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Polycystic Ovary Syndrome: Effect and Mechanisms of Acupuncture for Ovulation Induction

Polycystic ovary syndrome (PCOS), the most common endocrine disorder among women of reproductive age, is characterized by the coexistence of hyperandrogenism, ovulatory dysfunction, and polycystic ovaries (PCO). PCOS also represents the largest part of female oligoovulatory infertility, and the management of ovulatory and menstrual dysfunction, comprises a third of the high costs of PCOS treatment. Current pharmacological and surgical treatments for reproductive symptoms are effective, however, associated with negative side effects, such as cardiovascular complications and multiple pregnancies. For menstrual irregularities and ovulation induction in women with PCOS, acupuncture has indicated beneficial effects. This review will focus on the results from randomized controlled acupuncture trials for regulation of menstrual dysfunction and for inducing ovulation in women with PCOS although there are uncontrolled trials with nonetheless interesting results. Animal experimental studies will be further discussed when they can provide a more mechanistic explanatory view.

Introduction
Although polycystic ovary syndrome (PCOS) has been recognized for more than 70 years there is no cohesive definition, and the diagnosis still causes debate. The most recent and used definitions are the National Institute of Health (NIH), Rotterdam, and Androgen excess and PCOS society (AES) criteria, where all are presently used in clinic (Table 1). In consequence, prevalence is difficult to conclude and depends on the used definition, as well as the ethnicity of the measured population. Most studies report prevalence between 6-15% but sometimes even up to 20%, depending on used criteria [1][2][3]. The different definitions also result in several PCOS phenotypes with a wide range of severity of the syndrome (Table 1), which comprises concerns when comparing clinical studies using different definitions. A recent NIH workshop on PCOS recommended to explicitly report PCOS phenotypes in all future clinical studies [4, 5].

Pathophysiology and Etiology of PCOS
Despite high incidence, the etiology of PCOS remains unknown. Due to the heterogeneity in the representation of clinical and biochemical features, it has been debated whether PCOS actually represents one single disorder or several ones. Symptoms of PCOS often manifest around puberty, but the origin may be programmed already as early as during fetal development [6][7][8]. One of the most common features of PCOS is insulin resistance, represented in 85% of PCOS women [9]. The other common feature of PCOS, elevated androgen levels, affects around 60-80% of PCOS women and can result in the clinical signs: hirsutism, acne, and, to some extent, alopecia [10]. In PCOS, high circulating levels of androgens, estrogens, sex steroid precursors, and glucuronidated androgen metabolites have been demonstrated by gas chromatography tandem mass spectrometry (GC-MS/ MS) and liquid chromatography tandem mass spectrometry 2 Evidence-Based Complementary and Alternative Medicine  (LC-MS/MS) [11]. The major androgen excess in PCOS originates from the ovaries, but the adrenals also contribute to some part [12]. Hyperinsulinemia, often found in PCOS, inhibits the production of sex hormone binding globulin (SHBG), thereby further contributing to levels of free circulating androgens [13]. Figure 1 summarizes the PCOS pathophysiology described below. Androgens possess a central position in PCOS, are closely related to the ovarian morphology, and are sufficient to cause PCOS-like states in both animal models and femaleto-male transsexuals [14,15]. Both androgens and insulin have therefore been presented as a key underlying cause of PCOS. Since it is not known when or where the pathology actually begins, several different hypotheses are presented. Prenatal androgenization represents an established hypothesis of PCOS etiology and is based on animal models including monkeys, sheep, and rodents where prenatal androgenization inflicts several features of PCOS in the offspring [16][17][18][19]. However, in humans, although increased levels of androgens have been found in pregnant PCOS women [20], only one study has found increased levels of testosterone in umbilical vein blood in infants of PCOS mothers although these levels were measured by immunoassays and not mass spectrometry [21]. Prepubertal exposure to androgens is yet another hypothesis that originates in the pubertal start of symptom manifestation [7,8], from which several animal models of PCOS have been developed [22][23][24].
Although it is not a part of the diagnosis, PCOS is also strongly associated with insulin resistance and compensatory hyperinsulinemia, type 2 diabetes, and dyslipidemia [25,26] and the prevalence of women with PCOS being either overweight or obese is high [27]. Both androgens and insulin, which both increase during puberty, are therefore considered to be two key players in PCOS. Most likely both contribute, but it is still unclear which one of these that is most related to the etiology [7,8].
The cause of LH hypersecretion in PCOS is probably due to enhanced pituitary sensitivity to gonadotropin releasing hormone (GnRH) or to changes in GnRH secretion patterns rather than increased GnRH secretion [35,39,40]. It appears to be a result of an acquired impaired sensitivity of the hypothalamic pulse generator to the negative feedback of estrogen and progesterone in PCOS, possibly by chronic estrogen exposure [35,38,39,41]. Levels of follicle stimulating hormone (FSH) in PCOS appear to be low or within the lower follicular range, and response to GnRH is relatively similar to ovulatory controls [35]. Altered sex steroid production, metabolic dysfunction, and obesity may all contribute to the changes in LH secretion pattern. Hyperandrogenemia itself may cause hypothalamic desensitization to progesterone/estrogen negative feedback that further increase gonadotropin secretion and hence ovarian androgen production, causing a self-driven viscous circle [42,43]. Although increased body mass index (BMI) has a blunting effect on LH secretion [36,39], hyperinsulinemia and insulin resistance may directly or indirectly (by enhancing ovarian gonadotropin stimulated sex steroid production) contribute to the abnormal gonadotropin secretion, although the mechanism is not clear [44][45][46]. All these factors increase free androgen levels and contribute to anovulation.

