10 July 2021, with several events of 3.1 ≤ M<sup>L</sup> ≤ 3.4 (red group #2 and cyan group #3), which compose the western half of the 2021 swarm, also initiating seismic activity at group #6 (dark gray) at the midnorthern part with a rate of 0.3–0.2 events/day. A stronger event of M<sup>w</sup> = 4.2 occurred on 11 July, 00:00:17 UTC, in the group #2 area, followed by another event of similar magnitude on 20 July in the same area. The latter earthquake increased the seismicity rate of groups #2 and #3, but also triggered seismicity towards other areas, mainly group #4 (purple) and, secondarily, group #5 (green), while a few events were located at groups #7 (yellow) and #11 (beige). Minor outbreaks, causing abrupt increase in the seismicity rate, occurred on 25 July (group #2, including an M<sup>L</sup> = 3.6 event, and group #3), 28 July (including an M<sup>L</sup> = 3.5 event at group #2), 4 August (group #5), 6 August (groups #2 and #5), and 12 August (group #7). A small isolated cluster of deeper events (~12 km; group #9) occurred on 29–30 August, ~4 km ESE of group #1, at the eastern end of the study area, initiated by an M<sup>L</sup> = 3.2 earthquake. On 1 September 2021, an increase in the seismicity rate was observed in the group #6 area (dark gray), followed by an M<sup>w</sup> = 4.1 event on 2 September in the same area. The latter also caused an increase in the seismicity rate, mainly at groups #2, #4, and #5 and less at groups #3 and #11, while events began occurring at group #10 (light gray). An abrupt increase in the seismicity rate of group #11 followed the occurrence of two M<sup>L</sup> = 3.5 events on 10 and 11 September in the same group, which also triggered events at group #10. A small cluster was triggered between 16 and 19 September at group #3, including four events with 2.5 ≤ M<sup>L</sup> ≤ 2.8. Another outbreak at group #11 occurred on 20 September, including an M<sup>L</sup> = 3.2 event on 21 September and an M<sup>L</sup> = 3.4 event on 22 September. A significant burst of seismicity at group #10 was triggered by an M<sup>L</sup> = 3.4 event on 23 September. Lastly, group #12 (brown) at the eastern end of the main part of the 2021 sequence increased seismic activity after 3 October without any major event. The strongest event since then was an M<sup>L</sup> = 2.8 earthquake on 10 October 2021 at group #11. The spatiotemporal projection in Figure 5, along the WNW–ESE-oriented profile A–B in Figure 4a, roughly parallel to the main direction at which the epicenters are aligned, reveals the complex evolution of the 2021 Thiva earthquake sequence. The swarm was initiated on 10 July 2021 at the western end of the activated zone, spanning along groups #2 and #3, with a few events belonging to the neighboring group #6 at the eastern edge of the activated patches. On the outbreak of 20 July, the major Mw = 4.2 event occurred at group #2, but seismicity was also triggered eastwards to group #4, while seismic activity began at group #5, which presented some more bursts between 28 July and 8 August. The small isolated cluster #7, north of the main seismic cloud and slightly to the west of group #5, was activated between 12 and 15 August. A 2-week period of relative quiescence followed. Then in September, the outbreak at group #6 began with some foreshocks, followed by an Mw = 4.1 event on 2 September, mainly triggering aftershocks to the eastern half of the activated zone, including group #5 and a few events at group #2, at which the activity ceased later. Seismicity continued to spread eastwards, with few events at group #4. The next seismic burst occurred at groups #10 and #11 between 10 and 13 September. A last temporal cluster at group #3 took place between 16 and 19 September, followed by reactivation of groups #11 on 20 September and #10 on 23 September. Lastly, cluster #12 occurred at the easternmost end of the 2021 Thiva swarm. Overall, the seismicity presents a general tendency for spatiotemporal migration towards ESE. This is not straightforward, as the early outbreaks are mainly confined at the western half, between groups #2 and #3, then spread suddenly to group #4 and later group #5. Since September 2021, however, seismicity mostly occurs at the eastern half of the activated zone. Foreshock seismicity near the origin of the Mw = 4.1 event of group #6 spreads towards WNW at a distance of ~1.5 km within 1 day, where it triggered an M<sup>L</sup> = 3.5 event at group #2. However, a slower seismicity triggering front can also be observed, spreading towards the east, including some events at group #4, then passing by the eastern edges of group #11 and later group #12, at a migration speed of ~70 m/day (Figure 5). Considering the possibility that this part of the seismicity is induced by the fluid-driven diffusion of pore pressure through the fracture network, a parabolic seismicity triggering front can be drawn according to the relation of [68]: $$r(t) = \sqrt{4\pi Dt} \tag{1}$$ where r(t) is the distance from the beginning of fluid injection at the origin and t the time elapsed since then, while D is the hydraulic diffusivity. This front would begin on ~1 September near the origin of the Mw = 4.1 event at group #6 and spread eastwards through mid-October, with a hydraulic diffusivity value between 0.20 and 0.25 m2/s (Figure 5). It can be observed that most of the eastward migrating seismicity is located behind the proposed parabolic triggering-front envelope, although some sparse events, mainly those of groups #4 and #8, are located on the outside, possibly triggered due to pore-fluid diffusion following paths of higher hydraulic diffusivity values. #### 3.3. Focal Mechanisms and Activated Structures at Depth Focal mechanism data for the 4 larger events of the sequence (Mw > 4.0) are available from routine waveform modelling and moment tensor inversion analysis performed at SL-NKUA and GI-NOA. The fault-plane solution of the 2 December 2020 Mw = 4.6 Kallithea event is adopted from [36]. In addition, first-motion polarities (FMPs) were manually picked for 20 significant events of smaller magnitude (2.8 ≤ M<sup>L</sup> ≤ 3.6) from visual inspection of their recordings at local and regional stations of HUSN (Table A1 in Appendix B and Excel File S2 in the Supplementary Material). For each event, taking into account the relocated foci and the respective azimuth and angle of incidence corresponding to each station, a grid search was applied in the whole range of strike, dip, and rake (ϕ, δ, λ) values to find solutions compatible with the observed FMPs. In cases where no such solution was available, solutions with a lower percentage of compatibility with the FMPs were sought, considering the analyst's quality estimate of each FMP, with a lower weight applied to measurements of emergent P arrivals or uncertain polarities. Each individual solution compatible with a minimum percentage of polarities was converted into a unitary doublecouple moment tensor from the respective ϕ, δ, and λ parameters, and their sum provided an average focal mechanism for each event. Then, the RMS angular difference between average and individual solutions was measured in terms of their Kagan [69] angle, and solutions with an RMS < 25◦ were retained. The accepted focal mechanisms have at least 13 FMP measurements with over 90% compatibility. The fault plane solutions of the major events, presented in Figure 6, indicate dominant normal faulting in an E–W to WNW–ESE direction, with few events presenting obliquenormal kinematics. The spatial distribution of the relocated hypocenters and the acquired focal mechanisms were examined in a series of cross sections, oriented N200◦ E (Figure 7). This direction is roughly perpendicular to the general trend of the mapped faults on the surface, which are in line with the focal mechanisms and with the main axis of the distribution of epicenters (i.e., N 110◦ E-oriented profile line A–B in Figure 4a). Starting from the westernmost cross section, a1–a2, groups #2 and #3 appear to correspond to the deeper and shallower parts, respectively, of a steep, south-dipping structure. This is represented by the red dashed line, which is the cross section of a least-squares plane determined from the joint distribution of groups #2, #3, #5, and #10 (see also the 3D model of Interactive Matlab Figure S1 in the Supplementary Material). The same pattern persists in section b1–b2, where, in addition, a few events of group #5 appear below group #2. The small isolated cluster #7 is also presented, with a dashed line showing the cross section of the least-squares north-dipping plane determined from this group and extrapolated a few km updip. Figure 6. Map of relocated epicenters of the 2020–2021 Thiva earthquake sequence and focal mechanisms of the major events. The labeled (a1–a2 to h1–h2) dashed rectangles indicate the boundaries and orientation (N 200◦ E) of the respective cross sections of Figure 7. The red dashed line is the proposed eastward extension of the Yliki fault. Figure 7. Vertical cross sections drawn in a N 200◦ E direction along the profiles in Figure 6. Dashed lines at depth are inferred planes from the distribution of relocated hypocenters, whereas dashed lines at the top indicate the planes of known faults on the surface, that is, the eastward extension of Yliki fault (red dashed line in Figure 6) and Kallithea fault. See also Interactive Matlab Figure S1 in the Supplementary Material for a 3D model of the earthquake sequence and the activated structures. In the cross section c1–c2, the distribution of hypocenters becomes more complex, with the introduction of more spatial groups. The dark green dashed line, at a lower dip angle than the red one, represents a least-squares plane determined from the distribution of groups #5, #6, and #10. This plane seems to be consistent with the eastward extension of the Yliki fault when extrapolated downdip at an angle of 65◦. Sparse seismicity belonging to group #4 can be observed in this slice. The cross section d1–d2 marks the beginning of the eastern half of the 2021 sequence, mainly activated after September. The dashed green line is consistent with the distribution of groups #6 and #10, as well as the remaining events of group #5, whereas groups #2 and #3 are not present in this slice. In the cross section e1–e2, a dense cluster of group #4, activated on 20 July 2021, can be observed, apparently belonging to a north-dipping structure (purple dashed line). The brown dashed line is the cross section of a least-squares plane determined from the distribution of groups #11 and #12, also north-dipping, but at a steeper dip angle. The latter structures can also be observed in the cross sections f1–f2 and g1–g2. The plane determined from groups #11 and #12 is oblique to the direction of the cross sections, increasing its distance from the plane of group #4 toward the east. Finally, the cross section h1–h2 shows the distribution of group #1, which is related to the shallow 2 December 2020 Mw = 4.6 event associated with the south-dipping Kallithea fault. The relocated epicenters of the mainshock and most aftershocks (Figure 6) are clustered south of the Kallithea fault trace with the hypocentral distribution at depths shallower than 4 km being roughly subvertical but apparently favoring a south-dipping structure when considered together with the mainshock in agreement with [36]. The absence of a local seismological network in the area during the occurrence of the 2020 sequence limits our capacity to constrain the hypocentral depths for this group; however, the shallow depth of the rupture (or deformation source) is supported by the observed surface displacements given that almost two fringes are visible in Sentinel-1 coseismic interferograms [36]. #### 4. Coulomb Stress Transfer Due to the 2 December 2020 Mainshock In the following, we calculate the effect of the 2 December 2020 Mw = 4.6 earthquake at Kallithea on the redistribution of stress in the study area to examine possible stress load to the zone that was activated after 10 July 2021 near Thiva. For this purpose, we adopt the geodesy-favored fault model in [36], determined through modeling of the observed surface deformation together with seismological data, which associated the earthquake with a shallow rupture of the Kallithea fault. Taking into account the abovementioned fault geometry and kinematics, with strike = 120◦, dip = 48◦, rake = −74◦, and net slip of 240 mm, homogeneously distributed on the fault plane with a rupture top at 0.70 km and bottom at 1.44 km (fault length L = 2 km, width W = 1 km), we calculate the Coulomb stress transfer using the Coulomb 3.3 code [70] with an effective coefficient of friction μ = 0.4. The Coulomb stress transfer model, determined for optimally oriented normal faults according to the regional stress field, which is dominantly extensional with a subhorizontal S<sup>3</sup> axis oriented ~N186◦ E [71], is presented in Figure 8. The calculations indicate that a stress load greater than +0.1 bar is mainly confined at depths shallower than 6 km, whereas no significant stress transfer can be inferred at the seismogenic depths of the 2021 seismic swarm, which are between 7 and 12 km (Figure 7). Even at shallower depths, where the main rupture occurred, the +0.1 bar contour hardly reaches the eastern edge of the 2021 seismic sequence epicentral area. Furthermore, the nucleation of the 2021 sequence occurred at its western edge, that is, groups #2 and #3 (e.g., white star with blue outline in Figure 8), located at an even further distance from the Kallithea fault. This suggests that static stress transfer due to the 2 December 2020 mainshock could not have possibly triggered the 2021 seismic swarm. Figure 8. Coulomb stress transfer due to the 2 December 2020 mainshock at Kallithea (yellow star with red outline), following the fault model in [36], marked with a red rectangle, for normal faults optimally oriented to the regional stress field, at different depths Z: (a) Z = 0 km, (b) Z = 3 km, (c) Z = 5 km, and (d) Z = 8 km. Gray circles represent the relocated epicenters of the 2020–2021 Thiva seismic sequence, with the white star with blue outline marking the location of the 11 July 2021 Mw = 4.2 event. The dashed line shows the trace of the fault plane (extended downdip) at the depth of the respective horizontal slice. Black lines are fault traces after [35], the NOAfaults database [15,16], and the eastward extension of Yliki fault (Figure 6, dashed red line). #### 5. Instrumental and Imaging Geodesy to Map Local Deformation Over the past 30 years, Interferometric Synthetic Aperture Radar (InSAR) and in particular Multi-temporal InSAR (MT-InSAR), as well as Global Navigation Satellite System (GNSS) networks, have been established as methodologies capable of monitoring surface deformations in urban and rural environments with millimeter resolution [72–77]. However, a GNSS network provides information only about a few points, but is characterized as a dynamic real-time monitoring system of high precision in all three motional components. On the other hand, spaceborne SAR interferometry provides a large number of measured points over long time periods. Moreover, GNSS systems have a big economic cost. In addition, SAR interferometry has the capability to remotely monitor areas much wider than traditional surveying techniques without the necessity to install in situ ground instrumentation. The pre- and coseismic deformation of the Thiva area was investigated based on MT-InSAR results using interferometric data from the Sentinel-1 A and B satellites of the Copernicus program. Copernicus Sentinel-1 A and B SAR scenes are freely available from the Sentinel Hub portal [78] and can provide improved SAR SLC (Single Look Complex) data, ensuring (a) continuous, all-weather, day and night imagery; (b) rapid revisit period in the same imaging mode (6 days); (c) constant and regular acquisition to build a large global archive; and (d) wide area coverage, thanks to the 250 km image swath width. Satellite geodetic data (GNSS) were used to calibrate and validate the interferometric results, while the location of the geodetic device acted as a reference point during the interferometric processing. #### 5.1. GNSS Geodetic Data to Map Local Deformation GNSS data from continuous stations (Figure 1, inverted yellow triangles) were processed for the period of January 2017 to November 2021. The available data cover the time prior to the outbreak of the seismicity, as well as the coseismic period. One of the analyzed stations, THIV (Figure 3), belonging to the HexagonSmartNet commercial network by Metrica S.A. [79], is located at Thiva and was operating continuously during the 2020–2021 earthquake sequence. Partial data from two more sites in Thiva were processed for the year 2018 and for the period 2019 to 2020 (belonging to the Uranus network by TreeComp [80]). The processing of the raw GNSS data was performed using the precise double-difference method with the Bernese v5.2 GNSS software [81]. The precise orbital solutions from the Center for Orbit Determination in Europe (CODE) were introduced in the estimation of the station's coordinates. The time series of all three components of the daily coordinates of the station THIV on the global ITRF2014 reference frame area are presented in Appendix C (Figure A6). The velocity vector of the THIV station was estimated to be VNorth = −8.97 ± 0.04 mm/yr, VEast = 9.29 ± 0.04 mm/yr, and VUp = 1.16 ± 0.08 mm/yr. For the two other GNSS sites in Thiva, a similar velocity vector was estimated, although they refer to a slightly different time period. These velocity values are in accordance with previous estimations for the station THIV [5,82]. Based on the continuous daily solutions of the site THIV, it appears that the increased seismicity in the area in 2021 has not caused any observable static ground deformation or alteration of the velocity vector. The only noticeable change that was identified was a small alteration (changing point of the rate) on the vertical component since May 2019. More specifically, a slightly increased uplift motion was detected, from 0.09 ± 0.23 mm/yr prior to May 2019 to 1.18 ± 0.39 mm/yr, for the rest of the period. Moreover, a distinctive seasonal signal is emerged in the vertical component that could be associated with the seasonal fluctuations of the groundwater horizon. Calculation of the baseline changes between the THIV GNSS site and stations located in Chalkida (~30 km NE from Thiva) and Corinth (~54 km SW of Thiva), with both sites belonging to HexagonSmartNet, showed a linear increase during the whole time period of the distance between Thiva and Chalkida, as well as between Thiva and Corinth, equal to 0.8 ± 0.1 mm/yr and 4.0 ± 0.1 mm/yr, respectively. These results are compatible with the extensional regime of the broad area that has been previously described [5,82]. The time series of the Uranus stations was similar to the one presented here. These data were acquired at a high rate (1 Hz). A close examination during the occurrence time of the three earthquakes with M<sup>w</sup> ≥ 4, with their epicenters located 2.8 km NE, 1.1 km NNE, and 0.6 km NW of the THIV GNSS station, respectively, yields that they caused no displacement waveform. In an effort to define the source of the fluctuations in the vertical component, a joint interpretation of the local rainfall data and the geodetic data was performed. Rainfall data were recorded from a local meteorological station within Thiva. This station is operated by the METEO unit at the National Observatory of Athens and is part of the NOAAN network, consisting of 430 automatic surface weather stations in Greece, which monitors all basic meteorological variables, including rainfall, at 10 min intervals [83]. In Figure 9, the accumulated rainfall time series is shown upon the vertical THIV GNSS displacement time series, the latter being temporally shifted, after visual inspection, by 3 months towards the past, in order to minimize the difference between the rainfall variation and its impact to the ground observation. That is why the date of the alteration shown was previously mitigated from May 2019 to February 19 of the same year. In the diagram, a correlation of the peaks and valleys between the two time series within ±2 months is observed. Moreover, a similar correlation of the amplitudes of the two time series can be identified until October 2019. Then, there is no clear connection between the two datasets, except in October 2020. Towards the end of the examined period, we observe an intense decrease in the values of both curves. Thus, the vertical component of GNSS seems to be driven mainly by the water withdrawal to recovery equilibrium. From this, it can be inferred that the uplift observed after May 2019 can be attributed to the increase in the rainfall. The latter indicates that the observed vertical ground deformation is mainly controlled by the changes on the groundwater level horizon in the broad Thiva area and cannot directly be associated with the recorded seismic activity or be considered as a tectonic result. Figure 9. Two-month moving average time series of the vertical component of the THIV GNSS station (black) temporally shifted by 3 months towards the past and detrended accumulated rainfall time series (orange) after removal of its linear regression. The origin times of the Mw > 4.0 earthquakes are depicted with dashed red lines. #### 5.2. Imaging Geodesy For the InSAR analysis, 177 acquisitions of Copernicus European Sentinel-1 SAR SLC over Thiva were used, available on the Copernicus Open Access Hub [78]. For the processing, a 90 m/pixel digital elevation model (DEM) was used [84]. The Sentinel-1 products were acquired in interferometric wide (IW) swath mode consisting of three subswaths with a series of bursts covering a swath of 250 km. Specifically, 89 acquisitions on ascending (track 102) and 88 images on descending (track 09) were used, covering the period from January 2018 to September 2021. The Multi-temporal InSAR data processing was carried out following the Small BAseline Subset (SBAS) MT-InSAR approach [85] due to the characteristics of the study area (mostly covered by rural areas) using the ENVI SARscape® software (L3Harris Geospatial, Boulder, CO, USA). The preprocessing phase includes the orbit correction of every image, the burst selection over the study area, and the image coregistration using a master image in order to generate interferometric pairs. On the main processing, wrapped interferograms were generated with a multilook range and azimuth (5 × 1), and 90 m/pixel DEM was used to subtract the topographic phase. The wrapped interferograms were unwrapped using the minimum cost flow (MCF) method [86] and converted into line-of-sight (LOS) displacement. The reference for both tracks was set up in the location of the GNSS station THIV. Considering that InSAR displacement in LOS only measures the path length difference between the earth surface and the satellite, displacement decomposition was carried out, exploiting the ascending and descending sensing trajectories to recover the vertical (up–down) and horizontal (east–west) deformation. The vertical displacement map over the study area (Figure 10) shows mainly subsiding sites with a maximum cumulative displacement of about −40 mm. Nevertheless, there are areas where localized uplift took place with maximum values of 20–30 mm. On the time series plot (Figure 10, inset panel), 12 scatterers were selected to observe the vertical deformation pattern during the period of satellite acquisition (June 2018 to September 2021). As observed in the plot, the scatterers that were located inside Thiva (9, 11, and 12) are relatively stable, compatible to the GNSS result. The same pattern is followed by those that are located south of Thiva (2, 3, and 4), while scatterers 1 and 5 show a subsidence trend, especially after the 2 December 2020 mainshock, up to −10 and −19 mm, respectively. The highest uplift deformation is observed on scatterer 4, up to 15 mm, not associated to the earthquake (Mw > 4.0) occurrences. A different pattern is recorded in the vicinity of Kallithea (scatterers 6, 7, 8, and 10) characterized by subsidence trends. The deformation pattern in these scatterers is altered significantly after the occurrence of the Mw > 4.0 earthquakes during the processing period, as can be seen in Figure 10 (red lines). Scatterers 6, 7, and 8 show an increased rate of subsidence caused by the 2 December 2020 event, while scatterer 10 is characterized by a smaller subsidence rate. Figure 10. (Map) Vertical cumulative displacement inferred by the SBAS method. The relocated epicenter of the 2 December 2020 earthquake is presented with a white star, while black stars depict the relocated epicenters of major events (M ≥ 3.5) of the 2021 seismic swarm. Faults (dashed black lines) after NOAFAULTS [15,16] as Sr: Soros; Ka: Kallithea; K8: Kallithea 8. (Lower-right inset panel) Diagram of the cumulated vertical SBAS time series for 12 points distributed over the study area (red dots with numbers on the map). Red vertical lines mark the origin time of the major events (Mw > 4) of the 2020–2021 sequence, also marked on the map as follows: a: 2 December 2020; b, including b1: 11 July 2021 and b2: 20 July 2021; and c: 2 September 2021. The reference point for both the displacement and the inset time series is the GNSS Station THIV (black triangle). #### 6. Discussion The 2020–2021 seismic sequence at Thiva, Central Greece, is a peculiar case of an Mw = 4.6 earthquake (on 2 December 2020) occurring at a relatively small distance (6–12 km) from a quite productive seismic swarm, which started ~7 months later. The 2 December 2020 event was interesting on its own, given that it was an unusually shallow earthquake, with its rupture expanding downdip, which produced observable deformation of about two fringes, on Sentinel-1 interferograms, on the surface, permitting its association with the Kallithea fault [36], despite its relatively small magnitude. It is worth noting that earthquakes with similar shallow depths have been reported worldwide, as the 2014 M<sup>w</sup> = 5.1 La Habra earthquake in California at a depth of 2 km [87], a Mw = 4.9 earthquake in France at a depth of 1 km [88], and a M<sup>w</sup> = 5.0 earthquake in Ecuador at a depth of 1.5 km [89]. At the time, there was no indication that this earthquake would be followed by an intense swarm near Thiva, as previous seismicity in the area was generally low and no increase in the seismicity rate could be observed until July 2021 (Figure A4). In the seismic catalogue in [6] for the periods of 1900 to 2009, only few events with M<sup>w</sup> ≥ 4.0 are located in the broad area of Thiva; few sparse epicenters to the south are mainly related to aftershocks of the major earthquakes of 1981 in the eastern Gulf of Corinth owing to significant location uncertainties. In this work, the parameters of four destructive events that have affected Thiva in the period of 1853–1917, all without any surface fault traces, were recalculated using macroseismic data (Table 1). These indicated seismic activation a few kilometers west of Thiva in 1853 and 1893, both of equivalent moment magnitude of M<sup>w</sup> = 6.2, as well as an M<sup>w</sup> = 4.5 event near Thiva in 1917. The epicenter of the M<sup>w</sup> = 6.0 1914 event is located between Thiva and Kallithea, possibly within the area covered by the recent 2021 spatial groups in Figure 4. Coseismic effects, including ground effects, were reported for previous earthquakes with I > 8 in Thiva and its vicinities, in accordance with the estimated macroseismic parameters presented in Table 1. A series of small shocks, of intensity I < 5, originating from the study area throughout 1915 (Figure 2b) were also recorded, likely attributed to a seismic swarm or to aftershocks triggered by the 1914 mainshock. Such activity, characterized by small events of intensity I < 5, has similarities to the recent seismicity. Since the beginning of the enhanced monitoring by the HUSN in 2007, only sparse seismic activity has been detected near Thiva with magnitudes mainly M<sup>L</sup> < 3, with the largest event (M<sup>w</sup> = 4.0) occurring near Yliki Lake on 25 June 2017 (see also the New Seismotectonic Atlas of Greece [13] for an overview of previous activity in the area). The application of double-difference relocation in this study produced a high-resolution catalogue that permitted the distinction of several clusters within the seismicity cloud of the 2021 swarm (Figure 4). The spatiotemporal distribution of the sequence suggests that these spatial groups also exhibit strong temporal clustering, with a series of outbreaks related to the larger events of the sequence. The resolved focal mechanisms indicate mainly normal dip-slip and, to a lesser extent, oblique-normal faulting (Figure 6), compatible with the regional stress of the area [71]. Through detailed cross sections and a 3D model of the spatial distribution of hypocenters (Interactive Matlab Figure S1 in the Supplementary Material), it was possible to identify the likely geometries of the activated structures at depth. In contrast to the shallow 2 December 2020 mainshock, which is associated with the Kallithea fault by straightforward interpretation of the observed displacement pattern [36], the deformation due to the 2021 swarm did not reach the surface. This is explained by the larger focal depths of the major events of the swarm, combined with their low magnitudes. Furthermore, the epicentral area of the 2021 swarm is void of mapped faults (Figure 1). Specifically, the traces of known south-dipping faults in the east, including Kallithea fault, having a WNW–ESE trend, compatible with the focal mechanisms and general geometry of the structures involved in the 2021 seismicity, are terminated ~1–2 km before reaching the first epicenters of the swarm. The same is true for the mapped faults west of the swarm, which are less compatible in terms of their strike. To the south, a south-dipping fault is mapped near the edge of the southern cluster #4, which is associated with a north-dipping structure. To the north of the epicenters, there is a plain of agricultural land without mapped faults or any apparent anomaly in the topography. The only likely candidate fault that could be associated with some of the activated structures at depth is the south-dipping Yliki fault at the southern coast of Yliki Lake. Even so, the fault trace would have to be extended by a few km towards ESE (Figure 6) in order to match the updip extension of some of the activated structures at depth, particularly those related with the major (M<sup>w</sup> > 4.0) events of the sequence. Other identified structures at depth seem to correspond to steeper faults that would outcrop south of Yliki fault. Two of the identified planes at the eastern part of the swarm suggest a north-dipping fault. The latter cannot be associated with any of the mapped faults on the surface. The initiation of the 2021 seismic swarm took place near its western extreme, about 10 km WNW of the 2 December 2020 mainshock's relocated epicenter. In the constructed Coulomb stress transfer model for this earthquake, it became apparent that, even with a fault model derived by geodetic data [36], which produced a slightly overestimated moment magnitude (Mw = 4.7), stress loading for optimally oriented normal faults under the concurrent regional stress regime drops below +0.1 bar, which is regarded as the lower threshold capable for stress-trigger earthquakes [90], near the eastern margins of the swarm (Figure 8). Given that the nucleation of the 2021 sequence took place at its western end, with an Mw = 4.2 event on 11 July 2021, the Coulomb stress transfer due to the 2 December 2020 mainshock is not considered sufficient to trigger the 2021 swarm. This is further justified by the shallow depth of the 2 December 2020 event, but the result, concerning stress transfer at the seismogenic depths of the 2021 swarm, would be the same even if the 2020 mainshock's fault model was placed ~5 km deeper. Therefore, despite seeming counterintuitive, there appears to be no causative link between the 2 December 2020 mainshock and the occurrence of the 2021 swarm; that is, there is no evidence that the former accelerated the occurrence of the latter. To further examine other possible triggering mechanisms, we employed instrumental and imaging geodesy to investigate pertinent local deformation transients that may have played a role in the stress loading of the area. As in reported cases in the literature, a slow slip event (e.g., [91]) or uplift/subsidence due to groundwater level changes (e.g., [92]) could have increased the applied stresses to the faults of the area, including Kallithea fault and the fault system that hosted the 2021 swarm. This would have the potential to induce seismicity at both places and indicate a common cause for their seismic excitation. However, no significant anomalies in the displacement components of the GNSS station THIV, located at Thiva, could be detected (Figure A6), with the station presenting the anticipated regional horizontal motion and a steady long-term uplift at a slightly increased rate of 1.18 ± 0.39 mm/yr since May 2019. It is noted that the variability of vertical displacement at THIV was found to partially correlate with the detrended variability of accumulated rainfall at a local meteorological station with a lag of ~3 months (Figure 9). This likely indicates a delayed response of the vertical ground displacement due to the groundwater equilibrium, but the uplift trend prevails. Similar cases of uplift incidences due to recharging of the aquifer after near-record rainfall were documented by [93] using both GNSS and InSAR, supported by well level signals. The seismic swarm was initiated at the time of a highly decreased rate of the detrended accumulated rainfall that started declining 1 year before. This observation alone cannot support the groundwater level changes as a triggering cause of the seismic activity. By employing InSAR data and through the application of the SBAS method, downlift or uplift, cumulated through the period between 2018 and September 2021, with reference to the GNSS station THIV, were detected at several localities in the study area. The examination of the temporal variation of vertical displacement at selected scatterers (Figure 10) showed subsidence associated with the Mw = 4.6 2 December 2020 earthquake in the vicinity of Kallithea fault. On the other hand, no significant anomalies in the vertical displacement were recognized at scatterers near or south of Thiva, that is, the epicentral area of the 2021 swarm. Although coseismic displacements on the surface were not expected to be detected for the major events of the 2021 swarm, due to the small magnitudes and larger focal depths compared with the 2 December 2020 mainshock, the lack of preseismic deformation indicates no detectable changes that could be associated with slow slip or groundwater level changes, affecting the local stress. Although the initiation of the 2021 swarm could not be explained by Coulomb stress transfer due to the 2 December 2020 mainshock, its evolution can be partly attributed to stress triggering. The major earthquakes of the swarm in July 2021 occurred at its western end, and the swarm progressed eastward in a cascade of outbreaks related to major events. Following a small interval of low seismic activity in the second half of August 2021, a second stage of the swarm was initiated by an M<sup>w</sup> = 4.1 event on 2 September. However, part of the continuing eastward migration of seismicity could possibly be attributed to porepressure diffusion, as seismicity is roughly bounded by a parabolic envelope of hydraulic diffusivity D = 0.25 m2/s (Figure 5). On a larger temporal scale, seismicity tends to spread eastward at a rate of ~70 m/day since August 2021. During the continued monitoring of earthquake activity in the study area at SL-NKUA, after 16 October 2021, that is, the end of the period studied in this work, the seismic swarm gradually diminished. Fewer than 300 events of M<sup>L</sup> ≤ 2.6 occurred until the end of January 2022 east of Thiva, where seismic activity was already observed in the first half of October 2021. Both the abovementioned hydraulic diffusivity and the migration rate are comparable with the respective values previously measured in fluid-associated earthquake triggering at the Western Corinth Gulf (e.g., [9,10,12,94,95]). Seismic swarms are commonly observed in areas related to volcanic activity, involving magmatic or hydrothermal fluids (e.g., the 2008– 2009 swarm at Yellowstone Lake [96] or the 2008 swarm at Vogtland/NW Bohemia [97]), or induced by water injection in geothermal fields (e.g., the 1993 swarm in Soultz-sous-Foret, France [98]). In tectonic environments, where seismic swarms are less frequent, the fluids that drive seismicity can be of meteoric origin, as has been suggested for cases such as the 2003–2004 swarm in the Western Corinth Gulf [99] or the 2011 swarm in Oichalia, Southern Greece [100], as well as for the 2002 swarm in Hochstaufen Mountain, SE Germany [101], and the 2003–2004 swarm in the Ubaye-Argentera area in the southwestern French–Italian Alps [102]. Another possible source could be the subducting oceanic slab through dehydration, with fluids migrating upwards (e.g., [103]). The intrusion of fluids through fissures or the fracture network can induce seismicity either by localizing stress or by increasing pore pressure, reducing the effective normal stress, and facilitating aseismic creep. During the evolution of fluid-driven swarms, small asperities embedded within the faults are breaking, as creeping of the surrounding surface proceeds, generating multiplets, caused by repeated slip on the same fault patch. Larger asperities may generate the major events of a swarm, transferring static stress to neighboring fault patches where aftershocks are triggered, causing the seismicity rate to increase abruptly, then to gradually decay following Omori's law. Therefore, swarms evolve as a consequence of both fluid intrusion and stress transfer, as has been suggested for the case of the seismic swarm of 2000 in Vogtland/NW Bohemia [104], and even involve episodes of aseismic slip, as in the October 2015 seismic swarm in Malamata, in the Western Corinth Gulf [12]. Spatial leaps in the triggering of earthquake clusters (e.g., group #4 of the 2021 Thiva swarm) or delayed major events (e.g., the 2 September 2021 event in group #6) could also be explained by subcritical crack growth due to stress corrosion attributed to chemical action at the crack tips (e.g., the earthquake swarm at Hida Mountains, Central Japan [105]). Another possible mechanism for gradual fault weakening is the erosion of the fault walls by fluids, which has been suggested for the 2008 swarm in Vogtland/NW Bohemia [106]. The short time period of relative seismic quiescence between 15 and 31 August in the Thiva area suggests that stress-transfer-related triggering effectively ceased in the western part of the swarm, but stress-induced corrosion, fluid erosion, or another aseismic factor, such as creeping or intrusion of subsurface fluids, were undermining the faults of the eastern half. The signature pattern of fluid-driven pore-pressure diffusion is the observed parabolic envelope in the spatiotemporal projection of Figure 5, indicating an expanding triggering front, starting from an injection point that roughly coincides with the location of the 2 September 2021 event. It is likely that the latter earthquake created a new pathway for the propagation of pressurized fluids, which caused seismicity to migrate further eastwards. As evidenced by historical data, Thiva has experienced destructive M<sup>w</sup> ≥ 6.0 earthquakes in the past. Even though there are no significant recent events in either Kallithea or Thiva, macroseismic data suggest a past activation of the area in between. This points out that the 2020–2021 earthquake sequence does not reflect the full seismic potential of the study area. The local densification of the seismological network has permitted the detailed mapping of the activated structures at depth and the enhancement of the detection threshold. The fault segmentation, leading to a cascade of small outbreaks, and the b-value of the Gutenberg–Richter law of the 2021 seismic swarm, which is near or slightly above unity (b ≈ 1.07 by the least-squares method, or b ≈ 1.14 by the maximum likelihood method for a magnitude of completeness of M<sup>c</sup> = 1.6; Figure A5), indicates that this particular seismogenic volume is unlikely to produce a large event at this time. However, other neighboring fault zones could possibly be triggered due to the cumulative stress transfer caused by the major events of the sequence. This could have potential implications to the seismic hazard of the area, as several seismogenic sources have been recognized in the vicinity of Thiva (e.g., [107]). It is also worth noting that the evolution of a seismic swarm is not always similar to the one of the present study. Recently, on 27 September 2021, while the Thiva swarm was still active, a destructive earthquake (Mw = 6.0) occurred in Arkalochori in central Crete [31,32]. In Arkalochori, the seismic activity was initiated about 4 months before the mainshock, in early June 2021, as an earthquake swarm. However, the evolution of the Arkalochori sequence was dramatically different, culminating in the M<sup>w</sup> = 6.0 mainshock that caused one fatality and hundreds of damaged buildings. In that instance, the seismic swarm was characterized, retrospectively, as a foreshock sequence. It is, therefore, important to continue monitoring the microseismic activity of the area of Thiva, in case seismicity migrates to neighboring structures. #### 7. Conclusions We performed a detailed seismotectonic analysis of the 2020–2021 earthquake sequence in Central Greece involving a mainshock–aftershock sequence in December 2020 at Kallithea fault and a seismic swarm in July–October 2021 near Thiva. Through highresolution double-difference relocation of the earthquake catalogue, we identified a system of conjugate WNW–ESE-trending normal faults below Thiva with several fault segments being triggered at different times, producing a complex seismic swarm that evolved in a series of bursts. Seismicity mainly migrated from west to east, with triggering mainly attributed to stress redistribution due to the larger events of the sequence. The swarm could be divided in two stages, the first concerning its western half, initiated by an M<sup>w</sup> = 4.2 event on 11 July 2021, and the second triggered by an M<sup>w</sup> = 4.1 event on 2 September 2021, activating its eastern half. In the former stage, the seismic activity can be partly related to the, inferred, eastward extension of the south-dipping Yliki fault at the southern margin of Yliki Lake, north of Thiva. However, activity at more steeply dipping structures could also be identified. The second stage, likely related to the eastern termination of Yliki fault, also generated seismic activity on some conjugate north-dipping structures with some indications of triggering by overpressurized fluids, with seismicity migrating eastwards following a pore-pressure diffusion front. We applied a multidisciplinary approach to investigate possible triggering mechanisms of the swarm. Coulomb stress transfer due to the M<sup>w</sup> = 4.6 2 December 2020 earthquake was considered insufficient to trigger the July–October 2021 swarm near Thiva, as stress loading much lower than +0.1 bar was measured at the nucleation site. Furthermore, surface deformation measurements through GNSS and InSAR did not show any significant anomalies, which could be related to phenomena such as subsidence due to groundwater withdrawal or slow slip, which could have triggered the 2021 seismic swarm. Despite the apparent association of the December 2020 event with the July–October 2021 swarm, due to their proximity in both space and time, no causal relation could be derived between the two sequences. Subsurface fluids under pressure seem to have played a significant role in the evolution of the 2021 seismic swarm, either causing local stress concentrations or facilitating slip by lowering the effective normal stresses or weakening the faults' walls through erosion. The rich seismic activity in the conjugate normal fault system below Thiva likely released the accumulated stress in this particular area. However, considering the previous historic records of major M > 6 events in the vicinity, the occurrence of a significant earthquake at the unruptured downdip extension of Kallithea fault or in the seismic gap between Kallithea and Yliki faults cannot be excluded. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/app12052630/s1, Excel File S1: Relocated catalogue of the 2020–2021 earthquake sequence in Thiva; Excel File S2: Focal mechanisms determined by first-motion polarities (FMPs); Interactive Matlab Figure S1: 3D model of the 2020–2021 earthquake sequence at Thiva. Author Contributions: Conceptualization, G.K., P.E., V.S. and I.P.; methodology, G.K., V.K. (Vasilis Kapetanidis), I.S., V.K. (Vasiliki Kouskouna), P.E. and V.S.; software, V.K. (Vasilis Kapetanidis), N.S., P.E., A.K. (Andreas Karakonstantis), A.K. (Andreas Karavias), V.S. and T.G.; validation, G.K., I.S., V.K. (Vasiliki Kouskouna), P.E., I.K., J.D.A., P.P., N.V. and I.P.; investigation, V.K. (Vasilis Kapetanidis), I.S., N.S., A.K. (Andreas Karakonstantis), A.K. (Andreas Karavias) and T.G.; resources, G.K., V.K. (Vasiliki Kouskouna), P.E., V.S., I.K., J.D.A., P.P., N.V. and I.P.; data curation, G.K., V.K. (Vasilis Kapetanidis), I.S., N.S., V.K. (Vasiliki Kouskouna), P.E. and V.S.; writing—original draft preparation, G.K., V.K. (Vasilis Kapetanidis), I.S., N.S., A.K. (Andreas Karakonstantis), V.K. (Vasiliki Kouskouna), P.E., V.S. and I.P.; writing—review and editing, G.K., V.K. (Vasilis Kapetanidis), I.S., N.S., V.K. (Vasiliki Kouskouna), P.E. and V.S.; visualization, V.K. (Vasilis Kapetanidis), P.E., A.K. (Andreas Karavias) and V.S.; supervision, G.K.; project administration, P.P. and N.V.; funding acquisition, G.K. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The initial earthquake catalogues and arrival-time data are available at the database of SL-NKUA (http://www.geophysics.geol.uoa.gr/stations/gmapv3\_db/index.php? lang=en; accessed on 31 January 2022) and GI-NOA (http://bbnet.gein.noa.gr/HL/databases/ database; accessed on 31 January 2022). The relocated catalogue of this study is available in the Supplementary Material (Excel File S1). Seismological waveform data from permanent and temporary stations of the HUSN are available at the European Integrated Data Archive (EIDA) node hosted at GI-NOA (http://eida.gein.noa.gr/; accessed on 31 January 2022) [56]. Data of the meteorological station at Thiva are operated by the METEO unit at the National Observatory of Athens (https: //penteli.meteo.gr/stations/thiva/; accessed on 28 January 2022). The Sentinel-1 SAR SLC images are available on the Copernicus Open Access Hub (https://scihub.copernicus.eu/dhus/#/home; accessed on 28 January 2022) [78]. The digital elevation model was extracted by the NASA's Shuttle Radar Topography Mission (SRTM) 3 arc-second (90 m/pixel) database (https://srtm.csi.cgiar.org/10; accessed on 10 October 2021) [84]. The GNSS data used in this study are provided by the private sector and are not available. Acknowledgments: The authors would like to thank the three anonymous reviewers and the academic editor for the constructive and detailed comments that contributed to the improvement of the manuscript. We would like to thank the scientists and personnel who participated in the installation or maintenance of the permanent and temporary stations belonging to the HUSN. Sentinel-1 satellite imagery was acquired through the European Space Agency and Copernicus. We are grateful to Metrica S.A. and TreeComp for kindly sharing their GNSS data and M. Chanioti (sales manager of Inforest Research O.C.) for SARSCAPE s/w availability. We would also like to thank Charalambos Georgiou for the valuable discussions. Most maps and cross sections were drawn with the Generic Mapping Tools (GMT) software [108]. The Coulomb stress transfer model for the 2 December 2020 mainshock was constructed using the Coulomb 3.3 software [70]. Conflicts of Interest: The authors declare no conflict of interest. #### Appendix A. Additional Macroseismic Data Macroseismic intensity data points (IDPs) were re-evaluated to determine the macroseismic epicenter using the MEEP software [50]. Figure A1. EMS98 intensity data points (IDPs) of the 17 October 1914 earthquake and the calculated macroseismic epicenter (star). #### Appendix B. Seismological Methods Statistics and additional information concerning the seismological data. Figure A2. Chatelain [64] diagram for the determination of the average ratio Vp/Vs = 1.79 using P and S travel-time data from the 2020–2021 Thiva earthquake sequence. Figure A3. Histograms of location statistics and uncertainties for the 2020–2021 Thiva earthquake sequence: (a) root mean square (RMS) travel-time residuals, (b) horizontal (ERX, ERY) and vertical (ERZ) formal uncertainties reported by the location algorithm HypoInverse, (c) focal depths from the relocated catalogue with the HypoDD code, (d) azimuthal gap, (e) epicentral distance to the closest station, and (f) number of P- (blue) and S-wave (red) arrival times. Figure A4. Cumulative number of events with M ≥ M<sup>c</sup> = 1.6 per spatial group, represented by different colors and numbers, for the period between 25 November 2020 and 16 October 2021. The occurrence of events with M ≥ 3.0 is marked with circles with size proportional to magnitude. A close-up of the period between July and 16 October 2021 is presented in Figure 4b. Figure A5. Gutenberg–Richter law of the 2021 earthquake swarm (groups #2–#12, excluding group #9; Figure 4) with blue dots showing the log10 number N of events with magnitude M > m, whereas red dots show the reverse relation. Open triangles show the number of events with magnitude M in the range m − 0.05 ≤ M ≤ m + 0.05. The straight red line shows the linear fit corresponding to a b-value of 1.072. The magnitude of completeness is Mc = 1.6. The b-value determined from the maximum likelihood method is bMLE = 1.14. The red dashed line is the extension of the linear fit to log10N = 0, with m\* = 4.51 the largest expected magnitude according to the modified Bath's law [109]. Table A1. Focal mechanisms of major events determined by first-motion polarities (FMPs). CLID is the spatial group number. For more details, see Excel File S2 in the Supplementary Material. #### Appendix C. Geodetic Methods Daily data from the continuous GNSS station THIV located in Thiva were processed over a period of 5 years, 2017 to 2021. Time series were produced, and the velocity vector was calculated. Figure A6. Time series of the three components for the THIV GNSS site. The fit (red line) produced by the software in [110]. #### References #### Article ### The 27 September 2021 Earthquake in Central Crete (Greece)—Detailed Analysis of the Earthquake Sequence and Indications for Contemporary Arc-Parallel Extension to the Hellenic Arc *Emmanuel Vassilakis 1,\, George Kaviris 2, Vasilis Kapetanidis 2, Elena Papageorgiou 3, Michael Foumelis 3, Aliki Konsolaki 1, Stelios Petrakis 1, Christos P. Evangelidis 4, John Alexopoulos 2, Vassilios Karastathis 4, Nicholas Voulgaris <sup>2</sup> and Gerassimos-Akis Tselentis 1,4 #### Featured Application: Field validation of combined remote sensing and seismological data after a large earthquake. Abstract:** The Arkalochori village in central Crete was hit by a large earthquake (Mw = 6.0) on 27 September 2021, causing casualties, injuries, and severe damage to the infrastructure. Due to the absence of apparent surface rupture and the initial focal mechanism solution of the seismic event, we initiated complementary, multi-disciplinary research by combining seismological and remote sensing data processing, followed by extensive field validation. Detailed geological mapping, fault surface measuring accompanied with tectonic analysis, fault photorealistic model creation by unmanned aerial system data processing, post-seismic surface deformation analysis by DInSAR image interpretation coupled with accurately relocated epicenters recorded by locally established seismographs have been carried out. The combination of the results obtained from these techniques led to the determination of the contemporary tectonic stress regime that caused the earthquake in central Crete, which was found compatible with extensional processes parallel to the Hellenic arc. Keywords: Arkalochori; Messara Basin; Heraklion Basin; Kastelli fault zone; supra detachment basin; fault segmentation; double-difference relocation; DInSAR #### 1. Introduction The broader area of central Crete represents a neotectonic structure in the vicinity of the Hellenic Trench, which comprises two post-orogenic basins with trending orientations normal to each other, forming a uniform basin complex [1]. The northernmost part of the latter, the Heraklion Basin (HB), trends approximately N-S, whereas adjacent to its southern margin, the Messara Basin (MB) developed trending E-W [2,3]. We refer to this area as the Heraklion–Messara Basin complex, as it is covered by the same Miocene formations, implying that it shares a common paleo-environmental history, even though there are significant structural differences between the two basins. Citation: Vassilakis, E.; Kaviris, G.; Kapetanidis, V.; Papageorgiou, E.; Foumelis, M.; Konsolaki, A.; Petrakis, S.; Evangelidis, C.P.; Alexopoulos, J.; Karastathis, V.; et al. The 27 September 2021 Earthquake in Central Crete (Greece)—Detailed Analysis of the Earthquake Sequence and Indications for Contemporary Arc-Parallel Extension to the Hellenic Arc. Appl. Sci. 2022, 12, 2815. https://doi.org/10.3390/app12062815 Academic Editor: Valerio Comerci Received: 26 January 2022 Accepted: 4 March 2022 Published: 9 March 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). In particular, MB (Figure 1) acts as a supra-detachment basin [2,4] which lies on top of the hanging wall of the south Cretan extensional detachment [3]. The latter is located at the active margin of the rapidly southwestwards-moving Aegean micro-plate [5–7]. It is an extensional, south-dipping structure, similar to the north-dipping Cretan Detachment [8–10]. Figure 1. (a) Location of Crete (red rectangle) in southern Greece. Bold lines marked with dents or arrows indicate major tectonic structures in the vicinity of the Hellenic Arc; ACZ: Apulian Collision Zone, CTFZ: Cephalonia Transform Fault Zone, NAT: North Aegean Trough, NAF: North Anatolian Fault. Lines indicate the traces of seismogenic sources from the Euro-Mediterranean Seismic Hazard Model 2013 (ESHM13; [11]). The NNE-pointing vector at the lower-left indicates the direction and convergence rate of the Nubian plate [12]. (b) Seismotectonic map of Crete (red rectangle in panel (a). Black lines are active faults from the NOAfaults v3.0 database [13]. Beachballs indicate focal mechanisms of earthquakes (M<sup>w</sup> ≥ 4.0) from the compilation of [14] and sources referenced therein (see also Data Availability). Circles (M < 5.0) and stars (M ≥ 5.0) represent past seismicity of years 1000–1899 (SHEEC; [15]) and 1900–2009 [16] for events with focal depths down to 70 km. The maximum (S1) and minimum (S3) principal stress axes of the regional crustal stress field [14] are depicted by blue and red arrows, respectively, with arrow length proportional to the cosine of the plunge angle. The epicenter of the Arkalochori earthquake is presented by a star within MB and HB areas. MB has been considered an E-W-trending, typical graben structure [17]; however, it is characterized by the absence of classic continuous bounding faults at the north and south margins of the basin. Its northern margin is probably covered with Miocene and/or Pliocene sediments. Therefore, the exact boundaries between MB and HB can hardly be observed and, consequently, several studies, with different arguments [18–20], implicate arc-parallel extension dynamics, trending along the local Hellenic trench axis (Figure 1). Intense historical and recent seismicity reveal the continuous contemporary tectonic activity of the entire Cretan territory (e.g., [21]). Destructive earthquakes have occurred in the broader Crete region since antiquity and during the pre-instrumental era, until 1900 AD. The earthquake of 8 August 1303 [15,22], with an estimated magnitude of 8.3 [23], is one of the largest that has ever been reported in the entire Mediterranean region. This earthquake also caused a large tsunami that was observed at Crete, Rhodes, Peloponnese, Egypt, Syria, the Adriatic Sea, and along the eastern Mediterranean coast. In the vicinity of the study area, two major historical earthquakes have been reported (Figure 1). The first is the 1 July 1494 (M = 5.4) earthquake [15] that occurred close to Heraklion and caused damage to church towers and private buildings, while large waves were observed in the harbor [22]. One century later, on 26 November 1595, another earthquake (M = 6.4) took place a few kilometers off the Heraklion city, causing severe damage and destruction in the entire Crete island [15,22]. It is worth noticing that no large events (M ≥ 6.0) occurred in the study area during the instrumental period (after 1900), with the nearest one being recorded on 23 May 1994 (Mw = 6.1) and that was an intermediate depth (60–70 km) event (Figure 1b) located approximately 40 km WNW of Heraklion [16]. Tsapanos [24] assessed the seismic hazard for the three largest cities of Crete and obtained maximum peak ground acceleration (PGA) values for Heraklion between 0.130 g and 0.165 g for rock and soft soil conditions, respectively. One of the major structures that seem to control the HB-MB complex is the NNE-SSW fault zone that controls its eastern margin, located only a few kilometers east of Arkalochori village (Figure 2). It bounds the easternmost post-Alpine sediment outcrops of the HB-MB in a morphologically clear way, even though several active structures seem to intersect. It was only recently (27 September 2021) that the lethal "Arkalochori earthquake" occurred in this area, causing one fatality, and resulting in hundreds of damaged buildings, mainly of old stone-built masonries. As the authorities reported, more than 6150 houses have been declared uninhabitable due to excessive damage in the mainly affected municipality of Minoa Pediadas (Figure 1), as a result of the mainshock and the major aftershocks. The earthquake of 27 September 2021, 06:17:21 UTC, occurred near Arkalochori, Central Crete, ~25 km SSE of Heraklion city [25,26]. Its focal mechanism is characterized by an SSW-NNE to SW-NE-trending, nearly dip-slip normal faulting. Considering the solutions proposed by different agencies (Table S1), except for a few outliers, its strike generally ranges N200◦ E–N230◦ E and its dip angle varies between 40◦ and 60◦. The magnitude of the main event was initially determined as M<sup>L</sup> = 5.8 by the Seismological Laboratory of the National and Kapodistrian University of Athens (SL-NKUA) and the Geodynamic Institute of the National Observatory of Athens (GI-NOA), but later measurements of seismic moment upgraded its value to M<sup>w</sup> = 5.9–6.0. The largest aftershock (M<sup>w</sup> = 5.3) occurred on 28 September 04:48:08 UTC, one day after the mainshock, with a similar focal mechanism. According to the report of ITSAK [27], the recorded PGA at station ARK1 in the epicentral area (Arkalochori) was 609 cm s−<sup>2</sup> in the horizontal component (N-S) and 806 cm s−<sup>2</sup> (~0.82 g) in the vertical one, with the duration of strong ground motion (>0.1 g) being 6 s. The horizontal to vertical spectral ratio reached 8 at the dominant frequencies of 0.65Hz and 1.4 Hz. The accelerometric recordings at station ARK1 far exceeded the provisions of the National Building Code [28] for effective PGA = 0.24 g at the epicentral region in the seismic hazard zone II and for soil classes A and B. Herein, we present a multi-disciplinary work, based on extensive fieldwork and detailed geological mapping of the affected area at central Crete, combined with results from seismological and geodetic imaging techniques. Our aim is to understand the contemporary seismotectonic regime of a densely populated area close to the active Hellenic arc and provide useful data for a realistic model concerning the neotectonic evolution of Crete. We intend to examine whether arc-parallel extension is a temporal phenomenon that is related to the Arkalochori earthquake or a dominant contemporary stress component. Figure 2. Close up of the study area, marked with a red rectangle in Figure 1b. Black lines show the mapped tectonic structures. Seismicity of the period from 2008 to May 2021 is from GI-NOA. The Arkalochori earthquake is represented by a star with bold outline. Note the offset beachball linked with a dashed callout line representing its focal mechanism solution from GI-NOA (see Table S1 at Supplementary Materials) and the faults, described in this work, are drawn in red (KF: Kastelli Fault, NF: Nipiditos Fault, LF: Lagouta Fault). Additional focal mechanisms are from [29]. #### 2. Geological Setting The study area is located at central Crete (Figure 1b) and, specifically, at the easternmost part of the supra-detachment MB, which is bounded by active fault systems producing high relief landscape [3]. It is the area where HB shares significant structures with MB and, consequently, it is quite complex to distinguish the margin between these Neogene basins with a single discipline method. In the following sections, we present interpretations on the geological setting of the study area, based on geomorphology, lithology, and seismotectonics. #### 2.1. Geomorphology The general morphology of the wider area is rather mountainous, with steep slopes which very often exceed the order of 45◦, due to active tectonic structures that divide it into fault blocks that move either independently or in groups, depending on the local dominant tectonic regime [3]. The latter seems to have been changing since Miocene and is either expressed with the change of sediment facies or imprinted on the drainage network patterns, which are developed within a hilly area, with tilted monocline blocks, between higher relief mountains, implying a listric geometry of the involved faults (e.g., [30–32]). The highest mountain is Dikti Mt which exceeds an elevation of 2100 m and bounds both HB to the east and MB to the N-NE (Figure 2). The much lower elevation E-W-trending Asteroussia mountain chain (~1200 m) is the geomorphological structure that acts as the southern margin of MB (Figure 2). #### 2.2. Lithology The geology of the area consists of Alpine (mainly Triassic to Late Jurassic limestones/dolomites, flysch, marbles, and ophiolites) and post-Alpine formations (silts, clays, conglomerates, and sandstones of Middle-Upper Miocene). In many places, they are covered by Quaternary alluvial sands and conglomerates of small consistency, as a result of the erosion of the aforementioned formations. More specifically: - Limestones and Dolomites (Mani Unit). Carbonate rocks consisting of thinbedded crystalline marbles with layers of siltstones (L. Jurassic—L. Eocene). They appear heavily folded with isoclinal asymmetric folds. - Limestones and Dolomites (Tripolis Unit). Intercalations of unbedded to thick-bedded neritic dark-colored and partially bituminous Limestones and Dolomites, with sparse appearances of evaporites. - Flysch (Pindos Unit). It consists of heavily folded layers of Sandstones, Schists, and Conglomerates, with the characteristic appearance of large carbonate olistoliths that originated from inner units. - Ophiolitic nappe. Limited appearance at the top of E. Asteroussia Mt, emplaced between the Flysch and the Viannos formation sediments as its basement rock. The entire nappe pile behaves homogenously till after the Middle Miocene, when the compressional, almost N-S-trending, orogenic stress field is converted into an extensional one due to the African plate slab roll-back [9,34–36], which happened at the edge of the Aegean microplate [37]. Within this context, Crete is a large part of the Mid-Miocene Southern Aegean core complex exhumation that took place between 24 and 15 Ma [9] which hosts several supra-detachment depositional areas [3,4], with MB being one of them. - Viannos Formation (Mid. Miocene). Sandy/silty deposits of lacustrine origin, coexisting with alluvial conglomerates. The average thickness of the formation is about 400 m. Its outcrops may be found at most of the eastern MB margin, either covering unconformably the basement rocks or in tectonic contact with them through ruptured fault zones. #### 2.3. Seismotectonic Setting The vast majority of the large events affecting Crete, before the 2021 Arkalochori sequence, have occurred offshore, either to the north or to the south, with the latter probably related to the intra-crustal graben system, i.e., the Ptolemy and Pliny trenches [39] (Figure 1b). The only knowledge regarding the local seismic activity in the study area comes from a temporary seismological network, which consisted of 10 analog stations, installed in the area of Heraklion during the period of September to December 1995 [29]. Data recorded by this network revealed the existence of shallow onshore activity. During that period, the seismicity was mainly concentrated along the eastern margin of the Heraklion Basin, decreasing from east to west, with epicenters also located in the vicinity of Arkalochori. Furthermore, a W-E-trending decrease in the sedimentary cover has been identified offshore, north of Crete [40]. In a broader view frame, the location of Crete Island, north of the Hellenic Arc (Figure 1a), comprises an area of complex geodynamics, with transpressional tectonics dominating the south of Crete, in contrast with normal and oblique-slip faulting onshore, yielding a heterogeneous stress field [14,41]. On the other hand, an extension has been observed to the north, between Santorini and Crete [42–45]. The latter reveals extension in a general E-W direction, with NNE-SSW shortening observed in western Crete [46]. According to [14], the sub-horizontal S1 axis is in an SW-NE (in the east) to the N-S direction (in the west), likely affected by the proximity of the region to the subduction interface and compression parallel to the direction of convergence, which becomes more oblique to the arc towards the east. However, the sub-horizontal S3 axis also reveals E-W (in the west) to WNW-ESE (in the east) extension onshore, even in the region where the 2021 Arkalochori earthquake occurred, in accordance with the observed normal fault scarps, trending in a general N-S direction [19]. The resulting regional shear stress favors SW-NE left-lateral faults, mainly offshore, in agreement with [41,47]. On the other hand, fault sources, determined by [11], indicate mainly E-W normal faulting to the north and south of Crete, incompatible with the resolved stress. It should be taken into account that the aforementioned stress field was determined using offshore earthquakes at crustal depths, both to the north and the south of Crete, as no large onshore events were located onshore until recently. The stress field in the region of Crete was characterized as transpressional by [14] due to the involvement of shallow events related to strike-slip (primary stress state) or reverse faulting (secondary state). In central Crete, due to the absence of focal mechanism data for shallow onshore events, the stress field is largely a result of interpolation from resolved nodes of neighboring regions. The stress shape value of 0.5 indicates instability in both S1 and S3 axes. However, the direction of S3 seems to be generally stable in a WNW-ESE direction in the region from central Crete to the volcanic arc in the north. This complex stress regime is also revealed by the focal mechanisms, as all types (normal, strike-slip, and reverse) can be observed offshore, either to the north or to the south (Figure 1). Focal depths increase towards the north, due to the African slab rollback phenomenon. Onshore focal mechanisms at shallow depths are few. De Chebalier et al. [48] worked on the western edge of Crete and obtained fault-plane solutions of shallow events, revealing either approximately N-S normal or strike-slip faulting. Delibasis et al. [29] constrained 29 fault-plane solutions close to the 2021 Arkalochori earthquake sequence, which also indicate normal or reverse motion with, in some cases, a significant strikeslip component. #### 3. Materials and Methods We combined data from different sources, performing a multidisciplinary study, to further understand the geotectonic regime that caused the 2021 earthquake activity and possibly reveal its association with the mapped fault pattern. Therefore, our methodology included field mapping accompanied by the necessary laboratory work (e.g., GIS techniques for tectonic geomorphology, photogrammetric processing of unmanned aerial system images), seismological data analysis and interpretation, as well as SAR interferometry processing. #### 3.1. Field Mapping The fieldwork included detailed mapping of the outcrops, especially the post-Alpine sediments, as well as of the fault surfaces that crosscut the stratigraphic contacts between them. At several places, a drone was used for image acquisition, followed by photogrammetric processing and the generation of high-resolution digital surface models and ortho-photographs. A quite clear marginal structure, trending NNE-SSW, which delineates the eastern side of the MB-HB, can be identified, even on a medium resolution shaded relief of the study area in central Crete (Figure 2). This represents the major Kastelli fault zone, which constitutes the western boundary of Dikti Mt that lies to the east [3]. One of its segments, just uphill the new international Kastelli Airport (Figure 2), was captured by the UAS and modeled through structure-from-motion techniques, aiming to map its trace with the highest possible detail, especially at areas where the morphology is relatively smooth. About 320 images were processed and a point cloud of more than 100 million points was generated for the construction of a photorealistic 3D model, with a spatial resolution of 2.5 cm (Figure 3). Figure 3. Photorealistic 3D model of a Kastelli fault segment, which, at some places, can be clearly identified by a morphological discontinuity (white arrows) or is quite difficult to distinguish where the morphology has been smoothened by the erosion. The mapped faults were measured along with the kinematic indicators that were found on their surfaces and interpreted within the frame of kinematic analysis. The classification and clustering of the field measurements were performed using algorithms and methods already published and incorporated in software packages, such as M.I.M. v.4.0.1 [49]. #### 3.2. Seismological Data and Methods The 2021 seismic crisis in the wider area of Arkalochori began in the form of an earthquake swarm in early June 2021. The situation, however, changed dramatically after the occurrence of the M<sup>w</sup> = 6.0 mainshock of 27 September 2021. Although several permanent stations of the Hellenic Unified Seismological Network operate on the island of Crete, at the time of the mainshock, the closest station to the epicentral area was KNSS of the Hellenic Mediterranean University (HC network at formerly Technological Educational Institute of Crete [50]), at a distance of about 12–22 km (Figure S1). As a result, the seismicity of the first stage of the sequence (foreshock swarm) could not be determined with high precision, due to the lack of data from stations at local distances, which would help constrain the focal depths. Resolving hypocentral locations with data from more distant stations is further problematic due to the inhomogeneity of the velocity structure, because of the proximity of the area to the Hellenic subduction zone. The day after the mainshock, four temporary seismological stations (CRE1-4) were deployed by GI-NOA (HL Network [51]) around the aftershock zone (Figure S1b). The inclusion of data from these stations drastically improved the hypocentral locations, even with the use of a generic velocity model. We collected the available catalogues, including manually analyzed phase data from SL-NKUA and GI-NOA at all operational stations, and compiled a dataset of ~2500 events for the period between 1 June and 18 October 2021. We divided the data to be processed in two periods, one before (Period A) and one after (Period B) 28 September, when the temporary local network was deployed. We used the HypoInverse code [52] to locate the sequence, initially with a generic model used by routine analysis at SL-NKUA, as well as with the local velocity model of [29] for Central Crete. The latter model was preferred, as it yielded lower root mean square (RMS) travel-time residuals and locations errors, while providing a better general image regarding the distribution of epicenters, in terms of scattering, especially between events that form spatial clusters. Another issue concerns the distance weighting parameters for the two periods, i.e., the maximum epicentral distance of stations to be used. After several tests, the maximum distance was set to ~280 km for the first period and ~120 km for the second one. A VP/VS ratio of 1.78 was measured using the Chatelain [53] diagram for the first period, whereas a VP/VS = 1.76 was preferred for the second period with the shorter distance limit. The application of station corrections, especially for the first period, plays an important role to reduce artifacts of the initial epicentral distribution, where spurious epicenters of small magnitude events seemed to spread northwards of the main seismicity cloud. To further improve the hypocentral distribution, with emphasis on the second period, we applied a relocation procedure using the HypoDD code [54]. The algorithm works by minimizing the double difference between calculated and observed travel times in combinations of event pairs with neighboring hypocenters, at inter-event distances much smaller than their respective hypocentral distance from a station. This method enhances the hypocentral distribution by minimizing relative location uncertainties between correlated events, caused by discrepancies between the velocity model and the real structure. Furthermore, waveform cross-correlation data are used to minimize uncertainties due to arrival-time reading inconsistencies in groups of events with similar waveforms, called multiplets. We used station KNSS (Figure S1b) as the main reference station for the identification of multiplets, as this is the closest station that was operational during both periods, with the exception of its N-S component that was damaged after the occurrence of the 27 September mainshock. Recordings of the temporary station CRE1 (Figure S1b) were also used to enhance the reference cross-correlation measurements, particularly those of smaller events, during the second period. The waveforms were band-pass filtered in the range 2–8 Hz, for KNSS, and 2–15 Hz, for CRE1, before cross-correlation was calculated for the full P and S waveform signals in all combinations of event pairs with available data. The crosscorrelation maximum (XCmax) of every pair was registered in a matrix for every component. The matrices produced for different reference stations and components were then combined, retaining the RMS value of the XCmax determined for each pair. The nearest neighborhood linkage was applied and a Cth = 0.81 optimal threshold was selected, maximizing the difference between the size of the largest multiplet and the sum of clustered events [55]. This procedure created 234 multiplets containing a total of 1725 events. The P- and S-wave windows for each pair of events within the same multiplet were then cross-correlated for all stations with available picks, measuring the XCmax and its respective time lag to be used as input for HypoDD. The relocation procedure was applied separately for the two periods, acknowledging issues due to the different network geometry, so as not to degrade the data quality of the second period. On the other hand, we chose to relocate the mainshock and its early aftershocks (roughly a hundred), which occurred before the deployment of the temporary local stations, together with events that were located with data from the local network. This manages to improve the relative locations of the first aftershocks by exploiting their links with the rest of the events of the second period. Each relocation procedure was mainly divided into two types of sets, one with stronger a priori weights to the catalogue data and the other with stronger weights to the cross-correlation data. #### 3.3. SAR Data and Interferometric Processing Space-born synthetic aperture radar (SAR) imagery is routinely utilized for measuring co-seismic surface displacements based on the DInSAR technique. DInSAR allows mapping the co-seismic motion by using two satellite images taken before and after an earthquake [56–58]. The systematic availability of SAR data from the Copernicus Sentinel-1 mission, with a revisiting time of 6 days over Europe (12 days globally), as well as the wide swath coverage of 250 km, enables the near-real-time response to seismic events [58–63]. The contribution of platform-based solutions in the early response phase of an earthquake has been well demonstrated [64,65]. The Geohazards Exploitation Platform (GEP; https://geohazards-tep.eu, accessed on 23 January 2022) focuses on mapping hazard-prone land surfaces and monitoring terrain motion, providing access to several Earth Observation (EO) missions and a broad range of relevant online services and development on cloud processing resources [66]. The present work utilized Sentinel-1 data acquired during the period from 23 September to 1 October 2021, with different viewing geometries to optimize the displacement map generation. For the interferometric processing, the GEP DIAPASON service was exploited, considering precise orbit state vectors and SRTM Digital Elevation Model 3 arcsecond (≈90 m) data to remove the topographic phase. Interferometric measurements correspond to movements detected along the Line-of-Sight (LoS) of the satellite, i.e., the oblique direction between the satellite and the Earth's surface. Positive LoS values indicate uplift or motion towards the satellite, whereas negative ones correspond to subsidence or motion away from the sensor. The DInSAR processing scheme implemented herein is described in detail in [67,68]. Four differential interferograms were generated for the descending 036 (D036) and 109 (D109) orbital tracks, as well as for the ascending 102 (A102) and 029 (A029) ones, with temporal separation of 6 to 12 days and spatial resolution of approx. 45 m. Figure 4 and Figure S2 in the Supplementary Materials show the obtained displacement fields, while Table 1 details the interferometric pairs considered for the various geometries. Based on their temporal coverage, it is important to note that each co-seismic displacement map is affected by a different contribution of post-seismic motion. The interferograms for tracks A102 and D036 represent those with the smallest and largest post-seismic contribution, respectively (Figure 4). Figure 4. Co-seismic LoS displacement maps, as derived using GEP DIAPASON service, for ascending track A102 (23 September 2021–29 September 2021) and descending D036 (25 September 2021–1 October 2021) (c,d) and the corresponding wrapped differential interferograms (e,f). The spatial extend of Sentinel-1 acquisition frames for both geometries are also indicated (a,b). The difference in the temporal span following the mainshock (27 September 2021) for the two opposite geometries is worth noting. While A102 spans approx. two days, D036 extends over five days after the earthquake, denoting larger contribution of post-seismic motion. Black lines correspond to fault zones (after [3]). The relocated epicenter of the 27 September 2021 mainshock is marked with a star. Table 1. Overview of the interferometric pairs considered in DInSAR processing, including the LoS incidence angles of the measurements. The time span of each DInSAR pair following the mainshock is also indicated. Furthermore, due to the opposite geometries, independent LoS measurements were decomposed into vertical (Up) and E-W-motion components, to facilitate the optimal interpretation of motion patterns. The horizontal and vertical components were calculated using both Sentinel-1 geometries in a comprehensive decomposition scheme [69], considering the variability of the incidence angles within the scenes. In order to simplify the geometrical assumptions, only measurements of similar incidence angles were considered in the decomposition (i.e., orbital tracks A102—D036 and A029—D109) (see Table 1). #### 4. Data Analysis and Results The combination of data from various and heterogeneous sources is a complex task that needs to be evaluated and prioritized to examine their compatibility grade. The field data analysis provides us with strong arguments for establishing plausible planes of movement, which host the hypocenters of an earthquake. Therefore, it offers a tool for filtering them with higher relevance to the contemporary tectonic regime. In addition, the field data were used to validate the surface deformation detected through DInSAR data and attribute it to specific geological structures (e.g., active faulting). #### 4.1. Field Data Analysis The length of the Kastelli fault zone exceeds 30 km since it can be traced from the northern coast of Crete to Asteroussia Mt to the south, within the recent sediments of MB. It was mapped and measured at several places, especially where the Alpine basement outcrops hosted the fault plane. It was quite frequent that striations along with other kinematic indicators were overprinted on the calcite coating of the fault plane (Figure 5). Figure 5. Fault plane of a Kastelli fault segment measured at 74/263, on which striations measured at 61/220 were found (parallel to the pen), confirming the oblique normal movement with left-lateral component. Measurements at several places are plotted in the inset diagram. The fault slip data recorded in the field were used as inputs within the stress inversion method TRM [70] and a stress tensor with S1: 90/079, S2: 00/349, and S3: 00/079 (R = 0.25) was defined. The resolved stress tensor, although calculated with fault planes that do not deviate strongly from each other, is rather compatible with the E-W-trending extension, which in turn agrees with the Arkalochori earthquake focal mechanism. During the fieldwork, it was rather obvious that the morphological discontinuity, which the Kastelli fault zone has generated through its activity, is gradually eliminated and finally disappeared within the early Miocene lacustrine sediments of the Viannos, Skinias, and Ampelouzos units [71] and, hence, the fault trace which comprises its southern segment (Lagouta fault, according to [3]) is not very clearly delineated. Despite this, a significant number of smaller fault traces that were mapped around Lagouta and Kassani villages (Figure 6a) yield that the Kastelli fault zone continues to the south and ends up to an E-W-trending fault, almost parallel and antithetic to the south Cretan detachment [3]. In addition, a pair of conjugate syn-sedimentary faults was located, measured, and analyzed (Figure 6b) from a kinematic and dynamic perspective. The measurements of the two faults (p1 88/114, p2 72/150) and the sliding directions recorded on them (s1 27/025, s2 19/066), respectively, were found to be compatible—after tectonic analysis—with left-lateral strike-slip faulting and, specifically, with a stress field which is defined by the axes S1: 24/046, S2: 63/200 and S3: 11/311 [3]. Figure 6. Field photographs of (a) the Lagouta normal fault, which comprises the southern segment of the Kastelli fault zone, and (b) a pair of conjugate syn-sedimentary faults, within Tortonian deposits, compatible with left-lateral strike-slip faulting. The left-lateral component of this movement is in agreement with the en-echelon formation of numerous faults found along the previously activated E-W-trending, MB marginal fault zone, at the southern edge of this segment at Asteroussia Mt [3]. The monoclinal stratigraphy of the area yields that the same west-dipping fault zone has also a strong normal component. This conclusion is based on the fact that the outcrops of the older post-alpine sediments (Viannos formation) are found beneath more recent successions that have been mapped in detail on the western fault block and simultaneously at higher altitudes at the eastern one, yielding that the latter has been relatively uplifted (Figure 7). Figure 7. Simplified 3D block diagram of the eastern marginal area of MB. Arkalochori village is located at the hanging wall of Lagouta fault but also at the hanging wall of Nipiditos fault, which is segmenting the Kastelli fault zone. #### 4.2. Seismological Results Regarding the initial locations of earthquake hypocenters (Figure 8a) of the first period, before 28 September 2021, focal depths remain uncertain, due to the lack of local stations, mainly ranging between 3 and 20 km (Figure S3c), with the majority of events being concentrated near a discontinuity of the velocity model at ~6 km [29]. On the other hand, more constrained focal depth values, between 5 and 13 km (Figure S3d), are determined for the second period. Location statistics for the two periods are presented in Table 2 and Figures S3 and S4. Average vertical location errors are nearly halved during Period B and horizontal location errors are reduced by ~26%, as a result of the deployment of stations at local distances, which also reduced the azimuthal gap (Figure S4). Figure 8. Seismicity of the study region for the period between 1 June and 18 October 2021. (a) Initial locations with HypoInverse, (b) results of double-difference relocation with HypoDD. Colors and numerical labels in panel (b) represent the foreshock swarm activity (group #1, red) and the three spatial aftershock groups: #2 (cyan) middle, sparse group, #3 (blue) northern cluster near Kastelli airport, #4 (green) southern group, including the mainshock. Major events (M ≥ 4.5) are depicted as stars. Profile line A-B in panel (b) is used for the spatiotemporal projection of Figure 9. The location of Arkalochori village is marked with a black square. Table 2. Initial location statistics for Period A (1 June–27 September 2021, VP/VS = 1.78, long-distance weighting) and Period B (28 September–18 October 2021, VP/VS = 1.76, short-distance weighting). ERH, ERZ are horizontal and vertical location errors, "min.stat.dist" denotes the epicentral distance of the closest station with available arrival-time data (see also Figures S3 and S4 in the Supplementary Materials). In order to assess the 2021 Arkalochori earthquake sequence in more detail and to identify the activated tectonic structures, a relocation procedure was applied, as previously described in Section 3.2, to both periods (Figure 8b). The events distribution was divided into four groups. Group #1 (red) is a temporal cluster that concerns the first period of the foreshock swarm activity, whereas groups #2, #3, and #4 (cyan, blue, and green, respectively) were determined by spatial clustering of the events of the second period. This was achieved by applying Ward's linkage to the matrix of inter-event epicentral distances of the relocated hypocenters [72]. This procedure of agglomerative hierarchical clustering, also known as the "minimum variance method", fuses two clusters into a new one, provided that the resulting increase in the sum of squares (i.e., its objective function) between the objects of the new cluster is minimized, compared to the outcome of any other potential combinations of cluster fusions. After creating the clustering hierarchy with Ward's linkage by setting an appropriate threshold to the fusion level, we divided the spatial distribution into three clusters (i.e., groups #2, #3, and #4) to aid the description. Figure 9. (Top) Stacked histogram of the number of seismic events per day in the study area between 1 June and 18 October 2021. (Bottom) Spatiotemporal projection along the SW-NE-oriented profile A-B of Figure 8b. Colors and numbers (CLID) represent groups 1–4 of Figure 8b. Events with M ≥ 4.5 are depicted as stars. The evolution of the earthquake sequence is examined through the spatiotemporal projection of Figure 9, along the SW-NE-oriented, 16 km-long profile A-B of Figure 8b. The foreshock swarm began in June 2021 with a few small events (M<sup>L</sup> ≤ 3.0) and an M<sup>L</sup> = 4.2 event on 4 June which did not, however, cause any significant outbreak. Minor bursts occurred on 8 June and then on 18 July including three M<sup>L</sup> = 4.1–4.2 events. During that period, a slow migration of seismicity towards the southwest can be observed in Figure 9. Then on 24 July 02:07:37 UTC, an M<sup>L</sup> = 4.8 occurred, triggering a subsequence. Afterwards, apart from a few more minor bursts, the seismicity rate gradually diminished, notably to the point where only 17 events were located in the period between 16 and 25 September, with a complete absence of detectable events for about 41 h preceding the Mw = 6.0 mainshock of 27 September 2021, 06:17:21 UTC. The whole foreshock group #1 is roughly distributed in an area of 3.0 km × 1.7 km, elongated in an N-S direction, with its centroid ~2.5 km WNW of Arkalochori. The main event's epicenter is located adjacent to the foreshock swarm, near its eastern edge. The occurrence of the mainshock, near the middle of profile A-B, caused seismicity to spread rapidly nearly through the whole length of the aftershock zone, both towards the southwest (group #4) and the northeast (group #3). The aftershocks appear to have little overlap with the foreshock zone, specifically with the sparser middle group #2 (cyan) and the northern margin of the southern group #4 (green). The two major groups, #4 south-west of the foreshocks and group #3 (blue) towards the NE, the latter with epicenters near the Kastelli airport, are separated by the less populated group #2 (cyan). This deficit of aftershocks between groups #3 and #4 is likely associated with an area of maximum coseismic slip during the mainshock. The rupture of a large asperity in that region apparently caused redistribution of stress towards the northern and southern margins of the main fault, while the central part was relaxed due to stress drop. The largest aftershock was an M<sup>w</sup> = 5.3 event that occurred on 28 September 04:48:08 UTC, about 4 km west of the mainshock. The epicenter of the main event is located at the NE edge of group #4, whereas the largest aftershock occurred towards its western part, near the southwestern tip of the foreshock group. The major aftershocks include 11 more events with M<sup>L</sup> ≥ 4.0 between 27 and 29 September, with only smaller events being detected through the rest of the sequence during the period of study (up to 18 October 2021). Regarding the period after 18 October, routine monitoring of seismicity at SL-NKUA detected five more events with 4.0 ≤ M<sup>L</sup> ≤ 4.5 between 20 and 22 of October, two associated with cluster #3 and three with cluster #4, while another M<sup>L</sup> = 4.2 event occurred on 29 December associated with the latter cluster. However, no significant changes in the overall spreading of the aftershocks' distribution were observed, besides a small, isolated cluster that was detected ~10 km NNW of Kastelli, activated between 16 and 18 January 2022, with the largest event having M<sup>L</sup> = 3.6. The three groups of the relocated aftershocks, if considered as a whole, appear to be well distributed on a plane, striking ~N216◦ E and dipping ~45◦ (Figure 10, red-dashed line), as determined by the least-squares fit on the hypocenters. This is quite compatible with focal mechanism solutions for the mainshock reported by most agencies, taking into account the WNW-dipping nodal plane (Table S1). Regarding specific spatial groups of the aftershock sequence, the best-fit plane for the northern cluster #3 strikes N181◦ E and dips 49◦ westwards (Figure 10, blue dashed line in profile c1-c2), whereas the mid-southern part (groups #2 and #4) has a least-squares plane that trends N216◦ E and dips 53◦ WNW (Figure 10, green-dashed line in profiles a1-a2 and b1-b2). The more north-southwards orientation of the northern cluster's plane makes it more compatible with the Kastelli fault. The fault is steeply dipping at the surface (74◦), but the aftershocks' distribution indicates a lower dip angle at the hypocentral depths of 6–9 km, implying listricity of the fault. Cluster #3 is fairly more concentrated than group #4 in the south, which appears to include several smaller sub-clusters, some of which may be related to the activation of antithetic structures (e.g., Figure 10, black-dashed lines with a question mark in profile a1–a2). Normal faulting is indicated for the mainshock, the major aftershock, and several other aftershocks and foreshocks. However, oblique and strike-slip faulting is also observed for several events, including some foreshocks (Figure 10). Although the mainshock's focal mechanism is in line with the rest of the aftershocks, it is noted that its hypocenter, along with those of aftershocks that occurred before the deployment of the local network, is not well constrained at depth. Hence, it is reasonable to assume that its true focal depth should be deeper, i.e., between 6 and 14 km, which is an estimated range for its centroid (Table S1). Figure 10. Map and cross-sections of the relocated 2021 Arkalochori aftershock sequence (period 27 September–18 October 2021). Colors and numbers correspond to the aftershock spatial groups 2–4 presented in Figure 7b. The cross-section profiles are drawn in an N103◦ E direction, roughly perpendicular to the strike of the mapped Kastelli and Lagouta faults, whose downdip extension is presented in the cross-sections with gray-dashed lines. The red dashed line is the section of the least-squares plane fitted to the relocated hypocenters of period B, whereas the green (a1–a2, b1–b2) and blue (c1–c2) dashed lines are the planes fitted to groups #2 and #4 (green), and group #3 (blue), respectively. The possible antithetic structures are marked with black-dashed lines (a1–a2). Focal mechanisms of the mainshocks and other major events of the sequence, including foreshocks (red beachballs, on the map only) are from the database of GI-NOA. The location of Arkalochori village is marked with a black square on the map. #### 4.3. DInSAR Analysis In terms of satellite observations, the systematic acquisition strategy of the Copernicus Sentinel-1 mission ensured the availability of SAR data to investigate the earthquake sequence. Interferometric coherence levels for 6- to 12-days pairs are maintained, allowing for the extraction of reliable DInSAR ground motion measurements. Independent results from four different satellite tracks (Figures 4 and S2) are consistent, indicating co-seismic downthrow of the epicentral area, extending over an area of about 38 km2 to the east of the Lagouta and Kastelli faults. The ground displacement patterns are comparable, retaining an elliptical shape of the co-seismic fringes within the epicentral area. The increased ellipticity of the fringes for pairs covering a larger time span after the mainshock (tracks D036, A029, and D109) implies the contribution of post-seismic motion, compared to the more concentric fringes of the A102 track, containing only 2 days of postseismic activity. In addition, the direction of the major axis of the elliptical fringe pattern is following the aftershock distribution, retaining roughly an NE-SW trend. The spatial continuity of the interferometric fringes yields that the rupture did not reach the surface. The higher density of fringes at the eastern part of the epicentral area implies higher ground displacement gradients, and thus, proximity to the upper part of the activated rupture zone (i.e., NNE striking, west-dipping fault). However, examining in more detail the DInSAR displacement fields for the various independent measurements, variability arises both in terms of magnitude and location of ground motion maxima (Figure 4). Specifically, for the pair with the shortest temporal extent (2 days) after the mainshock (A102, 23 September 2021–29 September 2021), LoS ground displacement is located in the vicinity of the relocated mainshock epicenter, with values reaching −18 cm. On the contrary, for the pair with the largest temporal extent (track D036, 5 days), the location maximum has been shifted approx. 2.5 km towards ENE and the observed ground displacement increased to −20 cm (Figure 4). The other two displacement pairs (A029 and D109), spanning 3 days within the post-seismic period, show comparable motion (downlift of about −20 cm) with the 5-days D036 pair (Figure S2). The shift of the displacement maxima can be attributed to the amount of post-seismic motion contained in the DInSAR measurements (see Table 1), and it is consistent with the seismological observations indicating migration of aftershocks towards the northern margins (cluster #3) of the mainshock epicentral area. It can also be assumed that no surface deformation occurred, or at least with a magnitude detectable by DInSAR, after three days of post-seismic activity. In fact, a few centimeters of variation of LoS displacement measurements (still within the error budget of the conventional DInSAR technique) might also be related to the difference in viewing the angle between the ascending and descending satellite observations, and the contribution of the horizontal (mainly E-W) motion component. Therefore, the interpretation of the observed LoS displacements is in advance favored by the decomposition of the ascending and descending interferograms (Figures 11 and S5). The estimated vertical motion indicates down throw reaching −22 cm (Figure 11a), concentrated within the mainshock epicentral region, whereas a more complex pattern, with a maximum of +8 cm, is presented by the E-W horizontal component (Figure 11b). Figure 11. Vertical (a) and E-W (b) ground displacement maps as decomposed using Sentinel-1 DInSAR LoS observations from ascending A102 and descending D036 tracks (see Table 1). Black lines correspond to fault zones (after [3]). The relocated epicenter of the 27 September 2021 mainshock is marked with a star. #### 5. Discussion and Conclusions The recent Arkalochori earthquake sequence, which was recorded before and after the mainshock (27 September 2021), seems to be a quite prominent opportunity to discuss the contemporary tectonic regime of MB. The latter has a very significant geotectonic placement in the context of the African plate subduction that takes place under the Aegean microplate, as it is an onshore post-orogenic basin in the vicinity of the active Hellenic Arc [4,73]. Crete is at a crucial location right above the subducting slab and it is more than certain that the impact of the plate convergence is imprinted on the island, at all scales [74]. The fault pattern that was mapped in detail revealed the different generations of fault-block movements, as a result of the stress field changes since the integration of the Alpine nappe pile buildup [1]. Several direction changes of the main stress axes have been imprinted on the fault structures and the surface morphology [3]. It seems that the easternmost area of MB had been under N-S-trending compression during the orogen buildup, followed by an N-S-trending extension during the slab rollback and the orogen collapse through the detachments' operation (either north or south dipping) [1,8,10,75]. From an evolutionary point of view, we argue that after the generation of the nappe pile on Crete and the south-dipping detachment fault, several almost E-W-trending fault zones were activated (as segments of the south-dipping detachment) since they crosscut rocks that belong to different Alpine units. These fault zones remained active until after the end of late Serravallian, when the deposition of the lacustrine Viannos sediments was completed, as large fault blocks comprising Serravallian age successions are found tilted towards NNE. A transtensional geotectonic regime, which seems to be dominating the entire Aegean microplate [76], is expressed on the Cretan territory by nearly NNE-SSW-trending strike-slip fault zones [20,77] and one of them crosscuts the central area of the island and simultaneously was acting as the marginal structure of the HB-MB system [2]. It should be mentioned that the main fault zones that were found along the eastern margin of MB are striking NNE-SSW, NW-SE, and E-W. The kinematic analysis of these data showed that most of the faults that were activated after the Early Miocene were either normal or strike-slip faults with left-lateral movement components [3]. According to this analysis, the NNE-SSWtrending shear zone was also affecting the previously generated E-W-trending fault zones, which were inherited by the Early Miocene N-S extensional period, when the south Cretan detachment was still active [1]. The impact on these E-W-trending structures is crucial as they seem to have been segmented and some of the segments have been reactivated during or shortly after the Pliocene (e.g., Nipiditos fault). Specifically, during Tortonian, while the sediments of the Ampelouzos formation were still depositing, the dominating stress field had a strong left-lateral component, which is evidenced by the syn-sedimentary structure analysis (Figure 6b). The stress field seems to be active during the Tortonian since the syn-sedimentary faults were deforming the Ampelouzos unit strata (see Section 2.2 for lithology description), which were deposited before 10 Ma in a coastal to shallow depth marine environment [78]. A strong case scenario, considering both tectonic and stratigraphic data, is that the same fault zone seems to have a larger left-lateral component during Tortonian and the stress field gradually changed during Messinian, when the normal component dominates, increasing the compatibility of the stress field with arc-parallel extension. The latter seems to be active up to the contemporary era and is tectonically responsible for the Arkalochori earthquake. The tectonic analysis of the collected measurements during the fieldwork provided strong arguments of stress field changes along the NNE-SSW-trending fault zones, as two groups of structures were found on fault surfaces revealing lateral movement and extension along the NW-SE direction. As explained above, the lateral movement on the same fault surfaces is older than the normal one, which in turn coincides with arc-parallel extension. Finally, we refer to the Kastelli fault zone and in particular its southernmost segment (Lagouta fault), which is a west-dipping structure hosting a normal displacement movement with a strong left-lateral oblique component inherited by the dominating stress field during Tortonian. It was exactly this structure that was activated during the Arkalochori earthquake, as the hypocenters of the sequence clearly delineate the above-mentioned fault surface. It is obvious that the two hypocentral clusters that have been described, form two discrete segments that belong to the same fault zone. It is the WNW-ESE-trending Nipiditos fault that seems to be the structural reason for this segmentation. The northern cluster is formed on the surface of the Kastelli fault and the southern cluster on the Lagouta fault (Figure 10). Even though all events belong to the same sequence, it is rather clear that the northern cluster is located at more shallow depths compared to the southern cluster. Concerning the seismological data, the 27 September M<sup>w</sup> = 6.0 mainshock was a reminder that strong earthquakes do occur onshore Crete. It was preceded by a rich sequence of over 700 foreshocks that was considered a swarm, due to its largest event occurring halfway through 24 July. However, it did not cause much concern at the time, as similar swarms have been known to occur elsewhere in Greece, without leading to a strong event of M<sup>w</sup> ≈ 6.0, unlike the case of the Arkalochori earthquake. The herein analysis highlights the importance of local seismological networks to the reduction in location uncertainties. The applied double-difference relocation has managed to improve the relative locations of the foreshock epicenters, which are concentrated in the vicinity of the 27 September mainshock, although their hypocenters could not be adequately constrained. The mainshock apparently broke a large asperity of a west-dipping normal fault and distributed stresses towards its northern and southern edges, triggering aftershocks mainly at two large groups, separated by a spatial gap, where the asperity was located. Similar cases have been previously reported in other significant earthquakes on normal faults in Greece, including the 1999 Athens [79,80], the 2017 Kos [81,82], and the 2020 Samos [83–86] earthquakes. The herein presented relocation analysis of the sequence during its second period, i.e., with data from the temporary local network that was deployed by GI-NOA, permitted the detailed delineation of the main activated structures which could be related to mapped faults on the surface, allowing for a small degree of listricity, as well as contingent smaller antithetic, conjugate faults at depth. The spatiotemporal evolution of the sequence indicated triggering of seismicity throughout most of the aftershock zone soon after the mainshock, attributed to coseismic stress transfer, followed by slower migration towards its outer edges, indicating possible after slip. Based on the post-earthquake fieldwork, both activated fault segments (Kastelli and Lagouta) did not show any signs of surface raptures along their traces, which agrees with the interferometric findings. A straightforward interpretation of horizontal motion patterns derived from the DInSAR analysis points out that the observed migration of motion during the post-seismic period (interferograms of different temporal spans) is mainly in agreement with the aftershock distribution and clustering, and to a lesser extent linked to actual co-seismic motion patterns. This is more evident by the collocation of the horizontal motion lobes to the aftershock clusters #3 and #4, as well as the absence of motion in the central part of the epicentral area where the mainshock nucleated. The vertical down-throw at the Arkalochori village area is also in accordance with the local stress field, being attributed to the hanging wall of the normal westward dipping Lagouta and Kastelli faults (Figure 6). The activation of the specific fault zone is also implied by the increased displacement gradients at the easternmost part of the epicentral area. However, the −22 cm of vertical motion, based on the decomposition of opposite DInSAR geometries (Figure 10), appears to be contaminated by post-seismic deformation. A more conservative estimate would attribute −18 cm of LoS motion to the main earthquake event, as calculated by the interferometric pair of the shortest temporal span after the mainshock. Additional displacement detected by other DInSAR pairs, also present in the decomposition, is attributed to the migration of motion towards NE, in accordance with the clustering and the shallower aftershocks' depth nearby the northern Kastelli fault segment. In conclusion, the 2021 Arkalochori earthquake is a characteristic implication of the contemporary existence of a local arc-parallel extensional regime adjacent to major arcnormal strike-slip zones on the forearc region of the Aegean microplate. The consistency between seismological, geodetic, and field observations has been well demonstrated, highlighting the complementarity of multi-disciplinary approaches. The availability of dense seismological recordings and the contribution of Earth Observation platform-based solutions, validated, when possible, with field observations ensure proper mapping and tectonic interpretation of seismic events. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/app12062815/s1, Table S1: Fault plane solutions for the mainshock of the 2021 Arkalochori sequence, as determined by different agencies. Figure S1: Seismological and accelerometric stations of the Hellenic Unified Seismological Network (HUSN) with available data during the study period (1 June–18 October 2021). (a) Stations in the broader area of Southern Greece, (b) zoom in the area marked with a red rectangle in panel (a), showing stations at distances less than 60 km from the epicenter of the 27 September 2021, mainshock at Arkalochori, Central Crete, marked with a star. Figure S2: Co-seismic LoS displacement maps, as derived using GEP DIAPASON service, for ascending track A029 (18 September 2021–30 September 2021) and descending D109 (24 September 2021–30 October 2021) (c,d). The spatial extend of Sentinel-1 acquisition frames for both geometries are also indicated (a,b). Black lines correspond to fault zones (after [3]). The relocated epicenter of the 27 September 2021, mainshock is marked with a star. Figure S3: Initial location statistics (as reported by HypoInverse). (a,b) RMS travel-time residuals, (c,d) focal depth, (e,f) horizontal (ERX, ERY) and vertical (ERZ) formal location uncertainties, (a,c,e) for Period A, between 1 June and 27 September 2021, and (b,d,f) for Period B, between 28 September and 18 October 2021. Figure S4: Initial location statistics. (a,b) Distribution of distance from the closest station with available data, (c,d) azimuthal gap, (a,c) for Period A, between 1 June and 27 September 2021, and (b,d) for Period B, between 28 September and 18 October 2021. Figure S5: Vertical (a) and E-W (b) ground displacement maps as decomposed using Sentinel-1 DInSAR LoS observations from ascending A029 and descending D109 tracks (see Table 1 in manuscript). Black lines correspond to fault zones (after [3]). The relocated epicenter of the 27 September 2021, mainshock is marked with a star. File E1: Relocated earthquake catalogue (1 June–18 October 2021). References [50,51,87–89] are cited in the Supplementary Materials. Author Contributions: Conceptualization, E.V., J.A. and G.K.; methodology, E.V., G.K., V.K. (Vasilis Kapetanidis), J.A., N.V. and M.F.; software, E.V., V.K. (Vasilis Kapetanidis) and E.P.; validation, E.V., A.K., C.P.E. and S.P.; resources, E.V., G.K., V.K. (Vasilis Kapetanidis), E.P., M.F., C.P.E., V.K. (Vassilios Karastathis), G.-A.T. and J.A.; data curation, A.K., V.K. (Vasilis Kapetanidis), C.P.E. and E.P.; writing original draft preparation, E.V., G.K., V.K. (Vasilis Kapetanidis), E.P., M.F. and J.A.; writing—review and editing, E.V., G.K., V.K. (Vasilis Kapetanidis), M.F., J.A. and N.V.; visualization, E.V., V.K. (Vasilis Kapetanidis) and M.F.; supervision, E.V.; project administration, J.A. and N.V.; funding acquisition, E.V., M.F., J.A., G.-A.T. and N.V. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Initial earthquake catalogues and arrival-time data are available at the database of SL-NKUA (http://www.geophysics.geol.uoa.gr/stations/gmapv3\_db/index.php?lang= en; accessed on 7 November 2021) and GI-NOA (http://bbnet.gein.noa.gr/HL/databases/database; accessed on 7 November 2021). Waveform data from permanent and temporary stations of the HUSN are available at the National EIDA Node hosted at GI-NOA (http://eida.gein.noa.gr/; accessed on 7 November 2021; [90]. Focal mechanisms of the mainshock from various agencies (Table S1) were acquired from https://www.seismicportal.eu/mtws/ (accessed on 22 January 2022). Focal mechanisms for the aftershocks are from GI-NOA (http://bbnet.gein.noa.gr/HL/seismicity/mts/ automatic-moment-tensor-gisola; accessed on 7 November 2021). The relocated catalogue of this study is available in the Supplementary Materials. Acknowledgments: The authors would like to express their appreciation to the three anonymous reviewers for their constructive comments that helped improving the initial version of the manuscript, as well as the editors for giving us the opportunity to publish our work. We thank the scientists and personnel who have participated in the installation or maintenance of the permanent and temporary stations belonging to the HUSN and routine analysis of earthquake data. Conflicts of Interest: The authors declare no conflict of interest. #### References #### Article ### Inventory of Historical and Recent Earthquake-Triggered Landslides and Assessment of Related Susceptibility by GIS-Based Analytic Hierarchy Process: The Case of Cephalonia (Ionian Islands, Western Greece) *Spyridon Mavroulis 1,\, Michalis Diakakis 1, Haralambos Kranis 1, Emmanuel Vassilakis 2, Vasilis Kapetanidis 3, Ioannis Spingos 3, George Kaviris 3, Emmanuel Skourtsos 1, Nicholas Voulgaris <sup>3</sup> and Efthymis Lekkas <sup>1</sup> Abstract: Cephalonia, located in the middle of the central Ionian Islands, has been affected by destructive earthquakes during both the instrumental and the historical period. Despite the fact that it is widely studied from several scientific viewpoints, limited research has been conducted so far regarding the earthquake-triggered landslides (ETL) and the related susceptibility. In the context of the present study, an inventory with 67 ETL from 11 earthquakes that occurred from 1636 to 2014 is presented. Given this record, the study further examines the ETL susceptibility exploiting 10 landslide causal factors in the frame of a GIS-based Analytic Hierarchy Process (AHP). Four factors (i.e., slope, PGA, tectonic structures and lithology) were associated in a higher degree to the locations where ETL occurred on the island. Based on the comparison of the ETL inventory and the landslide susceptibility index (LSI) map, the distribution of ETL in Cephalonia is not random, as their majority (82%) were generated within high to critically high susceptible zones. This fact, along with the AUC values of 80.3%, reveals a fair-to-good accuracy of the landslide susceptibility assessment and indicate that the contribution of the studied variables to the generation of ETL was effectively determined. Keywords: earthquake-induced landslides; landslide inventory; event inventories; rockfalls; contemporary sources; landslide susceptibility; earthquake-induced landslide susceptibility; Analytic Hierarchy Process; Ionian Sea; Cephalonia #### 1. Introduction Among the earthquake environmental effects (EEE) as defined by Michetti et al. [1], the earthquake-triggered landslides (ETL) prevail in a variety of environments from coastal to mountainous areas. They are characterized by high potential to cause not only economic but also human losses [2–5]. Casualties due to ETL are attributed to the collapse of buildings and other infrastructure due to slope failures and mobilization of unstable geological formations [6], as well as to the transport of material with high velocities over terrains with gentle slopes [7]. ETL are responsible for the 70% of all earthquake-induced human losses, which are not directly attributed to the earthquake ground motion [2], but to secondary phenomena. At least 90% of the ETL fatalities are attributed to rockfalls, rock avalanches Citation:** Mavroulis, S.; Diakakis, M.; Kranis, H.; Vassilakis, E.; Kapetanidis, V.; Spingos, I.; Kaviris, G.; Skourtsos, E.; Voulgaris, N.; Lekkas, E. Inventory of Historical and Recent Earthquake-Triggered Landslides and Assessment of Related Susceptibility by GIS-Based Analytic Hierarchy Process: The Case of Cephalonia (Ionian Islands, Western Greece). Appl. Sci. 2022, 12, 2895. https://doi.org/10.3390/ app12062895 Academic Editor: Saro Lee Received: 18 February 2022 Accepted: 9 March 2022 Published: 11 March 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). and rapid soil flows, despite the fact that rock avalanches and rapid soil flows are neither common nor frequent events [7]. The consequences of ETL on society are not only direct, i.e., destruction of buildings and infrastructure, but also indirect, being associated with failures and damage to lifelines including road network, dams and utilities (e.g., [8–10]). This damage has high potential to cause disruption of various socio-economic activities including transportation as well as communication breakdown. This, in turn, can result in a delay in the emergency response and recovery actions and an increase in the number of casualties, due to delayed first aid and transport of the injured to health facilities. ETL have been reported since 1789 BC in China [11]. The most used ETL classification has been provided by Keefer [7], who has analyzed data from a set of 40 historical earthquakes worldwide having a magnitude ranging from 5.2 to 9.5 and generating in a variety of geographic, geological and seismotectonic environments. This classification is based on the nature of the movement, the degree of the internal disruption of the mobilized material and the morphological, lithological and geological properties of the affected areas. Fourteen individual types of ETL are classified into three main categories: (i) disrupted slides and falls, (ii) coherent slides and (iii) lateral spreads and flows [7]. The first category comprises falls, slides and avalanches in rocks and soils, the second one slumps and block slides in rocks and soils as well as slow earth flows and the third one soil lateral spreads, rapid soil flows and subaqueous landslides [7]. The most common type of ETL comprises highly disrupted landslides, which includes falls, slides and avalanches involving either rock or soil, generated along steep source slopes and extending further on relatively gentle slopes [7,12]. The total number of ETL generally increases with earthquake magnitude [7] ranging from a few tens for the majority of M < 5.5 earthquakes to several thousand in M > 8.0 earthquakes. Since the extensive research on ETL worldwide conducted by Keefer [7] and Rodriguez et al. [13], several ETL studies have been performed both at national and local level. Among others, Hancox et al. [14,15] and Rosser et al. [16] have published relevant research and databases for New Zealand, Papadopoulos and Plessa [17] for Greece, and Prestinizi and Romeo [18] and Martino et al. [19] for Italy. Taking into account the existing literature on seismicity and EEE in the Mediterranean, we see that the Lefkada, Cephalonia, Ithaki and Zakynthos islands have been repeatedly hit by strong and destructive earthquakes with many triggered EEE (e.g., Rondoyanni et al. [20] for Lefkada Island and Mavroulis et al. [21] for Zakynthos Island). This strong seismicity is attributed to the proximity of the southern Ionian Islands to the: (i) Cephalonia Transform Fault Zone (CTFZ), which is a NE-SW striking and SE-dipping right-lateral strike-slip fault zone, the most seismic active structure in Greece and the Eastern Mediterranean and (ii) the northwesternmost part of the Hellenic Trench, along which the African plate is subducting beneath Eurasia (Figure 1). Figure 1. The Cephalonia Island is located at the northwestern part of the Hellenic Arc, east of the Cephalonia Transform Fault Zone (CTFZ). The island belongs to the zone III of the current Greek Building Code with a Peak Ground Acceleration (PGA) value of 0.36 g for a return period of 475 years [22]. However, there is a gap regarding the inventory of ETL in Cephalonia, especially the historical ones, which are poorly recorded and mapped. The filling of this gap is considered to be of high importance since this area has suffered from the strongest and most destructive earthquake in the Ionian Sea and from the most destructive earthquake sequence in recent history of Greece: the 1867 earthquake and the August 1953 seismic sequence, respectively, with adverse effects on the local population, the building stock and the natural environment [21,23]. Moreover, Cephalonia is a highly touristic island, with millions of visitors spending time in various landslide-prone locations, especially along the coast. In this context, we used all available sources of information to create an inventory of landslides, which have been induced from destructive earthquakes generated not only during the instrumental period, but also during the historical pre-instrumental period in Cephalonia (Figure 2). The compilation of an ETL inventory constitutes a preliminary step toward the assessment of the landslide susceptibility in one of the most seismic active areas of the world, for which, however, no relevant information has been published so far. Landslide susceptibility mapping is an important tool that contributes to the mitigation of the adverse effects of disasters induced by ETL not only in Cephalonia but also worldwide. In addition, it is another significant step towards more effective and responsible urban and land-use planning and related policies, procedures, guidelines [24] and improvements in seismic and landslide hazard and risk assessment and mitigation. In this study, we assess the landslide susceptibility of the island in order to initially detect and then highlight the areas susceptible to ETL. This assessment is composed of several steps, including the development of landslide causal factors datasets, the implementation of the Analytic Hierarchy Process (AHP) adapted to the needs of the study, the calculation of the landslide susceptibility index (LSI) and finally the validation of the resulting LSI map (Figure 2). Figure 2. Flow chart showing the approach followed for the compilation of the ETL inventory and the respective landslide susceptibility assessment in Cephalonia. #### 2. Geological and Seismotectonic Setting The geological structure of Cephalonia comprises alpine formations that belong to the Paxi and Ionian geotectonic units and post-alpine deposits of Pliocene to Quaternary lying uncomformably on the alpine basement [25–33] (Figure 3). The Paxi unit occurs in the largest part of Cephalonia (Figure 3) and includes mainly carbonate rocks from Triassic to Middle Miocene and a Middle Miocene–Early Pliocene clay-clastic sequence, which includes marls, clays and limestones [32,33] (Figure 3). Figure 3. The neotectonic map of Cephalonia along with epicenters of historical and recent earthquakes that triggered landslides in the island. Fault blocks are also noted (EP: Erissos peninsula, AM: Aenos Mt fault block, AP: Argostoli peninsula, PP: Paliki Peninsula) along with their marginal faults (AEF: Ayia Efimia Fault, KAF: Kontogourata–Agonas Fault, CTFZ: Cephalonia Transform Fault Zone) and other major structures (IT: Ionian Thrust). The Ionian unit is the allochthon tectonic nappe, which crops out in the eastern part of Cephalonia emplaced on top of the Paxi unit (Figure 3). It includes a sequence of Triassic evaporites and limestone breccia as well as Jurassic–Cretaceous fine-grained limestones, red nodular limestones, and shales with the Upper Eocene–Lower Miocene flysch at the top of the sequence [32,33]. The western boundary of the Ionian unit is defined by the Ionian thrust (IT, Figure 3), which is the most external tectonic structure of the Hellenides. The Paxi and Ionian geotectonic units are affected by major reverse faults [31] (Figure 3). The Pliocene–Quaternary deposits include the Pliocene–Calabrian sequence and the Pleistocene–Holocene formations [32,33]. The Pliocene–Calabrian sequence is a marine sequence, observed in an elevated coastal area with width ranging from 2 to 10 km, especially in the largest part of the Argostoli peninsula (AP) and in the eastern and western part of the Paliki peninsula (PP) (Figure 3). It has developed over Paxoi formations, with thickness ranging from 200 to 500 m. The lower part of the sequence is composed of conglomerates, breccia, calcarenites and limestones of the Lower Pliocene. The intermediate part includes yellowish marls with siltstones, sandstone intercalations, sands and conglomerates of the Middle-Upper Pliocene. The upper part consists of marls with intercalations of sands and coarse-grained calcarenites [29,32,33]. The Middle-Upper Pleistocene–Holocene formations occur in several places, mainly in the Argostoli peninsula (Figure 3). These are continental deposits and are classified into scree, marine calcarenites of the Middle Pleistocene, Pleistocene interglacial deposits, Pleistocene scree and Holocene deposits [32,33]. Recent deposits also occur in many sites. Red terrestrial clay sands and coastal conglomerates as well as red calcareous crusts are found in the southwestern part of AP and in the area located WSW of Skala (Figure 3). Scree and alluvial fans cover the Pliocene formations along the foothills of Aenos Mt. These formations cover many limestone slopes. Loose fine-grained and coarse-grained alluvial deposits consist of sands, cobbles and clays on narrow beaches and wide valleys of Cephalonia. Regarding its neotectonic structure, the island is composed of the following fault blocks with different kinematic evolution [33] (Figure 2). The fault block of Aenos Mt (AM) is located in the central and eastern part of Cephalonia. It is bounded to the southwest by the eponymous fault (AFZ—Aenos fault in Figure 3), to the northwest by the Kontogourata-Agon fault (KAF—Kontogourata-Agon fault in Figure 3), to the northeast by the Ayia Efimia fault (AEF—Ayia Efimia Fault in Figure 3) and to the southeast by the Palaeokastro fault (PF—Palaeokastro fault in Figure 3). The Aenos fault block is characterized by intense uplift and intense incision since the Lower Pliocene [32,34]. It comprises an anticline with a NW–SE to N–S trending and westward plunging axis extending for a distance of about 20 km from Myrtos in the north to Kolaitis area in the southeast. The fault block of Erissos peninsula (EP) is located in the northern part of Cephalonia. It is bounded to the south by the Ayia Efimia fault, and it is characterized by uplift and erosion. Since the Pleistocene, the Erissos peninsula presents the same evolution as the Aenos fault block. The Paliki peninsula is located in the western part of Cephalonia (Figure 3). It is bounded to the west by the CTFZ and especially by its southern segment, located offshore western Cephalonia. The evolution of this fault block is affected by the CTFZ throughout Pliocene–Pleistocene, an impact which is expressed through intense uplift [34]. Complex movements attributed to evaporites of the Ionian unit control local vertical movements [35,36]. The Argostoli peninsula is extended southwest and south of Aenos Mt (Figure 3). It is bounded to the east and south by the Aenos fault (Figure 3). A possible increase in compression on the Argostoli peninsula during the Pleistocene resulted in the development of back-thrust faults, exclusively in this fault block. The main seismogenic structure in the Cephalonia area is the Cephalonia Transform Fault Zone [37–39] (Figure 3). It is the most seismically active structure in the Ionian Sea and one of the most active in the Eastern Mediterranean region. A large proportion of the high seismicity in the studied part of the Ionian Sea has been attributed to this structure. Based on the historical and recent seismicity, Cephalonia has been frequently stricken by large and destructive earthquakes [40] with significant impact on the local population, buildings and infrastructures (e.g., [41,42]). A special characteristic of the large earthquakes in the southern Ionian Islands is that they are usually generated as twin or cluster events with occurrence period ranging from a few days to 5 years [43]. Typical examples from Cephalonia are the 1953 earthquakes on 9–12 August and the 2014 earthquakes on 26 January and 3 February (e.g., [44]). #### 3. ETL Inventory in Cephalonia Island 3.1. Recent Studies on Landslide Inventory in Greece The research for the systematic inventory of landslides in Greece is not so widespread. It mainly includes studies presenting maps with landslides throughout Greece [45–49] and maps of landslide-affected settlements [50] as well as several studies with results from field reconnaissance surveys in several earthquake- and landslide-affected areas, where ETL are reported and recorded among other effects. The first attempt for the development of a complete recording system of the landslides in Greece was made by Koukis and Ziourkas [45]. They conducted a statistical analysis on 800 case histories of landslides in Greece from 1949 to 1986. The related information was retrieved from 1500 coded engineering reports and studies conducted by different public corporations including the Hellenic Authority of Geological and Mining Research (former Institute of Geology and Mineral Exploration of Greece) and the Central Union of Greek Municipalities. Their study includes cases in villages and their surroundings as well as along the road network and refers to the landslide frequency distribution and their consequences on different geological formations, altitudes, rainfall, slope angle, etc., while certain interrelations were studied and an engineering geological map as well as landslide distribution and frequency zone maps were compiled. A similar approach was followed by Koukis et al. [46], who further evaluated data from 1116 case histories of landslides from 1949 to 1991, expanding the dataset of Koukis and Ziourkas [45]. They investigated the frequency distribution of landslide control parameters, and their results were presented in tables and graphs. Analogous extensions of the number of landslides and the time period and update of the results of the statistical analysis were conducted by Koukis et al. [47] with 1200 landslides from 1949 to 1995, by Koukis et al. [48] with 1300 landslides from 1950 to 2004 and by Sabatakakis et al. [49] with 1635 landslides up to 2010. The landslide inventory map of Greece [50] and the above works present the landslide locations, but no additional qualitative or quantitative characteristics of the landslides, or information on their causes and time of occurrence. As a result of this approach, the marked landslides could not be associated with specific triggering events, e.g., earthquakes, rainfall or even human activity. For the ETL in Greece in general and for the Cephalonia in particular, the maps of the above studies are not significantly useful as it is not possible to determine whether the eight recorded landslides on the island were caused by earthquakes or rainfall or another triggering factor. Furthermore, Papadopoulos and Plessa [17] collected data on 47 landslides induced by earthquakes from 1650 to 1995 with magnitude ranging from 5.3 to 7.9. Based on the ETL data, they presented magnitude-distance relations for ETL in Greece, which are compatible with curves proposed worldwide. They also examined the spatial distribution of ETL, the size distribution, and the maximum landslide distance from the epicenter. However, the collected data was not sufficient or accurate to be used for studying other parameters, such as the area affected by landslides and correlation with earthquake magnitude, as well as the minimum intensity for generating ETL. According to Papadopoulos and Plessa [17], the historical descriptions of ETL in Greece are usually not precise enough to provide a reliable basis for a modern scientific analysis. Furthermore, after examining hundreds of documents, they supported that a database with data from the 16th century onwards can be exploited. From these 47 landslides presented by Papadopoulos and Plessa [17], only 4 were generated in the seismically active area of the Ionian Islands: a rockslide in Zakynthos from the October 30, 1840, Ms = 6.7 earthquake; a rockfall in Keri (southern Zakynthos) from the April 17, 1893, Ms = 6.4 earthquake; a rockfall in Lefkada from the 27 November 1914, Ms = 6.3 earthquake; a rockfall in the settlement of Vassiliki in southern Lefkada from the April 22, 1948, Ms = 6.5 earthquake. In addition to the above studies, the Emergency Events Database (EM-DAT) of the Center of Research on the Epidemiology of Disasters (CRED; [51]) has also been taken into account. Based on a search for disasters induced by geophysical hazards in Europe from 1920 to present, it is concluded that the EM-DAT comprises records of ground movements caused by the 12 August 1953 and the early 2014 Cephalonia earthquakes and the 14 August 2003 Lefkada earthquake. However, there is no further information on the sites of these landslides and their qualitative and quantitative properties. From the aforementioned, the importance of the present study is ascertained by highlighting the historical and recent ETL in Cephalonia and by presenting all available qualitative and quantitative characteristics of these phenomena on the island and the results of their analysis. #### 3.2. Methodology Landslide inventories are essential for predicting future landslides generation on the basis of past conditions [52]. In order to compile a landslide inventory, various mapping approaches can be applied comprising visual interpretation of aerial and satellite imagery and field surveys and mapping, supported by investigation of contemporary sources and post-event field survey reports [52,53]. The resulting landslide inventories are classified into (i) geomorphological inventories, resulted from respective features of the study area, (ii) event inventories, associated with a specific causative event including mainly earthquakes and rainfalls, (iii) multi-temporal inventories of an area for different time periods and (iv) historical inventories for respective time periods and events [52,53]. We conducted a re-examination of the available scientific literature for historical earthquakes of Cephalonia Island with emphasis on their environmental effects and especially on the ETL. Due to the limited related information in the existing scientific literature (e.g., [41,42,54]), we expanded our search to contemporary sources, including not only post-event field survey reports (e.g., the report of the Institute of Geology and Underground Research [55]), but also daily press databases comprising newspapers of local and national circulation. The newspaper databases comprised (Table 1): The Press Museum was founded by the Association of Editors of Daily Newspapers of Peloponnese-Epirus-Islands, was based in Patras (Northwestern Peloponnese) in 1956 and has been operating since 28 May 1957. Its purpose is the collection and preservation of newspapers, magazines, manuscripts and other publications from previous centuries to present, which constitute knowledge and information sources about the history of the area. Through this website, we were given the opportunity to search for newspaper clippings about the August 1953 earthquakes. In particular, four newspapers were found (Table 1), which provided important information about the environmental effects triggered by the August 1953 earthquakes: The Digitized Historical Archive of the Lambrakis Press Group comprises black and white as well as colored newspapers and magazines, volumes and microfilms in various dimensions (from A2 to A4) from 1922 to present. From this archive, we managed to retrieve related information from two newspapers of national circulation (Table 1), which provided information about the August 1953 earthquake sequence: • TO VIMA (Greek: TO BHMA, lit: The Tribune, abbreviated by TV) #### • TA NEA (Greek: TA NEA, lit: The News, abbreviated by TN) Table 1. Contemporary sources comprising newspapers and magazines of local and national circulation used in this study for revisiting landslides induced by historical and recent earthquakes generated in Cephalonia. The Digital Library of Newspapers and Magazines of the National Library of Greece includes digitized archives of newspaper sheets. Through the website of the digital library, we searched information for the post-earthquake period from two newspapers (Table 1): The information retrieved from the AN, AV and ME newspapers refers to the 1867 earthquake and from the EL and EM to the seismic sequence of 1953. The Greek Parliament Library houses one of the richest collections of Greek and foreign newspapers and magazines from the 18th century to present. Due to the sensitivity of the material and the large number of visitors, the Library has been utilizing digitization and microfilming since the 1980s. Today, about 25,000 microfilms have been digitized to make their content accessible to all. We managed to retrieve related information from three newspapers of national circulation (Table 1): The information retrieved from the EL and ZN refers to the 1912 earthquake and from TD to the 1953 seismic sequence. Furthermore, we also retrieved information included in several papers of a local journal titled "I Kefalonitiki Proodos" (English: "The Cephalonian Progress", abbreviated by KP), which referred to the 1867 earthquake and the 1953 sequence [60,61]. The related information for the recent earthquakes has been retrieved from already published scientific papers depicting the impact of the earthquakes on the natural environment [10,62,63] and from official reports of post-event field surveys [64]. Mavroulis and Lekkas [10] revised the August 1953 earthquake sequence, which comprises the most destructive events in the recent history of Greece. In particular, the latter study reconstructed a complete picture of the primary and secondary effects on the environment of the Cephalonia, Ithaki and Zakynthos Islands induced by the mainshock of 12 August, and its large foreshocks that occurred on 9 and 11 August. Eleftheriou and Mouyiaris [64] reported detailed macroseismic observations of the earthquake in Argostoli, Sami, Lixouri and Fiskardo and their surroundings and presented environmental effects triggered by the earthquake comprising rockfalls and ground failures related to liquefaction phenomena. Lekkas and Mavroulis [62,63] presented the environmental effects triggered by the early 2014 Cephalonia earthquakes (Mw = 6.1 on January 26 and Mw = 5.9 on February 3) along with the ESI-07 intensities based on the qualitative and quantitative information of the generated coseismic and secondary phenomena. The majority of the extracted information was also verified during field surveys and campaigns held in Cephalonia after their extraction from the aforementioned contemporary sources. #### 3.3. Historical and Recent ETL in Cephalonia Island Landslides in Cephalonia have been triggered by earthquakes generated in 1636, 1658, 1767, 1867, 1912, 1953, 1983 and 2014 (Figure 4). The focal parameters of these earthquakes are presented in the following Table 2. Figure 4. The neotectonic map of Cephalonia along with the historical and recent ETL in the island. Legend of the geological formations in Figure 3. Table 2. Focal parameters and impact on the local population of the studied earthquakes that triggered landslides in Cephalonia Island. <sup>1</sup> Sources: Refs. [40,54,65] for epicenter coordinates, occurrence date and magnitude, Ref. [41] for intensities, fatalities and injured people, and [66] for intensities. #### 3.3.1. The 30 September 1636 Earthquake The 30 September 1636 earthquake caused severe structural damage to all buildings of Cephalonia, resulting in 520 fatalities and about 1500 injured people [41,42,67]. Heavier damage was reported in the southern part of Cephalonia, where many villages were completely destroyed. Regarding the environmental effects, the earthquake affected the southeastern part of the island. Failures, comprising slides and rockfalls, were triggered along the south facing southern slopes of Aenos Mt. Unstable limestone boulders were detached from slopes in Eleios area (Figure 4) and rolled down resulting in effects on vegetation, buildings and livestock. In particular, a part of the forest on the slopes was destroyed [42], buildings collapsed after crushing and animals were buried under debris. #### 3.3.2. The 24 August 1658 Earthquake This earthquake affected the western part of Cephalonia [41,42]. It caused the destruction of 500 houses, resulting in 20 fatalities [67–69]. As regards the induced earthquake environmental effects, Tsitselis [69] referred to the triggering of landslides in the coastal eastern part of Paliki peninsula (Lixouri) resulting in submergence of two coastal sites. Furthermore, ETL were induced, resulting in the collapse of a church founded on the top of a hill. Unfortunately, there is no more information available for determining the exact location of the induced phenomena. #### 3.3.3. The 22 July 1767 Earthquake Cephalonia was also affected by the 22 July 1767 earthquake [41,42,67,70,71]. Lixouri, in the eastern part of Paliki peninsula, was completely destroyed, Argostoli, in the northern part of the eponymous peninsula, was less affected, while Sami, in the eastern part of the island, and Fiskardo, in the northern part of Erissos peninsula, were extensively damaged as all churches and monasteries were razed to the ground [72–74]. As a result, 303 fatalities were reported, with 50 of them in Paliki peninsula [69]. Concerning the ETL, rockfalls were generated in Assos area, located in the central-western part of Erissos peninsula [75,76] (Figure 4). #### 3.3.4. The 4 February 1867 Earthquake This earthquake is the largest that ever hit the Ionian Sea. The greatest damage was observed in the western part of Cephalonia, especially in Paliki peninsula, where Lixouri and the surrounding villages as well as residential areas in Thinia valley were completely destroyed ([67]; AN, 04.02.1867; AV, 06.02.1867; ME, 07.02.1867). As a result, 3200 houses were destroyed and 2600 were damaged, resulting in 224 fatalities [42]. Concerning ETL, rockfalls and slides were reported in Paliki peninsula. Large volume of clay material failed resulting in a large ground crack with depth of 8 m and width of 3 m in Ayios Stefanos area in southern Paliki [76]. In the same area, and particularly in Soulari village, landslides were also induced during the 2014 earthquake [62]. In addition, debris slides and rockfalls were triggered by the 1867 earthquake in Parissata, Monopolata, Vilatoria, Vovikes and Kontogenada villages, all in the central western part of Paliki peninsula, without causing structural damage [60,68] (Figure 4). #### 3.3.5. The 24 January 1912 Earthquake The 1912 earthquake destroyed what had remained intact during the great 1867 earthquake. The human losses were minimal compared to the 1867 disaster, comprising seven fatalities in Asprogerakas village, located between Eleios and Anninata villages in southeastern Cephalonia, attributed to house collapse and one in Skala village from falling debris. In Skala area, rockfalls were reported and the mobilized material ended up in the adjacent roads and fields (ZN, 28.01.1912). At a distance of 400 m from Skala village, large limestone boulders were detached from the slopes, rolled down and ended up to adjacent road resulting in traffic disruption (ZN, 28.01.1912) (Figure 4). #### 3.3.6. The August 1953 Seismic Sequence The August 1953 seismic sequence comprised the mainshock on 12 August and its large foreshocks on 9 and 11 August. All earthquakes triggered landslides in Cephalonia with considerable impact on the local population and on the natural and the built environment [10]. All affected sites described below are presented in Figure 4. • The 9 August 1953 earthquake This earthquake induced rockfalls in the coastal quarries of Argostoli town and along the western slopes of Ayia Dynati Mt (EK, 10.08.1953; NP, 11.08.1953; PL, 11.08.1953; EM, 11.08.1953). • The 11 August 1953 earthquake This seismic event generated rockfalls and slides (TV, 12.08.1953; IM, 12.08.1953; NP, 12.08.1953; PL, 12.08.1953). Rockfalls were generated in Sami area (NP, 12.08.1953), in Tzanata village (PL, 12.08.1953) in Lourdata village (TV, 12.08.1953; IM, 12.08.1953) and along road slopes leading from Sami to Argostoli (TV, 12.08.1953), from Argostoli to Eleios area (IM, 12.08.1953) and along the road network leading to Chionata, Skala and Poros villages (TV, 12.08.1953). The unstable material was accumulated on adjacent roads, resulting in traffic and communication disruption. In addition, the detached limestone boulders crushed buildings in residential areas, resulting in heavy structural building damage ranging from partial to total collapse and subsequently increased related fatalities and injuries. A characteristic example of such impact is Tzanata village, which suffered rockfall-induced complete building destruction, uprooting of trees and 14 fatalities (PL, 12.08.1953). • The 12 August 1953 earthquake The mainshock of the 1953 earthquake sequence induced landslides in several sites of Cephalonia. The generated landslides are classified into detachment of boulders from abrupt slopes and consequent rockfalls and slides. The majority of them were rockfalls, which affected the adjacent infrastructures including the road network and buildings in villages and towns in the earthquake-affected area. The generated landslides are presented below along with the impact on the local population and on the built environment. Starting from northern Cephalonia, rockfalls were generated along a hill adjacent to Markoulata village, located in the northern part of Erissos peninsula [55]. In the southern part of the same fault block, rockfalls occurred along a gorge between Karousata and Lekatsata villages, causing destruction of olive trees, as well as in Logarata and Karousata villages founded along the southern slopes of Kalon Mt, resulting in damage to buildings. In the northwestern part of the transition zone between the Erissos peninsula in the north and the fault block of Aenos Mt in the south, Myrtos beach, one of the most impressive sites in Cephalonia, was also affected by rockfalls. A limestone boulder was detached from the slopes and ended up in the adjacent bay, while a church founded on the slope was completely destroyed by the generated rockfalls (EM, 14.08.1953). Rockfalls were also triggered along the eastern margin of Thinia valley, which has been formed along the transition between the northwestern Ayia Dynati Mt located east and the northeastern Paliki peninsula located west. The reported landslides affected the upper part of Agonas village [55], the coastal Ayia Kyriaki area, where debris reached the sea, and along the slopes over Sotira beach (TX, 16.08.1953). Rockfalls were also triggered along the abrupt slopes surrounding Kourouklata village [55] located south of the aforementioned sites. It was the worst affected village as it was completely destroyed by fallen boulders (PL, 16.08.1953). Similar phenomena were reported from north of Drapano village. The unstable blocks ended up in Argostoli Gulf without impact on residential areas [61]. Several areas were also affected by ETL in the eastern part of the fault block of Aenos Mt. Rockfalls were generated in Sami, Zervata and Katapodata villages along the western front of Avgo Mt (TN, 12.08.1953; IM, 13.08.1953; TX, 14.08.1953, 19.08.1953; [55]), in Dichalia village in the northern slopes of Avgo Mt [55], in Pyrgi village in the eastern part of Roudi Mt (TX, 19.08.1953), in Valsamata village west of Roudi Mt (TX, 14.08.1953), in Tzanata and Monastiraki villages in the southern part of the Atros Mt (TX, 14.08.1953; IM, 14.08.1953; [55]), and in Atsoupades, Kolaitis, Arginia, Chionata and Plateies villages along the southern front of Aenos Mt (IM, 13.08.53; IGUR, 1954). Similar phenomena were also induced in the southeastern end of Cephalonia, in particular in Skala and Anninata villages (TX, 14.08.1953; NP, 14.08.1953; TN, 19.08.1953). The Argostoli and Paliki peninsula were also affected by rockfalls. In Argostoli area, rockfalls were generated along the slopes of kilns close to the town (IM, 14.08.1953, 16.08.1953) and in Faraon hill, located in Lassi area (NP, 14.08.1953, 15.08.1953; PL, 14.08.1953; IM, 15.08.1953). In Paliki, rockfalls were generated in Ntouri (Paliokastro) hill, located north of Lixouri, as well as at a cape located at the southeastern end of the peninsula. Moreover, landslides were observed along slopes in the road network leading from Sami to Argostoli and from Argostoli to Livadi, located north of Ntouri site. The ETL had considerable impact on the local population and the natural and built environments. They affected vegetation, caused damage to infrastructures including the road network and to buildings in residential areas resulting in increasing fatalities and injuries. Rockfalls caused damage to olive groves along the abrupt slopes of the gorge in Karousata and Lekatsata [55], in Agonas area [55] and in Ntouri area of Paliki as well as to olive groves and vineyards in Zervata area (TX, 14.08.1953). The vegetation acted as protection barrier for the residential areas, for example, in the case of Agonas, where olive trees protected its lower part from rolling boulders and subsequent destruction [55]. As regards the building damage, the ETL caused heavy structural damage in several villages and led to more fatalities and injured people. Characteristic examples of such impact are the villages of Tzanata (PL, 12.08.1953), Chionata (IM, 13.08.53), Anninata (TX, 14.08.1953) and Plateies [55], where rockfalls claimed the life of residents and increased the number of injuries, Kourouklata (PL, 16.08.1953; [55]), Valsamata (TX, 14.08.1953) and Kolaitis [55], which were totally destroyed by rockfalls, and Zervata (TX, 14.08.1953), Katapodata [55] and Atsoupades [55], where buildings suffered extensive damage by rockfalls. Rockfalls affected several parts of the road network in Cephalonia. The main effect was the accumulation of the mobilized material on the adjacent roads resulting in temporary traffic disruption and communication breakdown between the affected residential areas. In particular, traffic disruption due to proximity to high steep slopes and to rockfalls triggered by the 12 August mainshock and the foreshocks on 9 and 11 August was reported for the road leading from Argostoli to northern Cephalonia (NP, 11.08.1953; PL, 11.08.1953; EM, 11.08.1953), from Argostoli to Sami (TV, 12.08.1953), from Argostoli to Eleios-Pronnoi area (IM, 12.08.1953) and from Sami to the surrounding villages (NP, 12.08.1953). In addition, roads connecting villages in several parts of the island were also affected. Characteristic examples are the roads in Pyrgi village (TX, 19.08.1953), in Sami and Zervata village (TN, 12.08.1953; IM, 13.08.1953; TX, 14.08.1953, 19.08.1953), in Atsoupades village (IM, 13.08.53) and in Skala area (TX, 14.08.1953) among others. #### 3.3.7. The 17 January 1983 Earthquake The 1983 Ms = 7.0 earthquake [37] caused slight damage to Cephalonia, due to the fact that its epicenter was located offshore between Cephalonia and Zakynthos Islands. It mainly generated non-structural damage to buildings with reinforced concrete frame and infill walls in Lixouri, Argostoli, Sami and Fiskardo areas [64]. The largest aftershock of M = 6.2 occurred on 23 March [37], but the M = 6.0 aftershock that was generated the same day as the mainshock resulted in larger damage than the mainshock [64]. Along the road to Fiskardo, at Charakas site, rockfalls were triggered along the subvertical artificial road cut [64] (Figure 4). Limestone boulders and debris from the slope were concentrated on the adjacent road and resulted in traffic disruption. Downstream of Charakas road, on the steep coastal slope, rockfalls were also generated, resulting in sea water turbidity [64] (Figure 4). #### 3.3.8. The Early 2014 Cephalonia Sequence The early 2014 Cephalonia sequence comprised two earthquakes generated on 26 January (Mw = 6.1) and 3 February (Mw = 5.9), with considerable impact on western Cephalonia [62,63]. Among other effects, the 2014 earthquakes triggered landslides mainly in Paliki peninsula (Atheras, Petanoi, Kipouraeoi, Soullari and Xi areas), along the margins of Thinia valley (Agonas and Ayia Kyriaki areas), in Myrtos coastal area, along slopes in the western part of Ayia Dynati Mt and in the southern part of Argostoli peninsula (Vlachata) [62,63]. It is significant to note that such failures were not generated in Erissos peninsula. All affected sites described below are presented in Figure 4, while representative views of the triggered effects and the affected sites are presented in Figure 5. In the Myrtos area, two limestone boulders were detached from the eastern steep slope, they rolled down and stopped in the beach at about 100 m from the shoreline (Figure 5a–c). During rolling down the boulders resulted in destruction of the road leading from the top of the slope to its base. Furthermore, extensive rockfalls and rockslides were also triggered along the coastal slopes close to the beach, particularly in Charakas (Figure 5a), a site which was also affected by the 1953 and 1983 Cephalonia earthquake [55,64]. Rockfalls and slides were generated along the margins of Thinia valley, mainly in the steep slopes over Agonas village and in Ayia Kyriaki coastal area in its eastern margin and secondarily along slopes adjacent to the road leading to Zola village in its western margin (Figure 5d). Rockfalls also affected several parts of the road network leading from Argostoli town to Livadi area located northwards. Unstable material was mobilized by the earthquake along steep road slopes and accumulated on the road asphalt surface resulting in some cases to temporary traffic disruption. Extensive landslides were triggered on slopes of Paliki peninsula by the first earthquake on 26 January. Rockfalls were generated in its northern part, where a building in Atheras village was crushed by a rolling boulder and partially collapsed (Figure 5e,f), fortunately without casualties and injuries. During the second earthquake on 3 February, boulders in the same site were again detached from the slopes, but they did not reach the village. Figure 5. Cont. Figure 5. The 2014 ELT in Cephalonia: (a) The landslide-prone Myrtos coastal area. (b) Water turbidity in Myrtos Bay after the 26 January 2014 earthquake attributed to rockfalls along the adjacent coastal slopes. (c) Rockfalls by the early 2014 Cephalonia earthquakes over the Myrtos beach and at Charakas site. (d) The landslide-prone eastern margin of Thinia valley, where the Kontogourata– Agonas fault occurs. Rockfalls and slides triggered in Cephalonia by the early 2014 earthquakes: (e,f) Unstable limestone boulders in Atheras village in north Paliki crushed trees and a building. (g,h) Slides in Petanoi coastal area resulting in traffic disruption. (i–k) Rockfalls close to the Kipouraioi monastery affecting its yard. Landslides were also triggered along the western coastal part of Paliki, which is characterized by high coastal fault scarps and steep slopes. Characteristic examples were recorded in Petanoi and Kipouraioi coastal areas. In Petanoi area, the second earthquake triggered a large slide (Figure 5g,h), which reached the beach and partially covered it. In addition, the slide material blocked the road leading to the beach, resulting in traffic disruption for 3 days. In the Kipouraeoi area, landslides were generated by both 2014 earthquakes along the impressive steep coastal fault scarps (Figure 5i–k). In the area of Kipouraeoi monastery, rockfalls and rockslides occurred along an abrupt slope, at the top of which the monastery is constructed. The crown of the slide was very close to the monastery and affected its yard (Figure 5j,k). Additionally, slides also occurred along road slopes in the area surrounding the monastery. In the Xi coastal area, in the southern part of Paliki peninsula, small rockfalls were generated by both 2014 earthquakes. In Soulari area, an impressive slide was triggered by the 26 January earthquake on the top of a hill. A large fragment composed of silt and clay moved downhill and formed a large main scarp, while the main body of the slide was split into smaller sections. With the contribution of heavy rainfalls that followed the first earthquake, the main scarp of the slide further collapsed during the second earthquake and the pre-existing cracks were enlarged, resulting in larger gaps in the area. The list of landslides induced by the 2014 earthquakes also includes the rockfalls triggered in the limestone quarry situated west of Vlachata village. These rockfalls temporarily blocked the road leading from Argostoli to Poros port. #### 3.4. Maximum Epicentral Distances as a Function of Earthquake Magnitudes The epicentral distances of the landslides, which were induced by the aforementioned historical and recent earthquakes in Cephalonia, were calculated, taking into account the locations of the landslides, as obtained from the ETL inventory of the island, and the earthquake magnitudes, as obtained: (a) for the 1636, 1767 and 1867 earthquakes from the SHARE European Earthquake Catalogue (SHEEC) 1000–1899 [54], (b) for the 1912, 1953 and 1983 earthquakes from the updated and extended earthquake catalogue for Greece and adjacent areas since 1900 compiled by Makropoulos et al. [40] and (c) for the early 2014 earthquakes from the relocated catalogue for the 2014 Cephalonia aftershock sequence compiled by Papadimitriou et al. [77], Kapetanidis [78] and Sakkas et al. [65]. The maximum epicentral distance of landslides for each earthquake was then determined. The pairs of maximum epicentral distance of landslides and the magnitude of the causative earthquakes were plotted on a graph (Figure 6). The distribution of these points was also compared with the upper bound limit/curve for the maximum observed epicentral distances of disrupted slides and falls proposed by Keefer [7], which was based on a dataset of 40 worldwide earthquakes. This curve represents the maximum distances at which ETL can be expected to be generated for specific Mw values. The graph in Figure 6a shows how the distribution of Cephalonia ETL data generally fit well the Keefer's relationships. In the graph in Figure 6b, together with the points for Cephalonia, the pairs of maximum epicentral distances for landslides and magnitudes for earthquakes in Greece, as presented by Papadopoulos and Plessa [17], are also shown. From these graphs, it is also concluded that outlier events attributed to various triggering factors and local seismic amplification were not recorded in Cephalonia. Figure 6. (a) Maximum distance from earthquake epicenter of disrupted slides and falls induced by historical and recent earthquakes of different magnitudes generated onshore and offshore Cephalonia versus earthquake magnitude. The dashed line is the upper bound for disrupted slides or falls by Keefer [7]. (b) Maximum epicentral distances of landslides triggered by historical and recent earthquakes of different magnitudes versus earthquake magnitude. The red subset comprises data from Cephalonia and the blue subset from around Greece published by Papadopoulos and Plessa [17]. The dashed line is the upper bound for disrupted slides or falls by Keefer [7]. #### 4. ETL Susceptibility Assessment in Cephalonia Based on GIS-Based Analytic Hierarchy Process #### 4.1. ETL Susceptibility Model In the context of landslide susceptibility assessment research, 596 thematic parameters have been used so far worldwide, which are classified into five main categories, as shown in the review by Reichenbach et al. [53]: (a) geological parameters, (b) hydrological parameters, (c) parameters related to land use, (d) morphological parameters and (e) other parameters not included in the previous categories. In the case of the assessment of landslide susceptibility in Cephalonia, the following parameters were used: (a) morphological parameters, including slope, slope aspect and slope curvature; (b) geological parameters, such as lithology of geological formations and distance from tectonic structures (faults and thrusts); (c) seismological parameters, including the peak ground acceleration (PGA); (d) environmental parameters including vegetation, derived from the NDVI index and the distance from the road network; (e) hydrometeorological parameters, such as the spatial distribution of rainfall and the distance from the drainage network (rivers and streams) (Figures 7 and 8; Table 3). Figure 7. Thematic maps illustrating the morphological, geological and seismological causal factors taken into account for the landslide susceptibility calculations. (a) slope, (b) aspect, (c) curvature, (d) lithology, (e) tectonic structures and (f) PGA. Figure 8. Thematic maps illustrating the environmental and hydrometeorological causal factors taken into account for the landslide susceptibility calculations. (a) NDVI, (b) yearly rainfall, (c) drainage network and (d) road network. Morphological slope controls the balance of restraining and destabilizing load forces acting on a slope [79,80], while forces are developed to maintain a steep rather than a gentle slope. From the statistical analysis of recorded landslides throughout Greece [81], it appears that the 16–30◦ class is the most susceptible one. Among the morphological parameters, slope is widely used in landslide susceptibility models, as it is the most critical parameter that contributes to the reliable estimation of susceptibility [24,82–84] and to the definition of non-susceptible areas [85]. Slope aspect is a very important parameter as it affects the exposure of the surface to sun and dry winds and indirectly to the flora as well as to the degree of saturation and evapotranspiration of the soil. According to Guzetti et al. [86,87], at northern hemisphere latitudes, where Ionian Islands are located, N- and NW-facing slopes are more prone to failures due to lower temperatures and more shade, factors that favor soil moisture retention. Table 3. Thematic layers used in the study and their sources. A concave surface is more likely to suffer landslides, as after prolonged rainfall it can retain and store rainwater for longer time periods, unlike a curved surface. Additionally, a curved slope can delineate the rocky bedrock that is likely to be present in the area. Areas with positive curvature values identify curved surfaces and correspondingly negative values indicate concave surfaces. A zero curvature value indicates a flat surface. The more negative a value is, the higher the probability of landslide, while on the contrary, a surface with a positive curvature value (curved surface) is less likely to experience landslide phenomena. The maps of slope, aspect and curvature (Figure 7a–c, respectively) were derived from the digital elevation model (DEM) of Cephalonia generated from the TanDEM-X mission, which was based on very high resolution (VHR) synthetic aperture radar (SAR) X-band data [94]. The TanDEM-X global DEM was initially introduced in 2016, based on the processing of SAR data, which were acquired within the years 2011 and 2012, succeeded a spatial DEM resolution of 0.4-arcsecond (ca. 12 m) with the aim of establishing a 2 m relative height accuracy [95–97]. Independently published comparisons prove an of TanDEM-X DEM smaller than ±0.20 m, a root-mean-square error (RMSE) smaller than 1.4 m and an excellent absolute 90% linear height error below 2 m. The assessed accuracy of these data sets is rather excellent and are used quite often for geomorphological and hydrological modeling [98,99]. Concerning the geological parameters used for the assessment of landslide susceptibility, the bedding of formations [100,101], the presence of faults and especially the presence of active faults in seismic active areas [102–106] and local hydrogeological features [24,107] are common in relevant models. The distance to tectonic structures and especially to faults is the most widespread parameter, derived from simple mapping of geo environmental and other data [53,108,109]. Consideration must be given to the fact that the faulted rock along the zone and the surrounding unfaulted rock behave differently during the occurrence of an earthquake, and each fault responds differently to each seismic event, even within the same fault zone. Regarding the geological parameters used for assessing landslide susceptibility, the presence of scree, talus cones and clastic formations of various age is an aggravating factor for the occurrence of slope failures by earthquakes. Alluvial deposits have little or no influence on the occurrence of these phenomena, while carbonate rocks are susceptible to slides and rockfalls in cases where a steep slope has been formed either by geological processes (e.g., fault zones, incision, erosion, etc.) or by human intervention (e.g., steep road slopes, etc.). Both geological parameters, lithology and tectonic structures, in the case of the landslide susceptibility in Cephalonia were derived from the Neotectonic Map of Cephalonia [32] (Figure 7d,e). Earthquakes are considered to be a main factor in landslide susceptibility, which is why they are taken into account in relevant studies worldwide, as in Greece [110,111] and Central Asia [112]. In seismic hazard calculations related to landslide susceptibility studies, co-seismic strong ground motion, which is the cause of damage after the occurrence of large earthquakes, is most often expressed in terms of peak ground acceleration (PGA). PGA is often obtained from probabilistic seismic hazard assessment (PSHA). It is mainly preferred as an earthquake metric, as it refers to the overall seismicity of the study area [110]. Another reason is that PGA has been used in many studies in order to determine earthquake-induced displacements [49,113–115]. PSHA in the framework of the present study, in terms of PGA, was adopted by Sakkas et al. [65], who applied the Cornell and McGuire method [116,117] that requires a seismotectonic model. Regarding the latter, the seismogenic crustal area source model of the European Seismic Hazard Model (ESHM), proposed by the Seismic Hazard Harmonization in Europe (SHARE) project [118,119], was used. The required seismicity parameters for PSHA, i.e., the b-value, earthquake rate and maximum magnitude for each zone, were taken from Sakkas et al. [65]. The Modified Gutenberg–Richter (MG-R) model [120,121] was adopted as the earthquake occurrence model. The Ground Motion Prediction Equation (GMPE) of Danciu and Tselentis [122] was used for the prediction of the PGA distribution. It is worth noting that this GMPE, obtained using Greek data, can be considered as reliable, as it has been applied in many PSHA studies in Greece (e.g., [123–125]). The R-CRISIS software [126] was utilized to obtain PGA for a return period of 475 years for Cephalonia Island, considering a rock basement and faulting types corresponding to reverse or strikeslip focal mechanisms (Figure 7f). In terms of land use, the majority of researchers use combinations of vegetation, land cover and land use from existing maps prepared after visual interpretation of aerial photographs and more recently from automatic or semi-automatic processing of satellite imagery at various scales [127,128] and in different regions, maps showing changes in vegetation or land use [129–132]. The rationale behind incorporating vegetation into landslide susceptibility models is that vegetation depends on slope stability. The normalized difference vegetation index (NDVI) gives a quantitative estimation of the vegetation growth and biomass in the study area and constitutes an index used in landslide susceptibility studies worldwide. NDVI values range from +1.0 to −1.0. Very low NDVI values (for example, 0.1 or less) correspond to no vegetation cover and exposure of the unvegetated area to surface erosion processes and subsequent slope failures [133]. Moderate NDVI values ranging approximately from 0.2 to 0.5 correspond to sparse vegetation, while high NDVI values ranging approximately from 0.6 to 0.9 correspond to dense vegetation [133]. Thus, the lowest NDVI value has the highest impact on the occurrence of landslides. In this study, Sentinel 2 satellite images were used (acquisition date 21.04.11) and the index was calculated by using the red and near infrared spectral bands [134] (Figure 8a), according to the following formula NDVI = (NIR − R)/(NIR + R), where NIR and R are the observed reflectance in the near infrared and red portions of the electromagnetic spectrum, respectively. Regarding the hydrometeorological parameters, the average annual precipitation, leads to an increase in landslide susceptibility by affecting the water content of rock/soil. In this case, gridded precipitation data were collected from the Hellenic National Meteorological Service [88], based on which the spatial distribution of the average annual rainfall in Cephalonia was modelled in raster form (Figure 8b). Concerning the environmental parameters and the influence of the drainage network, the distance from drainage network is used according to the following approach: rivers and streams can influence the probability of landslide occurrence, as their erosive processes especially at the base of the slopes and the intense incision across active faults and increase the instability conditions of the slopes and the susceptibility of landslides. As the distance from the branches of the drainage network increases, the susceptibility to landslide events decreases. Rivers and streams in the study area were derived from the topographic maps of the Hellenic Military Geographical Service in 1:50,000 scale [89–92] (Figure 8c). Due to inadequate drainage and destabilization of hillslopes by undercutting and overloading, landslides are more frequent near the road network [135]. With increasing distance from the road network, the landslide susceptibility in an area decreases. Important information on landslide susceptibility along the road network is obtained from the study of the history of such events. The road network in the study areas was derived from the topographic maps of the Hellenic Military Geographical Service in 1:50,000 scale [89–92], which was then updated by using OpenStreetMap [93] (Figure 8d). After the factors were selected, they were classified into classes and rank values and standardized ratings were assigned for these classes (Table 4). In order to evaluate the relative weight of each factor, the Analytic Hierarchy Process (AHP) developed by Saaty [136] was selected as a decision tool. In the frame of the AHP, all factors are compared pairwise by taking into consideration the intensity of their significance and contribution to the generation of ETL, as it is derived from expert opinions, experience and knowledge gained from previous events. In the current study, the SpiceLogic Inc. Analytic Hierarchy Process Software (trial version 3.4.5) [137] was used. In total, 45 pairwise comparisons undertaking an approximate eigenvector calculation were conducted resulting in the pairwise comparison matrix, the consistency ratio and the multi-criteria utility function (Table 5; Figure 9). For the consistency ratio, Saaty [136] suggested that it should be less than or equal to 0.1, to accept the weights. The consistency ratio in this case, as derived from the software, is equal to 0.071, which reveals a suitable and reasonable level of consistency in the pairwise comparison that is a satisfactory value to recognize and estimate the factors weights. Table 4. Landslide causal factors, classes, rank values, standardized ratings and weights used in the landslide susceptibility assessment in Cephalonia. #### Table 4. Cont. Table 5. Pairwise comparison matrix regarding the landslide causative factors as required for applying the AHP method. Consistency Ratio calculated as 0.071. Multi-Criteria Utility Function = 0.22 × [Slope] + 0.03 × [Aspect] + 0.04 × [Curvature] + 0.14 × [Lithology] + 0.18 × [TectonicStructures] + 0.03 × [NDVI] + 0.2 × [PGA] + 0.06 × [Rainfall] + 0.04 × [Rivers] + 0.06 × [Roads]. Next, the thematic layers of the distance to roads, to rivers and to faults were calculated with the use of the Euclidean distance tool included in the Spatial Analyst toolbox of ArcGIS 10.7 [138]. The thematic maps were converted to raster datasets, which were then reclassified based on the aforementioned classes, ranking and standardized ratings. The resulting weighted raster thematic maps were multiplied by the corresponding weights based on the multi-criteria utility function (Table 5) with Raster Calculator tool of the Spatial Analyst toolbox in ArcGIS 10.7. This tool allows researchers to create and execute Map Algebra expressions for outputting a raster. The zonation map is obtained from the sum of the weighted map. Each cell of the map resulted from the aforementioned calculation is characterized by a certain landslide susceptibility index (LSI) value. The final earthquake-triggered landslide susceptibility map of Cephalonia Island comprises seven susceptibility zones (non-susceptible, very low, low, moderate, high, very high, and critically high susceptibility) (Figure 10) according to the Jenks natural breaks classification method [139]. The area of each zone as well as the percentage on the total area of the studied island is presented in Table 6. Figure 9. Ranking of weights for the 10 landslide causative factors. Figure 10. The landslide susceptibility index (LSI) map of Cephalonia Island, against the recorded ETL. Table 6. Areas of susceptibility map classes. #### 4.2. Validation of the Model The creation of the landslide susceptibility map was followed by the verification of the accuracy of the landslide susceptibility model. The validation of the landslide susceptibility results is usually carried out by applying two methods in the frame of the relevant studies. The first approach involves the comparison of the landslide susceptibility map with an inventory map, which includes landslide events in the study area (e.g., [110,111,140–142]). The second approach involves the use of the receiver operating characteristic (ROC; [143–145]). The result of this approach is a success rate resulting from the comparison between the earthquake-triggered landslide grid cells and the earthquaketriggered landslide susceptibility grid (e.g., [146]). Both approaches were used in this study for Cephalonia. For the first one, the ETL inventory in Cephalonia from 1636 to 2014 was used. The majority of the 67 landslides occurred in areas of high (24/67, 35.82%), very high (16/67, 23.88%) and critically high (15/67, 22.39%) susceptibility (Table 7). In total, 55 out of 67 events (82.09%) occurred in high to critically high susceptibility zones, while 12 occurred in low to medium susceptibility zones (3 in low and 9 in medium susceptibility zones, with a percentage of 4.48% and 13.43%, respectively) (Table 7). Table 7. Distribution of the recorded historical and recent ETL in the detected landslide susceptibility zones of Cephalonia Island. For the second approach based on the ROC curve, the curves are obtained by plotting in a graph with both axes having values ranging from 0 to 1. The x-axis comprises values of false positive rate based on the resulting earthquake-triggered landslide susceptibility map and the y-axis comprises values of true positive rate based on the ETL sites. The accuracy of the model is related to the area under the curve (AUC). The AUC ranges from 0.5 to 1.0 [147,148] with a value equal to 1.0 corresponding to a perfect model prediction accuracy. A value found along the diagonal line of the graph implies a 50% probability of predicting the generation of landslides. The larger the value of the AUC, the better the landslide susceptibility model predicts susceptible areas and landslides. In the case of Cephalonia, the ROC graph and the AUC have been derived by using the ArcSDM tools in ArcGIS 10.7. The AUC was calculated as 0.803 (Figure 11), which reveals fair to good model prediction accuracy. Figure 11. ROC graph and AUC for the landslide susceptibility model applied for Cephalonia. #### 5. Discussion This work explores earthquake-triggered landslides (ETL) on the island of Cephalonia, one of the most active areas in the Mediterranean region. The study addresses the gap in the record of past events and examines the ETL susceptibility of the island exploiting the long record that has been created. Given the rich record of the identified ETL phenomena, the island is considered to be particularly prone to slope failures due to earthquakes. From the implemented inventory, it is concluded that 67 cases were induced by earthquakes in Cephalonia from 1636 to 2014, with most of them being triggered by the 12 August 1953 earthquake (30 cases, 44.78%). The majority of failures have been reported from earthquakes after 1900 (Figure 12). Although historical earthquakes that have affected Cephalonia had magnitudes larger than 5.9, information on environmental effects, despite their important impacts, is limited. This does not mean that these events did not cause such effects, but it is probably due to the fact that the interest of residents, authorities and researchers at the time was mainly focused on damage to buildings which were mainly associated with unpleasant effects on the local population. In addition, the earthquake effects resulted usually in heavy damage of the road network, disrupting transportation between local villages, making it impossible for researchers to record the complete impact of earthquakes on both the natural environment and the building stock. This situation seems to have changed after the first half of the 20th century, as evidenced by the plethora of recorded landslides caused by the 1953, 1983 and 2014 earthquakes. Based on the rich record of ETL developed, it was possible to validate the result of landslide susceptibility analysis in terms of spatial distribution. In the course of this analysis, we used not only morphological and geological variables as the causative factors, but also seismological, hydrometeorological and environmental parameters, in order to effectively identify and precisely map the most susceptible zones in the island. Both from the landslide susceptibility map individually and from the comparison of the historical and landslide susceptibility maps, it was found that the spatial distribution of the studied effects in Cephalonia is not random. They were mainly generated in certain zones with high to critically high susceptibility to landslide triggering in the case of strong earthquake ground motion. These zones include the following from north to south: to the eponymous fault zone, which bounds the mountain to its south and west. As a result of the presence and activity of the zone, extensive outcrops of scree and talus cones as well as fragmented limestone of the Paxi unit participated in the geological setting and led to the generation of landslides during the 1636, 1912 and August 1953 earthquakes. In particular, the slope failures of 1953 had considerable impact on settlements in the area, which were founded on the above-mentioned formations, resulting in their partial destruction and human losses. Concerning the ETL distribution in the fault blocks of Cephalonia, the most affected in terms of ETL is the fault block of Aenos Mt and the eastern Cephalonia followed by Paliki, Erissos and Argostoli peninsulas. #### 6. Conclusions The landslide susceptibility map was classified according to the natural break method into seven classes of critically high, very high, high, moderate, low, very low, and no susceptibility (Figure 10) corresponding to 3.48%, 9.76%, 14.97%, 19.57%, 23.16%, 20.60% and 8.46% of the total study area respectively (Table 6). Overall, from qualitative assessment of the distribution of the landslides, illustrated in the description of the aforementioned most susceptible zones, it was concluded that four factors (i.e., slope, PGA, tectonic structures and lithology) were associated in a higher degree to the locations where ETL occurred on Cephalonia Island. This supports the difference between this group of parameters and the rest in terms of weighting factors estimated in AHP. It should be also noted that despite the limited extent of high to critically high susceptible zones in the island (28.21% of its total area based on Table 6) they host 82% of the ETL phenomena included in the inventory. This fact along with the AUC values of 80.3% reveals a fair-to-good accuracy of the landslide susceptibility assessment in the island. Furthermore, the validation results indicate that the geoenvironmental conditions and the contribution of the studied variables to the generation of the landslides were effectively detected and determined. Such landslide susceptibility assessment approaches are interesting from the scientific viewpoint, but mostly they are useful and necessary for decision making regarding effective and responsible land-use planning and environmental development. The combined use of ETL inventories and susceptibility maps maximizes the information content provided to the involved decision-makers, planners and other stake-holders. Such applied studies give the opportunity and the possibility to all involved parties from the research community and the risk professionals to further and in detail identify the most important landslide triggering factors, to understand the contribution of several parameters to the generation of landslides to seismically active areas and to interpret the generation mechanisms and processes of landslides. Furthermore, by taking into account the results of such studies, the authorities involved in disaster risk reduction acquire related knowledge in order to adopt preventive measures and site-specific mitigation strategies. These strategies have the potential to mitigate the adverse effects of ETL to the natural environment and the building stock and eliminate and reduce human and economic losses, respectively. The application of similar approaches is expected to be extensive in the future as urbanization and development have already expanded and will further expand to areas prone to landslides, while the simultaneous presence of landslide causal factors will be continuous and the occurrence of landslide triggering events constantly increasing. Furthermore, it is also expected that the processes applied in the landslide susceptibility models will also be used to other fields of assessment of related hazard and disaster risk reduction. Author Contributions: Conceptualization, S.M.; methodology, S.M. and M.D.; software, S.M. and M.D.; validation, S.M. and M.D.; formal analysis, S.M. and M.D.; investigation, S.M. and M.D.; resources, N.V. and E.L.; data curation, S.M., M.D., V.K. and I.S.; writing—original draft preparation, S.M., M.D., V.K. and I.S.; writing—review and editing, S.M., M.D., H.K., E.V., V.K., I.S., G.K., E.S., N.V. and E.L.; visualization, S.M. and M.D.; supervision, S.M.; project administration, N.V. and E.L.; funding acquisition, N.V. and E.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially funded by the "Telemachus—Innovative Operational Seismic Risk Management System of the Ionian Islands" project, included in the Priority Axis "Environmental Protection and Sustainable Development" of the Regional Operational Programme "Ionian Islands 2014–2020", grant number MIS 5007986, which is funded by the European Regional Development Fund (ERDF) and National Resources under the National Strategic Reference Framework NSRF 2014–2020. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. #### References ### Article Reconstructing the 26 June 1917 Samoa Tsunami Disaster *Laura Sischka 1, Cyprien Bosserelle 2, Shaun Williams 2,\, Josephina Chan Ting 3, Ryan Paulik 2, Malcolm Whitworth 1, Lameko Talia <sup>4</sup> and Paul Viskovic <sup>5</sup> Abstract: The 1917 Samoa tsunamigenic earthquake is the largest historical event to impact this region. Over a century later, little is known about the tsunami magnitude and its implications for modern society. This study reconstructs the 1917 tsunami to understand its hazard characteristics in the Samoan region and assesses the risk implications of tsunamis sourced from different locations along the subduction zone bend of the Northern Tonga Trench (NTT). We model the event from its origin to produce outputs of tsunami inundation extent and depth at spatially flexible grid resolution, which are validated using available runup observations and Apia harbour tide gauge records. We then combine the inundation model with digital distributions of buildings to produce exposure metrics for evaluating the likely impacts on present-day coastal assets and populations if a similar tsunami were to occur. Results exhibit recorded and modelled wave arrival time discrepancies in Apia harbour of between 30–40 min, with runup underestimated in southeast Upolu Island compared with the rest of the country. These differences could reflect complexities in the tsunami source mechanism that are not represented in our modelling and require further investigation. Nevertheless, our findings suggest that if a characteristic 1917-type event were to occur again, approximately 71% of exposed people would reside in Savai'i. Overall, this study provides the first detailed inundation model of the 1917 tsunami that supports an appreciation of the regional risk to local tsunamis sourced at the subduction zone bend of the NTT in Samoa. Keywords: tsunami inundation; historical records; hazard risk exposure; Pacific; BG-Flood; RiskScape #### 1. Introduction More than 700 million people live in island states, most of them developing countries, and low-lying areas at the coast are under constant risk from tsunamis, storm surges, or severe fluctuations of sea levels. In the Pacific, small island developing states (SIDS) represent a collection of remote island communities with developing economies that are often at elevated risk from climate change, sea level rise, coastal erosion, and both natural and anthropogenic hazards [1,2]. It is often the case that due to their geography and the relatively small size of these islands, a significant proportion of the population, infrastructure, and commercial and industrial activity are concentrated in low lying areas, typically in a strip close to the coasts, which render them at considerable risk from coastal inundation from tsunamis [3]. Citation:** Sischka, L.; Bosserelle, C.; Williams, S.; Ting, J.C.; Paulik, R.; Whitworth, M.; Talia, L.; Viskovic, P. Reconstructing the 26 June 1917 Samoa Tsunami Disaster. Appl. Sci. 2022, 12, 3389. https://doi.org/ 10.3390/app12073389 Academic Editors: Spyridon Mavroulis and Efthymios Lekkas Received: 28 February 2022 Accepted: 24 March 2022 Published: 26 March 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The central south Pacific region is frequently affected by tsunamis generated from earthquakes centred on the Tonga Kermadec Trench, including 39 events between 1837 and 2009 that included the 1917 and 2009 tsunamis that affected the Samoan islands [4]. These two nearly identical events suggest that tsunamis in this region are relatively common, and, as a consequence, the SIDS in this area are exposed not only to global tsunamigenic events, but also to frequent locally derived tsunamis triggered by earthquakes, volcanic eruptions, and submarine landslides [5]. Indeed, the recent submarine volcanic eruption of Hunga-Tonga Hunga-Ha'apai (HTHH) in Tonga on 14 January 2022 [6,7], and the resulting tsunami along with potential inferred predecessors (e.g., [8]), illustrate that there is a high degree of residual risk to these islands from local tsunami generating events. Local tsunamis with less than 30 min impact time are extremely hazardous to the island communities, due to the limited warnings and response times between the triggering event (earthquake or eruption, for example), and the tsunami wave making landfall. Consequently, when trying to understand current tsunami risks and the nature and extent of exposure to these islands, it is common to use data or records from historical events to constrain the likely intensity (inundation extent and flow depths, for example), and then use this within a scenario-based context to understand the present-day exposure if such an event was to occur today. The focus of this study is to reconstruct the 1917 tsunami that struck the islands of Samoa, which was the second most deadly tsunamigenic event on record to affect this region after the fatal 2009 tsunami that devastated the southeast coast of Upolu [9,10] (Figure 1). Although the 2009 event provides a benchmark to help plan for and mitigate the impacts of local tsunamis sourced at the Northern Tonga Trench (NTT) in Samoa, areas that experienced little or no impact give rise to a public perception that these areas are at minimal threat from tsunamis. This is particularly the case for coastal areas in Savai'i island to the west of Upolu. However, very little is known about the impacts of the 1917 tsunami predecessor, despite the earthquake source magnitude being the largest ever recorded in this region [11]. Figure 1. Location of the 1917 earthquake epicentre (star) and 2009 earthquakes (circles) relative to Samoa. The 1917 earthquake epicentre was located along the transform segment of the NTT margin about 100 km south of west Savai'i, and approximately 150 km west of the 2009 earthquake. Preliminary modelling by Okal et al. (2011) [12] suggests a focusing of tsunami flux northwards toward the west and southern areas of Savai'i. This implies that these areas would have been severely impacted compared with what they had experienced in the 2009 event. Paradoxically, the evidence presented in [12] does not seem commensurate with the lack of documented impacts for the event, which led them to hypothesize that the scale of impacts was masked by the impacts of the 1918 influenza pandemic, which occurred approximately 1 yr later. The 1918 pandemic is Samoa's deadliest disaster in history, which saw the loss of between 20 and 25% of the country's population at the time [13,14], most of whom were adults or knowledge holders. This is a gap in our current understanding of the scale and magnitude of the 1917 tsunami disaster within the context of regional hazard risk resilience planning. To help elucidate this enigma, we model the tsunami from source to inundation and use this scenario to evaluate the exposure characteristics of a similar event-type on present-day distributions of buildings and people. We provide an overview of the geographical and historical event context in Section 2, and describe the data and analytical methods in Section 3. Findings of the analysis are presented in Section 4, which includes a comparison with observations of the 2009 event. The uncertainties and implications of our results in understanding the regional hazard characteristics that tsunamigenic-earthquakes occurring on different segments of the NTT have on the distribution of exposed areas are discussed in Section 5, with conclusions and suggestions for future research provided in Section 6. #### 2. Geographical and Historical Context Samoa consists of two main islands, Upolu and Savai'i, with several smaller inhabited and uninhabited islands between them (e.g., Manono and Apolima), as well as east and south of Upolu (e.g., Fanuatapu, Namu'a, Nu'utele, Nu'ulua, and Nu'usafe'e) (Figure 1). Comprising part of a larger archipelago encompassing the geologically younger islands of American Samoa to the east, the island chain originated from hotspot volcanic activity and is fringed by coral reefs [15–17]. The geology of Samoa largely consists of mafic material (e.g., basalt and gabbro), due to its oceanic intraplate volcanic hotspot origins [18]. Savai'i is the bigger island with an area of 1820 km2, whereas Upolu has an area of 1114 km2 and accommodates over 67% of the total population of approximately 200,000 people [19]. Apia, the capital of Samoa, is located in the central north of Upolu. In 1902, a temporary geophysical observatory was established in Apia on the then-German administered island of Upolu, whereby it was initially set up to obtain baseline earth observations to compare with the British and German south polar expeditions of 1902–1903. Meteorological instruments and seismographs were installed in 1902 and magnetic instruments in 1905. These enabled studies in geomagnetism, seismology, meteorology, tidal variations, and atmospheric electricity and were so productive that in 1908 the observatory was established on a permanent basis. In August 1914, troops of the New Zealand Expeditionary Force seized control of German-controlled Samoa and the observatory. Its operations were much curtailed during the World War 1 (WW1) years, but the German Director (G. Angenheister) continued observatory operations until it was formerly taken over by the New Zealand government in 1921 [20–22]. New Zealand administration of the observatory continued until the handover in 1963, shortly after Samoa achieved political independence and control of the observatory. Between 1917 to 1919, four tidal waves were recorded by the observatory on continuously recording tidal gauges that correlated with four earthquakes recorded by the seismograph installed at the observatory, with observations reported in [23]. Situated approximately 100 km north of the Tonga Trench, Samoa is exposed to a range of local geophysical hazards (e.g., earthquakes, volcanoes, landslides, tsunami). For example, the subaerial volcanic eruption from 1905 to 1911 on northeast Savai'i caused displacement/relocation of affected villagers to neighbouring Upolu [24] and generated several small tsunamis during this period, with the most damaging occurring in 1907 [4]. Indeed, the recent 2009 complex earthquake sequence [25] and consequent tsunami that resulted in severe casualties and livelihood destruction in southeast Upolu reinforces this vulnerability [26,27]. The lesser known predecessor to the 2009 event, the 26 June 1917 UTC (local time in 1917 = UTC-11) earthquake and tsunami that originated in a proximal source region northwest of the 2009 epicentre (Figure 1), is arguably considered the largest earthquake to have occurred in this region in terms of magnitude (i.e., Mw 8.3 compared with Mw 8.1 for the 2009 earthquake sequence) [11,28]. However, the scale of impacts from the resulting tsunami appear to have paled in comparison with the devastation observed in the 2009 event (e.g., [12,29,30]). Although anecdotal records indicate that the 1917 tsunami inundation had flooded several villages and caused damage to buildings and infrastructure (e.g., Satupaitea in southeast Savai'i and Lotofaga in southeast Upolu) [4,31,32], there are virtually no accounts of any casualties. Available modern interpretations assume at least two people lost their lives based on generic descriptions of damage recorded after the event (e.g., [32]). Here, we use our inundation model for the 1917 tsunami along with present-day patterns of inundation exposure as a proxy to discuss and offer alternative views to help elucidate this enigma. #### 3. Methods and Data The methods used in this study are described in two sections: (1) tsunami modelling, which describes the process from the initial earthquake to the benchmarking process and inundation on land; and (2) tsunami exposure and damage analysis used in quantifying the hazard exposure on present-day buildings and population. #### 3.1. Tsunami Modelling #### 3.1.1. Model Setup and Configuration This tsunami modelling analysis adapts a similar approach used by Bosserelle et al. (2020) [27] to model the inundation of the 2009 event. For generation of the tsunami from the initial earthquake through to propagation and inundation, the BG-Flood software was used. BG Flood (Block-adaptive on Graphics processing unit Flood model) is suited for the simulation of flooding and/or inundation caused by rivers, rain, tides, or tsunamis. It is based on the formulation of Basilisk as well as on the memory structure on the GPU of block uniform quadtree of Vacondio et al. (2017) [33]. The block uniform quadtree structure enables various resolutions with the same memory size but with different physical sizes [33]. For tsunami initialization, we used the earthquake parameters for the 1917 event presented by [12] to configure the source model. To start generating the initial earthquake, the following values were needed: fault parameters (strike, dip, rake, slip), the dimensions of the rupture (length and width), the hypocenter of the earthquake (coordinates and depth), and the timing of the rupture. The rupture length was 150 km with a rupture width of 50 km. The earthquake epicentre was located at 15.13◦ S and 173.28◦ W on 26 June 1917 at 05:49 UTC (i.e., 25 June 1917 at 18:49 local time) at a depth of 10 km. After applying vertical deformation to the fault, the initial water displacement was calculated as a theoretical visco-elastic fault displacement using the formulation of [34]. Figure 2 displays the water displacement after the 1917 earthquake. For the tsunami modelling, a fault length of 150 km was used to match the runup observations. As the model uses an adaptive grid (more than one resolution), three output NetCDF files were created with resolutions of 10 m, 20 m, and 40 m. Figure 2. Tide gauge records for the 1917 tsunami in Apia harbour. (a) Original maregram taken from Angenheister (1920) [23]; (b) inverted and reprojected maregram for digitization; (c) digitized tide record (grey solid line) and predicted tide (black dashed line). The maregram shows the fluctuations of the sea in Apia harbour arriving only a few minutes after the earthquake. The earthquake occurred at 05:49 UTC (dashed red line), with the first noticeable sea level peak at 06:03 UTC. The maregram record prior to the earthquake better matched the predicted tide when shifting the record by 20 min (black solid line), which also produces more consistent arrival time for the tsunami. #### 3.1.2. Tide Gauge and Runup Observations Available tide gauge records from Apia harbour as well as runup observations from different parts of Upolu and Savai'i were used to validate the inundation modelling and subsequent assessment of present-day exposure and impacts in the runup zone. Tide gauge readings for the 1917 event measured in Apia harbour were digitized using the analog maregraph provided in [23]. The maregram in Figure 2 shows the fluctuations of the tsunami waves within the harbour. The digitized maregram generally matches the predicted tide at Apia (predicted by analyzing tide constituent from recent tide record). However, the tide time reference given in [23] cannot be reconciled with an expected 30–40 min travel time for the tsunami to reach Apia. Therefore, either the tide time reference or earthquake time/location is inaccurate. Moving the tide time reference given in [23] to 20 min later improves the correlation between predicted and measured tide and resolves the arrival time inconsistency. Although there is no clear evidence that the tide time reference from [23] is incorrect, it appears to be the simplest explanation of the inconsistency, and it still provides the first indication of the likely tsunami arrival time in Apia. Runup observations derived from historical records of eyewitness accounts documented in [4,30,31] provided benchmarks to infer the extent of wave runup onto land. These observations were digitized to help validate the tsunami runup modelling. #### 3.2. Tsunami Exposure and Damage Analysis #### 3.2.1. Building and Population Exposure Data Buildings on Savaii and Upolu that were located within the maximum tsunami inundation extent were remotely digitized from aerial and Google satellite imagery captured between 2016 and 2020. Buildings were manually digitized in GIS software, using roof outlines to create a vector polygon layer. Physical and non-physical attributes including use category and construction frame were assigned to each building object (Table 1). Samoan building construction frame typologies defined by [9] were attributed to features based on their size (i.e., outline area), roof shape, and use category. In the absence of resources such as Google street view to visually validate use category and construction frame, these attributes were confirmed by local engineers and disaster risk management experts. The outline area (m2) for confirmed buildings was calculated in GIS software (Esri, Redlands, CA, USA). Table 1. Summary of attributes represented in the building exposure data. Samoa's usually resident population was obtained from the 2016 national census [13]. Descriptive statistics for 'usually-resident population' at their residence on census day are aggregated and publicly available at national, district, and village levels. Here, we apply usually resident population at village levels (VPop) to determine a residential building-object population rate (BPRate) as follows: $$VB\_{area} = \sum\_{j=1}^{n\_i} BA\_i \tag{1}$$ $$VBP\_{\text{Rate}} = VP\_{\text{j}} \, / \, VB\_{\text{area}} \tag{2}$$ $$BP\_{\text{Rate}} = VBP\_{\text{Rate}} \; / BA\_i \tag{3}$$ where BAi is the outline area (m2) for a residential building located in village j, with ni, the number of residential buildings within village j. The variable VBarea is the total residential building outline area within village j. Residential building-object VBPRate is the per m2 residential building population based on the usually resident population (VP) of village j. #### 3.2.2. Building Fragility Model Fragility functions relate tsunami hazard intensity (e.g., flow depth) to the conditional probability of a building reaching or exceeding a given damage state [35]. Here, physical building damage is measured from empirical fragility curves representing Samoan buildings damaged in the 2009 SPT [9]. The fragility curves apply a cumulative lognormal function for 'timber', 'masonry', and 'reinforced concrete' construction frame buildings to determine the conditional probability (0–1) of "light", "minor", "moderate", "severe", and "collapse" damage states (DS) being reached or exceeded for a maximum tsunami inundation depth (Table 2). In the absence of representative fragility curves for some building typologies, 'masonry' curves are applied for 'steel' construction frame buildings, and DS1 and DS2 fragility curves are applied for timber and reinforced concrete building typologies. Table 2. Building fragility model parameters applied in this study. #### 3.2.3. Tsunami Inundation Exposure and Damage Model A deterministic model is applied to quantify the present-day building and population exposure as well as damage from tsunami inundation. To this end, we use RiskScape, an open-source software that provides a multi-hazard risk modelling framework for deterministic analysis of tsunami impacts [36]. Here, a deterministic model 'pipeline' is developed to analyze the exposure and damage based on the tsunami model, exposure inventory, and fragility model components described in Sections 3.2.1 and 3.2.2. These components formed the 'input data' for the model pipeline used, which sequences a series of steps and step-functions to sample and analyze deterministic tsunami impacts (Figure 3). Figure 3. A schematic representation of the RiskScape model pipeline steps and functions applied in this study. The 1917 event input hazard data layer represented at the adaptive grid resolutions 10 m, 20 m, and 40 m were segmented using a geoprocessing step-function (i.e., 'cut by segment') to extract all tsunami inundation grid cells within the exposure data layer (building outlines). Extracted grid cells were then spatially sampled (i.e., 'all intersections') to determine the maximum inundation flow depth (MaxD) at each building location. The consequence analysis applies MaxD to determine: (1) building and population exposure to tsunami inundation; and (2) building damage state. Individual building exposure (Bldexp) to inundation is quantified using a simple binary function: $$Bld\_{\text{exp}} = \begin{cases} \begin{array}{c} 1, \MaxMaxD < 0 \; m \\ 0, \; MaxD \ge 0 \; m \end{array} \end{cases} \tag{4}$$ When inundation is present or not at a building location, the corresponding binary value is assigned to the building in the 'event impact table' (EIT). Where inundation is not present (i.e., '0'), no damage (DS0) is assumed. Where inundation is present (i.e., '1'), conditional probability (i.e., 0–1) of damage states DS1 to DS5 based on fragility curves from [9] is calculated in response to the independent variable MaxD. Fragility curves scripted in Python using nested statements apply a lognormal function for each curve based on the dependant variables shown in Table 2 for the corresponding building construction frame in Table 1. The conditional probability determined from each fragility curve is then reported in the EIT for each building exposed to tsunami inundation. The resulting EIT contains tsunami exposure and damage information for model output reporting. In this study, the EIT includes attributes, hazard intensity (i.e., MaxD), exposure (i.e., Bldexp), and damage state information for each building object. The 'output data' pipeline step-function 'group results by attribute' is applied here to report descriptive statistics of model results. Building 'count' and population 'sum' exposure to tsunami inundation is enumerated and reported at national and village scales and by hazard intensity (flow depth) bins of 0.5 m. Building damage states are also reported by building count for 0.1 conditional probability bins between 0 and 1. The step-function 'results output file format' outputs this information as spatial file formats (e.g., GIS shapefile, commaseparated value) for national and sub-national spatial analyses of present-day building and population exposure, as well as damage from the 1917 tsunami event. #### 4. Results #### 4.1. Tsunami Inundation and Validation Figure 4 shows the modelled maximum tsunami wave heights for the 1917 tsunami event. Of particular note is that the southwest side of Savai'i is mostly affected where wave heights of 2 m appear to have impacted the coast. Interestingly, the arrival of the simulated wave in Apia harbour suggests that it took approximately 34 min travel time to this location. This is consistent with the observed tide gauge record (Figure 5) when using a 20 min shift in the reference time. Figures 6 and 7 show which parts of Upolu and Savai'i were most affected by the tsunami, which are generally consistent with available runup observations [4,31] as well as sedimentary evidence presented in [30]. Inundation on Savai'i mostly affects the south western side of the island with much higher flow depths compared with eastern parts of the island including Upolu. Northern Upolu appears unaffected except in areas near Apia. Observed runup points derived from historical records identified in Savai'i (Figure 6) and Upolu (Figure 7) highlight the limited runup observations available for this event. #### 4.2. Damage to Present-Day Buildings and Population Exposure If a characteristic 1917-tsunami event scenario were to occur in the near future, we estimate that approximately 2295 buildings would be affected by the inundation (based on present-day building stock). Most exposed buildings on both Savai'i and Upolu are subjected to flow depths >0.0 m to ≤0.5 m (Figure 8). As flow depth increases, the number of buildings in each category decreases. However, it is worth noting that 206 buildings on Savai'i are exposed to flow depths >3.0 m, which generally means these buildings are most likely to experience moderate to severe damage. Construction frames made of timber have a higher probability of suffering from severe damage or undergoing complete collapse (Figure 8b). Figure 4. Maximum wave height offshore for the modelled 1917 tsunami. Figure 5. Detided water level recorded in Apia (black line) and simulated (red line). Earthquake time is also given. Figure 6. Inundation on Savai'i in close-ups (10 m horizontal resolution). The map shows the 10 m resolution run-up on Savai'i (a) and close-up of inundation in Satuiatua (b), and in Palauli and Satupa'itea (c). The total number of people living in residential buildings within the modelled 1917 tsunami inundation zone are exhibited in Figure 9. In total, approximately 7919 people across 1074 residential buildings (71% of which are in Savai'i) would be affected by the tsunami, which amounts to approximately 4% of the total population in 2016 [37]. It is worth noting that on Savai'i, 13% of the affected inhabitants live in buildings that are estimated to sustain damage states of DS5 (i.e., complete building collapse). This is particularly the case for the district of Palauli West. On Upolu, more than half of the affected population live in buildings estimated to sustain damage states DS0 (i.e., no damage). The more inundated part of Upolu in the west of the island exposes approximately 77 people who live in buildings likely to sustain damage state DS3 or greater. #### 4.3. Comparison with the 2009 Tsunami The 2009 event is the most devastating tsunami to have affected Samoa in recent history. Occurring on 29 September, at 06:48 a.m. local time, two earthquakes only minutes apart shook the ground and caused large waves to travel quickly through the ocean, which caused major destruction and loss of life in less than 30 min after the earthquake rupture [26,38,39]. As the 1917 event can be considered a historical predecessor to the 2009 event in terms of source region [12], a comparison between the two events is made. Apart from the obvious differences in instrumental monitoring quality in 1917 compared with 2009 [26,40], the main differences between the two events in terms of the distribution of affected coast are highlighted in Figure 10. The main energy beam for the 1917 tsunami was focused towards west and south Savai'i, whereas for the 2009 event, energy was focused towards American Samoa and southeast Upolu, which reflects the proximal epicentral locations of the generating earthquakes, respectively, which were about 150 km apart. Figure 8. Cont. Figure 8. (a) Present-day buildings categories on Savai'i and Upolu. (b) Exposure of buildings in Savai'i and Upolu to the 1917 modelled scenario event. (c) Close up of Sala'ilua village on southwest Savai'i showing the modelled tsunami inundation and exposed buildings. These hazard characteristics help to explain the observed variations in exposure impact distributions between the modelled 1917 and benchmark 2009 events. A larger proportion of people would be exposed in Savai'i island (71% of the total exposure) compared with Upolu island if a characteristic 1917-type event were to occur. In contrast, coastal areas in southeast and east Upolu were most severely affected in the 2009 event, which reflects the epicentral location of the generating earthquake at the subducting bend of the NTT terminus and main direction of tsunami flux east/northeast towards southeast Upolu. The 1917 epicentre 150 km to the west at the transform segment of the NTT results in the main direction of tsunami flux northward towards Savai'i. This implies that the distribution of the relative exposure of elements at risk to NTT-sourced tsunamis depends on the location of earthquake origin along the subduction zone bend of the NTT. Notwithstanding the spatial differences in exposure relative to the location of tsunami origin at the NTT, of note is the time of day that each event occurred. Both events struck either in the morning (2009 tsunami) or in the evening (1917 tsunami) during times when the residential population was not at maximum capacity. Had they occurred in the middle of the night, for example, then the scale of human losses might have been significantly greater in each event; this is a key consideration in resilience planning for future NTT sourced tsunamis. Figure 9. Number of people affected in residential buildings in the six damage states on: (a) Upolu, and (b) Savai'i. Figure 10. Epicentres of the 1917 and 2009 earthquakes and corresponding extents at the coast that were predominantly affected by each respective tsunami according to the models. The red crosses indicate the 1917 runup benchmarks shown in Figures 5 and 6 showing locations that should be inundated. #### 5. Discussion Our results suggest that the south coast of Savai'i including parts of south Upolu were affected by the 1917 tsunami. A comparison of the historic evidence with the modelled inundation shows some inconsistencies. For example, observations of runup in the village of Lotofaga in southeast Upolu indicate that half of the village was submerged [4,31], which suggests significantly greater inundation extent and flow depth in Lotofaga than what our model reproduces. That is, the modelled inundation suggests that Lotofaga was not severely impacted. This inconsistency likely reflects the simplicity in our tsunami source model in the light of recent evidence of complex behaviour at 'subduction-zone bends', as is the case at the northern Tonga Trench [26]. In addition, records of the maregram of Apia harbour indicated initial water level fluctuations only 5–10 min after the earthquake compared with approximately 34 min modelled wave arrival time in Apia. The observed fluctuations of 5–10 min in Apia after earthquake initiation is highly unlikely if considering that the tsunami was generated/influenced by the earthquake source alone, which was located further west of the 2009 epicentre and farther away from Apia. However, the maregram accurately recorded the normal tides at the time of the event, which were within 14 min of tide predictions. This suggests that either the recorded timing of the tsunami or earthquake were inaccurate, or that there is more complexity in the tsunami source mechanism than what is currently captured in our modelling. For example, the possibility of complex near-simultaneous faulting source comparable with that seen for the 2009 event [25,38,39] has not been assessed in this study. It is also probable that the earthquake could have caused co-seismic submarine landsliding close to or along the north coast of Upolu, which might explain the early fluctuations observed in Apia harbour that cannot be accounted for using our current earthquake source mechanism (e.g., [41]). Further investigations are needed to unravel this dilemma. Of particular note is the comparison in total number of casualties between the two events. In the 2009 tsunami, 146 people lost their lives [26] and several thousand were displaced, compared with only two casualties inferred from damage descriptions in the 1917 event [32]. In contrast, the extent of inundation inferred from our modelling of the 1917 tsunami, particularly on Savai'i, suggests that damage to property, threat to life, and displacement would have been more severe than what is currently inferred from the historical records. It is plausible that loss of life may have been minimal based on the modern analogy presented by the devastating Hunga-Tonga Hunga-Ha'apai volcano-generated tsunami where three casualties along with major destruction to property, homes, and businesses, and displacement of approximately 1500 people were observed [42]. However, it is equally plausible that loss of life and human displacement far exceeded that which has been documented in historical records. Nevertheless, the hazard risk patterns presented in this study when compared with the 2009 event indicate that the 1917 tsunami would have been more severe on the island of Savai'i compared with Upolu. However, the absence of verifiable records pertaining to loss of life in the 1917 event suggest that either: (1) inundation from the 1917 event, on balance, was less destructive than the inundation caused by the 2009 event; (2) the occurrence of the 1917 tsunami in the evening meant that people who felt the earthquake shaking might have been more aware of the potential tsunami threat and self-evacuated, which helped to minimize/avoid loss of life; or (3) potential casualties from this event were simply not accurately reported and documented in the historical literature. The latter likely reflects a probable association with the 1918 influenza pandemic that overwhelmed Samoa and resulted in the loss of close to 25% of the population, most of them adults and knowledge holders [13,14]. Coupled with the impact of post-WW1 colonialism in Samoa and a shift in political administration could have resulted in the inaccurate reporting of total loses in the event. An example where this is potentially evident is the significant mis-representation of post-colonial population decline in Samoa of up to 80–90% compared with previous estimates of only 20–50% [43]. #### 6. Conclusions This study aimed to reconstruct the 1917 tsunami in Samoa and assess the impacts of inundation on present-day buildings and population exposure. Our findings show variable consistency between modelled-to-observed event reconstructions, which are exemplified by the inconsistency in the wave arrival time in Apia and underestimation of inundation extent/intensity in southeast Upolu. The observed discrepancies are probably due to: (1) earthquake source model and geometry configuration; (2) instrumental seismic and/or tide gauge record uncertainties for Samoa in 1917 that might explain the 20 min anomaly in the tidal reference time; (3) limited records of runup observations for validation; (4) uncertainties in potential source and/or co-seismic mechanisms that might have exacerbated the observed characteristics of the tsunami (e.g., in Apia harbour). These uncertainties represent areas for further modelling investigation. Notwithstanding these discrepancies, our modelling provides a first-order estimation to better quantify the magnitude of impacts for the 1917 tsunami inundation in Samoa that can support scenario-based hazard risk assessment. Our modelling suggests that the scale of impacts, in particular on Savai'i and with regards to potential casualties and human displacement, likely exceeded that which was recorded in historical records. However, it is equally plausible that, although the extent of property damage and displaced peoples was likely severe, the casualty rate may have been low comparable to the death toll observed in the January 2022 Hunga-Tonga Hunga-Ha'apai tsunami in Tonga. Nevertheless, comparison between the 1917 and 2009 events suggests that the extent of present-day exposure distribution around the two main islands of Samoa from local tsunamis originating at the Northern Tonga Trench is highly influenced on the earthquake epicentre and location/orientation of co-seismic displacement. That is, Savai'i Island is more exposed to tsunamis originating along the western segment of the NTT (e.g., 1917 event), compared with Upolu in the east, which exhibits greater exposure to outer-rise events originating along the east NTT segment. Author Contributions: Conceptualization, L.S., C.B., S.W., R.P., J.C.T., M.W., L.T. and P.V.; methodology, C.B., S.W., R.P. and L.S.; software, C.B., R.P., L.S., S.W. and M.W.; validation, L.S., C.B., S.W., R.P., J.C.T., M.W., L.T. and P.V.; formal analysis, L.S., C.B., S.W., R.P., J.C.T., M.W., L.T. and P.V.; investigation, L.S., C.B., S.W., R.P. and P.V.; resources, S.W. and M.W.; data curation, L.S., C.B., S.W., M.W., J.C.T. and L.T.; writing—original draft preparation, L.S., S.W., C.B., R.P., M.W. and P.V.; writing—review and editing, L.S., C.B., S.W., R.P., J.C.T., M.W., L.T. and P.V.; visualization, L.S., C.B. and R.P.; supervision, S.W., M.W. and J.C.T.; project administration, L.S., S.W. and M.W.; funding acquisition, S.W., L.S., M.W., P.V., J.C.T. and L.T. All authors have read and agreed to the published version of the manuscript. Funding: The APC was funded by NIWA Taihoro Nukurangi Project No: CARH2206. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Baseline topography, bathymetry, and exposure datasets used in this study as well as raw results are accessible via formal request to the Samoa Ministry of Natural Resources and Environment. The BG-Flood hydrodynamics modelling software is available on the GitHub: https://github.com/CyprienBosserelle/BG\_Flood (accessed on 1 June 2021), and the RiskScape multi-hazard impacts modelling software is available at: https://riskscape.org.nz/ (accessed on 28 June 2021). Acknowledgments: This research was supported via collaboration between NIWA Taihoro Nukurangi Natural Hazards National Science Centre (S.W.; C.B. and R.P.), the University of Portsmouth (L.S. and M.W.), the Samoa Ministry of Natural Resources and Environment (J.C.T. and L.T.) and GNS Te Pu Ao National Earthquake Information Database (P.V.). Three anonymous reviewers are thanked for the helpful comments which significantly improved the paper. Conflicts of Interest: The authors declare no conflict of interest. #### References ### Article Developing a Guideline of Unmanned Aerial Vehicle's Acquisition Geometry for Landslide Mapping and Monitoring *Konstantinos G. Nikolakopoulos \, Aggeliki Kyriou and Ioannis K. Koukouvelas Department of Geology, University of Patras, 265 04 Patras, Greece; [email protected] (A.K.); [email protected] (I.K.K.) \* Correspondence: [email protected] Abstract: Remote sensing data and techniques are widely used for monitoring and managing natural or man-made disasters, due to their timeliness and their satisfactory accuracy. A key stage in disaster research is the detailed and precise mapping of an affected area. The current work examines the relationship that may exist between the acquisition geometry of Unmanned Aerial Vehicle (UAV) campaigns and the topographic characteristics of an investigated area, toward landslide mapping and monitoring that is as accurate as possible. In fact, this work, concerning the systematic research of the acquisition geometry of UAV flights over multiple active landslides, is conducted for the first time and is focused on creating a guideline for any researcher trying to follow the UAV photogrammetric survey during landslide mapping and monitoring. In particular, UAV flights were executed over landslide areas with different characteristics (land cover, slope, etc.) and the collected data from each area were classified into three groups depending on UAV acquisition geometry, i.e., nadir imagery, oblique imagery, and an integration of nadir and oblique imagery. High-resolution orthophotos and Digital Surface Models (DSMs) emerged from the processing of the UAV imagery of each group through structure-from-motion photogrammetry (SfM). Accuracy assessment was carried out using quantitative and qualitative comparative approaches, such as root mean square error calculation, length comparison, and mean center estimation. The evaluation of the results revealed that there is a strong relationship between UAV acquisition geometry and landslide characteristics, which is evident in the accuracy of the generated photogrammetric products (orthophotos, DSMs). In addition, it was proved that the synergistic processing of nadir and oblique imagery increased overall centimeter accuracy. Keywords: photogrammetry; viewing angle; oblique; nadir; mapping; geomorphology #### 1. Introduction Remote sensing has emerged as an important and valuable tool for various earth observation applications. In particular, remote sensing data and techniques are widely used for monitoring and managing natural or man-made disasters, due to their timeliness and their satisfactory accuracy [1]. The development of UAVs has opened up new possibilities in hazard assessment and disaster risk management [2–4]. In fact, these low-cost remote sensors have proven their effectiveness in mapping hazards and disasters (earthquakes, floods, landslides, etc.), monitoring human activity during emergencies, and protecting and preserving cultural heritage sites affected by geo-hazards [5–10]. A key stage in disaster research is the detailed and precise mapping of an affected area. In this framework, scientists have developed several new processing methodologies, in which different parameters were assessed in achieving the most efficient results. Specifically, the selection of an appropriate number of ground control points (GCPs) during the georeferencing procedure of the obtained UAV imagery was one of the primary parameters under investigation. Thus, various photogrammetric campaigns, consisting of different combinations of GCPs (varying from 4 to up to 20), were evaluated using root mean square Citation:** Nikolakopoulos, K.G.; Kyriou, A.; Koukouvelas, I.K. Developing a Guideline of Unmanned Aerial Vehicle's Acquisition Geometry for Landslide Mapping and Monitoring. Appl. Sci. 2022, 12, 4598. https://doi.org/ 10.3390/app12094598 Academic Editors: Spyridon Mavroulis and Efthymios Lekkas Received: 21 March 2022 Accepted: 29 April 2022 Published: 2 May 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). error (RMSE) values extracted from the respective DSMs [11–13]. The results demonstrated a clear influence of the number of GCPs on the accuracy of UAV photogrammetry. Although the selection of about 20 GCPs (defined according to the study area) managed to reduce the RSME by 50%, it was proved that a higher number of GCPs slightly improved the accuracy. Moreover, other studies dealt with analyzing the distribution of GCPs within the study area and the effect of such distribution on the accuracy of the derived DSMs, which decreases as the distance to the nearest GCP increases [14,15]. In general, the accuracy of photogrammetric products increases asymptotically as the number of GCPs increase, until an optimal GCP density is reached. The collection of GCPs is a time-consuming task. Thus, the development of UAVs with onboard Global Navigation Satellite System Real Time Kinematic (GNSS RTK) positioning in which the georeferencing of the images does not require GCPs—constitutes a promising solution [16,17]. The main disadvantage of the specific approach is the presence of systematic elevation errors, which emerge from the incorrect determination of the interior orientation parameters estimated during bundle adjustment [18]. A recent study proposed that a combination of two UAV flights at the same altitude, consisting of nadir and oblique imagery, was able to reduce the systematic elevation error to less than 0.03 m [19]. Other approaches focused on evaluating the effect of different scenarios, consisting of multi-view camera combinations, on the accuracy of UAV photogrammetry. Specifically, the combination of non-metric oblique and vertical views, along with an appropriate collection of GCPs, has significantly enhanced the accuracy of the photogrammetric procedure, extracting DSMs comparable to those derived by Light Detection and Ranging (LiDAR) [20]. Indeed, this integration of nadir and oblique imagery has proven its effectiveness in numerous studies, including the reconstruction of the surfaces and the determination of the main geometries in a quarry environment, the geomorphological mapping of landslides, and geotechnical/ hazard mapping [21–23]. In addition, it has been proven that the reconstruction of a topographically complex terrain requires the synergy of oblique and facade images to complement nadir views [24]. This synergistic use significantly improved the geometric accuracy of topographic reconstruction, by approximately 35%. Although the combination of multi-view UAV imagery is a very successful approach in a variety of applications, nadir images combined with a dense distribution of GCPs constituted an ideal solution for the reconstruction of 3D agricultural surfaces [25]. On the other hand, oblique UAV imagery was considered particularly suitable for the threedimensional modeling of buildings, cities, or urban settlements, presenting an enhanced achievable accuracy [26]. Moreover, oblique-viewing images can be utilized effectively for the 3D modelling of historical architectures and cultural heritage research, especially in areas characterized by limited accessibility [27]. In addition, oblique images increased by 50% the accuracy of 3D representations of topography in areas with high and steep slopes [28]. The current study aims to create a guideline for researchers who try to follow the UAV photogrammetric survey method for landslide mapping and monitoring, by exploring derived data quality (orthophotos and DSMs) in reference to data acquisition geometry. There are previous studies on the influence of the ground control number to the horizontal or vertical accuracy of orthophotos and DSMs, respectively [12–15]. However, the current study is the first to conduct systematic research on UAV data acquisition methodology applied on multiple active landslides. UAV flights were performed over four landslide areas with different characteristics (land cover, slope, volume, dimensions, etc.), and the collected data from each area were classified into three groups, depending on UAV acquisition geometry: (a) nadir-only images, (b) oblique-only images, and (c) an integration of nadir and oblique images. Moreover, a flat urban area was also surveyed for validation purposes. The processing of UAV imagery in each category was based on structure-from-motion (SfM) photogrammetry, leading to the creation of high resolution orthophotos and digital surface models (DSMs). Accuracy assessment was carried out using quantitative and qualitative comparative approaches, such as root mean square error calculation, length comparison, and mean center estimation. The derived results demonstrate the benefits of combining oblique and nadir images in order to reduce systematic errors and increase the overall accuracy of orthophotos and Digital Surface Models. #### 2. Materials and Methods #### 2.1. Case Studies The selection of research areas was based on the criterion of heterogeneity in terms of slope, land cover, vegetation coverage, etc. Thus, four different landslides were set as case studies. We also surveyed a flat industrial area for validation purposes (Figure 1) (Table 1). In order to eliminate the influence of vegetation height on DSM production, the study areas presented, in general, with low and sparse vegetation, as described in Table 1. Figure 1. Location of the five case studies within Greece and UAV orthophotos of each area of interest. <sup>1</sup> Based on coordination of information on the environment (CORINE) land cover classification. The first landslide area is located close to the village of Moira, within Western Greece. The landslide occurred on 20 January 2017 on a mountainous, steep slope, covering an area of approximately 65,569.20 m2. The landslide material was spread 300 m in length and 300 m in width and it was geologically structured from flysch, limestone, and loose cherts. It was characterized as a complex slide, based on the different lithologies—silicate and/or silicate lithology was presented in the northwestern part, while limestones were detected in the southeast. The occurrence of the landslide was related to geological factors (flysch lithology), as well as to the rapid snow melting. It has been demonstrated that the increase of the water content of clay soils (flysch) reduces the shear strength of soils, acting as a triggering factor for the occurrence of landslides [29]. The destruction of the road connecting the village of Moira with the city of Patras, as well as a significant change in the local landscape, were the main consequences of the landslide. The second landslide took place in November 2015 on the Egkremni beach on the island of Lefkada; it was classified as a debris slide . The Ionian Islands constitute the northwestern part of the Hellenic arc, which is considered as a site of complex continentcontinent to continent-ocean convergent plate margins. The Cephalonia Transform Fault Zone (CTFZ) is recognized as the major tectonic structure of the site and is responsible for the particularly active seismicity in the wider area. In addition, another fault, named Athani, is detected ashore, shaping the west coast of Lefkada island [30,31]. The Athani fault, along with smaller, parallel faults with similar kinematics, form steep slopes, which are an ideal site for the occurrence of landslides during seismic events [32]. On 17 November 2015, a 6.5 magnitude earthquake struck the west coast of the island, causing a series of landslides on the cliffs [33,34]. Another active continental region, in terms of tectonics and seismicity, is Northern Peloponnese, due to the fast-extending rift of the Gulf of Corinth. This third area of interest is located close to the village of Kato Zachlorou within Western Greece. The first mass movements at the specific area occurred in April 2019, while the main event, consisting of rock falls and earth flows, took place on 4 April 2020. The repetitive sliding episodes were strongly associated with the increase of the water content of the clay soils, due to intense rainfall [29]. The fourth area of interest includes a landslide located in a semi-mountainous village of Western Greece named Messarista. Extremely heavy and prolonged rains hit the wider region on 11 December 2021, acting as a main triggering factor for the occurrence of a series of landslides in different places throughout the region, as well as within the village of Messarista. The phenomenon is still ongoing and is being monitored by local authorities, while the landslides were classified as earth slides and earth flows. Finally, a flat industrial area, covering part of the new port of the city of Patras, was chosen for the validation of the results, due to the past view on photogrammetry, in which flat areas were surveyed using nadir imagery solely. #### 2.2. Data Collection UAV imagery was obtained using a DJI Phantom 4, which is equipped with a built-in GNSS system and a CMOS camera (12.4 MP) with 4000 × 3000 resolution. Each case study consisted of three different flight grids (nadir, oblique, and nadir and oblique). During these distinct campaigns, flight characteristics were kept the same (Table 2). Specifically, flights were executed with a 90% along-track overlap and a 75% cross-track overlap. However, the flight acquisition altitude was adapted to the topography of each study area, with the aim of extracting photogrammetric products with spatial resolution between 2.3 and 3.5 cm (Table 2). The collected UAV imagery of each area was classified into three groups of data. The first group included nadir-only images with a 90-degree gimbal pitch angle; the second group consisted of oblique-only images with a 65-degree gimbal pitch angle; and a synergistic use of nadir and oblique imagery was selected for the third group. Table 2. Characteristics and parameters of UAV campaigns. In addition, GCPs were distributed throughout the survey areas in order to orient and match the aerial imagery to data measured terrestrially. GCPs were collected using a Leica GS08 GNSS receiver. Coded targets were created according to the general recommendations of Agisoft Metashape software [35], and were printed on matte finish plastic boards. These targets were used as GCPs (Figure 2). Furthermore, large rectangular aluminum targets were placed in the survey areas for comparison and validation of the photogrammetric outputs (Figure 3). These aluminum targets had specific dimensions and a hole in each corner, in order to be accurately measured with a GNSS sensor. Figure 2. Printed GCP pattern, distributed throughout the survey area during UAV campaigns. Figure 3. Aluminum targets, as captured in orthophotos during UAV campaigns over the landslide of Zachlorou. #### 2.3. Methodology The aim of the current study was to examine whether the geometry of UAV acquisition plays a key role in the accuracy of derived photographic products that are subsequently used as high-precision data for landslide mapping and monitoring. A schematic illustration of the applied methodology is displayed in Figure 4. Figure 4. A flowchart showing the processing steps of the research methodology. In more detail, UAV surveys were conducted over five study areas, presenting different characteristics (topography, land cover, etc.). Three UAV flights, consisting of: (a) nadironly acquisitions, (b) oblique-only acquisitions, and (c) an integration of nadir and oblique acquisitions, were executed for each case study. The different UAV acquisition trajectories of nadir- and oblique-viewing campaigns over the landslide of Messarista (case study) are depicted in the Figure 5. It is worth mentioning that the collection of the oblique-viewing imagery was implemented as the UAV moved forward and backward, following line paths that were perpendicular to the slope of each study area. Furthermore, photogrammetric GCPs were collected during the UAV surveys. Figure 5. UAV acquisitions over the landslide of Messarista. (a) Nadir-viewing acquisition trajectory; (b) Oblique-viewing acquisition trajectory. The processing of the collected UAV imagery was carried out through SfM photogrammetry. The specific technique contributes to the three-dimensional reconstruction of the topography, using the basic principles of photogrammetry along with computer vision algorithms [36–39]. The main advantage of this functional and low-cost method is that it allows the automatic and simultaneous determination of scene geometry, camera positions, and orientation, without requiring pre-existing known points. Thus, multiple, overlapped and shifted 2D images are transformed into 3D representations using an automatic, featurematching algorithm. The photogrammetric processing of UAV imagery took place in Agisoft Metashape software. The calibration and optimization of the camera took place in accordance with Agisoft's default values, as set for the DJI Phantom 4 camera. Internal orientation parameters were estimated automatically, due to the ability of the software to recognize the model of the camera and to specify the appropriate settings. The high-quality option was selected for the alignment of the images, aiming at a more precise estimation of the camera positions [40]. At the same time, the processing of UAV imagery was implemented using the original image size. The quality option was closely linked to the quality of the topographic reconstruction. Moreover the, ultra-high setting was defined as the parameter during "build dense cloud" and "build mesh" procedures. Orthophotos and DSMs emerged from the processing of the collected UAV imagery. These products were projected into the Hellenic Geodetic Reference System 1987. Specifically, three orthophotos and three DSMs were created for each study area, corresponding to the three different acquisition geometries (i.e., nadir-viewing images, oblique-viewing images, and an integration of nadir and oblique imagery). The evaluation of the accuracy of the generated orthophotos and DSMs was based on qualitative and quantitative comparative approaches, including RMSE calculation, length comparison, and mean center estimation. #### 3. Results #### 3.1. Accuracy Asssessment of Orthophotos Three high resolution orthophotos were extracted from the photogrammetric processing for each case study, consisting of UAV data from the three different acquisition groups, i.e., (a) nadir-viewing imagery, (b) oblique-viewing imagery, and (c) the integration of nadir and oblique imagery. The aforementioned orthophotos, covering the area of case study 4 (Messarista), are depicted in Figure 6. As can be observed, a visual comparison between the orthophotos created by data for the different groups of geometry acquisitions is a particularly difficult task, due to apparent similarity. Figure 6. UAV orthophotos of the landslide of Messarista, emerging from the processing of (a) nadirviewing images, (b) oblique-viewing images, and (c) synergistic use of nadir and oblique images. The scale of the orthophotos was set to 1:1500. In this context, the evaluation of the accuracy of the derived orthophotos was performed using comparison approaches in an ArcGIS environment. GIS applications have been utilized in respective studies for the characterization of geological features and geological mapping [41]. In this study, GCPs were utilized as precise reference positions. Then, two geodetic lines were shaped from the x-y coordinates of the GCPs of each study area. Figure 7 displays the reference geodetic lines, as formed for each case study. It is important to highlight that we tried to create the geodetic lines in different topographic reliefs, varying from steep slopes to flat regions. Subsequently, we digitized the same lines in all orthophotos, derived from the processing of the UAV imagery of all groups of acquisition geometry. The digitization of the lines was based on the visual identification of the respective GCPs, which were used to shape the reference geodetic lines. The length of each digitized and reference geodetic line were calculated and compared (Table 3). The length variations were significantly small, which proved the high accuracy of the photogrammetric products. In addition, the integration of nadir and oblique viewing acquisitions displayed the smallest length variations in eight out of ten comparisons, demonstrating the positive influence of the specific acquisition geometry on the enhancement of the accuracy of the derived orthophotos. Moreover, oblique-viewing geometry showed a better performance, compared to nadir-viewing acquisitions. In particular, the difference in the length of line 1, in comparison to the corresponding reference line in the case study of Zachlorou, was zero for the orthophoto resulting from the integration of nadir and oblique imagery. In addition, the variation for the nadir-viewing and oblique-viewing orthophotos was estimated at 0.190% and 0.067%, respectively. In the case of Egnemni, the percentage variations in line 2 were calculated at about 0.034% for the orthophotos that emerged from either the synergistic use of nadir and oblique images or the nadir-viewing imagery. In contrast, the corresponding difference for the nadir-viewing orthophoto was obviously larger (i.e., 0.228%). Figure 7. Reference geodetic lines as created in: (a) case study 1, (b) case study 2, (c) case study 3, (d) case study 4, and (e) case study 5. Table 3. Comparison of the length of the geodetic lines of each case study in accordance with the respective reference length. The processing of nadir-viewing images, along with oblique imagery, enhanced the overall accuracy in all the investigated areas. In more detail, for nine of ten cases (Table 3) the length difference between the GNSS measurements and the measurements on the orthophotos was less than 0.05 m (5 cm). Taking into consideration the terrain steepness and the dense vegetation, we assume that the overall accuracy is excellent. It is worth mentioning that even in the fifth test area (Patras Port), the combined processing of nadir and oblique images gave results that were more accurate than the single processing of nadir or oblique data. In detail, the length difference of the geodetic lines for the common data set was only 0.012 m (centimeter accuracy), while the respective accuracies for the nadir or oblique data set ranged between 0.12 m and 0.31 m (Table 3). Another method, utilized for the evaluation of the accuracy of orthophotos resulted from the photogrammetric processing of each acquisition geometry, was based on the comparison of the mean center of the reference geodetic lines and the respective digitized ones. The mean center constitutes the geographical center of a set of features resulting from average x and y coordinates [42,43], which is calculated as: $$\overline{X} = \frac{\sum\_{i=1}^{n} x\_i}{n} \text{ and } \overline{Y} = \frac{\sum\_{i=1}^{n} y\_i}{n} \tag{1}$$ where xi and yi are the coordinates for a feature i and n is the total number of features. This approach is widely used for the identification of distribution changes or for the comparison of the distributions of features. The mean centers of the reference geodetic lines of each case study are depicted in Figure 7. The Near tool [44] was applied for the determination of the distance between the reference mean centers and the respective mean center of the digitized lines. Distance variations are displayed in Table 4. As can be noted, the multi-acquisition geometry (nadir and oblique imagery) presented the shortest distances from the reference mean centers in 50% of the UAV campaigns. Table 4. Near distances of mean centers resulting from the reference mean centers of geodetic lines 1 and 2. As a final comparison of the orthophotos derived from different acquisition geometries, we utilized the variations of the dimensions of the aluminum targets. These dimensional variations, regarding the orthophotos covering the landslide of Zachlorou, are presented in Table 5. As can be observed, the synergistic use of nadir and oblique photos increase the accuracy of the orthophoto. The true length of the aluminum target is 0.538 m and the respective measured value from the oblique and nadir imagery is 0.542 m, meaning that the overall difference is only 4 mm. The difference value for the nadir imagery is 8 mm and for the oblique imagery is 10 mm. Table 5. Comparison of the dimensions of the aluminum target between the different acquisition geometries. #### 3.2. Accuracy Asssessment of DSMs The evaluation of the accuracy of the extracted DSMs was executed through the computation of RMSE, which estimates the differences between the values of a DMS and the reference high-precision values. The computation is performed by the following equation: $$RMSE = \sqrt{\frac{1}{n} \sum\_{i=n}^{n} \left( h\_{ref} - h\_i \right)^2} \tag{2}$$ where href is the reference elevation, hi is the DSM elevation at point i, and n is the number of GCPs. A basic condition for the performance of the calculation is that the reference data should be an order better than the data to be evaluated. Thus, GCPs with sub-millimeter accuracy were utilized as reference points in the current study. The variations in the elevation values between the reference data and the respective photogrammetric DSMs, acquired by different viewing geometries over all case studies, are presented in Table 6. It is obvious that the synergistic use of nadir- and oblique- viewing images during UAV campaigns managed to minimize the elevation errors in most DSMs. In particular, the RMSE for the DSM arising from the combined use of nadir and oblique imagery in the case study of Egnemni, was calculated at 0.083 m, while the respective errors for the nadir-viewing and oblique-viewing DSMs were estimated at 0.092 m and 0.085 m, respectively. Furthermore, in the case study of Messarista, the RMSE for the DSM that emerged from the simultaneous processing of nadir and oblique imagery was calculated at 0.090 m. The respective RMSE for the nadir-viewing DSM was computed at 0.162 m, while the error of the oblique-viewing DSM was notably larger (i.e., 0.357 m). Table 6. RMSE values emerging from the generated DSMs. Although oblique-viewing images demonstrated an overall good performance in the generation of DSMs, we noticed a correlation between the UAV acquisition geometry and the topographic characteristics of the survey area. Specifically, nadir-viewing geometry was considered more suitable for DSM generation in flat urban (case study 5) or densely built-up (case study 4) areas, since it revealed smaller values in elevation variations than oblique imagery. #### 4. Discussion The capability of performing a high accuracy field campaign with excellent repeatability is the basic demand [45–47] for landslide mapping and monitoring for assessing any deformation, usually within a steep and possibly dangerous environment. Sub-centimeter accuracy is a prerequisite in both horizontal and vertical axes in order to assess the activity of a landslide. Aiming to develop a guideline for accurate landslide mapping, we conducted four repeated tests within four active landslides with diverse characteristics. Both the horizontal and the vertical accuracy of the orthophotos and the DSMs were examined. It was proved that the combination of nadir and oblique imagery produced more accurate results in all the studied cases. This output was in full accordance with the results of a previous study [28] that mentioned that overall accuracy is increased by 50% when nadir and oblique imagery are combined. The statistical comparison of the length of the digitized geodetic lines demonstrated that the integration of nadir and oblique images provides orthophotos with higher accuracy. Oblique imagery seems to provide more accurate results than nadir imagery. The only exception was the Messarista case study, where the processing of the oblique images yielded the worst results, compared to the respective result from the nadir images. This can be justified, as Messarista is a densely built-up village on a steep hill with very narrow backstreets. These narrow side streets cannot be clearly mapped in oblique imagery. Furthermore, the houses are built side-byside and the front buildings hide the back ones. As a result, nadir imagery provides useful information that cannot be derived from oblique imagery alone. Similar results were extracted from the measurements of the near distances of the mean centers that were calculated in the ArcMap environment. In almost all the cases, the simultaneous processing of nadir and oblique imagery provided more accurate results. The same combination produced more accurate DSMs in all the study areas. As presented in Table 4, in very steep environments (the four landslide areas) and in a flat area (Patras Port), the vertical accuracy of the oblique-nadir DSM was higher in comparison to the accuracies of either nadir or oblique DSMs. Summarizing the overall assessment of the results, based on the application of simple statistical calculations, it was proved that the synergistic use of nadir and oblique imagery was considered to be the most appropriate geometry at 80% (i.e., in eight cases), according to the comparison of the length of the two lines of the five study areas (i.e., 10 cases) (Figure 8a). Moreover, comparing only nadir-viewing and oblique-viewing geometry, oblique acquisitions provided more accurate results by 70% (Figure 8b). Figures 9 and 10 display the corresponding evaluations using the comparison of near distances and RMSE calculation. Figure 8. (a) Overall assessment of the accuracy of the photogrammetric products in accordance with UAV acquisition geometry using length comparison; (b) assessment of the accuracy of the photogrammetric products using length comparison between nadir- and oblique-viewing geometry. Figure 9. (a) Overall assessment of the accuracy of the photogrammetric products in accordance with UAV acquisition geometry using near distances; (b) assessment of the accuracy of the photogrammetric products using near distances between nadir- and oblique-viewing geometry. Figure 10. (a) Overall assessment of the accuracy of the photogrammetric products in accordance with UAV acquisition geometry using RMSE; (b) assessment of the accuracy of the photogrammetric products using RMSE between nadir- and oblique-viewing geometry. Our findings are in accordance with those of previous studies [20,21,24–26]. Specifically, nadir and oblique images were compared with TLS data for the mapping of a fault plane [20]. The tests proved that both precision and accuracy have been increased when the two sets of images were combined. It was marked [21] that the combined use of nadir and oblique imagery offers a valued methodology for open-pit operations and for the continuous monitoring of the exploitation by managers. UAV data acquired from oblique and facade viewing angles were compared with nadir images in a steep and complex mountainous terrain, and the produced point clouds were compared with TLS data [24]. It was proved that the combination of oblique and façade images, compared to nadir data, increased the geometric accuracy of the derived point cloud data [24]. In a respective test [25], it was demonstrated that the combination of nadir and oblique images ameliorates the accuracy of 3D surface models in an agricultural area when no ground control points or a small number of ground control points are used. Nadir and oblique imageries were compared for 3D building representation [26]. The results proved that oblique data ameliorates the obtainable accuracy of the derived point cloud and of the produced 3D model. As described in [28], supplementing nadir image blocks with oblique images consistently mitigates the systematic error patterns within complex topography. The results from many scenarios proved that the combination of nadir and oblique images increased precision and accuracy, and reduced data gaps. Those previous results are in full accordance with our results. In all the studied areas, it was also proved that the simultaneous processing of the nadir and oblique imagery decreases the RMSE error and increases the geometric precision. #### 5. Conclusions The objective of the current study was to develop a guidance for accurate landslide mapping in steep terrains. In this context, we performed four identical tests within four active landslides spreading throughout Western Greece. The main conclusions that emerged from this research included the following: Author Contributions: Conceptualization, K.G.N.; methodology, K.G.N. and A.K.; software, K.G.N. and A.K.; validation, K.G.N., A.K. and I.K.K.; formal analysis, A.K.; investigation, K.G.N., A.K. and I.K.K.; resources, K.G.N.; data curation, K.G.N., A.K. and I.K.K.; writing—original draft preparation, K.G.N. and A.K.; writing—review and editing, K.G.N., A.K. and I.K.K.; project administration, K.G.N. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available, due to privacy considerations. Conflicts of Interest: The authors declare no conflict of interest. #### References ### Article Operational Mapping and Post-Disaster Hazard Assessment by the Development of a Multiparametric Web App Using Geospatial Technologies and Data: Attica Region 2021 Wildfires (Greece) *Triantafyllos Falaras 1,\, Ioanna Tselka 1,2, Ioannis Papadopoulos 1, Maria Nikolidaki 1, Andreas Karavias 1, Despoina Bafi 1, Aliki Petani 1, Pavlos Krassakis 1,3 and Issaak Parcharidis <sup>1</sup> Abstract: The environmental effects of wildfires are a hot issue in current research. This study examines the effects of the 2021 wildfires in the Attica region in Greece based on Earth observation and GIS-based techniques for the development of a web app that includes the derived knowledge. The effects of wildfires were estimated with the use of Sentinel-2 satellite imagery concerning burned area extent and burn severity using a NBR-based method. In addition, the erosion risk was modeled on a pre-fire and post-fire basis with the RUSLE. This study highlights the importance of assessing the effects of wildfires with a holistic approach to produce useful knowledge tools in post-fire impact assessment and restoration. Keywords: wildfires; geospatial intelligence; web app; operational mapping; burn severity; soil erosion; Attica; RUSLE (revised universal soil loss equation); NBR (normalized burn ratio); remote sensing (rs) #### 1. Introduction Greece in the 2021 fire season suffered from one of the greatest ecological disasters caused by wildfires. Especially in the first days of August, an intense prolonged heatwave struck Greece making it the worst that the country faced in almost 34 years. During this period of extreme conditions (27 July to 16 August 2021) in the Greek territory, many wildfires broke out due to the drought that increased wildfire vulnerability. All the means available, from the fire brigade to the local authorities and support from other countries, were used to fight these fires. In addition, evacuations of settlements were performed alongside the use of 112 to send alert messages to citizens, while the Copernicus Emergency Management Service (EMS) was also activated for rapid mapping of the wildfires in most cases [1]. Over the last couple of years, climate change has dramatically affected the Mediterranean region. The vulnerability of this region to drought and rising temperatures has led to severe ecological disasters annually. Specifically, Greece has been dealing with great environmental catastrophic events due to wildfire incidents and remarkably strong heatwaves [2]. The assessment of the wildfires' effect spatially provides a leading role in the documentation of vulnerable areas in different timescale evaluations [3,4]. Fire incidents constitute a dynamic process in planet Earth alteration affecting the global climate system [5]. Lately, wildfires show an increased occurrence, especially during Citation:** Falaras, T.; Tselka, I.; Papadopoulos, I.; Nikolidaki, M.; Karavias, A.; Bafi, D.; Petani, A.; Krassakis, P.; Parcharidis, I. Operational Mapping and Post-Disaster Hazard Assessment by the Development of a Multiparametric Web App Using Geospatial Technologies and Data: Attica Region 2021 Wildfires (Greece). Appl. Sci. 2022, 12, 7256. https:// doi.org/10.3390/app12147256 Academic Editor: Edoardo Rotigliano Received: 19 May 2022 Accepted: 16 July 2022 Published: 19 July 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). the summer months, causing a rise in the overall period of the fire season [6]. The Mediterranean countries have been damaged over the last couple of years, due to extensive fire incidents causing irreversible damage to the environment [7]. Especially, high mountain regions characterized by Mediterranean climates seem to be more vulnerable to wildfire incidents due to the occurrence of high rainfall intensity in sparse vegetation cover areas [8]. Fire impacts on vegetation, soil, atmosphere, and society are dependent on fire characteristics such as severity and size [9]. Particularly, in Mediterranean ecosystems where fires occur in summer, the increased temperatures and evapotranspiration succeeded by the increased precipitation in autumn cause alterations to the ecosystems including increased soil loss [10]. Geospatial intelligence (GEOINT) has as a basic principle, the collection, analysis, and combination of all the available data, both geospatial and satellite imagery for Earth's geographical area, so as to utilize them in creating useful products in planning, decision making, and emergency response [11]. The integrated use of remote sensing and GIS proved very important in disaster management, with satellite imagery providing a synoptic view, spatiotemporal changes monitoring, and of course sufficient spatial coverage alongside GIS, which enables the utilization of all the geospatial information available. The capabilities of remote sensing for this purpose can be found in their operational use and their damage assessment after a wildfire event [12]. The use of satellite-based remote sensing in wildfires is well-known in the literature. For the mapping of burned areas, plenty of methods have been developed [13] with the use of multispectral data through the decades, including spectral indices among them such as the burn area index (BAI) [14] and normalized burn ratio (NBR) [15]. Additionally, with the use of satellite imagery, not only the burned area but also further information regarding the impact can be retrieved, as in the case of the dNBR [16], where burn severity can also be assessed. In addition, the combination of remote sensing with GIS in the case of wildfires provides a wide range of geospatial information regarding the post-fire period including the mapping of burned areas, soil erosion estimation, and recovery of the ecosystems [17]. Soil erosion caused by land degradation affects both natural and human resources, resulting from insufficient agricultural productivity, and is characterized as one of the most important threats worldwide [18]. Forest fires constitute one of the most critical causes of soil erosion due to their ability to burn large amounts of vegetation cover leading to an increase in runoff and sediment transfer [19,20]. For this purpose, several indices have been developed so as to assess the damage caused by wildfires on soil properties, such as the dNBR (differential NBR), which [8] is calculated based on pre- and post-fire satellite images [21]. Research on soil erosion has been extensively carried out over the past few decades since it is inextricably linked to the world's most serious environmental problems [8]. There are a wide variety of models that have been developed throughout the years considering soil loss assessment in several regions around the world [22]. The most commonly known models are the Universal Soil Loss Equation [23], its revised version (RUSLE) [24], and the Water Erosion Prediction Project (WEPP) [25]. Those models are used for the estimation of long-term average soil erosion values based on information extracted from rainfall data, soil properties, topography, and land cover management. A recent study [2] assessed post-fire effects by mapping burned areas and their burn severity and also the soil erosion for an area within the Lokroi Municipality in Central Greece. This study made use of NBR to map the burned areas and to assess the burn severity with the use of Sentinel-2 and Landsat-8 imagery. In addition, to this affected area, the RUSLE was applied to estimate soil erosion. The combined use of these Earth observation and GIS-based methodologies was important before and after the wildfire and led to the identification of the vulnerable areas within the affected region. Another study was conducted [26] regarding the 2021 wildfires in Greece in Attica, Evia, and Peloponnese. In that study, the erosion vulnerability to these areas was evaluated after the fire events with the use of a GIS Boolean logic-based model. The results of the study led to the erosion risk assessment of these areas, making evident the increased susceptibility to surface runoff erosion. A similar study [27] studied southern Italy's Basilicata region, following an integrated approach with GIS and remote sensing regarding post-fire erosion risk. The soil fire severity was estimated by the use of Sentinel-2 images while the RUSLE was applied for the modeling of pre-fire and post-fire erosion for the first year after the events. Results of the study revealed the complex relations between fire severity and soil erosion factors while helping with soil loss estimation after the fire events. Based on all the above-mentioned, this study aims at the assessment of the impact that the 2021 fire season wildfires had in the region of Attica, Greece. More analytically, four major wildfires of Schinos, Varympompi, Lavreotiki, and Vilia were mapped in an operational way with the use of Sentinel-2 imagery with the normalized burn ratio spectral index-based approach, thus extracting the burned area extent and assessing the burn severity while Corine Land Cover data was also used to analyze the consequences each fire caused. Furthermore, the soil erosion in the affected areas via the implementation of the revised universal soil loss equation (RUSLE) was assessed to estimate the soil loss and potential high-risk areas. In this way, to meet the purpose of this study effectively to gain geospatial intelligence on the effects of these wildfires, the results of all the applied methodologies were used to develop a web app that constitutes an invaluable tool in post-disaster hazard assessment. The results of the study showed the spatial damage that the wildfires caused in Attica in 2021 and they also demonstrated a significant increase in soil erosion in the affected areas. #### 2. Wildfires in Attica Region 2021 In the region of Attica, which is located in Central Greece where the capital of Greece, Athens, is, during the 2021 fire season four major wildfires occurred with three of them happening in the above-mentioned August period resulting in extensive damages. To all the four wildfires the Copernicus EMS (Emergency Management Service) was activated with the activations EMSR51O, EMSR527, EMSR540, and EMSR542. Spatially, according to Figure 1, these wildfires were located in western, north-eastern, and south-eastern Attica with the closest to the metropolitan area of Athens being the fire in Varympompi [1,28,29]. Figure 1. Areas of study. Starting with the May 19th, 2021 wildfire in Schinos that broke out in the evening of that day close to the Schinos settlement in Korinthia. The fire moved from the east to western Attica, reaching to the north of the Kineta settlement during almost five days of activity. Damages were limited to the build-up environment and extended to the forests of the area. The affected area includes the northern slopes of the Geraneia Mountains, which includes a Natura 2000 site, and the geomorphological status of the area with high slopes aids the development of floods and other post-fire hazards to the region [28]. The next fire is that of Varympompi, which broke out on the midday of August 3rd, 2021, in the Ano Varympompi area, and lasted for the next few days. More precisely, the northern part of the Attica region was affected from Adames in Kifisia to Ippokrateios, Politeia, mainly within the base of Parnitha Mountain. Although the impact included the forest vegetation of the area, an important part of the urban areas of settlements was impacted due to the complexity of the forest and building mixture [1,26]. The wildfire in Lavreotiki started on the morning of August 16th in Markati in the northwest of Lavreotiki. Close-to-settlement areas such as coniferous forests, natural vegetation, and agricultural land were affected by the wildfire. The meteorological conditions made it difficult for the fire brigade to put the fire under control despite the smaller extent than the previously mentioned wildfires [1,26]. Lastly, the wildfire in Vilia also broke out on August 16th at midday a few hours after this in Lavreotiki in Pateras Mountain close to the Vilia settlement. The wildfire was burning an area predominantly consisting of sclerophyllous vegetation and coniferous forests for more than five days with a rapid spread day by day. The above-mentioned conditions in that period led to the devastation of a large area between Vilia and Nea Peramos despite the huge effort and the increased ground and aerial support [1,29]. #### 3. Materials and Methods 3.1. Data and Software In Table 1, the used datasets are briefly presented. Free and open accessible datasets were selected to be used for each methodological part described in the following subsections. Table 1. Utilized datasets. <sup>1</sup> The used Sentinel-2 acquisitions are presented in detail in Table 2. For the operational wildfire mapping, optical/multispectral Sentinel-2 mission satellite images of the ESA Copernicus program were utilized which are open and accessible from the Copernicus Open Access Hub [30]. The products used were the atmospherically corrected and scene classified Level-2A Bottom-of-Atmosphere (BOA) [31]. A key characteristic of Sentinel-2 images important in operational purposes is the 5-day interval between satellite acquisitions over an area and their availability on the Copernicus Open Access Hub a few hours after their acquisition. In Table 2, the full list of Sentinel-2 acquisitions is given for each purpose of this study regarding the mapping of wildfires and the C factor of the RUSLE. The image selection was redundant to the cloud-free scene availability, taking into consideration the temporal coherence for each purpose. Table 2. List of used Sentinel-2 acquisitions for each area and purpose. Land cover information for the affected study areas was retrieved with the use of the Corine Land Cover 2018 (CLC 2018) in vector polygon format from vector geodatabase open available from Copernicus Land Monitoring Service [32]. The CLC 2018 is highly accurate (≥85% accuracy) with a minimum mapping unit (MMU) of 25 hectares and a mapping minimum width (MMW) of 100 m, while it is detailed enough by having 44 land cover classes at its most analytical level-3 [33]. In this study, it was used to map the affected area of the wildfire land cover and for the RUSLE's P factor. Regarding the meteorological data required for the estimation of the R factor of the RUSLE, these were retrieved from the Meteo.gr of the National Observatory of Athens (NOA) [34]. Data regarding the precipitation for the examined period were obtained from the respective meteorological stations of the Meteo.gr network within and around of each area of study. The EU-DEM v1.0 is a digital elevation model (DEM) that was obtained freely from the Copernicus Land Monitoring Service for the application of the RUSLE's LS factor. With a resolution of 25 m, it was produced with the fusion of SRTM and ASTER GDEM datasets [35]. The last dataset used refers to the topsoil properties necessary for the K factor calculation of the RUSLE. This dataset consists of 500 m rasters acquired from the European Soil Data Centre (ESDAC) of the Joint Research Centre (JRC). More specifically, the dataset was derived from the topsoil data collected during the Land Use and Cover Area frame Statistical survey (LUCAS). It includes the content percentage (%) of clay, sand, silt, and the predicted topsoil soil organic carbon content in g C kg−<sup>1</sup> [36,37]. The software used includes the free and open ESA STEP SNAP v8.0 remote sensing software for the processing of the satellite images and the commercial GIS suite of ESRI that includes ArcGIS Desktop v.10, ArcGIS Pro v2.8–2.9, and ArcGIS Online with the Web AppBuilder. #### 3.2. Methodology The methods applied for the purposes of this study are described in the following subsections. In Figure 2, the flowchart presents all the steps followed for each part of the methodology with the mapping of the wildfires presented on the left and the implementation of the RUSLE on the right part. In the middle, the different products are presented with the following addition of them to the web app. Figure 2. Methodology flowchart. #### 3.2.1. Operational Wildfire Mapping The operational mapping of the previously mentioned wildfires was performed with the use of the Sentinel-2 L2A imagery listed in Table 2. In order to have the most accurate and timely available results, cloud-free images acquired immediately before and after each wildfire event were carefully selected. These images were imported in SNAP and then preprocessed with nearest neighbor resampling of the spectral bands to 10 m resolution and then subset to the area of interest (AOI) extent to enhance processing performance. The next steps included the creation of a cloud and water mask with band maths operations based on the available scene classification from the L2A product and the estimation of the water mask with the normalized difference water index (NDWI) [38] and the collocation of the pre-fire and post-fire event imagery followed to merge them into one product. For this study, for the wildfire mapping, the related spectral indices were applied. More analytically, the normalized burn ratio (NBR) is the used index that utilizes the spectral bands of near-infrared (NIR) in which burned areas present low reflectance and shortwave-infrared (SWIR) in which burned areas show high reflectance for the mapping of burned areas and the assessment of burn severity based on Equation (1). The index has high values where healthy vegetation exists and low values in burned areas and areas with low or no vegetation presence [15,16,39,40]. $$\text{NBR} = \frac{\text{NIR} - \text{SWIR}}{\text{NIR} + \text{SWIR}} \tag{1}$$ The NBR index was calculated before (pre-fire) and after (post-fire) the fire event and then the difference between the two, the dNBR (Equation (2)), results in a better distinction of the burned areas while the burn severity is also assessed. Burn severity is a term used to express the degree of a wildfire's impact on an area's ecosystem. The assessment of burn severity contributes to quantifying the impact of a wildfire and aids restoration attempts and natural disaster management. Based on the dNBR values, the burn severity is classified into categories as is shown in Table 3 with the higher values corresponding to higher severity and thus the more severe impact of the wildfire on an area [40–44]. $$\text{dNBR} = \text{PrefireNBR} - \text{PostfireNBR} \tag{2}$$ After the dNBR estimation, the relativized burn ratio (RBR) was calculated based on Equation (3). This index aids the assessment of burn severity in a diversity of ecosystems and regions by enhancing accuracy. Additionally, it aids the identification of the changes after a fire in areas with low vegetation [45]. $$\text{RBR} = \frac{\text{dNBR}}{\text{prefireNBR} + 1.001} \tag{3}$$ *dNBR Value \ Burn Severity 0.100–0.269 Low severity 0.270–0.439 Moderate-low severity 0.440–0.659 Moderate-high severity 0.660–1.300 High severity Table 3.** Burn severity classification according to dNBR values [16,46]. \* Values ≤ 0.099 represent unburned areas. The final RBR raster was masked using the above-mentioned cloud and water mask in order to be not only clear from any possible clouds, but also to eliminate existing water surfaces due to their spectral characteristics which make them attributed falsely as burned areas. Results production followed with the masked RBR raster import to the GIS software and its reclassification according to the Table 3 burn severity classes. Conversion to vector polygons, burned area extraction, and clipping of CLC 2018 to the burned area extent were the last steps including the area estimations. #### 3.2.2. Revised Universal Soil Loss Equation (RUSLE) The Revised Universal Soil Loss Equation (RUSLE) model provides a widely known and useful tool for soil erosion assessment [2,22,47,48]. Developed in the late 1970s [23], it was formulated so as to conclude a better estimation of the initial parameters defined by the Universal Soil Loss Equation (USLE) [49]. The derived methodology represents the influence of topography, soil properties, meteorological parameters, and land cover on surface and rill erosion [2,47,48]. The average soil loss assessment is enabled by the RUSLE according to the specific study area characteristics [2] and it is based on an empirical equation (Equation (4)), constituting of five factors [24], which can be easily implemented through a GIS framework: $$A = R \times K \times LS \times \mathcal{C} \times P \tag{4}$$ where A is the estimated mean seasonal soil loss (ton h−<sup>1</sup> Season−1), R represents the rainfall erosivity factor (MJ mm ha−<sup>1</sup> h−<sup>1</sup> Season−1), K is the soil erodibility factor (ton h MJ−<sup>1</sup> mm−1), LS is the slope length and steepness factor (dimensionless), C is the cover management factor (dimensionless), and P represents the conservation practice factor (dimensionless). These factors are of vital meaning in soil erosion assessment, and they should be very carefully estimated in order to lead to highly accurate results (source). R factor plays a crucial role in RUSLE modeling since it defines the possibility of erosion risk [49] and it is estimated based on meteorological data from meteorological stations surrounding the study areas as previously mentioned [50]. Using the empirical formulation (Equation (5)) developed by Loureiro and Couthino [50], monthly rainfall data concerning seasonal time periods of 2021, were used to calculate the R factor: $$R = \frac{\sum\_{i=1}^{12} (7.05 \times r\_{10} - 88.92 \times d\_{10})}{N} \tag{5}$$ where R represents the rainfall erosivity factor (MJ mm ha−<sup>1</sup> h−<sup>1</sup> season−1), N is the total months calculated annually, r<sup>10</sup> is the monthly rainfall exceeding 10 mm, and d<sup>10</sup> is the number of days when daily rainfall exceeded 10 mm per month. Specifically, rainfall data acquired from each weather station were implemented using a spatial interpolation technique, inverse distance weighting (IDW), so as to define unknown meteorological values within the study areas. K factor refers to the rainfall impact on soil properties resulting in soil erosion due to sediment penetration, detachment, and transport [23,24,27]. The soil erodibility factor is affected by soil properties such as structure, organic matter, permeability, and soil texture [48]. In this study, the estimation of the K factor was based on Equation (6) developed by Williams and Renard [51]: $$=0.2+0.3\exp\left(0.0256\times\text{Sa}\times\left(1-\frac{\text{Si}}{\text{FD}}\right)\right)\times\left(\frac{\text{Si}}{\text{Cd}+\text{Si}}\right)^{0.3}\times\left(1.0-\frac{0.25\times\text{C}}{\text{C}+\exp(3.72-2.95\text{C})}\right)\tag{6}$$ $$\times\left(1-\frac{0.7\times\text{SN}}{\text{SN}+\exp(-5.51+22.98\text{N})}\right)$$ where K is the soil erodibility factor (ton h MJ−<sup>1</sup> mm−1), Sa represents the percentage of salt, Si is the percentage of silt, Cl is the percentage of clay, C is the percentage of organic carbon, and SN stands for SN = 1 − (Sa/100). Soil data acquisition was based on datasets provided by the ESDAC database [37]. The resulted K values were implemented on the spatial interpolation method Kriging to produce values covering the total surface of the study areas. The topographic effect on soil erosion is determined by the impact of the slope length and steepness factor (LS factor) [27]. LS factor constitutes the result of slope length (L) and slope steepness (S) multiplication. Increased slope steepness values determine increased soil erosion risk due to the increase in the velocity and erosivity of the accumulated runoff [2]. The calculation of the LS factor was based on Equation (7) of Moore and Burch [52]: $$LS = \left(\frac{\mathcal{U}}{L\_0}\right)^m \times \left\{ \left[ \sin \left( \frac{\beta \times 0.01745}{S\_0} \right) \right]^n \right\} \times (m+1) \tag{7}$$ where LS is the topographic factor, U is the flow accumulation multiplied with the pixel size, L<sup>0</sup> is the slope length (22.13 m), β is the slope in degrees, S<sup>0</sup> is the slope percentage (9%), m is sheet erosion ranging from 0.4 to 0.6, and n is rill erosion ranging from 1 to 1.3. The LS factor was created based on data derived from a digital elevation model (DEM) into a GIS setting. The rill erosion values corresponded to n = 1.1 while the sheet erosion values ranged according to the examination area each time. Specifically, for the regions of Schinos, Vilia, and Varympompi, the sheet erosion value was set as m = 0.45, whereas for the Keratea region the corresponding value was m = 0.5. C factor demonstrates the cover management factor providing a measure of soil erosion rate as controlled by the cropping and management practices within a region [23,24]. The cover management factor was calculated based on satellite data acquisition corresponding to specific time periods. Specifically, the satellite data consisted of Sentinel-2 images for the regions of Schinos, Vilia, Varympompi, and Keratea, corresponding to time periods before and after the fire. Particularly, through the acquired data, normalized derived vegetation index (NDVI) images were generated to produce the factor's values based on the formulation (Equation (8)) developed by Durigon et al. [53]: $$C = \frac{1 - NDVI}{2} \tag{8}$$ The support practice P factor represents the practices on agricultural land that affect the soil erosion processes [54] P factor was estimated based on the Corine Land Cover 2018 dataset. According to Yang et al. [55], all Corine Land Cover classes were assigned the value of 1, except in agricultural regions where the P factor was given the value of 0.5. #### 3.2.3. Web App Development The final processing stage includes the preparation of the results layers to be suitable to be published as web layers on ArcGIS Online. ArcGIS Online is a cloud-based web GIS Software as a Service Software (SaaS) that is accessible from any device with internet access and which is interactive and enables web map creation among other capabilities. After the publication of the web layers, the web map needed for the web app was created. Finally, the web app in the Web AppBuilder was then developed with the addition of the previously mentioned map, the setting of the user interface (UI), parameters, and widgets [56,57]. #### 4. Results #### 4.1. Burned Areas and Burn Severity The total burned area of the four wildfires in Attica during the 2021 fire season reached 243.98 km<sup>2</sup> making evident the large extent of damage caused to the region. As Figure 3 presents, the wildfire in Vilia burned the largest area reaching almost 96 km2, followed by Varympompi with 78.95 km2, Schinos with 64.05 km2, and Lavreotiki that has a burned an area of 5 km2. Figure 3. Burn Severity of 2021 Attica region wildfires: (a) Schinos; (b) Varympompi; (c) Vilia; (d) Lavreotiki. The burned area classification based on burn severity is illustrated including the percentage of the burned area total of each area per class. Analyzing the burn severity of these wildfires, generally the impact was severe enough with the moderate-high severity accounting for 53.69% or 131 km<sup>2</sup> out of the total burned area with the moderate-low (28.36%) and low (17.8%) following, while high severity covers 0.14%. More specifically, considering each area based on Figure 3, it is evident that the moderate-high severity characterizes all the burned areas except Lavreotiki, where lower severity levels prevail, whereas in Schinos a mostly close-to-equal distribution is observed among burn severity levels. It should be noted that in the cases of Varympompi and Vilia, the percentages of burn severity levels coverage in each case follow an almost identical pattern in the moderate severity levels percentage. Regarding the affected land cover based on CLC 2018 as presented in Figure 4 and Table 4, the total numbers show that the third CLC category which includes forests, shrubs, and/or herbaceous vegetation is heavily impacted by the wildfires covering 82.71% or 201.8 km2 of the total burned area, thus highlighting the ecological disaster in the region. To put it another way, coniferous forests (72.48 km2), sclerophyllous vegetation (52.47 km2), transitional woodland-shrub (46.13 km2), and mixed forests (24.73 km2) comprised the most affected land cover. In Schinos, the burned area consisted of 62.80% of coniferous forests, in Varympompi transitional woodland-shrubs and mixed forests took up 46.60%, although it should be mentioned that a significant part of the artificial surfaces category was impacted in the region reaching 9.56% of the total burned area, including discontinuous urban fabric. Continuing with Lavreotiki, coniferous forests, natural grasslands, and sclerophyllous vegetation accounted for 64.44% of the total burned area, and lastly, in Vilia Sclerophyllous vegetation was 49.21% of the burned area. Figure 4. Burned land cover of Attica region wildfires based on Corine Land Cover 2018: (a) Schinos; (b) Varympompi; (c) Vilia; (d) Lavreotiki. The third level of Corine Land Cover 2018 was utilized and clipped to each burned area to assess the affected land cover. Table 4. Burned land cover of Attica region wildfires based on Corine Land Cover 2018 (% of the burned area total of each area per land cover category). The correlation between burn severity and land cover led to useful findings. More analytically, transitional woodland-shrubs were the main land cover category characterized by high severity along with broad-leaved forests. Moderate-high severity existed mostly in coniferous forests (41 km2), schlerophyllous vegetation (30.96 km2), and transitional woodland-shrub (28.65 km2). In Vilia, 28.49 km2 of schlerophyllous vegetation belonged to this burn severity level as well as 19.3 km2 of coniferous forests in Schinos, 12.31 km2 of mixed forest in Varympompi, and 0.79 in Lavreotiki accordingly. Regarding moderatelow severity, this was found in coniferous forests (18 km2), schlerophyllous vegetation (15.57 km2), and transitional woodland-shrubs (11.5 km2). In each area, the following prevailed: the schlerophyllous vegetation of Vilia (13.5 km2), coniferous forests of Schinos (10.27 km2), transitional woodland-shrubs of Varympompi (4.26 km2), and schlerophyllous vegetation of Lavreotiki (0.6 km2). Lastly, low severity is primarily met in coniferous forests (13.4 km2) and it mainly characterizes coniferous forests in Schinos (10.9 km2), sclerophyllous vegetation in Vilia (5.14 km2), and complex cultivation patterns in Varympompi (2.99 km2) and Lavreotiki (0.17 km2). #### 4.2. Soil Erosion Risk Regarding the soil erosion risk derived from the use of the RUSLE concerning the time periods before and after the fire incidents in the Attica region in 2021, the spatial distribution of soil loss is presented in the following Figures 5–8. Due to the assessed areas' heterogeneity of characteristics, the soil loss results in ton/ha/season were converted into a universal classification based on each area as presented in Table 5. More analytically, the 5 classes (from very low to very high erosion risk) were specified on an equal interval for each case estimated from the standard deviation average of soil loss before and after the fire events. In this way, a comparable classification, taking into consideration each area's soil loss results, was constructed which enables the identification of the change concerning erosion risk in pre-fire and post-fire results. Figure 5. Erosion Risk according to RUSLE soil loss for Schinos burned area: (a) before the fire; (b) after the fire. The erosion risk as classified is presented including the percentage of the burned area total of each area per class. Figure 6. Erosion Risk according to RUSLE soil loss for Varympompi burned area: (a) before the fire; (b) after the fire. The erosion risk as classified is presented including the percentage of the burned area total of each area per class. Figure 7. Erosion risk according to RUSLE soil loss for Lavreotiki burned area: (a) before the fire; (b) after the fire. The erosion risk as classified is presented including the percentage of the burned area total of each area per class. Figure 8. Erosion risk according to RUSLE soil loss for Vilia burned area: (a) before the fire; (b) after the fire. The erosion risk as classified is presented including the percentage of the burned area total of each area per class. Table 5. Erosion risk classification for RUSLE soil loss (ton/ha/season), presenting each class for each area. Soil loss results, in general, show a clear differentiation between the pre-fire and post-fire states in all four areas. More specifically, the pre-fire state is characterized by very low soil erosion risk, which is predominant in all cases. Regarding the post-fire soil erosion risk, it was clearly increased in the four areas of interest. Very low soil erosion risk was reduced to 45.76% whereas the low and moderate soil erosion risk constitutes a combined 40.12% of the burned areas. Moreover, the rest areas are characterized by high and very high soil erosion risk values of almost 7% each. It has to be mentioned that the results of the RUSLE cover a slightly reduced area than the original burned areas due to the slope estimation in the LS factor. To begin with, the analysis of Schinos burned areas (Figure 5). Figure 5a illustrates that the area has a very low soil erosion risk. In the post-fire state (Figure 5b) the increased soil erosion risk is evident in the area with a reduction of very low to almost 50% area coverage with a main concentration in an axis from the north to the southeastern parts of the region. To analyze more, the western part of the area is characterized by concentrations of moderate to very high erosion risk as well as the southern part with an important concentration on the east–west axis. Moderate (13.04% or 7.43 km2) and low (26.19% or 14.92 km2) are distributed over the area covering a total large extent. A further analysis associated with the burn severity shows that moderate-high severity prevails in all erosion risk classes while considering that land cover coniferous forests are the most affected. Regarding the burned areas of Varympompi in Figure 6a, the area in the pre-event soil erosion risk was very low. Analyzing the post-fire soil erosion risk as of Figure 6b, very low erosion risk covered 52.37% of the area, especially in the southern and eastern parts. Low erosion risk was distributed over the area characterizing 23.94% of it alongside moderate (11.71% or 8.14 km2). The higher erosion risk categories took up the rest, almost 12% of the area, with very high erosion risk covering important parts to the west in the Partnitha Mountain base and around the Afidnes area in the north-central to northern parts. The distribution of erosion risk based on burn severity followed the same trends proportionally as the predominant moderate-high severity category. Assessing erosion risk with land cover, very low erosion risk characterized mostly transitional woodland-shrubs and low mixed forests alongside moderate, and high while very high was mainly spread within mixed and coniferous forests and transitional woodland-shrubs. Proceeding to Lavreotiki, the pre-fire state as in previous cases showed a very low soil erosion risk (Figure 7a). In Figure 7b, the post-fire erosion risk presented an increase with very low and low erosion risk taking up 77.63% or 3.38 km2 of the area. In the rest of the area, the moderate risk was sparsely distributed (11.90%) while the almost equal coverage of high and very high erosion risk was mainly located in the north-northeastern part of the region. Concerning the burn severity, the erosion risk followed a distribution based on moderate-low severity which covered the larger area. From the CLC 2018 perspective, the very low and low erosion risk was found primarily in coniferous forests and schlerophylous vegetation while moderate, high, and very high was found in natural grasslands. In the last area, Vilia, the soil erosion risk is presented in Figure 8. During the pre-fire period, the area was at very low erosion risk (Figure 8a). The post-fire erosion risk was higher in all the previously mentioned areas as of Figure 8b. Starting with the very low erosion risk it only covered 38.57% of the Vilia burned area followed by the low with 27.63%. To continue with the moderate severity which was dispersed all over the area it encompassed 16.29% or 14.73 km2 of it. An important part covered the high (9.36%) together with very high (8.14%) soil erosion risk with the area's topography playing a key role in their spatial distribution located mainly in large parts in the central part of it. As already seen in the previous areas, the same pattern regarding burn severity was followed with the predominant moderate-high severity in this case covering the larger part of each risk category. Lastly, sclerophyllous vegetation, as of land cover, in every soil erosion risk class, was the major affected land cover. #### 4.3. Web App The web app is presented in Figure 9 and it includes all the above-mentioned results layers. Within the user interface, the various widgets are visible including map navigation ones and information, legend, layer list, and basemap selection. Others include location retrieval, search, drawing, sharing, measuring, and swiping between layers while coordinates can also be retrieved and converted. The web app is easily accessible from any device through the link: https://learn-students.maps.arcgis.com/apps/webappviewer/index. html?id=b55b196c8f464d28b18182809e590a33, accessed on 2 May 2022. Figure 9. Screenshot of the developed web app. The user interface is shown with the web map of the affected areas and the widgets. #### 5. Discussion This study achieved the target of assessing the impact of the wildfires that struck the Attica region in 2021 by combining remote sensing and GIS techniques and free open available datasets. The methods used led to the production of results which helped in understanding the effects of the four examined wildfires. The operational wildfire mapping with the use of Sentinel-2 optical imagery with the application of NBR was effective in mapping the burned areas and assessing their burn severity on a near-real-time basis and with a very good spatial resolution. The later soil erosion risk modeling derived from the RUSLE was sufficient enough to trace the soil loss before and after the wildfire events and understand the risk that each area faces after them. The highlight of this study is the addition of all these products to the web app developed for this purpose thus enabling the getting of an inclusive geospatial intelligence of the post-fire situation. As mentioned above, the wildfires of 2021 constitute one of the greatest disasters Greece has faced. The analysis that has been implemented in this manuscript was related to the wildfires that broke out in the Attica region. Specifically, the study areas consisted of the wildfire incidents that broke out in Varympompi, Schinos, Vilia, and Lavreotiki. Those regions had been extensively affected by the fires with the burned areas varying from 5 km2 to 96 km2. The burn severity demonstrated some very interesting patterns spatially, predominated by moderate-high burn severity values in all regions, except Lavreotiki, where moderate-low severity values presented high frequency as well. Based on the results, the fires affected mostly forestry area consisting of broad-leaved, sclerophyllous, and mixed vegetation. Wildfire incidents usually lead to post-catastrophic events such as the soil erosion phenomenon. In this study, advanced erosion modeling was applied to the above-mentioned regions in Attica in order to estimate the erosion risk resulting from the wildfire incidents. Particularly, RUSLE modeling was implemented so as to calculate the soil loss rates considering the study area. According to the results, erosion mapping after the wildfire incidents seemed to present high soil loss values in comparison to their pre-fire state. Post-fire mapping displayed very high erosion values according to RUSLE modeling in all four areas of interest. Analyzing the results of the burned areas, moderate-high severity characterized the burned areas which were also found in large concentrations in all of them except Lavreotiki. Within the boundaries of the affected areas, it could be observed that low severity values were present. Assessing the severity through the land cover, areas with moderate-high severity belong to the forest category of CLC 2018 thus making evident the ecological disaster but also the role of fuel that those areas constituted. The burned agricultural areas had moderate-low severity while the discontinuous urban fabric such as in Varympompi was characterized by mixed burn severity values. Another remark should be made about the terrain that affects the burned areas. More specifically, in most of the valley bottoms or higher altitudes, low severity values were found, or they were unburned due to their characteristics (e.g., low or no vegetation). We will proceed to the erosion risk analysis over the burned areas in which the soil loss and thus the erosion risk increased to a large extent, pinpointing areas that may face excessive soil loss in the future. The analysis of the factors has revealed the significant influence of topography on the erosion risk based on the LS factor, which makes clear the higher values of soil loss to the hydrographic network. Considering the K factor's influence, which is a factor that does not take into consideration the post-fire state, it showed some interesting patterns in Varympompi, where moderate to high values corresponded to high erosion risk. However, due to the fact that soil properties hardly ever face significant alterations over the years, the K factor is considered to contribute effectively in soil erosion assessment offering reliable results. With regards to the P factor, it did not play an important role in the model's results, making it evident that it is not taken into consideration in many studies [58]. The NDVI-based C factor before and after the wildfires clearly showed a homogenous increase within the affected areas contributing to the soil loss increase. In the last factor considered, the R factor, there was a moderate correlation with the erosion risk which in some cases was related to increased soil loss. Some comparisons can be made between burn severity and land cover with the erosion risk. In Schinos, higher erosion risk was found in coniferous forests, transitional woodlandshrubs, and mainly in areas of higher burn severity values. Passing to Varympompi, higher erosion risk values were located in the burned forest areas. It is worth mentioning that in the discontinuous urban fabric, in the north of the area, the risk was also high, presenting a trend of higher soil loss to the higher burn severity areas. In Lavreotiki, there was not a clear correlation between burn severity and erosion risk while the significant concentrations in high and moderate soil erosion risk were found in sclerophyllous vegetation and natural grasslands. Lastly, the soil erosion risk was higher in forest areas within the Vilia burned area and areas of moderate-high values. Lately, RUSLE modeling has been applied to several studies in the Mediterranean region regarding post-fire assessment [2,26,27,47]. Tselka et al. [2] implemented RUSLE modeling in Central Greece so as to detect spatial correlations regarding the erosion risk after a fire event. Results of this study demonstrated as well that there was a rise in soil loss values immediately after the fire incident. The outputs of this study were also formed mostly based on the LS factor, while there was also a correlation between factors R and C and the outputs which could be attributed to the total length of the study area. Another interesting study is the one of Polykretis et al. [47], in which RUSLE modeling was applied in Crete, Greece in order to evaluate the factor's influence in soil erosion mapping. The outputs showed that the R and C factors seemed to affect mostly the erosion risk assessment. In addition to our study, the LS factor was characterized as a static factor regarding its effect on soil loss mapping. Similarities were also detected in a study published by Evelpidou et al. [26] regarding the erosion risk after the wildfires that broke out in Greece in 2021. Results also demonstrated a significant increase in soil loss after the fire incidents using a Boolean logic-based model. Comparable patterns to our study have been also demonstrated in research carried out by Lanorte et al. [27], in which soil erosion risk was assessed after wildfire events in a southern Italy region. In this research, soil erosion risk is significantly increased after the fire, complying with the spatial patterns of our results. At this point, the empirical part of the study is evaluated. In the mapping of burned areas, satellite image availability plays an important role operationally. Sentinel-2 has a very good temporal resolution but all the optical sensors can be severely affected by cloud and smoke cover over an area of interest thus limiting the burned area mapping capabilities which is a factor that affected the image selection of this study. Although, despite the fact that the 10 m resolution of Sentinel-2 is sufficient, the availability of higher resolution datasets could enhance the mapping's detail. Proceeding to the burn severity, it is important that it is assessed also by field surveys to validate the results derived from the satellite imagery. An issue faced through the use of NBR is the misclassification of unburned areas as burned ones. This is caused by areas having similar spectral properties in the used spectral bands. In addition to the use of masks to minimize these effects, a manual selection of the burned consulting the post-fire Sentinel-2 natural and false-color images was also performed to maximize the accuracy. Continuing with the empirical part, generally, the datasets proved adequate to meet this study's purposes. The land cover retrieved from the CLC 2018 dataset gave a clear view of the affected burned areas and proved to be sufficient in in P factor estimation. However, a more detailed dataset such as Urban Atlas 2018 could further improve them. With regards to the used DEM, a high-resolution DEM like that of Hellenic Cadastre with a 5 m resolution [2] would have given higher detail in the RUSLE as well as a closer resolution to the burned areas. In this context, the difference in resolution between the 10 m of burn severity and the 25 m selected based on DEM of RUSLE soil loss creates a difference in detail. Considering the total extent of all four wildfires, a higher resolution could have caused processing volume-related problems. The meteorological dataset concerning rainfall data for the creation of R factor was based on a dense network of stations for the examined time periods. In general, dense point networks provide better outcomes in the implementation of spatial interpolation techniques. Soil-related datasets and, more specifically, the topsoil ones from ESDAC used in this study, led to the K factor estimation but their resolution is very low (500 m) thus degrading detail. In addition, quality of the ESDAC raster datasets in combination with their low resolution made it necessary to apply the kriging spatial interpolation technique in order to improve both quality and resolution. Finally, considering the RUSLE implementation, the C factor, which is based on NDVI, proved very useful in the RUSLE estimation before and after a wildfire event. However, it might present some issues in regards to distinguishing between burned and non-burned areas. Alternatively, for the K factor, the soil erodibility dataset by ESDAC would have been also suitable to be used instead of the previously mentioned ESDAC datasets as a ready-to-use solution. The RUSLE modeling validation could have included fieldwork after the fire event. Classification of the soil loss to create soil erosion risk categories was challenging and the final selection was performed after tests with other existing classification methods. The web app is maintained and updated through the ESRI's ArcGIS Online services while content updates can also be conducted if more products are added or by updating existing ones. In addition, any update to the UI or widgets can be conducted by taking into consideration end-users' feedback and when new or updated features are provided by ESRI. #### 6. Conclusions Taking into consideration the future work and potential based on this study, some remarks are presented. The use of higher resolution datasets could further improve the wildfire impact assessment detail and accuracy, such as high-resolution satellite imagery, DEM, more analytical land cover, and better soil properties datasets in terms of sampling and resolution. Improvements of the utilized methods and comparisons with other related ones could be a step forward in accuracy and validation assessment. Other steps may include a rethinking of the RUSLE erosion risk modeling approach concept based on the literature and a detailed analysis between burn severity and RUSLE erosion risk could aid in finding spatial relations between them [2]. A key future step is the conduction of in situ surveys for the validation of the results derived with the above-mentioned methods regarding burn severity [59] and soil loss. Finally, this work could also be expanded by considering more parameters and it could also be implemented in a similar way for future wildfire events constituting an important tool that not only permits post-fire impact assessment but also creates a useful archive for future studies and decision-making. To sum up, the findings of this study helped in gaining geospatial intelligence over the effects that the 2021 wildfires had in the Attica region. Results regarding burn severity, land cover, and erosion risk, combined in a usable web app, could give a significant advantage to impact assessment research along with the decision making and planning over natural hazards management by stakeholders over the restoration procedures as well as the needed measures in vulnerable areas. In other words, the knowledge that this tool encompasses could help in pinpointing problematic areas and making all the needed post-fire actions more focused and effective. In the region of Attica, the results of this study based on the four studied wildfires showed that in 2021, a large part of the region was burned severely losing important parts of natural ecosystems and these areas are at an increased risk of soil erosion. This situation highlights the need for immediate action that should be taken in those areas by the stakeholders. Lastly, this study, with the presented innovative workflow and holistic approach, tried to highlight the importance of assessing the effects of a wildfire event with the use of Earth observation and GIS-based techniques using a cloud-based platform, in an attempt to share and disseminate the knowledge gained about the impacts on the affected environment. Author Contributions: T.F. was involved in paper writing, data collection, methodology, visualization, and web app development; I.T. contributed to paper writing, data collection, and methodology; I.P. (Ioannis Papadopoulos) and M.N. were involved in methodology and assisted in paper writing; A.K., D.B., A.P. and P.K. were involved in methodology; T.F. and I.P. (Issaak Parcharidis), were involved in conceptualization and review; I.P. (Issaak Parcharidis) had the general supervision. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: The web app is accessible through the link: https://learn-students. maps.arcgis.com/apps/webappviewer/index.html?id=b55b196c8f464d28b18182809e590a33, accessed on 2 May 2022 It is open and accessible with a login not required. Data is available upon request or by login to the web app with an ArcGIS Online account. Acknowledgments: The authors would like to thank ESRI and its Learn ArcGIS Student Program for the initial access to ArcGIS Pro and Online for making the web app possible. In addition, the contribution to the wildfire operational mapping in the 2021 fire season in Greece of every one of our team, the HUA Earth Observation Team, should be acknowledged. Lastly, we are grateful to the editors of the special issue for the invitation and the opportunity to publish this work as well as the reviewers who reviewed our manuscript. Conflicts of Interest: The authors declare no conflict of interest. #### References ### Article Modeling the Ignition Risk: Analysis before and after Megafire on Maule Region, Chile *Gabriela Azócar de la Cruz 1,2,3,\, Gabriela Alfaro 3,4, Claudia Alonso 2,3, Rubén Calvo 3,5 and Paz Orellana <sup>3</sup> Abstract: Wildland fires are a phenomenon of broad interest due to their relationship with climate change. The impacts of climate change are related to a greater frequency and intensity of wildland fires. In this context, megafires have become a phenomenon of particular concern. In this study, we develop a model of ignition risk. We use factors such as human activity, geographic, topographic, and land cover variables to develop a bagged decision tree model. The study area corresponds to the Maule region in Chile, a large zone with a Mediterranean climate. This area was affected by a megafire in 2017. After generating the model, we compared three interface zones, analyzing the scar and the occurrences of ignition during and after the megafire. For the construction of georeferenced data, we used the geographic information system QGIS. The results show a model with high fit goodness that can be replicated in other areas. Fewer ignitions are observed after the megafire, a high recovery of urban infrastructure, and a slow recovery of forest plantations. It is feasible to interpret that the lower number of ignitions observed in the 2019–2020 season is a consequence of the megafire scar. It is crucial to remember that the risk of ignition will increase as forest crops recover. Wildland fire management requires integrating this information into decision-making processes if we consider that the impacts of climate change persist in the area. Keywords: wildfire; ignition risk; model; megafire; climate change; bagged decision tree; wildland urban interface #### 1. Introduction Wildfires are an extreme phenomenon of great global concern [1]. The frequency and intensity of these events have progressively increased in different areas of the world [2]. Studying the causes and risks of forest fires has become a field of broad interest. Various investigations and technical reports on wildfires agree that at least 90% of these have an anthropogenic origin [3–7]. It is not entirely clear, however, how many of the fires caused by human actions are due to negligence and how many are intentional. Identifying and punishing the people responsible for wildfires is a complex problem, given that the evidence is not recordable or disappears due to the fire [8]. A series of environmental factors favor the spread of wildfires, such as the combination of temperature, humidity, and wind [4,9–11]. Added to this are research results on the influence of productive activities and the characteristics of wildland–urban interface zones [6,12,13]. All the above indicates the need to deepen the analysis of the interaction between human action and its ecological environment to investigate the conditions that affect the fire origin and spread. In addition, there is a need to connect this with the impacts of socio-environmental disasters. Citation:** Azócar de la Cruz, G.; Alfaro, G.; Alonso, C.; Calvo, R.; Orellana, P. Modeling the Ignition Risk: Analysis before and after Megafire on Maule Region, Chile. Appl. Sci. 2022, 12, 9353. https://doi.org/10.3390/ app12189353 Academic Editors: Efthymios Lekkas and Spyridon Mavroulis Received: 15 August 2022 Accepted: 14 September 2022 Published: 18 September 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Climate change is a highly complex phenomenon that has become the focus of interdisciplinary studies on the relationship between society and the environment, particularly disaster risks. Its link to wildfires exemplifies this evident relationship [14–18]. Due to climate change, different areas of the world have been affected by the increase in heat waves, the increase in the magnitude of periods of drought, the decrease and absence of rainfall, and soil degradation [19–21]. All these phenomena are not direct causes of wildfires, but are factors that influence their recurrence and magnitude. Heat, drought, lack of rain, and land degradation affect the availability of fuel material, which favors the spread of wildland fires [4,22]. These factors also affect the intensity of fires and the damage they cause [13,23–25]. An interesting example of the interaction between climate change and wildfires is the increase in ignition points in mountainous areas that are difficult for people to access [26]. Lightning generated in dry electrical storms (without rain) can cause fires in native forests in these areas, where difficult access is an obstacle to their control [27–29]. The relationship between climate change and wildland fires increases concern about its social and environmental impacts. Wildland fires impact people's health due to burns or the large amount of CO2 they generate, produce irreparable damage to infrastructure and homes located in interface areas, and affect productive activities [3,30,31]. On the other hand, wildland fires destroy flora and fauna, generate desertification, and deteriorate biodiversity [20,31]. The interaction with climate change increases the damage capacity of these impacts. In this scenario, it is necessary to generate tools that allow technical organizations and communities to have information that enables better wildland fire risk management [20,32]. For this, it is essential to advance the study of the behavior of risk factors in particular territorial contexts. This will allow the delivery of valuable and valid information to territorial planners, risk managers, and community leaders about the prevention, preparation, firefighting, and mitigation actions they must promote in their environments. Forest fire risk analysis is, therefore, contextual, since it depends on the characteristics of the socioecological systems in which it is carried out. We understand socio-ecological systems as those that emerge from the relationship between biophysical and social factors, sustaining a set of human needs and environmental conditions in interaction [33–36]. From disaster risk management, the concept of risk integrates the following two main components: damage and future projection. Risk has been defined as the probability of occurrence of a future event that can cause possible damage [37,38]. Therefore, the risk of wildland fires refers to the negative impacts that these can have on communities and ecological environments [20]. The probability of wildland fire occurrence depends on the interaction between different geographic, topographic, climatic, land cover, and human action variables [11,39–41]. The interaction between these factors determines the magnitude of the projected damages [11,42]. The social theory of risk expands the analysis of this concept and its application in disaster risk management. This integrates the decision as a relevant component of the risk. The probability of damage is the consequence of an unwanted decision that wants to be avoided. For decisions to manage the risk, it is necessary to know what and how this damage is produced [43]. In the context of wildland fire risk, this means integrating knowledge about what makes wildland fires so that the decisions adopted for their management are those that prevent their occurrence or minimize their damage. In this sense, risk analysis implies anticipating possible negative results of a decision [44,45]. The study of fire risk can be divided between research on propagation and the ignition of forest fires. This work addresses the risk of wildland fire ignition due to its relevance in developing prevention and preparedness strategies. A fire occurs when an ignition source (human or natural) meets the available combustible material [3]. Ignition risk is the probability that a fire will start at a given point in the territory [46]. Depending on the conditions and characteristics of the space where the ignition point is located, the flames can increase in intensity and propagate, developing into a forest fire [5,47]. Ignition depends on a wide range of variables associated with the point where it occurs. These can be classified into (a) natural conditions, such as plant species and plantations height, humidity, temperature, topography, and local climate, [3,4,11,47,48] and (b) anthropogenic conditions, such as land use and cover, distance from roads, distance from urban or inhabited areas and urban infrastructure [6,20,23,26,47,49]. The ignition risk increases when the associated variables interact at a certain point. Wildland urban interfaces (WUI) are areas in which these factors interact and are enhanced. Research indicates that the risk of wildland fires is greater when human settlements mix with vegetation [40,50]. This acquires relevance in the context of megafires. The most significant damage caused by wildland fires is in the ecological environment in which they occur. The damage on urban surfaces is usually negligible if we compare it with vegetated or forested surfaces. In megafires, however, the risk of damage in inhabited urban areas increases [51–53]. Their high intensity characterizes megafires compared to the general pattern of wildland fires. Other characteristics are their broad ecological and social impact, the obstacles they impose on their management, and the danger of reactivation [19,54]. All these factors imply that these events tend to cross WUIs, affecting people and urban infrastructure more significantly than forest fires. Along with this, a megafire can change the land cover, reducing the ecosystem services that the land provides to the surrounding populated areas [51,55]. In this study, we analyze the risk of wildland fire ignition by modeling and mapping these events. Our case study is the Maule region in Chile, which was affected by the megafire of 2017. Considering the high vulnerability that this area presents to the impacts of climate change and the uncertainty about its future effects, we developed a model of ignition risk from historical fire data in the region. First, we identify the variables that best explain ignition. Based on these variable values, the model assigned a probability of fire occurrence to points on the map. We then validated the model with data from fires during the megafire and later years. Finally, we identify four interface areas affected by the megafire of 2017 and compare the state of the territory before and after this event. This last exercise aims to analyze the current fire risk conditions in a region that, due to its high exposure to climate change, may once again be affected by events of significant magnitudes, such as the 2017 megafire. #### 2. Case Background Between January and February 2017, the south-central zone of Chile experienced one of the largest megafires in history [56]. The amount of heat energy released during the months that this event occurred exceeded the scales used internationally until then [57]. Official figures indicate that the fire destroyed 529,974 hectares. Although the greatest damage occurred in forest areas, a set of WUIs was affected, with 3000 homes lost and 11 deceased persons [16,51]. Megafires are usually generated in Mediterranean landscapes, such as the affected area in Chile. The climatic conditions and homogeneous land cover, given the vast extension of forest plantations, were favorable conditions for this event [58,59]. These conditions are characteristic of the Maule region, one of the most affected by the 2017 event. In this region, the fire destroyed 252,556 hectares, equivalent to a third of the forest area of the region [51]. The mega-drought that has affected this region since 2010 led to the fire spread and a high level of damage [57,60,61]. Due to this disaster, the resources provided for firefighting were increased in the country, which positively increased the response capacity of specialized agencies [61]. Little has been addressed, however, in the prevention of wildland fires in science and public policy. We believe studying the risk of wildland fire ignition will contribute to the characterization of areas highly exposed to these fires, information that can be used in prevention policies and actions. On the other hand, the set of conditions that increased the chance of the development of the megafire in the Maule region has not changed in recent years, making it necessary to analyze the risk of ignition of wildland fires and the associated factors. #### 2.1. Study Area Region of Maule The study area is located in the Maule Region, in central Chile, between 34◦41 and 36◦33 south latitude. The surface area is 30,296 km2, equivalent to 10% of the national territory [62]. Its population is 1,044,950 inhabitants, with a density of 34.5 inhabitants per square km, with 73% of the population living in urban areas [63]. Its topography integrates mountains of 4000 m.a.s.l. in the Andes Mountains, an intermediate depression, and the Coastal Mountains with mountain ranges with moderate to steep slopes. The climate is a temperate Mediterranean climate, with a dry season of six months in the north and four months in the south. The primary use of the land is grassland and scrubland (25%), agricultural land (22%), and forest plantations (20%) [64]. It has an area of native forest of 581,515 hectares and 634,893 forest hectares [65]. Given the topographical and climatic characteristics, the forest crops are mainly located in the Cordillera de la Costa [64]. For the analysis of the changes in the landscape after the 2017 megafire, we selected the following three communes in the region: Constitución, Empedrado, and Cauquenes (Figure 1). The selected communes correspond to the populations most affected by the 2017 fires [66]. Figure 1. (a) Location of the study area in Chile. (b) Location of the study area on a regional scale. (c) Communal scale study area. It should be noted that in Empedrado, the town of Santa Olga was entirely consumed by the fire, becoming an emblematic case of megafires' impact on a population in a WUI [15,67]. The incident negatively impacted the water and electricity supply and generated contamination of water sources for human and animal consumption. In these communes, the labor sources associated with agricultural and livestock production and the forestry industry were also affected [67]. Constitución, located between latitude 35◦19 south and longitude 72◦24 west, is a coastal city with a total area of 1344 km<sup>2</sup> [68]. It is located on the south bank of the mouth of the Maule River in the Pacific Ocean. The maritime influence keeps its daily temperatures moderate, with an annual average of around 14 ◦C. It is characterized by hot summers and mild winters, with an average annual rainfall of 662 mm. Its population is 46,068, of which 19% reside in rural areas [63]. The main economic activity is forestry and agriculture. The use of the land is native forest (5%), plantations (41%), thickets (34%), crops, and grasslands (19%) [68]. In the south of the region is the commune of Empedrado, between the coordinates 35◦36 south latitude and 72◦16 west. Its surface is 565 km<sup>2</sup> and is located in the coastal mountain range. Its annual temperatures range between 10 ◦C and 33 ◦C [69]. Its total population is 4142 inhabitants, of which 27% live in rural areas [63]. The main economic activity is agricultural and forestry production, including agriculture, livestock, dairy production, wines, and liquors. Land use is divided into the native forest (17%), plantations (17%), bushes (17%), agricultural (17%), grasslands (17%), meadows (17%), grassland crop rotation (17%) and others (17%) [69]. Between 35◦58 south latitude and 72◦18 west longitude is the commune of Cauquenes, with an area of 2216 km2 [70]. Its population is 40,441 inhabitants, with a rural population of 18% [63]. It has a main body of water, the Cauquenes River, whose flow has decreased due to the drought affecting the region. Its climate is a Mediterranean climate, with average temperatures of around 25 ◦C in January and 7 ◦C in July. The economy of the commune is diverse and includes manufacturing activities (20.5%), forestry (17.3%), and electricity production (13.2%), among others. Land use is divided into the native forest (32%), forest plantations (32%), and thickets (31%), which together cover 95% of the surface [70]. #### 3. Materials and Methods We developed a model of the risk of ignition of forest fires in the Maule region through a machine learning model. For this, we defined the ignition of forest fires as a dependent variable and selected a set of independent variables as possible predictors of ignition. The construction of the database was in two stages. First, spatially represented points of the ignition variable (binary variable) were generated. Then, the values of the set of independent variables were estimated for each of these points. The dependent variable was generated from the Corporation Nacional Forestal (CONAF) data, published as official data on its website (CONAF https://www.conaf.cl/incendiosforestales/incendios-forestales-en-chile/estadisticas-historicas/ (accessed on 20 May 2022)). From this, we obtained information on the coordinates, start date, control, cause, and magnitude, of the fires that occurred in the Maule region between 2013 and 2015. The period defined for collecting information was due to the need to have a rich source of available data on fire ignition and independent variables before the megafire of the 2016–2017 season. On the other hand, we decided to generate the model with data before the megafire to contrast it with what happened during that event to validate its results. With this, we looked for a model that allowed us to explain the ex-post distribution of the ignition points of forest fires, while predicting the risk of ignition in the future. We assigned the value 1 (one) to the points where a fire occurred in the indicated years and a value 0 (zero) to the points where there was no ignition. The assigning points that represented areas with no ignition of wildland fires were carried out randomly. Points with the value 0 were assigned to located areas more than 500 m from an ignition point. For the construction of georeferenced data, we used the geographic information system QGIS (version 3.20.3. for Windows/Copyright © 2000, 2001, 2002, 2007, 2008 Free Software Foundation, Inc. <http://fsf.org/>). We obtained 3784 points, of which 1892 indicate the ignition of forest fires between 2013 and 2015 in the Maule region; these correspond to the red points in Figure 2. The 1892 blue points generated by the model represent areas with no ignition in the same period. The independent variables selected for the construction of the model were based on the work of Miranda et al. [40]. According to the authors, the scientific evidence indicates that this set of variables is the one that best represents the territorial characteristics of those places where wildland fires occur in interface areas. Based on the results of this group of researchers, we worked with 14 independent variables organized into the following 3 classes: (a) human activity, (b) geography and topography, and (c) land cover (Table 1). Each variable was spatially represented in a 30-m resolution raster. To assign the values of the independent variables at each point, we defined a zone of influence of 500 m (centered at the point of ignition). These buffers were built by taking a circumference of a radius of 500 m around each point of the raster. Each one was assigned the value (percentages, averages) of each explanatory variable to avoid bias. Figure 2. Binary dependent variable, ignition of wildland fires in the Maule region between the years 2013 to 2015. Each of the 3784 points was assigned values for these 14 variables. Thus, the model was trained with the values obtained between 2013 and 2015. We seek to identify the variables that most influenced the fire's start in the past. With this, we estimate what factors generate a greater probability of a fire. Based on the methodology used by Miranda [40], we use the bagged decision tree, BDT model [75]. This ensemble machine learning method combines different "weak" classification sub-models to obtain a "strong" one. The processing of the model was carried out using the MATLAB R2020a (see: https://www.mathworks.com/products/matlab.html?s\_tid=hp\_products\_ matlab (accessed on 10 April 2022)). Bagging (which is short for bootstrap aggregating) consists of building different submodels using random samples, with replacement, and then assembling the results. Various subsets of the training set data are created. The model has that name, since it trains the submodels using bagging. A model is trained with each subset, and the final predictions are averaged, making it more robust. An example of a bagging model is shown in Figure 3. Figure 3. Diagram of the bagged model. For this work, we use the bagged decision tree, where each model is a decision tree. The training set was built with 80% of the database, and the remaining 20% was used to test and verify the accuracy of the results. Once the model was trained, it generated the fire ignition susceptibility classification for each point on the 30 m × 30 m quadrant map. Finally, a comparative temporal matrix of four WUI was built to analyze changes in the distribution of wildland fire ignition. The matrix was elaborated through the QGIS software. The satellite images come from the Sentinel-2 mission. These images present an atmospheric correction at ground level, providing spectral radiance levels similar to reality. The vector layer of the fire scar was extracted from the Landscape Fire Scars database for Chile [76]. Previous layers corresponding to the ignition points of the 2016–2017 and 2019–2020 seasons were used on the satellite images. It should be noted that the 2020–2021 season was not considered in the analysis. We decided to exclude this season, since the lower number of wildland fires it registers is associated with fewer people in transit in the interface areas, due to the confinement measures adopted in the context of the COVID-19 pandemic. #### 4. Results #### 4.1. Model Fit The performance of the model was estimated through the global adjustment indicator AUC. This corresponds to the area under the curve ROC (relative operating characteristic), which represents the ratio between the true positives (TPR, true positive ratio) and the false positives (FPR, false positive ratio), as the model predicts values (Figure 4). When the AUC takes a value of 1, the model has perfect prediction. When it takes a value of 0.5, it is a model without explanatory power that does not discriminate between categories. For the model, an AUC value of 0.85 is obtained, which means that it has a high level of fit. Figure 4. ROC curve of the fit of the ignition model. Another measure of the model's effectiveness is the confusion matrix, which indicates the level of correct and incorrect classifications. In Figure 5, we can observe that 75.8% of values 1 (ignition) and 77.9% of values 0 (not ignition) were correctly classified. Figure 5. Confusion matrix. One of the advantages of decision tree models is that their results show the explicative importance of the variables. This importance represents the prediction model's error increase after the variable's value has been permuted (separated in a branch of the tree). This is the normalized average of how much this variable changes the final classification result. In this model, the importance of the variables is as follows, as we can observe in Table 2. Table 2. Variables' importance. In order of importance, the variables that best explain the ignition of wildland fire in the Maule region are the proportion of crops, the distance to the nearest road, and the distance to the nearest city. The second group of variables with a medium explanatory capacity integrates the proportion of forest plantations, the proportion of grassland, the proportion of scrub, exposure, and population density. #### 4.2. Ignition Risk Model Map One of the model results is the assignment of ignition risk levels to the different zones of the study area map. This is achieved due to the georeferencing of each point. Each pixel is classified according to its level of susceptibility to the ignition of a forest fire. In the map of the Maule region, we classify 33,696,273 pixels of a 30 m resolution raster. Each of these pixels is assigned the values of the independent variables of the trained model. The result of the classification delivers values between 0 and 1. It is a continuous variable that represents levels of probability of ignition of forest fires. This graphic representation is built on the map of the region. Figure 6 shows the result of the classification; the blue zones have a lower susceptibility to fire ignition, the yellow zones have a medium probability, and the red zones have a high probability. Figure 6. Ignition risk map in the Maule region, Chile. #### 4.3. Model Validation To verify the explanatory capacity of the model, we superimposed on the map that shows the results of the ignition risk model the ignition points recorded in the 2016–2017 season. These account for the distribution of the ignition points of the megafire in the Maule region (red points in Figure 7). In this comparison, it is important to consider that the modeling of the ignition risk was carried out based on data from 2013 and 2015. Figure 7 clearly shows that most of the points where a fire started are in areas the model classified as having a high probability of ignition. These results contribute to validating the model and provide useful information to risk management in the future. Figure 7. Ignition risk map and ignition points during 2016–2017 season. In addition, we compare the model results with the real ignitions between 2016 and 2020. Table 3 shows that 85% of fires in that period occurred in points classified as medium, high, and very high risk by the model. Table 3. Percentage of fire ignition during 2016–2020 according to model probability levels. #### 4.4. Temporal Comparative Matrix of Interface Zones To deepen the analysis of our model and generate guidance for decision-makers, professionals in charge of forest fire prevention and preparedness, and communities, we analyzed three interface zones severely damaged by the 2017 megafire (Figure 8). The matrix shows the results of the ignition risk modeling (a), the ignition points of the 2016– 2017 season (b), the scar of the 2017 megafire (c), and the ignition points of the 2019–2020 season (d). The results show that fire ignition in the two seasons under analysis occurred in areas the model classifies with a high probability (see columns a, b, and d). In these areas, crop areas of diverse composition can be observed—especially forestry—near the ignition points by roads and cities. These characteristics correspond to the variables with the most significant weight in the probability of ignition of our model. Comparative analysis of satellite images indicates that the number of ignitions in each WUI is lower in the 2019–2020 season. Changes in the territory's characteristics can also be observed, including a notable decrease in the density of forest crops in each of the analyzed areas (column d). This change in the territory's composition can be attributed to the 2017 megafire scar (column c). A large part of the forest plantations, grassland, and scrubs in these areas was destroyed by the fires of 2016–2017. Our model classifies these variables with a medium influence on the probability of ignition. It should be noted that the town of Santa Olga was destroyed by the fires of 2017. As a reparative measure, it was quickly rebuilt in the following years. The reconstruction of the town meant an improvement in the conditions and quality of life of its population, which received better quality housing and urban infrastructure that did not exist before the megafire [67]. These improvements, however, do not mean a reduction in the ignition risk in the area adjacent to this town, since it continues to be surrounded by extensive and dense forest plantations. #### 5. Discussion and Conclusions The ignition risk model generated in this work makes it possible to identify those sectors of the wildland urban interface zones with a higher probability of forest fire ignition. In turn, it tells us what characteristics of the study area make it more prone to ignition. Depending on their predictive capacity, two groups of variables allow us to understand why ignition is more probable in these sectors. The group of variables with the most significant predictive capacity includes the crop proportion, the distance to the nearest road, and the distance to the nearest city. These are variables of anthropogenic origin if we consider that they are associated with productive activities (crops) and urban characteristics (roads and cities). The second group of variables has a medium ability to predict ignition. Among these, we find variables of anthropogenic origin, such as the proportion of forest plantations and population density, and variables of natural origin, such as grasslands, scrubs, and exposure. The results coincide with previous studies that have verified the coincidence between forest fires' risk and high proportions of land use destined for crops [26]. In this regard, particular importance has been assigned to forest crops in the ignition of fires [22]. It has been shown that the uncontrolled growth of forest species increases the combustible material and the ignition risk [16,22,77–79]. On the other hand, it has been pointed out that replacing the native forest with homogeneous pine and eucalyptus plantations in the central-southern zone of Chile has led to a greater production of large-scale wildland fires [56]. Our results help confirm the strong influence of human activities on forest fire ignition [4,10,20,24,40]. As in our work, the proximity of ignition points to roads and human settlements are factors that previous studies highlight as risk factors [3]. The analysis of the WUI satellite images indicates that the territory's characteristics changed due to the scar from the 2017 megafire. The density of forest plantations is the most evident change. The images show that forest plantations are in the process of recovery. On the other hand, the urban infrastructure has recovered rapidly. This maintains the high levels of risk associated with the distance from roads and cities and population density. In particular, Santa Olga town reconstruction shows that the repair process focuses on restoring the urban infrastructure and improving the living conditions of the people affected by the fires. However, integrating the ignition risk prevalent in the Maule region into decisions is not evident. It is feasible to interpret that the lower number of ignitions observed in the 2019–2020 season is a consequence of the megafire scar. Variables associated with crops, scrubs, and grasslands decrease their influence on ignition, given that such combustible materials are less dense. However, it is crucial to remember that the risk of ignition will grow as forest crops recover. This information must be incorporated into the decisions adopted for the prevention and preparation of forest fires. Along with this, the authorities and communities must integrate into their decisions how climate change contributes to ignition probability. The decrease in rainfall, soil degradation, and heat waves are impacts of climate change related to the variables that the ignition risk model classifies with a strong and medium influence. Previous studies have confirmed that these impacts of climate change affect soil humidity, creating conditions for high flammability of combustible materials [4,10,11,56]. The exposure of the land to sunlight is another climate change variable that our model associates with the ignition risk. According to Maniatis (2021), in areas more exposed to sunlight, the vegetation loses humidity and becomes more flammable. The Maule region has been highly impacted by climate change. This condition is not expected to vary, at least in the short and medium term [60,61,80]. For this reason, it is imperative to consider the variables above in wildland fire risk management, especially given the uncertainty that a new megafire might occur in the region. The creation of models that make it possible to understand and make visible the risk of wildland fire ignition is necessary but not sufficient. If disaster risk is still understood only as the future projection of damage, these models only serve to identify the risk, not to manage it. Forest fire management requires integrating risk as a decision. This means improving the policies that regulate the density of forest plantations, the maintenance and expansion of firebreaks in interface zones, land use, and post-fire cleaning of areas where tree species' seeds are irregularly disseminated. It also means integrating the ignition risk into the measures to repair the damage caused by wildfires. Developing ignition models, such as the one in this work, is undoubtedly necessary to manage wildland fires. An example is the verification of sectors with a greater probability of ignition that, according to the model, are strongly associated with the areas where multiple fires occurred in 2017. This information will allow fire governance actors to implement better strategies to prevent wildland fire ignition. Nevertheless, we must be aware that, even so, forest fires will continue to occur. The risk of ignition is higher in regions such as the study area, where climate change has strongly impacted territory characteristics and conditions. For this reason, integrating the ignition risk into decisions also means implementing campaigns and actions to improve the ability to react to this kind of phenomenon. It should be noted that the model developed in this work accounts for the particularities of the study area. The model was trained with historical data from the Maule region in Chile. Therefore, the results obtained are valid only for the case analyzed. However, working with the bagged decision tree model has the following significant advantage: it is possible to replicate it in other areas with different characteristics. It is possible because the model learns from the data provided to it. In addition, variables such as human activity, geography, topography, and land cover can be obtained for other areas of interest. The only limitation is that these layers of information, particularly land cover, are scarce in many countries. One of the challenges that arise from this work is to continue deepening the characterization of the ignition risk. We believe that obtaining better views of the interface areas and the factors associated with risk is necessary. This will allow professionals and people to integrate this information. Visually identifying the components and characteristics of the territories where the ignition risk is higher can contribute to a better understanding of how to prevent and prepare for wildland fires. Author Contributions: Conceptualization, G.A.d.l.C.; methodology, G.A.d.l.C., G.A., R.C. and C.A.; software, G.A., R.C. and C.A.; validation, G.A and R.C.; formal analysis, G.A.d.l.C., G.A. and R.C; investigation, G.A.d.l.C., C.A. and P.O.; resources, G.A.d.l.C., G.A., C.A. and P.O.; data curation, G.A and C.A.; writing—original draft preparation, G.A.d.l.C.; writing—review and editing, G.A.d.l.C.; visualization, C.A.; supervision, G.A.d.l.C.; project administration, G.A.d.l.C.; funding acquisition, G.A.d.l.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Agency of Research and Development of Chile, ANID, Fondecyt N◦ 11190483. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The dependent variable was generated from the National Forestry Corporation of Chile (CONAF) https://www.conaf.cl/incendios-forestales/incendios-forestales-enchile/historical-statistics/ (accessed on 2 April 2022)). The independent variables can be found at the Instituto Nacional de Estadísticas, https://www.ine.cl/herramientas/portal-de-mapas/geodatosabiertos (accessed on 2 April 2022); Ministerio de Obras Públicas, https://ide.mop.gob.cl/geomop/ (accessed on 2 April 2022); Earth Resources Observation and Science Center (EROS), https://www. usgs.gov/centers/eros/data-tools (accessed on 22 May 2022). Sentinel-2 images for mapping-based use are located on the Copernicus Open Access Hub (https://scihub.copernicus.eu/dhus/#/home, accessed 22 May 2022). The vector layers used for the administrative political division can be downloaded from the Geospatial Data Infrastructure (IDE, Chile) (https://www.ide.cl/index.php/ limites-y-fronteras/item/1528-division-administrative-policy-2020, accessed on 11 April 2022). Conflicts of Interest: The authors declare no conflict of interest. #### References ### Article Landslides Triggered by Medicane Ianos in Greece, September 2020: Rapid Satellite Mapping and Field Survey *Sotiris Valkaniotis 1,\, George Papathanassiou 2, Vassilis Marinos 3, Charalampos Saroglou 3, Dimitrios Zekkos 4, Vasileios Kallimogiannis 3, Efstratios Karantanellis 5, Ioannis Farmakis 6, Georgios Zalachoris 7, John Manousakis <sup>8</sup> and Olga-Joan Ktenidou <sup>9</sup> Citation:** Valkaniotis, S.; Papathanassiou, G.; Marinos, V.; Saroglou, C.; Zekkos, D.; Kallimogiannis, V.; Karantanellis, E.; Farmakis, I.; Zalachoris, G.; Manousakis, J.; et al. Landslides Triggered by Medicane Ianos in Greece, September 2020: Rapid Satellite Mapping and Field Survey. Appl. Sci. 2022, 12, 12443. https://doi.org/10.3390/ app122312443 Academic Editors: Spyridon Mavroulis, Efthymios Lekkas and Valerio Comerci Received: 19 July 2022 Accepted: 2 December 2022 Published: 5 December 2022 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Abstract: Medicanes, a type of strong hurricanes/cyclones occurring in the Mediterranean, can be the source of major geohazard events in Mediterranean coastal and inland areas. Medicane Ianos that hit Greece during 17–19 September 2020 caused widespread damage, with numerous landsides and floods being the most prominent. Following the landfall of Medicane Ianos, a series of field surveys were launched together with rapid response through satellite imagery. We focused on two of the areas most affected by Medicane Ianos, Cephalonia island and Karditsa, Thessaly, both in Greece. A rapid landslide inventory for the Karditsa region was prepared using Copernicus Sentinel-2 satellite imagery, the first of its kind for a severe weather event in Greece. The mountainous area of Karditsa region in western Thessaly experienced the unprecedented number of 1696 landslides, mapped through satellite imagery and examined in the field. Cephalonia Island experienced a smaller number of landsides but damaging debris flows and severe structural damages. The rapid landside inventory was then compared to new methods of automated landslide mapping through change detection of satellite imagery. Keywords: landslides; landslide inventory; rapid mapping; remote sensing; Sentinel-2; Ianos; Medicane; Greece #### 1. Introduction As a distinctive part of the geomorphic evolution of active mountain belts, landslides play an important role in gradually changing the landscape. The triggering of landslides is related to earthquake, meteorological and human-induced factors. The former cases are reported in active tectonic zones and are characterized by the sudden occurrence of co-seismic landslides covering large areas close to the earthquake fault rupture. As it has been demonstrated by [1], fault rupture geometry and kinematics plays a significant role to the spatial distribution and density of the coseismic landslides. On the other hand, most of the landslide-related phenomena are generated by intense rainfalls. It is well known that long periods of low intensity rainfall can trigger deep-seated landslides while short duration heavy intensity rainfall is mostly related to shallow mass movements [2]. Climate change, caused by global warming in the recent years, is expected to lead to an increase of the rate of landslide phenomena in the near future [3]. One of the outcomes of climate change is a more frequent occurrence of tropical-like cyclones in areas of dry climate and relatively shallow seas. The Mediterranean Basin is one of the most cyclogenetic regions worldwide as a result of its characteristic morphology [4]. Medicanes, a concatenation of Mediterranean Sea with hurricanes, resemble tropical cyclones but present certain differences. The formation of Medicanes is highly controlled by the air–sea interaction [5]. Generally, high sea surface temperatures favor their formation especially when the air temperature is relatively low [6]. Tropical cyclones emerge when the temperature of the sea surface exceeds 26 ◦C. Nevertheless, the correspondent temperatures in the Mediterranean range between 18 to 23 ◦C [7,8], much lower than the threshold required for the tropical cyclone formation. Moreover, the expected lifespan of Medicanes is shorter than that of tropical storms. The return period of Medicanes is higher in the central and western Mediterranean Sea [9] despite the fact that the eastern basin is warmer (a favorable factor for the development of tropical-like cyclones). This phenomenon is probably related to the fact that the central and western Mediterranean is prone to cold upper-air intrusions from north and central Europe [6]. A Medicane usually carries enough energy to travel large distances (sometimes hundreds of kms) causing torrential rains and strong winds until its deterioration. Its environmental and socioeconomic impact can be devastating. One of these meteorological events is Medicane "Ianos" that occurred in September 2020 and formed as a result of a cluster of convection off the Libyan coast on 14 September 2020 [10–13]. Over the following days, it moved to the north and intensified before making landfall over Greece on 17 September 2020. After impacting western and central Greece, it changed its course and reached south Crete island by 20 September 2020. Medicane Ianos triggered intense rainfall at the central and southwestern part of Greece (Figure 1), including the two areas focused in this study; western Thessaly and Ionian Islands. Accumulated rainfall peaked at 769 mm for 17–18 September in Cephalonia island in the Ionian Sea, while Pertouli and Mouzaki stations in western Thessaly peaked at 317 mm and 268 mm [12]. This natural phenomenon induced thousands of mostly shallow landslides, debris flows and floods mainly at Central Greece and particularly at the area of Karditsa, Thessaly, Central Greece [14–16]. Landslides blocked most of the narrow mountainous roads and damaged numerous bridges [15,17]. This resulted in the isolation of communities located in higher elevations and delayed emergency response and recovery [14,17]. Figure 1. (a) Medicane Ianos over Greece, Sentinel-3 OLCI true color image acquired on 18 September 2020, one day after Medicane landing. (b) Satellite precipitation (NASA IMERG) of 18 September 2020. Overview boxes show the location of the two studied areas; 1—Cephalonia island, 2—western Thessaly. #### 2. Medicane Ianos-Induced Landslides The first step for understanding the mechanism of the cyclones-induced landslides and to mitigate in near future similar types of catastrophes is to rapidly document them. This can be achieved by immediately organizing a field survey after the event to acquire perishable field data before it disappears. Landslide debris and rockfalls are commonly cleaned out of the road network as part of the recovery operations, but their removal obscures critical information used to analyze the triggers for slope failures. Furthermore, physical processes such as weathering and erosion could additionally alter the landslide-formed landscape after the landslide event, especially concerning small size shallow landslides. Data recovered from landslide studies are essential to understanding landslide mechanisms and the associated risks, which greatly aids in identifying susceptible zones, augmenting emergency response, and mitigating potential property damage and loss of life. This research applies a mixed-method approach, primarily consisting of a desk study followed by a detailed post-event field survey aiming to report the failures in order to compile a landslide-event inventory map. In this study, the landslide phenomena triggered in the area of Thessaly and Ionian Islands are presented and preliminary analyzed with the aim to relate their spatial distribution with some basic geological and geomorphological parameters, i.e., geology, aspect, slope and elevation. Although large parts of central and western Greece were affected by Medicane Ianos, Karditsa and Cephalonia island are presented here in detail, as they experienced the most dense and severe landslide phenomena. In order to achieve this, we used information obtained during extensive field work and data provided by rapid satellite imagery mapping captured a few hours and days after the event. Satellite-derived precipitation (e.g., Global Precipitation Measurement–GPM) enables monitoring of rainfall amount and patterns in near real-time (Figure 1b), and precipitation maps can be derived for forecasting or study of landslide events such as hurricanes and Medicanes [18,19]. However, results from Medicane Ianos satellite-derived precipitation showed an inconsistency with ground station measurements, with significantly lower values measured from the satellite sensors [12]. This, in addition to the poor coverage of the affected area (mountainous western Thessaly) by ground stations does not permit us to recreate an accurate map of precipitation during Medicane Ianos. #### 2.1. Landslides in Thessaly #### 2.1.1. Geologic Setting Thessaly is the largest plain of central Greece surrounded by mountain ranges, most notably Pindos mountains in the west. The study area is part of the western Karditsa Prefecture and a smaller part of Evritania to the southwest. It includes Plastira dam lake at the center, and the high-elevation Agrafa Mountains (part of Pindos range) in the western part. The oldest post-alpine sediments in the area are the Molassic formations of the Mesohellenic trench [20,21]. The majority of the area is covered by the flysch formation of the Pindos geotectonic unit [21–23]. Flysch formation consists of alternations of shales, sandstones, and limestones that are highly susceptible to slope failures. Bedrock formations of the Alpine units also include limestones and cherts of Pindos, Koziakas and Sub-Pelagionian zones, while ophiolites can be found in the eastern and southwestern border of the area [21,23,24]. #### 2.1.2. Remote Sensing-Based Landslide Inventory To investigate and map the distribution of landslides in the Karditsa, Thessaly area, we used post-event acquisitions of Copernicus Sentinel-2 optical satellite imagery. Sentinel-2A/B multispectral imagery has a ground resolution of 10–60 m and covers wide swaths, thus enabling the rapid imaging of the affected area. Multiple Sentinel-2 frames were selected for the post-event period, and acquired 20, 25 and 30 September 2020, as large parts of the area were covered by clouds in the 20 and 25 September frames. Images acquired before the event, on 5, 10 and 15 September 2020, were used as references in order to visually identify and map landslides (Figures 2 and 3). The minimum size of landslides mapped was limited by the satellite imagery resolution (10 m for Sentinel-2), and consequently smaller-sized slides or rockfalls were not possible to identify and were not included. In addition, it was not feasible to classify most of the landslides into different types based on this remote sensing procedure. A small portion of slope failures was classified by the field surveys as presented in following section, validating the outcome of the statistical analysis presented in Figure 4. Figure 2. Example of manually mapping landslides within the study area using Sentinel-2 imagery. Pre- and post-event true color Sentinel-2 images (a,b) and an overlay (c) of mapped landslide points with red color (mostly headscarp points). Change detection MAD processing (d,e) assisted visual picking of headscarp points (f). Due to the small size of the majority of the identified landslides (most having a size of 2–4 pixels in Sentinel-2 imagery), landslide polygons were not digitized in this preliminary inventory. Digitizing was focused on marking the initiation point/headscarp (long avalanches or earthflows) or the approximate center of the landslide feature when the former was not possible due to small size and image resolution. For assistance during manual picking of landslides, we used additional analysis products, such as change analysis rasters (MAD-Multivariate Alteration Detection Transformation [25]) and multi-temporal single band RGB composites (Figure 2). Co-registration issues present between Sentinel-2 frames were resolved using GeFolki registration algorithm [26]. This preliminary inventory (Figure 3) includes 1697 landslides, limited to a narrow mountainous area of Karditsa and part of Evritania regions. Examination of Sentinel-2 imagery over the Pindos mountain region showed no or few sparse landslides, outside this area. The largest concentration of landslides was found around Amarantos village, southeast of Plastira lake, with up to 15 landslides per square kilometer (Figure 3). Figure 3. (a) Mapped landslides (black dots) triggered by Medicane Ianos during 17–19 September 2020, and simplified lithology of the area (see text for references). (b) Density map of Ianos landslides (using a radius of 1000 m); two major concentrations of landslides west of Lake Plastira and around Amarantos village. Figure 4. Statistical correlation of the spatial distribution of Ianos slope failures with: (a) elevation (histogram with area covered by each class in km<sup>2</sup> with a black line), (b) geology, (c) slope angles and (d) histogram of slope aspect values. #### 2.1.3. Statistical Analysis of Slope Failures The spatial distribution of the slope failures was correlated with topographic parameters e.g., slope angle, aspect and elevation and the geologic characteristics of the study area, as provided by the relevant 1:50,000 scale geological maps. In particular, information related to the spatial distribution of the geological formations acquired by the official geological map sheets in 1:50,000 scale available from the Institute of Geological and Mineral Exploration [27–32]. Afterwards, we combined those formations into a smaller number of generalized groups based on similar lithological features (Table 1). The topographic parameters were extracted from a digital elevation model (DEM) of 5 m resolution, provided by the Hellenic Cadastre. As shown in Figure 4, about 40% of the generated landslides were mapped on elevation ranging from 400 to 800 m; approximately 21% of the total number of landslides was documented from 400 to 600 m and 19% was reported on areas located between 600 and 800 m elevation. On higher elevation areas, the percent of slope failures was 13.91% between 800 and 1000 m, 15.26% between 1000 and 1200 m, and 10.49% for areas at elevation from 1200 and 1400 m. A lower percentage of slope failures has been identified at an elevation higher than 1400 m while a sharp decrease of landslides is related to areas located higher than 1800 m and lower than 400 m elevation. These statistics make sense as the areas located at high elevation are limited, while the latter outcome is due to the spatial distribution of geological units. More specifically, the lower parts of the study areas are mostly flat and covered by Quaternary sediments (loose clays, gravel and sand) and not by the flysch formation, which as it is demonstrated next is the predominant unit for landslide triggering among the geological units, along with slope gradient and relief. The largest part of the study area is covered by the flysch formation (Fo), a generally weak, complex, and of variable rock mass quality unit. Flysch is composed by different rhythmic alternations of competent/strong sandstone layers and is incompetent, of generally low-strength siltstone/clayey schist beds, while it is associated with intensive folding and fracturing. At the mountainous area, Cretaceous carbonates are mapped (K7-9.k), while Quaternary deposits (Q) are concentrated mostly in the basin of Thessaly. These three geological units cover almost 81% of the study area. In particular, the flysch and K7-9.k cover 63% and 9.41%, respectively, and the quaternary deposits 8.5%. As it was expected, most of the landslides, approximately 79%, were reported in the area that is geologically covered by the flysch formation, followed by the geological units of K7-9.k and K9.Pc with 5.6% and 4.37%, respectively. To facilitate the reader, only the geological affected by slope failures are plotted in the relevant diagram. Table 1. Description of geological formations (based on [26–31]). A strong correlation with landslide occurrence exists for the areas where the slope angle ranges between 25◦ and 40◦ (Figure 4c). In these areas, the identified cases of slope failures are 1167, which is approximately 69% of the total failures. On lower (20–25◦) and higher (40–45◦) slope angle areas, the reported landslides are 139 and 151, respectively and rapidly decrease for slope angles higher than 45◦ and lower than 20◦. The median slope angle of the landslides is approximately 32◦, which is in agreement with the one documented by [32] for the slope failures triggered by Hurricane Maria, Puerto Rico 2017. Analyzing the spatial distribution of slope failures with respect to the aspect of the slopes, a correlation between east-faced slopes and triggering of slope failures is observed (Figure 4d). In particular, considering the slopes with an aspect between 45◦ and 135◦, it was found that 587 (34.5%) cases are identified. A high number of failures (294) was additionally reported on slopes with aspects ranging from 135◦ to 180◦, while the lowest percentage of landslides (5.6%) was identified on areas oriented from 270◦ to 315◦. #### 2.1.4. Types of Landslides and Induced Phenomena Few days after the landfall and occurrence of Medicane Ianos, several teams of engineering geologists and engineers participated in field surveys aiming to map and document as many as possible slope failures, and validate the preliminary inventory produced based on remote sensing techniques [17]. This combination of desktop studies, remote sensing and field survey was also applied for documenting earthquake-induced secondary effects few kilometers to the east of the here study area, triggered by a seismic sequence, which occurred in March 2021 [33]. A total number of 73 landslides were documented during these field reconnaissance surveys in Karditsa Prefecture, which mainly caused partial or total damage to the roads and in some cases caused failures of riverbanks. This remote area is characterized by a combination of weak sedimentary sequences, high elevation deviations and steep slopes, as described in the previous section. The majority of remotely mapped landslides were found away from the local road network or in inaccessible areas, and thus the relative small number of field-surveyed landslides. The documented landslides were mostly found in flysch formations, validating the outcome of the remote sensing approach, as it was earlier presented. Following the information obtained by these surveys, the failure mechanisms are strongly related to the heterogeneity degree of the flysch formation (siltstone-sandstone participation), the intensity of tectonic disturbance and the weathering degree. In particular, the main types of landslides that were detected were: Based on the information obtained, the majority of landslides are classified as rotational (Figures 5 and 6), causing the most severe damage to the human-made environment in the wider investigated area. As it has been previously mentioned, these slides occurred mainly in the weathered flysch mantle and the siltstone flysch formation, due to their overall low rock mass strength. Figure 5. Types of landslides identified in the field reconnaissance surveys. Figure 6. Different lithology types affected by rotational and translational slides. Regarding the rotational landslides, most of them had a depth ranging between 10 and 15 m while a significant portion of failures was relatively shallow (<5 m). Their width ranges between 20 to 80 m, while the smaller slides have a width between 5 and 20 m. The width of the largest landslides was up to 200 m. Debris flows in most cases affected road sections with limited width, between 10 and 20 m, except a few cases where the affected width is almost 300 m. The most severe landslide-induced failures in Karditsa Prefecture were around Amarantos village, located to the south-east of Plastira lake. In Figure 7a, a landslide that occurred in siltstone flysch causing the road failure is shown. In particular, the road was totally covered by slipped material across a length of 200 m, while its depth was estimated at about 15 m. Its height is approximately 70 m. The slide failure plane was probably constrained by the presence of a deeper stronger flysch layer downslope. In Figure 7b, a rotational slide in the weathered mantle of siltstone flysch is also shown. This landslide resulted in the collapse of the road pavement for a length of 10 m. Figure 7. Characteristic rotational failures around Amarantos area. (a) large failure in siltstone flysch west of Itamos village and (b) failure in weathered flysch mantle material causing damage to the road. Extensive debris flows were mapped in Livadia and between the communities of Pefkofito and Vlassi (Figure 8). Specifically, the thickness of the debris at the road level is 5 m while the impacted area is 300 m long in the former case. The debris flow material mainly consisted of limestone fragments and clay materials (Figure 8a). Figure 8. (a) Characteristic debris flow at Livadia village; (b) extensive debris flows in gullies in the area between Pefkofito and Vlassi. Regarding the second case, located in the area between Pefkofito and Vlassi (Figure 8b), it should be pointed out that this area is characterized by steep morphology while the extensive amounts of debris have been transported via deep gullies from higher elevations. Moreover, the erosion and flooding of the Karitsiotis River damaged and blocked the road crossing south of Belokomiti village, Plastira Lake, as shown in Figure 9. Although the bridge itself did not collapse, road embankments on both sides were partially or completely washed out. The intersection of the roads west of the bridge was the most damaged part, blocking road traffic towards communities to the south and west of Lake Plastira. Riverbank erosion also caused several slope failures, while the flooding of the river caused slope undercutting that resulted in road failures (Figure 9). Figure 9. Damaged bridge crossing of Karitsiotis River near Belokomiti. Ground (a) and vertical aerial (b) images of the damaged bridge undergoing repairs on 1 October 2020. UAS orthophoto (c) and digital surface model (d) of the Karitsiotis River bridge area (surveys 1 October 2020). #### 3. Landslides in Cephalonia Island #### 3.1. Geologic Setting Cephalonia is part of the Ionian Islands and is located at the westernmost part of Greece. Geologically, the island is within the outermost edge of the ongoing subduction of the African plate under the Eurasian plate [34]. The bedrock of the island consists of two main formations: (i) the Pre-Apulian unit, which covers most of the island and consists mainly of a thick sequence of limestone and dolomite, overlain by a much thinner sequence of marl and pelite, and (ii) the Ionian unit, which covers part of the southeastern coastal areas and consists of limestone, shale, and breccia [35–37]. The basement is overlain by extensive sedimentary deposits in the southern and eastern part of the Paliki peninsula and the south part of the main island. These deposits consist of alluvial fans, mainly deposited along the stream channels (Figure 10). The dominance of carbonate rocks in combination with favorable climate conditions have facilitated the formation of karst units especially in the north and central part of the island. In particular, the Erissos Peninsula is dominated by an extensive, and partially karstified, planation surface. Cephalonia island is also characterized by steep slopes, especially along its western shoreline. Ridges are arranged in a NNW–SSE direction. The principal water divide has a NW–SE direction, with the main flow directions towards Sami Bay in the NE, and Poros in the SW. Figure 10. Lithological map of Cephalonia Island with observed landslides and related phenomena following Medicane Ianos. Modified from [35,37]. #### 3.2. Types of Landslides and Induced Phenomena Cephalonia Island was heavily impacted by Medicane Ianos. Ianos was the most damaging natural hazard event for Cephalonia island, since the earthquakes of 2014 that caused building damages, widespread landslides, rockfalls and liquefaction over the island [38–41]. Severe rainfall at Cephalonia peaked at 759 mm (17–18 September 2020) during the passing of Medicane Ianos from the Ionian Sea islands [12]. In particular, the highest severity of the Medicane-induced phenomena was reported at the northern-central part of the island towards Erissos peninsula, between Sami and Fiskardo. Debris flows and landslides occurred in several locations across the island (Figures 10 and 11). Often the debris flows covered wide areas (~40–70 m in width), while typically the average size of debris blocks reached 20–50 cm (Figure 11d). Moreover, the earth flows were often associated with road embankment failures (Figure 11c). The latter was also observed in cases of severe riverbank erosion/scour. The road network of the island was damaged and/or obstructed in several locations due to landslides, while a critical simple-span reinforced concrete bridge near the village of Agkonas collapsed due to scour of the west pier foundation by extensive gully flooding and debris (Figure 11e), heavily impacting the transportation network of the island. Figure 11. Characteristic damages in Cephalonia Island following Medicane Ianos: (a) Riverbank erosion, (b) debris flow within a rural community, (c) road embankment failure, (d) extensive debris flow, and (e) bridge collapse. Debris flows appeared to be the most common feature of the effect of Medicane Ianos across Cephalonia. Debris, collected or still in place, was present along gullies and riverbeds, across the island. Most notably, Assos village, situated in the north-west of the island, was severely impacted by a major debris flow, as it was covered by approximately 1.5 m of earth/debris material (Figure 11b). The source of the debris was traced to the hills east of the village, where significant surface erosion and ground cracking were observed. At places, eroded zones reached several meters in width. Eroded limestone boulders and residual soil material were present along the entire path of the flow. UAS-enabled mapping of the entire area indicates the change along the sea-shore due to material deposition (Figure 12). An initial, rough, estimation of the debris flow volume is 20,000 m3. Figure 12. UAS-enabled mapping of Assos village debris flow and surrounding area (3D model can be viewed at: https://skfb.ly/6WyTT (accessed on 2 July 2022)): (a) orthophotomap and (b) digital surface model (surveyed on 30 September 2020). Similar to Assos, just outside of the village of Fiskardo (Figure 13), which is situated at the northern part of Erissos peninsula, a debris flow was documented. The debris flow had a lesser extent than the one that occurred in Assos, but was still significant, with the flow reaching a run-out distance of approximately 400 m. Significant erosion features were observed near the source of the debris flow. Eroded zones reached, at places, a depth of 2 m. The debris flow is also visible in Sentinel-2 satellite imagery, as shown in the preand-post-event images (Figure 13). Moreover, several rotational landslides were identified along the steep central coastline, particularly near Myrtos beach. Sentinel-2 pre-and-post event images of landslides and debris flows near Myrtos beach, and the collapsed bridge at Agkonas village are shown in Figure 14. Figure 13. (Left): Sentinel-2 multi-temporal composite (Band 4) Fiskardo, Cephalonia. (Right): Sentinel-2 true color composite (Bands 4-3-2). Red colors mark the location of debris flows upstream of Fiskardo. Figure 14. Sentinel-2 multi-temporal composites (Band 4) of central Cephalonia. (Left): Mirtos beach area; (Right): Site of collapsed bridge on the E.O. Argostoliou-Fiskardou (38.3195◦ N, 20.5081◦ E). Red colors mark the location of debris flows, landslides and deposition of debris along the coast. #### 4. Discussion #### 4.1. Comparison with Automated Mapping Methods Rapid response to a major landslide event, such as hurricanes or earthquakes, is nowadays feasible due to the abundance of open remote sensing data available shortly after an event, from satellite imagery (e.g., Sentinel-1 & Sentinel-2, Landsat 8/9). The first satellite images available during or shortly after a meteorological event can provide a fast and comprehensive map of significant slides and effects, as described in the previous section. During the period that follows first response and early reconstruction, there is the need for more thorough and detailed landslide inventories. While this can be performed by manually digitizing landslides and effects using very high-resolution satellite imagery or other remote sensing data such as UAS surveys, new tools and a wealth of open satellite data can automate this workflow [42]. The ability to have access and process huge amounts of satellite imagery on the cloud through Google Earth Engine [43] and similar platforms has revolutionized remote sensing in geohazard response and analysis. We present a comparison of our manual rapid mapping using Sentinel-2 images, with a series of recent workflows and codes that use the multi-temporal analysis of satellite imagery in Google Earth Engine [44–47]. While this comparison is not straightforward due to the different workflow, data and time frame used by either rapid manual mapping or automated multi-temporal analysis, this is an interesting case study to compare them. The wide extent and large number of landslides in the area of Karditsa, Thessaly is suitable for this use and comparison (Figure 15). We selected four change detection techniques, that use multi-temporal analysis of open satellite data (Sentinel-1, Sentinel-2, Landsat); (a) method from [46] that calculates relative difference in the normalized difference vegetation index (rdNDVI) calculated from cloudfree composites of Sentinel-2, (b) HazMapper code by [44] that is based on the normalized difference vegetation index (dNDVI) calculated from cloud-free composites of Sentinel-2 [48], (c) ALDI, automated landslide detection index algorithm based on normalized difference vegetation index (NDVI) differencing of Landsat time series within Google Earth Engine taking into account seasonality [47] and (d) a SAR backscatter and amplitude change approach that uses multi-temporal stacks of Copernicus Sentinel-1 images [45]. Figure 15. Change detection results from HazMapper [44,48] utilizing multi-temporal Sentinel-2 imagery. Red colors show negative rdNDVI changes that correspond to land cover changes due to landslides and debris flows. Results were in par with manual mapping in the area of high-density landslides southeast of Plastira Lake (a), while in most areas change detection was more successful in depicting the major debris flows such as the ones upstream of Mouzakiotikos River (b). Treshold values for positive and negative landslide and debris flow identification was determined for each method results based on proposed values and also local conditions after cross comparing with mapped landslides and imagery (Table 2 and Figure S2). Examining the different change detection results we obtained from these four methods, we can observe that all methods captured the large debris flows that occurred along the mountainous watersheds such as Mouzakiotikos river (Figure 15) and Megalos river in Filakti, near Lake Plastira (Figure 16). Large landslides with significant dimensions and/or long run-out distance were also identified. As the majority of landslides triggered during Medicane Ianos were of small dimensions (less than 20–30 m), these were mostly missed or were undifferentiated from the background scatter noise. Table 2. Positive and negative detection of mapped landslides by the different change detection methods. A quick estimate of the positive or negative detection was performed by extracting pixel values on the mapped landslide inventory from the various methods. Table 2 presents the final percentage and number of positively and negatively detected landslides. Histograms of the detection results of Table 2 are presented in Figure 17. The highest success ratio is achieved by the methods of [44,46] with almost comparable results, as expected due to the similarities in the calculated change detection data (Sentinel-2 10 m) and parameters (time-series of normalized difference vegetation index). The worst percentage was achieved by ALDI [47], probably due to the lower resolution data used (Landsat multispectral bands with 30 m resolution instead of 10 m for Sentinel-2). SAR backscatter change [45] was close to the first two method results, with the difference attributed to the coarser pixel resolution of Sentinel-1 SAR imagery (20 × 4 m, translated into >15 m ground resolution when geometrically corrected to terrain) and the challenging oblique scanning of SAR satellites that can lead to terrain shadows. Figure 16. Debris flow and landslides along Megalos River in Filakti/Pezoula area (a). 3D view of the Megalos River watershed with debris flows along the main river course and its exit to Lake Plastira (red colors of ALDI change detection index values). Flooding, severe sedimentation and material transportation from upstream was mapped at its exit (photo (b), view towards east with location marked in (a)) and near Pezoula. Road bridges were destroyed in both locations, with positions labeled in (a). Figure 17. Histograms of pixel values by automated mapping methods corresponding to mapped landslides; (a) [46], (b) HazMapper by [44], (c) ALDI [47] and (d) SAR backscatter change by [45]. Yellow bars mark negative detections and blue bars positive detection, based on set threshold values for each method. In conclusion, rapid manual mapping of landslides for Ianos outperformed automated mapping methods based on Google Earth Engine time-series change detection of satellite imagery. The most prominent factor was the small size of the majority of landslides, which was barely close to the ground resolution threshold of 10 m for Sentinel-2, making it harder to identify by automated change detection algorithms. A large number of false positives were visually identified in the various change detection results, a factor that could negatively affect early response and mapping (Figures S1 and S2). Debris flow and large slides were identified in most automated mapping results (Figure S1), while smaller and shallow landslides were difficult to positively identify due to variations in image reflectance and terrain shadows. Surface manifestation of small shallow landslides kept being modified in the days and weeks following the landslide event, due to erosion and vegetation regrowth, a factor that affects the signal in the long time-series used by automated change detection methods. It is worth mentioning that a number of landslides occurred in the winter months following the Ianos landslide event. These landslides would be included in most automated change detection results due to the larger time frame examined. #### 4.2. Older Landslides/Hazard The mountainous area of Karditsa, Thessaly is known to be significantly affected by numerous landslides, many affecting communities and infrastructure [44]. The Agrafa mountain area, where the majority of landslides triggered by Medicane Ianos occurred, is one of the most hazardous areas in Greece for landslides. Landslide occurrence is mostly due to the factors of highly susceptible lithology (flysch and molassic sediments) and high precipitation rates of the Pindos mountain range. We compare the landslide inventory of Medicane Ianos with historically reported landslide locations in Figure 18. Historical locations were retrieved from [49,50] and official reports of the Institute of Geological and Mineral Exploration. A visual examination shows that Ianos landslides occurred along roughly the same locations of historical landslides, revealing a relation with long-term climatic and lithological/geomorphological conditions. Figure 18. Comparison of (a) Medicane Ianos triggered landslides (black dots) and (b) historical occurrences of landslides in the region (purple dots). #### 5. Conclusions Medicane Ianos in September 2020 caused one of the most widespread catastrophic events for Greece in the last few decades. While most of the areas affected were already susceptible to geohazard occurrences, the areal extent and scale of the almost simultaneous (within three days) occurrence of failures and damage over a large area, represent a unique challenge for future response and mitigation. The area of western Thessaly was the hardest hit by an unprecedent number of landslides and debris flows in the mountains, while the low plains were covered by 475.5 km2 of flood waters [14,17]. We examined in detail two of the most affected areas, Cephalonia island in the Ionian Sea and Karditsa area in Thessaly, using early remote sensing data and post-event field surveys. Karditsa suffered more than 1500 landsides and numerous debris flows that caused widespread damage and disruption. Cephalonia Island did not experience the number of landsides of western Thessaly, but was severely hit by landslides and debris flows in key location and settlements, destroying bridges and disrupting transportation within the island. Assos settlement was nearly buried by a major debris flow. A dominant factor in these effects was the heavy precipitation experienced by those two areas during Medicane Ianos, in combination with local conditions highly susceptible to mass wasting. The landside inventory acquired for Karditsa region is the first detailed inventory due to a major atmospheric event in Greece and a valuable asset for studying and mitigating future Medicane events in mainland Greece and surrounding regions. The detailed distribution of landslides can be studied along with meteorological and atmospheric observations, with possible implementation in the hurricane impact model development and forecasting. As more data sources (satellite imagery) and tools (UAS sensors and platforms, rapid remote sensing big data processing, etc.) will be available to scientists in the near future, response and rapid mapping during future landslide events will be more thorough and faster, based on the experience that data collection from events such as Medicane Ianos provide. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/app122312443/s1, Figure S1: Comparison of field surveyed landslides with automatic satellite mapping results (a–g). Left: field photos (acquired 1–3 October 2020); right; location map with automatic satellite mapping results (HazMapper). Blue dots show mapped landslides from Sentinel-2 images and triangles with letter marking field locations of surveyed landslides. Location map for sites a–g (h) with Sentinel-2 landslides (orange), Figure S2: Automatic satellite mapping results. Left: method results; middle: method results with mapped landslides (blue dots) and field survey locations (orange triangles); right: Sentinel-2 true color image, acquired on 30 September 2020. Author Contributions: Conceptualization, S.V. and G.P.; methodology, S.V. and G.P.; validation, S.V., G.P., V.M., C.S., V.K., E.K., I.F., G.Z., J.M. and O.-J.K.; formal analysis, S.V. and G.P.; investigation, S.V., G.P., V.M., C.S., V.K., E.K., I.F., G.Z., J.M. and O.-J.K.; data curation, S.V. and G.P.; writing—original draft preparation, S.V., G.P. and O.-J.K.; writing—review and editing, S.V., G.P., D.Z., G.Z. and O.-J.K.; visualization, S.V. and G.Z.; supervision, G.P., V.M., C.S., D.Z. and G.Z.; project administration, D.Z. and G.Z.; funding acquisition, D.Z. All authors have read and agreed to the published version of the manuscript. Funding: Part of this research (Geotechnical Extreme Events Reconnaissance-GEER Association work) was funded by the National Science Foundation through the Geotechnical Engineering Program under Grant No. CMMI-1826118. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: A detailed description of the field surveys and reconnaissance after Medicane Ianos can be found in the Geotechnical Extreme Events Reconnaissance Report https: //doi.org/10.18118/G6MT1T (accessed on 3 July 2022) and the companion web map: https:// elxisgroup.com/GEER-MedicaneIanos-FieldMap/ (accessed on 3 July 2022). Acknowledgments: We would like to thank Athanassios Ganas, Charalambos Kolovos and Vivi Diamantopoulou for discussions and assistance in the field surveys. Conflicts of Interest: The authors declare no conflict of interest. #### References MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com Applied Sciences Editorial Office E-mail: [email protected] www.mdpi.com/journal/applsci MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel: +41 61 683 77 34 www.mdpi.com	doab	2025-04-07T03:56:58.028342	7-3-2023 17:35	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001e4a90-6b26-4650-bf99-81d98b68a57f", "url": "https://mdpi.com/books/pdfview/book/6898", "author": "", "title": "Mapping, Monitoring and Assessing Disasters", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036565392", "section_idx": 1 }
001fcd9a-cd27-45e8-b23f-f447874e0974.0	Indoor Air Quality • Pasquale Avino and Gaetano Settimo	doab	2025-04-07T03:56:58.076460	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 0 }
001fcd9a-cd27-45e8-b23f-f447874e0974.1	Indoor Air Quality From Sampling to Risk Assessment in the Light of New Legislations Printed Edition of the Special Issue Published in Atmosphere Pasquale Avino and Gaetano Settimo Edited by www.mdpi.com/journal/atmosphere	doab	2025-04-07T03:56:58.076543	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 1 }
001fcd9a-cd27-45e8-b23f-f447874e0974.2	Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations	doab	2025-04-07T03:56:58.076589	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 2 }
001fcd9a-cd27-45e8-b23f-f447874e0974.3	Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations Editors Pasquale Avino Gaetano Settimo MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors Pasquale Avino University of Molise Italy Gaetano Settimo Istituto Superiore di Sanita` Italy Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Atmosphere (ISSN 2073-4433) (available at: https://www.mdpi.com/journal/atmosphere/special issues/indoor airquality). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Volume Number, Page Range. ISBN 978-3-0365-0946-4 (Hbk) ISBN 978-3-0365-0947-1 (PDF) Cover image courtesy of Pasquale Avino and Gaetano Settimo. © 2021 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.	doab	2025-04-07T03:56:58.076614	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 3 }
001fcd9a-cd27-45e8-b23f-f447874e0974.5	Kanittha Pamonpol, Thanita Areerob and Kritana Prueksakorn Indoor Air Quality Improvement by Simple Ventilated Practice and Sansevieria Trifasciata Reprinted from: Atmosphere 2020, 11, 271, doi:10.3390/atmos11030271 ................ 175 Tareq Hussein, Ali Alameer, Omar Jaghbeir, Kolthoum Albeitshaweesh, Mazen Malkawi, Brandon E. Boor, Antti Joonas Koivisto, Jakob L ¨ondahl, Osama Alrifai and Afnan Al-Hunaiti Indoor Particle Concentrations, Size Distributions, and Exposures in Middle Eastern Microenvironments Reprinted from: Atmosphere 2020, 11, 41, doi:10.3390/atmos11010041 ................ 191	doab	2025-04-07T03:56:58.076726	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 5 }
001fcd9a-cd27-45e8-b23f-f447874e0974.6	About the Editors Pasquale Avino is Associate Professor at the University of Molise in the field of analytical chemistry and environmental chemistry. After obtaining his doctorate in Chemical Sciences at the University of Rome "La Sapienza", he completed his professional training at the University of California, Irvine (USA), with Prof. F.S. Rowland (Nobel Prize in Chemistry in 1995) where he was interested in air pollution and related problems connected to sampling in remote areas (the South–East area of the Pacific Ocean) and at high altitudes (mountains and transoceanic air flights). He is the author or co-author of more than 160 peer-reviewed papers according to the Scopus database; he has coordinated several research projects in the environmental sector. He works in the context of regulatory proposals on air pollution for the transposition of EU directives on air quality by actively participating in national commissions on this issue. He is a member of several scientific associations/societies. For his studies, he received the NASA Award and the Sapio National "Environment and Health" Award. Gaetano Settimo is a researcher on air pollution (indoor and outdoor) at the Italian National Institute of Health (ISS). His degree is in Industrial Chemistry and he has a Ph.D. in Environmental Engineering. His working experience has been carried out in the area of environmental research, with a particular focus on the impact of chemical pollution on the environment and human health, and on the evaluation of exposure and risk assessment. This experience also includes the study and implementation of analytical methods for measuring certain pollutants that may occur in industrial and urban areas, and the evaluation of emissive sources and possible mitigation measures. He is in charge of various research groups and is currently coordinator for the National Study Group on Indoor Pollution established by the ISS. He belongs to various ministerial working parties regarding risks linked to the Environment and Health.	doab	2025-04-07T03:56:58.076784	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 6 }
001fcd9a-cd27-45e8-b23f-f447874e0974.7	Preface to "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations"	doab	2025-04-07T03:56:58.076962	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 7 }
001fcd9a-cd27-45e8-b23f-f447874e0974.8	1. Introduction Indoor Air Quality (IAQ) issues have been known to professionals for a long time but only recently has it found growing attention from European and national legislators. For the World Health Organization (WHO), it represents one of the main public health problems that must be addressed due to the impact it has on the population [1]. Some important milestones have been reached as regards the quality and role of sources, human activities and the often negative effects of the wrong interventions of energy saving measures adopted in buildings. In Europe (EU), in the light of what is historically happening in this period, great attention is paid to the IAQ issue, and the member states of the EU are at the forefront in adopting a series of improvement organic actions on several levels in promotion of prevention in the IAQ field, and training of operators who are responsible for the hygienic and health protection of the displays [2]. ### 2. Summary of This Special Issue Pending a community framework law for IAQ that considers the WHO guidelines, this Special Issue aims to make a concrete technical contribution to the solution of the various problems related to indoor air pollution. In 11 papers (nine regular papers and two reviews), international scientists report the most recent findings in this field from different points of view, including topics such as IAQ legislation [3], IAQ role in schools [4], hospitals and (micro)environments in general [5-9], the performance of an olfactometer system [10] or the impact of indoor malodor [11], BTEX measures in a fire station [12] and a chemical characterization of e-cigarette (e-cig) refill liquids (e-liq) [13]. IAQ is determined by a set of the presence of important sources of pollution, such as construction and insulation materials, furniture, furnishings, consumer products such as detergents, air fresheners, scented candles, diffuser liquids and electricals including incense sticks, maintenance and cleaning methods, etc. [14–17]. The polluting emissions from the sources depend very much on the poor quality of the raw materials used in the production [18], on the improper or incorrect use, on the state of conservation and maintenance, on the age and on the microclimatic factors [19]. Recently, the European legislators have intensified efforts to reduce and improve the quality of emissions from materials, with the study and development of standardized test methods and with the definition of common criteria for the labeling of emissions of materials. At the same time, the WHO has put a point of the IAQ guidelines [1], relating to a certain number of pollutants, present in indoor environments, for which the scientific knowledge relating to the effects on humans has been judged robust. For pollutants with carcinogenic action, a unitary risk is defined for the general population associated with their presence in the air. The substances considered are benzene, nitrogen dioxide, polycyclic aromatic hydrocarbons (especially benzo[a]pyrene), naphthalene, carbon monoxide, radon, trichlorethylene and tetrachlorethylene. Alongside these guidelines, we must remember those concerning the risks associated with the presence of humidity and biological agents. The WHO guidelines form a basis for establishing the relevant legislative standards (limits) adopted in the EU by various countries and must also be subjected to periodic review by the competent WHO office. The guide values of pollutants in the air are very low compared to the considerable number of pollutants found in indoor environments. Within the community, this has also contributed to the implementation of IAQ monitoring campaigns for the purpose of environmental and sanitation assessments as part of specific programs. In the European context, the activity of the WHO has tried to urge the various European countries with the aim of directing a series of legislative actions; this has led to the issuing of specific acts both on the fundamental role of guiding/reference values for some pollutants of particular hygienic-sanitary interest, and on the mandatory labeling of the emission levels produced by the materials previously with the aim of avoiding risks for health of the general population. Even the EU, while reaffirming the priority of energy efficiency measures, recommends greater healthiness of indoor environments and the development of a specific European strategy on the IAQ topic [20]. In some EU countries, legislative acts on IAQ have been drawn up that, if properly used, can allow a better assessment of the exposure of the general population and the related health risks [3]. #### 3. Conclusions Following the trend, now consolidated in several countries, it seems appropriate to encourage the development of reference values or specific action values in order to better manage particularly problematic situations in these environments. In the absence of national references to be used for a comparison, it is possible to use those reported in the legislation of other European countries or, by ad hoc working groups or by analogy, to other standards such as those relating to ambient air. Author Contributions: P.A. and G.S. contributed equally to the concept, creation and editing of this editorial. Acknowledgments: The authors would like to thank all the contributors to this Special Issue, as well as the Editorial team of Atmosphere. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:58.076987	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 8 }
001fcd9a-cd27-45e8-b23f-f447874e0974.10	Review Indoor Air Quality: A Focus on the European Legislation and State-of-the-Art Research in Italy Gaetano Settimo 1, Maurizio Manigrasso <sup>2</sup> and Pasquale Avino 3,\* Received: 1 March 2020; Accepted: 8 April 2020; Published: 10 April 2020 Abstract: The World Health Organization (WHO) has always stressed the importance of indoor air quality (IAQ) and the potential danger of pollutants emitted from indoor sources; thus, it has become one of the main determinants for health. In recent years, reference documents and guidelines have been produced on many pollutants in order to: i) decrease their impact on human health (as well as the number of pollutants present in indoor environments), and ii) regulate the relevant levels of chemicals that can be emitted from the various materials. The aim of this paper is to discuss and compare the different legislations present in the European Union (EU). Furthermore, a focus of this paper will be dedicated at Italian legislation, where there is currently no specific reference to IAQ. Although initiatives in the pre-regulatory sector have multiplied, a comprehensive and integrated policy on the issue is lacking. Pending framework law for indoor air quality, which takes into account WHO indications, the National Study Group (GdS) on Indoor Air Pollution by the Italian Institute of Health (IIS) is committed to providing shared technical-scientific documents in order to allow actions harmonized at a national level. An outlook of the main Italian papers published during these last five years will be reported and discussed. Keywords: indoor air quality; legislation; Europe; focus; residential; pollutants; TLV; health; workers; school	doab	2025-04-07T03:56:58.077404	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 10 }
001fcd9a-cd27-45e8-b23f-f447874e0974.11	1. Introduction Indoor air quality (IAQ) has been a well-known problem since the late 1970s. Its significant impact on human health has been addressed several times by the World Health Organization (WHO) in various documents and meetings, and has been carried out at various levels [1–3]. Further, economic studies and researches have highlighted the great importance that IAQ now has in all environments, e.g., houses, schools, banks, post offices, offices, hospitals, and public transport, just to name a few [4]. IAQ also has strong repercussions in the competitiveness of an organization, considering the increase in difficulty in carrying out its job in the best way, its performance, and the social and economic competitiveness between countries, due to the influence on the attention, degree, and number of days lost [5]. Scientific literature contains large documentation in terms of articles, conference papers, reviews, books, editorials, letters, and public articles on chemical contaminants in indoor environments. A search on the Scopus literature database, using the keyword "indoor air quality", led to a total of 7287 publications between 2000 and 2020 in the European Union (EU) (search executed on 19 January 2020), including Norway, Switzerland, and Turkey. According to this search, Italy and the United Kingdom (UK) are major contributors to this total amount of European publications, 12.3% and 10.4% of the total, respectively, followed by France and Germany with 9.5% and 9.1%. Figure 1 shows the relative percentage contribution of each Member State of the European Union (EU) including Norway, Switzerland, Turkey, as well as United Kingdom, which is expected to leave the EU on 31 January 2021. The United Kingdom, Italy, France, and Germany contribute more than 41% of the total amount of publications in the European IAQ field. Figure 1. Percentage of country contributions to the total amount of publications on indoor air quality (IAQ) in Europe from 2000 to 2020. (Source: Scopus, search: 19 January 2020); includes Norway, Switzerland, Turkey, and the UK, which is to be expected to leave UE on 31 January 2021. This continuous and growing attention on IAQ has evidenced, over time, the need for a profound cultural change, according to WHO indications, in order to develop organic health prevention and promotion actions, and cope with the complexity of such an issue. Noteworthy, at the European Community level, the resolution of 13 March 2019, defends clean air for everyone and highlights that people spend almost 90% of their time in indoors [6]. In these environments, the air can be significantly more polluted compared to outside [7] and, therefore, considered mandatory to issue indoor air quality certificates for both new and old buildings. From this perspective, it urges member states to adopt and implement measures to combat air pollution at the source. There are specific cases, such as schools, healthcare, or office environments, where the permanence of workers (e.g., medical, administrative, teaching, and non-teaching staff) is supposed to last for a relatively long period, and where "users", as well, are present (e.g., patients, students, vulnerable and/or fragile subpopulation, some of which with physical and psychological disabilities, etc.). In these situations, it is essential to consider the very close relationships between the various work activities and the quality of the building structure, finish, furnishings, and the degree of crowding of such environments. This includes the presence of technological systems or interventions for energy purposes only, without forgetting the ventilation needs of the environment for aspects related to health, performance, and staff and student performance [8]. The combination of these actions is fundamental for developing and implementing plans for the protection and promotion of health safety for citizens and workers [9]. This represents the priority and the common objectives of both national and European prevention plans (National Prevention Plans (NPPs) and programs from the United Nations (UN) Sustainability Development Agenda). Nonetheless, several European countries have had to overcome the absence of specific legislation, or legislative acts already developed, due to generic definitions of the characteristics of air quality. For instance, in closed workplaces, such as closed offices where employees have individual working areas that are distinctly divided—either by walls, cubicles, or panels—it is necessary to ensure that workers have healthy air in sufficient quantity, which is also obtained with ventilation systems [10]. It is necessary for updated laws and regulations to be adopted to improve the indoor air quality. Another fundamental requirement for correct understanding of the air quality pollution phenomena indoors is the availability of reliable (and systematically collected) information, according to well-established protocols, on the quality, quantity, and origin of the pollutants. In this regard, particular attention should be addressed to the activities of the European Committee for Standardization (CEN) and the International Organization for Standardization (ISO), which provide a series of specific indications on the operating procedures with which to carry out the checks. In recent years, several international organizations, e.g., the European Collaborative Action (ECA), the World Health Organization (WHO), and the International Agency for Research on Cancer (IARC) have produced reference documents, guidelines, agreements, and protocols. For example, the Parma declaration, the Children's Environment and Health Action Plan for Europe CEHAPE), European Union (EU) regulations (e.g., regulation 305/2011, which lays down harmonized conditions for the marketing of construction products); documents, and rules for characterization and determination on many pollutants (e.g., European Standards (EN) ISO 16000—Indoor air quality, European technical specification (CEN/TS) 16516: construction products—determination of emission into indoor air). The purpose of this documentation is to tend to the decrease in the number of pollutants present in indoor environments and to regulate the levels of chemicals that can be emitted from different materials, in order to contain the negative impacts on IAQ. In particular, the activities carried out by the European Committee for Standardization (CEN) and the International Organization for Standardization (ISO) represent important references, because harmonized methods of detection allow for better comparison between the different indoor air quality data produced at the European level. Such methods should be implemented by laboratories that carry out environmental surveys. Within this context, the aim of this paper is to summarize the entire legislation on IAQ present in the EU (the UK included), in February 2020, along with reference values, guide values, and unitary risks for many kinds of indoor air pollutants present. Particularly, the foundations of the different legislations will be compared for evidencing the main characteristics of each one, and the levels of the main pollutants will be presented and discussed. The focus is to highlight the strengths and weaknesses to deal with this important topic. According to the authors' knowledge, this is the first critical revision of the European legislation. Further, a section will be dedicated to the state-of-the-art research in Italy, from a legislative and scientific point of view. Although there have been many scientific papers and studies performed on IAQ, and a methodic and analytical review of the papers published in the last five years concerning the indoor field will be documented, it will be highlighted that, in Italy, the main problem is the lack of reference standards for residential indoor air quality.	doab	2025-04-07T03:56:58.077538	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 11 }
001fcd9a-cd27-45e8-b23f-f447874e0974.12	2. The Main European Legislation on Indoor Air Quality The WHO has developed guidelines for IAQ, relating to a certain number of pollutants, present indoor, for which scientific knowledge relating to human health effects were considered robust enough. The substances considered are benzene (C6H6, CAS number 71-43-2), nitrogen dioxide (NO2, 10102-44-0), polycyclic aromatic hydrocarbons (especially benzo[a]pyrene BaP, C20H12, 50-32-8) (PAHs), naphthalene (C10H8, 91-20-3), carbon monoxide (CO, 630-08-0), radon, trichlorethylene (C2HCl3, 79-01-6), and tetrachloroethylene (C2Cl4, 127-18-4). For carcinogenic pollutants (such as benzene, BaP, trichloroethylene), a unitary risk (UR) is defined for the general population associated with their presence in the air. Alongside these guidelines, mention should be made of those relating to the risks associated with the presence of humidity and biological agents. Furthermore, for the purpose of risk assessment, it is of particular importance to consider not only the guide value or reference parameter, but also other fundamental elements, such as the vulnerability of the population and the exposure conditions. There is no specific reference directive on IAQ in European legislation, although pre-legislative initiatives have multiplied over the years. For example, indoor air quality and its impact on human activities within the European Collaborative Action (ECA), e.g., Urban Air, Indoor Environment, and Human Exposure, as well as funded studies, EN standards, etc.); however, to date, there is still no integrated policy on indoor air quality in all of those indoor places. Some EU Member States, such as France, Portugal, Finland, Austria, Belgium, Germany, the Netherlands, and Lithuania, have started, through a series of actions, to adopt specific guide values, reference values, and action values for IAQ—in some cases enforced in the legislative acts of these countries. These actions can be summarized as follows: One particular aspect is to give indications for the IAQ evaluation at workplaces other than industrial ones. Currently, in order to evaluate the IAQ in environments where work is carried out (e.g., in offices, schools, hospitals, banks, post offices, etc.), the occupational exposure limit values (OELs) present in the regulations, or the threshold limit values (TLVs) of the American Conference of Governmental Industrial Hygienists (ACGIH), or the Scientific Committee For Occupational Exposure Limits (SCOEL-RAC) are used—albeit reduced by 1/10 or 1/100. This approach is overcome, as indicated, by specific documents elaborated by different national and European working groups on the indoor topic [11–14]. Such recommendations, as given by the WHO in the early 1980s in the document "Indoor air pollutants exposure and health effects" [15], reported that it was incorrect to use the industrial occupational exposure limit values for non-industrial indoor environments, and that for such environments, it was necessary to develop specific references. It should be remembered that these values represent the parameters to which references must be made for an assessment of the inhalation risk of workers and the population. They are not the only ones, because specific exposure and vulnerability conditions are fundamental elements to be considered for a correct risk assessment. In the document "Opinion on risk assessment on indoor air quality" [16], the Scientific Committee on Health and Environmental Risks (SCHER) of the European Commission recommends that risk assessment should always be focused on the most vulnerable groups, represented by children, pregnant women, elderly people (over 65), people suffering from asthma, and other respiratory and cardiovascular diseases, following a case-by-case approach. In fact, for groups of particularly sensitive and vulnerable individuals, who are potentially being exposed to the risk factors under consideration, the problem of the simultaneous presence of multiple risk factors may require the need to carry out specific in-depth assessments, which must be based on adequate knowledge of the context. In fact, it should be remembered that the reference values for confined spaces are more severe than the corresponding values in industrial environments (TLVs) whose hygienic-sanitary references are based on a working life of 8 h a day, 5 days a week, for a maximum period of 40 years, and are aimed at protecting workers against occupational diseases. In this context, the efforts carried out by bodies such as ISO and CEN, which have long been involved in the development of the specific standard "EN ISO 16000: Indoor air" [17], which describes the procedures for performing sampling activities and analyzes the main pollutants indoors, should not be forgotten. The adoption of these rules constitutes a significant improvement compared to what has been achieved so far in the study and control activities. The standardization of the methods also increases the possibility of a correct comparison between the different indoor air quality data produced at the European level [11]. The advantage is, also, in terms of the possibility of the correct comparison between the various IAQ data produced at the European level, underlining the need for timely application of the rules. This is particular so for the sampling phase (e.g., choice of the sampling point and height, distance from walls, preliminary activities, etc.), which represents the beginning of the control procedure and, therefore, conditions the final result. Table 1 shows the 40 parts of the ISO 16000 standard [17]. Table 1. List of International Organization for Standardization (ISO) 16000 series for IAQ. EN = European Standard. Table 1. Cont. In some EU countries such as France, Belgium, Portugal, etc., there are specific legislations for each pollutant and the relative reference ISO standards to be used. The great confusion of these years has been precisely the absence of sampling and analysis standards dedicated to IAQ. Standards for industrial environments were often used, i.e., National Institute for Occupational Safety and Health (NIOSH), Occupational Safety and Health Administration (OSHA), etc.), which have mg m−<sup>3</sup> sensitivities (and have nothing to do with indoor μg m−<sup>3</sup> concentrations). Against this background, the adoption of the ISO 16000 standard represented a significant improvement as to the study and control activities. Now, following behavior consolidated in several countries, it is, therefore, appropriate to develop indoor specific harmonized reference values in order to better manage particularly problematic situations in such environments. In the absence of specific national references to be used for a comparison, those reported by ad hoc working groups, or in the legislation of other European countries, are currently used. Several EU countries, in recent years, have set up working groups with a specific mandate to develop guide values for air quality in confined spaces. Table 2 shows a series of guide values, present in the official documents, for selected pollutants, including those considered in the WHO guidelines. For instance, Germany, by the German Working Group on Indoor Guideline Values of the Federal Environmental Agency and the States' Health Authorities (AG IRK/AOLG) [18], used a methodology starting from Lowest Observed Adverse Effect Level (LOAEL), or lower level of exposure to a toxic pollutant, for which negative health effects have been observed, introducing safety factors, such as inter- and intra-species. On the other hand, the UK adopted a different approach. In particular, the commission on the effects of air pollution on human health, i.e., the Committee on the Medical Effects of Air Pollutants (COMEAP) (updated in 2020) [19] and the Royal College of Pediatrics and Child Health (RCPCH) [20], developed guide values on the basis of WHO studies. France did the same, thanks to the collaboration between the French Scientific and Technical Center for Construction (CSTB) and the French Agency for Environmental and Occupational Health Safety (AFSSET) [21–23]. The working group developed a long series of studies to arrive at the elaboration of guide values for eight pollutants, such as hydrogen cyanide, carbon monoxide, benzene, formaldehyde, trichlorethylene, tetrachlorethylene, naphthalene, PM10 and PM2.5. This activity was part of the National Health and Environment Plan PNSE 2004–2008 [24], followed by the second National Plan for Health and Environment (PNSE 2) that was published for the period 2009–2013. Alongside the AFSSET indications, the authors would like to mention those identified by the High Council of Public Health-Haut Conseil de la Santé Publique (HCSP), which elaborated a series of documents on the values of action, and long-term for the evaluation of IAQ [25]. France implemented a plan of targeted interventions with the enactment of law no. 2010-788 of 12 July 2010, which is continuously updated (the last one in 2016), and establishes the obligation of periodic monitoring of the air quality in confined spaces, as well as the responsibility of the owners or occupants, gradually in force: For structures open to the public after these dates, the first periodic monitoring must be carried out no later than 31 December of the year, following the opening of the structure. Failure to comply with the terms of implementation of this obligation is punished with a fine. The control of the indoor environment through the monitoring of pollutants must be repeated every seven years, except in the case in which at least one of the pollutants measured during the monitoring shows levels higher than that foreseen in the aforementioned decrees. In this case, monitoring of the confined environment must be carried out within two years. In addition, the Netherlands, using the studies performed by the National Institute for Public Health and the Environment (RIVM) [26], achieved guiding values starting from the Maximum Permissible Risk (MPR), which represents the level of exposure to a toxic substance for which there are no negative health effects. Among the Nordic countries, in Finland, for example, the working group (coordinated by the Ministry of Social Affairs and Health (MSAH)), developed guide values for five pollutants: ammonia, carbon monoxide, carbon dioxide, hydrogen sulfide, and PM10. They were proposed in the decrees of the Ministry of the Environment Housing and Building Department D2 National Building Code of Finland—Indoor Climate and Ventilation of Buildings Regulations and Guidelines [27], which entered into force 1 October 2003. For the other pollutants, it is possible to derive guide values using 1/10 of the limits for industrial work environments (Occupational Exposure Limit, OEL). If more pollutants are present, the formula is to be applied: Σ (Ci/(HTP)i) > 0.1, where Ci is the measured concentration of a single pollutant and (HTP) is the occupational exposure limit of the pollutant in question. The guide values for confined spaces apply to buildings that are occupied for at least six months and where the ventilation system is kept constantly on. Alongside these references are those developed by the Finnish Society of Indoor Air Quality and Climate Classification [27]. It is an initiative desired and financed by the Ministry of the Environment, in collaboration with the experts of the manufacturers and stakeholders of the materials sector, which led to the identification of the target values defined as S1 (individual indoor environment), S2 (good indoor environment), and S3 (satisfactory indoor environment) categories [27]. Belgium, on the other hand, in the Flanders region, established by decree that entered into force on 1 October 2004, reference values for 15 pollutants: acetaldehyde, formaldehyde, total aldehydes, benzene, asbestos, carbon dioxide, nitrogen dioxide, toluene, ozone, carbon monoxide, volatile organic compounds, trichlorethylene, tetrachloroethylene, PM10 and PM2.5. For five of these pollutants, a category of concentration levels was also identified, defined as intervention values or concentrations of the pollutants corresponding to a level of maximum permissible risk that cannot be exceeded. Another interesting aspect present in the decree is that, in the event that an intervention on the field is requested by experts from the health inspectorate, and that the analytical results of this investigation highlights critical conditions linked to the negligence of the owner or occupant, the inspectorate charges the intervention costs to the applicant [28]. In 2019, further legislative acts were issued for office workplaces intended to welcome the public (decrees 31 January 2019, 2019/201064, and 21 May 2019, 2019/201857). In the late 1990s, in Austria, the Ministry of the Environment in collaboration with the Academy of Sciences established an interdisciplinary working group for the drafting of guiding values for indoor environments, using a methodology starting from No-Observed-Adverse-Effect-Level (NOAEL) [29]. Using this approach, guide values of six substances were developed: formaldehyde, styrene, toluene, carbon dioxide, volatile organic compounds (VOCs), and trichlorethylene. Portugal, in April 2006, by decree no. 79 of the Ministry of Public Works, Transport, and Communications [30], and in 2013 by decree no. 60 [31], set maximum reference concentrations for six pollutants: PM10, carbon dioxide, carbon monoxide, ozone, formaldehyde, total VOCs. The decree, in force since June 2006, also establishes the mandatory monitoring of the type and size of the building, and provides corrective actions within 30 days, if after the monitoring, the concentrations of pollutants present levels higher than reported in article 29 paragraph 8 of the decree. Further, the owner or tenant must also provide, within the following 30 days, the results obtained from the new measurements made. In case one of the above conditions is not met, the owner or tenant is subject to the penalties provided for in the decree, such as, for example, the immediate closure of the apartment or the payment of a fine. In all countries, the proposed guide values are correlated by the relative sampling and analysis methods developed or implemented by the various national training bodies for correct evaluation (e.g., sampling and analysis strategies). These training bodies include the German Institute for Standardization (Deutsches Institut für Normung, DIN), Association Française de Normalization (AFNOR), Bureau de Normalization (NBN), Finnish Standards Association (SFS), Austrian Standards Institute (ASI), Nederlands Normalisatie Instituut (NEN), and the British Standards Institution (BSI). It should be noted that the guide or reference value must always be related to the sampling and analysis method to be adopted for its verification. For all these countries, except Belgium, Finland, Lithuanian, Portugal, and France (for benzene, formaldehyde, carbon dioxide, and tetrachloroethylene), the recommended guide values have no legal value, even though, in practice they have reached considerable importance. These values, if properly used, can allow for better assessment of the IAQ. Finally, IAQ is also important for protecting vulnerable materials, including cultural heritage in museums. Inside museums, libraries, and cultural environments—or storage of materials of historical and artistic interest—the quality of indoor air, together with the microclimate (temperature and relative humidity, which must mainly take into account the nature of the materials and goods), and the lighting (another important parameter that can enhance the phenomena of degradation of materials and goods), is fundamental for the management, conservation, and enhancement of goods and finds, and for the choice of measures to contain energy consumption and improve the quality of museum environments, for the health of workers and visitors. There are several reference sources for museum environments, such as the United Nations Educational, Scientific, and Cultural Organization (UNESCO), International Council of Museums (ICOM), International Center for the study of the preservation and restoration of cultural property (ICCROM), National Information Standards Organization (NISO), Getty Conservation Institute, Environmental Conditions for Exhibiting Library and Archival Materials, WHO, and the Ministry of Cultural Heritage and Activities (MIBACT, Italy), just to cite a few. Among the different documents, the authors would like to highlight the following: The adoption of these rules constitutes a significant improvement compared to what has been achieved so far in the study and control activities; the standardization of the methods also increases the possibility of a correct comparison between the different data produced at the European level. 2.Indooraircontaminants:referencevaluesusedinEuropeancountries,guidevalues,andunitaryriskoftheWorldHealthOrganization Table 2. Cont. 1μg L−1 in water); the guide values for indoor environments apply to buildings that are occupied for at least six months and where the ventilation systemAbbreviations: UR unit risk; lt lifetime; RA rapid action; LP long period; RW I (all-day use) and RW II (danger threshold) German guide values (Richtwert);intervention value; RV reference value; PAHs Polycyclic Aromatic Hydrocarbons; BaP Benzo[a]pyrene; VOCs Volatile Organic Compounds; y year; d day; h hour; GV guideline value; IV m minute.	doab	2025-04-07T03:56:58.078166	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 12 }
001fcd9a-cd27-45e8-b23f-f447874e0974.13	3. The Italian Situation Among the Member States of the EU, Italy plays an important role, as evidenced by the 10.4% of publications in the IAQ field during the last two decades (Figure 1). The Italian situation is particularly interesting because, unlike the other countries, in the Italian legislation, there is no specific reference relating to residential IAQ, even if pre-regulatory initiatives have multiplied. In relation to IAQ, in almost all European countries, a legislative delay has been compulsorily and quickly filled. This delay has to be covered, with the issue of specific acts containing suitable references for chemical and biological pollutants, in line with those developed by the WHO, with the most recent and user-friendly specific protocols and procedures provided by the ISO 16000 indoor air standard in its various parts. For these reasons, in 2010, the National Study Group (GdS) on Indoor Air Pollution was established at the Italian Institute of Health (IIS), in which the various ministerial components are represented (Ministry of Health, Ministry of the Environment and Protection of the Territory and the Sea, Ministry of Labor and Social Policies), regions, local authorities and research institutes (IIS, National Research Council (CNR), Italian National Agency for New Technologies, Energy, and the Sustainable Economic Development (ENEA), Italian Institute for Environmental Protection and Research (ISPRA), National System for Environmental Protection (SNPA), and the National Institute for Insurance against Accidents at Work (INAIL). The GdS-ISS is working to provide shared technical–scientific documents in order to allow harmonized actions at national level in order to improve the correct assessment of indoor air pollution. The documents of the GdS-ISS, published as Rapporti ISTISAN, or dissemination documents, include recommendations to prevent indoor air pollution, to improve behavior, cultural awareness, training, to reduce exposure and effects on health, and to increase economic competitiveness. The GdS-ISS has developed eight reference documents for the monitoring strategies of the main indoor chemical and biological pollutants, the role of the different sources, the energy efficiency activities, and the different indoor combustion [38–48]. Table 3 shows the list of ISTISAN reports already published by the GdS-ISS. Some of the technical indications can already be used for the definition of a national plan on indoor air quality and constitute an important reference for the country. Table 3. Rapporti ISTISAN just published by the National Study Group (GdS) on Indoor Pollution. <sup>1</sup> This publication is not authored by the GdS, but it contains issues related to the IAQ. The results of this activity have been included in the Directive of the President of the Council of Ministers 1 June 2017 published in the Official Journal on 17 July 2017, among the mandatory training activities that the employer must provide to workers. Such activities of the GdS-ISS have also been taken up in the Air Pollution Strategy of the WHO Country Profile for Italy. In this way, an informative booklet entitled "Air in our home: how to improve it" was prepared, which illustrates the origin of indoor air pollution, the role of sources (household cleaning products, construction products, furniture, fabrics, incense sticks, scented candles, stoves, etc.), and the contribution of individual behaviors, providing specific recommendations to reduce indoor pollution levels. Work is currently in progress for the preparation of two new documents on indoor air quality in office environments and on contaminated sites. On the other hand, ISTISAN reports are being published that address the problems of indoor air quality in school and health facilities, with the identification of specific environmental detection methodologies and possible sanitary implications. In 2018, Pierpaoli and Ruello published a paper on the bibliometric study on the IAQ [49]: the authors asked the question "What are the actual trends in Indoor Air Quality (IAQ), and in which direction is academic interest moving?" Starting from that, the authors analyzed the worldwide literature from 1990 to 2018, using the Web of Science as a database. They identified past trends and current advances in IAQ, as well as the issues that were expected to be pertinent in the future. In this section, we would like to show state-of-the-art research in the IAQ sector in Italy from previous years, considering what is shown in Figure 1: 243 scientific papers in specialized journals have been published in Italy since 2015. The topics cover different subjects, i.e. environmental science, engineering, medicine, social sciences, energy, physics and astronomy, biochemistry, genetics and molecular biology, materials science, chemistry, chemical engineering, earth and planetary sciences, agricultural and biological sciences, immunology and microbiology, pharmacology, toxicology and pharmaceutics, computer science, mathematics, business, management and accounting, economics, econometrics and finance, arts and humanities, decision sciences, multidisciplinary, and nursing. This means there is a large interest by the scientific community in this field. Most papers are addressed to investigate the IAQ in schools. Such topics are important because, based on the subpopulation interested, such as suggested by Manigrasso et al. [50], which estimated the particle regional respiratory doses for both combustion and non-combustion aerosol sources currently encountered in microenvironments, with special regards to the age of subjects. Recent papers on school environments are related to monitoring PM, NOx, VOCs, and CO2, with regard to the ventilation efficiency and the energy consumption [51–56]. As to the radon exposure, according to two papers, the schools are vulnerable targets due to the long daily childhood presence, and the radon risk could be reduced by low-cost interventions (e.g., implementation of natural air ventilation and school maintenance) [57,58]. Over the last five years, several papers were published on residential IAQ: the authors would like to highlight the main papers of interest. Different research groups dealt the problems related to wood or biomass burning, evidencing the emissions and the related risk assessment [59–61]. Particular attention has been addressed to hospitals and healing places for defining protocol for inpatient rooms, to understand the state-of-the-art research and for suggesting design and management strategies for improving process quality [62,63]. Indoors, there are different combustion and non-combustion sources. Manigrasso et al. revised all of the possible sources and investigated the ultrafine particle emissions and relative doses deposited in the human respiratory tract [64–66]. The importance of the micro-climatic parameters was discussed by Zanni et al., which monitored the IAQ in the airport of Bologna (Italy) as a prototypal example of a large regional airport [67]. Siani et al. applied the cluster analysis on a long time series of temperature and relative humidity measurements for identifying the thermo-hygrometric features in a museum [68]. Cincinelli et al. characterized the IAQ in libraries and archives in Florence (Italy), evidencing that benzene, toluene, ethylbenzene, and xylenes (BTEXs) are the most abundant VOCs, along with cyclic volatile methylsiloxanes, aldehydes, terpenes, and organic acids. In particular, the authors detected presence of acetic acid, which is a chemical that can oxidize books and other exposed objects, and furfural, which is a known marker of paper degradation [69]. Tirler and Settimo discussed the increasing use of incense, magic candles, and other flameless products that may represent a health risk for humans. Pollutants, such as benzene and PM10 are mainly affected when these products are used indoors (for instance, the benzene concentration ranged from background levels to over 200 μg m−<sup>3</sup> after the incense sticks had been tested) [70]. As can be seen, one of the main focal points of the authors is the relationship between IAQ and energy consumption, which is very important. However, it should be considered that the plans and/or interventions of restructuring or renovation cannot be only oriented to the theme of insulation, containment, and energy efficiency, which can alter or worsen air quality, microclimatic conditions, and natural ventilation. They should follow approaches allowing an overall improvement in air quality, with criteria to promote and guarantee health, primarily, to offer all of the maximum benefits of the most current quality educational and training models, and to obtain savings in management costs. Similarly, the same approach should be followed in cases of complete plant adaptations or restructuring (water, electricity, heat, fire, etc.).	doab	2025-04-07T03:56:58.079332	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 13 }
001fcd9a-cd27-45e8-b23f-f447874e0974.14	4. Conclusions The IAQ determinants on human health and the potential presence of harmful contaminants released from indoor sources have always been stressed by WHO in its technical documents and position papers. In Europe, specific directive legislative framework on the quality of indoor air is not yet available. Despite an increasing number of pre-legislative initiatives, guidelines, and documents, a harmonized and global approach is still missing. Pending a European directive on indoor air quality, as already done with outdoor air (e.g., 2008/50), which takes into account the WHO indications, this paper aims to provide an overview of the main technical–scientific references in order to allow harmonized actions and to cope with the main issues in such environments. In fact, often in the absence of specific national references to be used for comparison, surveillance actions in indoor environments are limited. The paper, gathering the main references to be used (reported by ad hoc working groups, or in legislation of other European countries, or, by analogy, with other standards, such as those relating to ambient air), means to assist operators engaged in prevention actions to implement interventions in different indoor environments. It should be remembered that these values represent the parameters to which reference must be made for an assessment of the inhalation risk of workers and the population. They are not the only ones, because specific exposure and vulnerability conditions are fundamental elements to consider for a correct risk assessment. There is an urgent need for a change that is innovative, with a systemic, multidisciplinary approach based on skills. Nowadays, in the various member states, apart from the strong national differences, this situation entails a hygiene–health and environmental protection gap among the various countries (e.g., absence of standards and controls). To fill this gap, harmonization initiatives must be carried out, simultaneously establishing the elements (e.g., strategies, sampling, and analytical methods) and the parameters that must be considered for the control of pollutants indoors. There is no doubt that the heterogeneity of this current regulation system has led to a lack of comparability among the EU member states, both in terms of technical procedures and of health evaluation. Nonetheless, in some EU countries, regulations have been drawn up or recommendations have been developed on IAQ that can allow proper exposure assessment of the general population and the related health risks. Recently, the EU has also taken on a series of new commitments on the energy efficiency and the construction quality. In this regard, in the "Report from the Commission to the European Parliament and the Council: financial support for energy efficiency in buildings" [71] it is emphasized that improving the energy efficiency of buildings also entails important collateral benefits, including greater health. In Italian territory, there is no reference legislation, but several commissions and working groups are at work. Among these, there is the National Study Group (activated by the ISS), which is working to provide concrete technical contribution for operators in the public and private sectors engaged in the indoor theme, in order to allow a homogeneous action at a national level. The results may lead to appropriate public health strategies aimed at reducing exposure in indoor environments. Author Contributions: Conceptualization, G.S. and P.A.; methodology, G.S.; software, M.M.; validation, M.M.; data curation, M.M.; writing—original draft preparation, G.G. and P.A.; writing—review and editing, G.S. and P.A.; visualization, P.A.; supervision, G.G. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:58.080265	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 14 }
001fcd9a-cd27-45e8-b23f-f447874e0974.16	The Impact of Indoor Malodor: Historical Perspective, Modern Challenges, Negative Effects, and Approaches for Mitigation	doab	2025-04-07T03:56:58.080643	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 16 }
001fcd9a-cd27-45e8-b23f-f447874e0974.17	*Pamela Dalton 1,\, Anna-Sara Claeson <sup>2</sup> and Steve Horenziak <sup>3</sup> Received: 15 December 2019; Accepted: 21 January 2020; Published: 23 January 2020 Abstract: Malodors, odors perceived to be unpleasant or offensive, may elicit negative symptoms via the olfactory system's connections to cognitive and behavioral systems at levels below the known thresholds for direct adverse events. Publications on harm caused by indoor malodor are fragmented across disciplines and have not been comprehensively summarized to date. This review examines the potential negative effects of indoor malodor on human behavior, performance and health, including individual factors that may govern such responses and identifies gaps in existing research. Reported findings show that indoor malodor may have negative psychological, physical, social, and economic effects. However, further research is needed to understand whether the adverse effects are elicited via an individual's experience or expectations or through a direct effect on human physiology and well-being. Conversely, mitigating indoor malodor has been reported to have benefits on performance and subjective responses in workers. Eliminating the source of malodor is often not achievable, particularly in low-income communities. Therefore, affordable approaches to mitigate indoor malodor such as air fresheners may hold promise. However, further investigations are needed into the effectiveness of such measures on improving health outcomes such as cognition, mood, and stress levels and their overall impact on indoor air quality. Keywords:** malodor; indoor air; human olfaction; volatile organic comound (VOC); microbial volatile organic compound (MVOC); VOC; MVOC; health effects; smell; malodor mitigation; air fresheners; fragrance	doab	2025-04-07T03:56:58.080680	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 17 }
001fcd9a-cd27-45e8-b23f-f447874e0974.18	1. Introduction The sense of smell is a fundamental means of navigating the sensory world and orienting ourselves to ecologically and socially appropriate behavior. Evolutionarily, this chemical sense was the original means by which the earliest organisms achieved adaptive regulation of action and can be considered the origin of behavior [1]. For humans today, volatile molecules can travel for long distances and thus can provide important information about people, places, food and things that cannot otherwise be immediately detected by other sensory systems. Beyond its informational content, odor can attract, intrigue, impress and entice, as well as repel, offend, disgust, or evoke pity. Malodors are odors perceived to be unpleasant or offensive and, while not necessarily occurring at the known thresholds for direct adverse events, may elicit negative symptoms via the olfactory system's connections to other cognitive and behavioral systems. Malodors are sometimes depicted as an inconvenience or annoyance of relatively minor importance to human perception and experience [1]. An understanding of malodors as a merely "aesthetic" issue, however, ignores their potential for negative impact on human health and social relations [2]. Malodors propagate a variety of psychological, social and economic disturbances, many of which are preventable. As defined at the International Health Conference, "health is a state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity" [3]. Although crafted in 1946, this definition of health has remained in use by organizations such as the World Health Organization. Combating the sources and mitigating the impacts of malodors therefore represents an important public health undertaking. Throughout history, people have used perfumes, incense, herbs and other means at their disposal to rid their indoor environments of the malodors that occur in the course of human life and industry. However, within a social structure where people cannot always remove the sources of malodors or move themselves to avoid odors, or where odors are the result of industries that sustain food and energy supplies, frequent and intense malodors that are left unchecked can contribute to larger challenges. Malodors can directly affect physical health if the malodorous chemical represents an irritant or harmful airborne substance and occurs at a high enough concentration to exceed observable adverse effect levels. Additionally, malodors may act indirectly, as a mediator of mood, performance and health symptoms, effects which are the focus of this review [4]. Negative effects of low-level chemical exposures (e.g., malodor exposure) are further discussed in Section 5. As the World Sanitation Foundation notes, malodors that result from poor sanitation can compound sanitation issues in under-resourced and developing areas [5]. In rural India, malodors resulting from poor sanitation in pit latrines can indirectly result in open defecation and thus spark a variety of new community-wide health hazards, including compounded malodor issues [6–8]. While eliminating the source of indoor malodor can be a direct mode of intervening in odorous environments, it is often not achievable with the resources at hand. Even in today's urbanizing societies, where malodors now concentrate indoors and in private spaces, people, especially those in low-income communities, may not have the resources to remove the sources of malodors or to relocate their residence. In this review, we examine and discuss the current state of understanding on the role of indoor malodors for impacting human behavior, performance and health, including the individual factors that may govern such responses and identify research priorities to address the data gaps where they exist. Malodors have been reported to have a number of negative psychological, physical, social and economic consequences, as will be discussed herein. Conversely, removal of malodor by increased ventilation or filtering has been reported to increase performance and subjective responses in workers (e.g., ratings of air freshness and air quality), highlighting the potential benefits of mitigating malodors in indoor spaces [9,10]. However, such interventions may not be feasible in many indoor environments. Malodor is an important part of indoor air quality, and accessible and affordable malodor solutions such as air fresheners should be studied to determine if similar benefits are observed.	doab	2025-04-07T03:56:58.080823	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 18 }
001fcd9a-cd27-45e8-b23f-f447874e0974.19	2. A Historical Perspective Throughout history and across different parts of the world, malodors have varied in intensity and cultural impact as has the use of perfumes to mitigate malodors. Influenced by the Egyptians, the ancient Romans used perfumes intensely and even applied them to domestic animals to mitigate malodors. In the 4th century, the use of perfumes and pleasant aromas was condemned as an indulgence and idolatry by the Christian church [11]. Partially as a result of this policy, the European cities of the medieval and renaissance ages are known to be among the most foul-smelling environments in human history. Without proper sanitation infrastructures, their close quarters and high population density led to high concentrations of malodors [12]. Rotten food, excrement and slaughtered animal remains frequently littered the streets [11]. To mitigate indoor malodors, Medieval Europeans scattered herbs throughout their homes, sewed aromatic leaves into pillows, or polished wood with myrrh. To perfume themselves, they sprinkled rose water on their clothes or wore pomanders. Out of this environment emerged the beginnings of modern commercial perfumes. Perfumes with essential oils were made for royalty by Italian chemists in the 14th century. With Caterina de' Medici's marriage to Henry II in the mid-16th century, Renaissance Italy's perfumes traveled to France where they continued to flourish centuries later. Throughout these periods, fragrances were used to mitigate the negative impact caused by malodors and functioned as a social symbol of higher class [12]. Perspectives on odors have changed significantly since the Renaissance, though people today still seek out means of combating malodors and asserting control over unpleasant smells in their lives, often through the use of pleasantly scented products like air fresheners.	doab	2025-04-07T03:56:58.081163	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 19 }
001fcd9a-cd27-45e8-b23f-f447874e0974.20	3. Modern Indoor Malodor Challenges Associated with Urbanization As malodors in public spaces have generally decreased with post-industrialism sanitation improvements, the domestic household has become a prominent site for exposure to malodors. While malodors experienced in historical periods were concentrated in shared areas, contemporary experiences with malodors are frequently experienced in personal spaces [13]. Contemporary building and insulation techniques used in modern homes can allow malodors to concentrate within the household [14]. Household odors are a combination of external odors that enter the home and odors produced within the home. External odors that invade the home include emissions from industry and pollution. Odors produced within the home arise from aggregate effects of low concentrations of volatile organic compounds (VOC) caused by cooking, pet, and human body odors, and the use of personal and household cleaning products, among others. They can also arise from microbial volatile organic compounds (MVOC) formed by the metabolic processes of fungi and bacteria present on building materials [15]. Over time, these VOC will become absorbed by the porous surfaces in homes such as carpets, soft furnishings, curtains, wall paper and even the grout between tiles. The combination of the bouquet of VOC present in households imparts each home with its own unique smell [16]. Exposure to certain mixtures of MVOC and VOC has been shown to increase reports of poor air quality within indoor spaces [17]. However, it should be noted that VOC as a class of compounds are not inherently toxic or malodorous. With respect to establishing toxicity, one must measure the levels and refer to the known threshold for adverse effect for each specific VOC. Many indoor VOC are perceived to have a pleasant smell and can have positive associations, including the wide variety of VOC that are released during such activities as baking bread or cooking. Additionally, there is a certain amount of subjectivity in an individual's response to specific VOC, as one person may report a positive reaction to a certain VOC based on pleasant memories associated with that VOC while others may report it as a malodor. The perception of VOC also differs with respect to concentration and context. For instance, the substance skatole (3-methylindole) is present in flowers and essential oils and is frequently used in fine fragrances at low concentrations. However, at higher concentrations, it is perceived as having a distinct fecal odor, pointing to the importance of concentration with respect to malodor perception [18]. The context in which a VOC is interpreted is also critical to how it is perceived. Participants who were told that isovaleric acid (a cheesy-smelling fatty acid) was a body odor rated it as far more unpleasant than participants who were told it was a food odor [19]. Finally, genetic variation across the population has resulted in a 'highly personalized inventory of functional olfactory receptors' that not only determine what any individual can smell but how pleasant or unpleasant an odorant is perceived to be [20–22]. In today's urban societies, people can spend nearly 90% of their time in indoor environments [23,24]. As such, there has been investigation into whether the experience of indoor malodors should be regarded as a health issue or a merely aesthetic one [2,25,26]. This line of inquiry has encompassed field studies of industries that emit high quantities of malodorous compounds in residential areas, surveys of workplace productivity in specific chemical environments, psychological laboratory tests of odor exposure and case studies of heightened olfactory sensitivity, in addition to genetic and neurophysiological studies of olfaction and related biological systems. These types of studies have yielded important insights into understanding of the diverse effects of odors on health and social interactions.	doab	2025-04-07T03:56:58.081328	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 20 }
001fcd9a-cd27-45e8-b23f-f447874e0974.21	4. The Human Perception of Malodors Perception of a malodor occurs when a molecule activates receptor cells linked to one of several cranial nerves associated with chemoreception. The olfactory nerves of the nasal epithelium are the most significant in odor perception and transmit information from the nasal cavity to the olfactory bulb, which in turn transmits olfactory information to other areas of the brain. In addition, the trigeminal nerve transmits information about pungency from the mouth and eyes as well as the nose. The chorda tympani nerve, glossopharyngeal nerve or vagus nerve may additionally be activated if the compound enters via the mouth [27]. Pungency and odor perception have been determined to be separate chemical senses, as anosmics, who lack the ability to smell, can still sense chemicals through their pungency effects in the nose, mouth and elsewhere [28]. While unpleasant olfactory sensations define malodors, at sufficiently high concentrations these sensations can be further accompanied by unpleasant pungency sensations. The chemical senses of odor and pungency perception vary in several significant ways. For one, the threshold detection for pungency is generally several orders of magnitude higher in concentration than what is required to perceive the odor; people most often perceive a smell before it becomes so strong as to sting their eyes [29]. Though people may adapt to a constant odor in a matter of minutes or hours, adaptation to the perception of pungency occurs over longer periods [30]. Detection thresholds for malodors vary dramatically depending on the specific chemical in question, with thresholds generally declining with the carbon chain length of the compound [28,29]. Humans can detect common indoor malodors like hexyl acetate at concentrations as low as 2.9 parts per billion [30]. Malodors from sulfur compounds like isoamyl mercaptan can be detected at concentrations as low as 0.77 parts per trillion (ppt) [31], and MVOC can be detected as low as 0.2 ppt (i.e., from 2-Isopropyl-3-methoxy-pyrazine) [15]. From an evolutionary point of view, this ability to detect extremely low concentrations of chemical compounds in the air affords identification of various sources of danger, such as spoiled food or harmful chemicals. Like all senses, olfaction is a product of biological evolution whose features are linked to survival and adaptation [32]. To this end, major connections have been identified between the olfactory system and cognitive processes, such as associative learning [33] and emotional memory [34–36], as well as "fight or flight" response [37]. "Top-down" cognitive functions, such as risk and danger perception, can also influence "bottom-up" information from the odor stimulus by allocating greater attentional resources to malodors, thus increasing their negative impact [38]. While the perception of malodors across individuals follows the same physiological pathways, the intensity of and response to the perception can vary. Although there can be differences across individuals in their sensitivity to specific malodors, the more important drivers of an individual's hedonic response may be due to their expectations, past experiences and the context in which an odor is experienced. For instance, the smell of smoke around a campfire can evoke a positive scent experience. However, the smell of smoke within a home will evoke an entirely different response, as the smoke is a signal of danger in this context. ### 5. The Negative Effects of Malodors Utilizing the search term "malodor harm" or variations thereof (e.g., synonyms of "malodor" and "harm") via publicly available databases such as ScienceDirect (https://www.sciencedirect.com/) revealed scientific research across an array of disciplines documenting various negative effects associated with malodors. These negative effects were observed to cluster into six categories as identified by the authors of this review (Figure 1). Figure 1. Reported negative effects associated with exposure to malodors. Studies evaluating the negative effects of malodors have been conducted in both the field (observational) and the laboratory (experimental) employing a variety of dependent measures (Table 1). Table 1. Measurement of malodor effects conducted in the field via observations and in the laboratory (Lab) via experimental methods. Several studies have reported the negative effect of malodors on mood. Self-reported feelings of depression [39,40], fatigue [39,40], confusion [39,41], aggression [40,60,61], and tension [39,40] have all been positively correlated with malodor exposure, whereas subjective well-being [40] has been negatively correlated with such exposures. Even when no malodor is present, expectations of malodor exposure may cause negative effects on mood [43]. Malodors may cause individuals to feel a lack of control over their environment, adversely affecting stress levels. Indoor household malodors of external origin that are consistent and uncontrollable may produce feelings of helplessness [39,62]. Perceived control has also been shown to affect tolerance of a given malodor [44]. Individual coping style, however, may also affect odor annoyance and symptom prevalence. Studies have suggested that those who have "palliative" or avoidance coping styles generally report less annoyance and symptoms than those with "instrumental" or problem-oriented coping styles [52]. Stress about the perceived toxicological effects of malodors may further allow odors to act as a trigger for other symptoms and behaviors [48–50]. Malodors have been shown to have detrimental effects on cognition. Rotton [44] has shown that exposure to malodor does not affect simple cognitive tasks, but that it has a detrimental effect on more complex tasks, such as proofreading. Cognitive deficits resulting from malodor exposure may be due to their negative effect on focus [45]. Malodors have been shown to elicit somatic symptoms. Somatic symptoms that have been reported with malodor exposure include vomiting, nausea, dizziness, headache, loss of appetite, sleep disorders and irritation of eyes, throat and nose [2,63]. Malodors can also cause somatic symptoms via "odor-worry" and stress [46]. Asthmatics, for example, may experience exacerbation of symptoms from non-irritating odors that are perceived as harmful [51]. Others may experience the stress effects of malodors because of "environmental worry" [46], an association with the odor as socially taboo or by perceiving possible property devaluation resulting from the odor [39]. Exposure to certain malodors has also been shown to affect the immune system, an effect probably mediated by perceived stress [47]. Social relations are also threatened by indoor malodors. Habituation to the odors of one's background can make people acutely aware of intrusive malodors, which may cause a variety of problems in social settings. Subjective ratings of odor unpleasantness have been shown to correlate with perception of socially undesirable traits [54]. Odor perception may also integrate with higher-order visual processes, such as facial perception. Unpleasant facial expressions paired with malodors have been shown to increase people's ratings of odor intensity and decrease their respiratory amplitude [53]. Judgments of interpersonal attraction are also influenced by the presence of malodor [64]. Indoor malodors can also reduce social interactions by causing inhabitants to experience shame or embarrassment about the malodor, even when they are external in origin [55]. It is reasonable to assume that this occurs with household odors as well. Malodors can have economic effects. Unlike other "less visible" forms of pollution, malodors are readily identified and capitalized into property values [56]. Some industries, such as tanneries, paint factories, pulp mills and livestock operations, are regulated by legislated minimum "setback distances" that facilities must maintain from surrounding properties. Setbacks are used to minimize the economic effects of pollution, including malodors. Not only do properties surrounding these facilities decrease in value, but if the facilities' setback distance from surrounding properties is either unenforced or inaccurately determined [4,57], then malodors emitted from the facility can result in net economic loss, despite efficiency gains made by the offending firm [58]. Business and home owners alike can also suffer economic consequences. Malodors can affect car sales [59], worker productivity at call centers [9] and consumer satisfaction within the hospitality industry [65]. It is important to note that these negative effects are not necessarily independent measures and that individual effects can often act to compound economic ones. Levy and Yagil [66], for example, suggest that low Air Quality Index scores within stock exchanges may affect mood and risk aversion, thus resulting in lower stock returns. Fist, Black and Brunner [10] note that improvements to indoor environmental quality has a strong effect on workplace productivity and health, estimating a potential annual gain of \$20 billion from improvements in office buildings in the United States. #### 6. Contemporary Approaches and Benefits of Mitigating Malodors Efforts to protect the public from the adverse effects of outdoor malodors take the form of regulations in many jurisdictions. Regulations include concentration or exposure limits if the odors are produced by specific target pollutants, nuisance or annoyance laws and property setbacks. The odor impact criteria established by such regulations are most commonly based on field olfactometry-based concentration measurements, although instrumental concentration measurements or air dispersion models are also used [67]. In order to comply with odor regulations and to promote good relations with neighboring households, some facilities may install technology to control odors at the source. Control strategies include oxidation, adsorption, chemical reaction, chemical scrubbing, biofiltration/bioremediation and other methods [68]. To help households control indoor odors, product manufacturers often employ some of the same technical strategies used industrially to control odors. These include air filters and filter media, oxidizers, absorbents/adsorbents, surface and air sprays, and a variety of volatile ingredient diffusers. Air filtration to remove odors may be achieved with filters attached to heating, ventilation and air conditioning (HVAC) units or stand-alone filtering units. Such filters may utilize activated carbon or zeolite adsorbents, photocatalytic oxidants such as metal oxides (for example, patents US 8911670, US 8038935), or odor-reactive chemistry such as metallic salts or amine polymers (for example, patent US 4892719). Household consumer goods products designed to eliminate household and automobile odors include air fresheners, pump and spray aerosols and diffusers. Such products may contain technologies designed to: capture or alter the molecular structure of VOC responsible for the underlying malodor; prevent perception of the malodorous VOC (MOVOC) by the olfactory system; and/or mask MOVOC via fragranced ingredients. Technologies used in air freshening sprays and diffusers that are designed to capture or alter specific types of malodor molecules are summarized in (Table 2). Spray products may utilize one or a variety of such technologies to eliminate the molecular source(s) and/or perception of MOVOC. Table 2. Malodor classification and patented technologies that can be used in air fresheners to mitigate common indoor malodors. One such technology approach to mitigate indoor malodors is the use of cyclodextrin (CD) or cyclodextrin derivatives to trap MOVOC by complexation. Cyclodextrin has a macro-ring structure consisting of glucopyranose units. It is produced naturally by bacteria including Bacillus macerana and Bacillus circulans, and is made industrially in bioreactors utilizing engineered glucosyl transferase enzymes [81]. The cavity of the cyclodextrin ring is apolar, so that less polar MOVOC readily displace water and become "trapped" upon interaction with aqueous cyclodextrin. Once complexed, the volatility of the MOVOC is significantly reduced and the malodors remain trapped in the CD cavity as long as the complex stays dry [69,82]. Patent documents indicate that consumer product companies make use of cyclodextrin technology in spray air freshening and other consumer products (for example, patents US 5760475, US 6077318, US 6248135 and US 6451065). Spray air freshening products may also utilize pH buffers to neutralize acid or basic odors and convert them to non-volatile salts. Acidic odors include short chain fatty acids such as isovaleric acid, heptanoic acid and basic odors include amines such as ammonia, butyl amine, and trimethyl amine. Both types of neutralizable MOVOC are constituents of household odors such as food waste odors, and human odors [16]. Buffer systems used in spray air freshening products to neutralize odors may include for example, citrate or carbonate buffers. Neutralization of acid and amine odors to pH in the range of 5–8 by pH buffering converts these MOVOC to non-volatile salts, reducing or eliminating the odor [83]. Enzyme inhibitors may be used in consumer products, including air freshening products, to prevent production of odorous metabolites (patent US 9200269). For example, urease inhibitors and β-glucuronidase inhibitors have been described to prevent the formation MOVOC from urine by microorganisms on fibrous consumer products [84]. Unsaturated aldehydes are MOVOC components of household odors that are derived from the oxidation of skin oils or from oxidation of lipids during cooking [16,85,86]. Amine-functional polymers are known to bind with and capture aldehydes (including formaldehyde) through the formation of imine bonds [87,88], and have been used in air freshening products to bind with odors (patent US 9273427). Additional technologies used in air freshening sprays include anti-microbial agents such as quaternary ammonium compounds to eliminate odor-causing microbes, which use salts of transition metals, particularly zinc and copper to complex with odors (for example, patents US 5783544, US 6503413), and oxidizing agents such as chloramine (patent US 6743420) that eliminate MOVOC through both antimicrobial and oxidative mechanisms. Diffusion-type products, like heated or unheated fragrance diffusers, may typically contain reactive materials such as carbonyl compounds designed to react with nucleophilic or electrophilic malodorous molecules such as amines (for example, patents US 8992889, US 5795566, US 7998403) to form covalently bonded, non-odorous products. Technologies designed to prevent the olfactory perception of malodors based on mechanisms such as olfactory receptor antagonism have also been explored by consumer product companies (for example, patents EP 2812316, US 9526680, US 9254248). Such approaches target specific olfactory receptors known to be activated by malodorous agonists with antagonistic agents to block activation of these receptors by the malodor. Consumer products designed to control malodor often, but not always, contain fragrance in addition to the technologies described above, or may contain fragrance without additional technology. Fragrance can mitigate malodors by masking their smell. The mechanism by which fragrances mask malodors is not well understood and may be achieved through a combination of signal interference (such as by receptor antagonism as discussed above) and through top-down processing effects (e.g., blending malodor with other odors to create perception of a new, non-malodorous aroma). Additionally, as noted above, some fragrances may contain reactive materials, such as carbonyl compounds designed to react with nucleophilic or electrophilic malodorous molecules. Pleasant fragrances have also been shown to have beneficial effects by increasing positive emotions, decreasing negative mood states and reducing indices of stress [89,90]. It is postulated that fragrances exert these effects through emotional learning, conscious perception and belief/expectation [91]. While seeking solutions to mitigate malodors, consumers and regulators must balance the economic, environmental and health costs of indoor malodors against the benefits delivered by the odor mitigating approaches. Companies that manufacture odor control technologies that emit fragrance or odor mitigating molecules into the air follow safety assessment paradigms that are widely recognized to ensure consumer safety when used according to label instructions. These assessments are aligned with the process outlined by EU Scientific Committee on Consumer Safety and are based on an understanding of both the inherent hazards of any materials in a product formulation as well as the level of exposure to those materials based on usage scenarios including extreme consumer usage [76,92]. In addition, the Research Institute for Fragrance Materials (RIFM) has published extensive industry guidance for conducting safety assessments of fragrance ingredients [93]. A 2007 US Environmental Protection Agency (EPA) review found that 0.23% of reported air freshener exposures involved an adverse reaction and that the number of reported exposure incidents for air fresheners was relatively small when compared to the reported exposure incidents for other product categories [94]. Household consumer products designed to eliminate odors are widely used by consumers in the United States, with 75.9% of US households purchasing an air care product as of March 2019, according to Nielsen HomeScan panel data [95]. Air care products are broadly available at retail outlets at relatively low cost. Buying rates of air care products are highest in households with annual incomes less than \$20,000 [96]. This may be due in part because lower-income households are disproportionately affected by environmental odors, odors arising from crowded conditions, and by economic limitations on their ability to deal with odor sources, such as those associated with sub-standard housing. Despite the potential negative effects of malodors and the widespread use of consumer products designed to eliminate odors, the health and quality-of-life benefits of the use of such consumer products has not been widely studied. A review of published literature on the health impacts of using air cleaning devices was recently completed by Kelly and Fussell [97]. The studies reviewed focused mostly on indoor air cleaning devices that reduced particle and VOC concentrations by using filters, adsorbents, oxidative technologies or combinations thereof. The studies generally showed no or low levels of improvement in the health outcomes measured for households with good ambient air quality and modest improvements for households with very poor ambient air quality. However, none of these studies, and few other published studies, have specifically examined the impact of indoor malodor reduction on health outcomes such as cognition, mood, and stress levels, among others. It may be inferred that eliminating the perception of malodor can reduce psychological effects of malodors, such as the feeling of a lack of control [41,44,47]. While studies have shown that people with problem-oriented coping strategies experience more stress and stress-related symptoms due to malodor exposure [52], air care and cleaning technologies offer a solution that allows people with problem-oriented coping styles to directly address the problems caused by malodors. Air care products designed to eliminate malodors can provide a more widely affordable solution compared to more costly alternatives such as home filtration systems, especially for low-income households who are economically unable to purchase such systems, replace malodorous household structures or items, or relocate away from substandard housing or industrial sources of malodor.	doab	2025-04-07T03:56:58.081606	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 21 }
001fcd9a-cd27-45e8-b23f-f447874e0974.22	7. Conclusions While there are complex issues at play in the distribution and effects of malodors (e.g., pollution concentrated near low-income communities, lack of access to proper sanitation), malodors are a general fact of daily human life. Indoor malodors are particularly challenging for people in developing countries or in low-income communities [2], which may lack the financial resources or opportunities to directly change the sources or living situations that harbor malodors. In these scenarios, malodors ultimately contribute to broad issues of structural inequality [41,98]. Viewing malodors as a merely "aesthetic" issue ignores their potential for negative impact on human health, previously defined as a "state of complete physical, mental and social well-being and not merely the absence of disease or infirmity" [3]. Review of the current literature includes several studies from a diverse range of disciplines reporting negative psychological, physical, social and economic consequences of indoor malodor. Conversely, removal of malodor has been reported to increase performance and subjective responses in workers [10], highlighting the potential benefits of mitigating malodors in indoor spaces. However, there are several gaps in the current research. Specifically, there is a lack of understanding regarding the mechanisms by which malodors can elicit any adverse effects, whether through an individual's experience or expectations that provide the interpretative context in which a VOC is experienced or through a more direct effect on human physiology and well-being. Thus, well-controlled studies examining the emotional, behavioral and performance-related outcomes induced by exposure to malodors are needed, as are studies that include a formal examination of the individual variables (i.e., personality, gender, age, culture) that may influence the magnitude and direction of malodor effects. Eliminating the source of malodor can be a direct mode of intervening in odorous indoor environments, though it is often not achievable with the resources at hand, particularly in low-income communities. Therefore, easily accessible and affordable approaches to eliminate malodors such as air fresheners with odor eliminating technologies (Table 2) may hold promise for reducing some of the negative effects of indoor malodor. However, we found relatively few investigations into the effectiveness of such measures on improving health outcomes such as cognition, mood, and stress levels, among others. Therefore, further study is recommended on the impact of air fresheners and other odor mitigating products on health outcomes via malodor elimination and/or emission of pleasant fragrances, as well as their impact on measures of overall indoor air quality. Author Contributions: The authors (P.D., A.-S.C., S.H.) contributed equally to the work by providing substantial contributions to the conception and design of the work and the acquisition, analysis, and interpretation of data; drafting the work and revising it critically for important intellectual content; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript. Funding: The Household and Commercial Products Association (HCPA) provided funding for the preparation of this manuscript. Acknowledgments: The authors would like to thank Eric Moorhead of Spectrum Science Communications and Mary Begovic Johnson of The Procter & Gamble Company for their helpful reviews and comments during the writing of this manuscript. Conflicts of Interest: Pamela Dalton is a consultant/grantee or speaker for the following companies: American Chemistry Council, Ajinomoto Co., Inc., Altria Group, Campbell Soup Company, Church & Dwight, The Coca-Cola Company, Diageo, plc, Diana Ingredients, Estee Lauder Inc., Firmenich Incorporated, Fragrance Creators Association, Givaudan SA, GlaxoSmithKline, Intelligent Sensor Technology, Inc., Japan Tobacco Inc., Johnson & Johnson Consumer Products, Kao Corporation, Kellogg, Kerry, Mars, McCormick & Company, Inc., Mead Johnson Nutritionals, Mondel ez International, PepsiCo, Inc., Pfizer, Inc., Procter & Gamble, Reckitt Benckiser Group, Roquette, Royal DSM, Sensonics International, Suntory Holdings Ltd., Symrise, Takasago International Corporation, Tate & Lyle, Unilever Research & Development, Wm. Wrigley Jr. Company, Young Living Essential Oils and Zensho Holdings Co. Ltd. Dr. Dalton received an honorarium from the HCPA for the preparation of this manuscript. Anna-Sara Claeson has no conflicts of interest. Dr. Claeson received an honorarium from the HCPA for the preparation of this manuscript. Steve Horenziak is an employee of The Procter & Gamble Company. The funder (HCPA) had a role in the design of the review, the compilation of published work on the subject and the decision to publish the results. The funder had no role in the analyses or interpretation of the reported data. The authors had full editorial control over the content.	doab	2025-04-07T03:56:58.082982	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 22 }
001fcd9a-cd27-45e8-b23f-f447874e0974.24	Towards Sustainable Neighborhoods in Europe: Mitigating 12 Environmental Impacts by Successively Applying 8 Scenarios	doab	2025-04-07T03:56:58.083409	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 24 }
001fcd9a-cd27-45e8-b23f-f447874e0974.25	*Modeste Kameni Nematchoua 1,2,3,4,\, Matthieu Sevin <sup>2</sup> and Sigrid Reiter <sup>2</sup> Received: 20 April 2020; Accepted: 3 June 2020; Published: 8 June 2020 Abstract: The purpose of this research is to determine the most impactful and important source of environmental change at the neighborhood level. The study of multiple scenarios allows us to determine the influence of several parameters on the results of the life cycle analysis of the neighborhood. We are looking at quantifying the impact of orientation, storm water management, density, mobility and the use of renewable energies on the environmental balance sheet of a neighborhood, based on eleven environmental indicators. An eco-neighborhood, located in Belgium, has been selected as the modeling site. The results show that the management of mobility is the parameter that can reduce the impact the most, in terms of greenhouse effect, odor, damage to biodiversity and health. With the adaptation of photovoltaic panels on the site, the production exceeds the consumption all through the year, except for the months of December and January, when the installation covers 45% and 75% of the consumption, respectively. Increasing the built density of the neighborhood by roof stacking allows the different environmental impacts, calculated per inhabitant, to be homogeneously minimized. Keywords:** life cycle assessment; sustainable neighborhood; Belgium; urban scale; roof stacking	doab	2025-04-07T03:56:58.083466	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 25 }
001fcd9a-cd27-45e8-b23f-f447874e0974.26	1. Introduction From the 1970s, a general awareness had been created with regard to environmental problems. The first oil crises, the end of the Thirty Glorious Years and the emergence of mass unemployment were highlights that questioned the idealistic aspect of a "model society", which had been in force since the end of the Second World War in Western countries. People were beginning to notice the incompatibility between the well-being of the productivity system, which is infinite growth, and the survival of the ecosystem as we knew it then. Thus, many people had come to believe that it would be beneficial for everyone to change the way our society operated [1]. Between 1960 and 1971, two large non-governmental organizations had emerged to fight for the protection of nature—Greenpeace and the World Wildlife Fund (WWF). The following year, that is, in 1972, the Club of Rome published its report under the tutelage of the globally respected Massachusetts Institute of Technology. They warned that the frenetic development of major industrialized nations would lead to the depletion of the world's reserves of non-renewable resources. Many environmental disasters that were publicized a lot more played an important role in this awareness [2]. The oil spills, Chernobyl, hurricane Katrina, or the heat wave of 2003, which caused the death of 15,000 people in France, made their mark. At the beginning of the twenty-first century, a leap was made regarding the international awareness of environmental problems. The Communiqués of the United Nations Intergovernmental Panel on Climate Change (IPCC) were significant in making this happen [3]. These communiqués claim that humans are 90% responsible for the worsening of the greenhouse effect and that this could lead to a rise in water levels of more than 40 cm [4,5]. They point to the economic, environmental and social risks that global warming could create [6]. Even the Catholic Church has reacted by defining a new sin, the sin of pollution. Targets are set at the European level to address environmental issues. The 2030 Package fixed by all the member countries of the European Union revealed at least a 40% cut in greenhouse gas emissions (from 1990 levels); at least a 32% share in renewable energy; and at least a 32.5% improvement in energy efficiency. This objective can be reached if all these countries work in collaboration. In industrialized countries, the construction sector is responsible for 42% of the final energy consumption [7,8], 35% of greenhouse gas emissions [9] and 50% of greenhouse gas emissions from extractions, from all the materials combined [6]. In addition, the urban sprawl is increasing land use, and between 1980 and 2000, the built space in Europe has increased by 20% [10]. Buildings are responsible for different types of soil consumption: a so-called primary consumption, that is to say, their building footprint; and also a secondary consumption, that is, the extraction, production, transportation and end-of-life treatment of construction products [11]. This type of impact is minimally considered in most studies, if at all considered. However, the life cycle analysis also studies the environment around the built area [12]. The culmination of all thermal and energy regulations is the European Zero Energy Building (nZEB) goal [13]. It aims to ensure that all new buildings have a neutral annual energy consumption; that is, they produce as much energy locally as they consume over the course of a year. This concept can be extended to the neighborhood scale to target the zero-energy level at the community scale [14]. A life cycle assessment is a method, an engineering tool, initially developed for industry. It aims to quantify the environmental impact generated by a product, a system or an activity. This requires an analysis of material consumptions, energy and emissions in the environment, throughout the life cycle [15]. An environmental impact is considered to be any potential effect on the natural environment, human health or the depletion of natural resources [1,16]. Thus, LCA is an objective process that allows for the establishment of various means to ensure increased respect for the environment [17]. Nowadays, a life cycle assessment (LCA) is the most reliable method for assessing environmental impacts associated with buildings and materials. In 2002, Guinee et al. [18] stated that one of the first (unpublished) LCA studies on the analysis of aluminum cans was conducted by the Midwest Research Institute (MRI) for the Coca-Cola Company. Buyle et al. [19] showed that the first LCA in the construction sector was performed in the 1970s. In the early 1980s, life-cycle analysis widened its interest to the field of construction. Different studies used different methods, approaches and terminologies. There was a clear lack of scientific discussion and consultation on this subject [19]. In the 1990s, we saw many more multi-criteria approaches, such as environmental audits and assessments that studied the entire life cycle of products. These methods were beginning to get standardized; conferences were organized, and many more scientific publications were produced on the subject. From 1994, the International Organization for Standardization (ISO) was also involved in the field of life cycle analysis and in 1997 published, for the first time, its ISO 14040 and 14044 standards on the harmonization of procedures [20,21]. From the beginning of the twenty-first century, interest in LCA and reflections on the complete life cycle are increasing. Many more scientific studies are being published. Today, LCA is recognized as the most successful and objective multi-criteria assessment tool for environmental impacts, on the entire building scale [22,23]. In Belgium and several European countries, most of the studies on LCA in the construction sector concentrate on the building level [23–25]. In the literature, it is important to note that many studies on LCA have several limitations: some use a single scale when analyzing the reduction of the environmental footprint in the construction sector; others use only one indicator (energy demand) to conduct a study within a building; while others focus on a single stage of the life cycle (the occupation stage). To deepen this study, we carried out this work by pushing the reflection further. Thus, we will no longer work on the scale of a single building, such as many studies, but on the neighborhood scale. We will not study a single indicator, but more than ten. We will not focus on one step, but we will study the whole life cycle. In Belgium, the first thermal regulation was born in Wallonia in 1985. The EPB Directive—European Directive "Energy Performance of Building Directive" (EPBD) (European Parliament, 2002) —has been applied in Belgium since 2008 and was regularly reinforced in the years that followed. It is important to note that European regulations have the merit of reducing the energy consumption of buildings during their occupation phase. However, they focus only on this phase. All other phases of the life cycle—the extraction of raw materials, production, choice of building materials, their transportation and even their recycling at the end of their life cycle—are not taken into account. Furthermore, the there is a lack of integration and following of different requirements, which is responsible for an asymmetry of compliance in the member countries of the EU. Thus, our study has a goal to go beyond the occupation phase and take into account the complete life cycle. In addition, these regulations are interested in only one aspect, which is the consumption of energy. We want to study the set of different environmental impacts that are significant and known. Finally, the current studies on which the energy standards are based are often carried out only at the scale of the building. We want to broaden the reflection at the neighborhood level, as it is clear to us that the environmental issues of tomorrow will be resolved at the urban scale. We believe that this type of approach is the logical continuation of the current regulations and that it is important to take the plunge. It is thanks to this type of analysis, from the cradle to the grave, that one can judge the real and lasting aspect of a construction. Indeed, we can expect that the environmental cost of energy will decrease as well as the consumption in the building sector. As a result, the relative share of non-occupancy phases in the overall environmental balance will continue to increase. This research proposes a more efficient method for analyzing the life cycle at the scale of a neighborhood, and compares the results obtained with those of other existing research. The design and analysis of several scenarios allow us to assess several important characteristics of the LCA applied to an eco-neighborhood. ### 2. A Review on Current Researches Regarding Life Cycle Analysis in the Building Sector The life cycle assessment (LCA) method is a clearly validated scientific method and is even standardized at the European level [20,21]. The LCA allows one to carry out different types of comparative studies [23], for example: (i) comparison of two entire systems or part of their life cycle; (ii) comparison between different phases of the cycle life; (iii) comparison of two different versions of the same system; and (iv) comparison of a system with a reference. The method also makes it possible to quantify an environmental impact on the complete life cycle of a product or only on one stage of the cycle without necessarily making a comparison. Thus, it is a tool that can not only serve as a decision aid, but also allows targeting of the phases of the life cycle of a product, which would need to be reworked with attention paid to the environment. The normative framework of the LCA [20,21] defines four different steps to follow: (1) the definition of the objective of the study; (2) the "Life Cycle Inventory" (LCI); (3) the "Life Cycle Impact Assessment" (LCIA); and (4) the interpretation of results—all these phases are organized independently and iteratively. Specific standards were established for the LCA of the building sector by the European Committee for Standardization (CEN) in 2011: EN 15978 [26] and EN 15643-2 [27]. These standards are increasingly used to define and/or reduce the impacts of buildings on the environment. It is currently the only scientifically sound approach to carry out an environmental assessment at the building scale. It allows a quantitative study of the construction over their entire life cycle. However, its use at the urban or neighborhood scale is recent [28,29]. Despite the novelty of the LCA application at the neighborhood level, it is considered the most reliable method. It is a challenge and a fascinating research topic to test the application of the LCA method to an eco-neighborhood, especially since no other study to our knowledge has focused on the comparison of so many parameters and environmental indicators at the community level. Note also that many sustainable building certification schemes are based on the LCAs of the building materials [30], such as BREEAM, DGNB and Valid in Belgium.	doab	2025-04-07T03:56:58.083608	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 26 }
001fcd9a-cd27-45e8-b23f-f447874e0974.27	2.1. Building Scale Several studies in different countries studied LCA at the building level. In 2011, Rossi et al. [25] compared the LCA of the same building located in three cities distributed in three different European countries and climates: Brussels (Belgium), Coimbra (Portugal) and Luleå (Sweden). A difference of less than 17.4% was obtained on comparing the operational energy and carbon. Stephan et al. [31] analyzed the life cycle energy use in a passive building in Belgium and, then, carried out a comparison with other building types. The results showed that new techniques of construction had to be applied for improving the house energy efficiency. The passive house embodied energy accounts for 55% of the total energy on the 100-year life cycle. Cabeza et al. [32] gave a review of the life cycle assessment (LCA), life cycle energy analysis (LCEA) and life cycle cost analysis (LCCA) in numerous kinds of buildings, located in different countries with varied climates. The results showed that few of the LCA and LCEA studies were carried out in traditional buildings. In their research, Kellenberger and Althaus [33] carried out the LAC of many house components (roof, wall, etc.), with the purpose of evaluating the performance of the materials. The transportation of the building materials and other parameters were also studied. For deepening the knowledge of the environmental characteristics of the building materials and energy, Bribián et al. [34] applied three environmental impact categories for comparing the most used material in the new designs. The results showed that the impact of a material can be significantly reduced by applying the new methods of eco-innovation. Vilches et al. [35] showed that a majority of the LCA was based on energy demand compared at every stage of the life cycle. This research focused on the environmental impact of buildings system retrieval. A strong review on the life cycle energy analyses of buildings from 73 cases, applied in 13 countries and taking into consideration office and residential buildings, was shown by Ramesh et al. [36]. Rashid and Yusoff [37] assessed the phase and material that significantly affected the environment. In the research carried out by Chau and Leung [38], the results showed that the use of different functional units did not allow easy comparison of the studies found in the literature.	doab	2025-04-07T03:56:58.084448	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 27 }
001fcd9a-cd27-45e8-b23f-f447874e0974.28	2.2. Neighborhood Scale We will now broaden the field of study and move to the neighborhood scale as a whole. Now that much progress has been made on the energy consumption of new buildings, other issues emerge [39]. We emphasized the importance of focusing on phases other than occupation, whose relative impacts increase with a decrease in consumption [40]. It is also necessary to tackle the thermal renovation in a more serious way. Beyond the building scale, the concepts of a zero-carbon city, a city without CO2 or a post-carbon city are emerging around the world. Cities are now welcoming 50% of humanity. However, energy is not the only environmental problem. Cities are aware of the need to preserve biodiversity and green spaces [41]. To achieve these different objectives, new tools and methods are needed, to be able to measure, at an urban scale, the consequences of architectural and urban choices on the environment [41]. Many methodologies exist to quantify the environmental impacts at the city scale, but according to Anderson et al. [42], LCA is again the dominant method at the urban scale. Indeed, after the study of the different existing methods, Loiseau et al. [43] showed that LCA provides an appropriate framework and is the only method to avoid transferring environmental loads from one phase of the life cycle to another, from one environmental impact to another, or from one territory to another. There is currently a need at the neighborhood level to integrate reflections on bioclimatic design, shared facilities, urban density or mobility issues, in order to achieve better environmental performance. Olivier-Solà et al. [44] explained that it is highly likely that the environmental and energy issues we are currently dealing with at the building level will soon be transferred to the urban scale. Thus, neighborhood-level LCA is starting to get into practice. Some works and publications concerning this method have been written but they remain rare and heterogeneous [45]. Some studies carried out by Ecole des Mines ParisTech within the energy and process center aim to scientifically develop the LCA method at the neighborhood level. The goal is even more ambitious because this work aims to make the method a tool for decision support, from the design phase [41].	doab	2025-04-07T03:56:58.084625	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 28 }
001fcd9a-cd27-45e8-b23f-f447874e0974.29	2.3. Goal We want to study various parameters that impact the environmental balance of a neighborhood. We considered several environmental indicators that we detailed. We wished to identify the most important parameters that have the greatest impact on the environmental quality of a neighborhood. This may include, for example, orientation, the presence or absence of permeable soils, renewable energy sources or integration with public transit systems. Even if the general influence of some of these parameters was known, we wished to quantify precisely the environmental impacts and compare their importance with that of the other studied parameters. For this, we conducted the environmental analysis of a neighborhood and we varied the different design parameters to quantify their impacts. Thus, we were able to provide recommendations regarding certain design choices and their potential environmental impacts.	doab	2025-04-07T03:56:58.084906	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 29 }
001fcd9a-cd27-45e8-b23f-f447874e0974.30	3. Methodology This study is constituted of many important sections, such as (a) the survey; (b) modeling; (c) application of new scenarios; and (d) analysis.	doab	2025-04-07T03:56:58.084991	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 30 }
001fcd9a-cd27-45e8-b23f-f447874e0974.31	3.1. Location This study was carried out in a neighborhood located in Liege city in Belgium. This city is dominated by a continental climate in a temperate zone. During the year, we can note four seasons: winter, autumn, summer and spring. The neighborhood evaluated in this study is located nearby the University of Liege. This site is home to an extension of Liege University, and heavily dominated by green space, which is shown in Figure 1. Several categories of buildings are found in this new neighborhood. Notably, there are apartments with two, three and even four facades. Most of these surfaces were developed for social housing. Figures 2 and 3 show the location of this neighborhood. Figure 1. Cont. Figure 1. (a) Life cycle assessment (LCA) stage according to ISO 14044 [21]. (b) 3D modeling of some habitats in the studied site. Figure 3. Chaining analysis software. The neighborhood studied was newly built by the Belgian government by adopting the concept of sustainable development.	doab	2025-04-07T03:56:58.085028	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 31 }
001fcd9a-cd27-45e8-b23f-f447874e0974.32	3.2. Structure Analysis In this city, several residences were considerate with respect to the energy demand suggested by international standards [46,47]. A total of 50% of the studied residences are semi-detached. This eco-neighborhood was built on a 3.51 ha plot. It has 17,000 m<sup>2</sup> of totally green space, and a total of 219 people in the residences. The life cycle assessment of the neighborhood is fixed at 80 years in the case of this study. We have taken some environmental data from the ECOINVENT database.	doab	2025-04-07T03:56:58.085112	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 32 }
001fcd9a-cd27-45e8-b23f-f447874e0974.33	3.3. Simulation Tool In this study, we used Pleiades software, version (4.19). It is divided into six modules: Library, Modeler, BIM, Editor, Results and LCA. Indeed, these tools were applied in several publications [48–56]. The analysis chain was as shown in Figure 3. Other different details regarding these simulation tools are also given in [57–60]. Under the base of the modeler tool of this software, it is easy to model all the buildings with their main characteristics. It is also possible to make the first simulations. All the results are automatically saved in the "result module", which will be requested to evaluate the ACV of the neighborhood. The analysis of an LCA is not easy, because we must associate any constituent of the neighborhood in the software (buildings, roads, garden, water, people, climate, waste, energy mix, etc.). The environmental impact of all the main elements of the site is automatically added to form the global neighborhood. #### 3.4. Scenarios Some methods applied in this research were found in [54–60]. Globally, in this study, numerous scenarios were established, such as (1) building orientations; (2) water management; (3) mobility; (4) density; and (5) photovoltaic solar installation. It was very important to know the impacts of all these scenarios for improving the future planning of the new neighborhoods. #### 3.5. Modeling We began the modeling of our study area by studying the project's characteristics data and the graphic modeling of the buildings on the site. A note was made of the geometrical parameters attributed to each of the walls of the buildings and their thermal properties, and the zoning and scenarios of use were also defined. Once all the parameters were defined, the dynamic thermal simulation calculations were started. All the characteristics of the buildings are described in Table 1. It was necessary to model some elements of our buildings, such as the walls, joinery, surface conditions and thermal bridges. With regard to the walls, we not only reveal the materials and elements of construction, their thickness, and their characteristics, but also the possible thermal bridges. At this stage, we have modeled the actual walls of the project with their precise characteristics. It is also necessary to obtain information on the surface state of the different walls, in order to manage their behavior with respect to radiation. Tables 2 and 3 show the characteristics of the heat transmissions of the frame and glazing, as well as the thermal bridges. Table 1. Wall composition. Thickness (e), the mass per unit area (ρ\e), thermal conductivity (λ) and thermal resistance (R). Solar factors (Sw) and light transmission factors (Ti). The hourly temperature data, global and diffuse horizontal radiation, wind speeds, relative humidity, atmospheric pressure and precipitation of the studied sites, over the last forty years, were downloaded from American satellites by the Meteonorm software, and subsequently converted so as to implement them in the Pleiades software. We have modeled the walls, floors, slabs, roofs, openings and solar masks (Figure 4). The geometries of the buildings and the actual openings have been scrupulously valued. The significance of modeling the neighborhood in three dimensions is that we now take into account the orientation and the solar masks that different buildings make on each other. In this manner, we will be able to study the impact of a change in the orientation of the mass plan or the increase in height of certain buildings. Table 3.* Characteristics of the thermal bridges. Figure 4. View of the 3D model of the neighborhood as presented in the Pleiades software. #### 3.6. Other Input Data Several important results were obtained after simulation, such as (i) the detailed characteristics of all the simulated residences; and (ii) the different needs related to the consumption of water and energy. The lifespan of the different building materials was set at 80 years, such as those of the buildings. There were different impacts resulting from the renovation phase. This different energy data were analyzed under the reference of the Belgian energy mix integrated in the software. According to the report of the International Panel of Climate Change in 2016, the Belgian energy mix is set at 4% coal, 27% natural gas, 17% renewable and 52% nuclear. It was important to notice that the consumption related to heating and domestic hot water (DHW) were calculated using the most recent data. The supply system was a natural gas condensing boiler, having a 92% lower heating value (PCI) efficiency. The water consumption was estimated at 100 L/occupant/day. In the case of waste disposal, the new waste sorting policy was applied for this purpose (less waste.wallonie.be), which was set at 90% for glass waste and 75% for the paper and cardboard. This percentage of waste was applied as recycled, and not buried. With regard to the different Belgian statistics, it is found that 40% of the 1500 g of daily household waste per occupant are directly sent for incineration with an estimated yield of 85%, with the distance between the site and the landfill being 10 km, 100 km to the incineration plant and 50 km to the recycling site.	doab	2025-04-07T03:56:58.085169	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 33 }
001fcd9a-cd27-45e8-b23f-f447874e0974.34	3.7. Orientation Scenario We studied the different orientation effects of buildings at the neighborhood level. Initially, all the buildings were installed so as to more easily orient the different facades towards the south and the north. We have called this "scenario o". Subsequently, we tried this test on several other orientations by guiding the mass plane to successive rotations of 45◦, 90◦ and even 180◦. Subsequently, we calculated the standard deviation of all the buildings studied affected by each of its orientations. We chose the most unfavorable orientation to perform the LCA analysis of the neighborhood. Subsequently, we rigorously compared the different results of the new LCA in the neighborhood with that of the central neighborhood in order to evaluate the real effects of orientation on the LCA of a neighborhood.	doab	2025-04-07T03:56:58.085581	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 34 }
001fcd9a-cd27-45e8-b23f-f447874e0974.35	3.8. Water-Use Scenario The main objective of this new scenario is to collect all the rainwater and the discharged directly into a sanitary sewer network. If it were possible by pipeline to recover more than 95% of the rainwater in the different valleys, ditches, and cisterns, then it would be totally useless to evaluate the permeability of the different types of flooring. It is thus paramount to focus on two scenarios: one is based on the different rainwater collection systems and the other one is oriented towards the permeability of soils. ### 3.8.1. Rainwater Scenario In the specific case of this scenario, we modeled all the rainwater tanks. In summary, rainwater was used to clean the interior and exterior of buildings, cleaning instruments, etc. In addition, reclaimed rainwater was fed from a separate network of reservoirs, ditches, valleys and water bodies. Garden water was collected by several ditches and turned towards the water. Rainwater from the roof was directly poured into the tanks. We assumed that all the rainwater from this place of study was controlled by a separate network.	doab	2025-04-07T03:56:58.085678	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 35 }
001fcd9a-cd27-45e8-b23f-f447874e0974.36	3.8.2. Permeable Floors Analysis In this scenario, we implemented more permeable floor coverings than in the basic option. In this manner, aisles, squares and car parks are constructed with unrepaired concrete pavements and concrete–grass slabs. Thus, the total impermeability of the site goes from 66% in the initial state to 58% once the permeable coatings are implemented. This small difference between the average permeability of the two scenarios is explained by the high proportion of green spaces in our neighborhood that do not see the modified permeability between the two scenarios. The large proportion of green areas of the site explains its relatively high permeability from the initial state. In this scenario, we consider that no other rainwater harvesting system be implemented. All of the water does not infiltrate directly into the soil; it is sent to the wastewater network.	doab	2025-04-07T03:56:58.085787	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 36 }
001fcd9a-cd27-45e8-b23f-f447874e0974.37	3.9. Urban Mobility Impact Analysis Let us now look at the impact of mobility on the neighborhood's environmental record. In our basic scenario we considered a significant use of the car for daily commuting. We will compare this scenario with a second one, where the site is considered urban, perfectly integrated with public transport networks and at a short distance from the shops of primary needs. Let us recapitulate the mobility hypotheses: (i) Initial scenario: 80% of the occupants commute daily; the 20 km distance from home to work is carried out daily by car; and the 5 km distance from home to the shops is done weekly by car. (ii) "Urban Site" scenario: 100% of the occupants make the trip, daily; the 2.5 km distance from home to work is done daily by bus; the 300 m distance from home to the shops is carried out weekly by bike or on foot.	doab	2025-04-07T03:56:58.085881	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 37 }
001fcd9a-cd27-45e8-b23f-f447874e0974.38	3.10. Urban Density Impact Analysis The purpose of these scenarios is to analyze the different effects of density on the life cycle of a neighborhood.	doab	2025-04-07T03:56:58.085958	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 38 }
001fcd9a-cd27-45e8-b23f-f447874e0974.39	3.10.1. Vertical Scenario We introduce another floor to each building. The configuration of the neighborhood remains the same. Overall, we are increasing the number of occupants as well as the construction area of our new neighborhood. The new district will have 100 more inhabitants than the old district.	doab	2025-04-07T03:56:58.085999	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 39 }
001fcd9a-cd27-45e8-b23f-f447874e0974.40	3.10.2. Horizontal Scenario The objective is to assess the impact of horizontal densification. For this, we decided to add some residential buildings on the used area (see Figure 5). In total, four buildings were added. The total population is identical as in the reference scenario, which allowed us to keep the same configuration as that of the vertical density scenario. To achieve this goal, we had to occupy a small part of the public parking space. All the buildings added had the same characteristics as those existing; this in order to better compare these two methods and choose the best. Figure 5. View of the 3D model of the neighborhood in the "density +" (horizontal density) configuration as presented in the Pleiades software (openings only appear on the selected building). ### 3.11. Urban Renewable Energies Use Impact In the initial scenario, all the electricity used came from the Belgian electricity grid and the production impacts were taken into account. In this new configuration, we will have a photovoltaic system on all the roofs on the site, and we consider having a panel area equivalent to two-thirds of the roof area of each building. It must be noted that our homes use electricity only for light and to power household appliances. The installed installation will consist of mono crystalline photovoltaic solar panels. The sensors will be placed using a support on the roof terrace. They will be oriented south and inclined at 35◦, the optimal inclination in Belgium. We then performed the thermal simulation of each building and completed the final LCA of the neighborhood.	doab	2025-04-07T03:56:58.086056	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 40 }
001fcd9a-cd27-45e8-b23f-f447874e0974.41	4. Results In this study, we obtained a heating requirement of 15.4 kWh/m2·year. The main requirement for meeting passive standards is to have a heating requirement of less than 15 kWh/m2·year. We notice that some buildings do not respect the passive standard. This may be due to their wrong orientation. The average results of the LCA under building scales are shown in Table 4. These results showed that in the studied buildings, after 80 years, the average greenhouse gas was expected to be 2586.1 tCO2 eq, while the cumulative energy demand would be 73,935.2 GJ. Table 4. Average LCA results for the buildings in terms of calculated impacts. The radioactive waste would increase to 76.5 dm3. The waste water was from 58.593 m3 /inhabitant per year. According to the research of Marique and Reiter [24], heating energy was estimated to be between 190 and 200 kW h/m2, in the case of the conventional neighborhood. In this case, the heating energy is around 16 kW h/m<sup>2</sup> as requested by several international standards. Moreover, according to Lotteau et al. [29], the greenhouse gas is between 11 and 124 kgCO2/m2; in this research, it is around 35 kgCO2/m2. This means that the results found in this research are in the range given in the literature. The average odor concentration was 32.162 Mm3air/inhabitant per year. Table 5 shows some simulation results of the LCA on the neighborhood scale. It was seen that the total greenhouse gas was expected to be 21,733.64 tCO2 eq after 100 years, whereas the total cumulative energy demand was 532,385.49 GJ. The health damage was 22.29 DALYS (disability-adjusted life years), and the potential for degradation related to land use obtained was 28,630.00 m2/year. DALYs are the sum of the YLDs and YLLs, per disease category or outcome, and per age and sex class: $$\text{DALY} = \text{YLD} + \text{YLL} \tag{1}$$ where YLD (the morbidity component of the DALYs) = number of cases \* disease duration \* disability weight; YLL (the mortality component of the DALYs) = number of deaths \* life expectancy at age of death. It was interesting to notice that the total eutrophication assessed was 42,794.03 kg PO4 eq. The analysis of the most important sources of impact (greenhouse gas and cumulative energy demand) and their distributions according to the different stages of the life cycle is given in Figures 6 and 7. In Figure 6, we notice the strong predominance of the occupation phase, which concentrates on 93% greenhouse gas production. In this phase, mobility is strongly in the majority with 46% emissions. Heating and domestic hot water accounts for 24% of emissions, while waste treatment accounts for 15% of the emissions during the use phase. Only 1% of greenhouse gas comes from a "public space". It is interesting to note that emissions from the household waste management are equivalent to those from the production of hot sanitary water (15% of the use phase emissions), whereas the emissions from heating accounted for only two-thirds of the emissions, from the production of hot sanitary water. Table 5. LCA results at the neighborhood scale (initial case). Figure 6. Detailed results of the calculation of the "greenhouse effect" impact at the neighborhood level (initial case). Figure 7. Detailed results of the "cumulative energy demand" impact calculation at the neighborhood scale (initial case). Emissions due to the mobility of the inhabitants accounted for almost half of the emissions from the use phase. These characteristics were perhaps due to the fact that the high thermal performance of our buildings greatly reduced their heating consumption. Figure 7 showed the impact of cumulative energy demand defined in [61] .As in the previous Figure 6, it was seen in this figure that the use phase was predominant (96% of the cumulative total energy demand). This could be due to the accounting for mobility and waste management. The cumulative demand for energy due to waste management was almost identical to that due to the mobility of residents and was equivalent to almost one-third of the demand of the occupancy phase. In addition, this was because the cumulative energy demand from transportation and waste management during the use phase was 60% of the total cumulative energy demand of the neighborhood, over its entire life cycle. Meanwhile, the cumulative energy demand due to "the heating and domestic hot water" was only half of that required for the transport of inhabitants or the management of household waste. These different results show a very strong participation of the mobility component and the household waste management component in the LCA at the neighborhood level. #### 4.1. Orientation Impact Assessment This section studied the orientation impact assessment of the LCA outcomes at the neighborhood level. Figure 8 showed the comparison of the environmental impacts of the established scenarios to "0◦ orientation" and "90◦ orientation", in percentage. We noted that once all the neighborhood-level impacts were accounted for, the influence of the orientation became minimal. Indeed, it was mainly on the greenhouse effect, on the cumulative demand of energy, and on the depletion of the abiotic resources that the orientation had an important effect. This was due to the change in energy consumption due to heating. However, we observed only a relative increase of less than 1% of these impacts. Moreover, this evolution only affected the phase of use. On the other hand, we observed a 1% increase in greenhouse gas emissions, as well as the depletion of abiotic resources and cumulative energy demand during the use phase in the case of a rotation to 90◦. This is could be due to the increase in gas consumption caused by the increase in heating needs. Figure 8. Comparison of the environmental impacts of the "0◦ orientation" and "90◦ orientation" scenarios (functional unit: entire neighborhood), in percentage. Comparing the scores of the environmental indicators only for the heating items during the use phase, as also for both orientations, we notice an 11% increase in the greenhouse effect, as well as a cumulative energy demand and depletion of abiotic resources for the 90◦ orientation. Thus, the orientation has an impact on heating consumption and on environmental indicators relating only to these [62–65]. However, at the neighborhood level, this orientation has little impact on the overall results of the LCA. However, even if the orientation has little influence on the LCA results at the neighborhood level, at the building level it can be decisive, especially for obtaining the passive label. The different quantities of environmental impacts are shown in the Table 6. Table 6. Details of orientation scenario per square meter. (1) Greenhouse gas; (2) acidification; (3) cumulative energy demand; (4) waste water; (5) waste products; (6) depletion of abiotic resource; (7) eutrophication; (8) photochemical ozone production; (9) biodiversity damage; (10) radioactivity waste; (11) health damage; (12) odor. ### 4.2. Water Management Impact Assessment In Figure 9a, we note that setting up rainwater harvesting systems has a strong impact on certain environmental indicators. Indeed, collecting all the rainwater can reduce eutrophication by 32%. This significant decrease is due to the fact that the runoff water is entirely recovered on the site by the valleys and infiltration basins. Thus, the nutrients are not strained, but retained on the site. On the other hand, it was noticed that drinking water consumption is also strongly impacted. Indeed, with a well-sized tank, it is possible to use only rainwater to feed the washing machines and flushes with water. This will save drinking water up to 6000 L per person per year, which implies a 14% reduction in water consumption of the neighborhood on a scale of its total life cycle—a 7% decrease in waste produced over the entire life cycle of the neighborhood. Indeed, on the use phase, 15% less waste is produced. This is the runoff water that is no longer directed to the treatment plants, and therefore no longer needs to be treated. Moreover, we have observed a decrease of about 4% in damage to biodiversity, damage to health and acidification. The analysis in Figure 9b shows that the impact of soil permeability on the total LCA of the neighborhood is lower. In fact, the concerned indicators are still eutrophication and waste production. In this case, the use of permeable soils reduces the impact of eutrophication by 5% and the production of waste by 1% over the entire life cycle of the neighborhood. In fact, the amount of water that infiltrates into the ground, thanks to the permeable pavements, is less than the quantity that can be recovered by the recovery systems presented in the previous scenario. Figure 10 shows the comparison of the three scenarios (initial scenario, and with and without permeable floor coverings). The analysis of this figure shows that it is more efficient to install recovery systems like cisterns, valleys or infiltration basins at the neighborhood level. However, implementing permeable floor coverings on areas that cannot benefit from recovery systems will have a positive impact on the amount of wastewater to be treated and on eutrophication. Figure 9. Comparative diagram of the environmental impacts of scenarios with and without rainwater harvesting systems (a), and with and without permeable floor coverings (b) (functional unit: entire neighborhood), in percentage. Figure 10. Comparative diagram of the environmental impacts of the initial scenarios, with and without permeable floor coverings (functional unit: entire neighborhood), in percentage.	doab	2025-04-07T03:56:58.086200	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 41 }
001fcd9a-cd27-45e8-b23f-f447874e0974.42	4.3. Mobility Flow Impact Assessment This section analyzed the impact of mobility on the neighborhood's environmental record. Figure 11 shows an analysis of the environmental impact on the mobility scenarios. It is seen that all environmental impact indicators are reduced from 6% to 50%. Seven indicators out of 12 are reduced by more than 20%. Thus, it was concluded that mobility has a significant impact on the neighborhood's environmental record. "Photochemical ozone production" is reduced by more than 50% over the entire life cycle of the neighborhood. In fact, the combustion of fuels is the main source of nitrogen oxide production, which transforms into ozone under the effect of sunlight [64]. In our urban site scenario, 54% of photochemical ozone production in the use phase is avoided, by reducing the use of automobiles. Indeed, 95% of transport-related ozone production during the operational phase is avoided in this scenario. Another photochemical ozone production station is waste management. The previous figure (Figure 11) shows the same observation with the "greenhouse gases". Indeed, a decrease of 40% of the emissions is observed on the total life cycle of the neighborhood, thanks to a decrease of 93% transport emissions during the use phase. On the other hand, it is interesting to note that "acidification" has also been strongly impacted by the suppression of automobile use. We have observed a 35% decrease in this impact indicator over the entire life cycle of the neighborhood. It is the same for "depletion of abiotic resources" and "damage to health", which saw their score reduced by 34% and 32%, respectively. Indeed, much less fuel and fossil resources are consumed and the pollution responsible for many health problems is also greatly reduced. Figure 11. Comparative diagram of the environmental impacts of mobility scenarios (functional unit: entire neighborhood). For example, mobility and the use of personal vehicles to carry out daily commuting distances have a huge impact on the neighborhood's environmental record. Climate impact indicators are the most affected. It is possible to reduce them by half. The cumulative demand for energy, acidification, depletion of biotic resources and damage to health can be reduced by a third, thanks to a mobility scenario. Decreasing the use of cars can create huge savings in energy. Public transport uses the energy contained in fuels in a more efficient and rational manner. Thus, the cumulative energy demand is reduced by 28%. It is also shown that there has been a 23% decrease in damage to biodiversity, 17% in eutrophication, 15% in radioactive waste, 13% in odors and 6% in waste produced. Some data are showed on Table 7. (1) Greenhouse gas; (2) acidification; (3) cumulative energy demand; (4) waste water; (5) waste products; (6) depletion of abiotic resource; (7) eutrophication; (8) photochemical ozone production; (9) biodiversity damage; (10) radioactivity waste; (11) health damage; (12) odor. Detailed responses on the urban mobility are showed in Table 8. (1) Greenhouse gas; (2) acidification; (3) cumulative energy demand; (4) waste water; (5) waste products; (6) depletion of abiotic resource; (7) eutrophication; (8) photochemical ozone production; (9) biodiversity damage; (10) radioactivity waste; (11) health damage; (12) odor. ### 4.4. Density Impact Assessment Table 9 estimates the heating requirements of the various buildings in the study area. Table 9. Heating requirements of the different neighborhood buildings in the basic and high configuration of a floor. Analysis of this data showed that the heating requirements with an additional floor dropped slightly. We thought that the additional shading created should act as solar masks, which would reduce solar gain and increase heating needs. However, it seemed that the increase in compactness caused by the rise of the buildings was more impacting. Figure 12 shows the comparative diagram of the environmental impacts of the scenarios. In Figure 12a, the results are expressed on the basis of a functional unit encompassing the entire neighborhood. This is because the indicator scores had all increased in fairly similar proportions, from about 25% to 30%. Indeed, the share of the indicators related to the buildings was modified, but not that related to the district, which remained unchanged. This functional unit did not allow us to draw any interesting conclusions. This is why we are going to translate the results of the study into the "Occupant" functional unit, to be able to compare per capita impacts in both configurations. As shown in Figure 12b, if we compare the environmental indicators by reporting them to the number of inhabitants, we notice that the high-rise one-story has a better environmental performance. The odor indicator is reduced by 26% and eutrophication by 19%. The other ten indicators are reduced between 11% and 15%. Indeed, even the site welcomes more occupants and the consumption by these added to the initial consumption, all impacts from the site itself and public spaces remain unchanged. Thus, the built surface is more profitable. In the case of an increase in density built by adding buildings to the site (Figure 13a), the results were not as favorable as in the previous case. In fact, apart from odors, radioactive waste and eutrophication, the scores of which decreased by 21%, 3% and 10%, respectively, the other indicators had increased. They all earned between 1% and 5%. Indeed, we did not benefit here from a gain in compactness and we did not pool the networks. In addition, the construction of new buildings was greener in materials and energy than the rise of a floor. The analysis of Figure 13b showed that densifying the neighborhood vertically was more remarkable environmentally. The impact on the total LCA of the district was much more pronounced than during horizontal densification, for which the assessment was mixed. Figure 12. Comparative diagram of the environmental impacts of: (a) "Initial" and "Vertical Density" (functional unit: occupant); and (b) "Initial" and "Density +" scenarios (functional unit: entire neighborhood). Figure 13. Comparative diagram of the environmental impacts of the "Initial" and "Horizontal Density" scenarios (a); and "Initial", "Horizontal Density" and "Vertical Density" (functional unit: occupant) (b). Some results are showed on the Table 10. Table 10. Vertical and horizontal density scenarios. (1) Greenhouse gas; (2) acidification; (3) cumulative energy demand; (4) waste water; (5) waste products; (6) depletion of abiotic resource; (7) eutrophication; (8) photochemical ozone production; (9) biodiversity damage; (10) radioactivity waste; (11) health damage; (12) odor. #### 4.5. Impact of Renewable Energy Uses Taking into account the dynamic thermal simulation, the consumption and electricity production were calculated. For all buildings, production exceeded consumption throughout the year, except for the months of December and January, where the installation covered 45% and 75% of the consumption, respectively. In fact, the buildings consumed, on an average, 12 kWh/m2 of electricity per year. These results were consistent with the Belgian averages for dwellings that did not heat up with electricity. Photovoltaic panels produced an average of 26 kWh/m<sup>2</sup> over the year. Thus, except for the months of January and December, no electrical energy was drawn from the Belgian network. The effects on the LCA of the neighborhood are presented in Figure 14. Of all the configurations studied, the one comprising the addition of photovoltaic panels is the one that produces the most heterogeneous results on the neighborhood's LCA. Indeed, some indicators are greatly reduced, while others see their score increase considerably. The most affected impact is the production of radioactive waste. Over the entire life cycle, the production of radioactive waste is reduced by 102%. Indeed, even if this production of waste increases during the construction (9%) and renovation (1893%) phases, because of the impact of the manufacture of panels, the use phase makes up for this delay. The enormous increase in the usage phase score is explained by the fact that the panels are changed every 20 years and that in the previous scenario, the production of radioactive waste of this phase was insignificant. That being said, the production of radioactive waste during the use phase decreases by 127%. This is explained by the fact that production is higher than consumption. As a result, not only is the construction and maintenance of the system offset, but the production of radioactive waste from the use phase is also eliminated. Moreover, it allows other homes to benefit from the clean energy produced. Thus, our neighborhood reduces the production of radioactive waste from other neighborhoods, which gives a negative score for this indicator. The second-most impacted indicator is the cumulative demand for energy. The total energy needed by the neighborhood to operate over its entire life cycle is reduced by 37%. Once again, the construction and renovation phases are negatively impacted. The construction phase saw its energy consumption increase by 75% and the renovation phase by 978%, due to the manufacture of the panels. However, the occupation phase saw its demand decrease by 47%. The depletion of abiotic resources and the greenhouse effect also decreased by 14% and 12%, respectively, over the entire life cycle. The evolution of the indicators once again followed the same pattern: a significant increase in the construction and renovation phases. However, once again these increases are offset by a reduction in the environmental impact of the use phase, the most impactful phase of the life cycle. We observed a 25% drop in greenhouse gas emissions over this phase and a 26% decrease in the depletion of the abiotic resources. Conversely, some indicators see their score increase. This is the case of the production of waste. The renovation phase saw its waste production increase by 742%. In fact, 4400 m<sup>2</sup> of the panel area had to be replaced thrice over the neighborhood's life cycle and in addition included their initial installation. The 15% decrease in waste production during the use phase did not make up for this increase. As a result, the neighborhood's total waste generation over its entire life cycle was up by 21% (Figure 14). Some results are showed on the Table 11. Table 11. Impact of renewable energy. (1) Greenhouse gas; (2) acidification; (3) cumulative energy demand; (4) waste water; (5) waste products; (6) depletion of abiotic resource; (7) eutrophication; (8) photochemical ozone production; (9) biodiversity damage; (10) radioactivity waste; (11) health damage; (12) odor. Finally, paradoxically, the damage done to biodiversity is also increasing. It is again the manufacture and the replacement of the panels which is in question. The impact of the construction phase increases by 229% and that of the renovation phase by 849%. The 6% drop in impact during the use phase does not compensate for these losses. Thus, over the cycle, the damage to biodiversity increases by 18%. ### 4.6. Global Analysis of All the Scenarios In order to classify the different scenarios and define the design parameters to take into account their priority, we calculated the sum of the variations, as a percentage of all the indicators compared to the initial scenario. We chose to apply no weighting but will remove the indicator "odors", which distorts the results by its important variations. Table 12 shows some obtained results. It was noted by analyzing this table that mobility has an impact of 282% of the cumulative decrease on all indicators: vertical density (163%), renewable energies (138%), rainwater harvesting (76%), soil permeability (11%), orientation (4%) and horizontal density (−10%).	doab	2025-04-07T03:56:58.086799	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 42 }
001fcd9a-cd27-45e8-b23f-f447874e0974.43	5. Discussion Overall, it was seen in this study that the installation of photovoltaic panels has a mixed record. Indeed, the installation was heavily oversized. Several results found in this research are similar to those assessed by Lotteau et al. [65,66]. Indeed, those asserted by Lotteau et al. [65] and the divergence of methodology among different researchers with regard to LCA prevented an easy comparison of results at the neighborhood level. However, in the known research, several aspects common to the LCA were studied, such as (i) the operational energy consumption analysis of buildings; (ii) the quantitative analysis of the construction materials; and (iii) the transport requirement analysis and so on. The process ranged from statistical data collection from neighborhoods to detailed simulations based on physical modelling. Mobility management was the most significant element. Indeed, it was the parameter that allowed a reduction in most of the impacts in terms of greenhouse effects, odors, damage to biodiversity and health, acidification, depletion of abiotic resources and photochemical ozone production. The day-to-day use of individual transportation by local residents has a huge impact on the neighborhood's LCA. Eliminating the use of personal vehicles for the benefit of public transport makes it possible to limit the greenhouse effect four times more than to generate all the electricity of the district, thanks to the photovoltaic panels. Thus, mobility management must be one of the issues to be addressed as a matter of priority in any urban reflection. Designing a neighborhood that is sustainable and environmentally friendly, while being disconnected from public transport, is not always the ideal solution. In the past, Mohamad Monkiz et al. [67] also found that mobility management was one of the most important aspects in the LCA study. The criterion of vertical density was also a fundamental element. Increasing the built density of the neighbourhood by elevation of the buildings was environmentally very beneficial. This made it possible to pool many flows, to increase the energy and environmental efficiency of the neighbourhood, and, thus, homogeneously minimize the different environmental impacts. These results were almost similar to those of André Stephan et al. [16], who found that by replacing an area, part suburb, with apartment buildings, allowed to decrease the total energy consumption by 19.6%. An eco-district must therefore have a certain density. One of the criteria for a sustainable neighbourhood covers this aspect and imposes a density of 30 to 40 dwellings per hectare [68,69]. It was found in this study that the implementation of renewable energy production systems showed a significant environmental balance, as was seen in several research results [70]. This method was useful for limiting the production of radioactive waste and for the cumulative demand for energy. However, the manufacture of photovoltaic panel systems has a negative impact on the LCA in terms of damage to biodiversity and waste produced. Thus, their large-scale implementation does not necessarily seem to be a priority, at least not until their manufacturing and recycling processes are cleaner. On the other hand, integrating rainwater harvesting systems into the neighbourhood has been shown to have a strong impact on the results of an LCA, especially in terms of eutrophication and water use. Intelligent rainwater management should be a priority when designing a neighbourhood. Finally, soil permeability and orientation are parameters that can also improve the environmental record of a neighbourhood, but to a lesser extent. As for the choice of applying the concept of horizontal density to the neighbourhood, by adding more buildings, it can be counter-productive. In the studied neighbourhood, it is seen that the annual energy savings and avoided GHG emissions were less significant than those recorded in one neighbourhood of New York City (7.3 GJ and 0.4 metric tonnes). The main results of this research may be of interest to construction companies, public officials and decision makers for applying the environmental criteria to the planning process of new and existing neighborhoods.	doab	2025-04-07T03:56:58.087943	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 43 }
001fcd9a-cd27-45e8-b23f-f447874e0974.44	6. Limitations All scientific research has some limitations. In the case of this study, it was seen that	doab	2025-04-07T03:56:58.088263	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 44 }
001fcd9a-cd27-45e8-b23f-f447874e0974.45	7. Conclusions Despite the complexity and limitations of the LCA method, this tool has proven to fit the needs of this study perfectly. Even as the majority of the LCA study at the building level has been focused on a very limited number of indicators and often only one parameter, we have been determined in studying more than ten indicators and eight scenarios. This wide range of studied parameters has allowed us to make several interesting observations. First is the need to broaden the environmental thinking on the urban scale. The predominance of the impacts due to mobility and waste management in the overall environmental assessment of the district attests to this. We have shown that these typical problems of urban development are to be treated as a priority, given their considerable influence on the LCA of an already energy performing neighbourhood. Thus, once these urban issues are taken into account, the parameters influencing the scale of the building become insignificant. This is the case with guidance, which, as we have observed, has very little impact on a neighbourhood LCA. Given the internal design parameters of the neighbourhood, it is noted that some are more environmentally impacting than others. The density or management of rainwater parameters need to be carefully studied and prioritized, as they have a strong impact on the neighborhood's environmental performance. We have shown that it is highly preferable to densify the neighbourhood vertically rather than horizontally and that rainwater harvesting systems are more efficient than permeable soils. The installation of photovoltaic panels proved to be mitigated from the point of view of sustainability. This study focused on a theme that seemed most urgent in this line of study. However, many other parameters remain to be studied in order to provide designers with the complete lines of conduct. Thus, this study remains open and will be completed at the scale of a great metropolis and a country. Author Contributions: Conceptualization, M.K.N. and S.R.; methodology, M.S.; software, M.S.; validation, M.K.N. and M.S.; formal analysis, M.K.N.; investigation, M.S.; resources, S.R.; data curation, M.S.; writing—Original draft preparation, M.K.N.; writing—Review and editing, M.K.N.; visualization, M.K.N.; supervision, S.R.; project administration, S.R. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors would like to acknowledge and thank the AXA Company for their support of this study, as well as the LEMA laboratory team who helped conduct this study. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:58.088297	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 45 }
001fcd9a-cd27-45e8-b23f-f447874e0974.47	Exposure to Submicron Particles and Estimation of the Dose Received by Children in School and Non-School Environments	doab	2025-04-07T03:56:58.088546	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 47 }
001fcd9a-cd27-45e8-b23f-f447874e0974.48	*Antonio Pacitto 1, Luca Stabile 1,\, Stefania Russo <sup>2</sup> and Giorgio Buonanno 1,3 Received: 8 April 2020; Accepted: 7 May 2020; Published: 9 May 2020 Abstract: In the present study, the daily dose in terms of submicron particle surface area received by children attending schools located in three different areas (rural, suburban, and urban), characterized by different outdoor concentrations, was evaluated. For this purpose, the exposure to submicron particle concentration levels of the children were measured through a direct exposure assessment approach. In particular, measurements of particle number and lung-deposited surface area concentrations at "personal scale" of 60 children were performed through a handheld particle counter to obtain exposure data in the different microenvironments they resided. Such data were combined with the time–activity pattern data, characteristics of each child, and inhalation rates (related to the activity performed) to obtain the total daily dose in terms of particle surface area. The highest daily dose was estimated for children attending the schools located in the urban and suburban areas (>1000 mm2), whereas the lowest value was estimated for children attending the school located in a rural area (646 mm2). Non-school indoor environments were recognized as the most influential in terms of children's exposure and, thus, of received dose (>70%), whereas school environments contribute not significantly to the children daily dose, with dose fractions of 15–19% for schools located in urban and suburban areas and just 6% for the rural one. Therefore, the study clearly demonstrates that, whatever the school location, the children daily dose cannot be determined on the basis of the exposures in outdoor or school environments, but a direct assessment able to investigate the exposure of children during indoor environment is essential. Keywords:** exposure assessment; school; children; number concentration; lung-deposited surface area; dose	doab	2025-04-07T03:56:58.088581	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 48 }
001fcd9a-cd27-45e8-b23f-f447874e0974.49	1. Introduction Many studies highlighted the link between the exposure to airborne particles and health effects, such as respiratory diseases and inflammation [1], cardiovascular diseases [2,3], diabetes [4], higher systolic blood pressure and pulse pressure [5], and decreased cognitive function in older men [6]; in particular, the World Health Organization (WHO) estimated that the overexposure to particulate matter (PM) causes about 4.2 million deaths per year worldwide [7]. Moreover, the WHO has recently classified PM, referred to as outdoor pollution, as a carcinogenic pollutant for humans (group 1) [8–10]. The harmful potential of airborne particles is related to their ability to penetrate and deposit in the deepest areas of human respiratory tract (i.e., alveolar region), causing irritation, inflammation and possible translocation into the blood system, carrying with them carcinogenic and toxic compounds [11–14]. The inhalation and consequent deposition of these compounds are strictly related to the size of the carrying particles: higher deposition fractions in the lungs are characteristics of submicron and ultrafine particles [15]. Moreover, smaller particles are also recognized to translocate from lungs to the cardiovascular system and from there to other organs (liver, spleen, kidneys, brain) [16–18]. In the last years, the attention of scientific studies has shifted from super-micron particles (whose contribution is expressed in terms of mass concentrations of particles smaller than 10 and 2.5 μm, i.e., PM10 and PM2.5) [19,20] to submicron and ultrafine particles (UFPs, particles smaller than 100 nm) whose contribution is better related to particle number [21,22] and surface area concentrations [23,24] than mass concentration. In fact, many studies highlighted that dose-response correlation in terms of human health effects is better related to surface area of particles deposited in the lungs than other metrics of exposure. To summarize, particle surface area is the most relevant dose metric for acute submicron particle lung toxicity [1,25–32]. In light of this, to evaluate the health effect of the exposure to airborne particles, a critical factor that should be assessed and provided to medical experts is the dose of submicron particles received by individuals [33–35]. Moreover, the airborne particle dose is the main input data for human health risk model [36–39]. Airborne particle doses received by people can be evaluated on the basis of measurements obtained from ad-hoc exposure assessment research. Nonetheless, even though the scientific community is moving from particle mass-based (PM) to number- and surface area-based metrics (submicron particles), the current legislation is still limited to the outdoor concentration of PM10 and PM2.5; such measurements are limited to some outdoor fixed sampling points (FSPs) placed in specific points classified as a function of the type of site (rural, urban, suburban) and the type of station, i.e., proximity to main sources (background, industrial, or traffic) [40–42]. Moreover, PM10 and PM2.5 measurements at FSPs cannot be considered proxies for exposure to submicron and ultrafine particles since they present different dynamics (e.g., dilution, deposition) and origins/sources [43–50]. Indeed, differently from PM10 concentrations that are typically quite homogeneously distributed around the city, the concentrations of submicron particle metrics (number and surface area) are strongly affected by the proximity to the source [51,52]. Finally, the measurement at an outdoor FSP cannot take into account for the exposure in indoor environments; therefore, a proper evaluation of the overall human exposure to submicron and ultrafine particles can be only obtained through personal monitoring able to measure the exposure at a personal scale and also to include the exposure in indoor microenvironments [53–55]. One of the most vulnerable populations in terms of air pollution exposure is represented by children [56,57]. This is due, amongst other things, to their high inhalation rates, resulting in larger specific doses than adults [58–61]. Children use to spend a large part of their day in indoor environments, such as schools and homes. In our previous studies involving adults, we found that some environments and activities affect the total daily dose more than other ones: in particular, the indoor environments were recognized to contribute up to 90% of the total daily dose in terms of particle surface area, with cooking and eating activities alone accounting up to 50% [53,62,63]. Schools as well may be considered a critical indoor environment under certain circumstances, in fact, the long exposure time in schools (children spend from 175 to 220 days and from 5 to 8 hours at school [64]) could significantly affect the overall dose received by children. Actually, the exposure (and then the dose) in school environments is not affected by the presence of submicron particle sources (smoking is typically not allowed and cooking activities are in most of the cases no longer performed in the school) but mainly by the outdoor-to-indoor penetration of submicron particles produced outdoors, which depends on (i) airtightness of the building, (ii) type of ventilation and (iii) particle physical-chemical properties (e.g., size) [65–71]. Therefore, the location of the school, as highlighted in few previous studies [39,72–74], is the main parameter affecting the students' exposure to submicron particles leading to critical exposure scenarios for those attending schools located near highly trafficked urban roads. To the best of the authors' knowledge the dose of submicron particles received by children in school and non-school environments was investigated just in one (our) previous paper [55], but in this study the investigated schools (both in the rural and urban areas) were placed in the same city, thus the possible contribution of the outdoor concentration levels to the daily dose was not adequately deepened. Within this context, the aim of the present research is to evaluate the actual exposure to submicron particles of children attending schools located in different urban contexts and cities (urban, suburban rural sites) and to estimate the corresponding doses received both in schools and in other non-school environments where they spend time. To this end, an extensive experimental campaign was performed by measuring the personal exposure of 60 children (for 48 h each) attending three different schools in Italy, characterized by different outdoor concentration levels, using wearable monitors able to measure particle number and lung-deposited surface area concentrations.	doab	2025-04-07T03:56:58.088743	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 49 }
001fcd9a-cd27-45e8-b23f-f447874e0974.51	2.1. Study Area and Monitoring Site Children considered in the experimental campaign attended three naturally ventilated schools located in three different cities in Italy (Salerno, South of Italy, Roma, Central Italy, and Parma, North of Italy); the locations of the three schools within the urban contexts are completely different. In particular, the school in Salerno (S1) is placed in a suburban area as it is 1.6 km outside of the city centre, but quite close to a highway. The school in Rome (S2) is located in the urban area, and, in particular, in the proximity of highly trafficked roads, whereas, the school located in Parma (S3) is in the rural area, about 5 km from the city centre, and quite far from trafficked roads. The experimental campaigns in the three schools were performed from November 2018 to May 2019 for about two months in each school as summarized Table 1. Table 1. School sites, sampling periods and summary of the meteo-climatic conditions (temperature, T, relative humidity, RH) and air quality parameters (NO2, PM10 and PM2.5) measured by the closest fixed sampling stations of the Italian environmental protection agency. The data related to every single period of the campaign are expressed as daily average values and their ranges (min–max). In order to better describe the three sampling sites in terms of outdoor air quality, in Table 1, the distance of the closest fixed sampling point (FPS) installed by the Italian environmental protection agency to the schools are reported, as well as its definition in terms of type of station (background, industrial, or traffic) and type of site (urban, suburban rural sites). The closest FPSs to school S1 (100 m), S2 (1 km), and S3 (12 km), are defined as suburban/background station, urban/traffic station, and rural/background station, respectively. The parameters measured by the three FSPs during the three different sampling periods (November–December 2018, February–March 2019, and April–May 2019 for school S1, S2 and S3, respectively) clearly highlight the different outdoor air quality of the locations investigated: indeed, the highest NO2, PM10 and PM2.5 values were measured by the FSP close to the school in Rome (S2) (average values of 42, 29, and 16 <sup>μ</sup>g·m−3, respectively) whereas the lowest, as expected, were measured by the FPS close to the school in Parma (S3) (average values of 12, 12, and 7.5 <sup>μ</sup>g·m−<sup>3</sup> for NO2, PM10 and PM2.5, respectively). The authors, once again, point out that the concentration of the different PM fractions cannot be considered as a good proxy for ultrafine or submicron particles. Indeed, the latter, along with NO2, are good markers of the tailpipe emissions of the vehicular traffic, whereas PM10 is only partially due to tailpipe emissions of vehicles (a significant fraction is due to the traffic-induced particle resuspension) and it is a good marker, amongst others, of biomass combustion for residential heating [75]. Therefore, an overall correlation between outdoor concentrations of PM10 and submicron particles can be found, but, in some conditions (e.g., co-presence of other sources) these two metrics could be poorly correlated. Actually, since the FSPs close to S1 and S2 are strongly affected by traffic sources, a good correlation between PM10 and submicron particles is somehow expected; this is partially confirmed by the fact that NO2 and PM fractions data shown in Table 1 present very good correlations (linear regressions with r2 equal to 0.95 and 0.99 for PM10 and PM2.5, respectively). Finally, regarding the meteo-climatic parameters, temperature and relative humidity values were found to be roughly similar in the three sites during the three measurement periods. This is a not trivial aspect—indeed, generally, the time of the year (e.g., season) can affect the children's exposure and doses both in terms of time-activity patterns and ventilation of the microenvironments since warmer conditions would have increased the time spent outdoor and the manual ventilation in indoor environments (e.g., schools and homes). Thus, the similar outdoor meteo-climatic conditions had a relatively negligible effect on the time of the year on the results. ### 2.2. Study Design To evaluate the surface area dose received by children attending the three schools considered in the present study, particle number (PN) and lung-deposited surface area (LDSA) concentrations and average particle sizes (Dp) were measured by means of a personal monitor, which is a handheld diffusion charger particle counter (NanoTracer, Philips). The children were equipped with the mobile monitor fixed to a belt at the hip for 48 h. During the campaign, 20 children for each school (60 children in total) were monitored. In particular, children aged 6–10 years were monitored (both males and females). Measurements were performed only on school days; weekends were not considered in the study. The authors monitored such high number of children in each school in order to obtain sufficient data that could be representative of the exposure level in each microenvironment where they live/reside. Indeed, the exposure of the children in each microenvironment and during each activity was affected by several parameters, such as the outdoor concentration levels, the volume of the indoor environments, and the presence and the strength of indoor sources (e.g., cooking, smokers, incense, candles etc.). As an example, the children's exposure when they stay in the kitchen during parents' cooking activities is strongly affected by the kitchen volume and the different types of foods and stoves ([76–80]), thus having performed different measurements (on different children) allowed the authors averaging amongst all these influencing parameters. Similarly, the exposure during transport can vary significantly as a function of the transportation modes (i.e., car, walking, bus, etc.; [43,81]), thus, once again, multiple measurements allowed to take into account for all these conditions. In order to estimate the dose, the children, with the support of their parents, were asked to fill in an activity diary to take note about the place, time, and activity performed. A pre-compiled form of the activity diary was prepared by the authors and given to the children along with the portable instrument; the form was prepared considering 15-min time slots (e.g., 00:00–00:15, 00:15–00:30, etc.) in order to make it easy to fill in the forms with the required information. The diary was then used during the data post-processing in order to evaluate the time spent in each activity (i.e., the time-activity pattern) and to determine the exposure during each activity and in each microenvironment. The daily dose of the children under investigation in terms of particle surface area in the tracheobronchial and alveolar regions of the lungs (δ), was calculated as sum of the dose received during the activities performed in the j microenvironments: $$\delta = \sum\_{j=1}^{n} \left\{ IR\_{\text{activity},j} \cdot \text{LDSA}\_{j} \cdot T\_{j} \right\} \left( \text{mm}^{2} \right) \tag{1}$$ where IRactivity (m3·h<sup>−</sup>1) is the inhalation rate of the child, LDSA is the Lung-Deposited Surface Area concentration (μm2·cm−3), and Tj (h) is time spent in each microenvironment. The IRactivity is a function of the age and activity performed by the children; in particular, we have considered the IR data for 6–10-year-old children summarized in Buonanno et al., 2012 [63]. In Equation (1) the term "microenvironment" is used for the sake of simplicity: the activities performed by the children, obtained based on the time-activity patterns, were grouped in six main microenvironments, summarized in Table 2. Particular attention should be paid to the "Cooking & Eating" microenvironment; indeed, children do not perform cooking activities per se, thus, the exposure related to this microenvironment is due to cooking activities performed by the parents. To compare the received dose of the children in different microenvironments, the dose-intensity ratio (i\_δ, mm2·min<sup>−</sup>1), i.e., the ratio between the daily dose fraction and the daily time fraction characteristics of each microenvironment, was also evaluated [53]. Table 2. Classification of the activities performed by the citizens in seven main microenvironments. #### 2.3. Instrumentation and Its Quality Assurance As mentioned above particle number (PN) and lung-deposited surface area (LDSA) concentrations and average particle sizes (Dp) were measured by means of a hand-held diffusion charger particle counter (NanoTracer Philips). It measures the particle number concentration and the average particle size in the range 10–300 nm, with a sampling time of 10 seconds. The operating principle of this instrument is based on the diffusion charging technique. In particular, the sampled aerosol is charged in a standard positive unipolar diffusion charger imparting an average known charge on the particles that is approximately proportional to the particle diameter of the aerosol. The number of charges, and thus the number of particles, is then detected by an electrometer [82–84]. Since over 99% of total particle number concentrations in urban environments are due to particles below 300 nm in diameter [85,86], the instrument was considered adequate for the experimental campaign. Actually, the lung-deposited surface area (LDSA) concentration cannot be considered, strictly speaking, a direct measurement, since it is provided by the instrument on the basis of built-in semi-empiric relationships allowing calculating the particle surface area deposited in the alveolar and tracheobronchial through the PN concentration and average particle size (Dp) measured data as described in details in Marra, et al. [87] and Fierz, Houle, Steigmeier and Burtscher [82]. Then, the LDSA concentration was evaluated as sum of the alveolar- and tracheobronchial-deposited contributions. Nonetheless, in order to take into account for calibrated PN concentrations and Dp values, we have used the semi-empiric relationships to calculate the LDSA concentrations on the basis of the calibrated values. In particular, the calibration of the device was performed before and after each experimental campaign. To this end, both a Condensation Particle Counter (CPC 3775, TSI Inc., Shoreview, MN, USA) and a Scanning Mobility Particle Sizer (SMPS 3936, TSI Inc.) were used to compare the devices in terms of number concentration and particle size, respectively. The SMPS consisted of an Electrostatic Classifier (EC 3080, TSI Inc.), a Differential Mobility Analyzer (DMA 3081, TSI Inc.), and a CPC 3775. The SMPS 3936 was used, with an aerosol/sheath flow ratio of 0.3/3.0 L·min<sup>−</sup>1, thus measuring particle number distributions in the range 14–700 nm. The calibration was carried out at the European Accredited Laboratory of Industrial Measurements (LaMI) of the University of Cassino and Southern Lazio (Italy) in a 150 m3-room, with a conventional mechanical ventilation system guaranteeing constant thermo-hygrometric conditions (20 ± 2 ◦C and 50 ± 5% RH). Comparisons were performed for two different aerosols: aged indoor aerosol and freshly emitted aerosol produced by incense burning. Tests were conducted for 2 h performing simultaneous measurements with the Nanotracer, the CPC 3775, and the SMPS 3936. CPC and SMPS sampling times were set at 1 s and 135 s, respectively. SMPS measurements were corrected for multiple charge and diffusion losses. The correction factors obtained by averaging the results of the two aerosols investigated before and after each experimental campaign were applied as correction factors for each campaign. The differences in correction factors measured before and after the campaigns were found lower than 10%.	doab	2025-04-07T03:56:58.089219	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 51 }
001fcd9a-cd27-45e8-b23f-f447874e0974.52	2.4. Statistical Analysis of the Data In order to perform a statistical analysis of the concentrations experienced by the children in the different microenvironments (in terms of PN and LDSA) a preliminary normality test (Shapiro–Wilk test) was performed to check for the statistical distribution of the data. Since the data did not meet the assumptions of Gaussian distribution, non-parametric tests and further post-hoc tests (Kruskal–Wallis test [88]) were considered in the analysis. The statistically significant result was referred to a significance level of 99% (a p-value < 0.01). In particular, the Kruskal–Wallis tests were performed (a) amongst the six different microenvironments for each group of children separately (S1, S2, and S3; thus 3 non-parametric tests and further post-hoc tests) and (b) amongst the three groups of children for each microenvironment separately (six microenvironments plus the whole day data, thus seven non-parametric tests and further post-hoc tests). The PN and LDSA concentration data considered in the statistical analysis, and then shown in the result section, included all the data provided by the instrument (roughly 48 h of total sampling per each child with a sampling frequency of 10 s), thus, a huge number of values were available for each microenvironment of each children group. On the contrary, the dose values reported and discussed in the results represented the median values (and corresponding ranges) obtained from the 20-dose data (i.e., 20 children) per each microenvironment per each children group. Thus, due to the limited number of dose data, the statistical analysis on such values was not performed as it could led to misleading results.	doab	2025-04-07T03:56:58.090118	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 52 }
001fcd9a-cd27-45e8-b23f-f447874e0974.54	3.1. Time Activity Patterns In Table 3, data on time-activity patterns of the children under investigation are reported, which were obtained from the activity diaries filled in by children and parents during the measurements. The median data demonstrate that children spend the most significant time fraction performing indoor activities in indoor microenvironments: indeed, the median time spent by the children indoor, as sum of the microenvironments labelled as "sleeping", "indoor day", "cooking & eating", resulted equal to 68–69%, to which must be added the time spent at school (25%). On the contrary, the time fraction spent in "outdoor day" (2–3%) and "transport" (3–4%) microenvironments resulted very limited, likely due to the fact that just school days were included in the experimental analysis, thus, the time spent in "transport" microenvironment is mostly limited to the time to take children to school. The huge time spent in indoor environments is consistent with our previous studies analyzing western populations, in which emerged that also adults spend a significant time fraction (roughly 90%) performing indoor activities [53,62,63]. Amongst the indoor activities, the time spent in "cooking & eating" microenvironment (here 8%) is of particular concern since these activities were recognized in our previous papers as the most influencing in terms of exposure and health risk [36,89]. Table 3. Time activity pattern, particle concentrations (PN and LDSA) and dose received by children of the three schools in the different microenvironments expressed as median values and range (5th and 95th percentile). Total daily doses as sum of the median doses received in the different microenvironments are also reported as well as daily dose fractions and intensity–dose ratios.	doab	2025-04-07T03:56:58.090264	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 54 }
001fcd9a-cd27-45e8-b23f-f447874e0974.55	3.2. Exposure to Submicron Particles In Table 3 and Figure 1, the submicron particle concentrations, in terms of particle number and lung-deposited surface area, to which the children attending the three different schools (S1, S2, and S3) were exposed to in the different microenvironments (sleeping, indoor day, outdoor day, school, transport, cooking & eating) are shown. In the box plots of Figure 1, exposure data not statistically different amongst the six different microenvironments for each group of children separately (S1, S2, S3) and amongst the three groups of children for each microenvironment separately are also indicated (p > 0.01) as resulting from the statistical analysis explained in Section 2.4 (Kruskal–Wallis test). Due to the huge amount of data available for each microenvironment, most of the exposure received in the six microenvironments by the same group of children as well as those received in the same microenvironment by the three groups of children resulted in statistically different results. Figure 1. Statistics of (a) particle number and (b) lung-deposited surface area concentrations experienced by three groups of children (attending school S1, S2, and S3) in each microenvironment. Data was not statistically different within each group of children (S1, S2, S3) and amongst the same microenvironments of different groups (\,+) are also indicated (p* > 0.01). The children's exposure to submicron particles in the "school" microenvironment presents a significant deviation amongst the three schools. Indeed, children attending school S1, S2 and S3 were exposed to median PN and LDSA concentrations of 1.57 <sup>×</sup> 104 part. cm−3/66 <sup>μ</sup>m2·cm−3, 2.13 <sup>×</sup> 10<sup>4</sup> part. cm−3/89 <sup>μ</sup>m2·cm−3, and 3.39 <sup>×</sup> 103 part. cm <sup>3</sup>/14 <sup>μ</sup>m2·cm 3, respectively. In particular, the concentration levels in the school S3 were much lower than S1 and S2 ones. This is due to the different outdoor concentrations, indeed, if no indoor submicron particle sources are in operation in the schools (as mentioned in the methodology section), the indoor concentrations are just affected by the outdoor-to-indoor penetration factors [65,90,91]. Thus, the low concentrations measured in school S3 are just related to the low outdoor concentrations typical of the rural site under investigation and discussed in the methodological section (Table 1). Indeed, the median particle number and lung-deposited surface area concentrations in the "outdoor day" microenvironment were equal to 1.91 <sup>×</sup> <sup>10</sup><sup>4</sup> part. cm−3/<sup>79</sup> <sup>μ</sup>m2·cm<sup>−</sup>3, 2.58 <sup>×</sup> <sup>10</sup><sup>4</sup> part. cm−3/<sup>106</sup> <sup>μ</sup>m2·cm<sup>−</sup>3, and 4.22 <sup>×</sup> 10<sup>3</sup> part. cm−3/17 <sup>μ</sup>m2·cm−3, for children attending school S1, S2 and S3, respectively. The resulting "school"/"outdoor day" concentration ratios (considering the median concentrations) were equal to 0.80–0.83 and 0.82–0.86 in terms of PN and LDSA concentrations, respectively, then consistent with the typical penetration factors reported in the scientific literature for naturally ventilated schools [65,90,91]. The location of the children's schools and homes is then the most influencing parameters in their exposure to submicron particles in "outdoor day" and "school" microenvironments, in fact the highest correlations between average outdoor NO2 concentrations measured at the FSPs (Table 1) and PN concentrations measured during the experimental campaigns were determined for these two microenvironments (linear regressions with r2 >0.99). The correlation between outdoor and indoor concentrations gets weaker when it comes to non-school environments, indeed, here the possible presence of indoor sources (cooking, incense, candles, heating systems) can lead to high indoor concentrations. In this context, as expected, the most critical microenvironment is "cooking & eating" which presents median values of PN and LDSA concentrations of 4.20 <sup>×</sup> <sup>10</sup><sup>4</sup> part. cm−3/<sup>112</sup> <sup>μ</sup>m2·cm<sup>−</sup>3, 5.11 <sup>×</sup> 104 part. cm−3/136 <sup>μ</sup>m2·cm<sup>−</sup>3, and 4.91 <sup>×</sup> 104 part. cm−3/130 <sup>μ</sup>m2·cm<sup>−</sup>3, for children attending school S1, S2 and S3, respectively. The correlation with the average outdoor NO2 concentrations measured by the FSPs barely doesn't exist, indeed the concentrations are much larger than the outdoor ones, and also children attending school S3 are exposed to very high submicron concentrations in "cooking & eating" microenvironment and roughly comparable to the S1 and S2 ones despite the much lower outdoor concentrations. In regard to the other indoor environments labelled as "indoor day" microenvironment, the children's exposure resulted in lower statistical rates than the "cooking & eating" ones for all the three children groups. Nonetheless, the exposure in the "indoor day" microenvironment, when compared to the "outdoor day" one, varied amongst the different children groups. Indeed, the exposure in the "indoor day" microenvironment resulted statistically similar results, slightly lower, and much larger than the "outdoor day" environment for S1 (1.85 <sup>×</sup> 10<sup>4</sup> part. cm−3/84 <sup>μ</sup>m2·cm−3), S2 (1.79 <sup>×</sup> <sup>10</sup><sup>4</sup> part. cm−3/<sup>81</sup> <sup>μ</sup>m2·cm<sup>−</sup>3), and S3 group of children (1.32 <sup>×</sup> 104 part. cm−3/<sup>60</sup> <sup>μ</sup>m2·cm<sup>−</sup>3), respectively. The huge "indoor day"-"outdoor day" difference in the exposure detected for S3 group of children is related to the very low outdoor concentration level; thus, even a minor indoor source can easily increase the indoor concentration to values higher than the outdoor ones. Regarding the exposure in the "sleeping" microenvironment, the concentrations resulted in 0.5–0.6-fold of the "indoor day" microenvironment for all the three groups of children. Finally, during the "transport" microenvironment, higher concentrations were measured for children attending school S1 (2.38 <sup>×</sup> 104 part. cm−3/106 <sup>μ</sup>m2·cm<sup>−</sup>3) and S2 (1.93 <sup>×</sup> 10<sup>4</sup> part. cm−3/86 <sup>μ</sup>m2·cm<sup>−</sup>3), which are close to trafficked roads. On the contrary, children attending school S3 were exposed to quite low concentrations (6.58 <sup>×</sup> <sup>10</sup><sup>3</sup> part. cm−3/<sup>29</sup> <sup>μ</sup>m2·cm<sup>−</sup>3), likely due to the location of the schools (rural area). In summary, the daily exposure of the children is not only affected by the location of schools and homes, i.e., the proximity to outdoor sources, but also by the presence of indoor sources (mainly cooking); therefore, using outdoor concentration values as proxies of the daily exposure of the children could lead to serious under- or overestimation of the exposure. This is clearly highlighted by the daily median exposure data reported in Table 3; the concentrations, in terms of PN and LDSA, were equal to 1.44 <sup>×</sup> 10<sup>4</sup> part. cm−3/71 <sup>μ</sup>m2·cm<sup>−</sup>3, 1.55 <sup>×</sup> 104 part. cm−3/77 <sup>μ</sup>m2·cm<sup>−</sup>3, and 0.62 <sup>×</sup> 10<sup>4</sup> part. cm−3/34 <sup>μ</sup>m2·cm−3, for children attending school S1, S2 and S3, respectively. Indeed, such values were 0.75-, 0.60-, and 1.48-fold the outdoor PN concentration values and 0.90-, 0.73-, and 2.00-fold the outdoor LDSA concentration values for S1, S2 and S3 children groups, respectively.	doab	2025-04-07T03:56:58.090389	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 55 }
001fcd9a-cd27-45e8-b23f-f447874e0974.56	3.3. Particle Doses Received by Children Median values (and corresponding 5th–95th percentile ranges) of particle surface area doses received by the three groups of children investigated (attending school S1, S2 and S3) in each microenvironment are shown in Table 3, here the daily doses are also reported. The doses received in the different microenvironments were calculated through Equation (1) considering the above mentioned and discussed (i) time–activity patterns and (ii) exposure data, as well as the (iii) inhalation rates characteristics of the children age and activity as resulting from the activity diaries, whereas the total daily doses here reported represent the sum of the median doses received in the different microenvironments. The total daily doses for children attending school S1, S2 and S3 resulted equal to 1062, 1169 and 646 mm2, respectively. The higher doses received by children of schools S1 and S2 are mostly due to their higher median daily exposures discussed in Section 3.1, while the time activity patterns (and then the inhalation rates) were quite similar amongst the three children groups. The dose received in "school" microenvironment resulted equal to 163, 222 and 36 mm2 for school S1, S2 and S3, respectively; with contributions of 15%, 19%, and 6% to daily dose. The dose received by children in school S3 is extremely low due to the low outdoor concentration of that rural area, whereas, the more polluted outdoor environments of S1 and S2 lead to higher doses. Anyway, such doses can be considered not extremely high if compared to the important time fraction of the day spent in such environments (25% of the day): this is clearly confirmed by the dose-intensity ratio (i\_δ) summarized in Table 3; such ratios were lower than 1 for all the schools and, apart from "sleeping", they were the lowest values (0.45, 0.62, and 0.10 mm2·min−<sup>1</sup> for S1, S2, and S3, respectively) amongst the microenvironments investigated. Regarding the non-school environments, the contributions of "outdoor day" (2–5% of the daily dose) and "transportation" (3–6% of the daily dose) microenvironments are very limited due to the reduced time spent therein. As mentioned above, children attending school S1 and S2 were exposed to quite high concentrations in these two microenvironments then leading to dose-intensity ratios >1: this suggests that higher doses would be received in days and seasons characterized by different time-activity patterns with longer periods spent in such environments. The main contribution to the daily dose is obviously received in non-school indoor environments, indeed summing up the doses received by children in "sleeping", "indoor day" and "cooking & eating" microenvironments, total contributions of 74%, 73%, and 90% were estimated for children attending school S1, S2 and S3, respectively. The most important contribution is due to the "indoor day" environment (36–38%) due to the both the significant time fraction (19−24%) and the possible presence of other sources leading to concentrations higher than the outdoor ones: indeed, dose-intensity ratios close or larger than 1 were measured for that environment. The contribution of the "sleeping" microenvironment is quite low (15–17%) if compared to the huge time spent in such activities (dose-intensity ratios extremely low), whereas an important dose fraction is received by children in "cooking & eating" microenvironments due to the high concentrations to which children are exposed to. Indeed, despite the time fraction spent in "cooking & eating" microenvironment is about 8% for all the three children groups, the contributions to the daily dose resulted equal to 19%, 21%, and 35% for children attending school S1, S2 and S3, respectively. In fact, such microenvironment resulted the one with the highest dose-intensity ratios (1.66, 2.03, 1.94), then consistently exceeding the "transportation" and "outdoor" microenvironments typically affected by outdoor sources. In conclusion, the results on exposure levels in the different microenvironments confirm that indirect exposure assessments based on measurements at city scale or outdoor scale, typically adopted in cohort studies evaluating epidemiological effects on large populations [92,93] due to their easiness and cheapness, cannot provide a good estimate of the dose received by children whatever the location of their homes and schools. Thus, direct exposure assessment based on measurements at a personal scale, i.e., sampling aerosol from the breathing zone of the person using wearable instruments carried as personal monitors, is the only accurate experimental approach allowing proper dose estimates as it takes into account the different personal exposure of people moving between different microenvironments also including the indoor ones. Regarding the exposure assessment results shown here, some broader implications can be drawn from the paper. In particular, concerning the exposure in outdoor-driven microenvironments (e.g., schools, outdoors), it can be reduced just building the schools and performing outdoor activities as far as possible from main outdoor sources (e.g., vehicular traffic). The reduction of the exposure (and then the dose) in indoor microenvironments can be reached (i) mitigating the particle sources (e.g., using ad-hoc hoods during kitchen activities, avoiding the use combustion sources such as biomass burning, candles, etc.) and/or (ii) reducing the exposure (e.g., increasing the air exchange rates through proper ventilation approaches, using air purifiers).	doab	2025-04-07T03:56:58.090778	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 56 }
001fcd9a-cd27-45e8-b23f-f447874e0974.57	4. Conclusions In the present study, an assessment of the total daily dose in terms of submicron particle surface area received by children living in different Italian areas and attending different schools located in different urban contexts (rural, suburban and urban area), was performed. The study aimed at investigating the children daily doses received in different microenvironments (both school and non-school environments) also taking into account the impact of the outdoor concentration levels on the received dose. To this end, an experimental analysis using portable instruments able to measure the concentrations at personal scale of the children was performed. The findings of the study shown that the contribution of the school environment to the overall daily dose of the children is quite limited although they spent a significant time fraction of the day therein. Such dose is mainly affected by the outdoor concentrations; thus, schools placed close to main outdoor sources (e.g., trafficked roads) may results in higher rates of exposure and related doses then rural ones. Outdoor and transport microenvironments present an almost negligible contribution to the children daily doses, whatever the investigated sites, due to the reduced exposure time in such environments. Therefore, a child's daily dose is mainly affected by indoor non-school environments, e.g., homes. In particular, the contribution of non-school indoor microenvironments to the children's daily dose account for more than 70% of the data from the children and school locations. Such a high contribution is led by "cooking & eating" and other "indoor day" microenvironments. Indeed, the "cooking & eating" microenvironment contributes up to 36% of the daily dose despite the reduced time spent therein: this is due to the high levels of exposure from high-emitting cooking activities. In conclusion, the results of the study demonstrate that a proper evaluation of the submicron particle dose received by children cannot be performed only relying upon outdoor concentration data and that despite the location of the school and home, the contribution of indoor non-school environments is essential to properly assess the dose received by children. Author Contributions: Conceptualization, G.B. and S.R.; methodology, A.P.; investigation, A.P. and L.S.; data curation, L.S and A.P.; writing—original draft preparation, A.P.; writing—review and editing, L.S and G.B.; funding acquisition, S.R. and G.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the ANTER association and the company NWG Energia Srl Società Benefit. Acknowledgments: The authors want to thank the children and their families for their key contribution in the successful experimental campaign. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.	doab	2025-04-07T03:56:58.091095	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 57 }
001fcd9a-cd27-45e8-b23f-f447874e0974.59	Article Sniffin' Sticks and Olfactometer-Based Odor Thresholds for n-Butanol: Correspondence and Validity for Indoor Air Scenarios	doab	2025-04-07T03:56:58.091428	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 59 }
001fcd9a-cd27-45e8-b23f-f447874e0974.60	*Marlene Pacharra 1,2,\, Stefan Kleinbeck 2, Michael Schäper 2, Christine I. Hucke <sup>2</sup> and Christoph van Thriel 2,\* Received: 3 April 2020; Accepted: 4 May 2020; Published: 7 May 2020 Abstract:** Threshold assessments for the reference odorant n-butanol are an integral part of various research, clinical, and environmental sensory testing procedures. However, the practical significance of a high or low threshold for n-butanol beyond a particular testing environment and procedure are often unclear. Therefore, this study aimed to determine between-method correlations and to investigate the association between the n-butanol threshold and perceptual/behavioral odor effects in natural breathing scenarios in 35 healthy adults. The thresholds for n-butanol derived from the Sniffin' Sticks test and determined by the ascending limit dynamic dilution olfactometry procedure were significantly correlated (\|r\| = 0.47). However, only the thresholds determined by olfactometry were significantly correlated to the odor detection of n-butanol in an exposure lab. Moreover, participants with a higher sensitivity for n-butanol in the olfactometer-based assessment rated ammonia, during a 75 min exposure, to be more unpleasant and showed better performance in a simultaneous 3-back task than participants with lower sensitivity. The results of this study suggest that beyond the strict parameters of a certain psychophysical procedure, the threshold for n-butanol can be a meaningful indicator of odor detection and effects in some cases. Keywords: odor threshold; olfactometry; Sniffin' Sticks; chemosensory perception; validity assessment	doab	2025-04-07T03:56:58.091463	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 60 }
001fcd9a-cd27-45e8-b23f-f447874e0974.61	1. Introduction In clinical, research, and environmental assessment practice, odor sensitivity is currently determined almost exclusively with n-butanol (CAS: 71-36-3) as a reference odorant. As a consequence, parts of the clinical diagnosis of anosmia, the selection of panel members for sensory emission testing, and participation in olfactory research experiments can depend on an individual's threshold for n-butanol [1,2]. Moreover, n-butanol is one of the more abundant and relevant volatile organic compounds (VOCs) in indoor air environments. The German Environment Agency (UBA) mentioned in their indoor air guidance value document for 1-butanol (synonymical to n-butanol) that this VOC was found in 75–90% of indoor air samples in various databases and surveys [3]. Based on the developmental toxicity of 1-butanol, a health hazard guide value (RW II) of 2 mg/m<sup>3</sup> and a precautionary guide value (RW I) of 0.7 mg/m<sup>3</sup> were derived. The UBA report also stated that the RW I is above the odor threshold and that the olfactory perceptions need additional considerations. Regardless of the relevance of n-butanol as an indoor air pollutant, empirical evidence is lacking as to whether sensitivity to n-butanol is an adequate marker for sensitivity to other odorants as well as for n-butanol itself outside of a given lab environment and testing procedure [4,5]. Odor delivery methods and psychophysical testing procedures used to derive the odor threshold for n-butanol vary widely between areas of application. This may give rise to a between-method variability in thresholds. While the Sniffin' Sticks test [6] is very common in research and clinical practice, dynamic dilution olfactometry is the most common method in environmental practice (see DIN EN 13725 [7]). The single staircase, 3-alternative forced choice procedure used in the Sniffin' Sticks test adapts every subsequent step to the individual's previous performance [6]. As this technique is difficult to implement when testing several participants simultaneously, dynamic olfactometry, as used during environmental odor evaluation procedures [8], relies on an ascending limit procedure [2]. While a recent report indicated a non-significant correlation between n-butanol thresholds determined with the Sniffin' Sticks test and ascending limits olfactometry (r = 0.27) [4], another study comparing sniff bottles and olfactometry methods for n-butanol and ammonia (CAS: 7664-41-7) reported adequate between-method correlations (e.g., r = 0.78) [9]. With regard to the real-life impact of n-butanol thresholds, there is some indication that a lower Sniffin' Sticks threshold for n-butanol is associated with lower pleasantness ratings for different odors presented in glass jars [10]. However, necessary parts of olfactometry and the Sniffin' Sticks tests are (a) prompted sniffing at a clearly identifiable odor source and/or (b) artificial breathing rhythms. Thus, the association between the odor thresholds derived from these methods and the odor detection and evaluation of environmental odors presented more naturally in the ambient air is so far unclear. Given the practical importance of thresholds for n-butanol in clinical, research, and environmental assessment practice, the aims of the current study were threefold. Firstly, the between-method correlation (concurrent validity) was assessed for n-butanol thresholds determined with the very common Sniffin' Sticks test [6] and the established ascending limit dynamic dilution olfactometry procedure [2]. Secondly, the correspondence of these established threshold tests with the odor detection of n-butanol in indoor air scenarios was tested using an exposure lab. Thirdly, the association of these thresholds with odor effects caused by ammonia in an exposure lab was investigated. As the odors are presented in the ambient air, the exposure lab should more closely mimic the situation in the real world. Thus, the results of the here presented exposure lab experiments should be helpful in determining the ecological validity of the Sniffin' Sticks and olfactometry-based n-butanol thresholds. To this end, a novel ascending limits procedure presenting a stair-wise increasing concentration of n-butanol under normal breathing conditions in an exposure lab was conducted, and its results correlated with the results of the established methods (Sniffin' Sticks and olfactometry). Moreover, the transferability of the results to the malodorous compound ammonia and its odor effects was tested; it was investigated whether the n-butanol thresholds derived using Sniffin' Sticks or olfactometry are associated with the perceptual and behavioral odor effects of the malodorous compound ammonia in a well-controlled natural breathing scenario simulated by means of an exposure lab experiment [11,12]. To compare the results of individuals more and less sensitive to n-butanol during ammonia exposure and, in this way, to mimic the potential behavior of different selected panelists in real-world scenarios, subgrouping of the sample was performed using cut-off values from a large normative sample (Sniffin' Sticks) [1] or the DIN EN 13725 norm (80 ppb) [7].	doab	2025-04-07T03:56:58.091604	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 61 }
001fcd9a-cd27-45e8-b23f-f447874e0974.62	2. Experiments ### 2.1. Participants Thirty-nine non-smoking participants were recruited for this experiment. Exclusion criteria included pregnancy, asthma, and acute or chronic upper airway diseases. Four participants were excluded from the data analysis to avoid unclear or biased odor thresholds; three participants had increased false alarm rates during the olfactometer threshold test (>mean + 2 SD) (cf. [13]), and one participant indicated that he could not detect an odor at all in the exposure lab threshold test. Thus, the final sample comprised 35 participants. For descriptive details, see Table 1. To evaluate if the number of subjects was sufficient, a power analysis (G-Power; [14]) was conducted. The expected correlations should be in the range of the test-retest reliabilities of the established olfactory detection threshold tests (e.g., for Sniffin' Sticks, between 0.43 and 0.85 [15]; 0.61 [6]; 0.92 [16]). Thus, for the comparison of different methods, we expected a correlation (Pearson r) of about 0.60 (see also [9], r = 0.78 correlation between sniff bottles and olfactometry). With 35 subjects, a statistical power of 1 − β = 0.97763 could be achieved [17]. Table 1. Descriptive statistics for the total sample. Note: SD = standard deviation, SEM = standard error of the mean, CSS-SHR = Chemical Sensitivity Scale for Sensory Hyperreactivity, FEV1 = forced expiratory volume in 1 s. ### 2.2. Procedure The ethics committee of the Leibniz Research Centre for Working Environment and Human Factors (IfADo) approved the study protocol (approval date: 23 March 2016), and written informed consent was obtained from all participants. The participants received no feedback about their test performance in any of the performed tests at any point during the study. They were instructed not to talk to the other participants about their odor perceptions during any of the tests or during the ammonia exposure. The study procedure is depicted in Figure 1. Figure 1. Study procedure. \* blocks were switched randomly for half of the participants. LMS = labeled magnitude scale, LHS = labeled hedonic scale. After arrival in the lab and giving informed consent, groups of 3–4 participants were administered the first trial of the n-butanol threshold procedure in the exposure lab. After completion, a 15 min break followed. Participants were assigned according to an a priori computed randomization scheme to one of two groups, which differed in the order the following detection tests were presented (see Figure 1): half of the participants (Group 1) first completed the olfactometer threshold assessment in groups of two participants and answered the Chemical Sensitivity Scale for Sensory Hyperreactivity [18] and the trait version of the Positive and Negative Affect Schedule [19]. The other half of the participants (Group 2) were first administered, individually, the Sniffin' Sticks threshold test and a lung function test (VitaloGraph, Hamburg, Germany). In accordance with the GOLD guidelines [20] a forced expiratory volume in 1 s (FEV1) value ≤ 80% in the lung function test was used as an indicator of asthma and chronic obstructive pulmonary disease. Accordingly, subjects with lower FEV1 values would have been excluded from the experimental exposure to ammonia. As only non-smoking, young, and healthy volunteers were enrolled, none of the participants had a FEV1 value below 80% (see Table 1) [20]. Then, all participants completed the second trial of the threshold procedure in the exposure lab. After a 15 min break, participants completed either the Sniffin' Sticks and the lung function test or the olfactometer test and questionnaires, depending on which tests they had already been administered by this point. After a 15 min break, all participants underwent the 75 min ammonia exposure in the exposure lab. During ammonia exposure, cognitive testing, namely the n-back task [21] and flanker task [22], and perceptual ratings (via labeled magnitude scale, LMS; [23]) were conducted. The LMS is characterized by a quasi-logarithmic spacing of verbal labels and mimics the ratio-like properties of magnitude estimation scaling [23]. Furthermore, for hedonic scaling, the labeled hedonic scale was used (LHS; [24]) that is based on the LMS. The scale values for LHS and LMS in the computerized version used in this study ranged from 0 to 1000.	doab	2025-04-07T03:56:58.091953	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 62 }
001fcd9a-cd27-45e8-b23f-f447874e0974.64	2.3.1. Sniffin' Sticks-Based Threshold for n-Butanol The Sniffin' Sticks (Burghart, Wedel, Germany) subtest for the assessment of the n-butanol threshold was used [1,6]. Here, the threshold value is defined as the average Sniffin' Stick number (lower numbers indication higher concentrations) of the last four reversals in a single-staircase, 3-alternative forced choice procedure. Following the newest available norms of the test (see [25]), the cut-off score for individuals more and less sensitive to n-butanol using this test was 9 (median normative sample for age 21–30). Within this age range, there are only negligible differences between males and females, 8.75 for males and 9 for females (age 16–35; [1]), or more recently, 8.5 vs. 8.75 (age 21–30; [26]). As only non-smoking, healthy volunteers participated in the study, a cut-off value of 9 for males and females seemed to be appropriate.	doab	2025-04-07T03:56:58.092260	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 64 }
001fcd9a-cd27-45e8-b23f-f447874e0974.65	2.3.2. Olfactometer-Based Threshold for n-Butanol A dynamic dilution olfactometer TO 8 (ECOMA GmbH, Kiel, Germany) was used that complies with DIN EN 13725 [2]. N-butanol was injected into 25 L Tedlar®-bags filled with nitrogen. The mixture was homogenized by heating and rotating the bag. The standard procedure of the ascending method of limits with a 2-fold geometric dilution series was applied as in previous studies [13,27,28]. In short, the threshold measurement consisted of three trials in which increasing concentration steps of n-butanol were presented, interspersed with blank samples. Participants had to press a button whenever they thought they detected an odor. The lower of two subsequent correctly identified concentration steps represented the estimate of reliable olfactory detection in that trial. The detection threshold was defined as the geometric mean of the three trial estimates [13,27,28]. As in previous studies [13,28], the detection thresholds were subjected to log-transformations before data analysis. According to DIN EN 13725 [7], a panel member for environmental odor testing should have an n-butanol threshold between 20 and 80 ppb [2]. There are no established, published thresholds that differentiate between males and females for the here used olfactometry test for n-butanol. Thus, the cut-off value for individuals more and less sensitive to n-butanol using the ascending limits olfactometry test was set to 80 ppb in this study.	doab	2025-04-07T03:56:58.092335	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 65 }
001fcd9a-cd27-45e8-b23f-f447874e0974.66	2.3.3. Exposure Lab-Based Threshold for n-Butanol The threshold assessment took place in a 28 m<sup>3</sup> exposure lab with four PC workstations. This environmental chamber has been used in previous experimental exposure studies, i.e., [29]. The assessment followed the same general procedure of the ascending method of limits as used for the olfactometer-based assessment [2]. Due to the higher time and operating costs of the exposure lab compared to the olfactometer, the assessment in the exposure lab consisted of only two instead of three trials. In each trial, subjects were exposed over 30 min to an ascending concentration series of n-butanol (2-fold geometric series: 20, 40, 80, 160 and 320 ppb; see Supplement Figure S1). Every 5 min, subjects were prompted on a computer screen to indicate whether they detected an odor or not ("Odor? Yes/No"). Due to the technical restrictions in the lab, it was not feasible to insert randomly blank samples into the series. Thus, the first correctly identified concentration step represented the estimate of reliable olfactory detection in that trial. The detection threshold was defined as the geometric mean of the two trial estimates. Just as the olfactometer-based thresholds [13,28], the detection thresholds derived from the exposure lab procedure were subjected to log-transformations before data analysis. ### 2.3.4. Experimental Ammonia Exposure The procedure as described in previous studies [11,12] was applied. In short, subjects were exposed to an ascending concentration of ammonia (CAS: 7664-41-7) over 75 min. The maximum concentration after 75 min was 10 ppm (see Supplement Figure S4) corresponding to 50% of the German maximum workplace concentration (MAK value) [30]. This concentration is clearly above previously published odor thresholds but still well below the lateralization thresholds [28]. To estimate the odor effects of ammonia during the exposure, chemosensory perceptions were rated via the LMS [23] and the LHS [24]. Further, cognitive performance was assessed using a 3-back working memory and response inhibition task (see Supplementary Figure S3).	doab	2025-04-07T03:56:58.092444	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 66 }
001fcd9a-cd27-45e8-b23f-f447874e0974.67	2.3.5. Air Monitoring in the Exposure Lab The 28 m<sup>3</sup> laboratory was supplied with conditioned air by a climate control unit in a neighboring room (temperature, 24.4 ◦C; humidity, 46.0%). A predefined amount of n-butanol or ammonia (experimentally determined by volumetric analysis) was mixed into the inlet airstream of the climate control system. The conditioned air was dispersed throughout the laboratory by a branched pipe system, which was located on the floor. The outlet system at the ceiling of the laboratory was actively controlled through four outlets by an exhaust air ventilator; it maintained the laboratory at a negative pressure of 20–30 Pa. The air exchange rate was approximately 300 m3/h. Air samples were taken from the airflow of the inlet pipe and from the inside of the exposure laboratory quasi-continuously (every 80 s) during all exposure sessions. Photo acoustic IR spectroscopy was used to analyze the air samples (INNOVA, 1412i Photo Acoustic Field Gas-Monitor, LumaSense, Ballerup, Denmark). An overview of measured concentration values for n-butanol and ammonia is given in the Supplement (Supplementary Figures S2 and S4).	doab	2025-04-07T03:56:58.092574	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 67 }
001fcd9a-cd27-45e8-b23f-f447874e0974.68	2.4. Statistical Analysis The statistical analyses were performed in IBM SPSS Statistics 24. The level of significance for all statistical tests was set to 0.05. We checked for outliers by using the more liberal definition of extreme outliers ("outer fences": Q3 + 3 × IQR) [31], and according to this criterion, all participants could be included in the analysis. Based on the two thresholds, participants were classified into a 2 × 2 table below or above the respective cut-off values. Pearson's chi-square and exact tests were used to analyze the association of the grouping results. Moreover, the group differences for the Sniffin' Sticks scores and the olfactometer-based threshold were analyzed by Mann–Whitney U tests. A Pearson correlation was computed between the Sniffin' Sticks-based and olfactometer-based threshold for n-butanol to compare the methods. Next, the two established thresholds were correlated with the exposure lab-based threshold using further Pearson correlations. All correlations were adjusted (Bonferroni method) for the total number of computed multiple comparisons. Bonferroni-adjusted p-values are shown in addition to the non-adjusted correlations for these analyses. The experimental data from the ammonia exposure were analyzed using full-factorial analyses of variance (ANOVAs), with time as the repeated measures factor and group as the between-subjects factor. Models were calculated taking into account, on the one hand, the grouping factor Sniffin' Sticks threshold (cut-off value: 9, see Sniffin' Sticks norms) and taking into account, on the other hand, the grouping factor olfactometer-based threshold (cut-off: 80 ppb, see DIN EN norm 13725 [7]). If the assumption of sphericity was violated, Greenhouse–Geisser-corrected degrees of freedom were used. Significant interaction effects were further analyzed using Bonferroni-adjusted post hoc tests. ### 3. Results #### 3.1. Results of the Psychometric Threshold Assessments Table 2 presents the descriptive statistics of the three olfactory measures of n-butanol sensitivity for the total sample and after applying the respective cut-offs. Unsurprisingly, when a cut-off was applied based on one of the thresholds, Mann-Whitney U tests indicated a significant difference between resultant groups in this threshold. Moreover, participants more and less sensitive in the Sniffin' Sticks tests also differed significantly in their olfactometry-based threshold. Participants did not differ in relevant psychological variables for odor effects [29] such as negative affectivity and self-reported chemical sensitivity (see supplement Table S1). Table 2. Description of total sample and classified subgroups. Note. IQR = inter-quartile range, T = threshold, \* p ≤ 0.05 subgroup comparison using Mann-Whitney U tests.	doab	2025-04-07T03:56:58.092678	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 68 }
001fcd9a-cd27-45e8-b23f-f447874e0974.69	3.2. Results of the between-Method Correlations for n-Butanol Thresholds The correlation between the Sniffin' Sticks- and olfactometer-based threshold (see Figure 2) was significant (r = −0.47; p = 0.004, Bonferroni-adjusted p = 0.012). Figure 2. Scatter plot depicting the association between n-butanol Sniffin' Sticks- and olfactometer-derived thresholds. Note that in the Sniffin' Sticks test, a higher pen number corresponds to a higher n-butanol dilution and thus a lower threshold (higher sensitivity). Vertical and horizontal lines depict the respective cut-off values for high vs. low sensitivity groups (for details see text). When applying the cut-off values for the Sniffin' Sticks- (≥9) and olfactometer-based (≤1.9 log ppb) thresholds, nine participants (25.7%; lower right quadrant) were classified as individuals with high olfactory sensitivity in both standardized n-butanol threshold assessments (cf. Figure 2). Three of these participants were males (three out of 12; 25%) and the other six were females (six out of 23; 26%). However, the statistical analysis of the 2 × 2 contingency table yielded a non-significant Pearson chi-square value of 0.94 (p = 0.49). Thus, there was no significant overlap of the two olfactory sensitivity classification approaches. Both thresholds for n-butanol (olfactometer and Sniffin' Sticks) were correlated with the exposure lab-based threshold for n-butanol (Figure 3). Due to repeated computation of correlations with the same participants, a Bonferroni adjustment of p-values was conducted, resulting in a significant correlation between the olfactometer-based threshold and the exposure-lab based threshold (r = 0.41, p = 0.015, Bonferroni-adjusted p = 0.045, see Figure 3a). However, the Bonferroni-adjusted correlation between the Sniffin' Sticks-based threshold and the exposure lab-based threshold was non-significant (r = −0.34, p = 0.048, Bonferroni-adjusted p = 0.144, see Figure 3b). Figure 3. Scatter plots depicting the associations between the n-butanol thresholds derived with the exposure lab and the investigated methods, (a) Sniffin' Sticks and (b) olfactometer. Note that in the Sniffin' Sticks test, a higher pen number corresponds to a higher n-butanol dilution and thus a lower threshold (higher sensitivity). In a second step, it was investigated whether subgrouping the participants into more and less sensitive individuals based on detection thresholds was associated with odor effects for the compound ammonia. As only the olfactometry-derived thresholds showed a significant association with the exposure lab n-butanol detection threshold, a cut-off score of 80 ppb in the olfactometer-based assessment was used in the following analyses to indicate individuals more and less sensitive to n-butanol.	doab	2025-04-07T03:56:58.092856	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 69 }
001fcd9a-cd27-45e8-b23f-f447874e0974.70	3.3. Results of the Modulation of Odor Effects by n-Butanol Thresholds #### 3.3.1. Chemosensory Perceptions As expected, perceptual ratings were affected by the concentration of ammonia; participants perceived ammonia to be more unpleasant, intense, and pungent with increasing concentration (all main effects of concentration, p < 0.001; see Figure 4). Figure 4. Cont. Figure 4. Impact of different concentrations of ammonia on perceived (a) hedonic value, (b) odor intensity and (c) pungency (mean ± SEM). A significant main effect of the olfactometer-based threshold on pleasantness ratings emerged, F(1,33) = 4.2, p = 0.049. Participants with a lower olfactometer-based threshold (higher sensitivity) rated the exposure to be more unpleasant (mean = 426, SEM = 12; scale range: 0–1000) than participants with a higher olfactometer-based threshold (mean = 463, SEM = 13) (see Figure 5). Figure 5. Effect of olfactory sensitivity assessed via the olfactometer-based threshold on pleasantness ratings during ammonia exposure (mean ± SEM). Figure 5 indicates that the difference between the two groups increased with increasing ammonia concentration. However, the interaction of the sensitivity group and concentrations was not significant.	doab	2025-04-07T03:56:58.093006	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 70 }
001fcd9a-cd27-45e8-b23f-f447874e0974.71	3.3.2. Odor Effects on Behavioral Task Performance Participants improved their performance in the 3-back and response inhibition tasks over the course of the test session as indicated by an increase in the percentage of correct responses and a decrease in reaction times (all main effects of concentration, p ≤ 0.05). With regard to the olfactometer-based threshold for n-butanol, significant main effects on reaction times, F(1,33) = 19.7, p < 0.001, and error rates, F(1,33) = 5.4, p = 0.026, in the 3-back task emerged. Participants with a lower olfactometer-based threshold (higher sensitivity) had shorter reaction times and a higher percentage of correct responses in the 3-back task compared to participants with a higher olfactometer-based threshold (see Figure 6). Figure 6. Effect of olfactometer-based threshold on 3-back reaction times (left) and error rates (right) during ammonia exposure (mean ± SEM). Comparable to the rating data (see Figure 5), there was no interaction with the increasing ammonia concentration, indicating no additional impact of the increasing chemosensory perceptions. #### 4. Discussion Given the importance of n-butanol odor thresholds in many research, clinical, and environmental testing contexts, information on the practical significance of this particular odor threshold beyond the particular testing environment and procedure is scarce. This study sought to remedy that. In contrast to a previous report [4], a medium-sized, significant correlation between the thresholds derived from the Sniffin' Sticks test and the ascending limit dynamic dilution olfactometry procedure could be shown. This indicates that the determined sensitivity to n-butanol is associated between these two established methods of threshold assessment [2,6] and supports the good between-method correlations (concurrent validity) previously reported for other threshold assessment methods [9,28]. Beyond established threshold procedures, a novel exposure lab-based threshold assessment for n-butanol was proposed that more closely mimics odor detection during natural breathing. Measured concentration values showed that an ascending concentration series similar to the olfactometer-based method [2] could be generated in an exposure lab. After Bonferroni correction, only a significant medium-sized correlation between n-butanol thresholds derived using olfactometry and this novel method emerged. This indicates that olfactometry-derived thresholds can be meaningful indicators of odor detection in a more realistic context. While the Sniffin' Sticks threshold test requires artificial breathing (e.g., sniffing), the olfactometry and exposure lab scenarios have in common that they allow a more natural breathing pattern. Moreover, the results showed that a lower olfactometer-based threshold for n-butanol is associated with lower pleasantness ratings for ammonia during an exposure lab scenario. This further highlights the external validity of n-butanol thresholds with regard to perceptual effects during natural breathing of another odor and irritant (ammonia). Additionally, it is in line with a previous experimental finding [10], showing that the threshold for n-butanol is associated with lower pleasantness ratings for a range of odors presented in glass jars. An interesting, secondary finding in this study constitutes the better cognitive working memory performance in those with lower olfactometer-based thresholds irrespective of the ambient ammonia concentration. With regard to the Sniffin' Sticks threshold for n-butanol, Hedner, et al. [32] reported that the threshold is unrelated to cognitive factors such as executive functioning, semantic memory, and episodic memory. However, whether this is also the case for olfactometer-based thresholds is so far unclear. As the two "high odor sensitivity" groups showed only a weak overlap (25.7%), factors unrelated to olfaction but relevant for cognitive task performance (e.g., education and IQ) might have caused this general performance difference. In recent studies using gas chromatography-olfactometry, a coupling of gas chromatography analysis and human olfaction by panelists was employed to identify single VOCs in mixtures [33]. For n-butanol, a linear relationship was found between the modified detection frequency (frequency of detection × evaluation of intensity) of panelists and concentration of n-butanol as measured by gas chromatography (MS) in adhesives [34]. When humans inhale, ambient air is analyzed when reaching the olfactory epithelium. There, trace components of the air interact with receptor cells [35]. Thresholds and atmospheric lifetime are related in such a way that highly reactive odorants (short-lived molecules) are detected more sensitively [35]. N-butanol, belonging to the family of alcohols, therefore, has a relatively low odor threshold. All threshold assessments in this study indicated that the median olfactory threshold for n-butanol in the experimental sample was higher than what would be expected from norm values [1] or permissible for panel members during sensory emission testing according to DIN EN 13725 [7] (compare Table 2). This would suggest an overall lower than average sensitivity to n-butanol in the sample. This could be due to (1) a sampling error associated with the low sample size, (2) undetected nasal obstruction in the participants, or (3) olfactory adaptation due to multiple assessments of the odor threshold for n-butanol on the same day. Despite these possible confounding factors, the results showed that the threshold for n-butanol can be a meaningful indicator of odor detection and odor effects in natural breathing scenarios. This could be seen as a first step in providing much needed confidence in these thresholds [4,5] that are used daily in so many research and other application areas.	doab	2025-04-07T03:56:58.093186	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 71 }
001fcd9a-cd27-45e8-b23f-f447874e0974.72	5. Limitations of the Study Before coming to the conclusions, some limitations should be mentioned that need to be addressed in further studies. First, the sample size was sufficient to detect the association between n-butanol odor thresholds and the odor effects of another compound, but the sample was highly selective, and therefore, the transferability to the general population is somewhat limited. Here, a larger sample including older subjects, subjects with mild diseases of the upper respiratory tract (e.g., allergic rhinitis), and subjects reporting an increased odor sensitivity should be investigated. Second, the new method of the exposure lab-based threshold assessment should be tested with other odorants and compared to other threshold assessment procedures like squeezing and sniffing bottles [36–38] or the triangle bag method [39]. Third, odorants other and more pleasant than ammonia should be used to include the highly relevant dimension of pleasantness [40] into this branch of odor research. #### 6. Conclusions The results presented here provide further empirical evidence that the olfactory sensitivity of an individual may be an important predictor of odor perceptions in near to realistic scenarios of the human odor experience. The reference compound n-butanol seems to be an adequate choice as shown by the good cross-method correlations. Nevertheless, the role of suprathreshold olfactory functioning such as odor discrimination or identification has not been conclusively studied in this context. Moreover, other reference compounds for panelist selection are currently under discussion (DIN EN 13725:2019) [41]. With respect to the impact of environmental odors on cognitive task performance, our results showed that "high odor sensitivity" was not associated with worse performance in a challenging working memory task. The results were opposite to a distractive effect of malodors as proposed previously [42]. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4433/11/5/472/s1, Figure S1: Schematic overview of the experimental procedure during the exposure lab-based threshold assessment. Figure S2: Measured concentration values for n-butanol during the exposure lab-based threshold assessment. Figure S3: Schematic overview of the experimental procedure during the ammonia exposure (cf. [11,12]). Figure S4: Measured concentration values of ammonia during the experimental exposure. Table S1: Descriptive statistics for the total sample and subgroups. Author Contributions: Conceptualization, M.P. and C.v.T.; methodology, M.P. and M.S.; validation, S.K. and M.S.; formal analysis, M.P. and S.K.; investigation, M.P. and C.v.T.; resources, C.v.T.; data curation, M.S.; writing—original draft preparation, M.P. and S.K.; writing—review and editing, M.P., S.K., M.S., C.I.H. and C.v.T.; visualization, M.P., C.I.H. and S.K.; supervision, C.v.T. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors would like to thank Meinolf Blaszkewicz, Nicola Koschmieder, Eva Strzelec, Michael Porta, and Beate Aust for technical assistance. The publication of this article was funded by the Open Access Fund of the Leibniz Association. Conflicts of Interest: Marlene Pacharra, Stefan Kleinbeck, Michael Schäper, Christine I. Hucke and Christoph van Thriel declare that they have no conflict of interest.	doab	2025-04-07T03:56:58.093540	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 72 }
001fcd9a-cd27-45e8-b23f-f447874e0974.74	Article BTEXS Concentrations and Exposure Assessment in a Fire Station	doab	2025-04-07T03:56:58.093787	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 74 }
001fcd9a-cd27-45e8-b23f-f447874e0974.75	*Wioletta Rogula-Kozłowska 1, Karolina Bralewska 1,\ and Izabela Jureczko <sup>2</sup> Received: 9 April 2020; Accepted: 4 May 2020; Published: 6 May 2020 Abstract: The aim of this study was to evaluate benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS) concentrations in the changing room and garage in a fire station located in the Upper Silesian agglomeration (Poland), to compare them with the concentrations of the same compounds in the atmospheric air (outdoor background) and to assess the health exposure to BTEXS among firefighters and office workers in this unit. BTEXS samples were collected during the winter of 2018 in parallel in the garage, in the changing room, and outside, using sorption tubes filled with activated carbon. The average total BTEXS concentrations in the changing room and garage were over six times higher than those in the atmospheric air in the vicinity of the fire station. At each sampling site, toluene and benzene had the highest concentrations. According to the diagnostic indicators, the combustion of various materials and fuels was the source of BTEXS inside, while outside, the sources were the combustion of fuels and industrial activity. The carcinogenic risk related to benzene inhalation by the firefighters and office employees in the monitored unit exceeded the acceptable risk level value of 7.8 <sup>×</sup> <sup>10</sup>−<sup>6</sup> per 1 <sup>μ</sup>g/m3 by more than 20 times. Keywords:** BTEXS; health exposure; occupational risk; markers of exposure; air quality	doab	2025-04-07T03:56:58.093816	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 75 }
001fcd9a-cd27-45e8-b23f-f447874e0974.76	1. Introduction Firefighters are often exposed to very high concentrations of various products of combustion and pyrolysis, including substances in a gaseous phase adsorbed on ambient particulate matter (PM-bound). The toxic substances found in fire smoke are most often polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds (VOC) (including BTEXS), hydrogen cyanide (HCN), and several other organic and inorganic compounds [1–3]. Exposure to these compounds has been linked to a higher risk of specific cancers and cardiovascular diseases and thus acute and chronic effects that result in increased fire fighter mortality and morbidity [2,4–6]. The International Agency for Research on Cancer (IARC) assigns the profession of firefighter to group 2B, meaning "possibly carcinogenic to humans" [7]. Although firefighters use personal protective equipment during rescue and firefighting operations, such as gloves, coats, flash hoods, and breathing apparatus, this equipment can also serve as a secondary source of exposure. Some non- or semi-volatile compounds released during fires may settle and/or condense on protective equipment and exposed skin, leaving a greasy residue or film; then (e.g., when removing the equipment), these compounds may penetrate the body directly through the skin and eyes or through inhalation. Volatile organic compounds, including BTEXS, usually remain in the vapor phase. However, some of them can partition into a solid phase and condense onto the skin where they become available for deposition into the human body [8]. In addition, gaseous substances, especially VOCs such as HCN and the most volatile PAH, can penetrate into the interior space of the turnout gear [9] and then undergo the phenomenon of off-gassing in fire truck cabins and storage areas, such as changing rooms and garages [2,10,11]. In this way, firefighters can be exposed to these substances not only during firefighting operations but also during their return from action or while resting in their fire stations. Leaving clothes and equipment in the changing room or garage without first decontaminating them may facilitate the accumulation of toxic substances and transfer them to other fire station rooms, such as bedrooms or offices [12–14]. Accordingly, not only firefighters extinguishing fires but also dispatchers, commanders, and office workers working in fire stations can be exposed to toxic combustion products. Compounds of the BTEXS group are considered to be indicators of human exposure to volatile organic compounds [15]. Measuring the total concentration of BTEXS and the individual compounds from this group (as the main pollutants released during fires) is necessary to assess the threats to firefighter health, as well as the work environment. The most dangerous compounds from the BTEXS group are benzene and ethylbenzene, which are classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (Group 1). Exposure to these substances is linked to an increased risk of leukemia and hematopoietic cancers [7,16–18]. Toluene and xylene are non-carcinogenic, but they may produce adverse reproductive effects, especially when exposure is chronic at low to high concentrations [18,19]. While research on the concentrations of pollutants released during fires and assessments of the health risks to firefighters have been the subject of many global studies since the 1980s [2], the presence of combustion products in fire stations is a less well-trodden topic. There is little research in this area. Studies that are available focus on PAH and VOC concentrations in structural firefighting ensembles [10] as well as in turnout gear [3,20] at fire stations in Brisbane (Australia), Philadelphia, and Illinois (United States); and the concentrations of polybrominated diphenyl ethers (PBDEs), polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs) in dust samples collected by a vacuum cleaner at twenty fire stations in California [13]. These studies were among the first to indicate the problem of high concentrations of toxic combustion products in fire stations. The investigations described in this paper are the next stage of the multi-site study regarding the concentration of combustion products in fire station rooms. In the first stage of study, the concentrations of PAHs in garages and changing rooms at two selected fire stations belonging to the Polish National Fire Service were analyzed and compared to the concentrations of PAHs in the atmospheric air outside these units [14]. The goal of this work was to determine the ambient concentrations of BTEXS (benzene, toluene, ethylbenzene, xylene, and styrene) in a selected fire station in Poland, compare them with the outdoor background concentrations of BTEXS, and assess the health risks of the exposure to BTEXS (occupational carcinogenic and non-carcinogenic risks) between two groups of employees (firefighters and office workers) in the state fire service unit.	doab	2025-04-07T03:56:58.093949	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 76 }
001fcd9a-cd27-45e8-b23f-f447874e0974.78	2.1. Sampling Site The municipal headquarters of the state fire service, where the research was conducted, is located in the center of the Silesian voivodeship (50◦16 1.401" N, 18◦51 40.607" E). The building is located among old (100–150 years old) and low-rise (several-story) buildings. The apartments in the vicinity of the fire brigade building are mainly heated with hard coal and are largely inhabited by a low-income population. Therefore, a large number of people in the area burn poor quality fuel in their furnaces and produce waste. The property is located 50 m from voivodeship road no. 925 and about one kilometer from the A4 highway. The fire brigade building is a three-story building heated by a solid fuel stove that uses pellet fuel. The building was thoroughly rebuilt in the 1960s. The first floor of the building consists of a garage, changing rooms, a gym, and a workshop. The second floor contains commanders' offices, a common room, and social rooms for firefighters. On the third floor, there are a dispatch center and office rooms. The building also has two kitchens (with gas stoves) where the firefighters prepare meals. Each firefighter participating in fire-fighting operations has special clothing, gloves, and a balaclava, which offer external protection during all rescue and fire-fighting activities, as well as during exercises. Thus, this clothing is particularly exposed to the effects and absorption of all types of chemical compound. After a 24-h shift, the firefighter stores his or her clothes in lockers located in the changing room. Due to the relatively high cost of these clothes, firefighters usually have only one or two sets of clothes, and these clothes are washed no more than once a month. In the fire brigade building, the highest BTEXS emissions likely occur when parking fire vehicles in the garages due to the lack of engine exhaust hoods. An additional internal source of BTEXS could also be the building and its finishing materials (paints, wallpaper, and floor coverings), as well as the varnishes, glues, and solvents used during maintenance work, which also release compounds from the VOC group into the environment. The BTEXS concentrations were measured in November 2018. BTEXS samples were taken on five consecutive business days (Monday–Friday) simultaneously: Three GilAir Plus aspirators from the Gilian company were used to conduct this research. These devices are designed to sample air, dust, and gaseous pollutants. The aspirator has the function of regulating the air flow in the range of 20 to 5000 mL/min. The flow stabilization system allows one to maintain a constant flow with an accuracy of 5% in a temperature range of 0–40 ◦C. During the tests, especially at night, the ambient air temperature dropped a few degrees below zero. Therefore, adequate protection (the casing with the heater) of the aspirator used for sampling outside was provided. Anasorb CSC Lot 2000 sorption tubes were used to collect compounds from the BTEXS group. These tubes were filled with sorbent-activated carbon from coconut shells and were intended to take up a wide spectrum of organic compounds [21,22]. After breaking the glass protection on both sides, the measuring tube was placed in a silicone tube connected to the aspirator. The manufacturer of the sorption tubes requires an air flow of 200 mL/min. Considering the capacity of the sorbent used and the expected BTEXS concentrations (previous tests), it was assumed that the BTEXS intake per tube can last a maximum of 4 h. After this time, the tube was changed. In total, 6 tubes were used in one measurement place for one day, which provided 24 h of measurement per day. In total, during a continuous period of five days of measurements, the samples were collected in 90 tubes. After a measurement, each tube was sealed with special plugs on both sides, wrapped with aluminum foil, and stored in a refrigerator at about 2 ◦C until analysis.	doab	2025-04-07T03:56:58.094292	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 78 }
001fcd9a-cd27-45e8-b23f-f447874e0974.79	2.2. BTEXS Analysis Details of the BTEXS analysis can be found in [22,23]. Briefly, to isolate the BTEXS adsorbed on the activated carbon, carbon disulphide extraction (Sigma Aldrich, St. Louis, MO, USA; CS2 Chromasolv for HPLC; purity > 99.9%) was used. Sorbent from the 6 tubes used to collect BTEXS during one day was poured into a glass jar, and then 3 mL of solvent (CS2) was added and extracted in an ultrasonic bath for 10 min. The extract thus prepared was analyzed using a gas ionization detector (GC-FID) Trace 1300 GC from Thermo Scientific (Waltham, MA, USA). The separation was carried out using a Supelco Wax 10 capillary column (60 m <sup>×</sup> 0.53 <sup>×</sup> 10−<sup>3</sup> m <sup>×</sup> 1 <sup>×</sup> 10−<sup>6</sup> m). The carrier gas flow (helium) was 1 mL/min using the split function (split ratio 1:10), and the gas flow in the FID detector was as follows: air—350 mL/min; hydrogen—35 mL/min; and make-up (nitrogen)—40 mL/min. The detector temperature was 310 ◦C, and the dispenser temperature was 150 ◦C. In total, 1 μL of the sample was injected in each case for the GC-FID analysis. The determination of each sample was repeated twice. The difference in readings between the two measurements did not exceed 5%. The following temperature program was used in the chromatograph oven: 50 ◦C maintained for 10 min followed by a temperature increase at a rate of 30 ◦C/min up to 170 ◦C; the temperature was then maintained at 170 ◦C for one minute. The analysis time was approximately 15 min. The concentrations of individual BTEXS species (benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, and styrene) were quantified based on an external calibration standard mixture (Sigma Aldrich; MISA Group 17 Non-Halogen Organic Mix). A five-point calibration using standards between 1 and 25 μg/mL was performed for quantifying the BTEXS species in collected samples. These standard solutions were used to produce calibration curves. The correlation coefficients for standards curves were 0.99. The retention times were 5.234, 10.124, 14.754, 14.854, 14.982, 16.350, and 17.463 min, for benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, and styrene, respectively. The limit of quantification for the method was set to 0.05 μg/sample. The repeatability of the method was 5%; the expanded uncertainty was 25% (k = 2). The recovery values of the BTEXS constituents ranged from 90 ± 4% to 98 ± 3% (n = 10).	doab	2025-04-07T03:56:58.094590	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 79 }
001fcd9a-cd27-45e8-b23f-f447874e0974.80	2.3. Health Risk Assessment The health exposure assessment related to the inhalation of BTEXS compounds (excluding styrene) among the firefighters and office workers at a fire station in Poland was developed based on the methodology developed by the United State Environmental Protection Agency (US EPA) [24]. This assessment of the health exposure includes an assessment of the occupational carcinogenic risk (CR) associated with benzene inhalation, which was calculated according to Equations (1) and (2) [25], and an assessment of the occupational non-carcinogenic risk in terms of the threshold mechanisms of the toxic effects produced by compounds from the BTEXS group (expressed as hazard quotients, HQ), which were calculated according to Equations (1) and (3) [17]. $$\text{EC} = (\text{CA} \times \text{ET} \times \text{EF} \times \text{ED}) / \text{AT} \tag{1}$$ $$\text{CR} = \text{IUR} \times \text{EC} \tag{2}$$ $$\text{HQ} = \text{EC/(RfC} \times 1000 \text{ }\mu\text{g/mg)} \tag{3}$$ where CA is the chemical concentration (μg/m3). The other variables used in Equations (1)–(3) are explained in Table 1. In the calculations, we assumed that inhalation constitutes 50% of all intake [26]. The risk assessment was carried out for the period of professional activity of the two groups of firefighters: (1) active (i.e., those involved in firefighting) and (2) office workers. Exposure duration (ED) and exposure frequency (EF) were assessed on the basis of interviews and observations. It was assumed that firefighters participating in rescue and firefighting operations perform 24-h shifts three times a week, while office workers work eight hours a day, five days a week. In 2018, firefighters from the analyzed unit responded to 643 fires (33 times in November 2018) [27]. Moreover, we assumed that both firefighters and office employees spend 50% of the work shift in conditions such as in the garage, while the remaining 50% of time is spent in conditions such as in the changing room. During the day, firefighters use, among others, the workshop and gym, which are located next to the changing room and garage. Office rooms and a dispatch room are also located next to or directly above these rooms. Therefore, we have adopted a simplification that in these rooms, BTEXS concentrations are comparable to those in the changing room and garage. The values of the individual parameters used to calculate the carcinogenic and non-carcinogenic risks are presented in Table 1. \* The U.S. EPA provides two values of Inhalation Unit Cancer Risk (IURs) for benzene: 2.2 × <sup>10</sup>−<sup>6</sup> to 7.8 × <sup>10</sup>−<sup>6</sup> per 1 μg/m3. The higher IUR was used to obtain the maximum estimate of cancer risk from benzene exposure.	doab	2025-04-07T03:56:58.095054	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 80 }
001fcd9a-cd27-45e8-b23f-f447874e0974.82	3.1. BTEXS Concentrations The mean concentrations and standard deviations for the BTEXS compounds are presented in Table 2. The highest total BTEXS concentration averaged over the whole measurement period ( BTEXS) was recorded in the changing room (925.2 μg/m3). However, this value was only slightly higher than that in the garage (893.1 μg/m3). The average BTEXS in both the changing room and the garage was over six times higher than the BTEXS outside the unit (139.6 μg/m3). Most of the BTEXS compounds, except for xylenes, had higher concentrations in the changing room than in the garage. The concentration of individual BTEXSs in the changing room and garage on different measurement days varied notably. This is demonstrated by the high standard deviations of BTEXSs, especially toluene and ethylbenzene. However, every measurement day, the concentrations of benzene, toluene, ethylbenzene, xylenes, and styrene were many times higher both in the changing room and garage than in the atmospheric air (outdoors; the differences were statistically significant, p < 0.05; Table 2). Therefore, it can be assumed that the fluctuations in BTEXS concentrations may be related to the number and/or type of fires that the firefighters had to deal with on particular days. In November 2018, firefighters from the analyzed unit participated in extinguishing 33 fires and fighting 60 local threats [27]. In addition, the large differences between the BTEXS concentrations in the fire station rooms and in the surroundings suggest that the main sources of BTEXSs in the changing room and garage were the combustion products settled on uniforms, personal protective equipment, and the equipment used during firefighting. The standard deviations for the BTEXS concentrations in the garage were smaller than those in the changing room. Outside, the proportions of standard deviations relative to the average concentrations were very high—higher than those for the measurements inside. The BTEXS concentrations outside the fire station, although many times lower than those inside, were several times higher than, for example, the BTEXS concentrations observed in other urban areas, such as Tri-City, Tczew (Poland) [28], Hamburg (Germany) [29], or Pamplona (Spain) [30]. It is likely that this is related to the fact that the ambient air in the southern part of Poland (Upper Silesia) is more polluted than the air in other parts of the country. This pollution is caused by mining activities and intensified heating processes, especially since the measurements were conducted in winter [31]. Table 2. Mean concentrations of benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS) (μg/m3) in the changing room, in the garage, and outside the fire station in Poland. \* Indoor and outdoor concentrations of BTEXS in the case of changing room or garage are statistically significantly different (according to the Mann–Whitney U test; p < 0.05). The BTEXS profiles (i.e., the percentages of individual BTEXS compounds in the total BTEXS concentration) in the changing room and garage were very similar. However, they differed from the BTEXS profile for the atmospheric air outside the fire station (Figure 1). In both rooms and outside, toluene had the highest share in BTEXS (58% in the changing room, 59% in the garage, and 62% outdoors). In the whole measuring period, toluene concentrations were in the range 265–703 μg/m<sup>3</sup> in the changing room, 425–692 μg/m<sup>3</sup> in the garage, and 51–131 μg/m3 in the atmospheric air. Benzene had the second largest share in BTEXS (an average of 22% both in the changing room and the garage and 27% in the atmospheric air). It should be noted that toluene and benzene concentrations are reduced through their reactions with OH radicals, with the rate constant of toluene being approximately five times larger than that of benzene [32]. This explains the differences in the concentrations of these compounds. The average percentage of ethylbenzene and styrene among the BTEXSs inside the garage and the changing room was about four times higher than that outside. This suggests an internal source of these compounds in the analyzed rooms. Figure 1. Mean profiles of the BTEXSs in the indoor (changing room and garage) and outdoor air at the selected fire station in Poland. #### 3.2. Origin of BTEXs Inside and Outside the Fire Station Diagnostic indicators, which are the ratios of individual BTEXS concentrations, offer a preliminary assessment of the origin of individual compounds from the BTEXS group present in the indoor and outdoor air of the state fire service unit (Table 3). The ratio of toluene/benzene = 2.7 indicates local emissions of toluene and benzene in the changing room and garage, likely due to the combustion of liquid fuels [32–34]. However, the low values of the indicators m,p-xylene/benzene and o-xylene/benzene (<0.5) in comparison to [31,33,34] testify to other BTEXS sources in these rooms. Low m,p-xylene/benzene and o-xylene/benzene values indicate greater photochemical degradation and, therefore, suggest that a sampling site is being influenced by emissions that originated some distance away [32]. It can, therefore, be assumed that the main sources of BTEXS in the fire station rooms are the gases released during fires, which settled on the uniforms and equipment. For the outdoor measurements, the indicators toluene/benzene = 2.3 (outdoor) and m,p-xylene/ethylbenzene = 2 indicate the combustion of liquid fuels as a BTEXS source in the atmospheric air [32,34]. The obtained indicators are two times lower than other results in this field, such as those in Sarnia (Canada), where the impact of traffic emissions on BTEXS concentrations was clearly demonstrated [32]. High concentrations of benzene alongside relatively low indicators of m,p-xylene/benzene and o-xylene/benzene may also indicate additional industrial sources of BTEXS outside the fire station. An analysis of the environment of the sampling site indicates that these sources are likely related to the coal mining and storage processes taking place at a distance of about 5 km from the fire station, as well as the combustion of fuels in home boiler rooms [34,35]. It is difficult to clearly determine which of the sources listed, both inside and outside, has the greatest impact on shaping BTEXS concentrations. More detailed data could provide Pearson's correlations between the BTEXS concentrations, but this would require more measurements [32,36]. Table 3. Diagnostic indicators for the three sampling locations. Information on the origins of the BTEXS compounds is also provided by the I/O ratios calculated for the entire measurement period, presented in Figure 2. The average I/O ratios were in the range of 6.2–53.1. The values of the I/O ratios confirm that the BTEXS concentrations mainly originate from internal sources. The higher I/O ratios in the changing room than in the garage also suggest that the source of BTEXSs here could be residue on the uniforms, helmets, and gloves used during firefighting but also contaminated furniture (e.g., wardrobes for clothes and equipment shelves) [37]. The lower average I/O ratios obtained for benzene and toluene relative to the rest of the BTEXS compounds mean that, in addition to internal sources, the concentrations of these compounds are also likely affected by the infiltration of outdoor air, especially considering the fact that the changing room is located next to the garage, where the door is often opened. Ethylbenzene, styrene, and xylenes, whose I/O ratios are the highest and have the highest fluctuations relative to other BTEXSs, likely come from fires. The above observations are confirmed by the literature and other research conducted in this field around the world. Benzene is the second most frequently identified compound in over 80% of the fires tested, and the next most frequently occurring compounds during fires are toluene, xylenes, and ethylbenzene [3,10,38–40]. Furthermore, the presence of styrene may result from the thermal decomposition and combustion of polystyrene (plastics) [40]. The concentrations of individual compounds from the BTEXS group depend on the type of material burned, the phase of the fire [10], the type of fire (flame vs. flameless), the location of the fire, the meteorological conditions, and the distance from the fire [38,41]. The conducted research provides the basis for future research, which should also include an analysis of the ventilation solutions in fire stations and a study of other factors, such as PM concentration and the chemical composition of particulate matter, which would facilitate a more accurate assessment of the impact of the environment on BTEXS concentrations. Figure 2. Ranges and average values of the I/O ratios in the two rooms of the fire station calculated on the basis of the average set of five 24-h BTEXS concentrations. It is difficult to compare the obtained results with the results presented in the literature, where different methods were used, or sampling was conducted at different stages of the fire. Nevertheless, Table 4 summarizes several examples of such results from other researchers. The BTEXS concentrations in a fire station in Upper Silesia were several times higher than the BTEXS concentrations collected by Kirk and his team from the outer layer of the structural firefighting ensembles at various stages of a fire [10] and then from decontaminated and non-decontaminated turnout gear during the pre-fire and post-decon periods [3]. In addition, the toluene and ethylbenzene concentrations measured by these authors in both the garage and the changing room were higher than the concentrations of these compounds in the post-fire phase in Fent et al.'s study [3]. The BTEXS concentrations in the changing room and garage in the analyzed unit were also several dozen times higher than those in selected nursery schools in Poland [36] and Turkey [42], and the atmospheric air of urbanized areas in Poland, Germany, and Spain [28–30]. However, they were lower than the concentrations in the atmospheric air in the vicinity of waste dumps or at oil distribution stations [16,17]. Table 4. Concentration ranges of the BTEXS compounds (μg/m3) measured at different locations. Table 4. Cont. 3.3. Assessment of Occupational Carcinogenic and Non-Carcinogenic Risks Associated with Exposure to BTEXS The carcinogenic risk associated with the inhalation of benzene was calculated as 2.21 <sup>×</sup> <sup>10</sup>−<sup>4</sup> for firefighters participating in firefighting activities and 1.77 <sup>×</sup> <sup>10</sup>−<sup>4</sup> for office employees of the fire station (Figure 3). Both values are above acceptable cancer risk levels according to the Inhalation Unit Cancer Risk (IUR) (>7.6 <sup>×</sup> 10−6) [26,43,44]. The differences in risk values result from the different durations of working shifts between the individual exposure groups. Firefighters participating in rescue and firefighting operations perform 24-h shifts three times a week, while office workers work eight hours a day, five days a week. In the carcinogenic risk assessment, only benzene concentrations were used, while the concentrations of other pollutants, such as PAH or PM-bound substances, were not taken into account. In addition, the concentrations that prevail during fires were not taken into account. Firefighters use breathing apparatus and other personal protective equipment when extinguishing fires, but there are situations when they take that equipment off, such as during exterior operations (e.g., pump operations), immediately after extinguishing fires, when collecting equipment, or in fire truck cabins during their return from action. It can be assumed that then BTEXS concentrations are higher than in the garage or changing room [3]. Therefore, the risk may be even higher than calculated. Studies also show that the health exposure associated to hazardous combustion products does not only apply to firefighters extinguishing fires but also to dispatchers, commanders, and secretaries (i.e., people whose work rooms are often located near garages and changing rooms). Figure 3. Cancer risk assessment results for exposure to benzene for various groups of fire service workers. The occupational non-carcinogenic risk (adverse health effects)—expressed by the hazard quotient (HQ)—associated with exposure to compounds, for the BTEXS group of firefighters involved in firefighting and fire station office employees, was in the range of 0.01–0.76 (Table 5). This indicates an acceptable risk for non-carcinogenic effects in each scenario considered. However, as is the case for carcinogenic risk, risk modeling does not include the concentrations of other pollutants, such as PAH, which may also occur in fire station rooms [14]. Table 5. Non-cancer risk assessment results for exposure to BTEXS for various groups of fire service workers. The carcinogenic risk calculated for firefighters and office workers was about 10 times higher than the risk calculated for fuel workers and cashiers at gasoline stations in Thailand [24] but about two times lower for firefighters and three times lower for office workers than the average lifetime cancer risk calculated for petroleum product distributors working at stations belonging to an Iranian company [17]. The non-carcinogenic risk for fuel workers (HQ = 0.80 for benzene) is lower than that for firefighters and higher than that for office workers, while that for cashiers at the gasoline station (HQ = 0.01 for benzene) is many times lower than that for firefighters and office workers [25]. Two-times higher non-carcinogenic risk values were recorded for petroleum product distributors in Iran [17] than for firefighters. The above comparisons are for reference only due to the different periods of exposure and averaging.	doab	2025-04-07T03:56:58.095244	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 82 }
001fcd9a-cd27-45e8-b23f-f447874e0974.83	4. Conclusions The concentrations of individual compounds from the BTEXS in the changing room and garage are several to several dozen times higher than the concentrations of these substances in the atmospheric air outside the fire station. Both firefighters and office workers staying under measured conditions are at risk of carcinogenic exposure that exceeds an acceptable level. Among the entire BTEXS group, toluene and benzene had the highest concentrations. According to the diagnostic indicators, the combustion of various materials and fuels was the source of BTEXS inside, while the combustion of fuels and industrial activity was the source of those outside. This research provides the following conclusions: Author Contributions: Conceptualization, W.R.-K. and K.B.; methodology, W.R.-K.; software, K.B.; validation, K.B., W.R.-K.; formal analysis, W.R.-K.; investigation, I.J.; resources, I.J.; data curation, K.B.; writing—original draft preparation, K.B.; writing—review and editing, W.R.-K.; visualization, K.B.; supervision, W.R.-K.; project administration, K.B. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors would like to acknowledge the Ministry of Science and Higher Education for their financial support as part of the statutory works. Conflicts of Interest: The authors declare no conflict of interest.	doab	2025-04-07T03:56:58.096203	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 83 }
001fcd9a-cd27-45e8-b23f-f447874e0974.85	Chemical Characterization of Electronic Cigarette (e-cigs) Refill Liquids Prior to EU Tobacco Product Directive Adoption: Evaluation of BTEX Contamination by HS-SPME-GC-MS and Identification of Flavoring Additives by GC-MS-O *Jolanda Palmisani 1,\, Carmelo Abenavoli 2, Marco Famele 2, Alessia Di Gilio 1,\, Laura Palmieri 1, Gianluigi de Gennaro <sup>1</sup> and Rosa Draisci <sup>2</sup>* Received: 3 February 2020; Accepted: 6 April 2020; Published: 10 April 2020 Abstract: The present study focused on the determination of benzene, toluene, ethylbenzene and xylenes (BTEX) concentration levels in 97 refill liquids for e-cigs selected by the Italian National Institute of Health as representative of the EU market between 2013 and 2015 prior to the implementation of the European Union (EU) Tobacco Product Directive (TPD). Most of the e-liquids investigated (85/97) were affected by BTEX contamination, with few exceptions observed (levels below the limit of quantification (LOQ) of headspace-solid phase micro extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) methodology). Across brands, concentration levels ranged from 2.7 to 30,200.0 μg/L for benzene, from 1.9 to 447.8 μg/L for ethylbenzene, from 1.9 to 1,648.4 μg/L for toluene and from 1.7 to 574.2 μg/L for m,p,o-xylenes. The variability observed in BTEX levels is likely to be related to the variability in contamination level of both propylene glycol and glycerol and flavoring additives included. No correlation was found with nicotine content. Moreover, on a limited number of e-liquids, gas chromatography-mass spectrometry-olfactometry (GC-MS-O) analysis was performed, allowing the identification of key flavoring additives responsible of specific flavor notes. Among them, diacetyl is a flavoring additive of concern for potential toxicity when directly inhaled into human airways. The data reported are eligible to be included in the pre-TPD database and may represent a reference for the ongoing evaluation on e-liquids safety and quality under the current EU Legislation. Keywords: electronic cigarettes; flavoring additives; BTEX; contamination; headspace solid micro phase extraction; gas chromatography-olfactometry; human health; EU regulation	doab	2025-04-07T03:56:58.096331	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 85 }
001fcd9a-cd27-45e8-b23f-f447874e0974.86	1. Introduction Electronic cigarette (e-cig) use has increased extremely quickly worldwide over the last decade due to an intense marketing campaign aiming to advertise them as an aid to reducing and/or eliminating addiction to tobacco cigarette smoke [1]. Emerging in 2006 in China, e-cigs became widely available on the market throughout the world in 2008–2009. EU Commission public opinion surveys focused on the smoking attitudes of European citizens across 27 European Union (EU) member states highlighted that e-cig consumption increased from 7.2% to 11.6% between 2011 and 2014 and is expected to increase further [2]. Despite the claims of manufacturers and retailers advertising e-cigs as a healthier way to smoke nicotine and other chemicals in public places, to date reliable sociological data confirming the effectiveness of e-cigs use in changing smokers' behavior (e.g., smoking cessation and/or reduction) are not exhaustive enough to draw certain conclusions [3–5]. On one hand, public opinion surveys have provided data suggesting a relationship between e-cig consumption and quitting and significant reduction of traditional tobacco smoking [6]. On the other hand, however, scientific research still raises doubts regarding the role of e-cigs in smoking cessation and highlights the interchangeable and simultaneous use of e-cigs with tobacco cigarettes [7,8]. Moreover, a controversial debate is still ongoing within the scientific community on potential adverse effects on the health of both users and bystanders. Concerns about e-cig consumption, specifically related to e-liquids composition, are: (a) the potential inhalation exposure to chemicals of concern present in e-liquid formulations as contaminants of the main ingredients (i.e., aromatic hydrocarbons, aldehydes, PAHs, heavy metals); (b) the potential exposure to harmful by-products formed during the vaporization process; and (c) the unknown and unpredictable long-term health effects due to flavoring additive and main ingredient (i.e., glycerol and propylene glycol) inhalation exposure [9–12]. In view of the health-related concerns raised by the international scientific community and EU member states' competent authorities, specific provisions concerning e-cigs manufacture, labelling, and advertising were included in the EU Tobacco Products Directive 2014/40/EU (TPD), entered into force on May 2014 and fully implemented in EU countries between 2016 and 2018 [13]. E-liquids, available on the market in bottles or in replaceable cartridges, are basically a mixture of propylene glycol, glycerol, and water (the latter generally in smaller quantities). The inclusion of propylene glycol and glycerol in e-liquid formulations is common due to humectant and solvent properties, although the use of other chemicals, such as ethanol (EtOH), has been recently reported in literature [14]. This basic formulation may be enriched with nicotine (in variable and allowed quantities) and a wide selection of flavoring additives, in order to provide users a satisfying and enhanced sensory perception while vaping. ### 1.1. Flavoring Additives It is estimated that several hundred flavoring chemicals are currently used for e-liquid formulations, allowing consumers to choose on the market among several flavors belonging to menthol, tobacco, fruit (i.e., cherry, blueberry, strawberry, apple), sweets (i.e., caramel, vanilla, liquorice, chocolate) categories, to mention the most popular ones [15,16]. Scientific reports on addictive behaviors highlighted the key role of flavors in vaping initiation, especially among young adults, and the resulting addiction along to nicotine [17]. The inclusion of flavoring additives in e-liquids is one of the most debated issues. They are approved in foods, beverages, and cosmetics and included in the Generally Recognized As Safe (GRAS) list of the Flavors and Extracts Manufacturers' Association (FEMA); therefore their use is intended through ingestion and dermal contact routes, not for direct inhalation. As a result, both short- and long-term effects due to inhalation exposure cannot be predicted. Due to the lack of epidemiological data able to elucidate the issue and to be reliable foundations for human risk assessment, precautionary measures have to be taken. Moreover, besides this general precautionary principle, specific flavorings are worthy of further attention for their potential toxicity. For instance, 2,3-butanedione (usually named diacetyl) has been widely used in the past in microwave popcorn in the USA with the purpose to generate, depending on the concentration, buttery and caramel tastes. It is a chemical mentioned in the GRAS list and approved in certain limits for ingestion, therefore it is used as additive in foods [18]. Due to its flavoring properties it is also used in the manufacturing process of e-liquid formulations and its presence has been documented in previous investigations carried out in EU Member States, raising concerns in the scientific community regarding potential health implications [19,20]. In this regard, recently published scientific papers based on epidemiological data collected over recent decades have revealed that inhalation exposure to diacetyl is likely related to increased risk of a specific lung disease called bronchiolitis obliterans [21,22]. The use of flavoring chemicals for e-liquid manufacture stimulated scientists to focus on safety and quality aspects of the formulations. As a result, the number of scientific publications on the chemical characterization of e-liquids in terms of flavoring additives has recently increasing. To cite the most recent studies, in 2017 Aszyk et al. carried out a comprehensive determination of flavoring additives on 25 e-liquid samples highlighting that limonene and benzyl acetate were the two most frequently detected [23]. In 2018 Girvalaki et al. reported findings from qualitative and quantitative analysis performed on 122 of the most commonly sold e-liquids in 9 EU member states. Among the 293 flavoring chemicals identified, menthol was the most frequently detected compound, regardless the overall e-liquid flavor [24]. Specific flavoring chemicals with known respiratory irritant properties or identified as inhalation toxicants were detected in other studies in relevant amounts, i.e., benzaldehyde by Kosmider et al., methyl cyclopentenolone and menthol by Vardavas et al., diacetyl and acetylpropionyl by Barhdadi et al. [19,25,26].	doab	2025-04-07T03:56:58.096505	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 86 }
001fcd9a-cd27-45e8-b23f-f447874e0974.87	1.2. E-Liquids Contamination The attention of scientists to the chemical composition of e-liquids has not only been aimed at the identification of flavoring chemicals, but also to address the issue concerning the potential presence of compounds of toxicological concern, such as volatile organic compounds (VOCs), due to main component contamination and low purity level of nicotine and flavors [11,27–30]. Among VOCs, aromatic hydrocarbons have attracted remarkable attention in view of a toxicity assessment of refill liquids due to the recognized carcinogenic properties of benzene, classified as carc. 1A according to EU CLP regulation [31]. Specific investigations were carried out to perform both qualitative and quantitative characterization in terms of VOCs of e-liquids commercially available on the EU market prior to the EU TPD implementation and after 2016, in order to verify the compliance of e-liquids distributed over EU countries with the TPD in force, in terms of both chemical composition and classification/labelling [24,32]. With specific regard to aromatic hydrocarbons, BTEX contamination has been detected in e-liquids available on extra-EU markets. Lim et al. highlighted the potential health hazards for e-cig users reporting the results of investigations made on 283 flavored liquids, 21 nicotine-content liquids, and 12 disposable cartridges [33]. BTEX coexisted in most of the investigated samples at relevant concentrations (e.g., benzene concentration ranging from 0.008 to 2.28 mg/L) and the contamination was hypothetically related to the use of petrogenic hydrocarbons in the extraction process of nicotine and flavors from natural plants. BTEX contamination of liquid formulations was also previously observed by Han et al. in a study aiming to assess VOCs levels in 55 refill liquids of 17 different brands available on the Chinese market [34]. Benzene and m,p-xylenes were found in all of the samples investigated, whilst ethylbenzene and toluene were detected with different frequencies. They all were present at comparable levels in the concentration range 1.10–17.31 μg/g. In view of the findings obtained to date on e-liquids composition in terms of a broad range of chemicals, reported above, it appears clear that the attention on the issue has to remain high to ensure that consumers' health is safeguarded and that compliance to safety and quality standards is guaranteed. On one hand there is the need for a comprehensive database referred to e-liquids both manufactured and imported in EU member states before the implementation of the TPD allowing us to define a pre-TPD baseline reference useful for comparison. On the other hand, ongoing investigations into e-liquids currently on the market are necessary to evaluate the effectiveness of TPD provisions in EU member states with regard to the manufacture and labelling of e-liquids, and to formulate further recommendations to policymakers.	doab	2025-04-07T03:56:58.096992	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 87 }
001fcd9a-cd27-45e8-b23f-f447874e0974.88	1.3. Aim of the Present Study The aim of the present study was to evaluate BTEX contamination across a representative group of refill liquids for e-cigs (n=97) and to identify, in a selected sub-group (n=5), the main flavoring additives responsible for the flavor/taste perceived. BTEX quantification was carried out applying headspace-solid phase micro extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) methodology. The identification of flavoring additives was performed applying a hydrid analytical-sensory technique, the gas chromatography-mass spectrometry-olfactometry (GC-MS-O). This research activity has been carried out in the context of a more comprehensive national project supported by the Italian Ministry of Health and coordinated by the National Institute of Health aimed to evaluate in a comprehensive manner potential risks related to e-cig consumption. The refill liquids investigated were selected through a preliminary survey and were considered representative of the EU market between 2013 and 2015, prior to the implementation of TPD in most of EU member states. Therefore, the data here reported are eligible to be included in the pre-TPD database on e-liquids manufactured and/or imported in EU and may represent a useful reference for the ongoing evaluation on e-liquid safety and quality under the current EU Legislation.	doab	2025-04-07T03:56:58.097204	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 88 }
001fcd9a-cd27-45e8-b23f-f447874e0974.90	2.1. E-Liquids Selection In the framework of the national research project, the Italian National Institute of Health carried out a preliminary survey allowing to identify the most popular brands of e-liquids manufactured and imported in EU and representative of the EU market between 2013 and 2015. Ninety-seven e-liquids of 12 different brands, with and without Nicotine and characterized by different flavors, were purchased online from EU manufacturers and importers in 10–30 ml plastic bottles, as sold commercially. More specifically, the selected e-liquids were manufactured in Italy (n = 45), China (n = 28), France (n = 8), UK (n = 8), Germany (n = 4), and the USA (n = 4). E-liquid composition in terms of propylene glycol, glycerol, water content (expressed in %), nicotine content (expressed as mg/ml or mg/g) as well as characteristic flavor is reported in Table 1, as declared on product label. E-liquids belonging to different brands and within the same brand were classified with progressive letters and number, respectively (sample ID in Table 1). Moreover, three identical e-liquids in terms of brand, basic composition, flavor and nicotine content (e.g., samples 10, 11 and 12 C) belong to different production batches. Nicotine-containing e-liquids were 59 with variable content (11,14,16 and 18 mg/ml and 11, 18 mg/g), as reported on the product label. The remaining 38 e-liquids were declared nicotine-free. Most of the investigated e-liquids were flavored and may be included in the following typical flavor categories: tobacco (48), mint (17), sweets/candy (11), spicy (7), fruits (3), coffee (3), and alcohol (3). Before analysis, all e-liquids were properly stored at room temperature and kept away from direct sunlight, as recommended on the product label.	doab	2025-04-07T03:56:58.097288	11-1-2022 14:33	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "book_id": "001fcd9a-cd27-45e8-b23f-f447874e0974", "url": "https://mdpi.com/books/pdfview/book/3938", "author": "", "title": "Indoor Air Quality: From Sampling to Risk Assessment in the Light of New Legislations", "publisher": "MDPI - Multidisciplinary Digital Publishing Institute", "isbn": "9783036509464", "section_idx": 90 }

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