Ovarian Dysfunction in PCOS.
The ovulatory dysfunction in PCOS can be ascribed to disturbed follicular development with excessive early follicular growth and abnormal later stages of arrested follicle growth well before expected maturation [47]. This pattern of follicular growth with failure in the selection of a dominant follicle for ovulation results in one of the hallmarks of PCOS, PCO morphology. The ovarian dysfunction of PCOS involves both these morphological features of polycystic ovaries, described as an accumulation  (4) Insulin resistance with compensatory hyperinsulinemia enhances ovarian androgen production as well as (5) decreases production of SHBG in the liver, and both increase the pool of bioavailable androgens. (6) PCOS is also associated with increased muscle sympathetic nerve activity that is related to high testosterone, insulin resistance, and obesity. (7) Genetic defects probably contribute to the pathology of PCOS. LH: luteinizing hormone, FSH: follicle stimulating hormone, SHBG: sex hormone binding globulin, DHEA: dehydroepiandrosterone, and DHEAS: dehydroepiandrosterone sulphate.
of small antral follicles of size 2-9 mm, as well as the clinical consequence of oligo-/anovulation. The prevalence of menstrual irregularities oligo-/amenorrhea in PCOS depends on the used diagnostic criteria but is approximately 75% [10]. If we use the NIH criteria, of course all patients will experience menstrual irregularities. Ultimately, irregular ovulation can cause infertility due to difficulties conceiving and should be acknowledged.
Moreover, there are several other follicle abnormalities where the most consistent feature is androgen hypersecretion [48]. Ovarian steroid production is based on a close collaboration between theca and granulosa cells in the growing follicles and requires gonadotropin input ( Figure 2) [49]. Theca cells produce androstenedione from cholesterol, either by the Δ4 or Δ5 pathway, and the conversion to estrone and estradiol thereafter is exclusively acknowledged aromatase cytochrome P450 hydroxylase (CYP19) containing granulosa cells [50]. Women with PCOS appear to have theca interna hyperplasia, a thicker layer of the theca cells, which seem to be responsible for their increased androgen steroidogenesis.
Moreover, each theca cell has increased expression of LH receptors, with increased susceptibility to LH stimulation [33,50,51].
There are local selectors for follicle recruitment and growth within the ovary that might contribute to the impaired follicle development in PCOS. AMH is expressed by early antral and preantral follicles, but not in later stages of development, and reflects the size and activity of the follicular pool [52,53]. There is also evidence of its involvement in the regulation of recruitment of primordial follicles into the growing pool, presumably by decreasing the granulosa cell sensitivity to FSH [54]. Adding antimüllerian hormone (AMH) to ovarian cultures reduces the number of growing follicles, while removing the gene increased the proportion [55,56]. In the small primordial and transitional follicles of anovulatory PCOS, AMH protein expression is reported to be reduced [57]. This may contribute to the inappropriate recruitment of growing follicles. Additionally in both circulation and antral follicular fluid of PCOS women, AMH levels are increased, and these are associated with poor reproductive responsiveness to treatment [58][59][60][61][62][63]. These high circulating levels may be a reflection of the increased pool of granulosa cells instead of an increased expression. Since high levels of AMH are associated with lower levels of FSH and estradiol, it has been suggested that the AMH excess is involved in the lack of FSH-induced aromatase activity that is characteristic of follicular arrest in PCOS [62,64]. In addition, testosterone exposure downregulates AMH expression in granulosa cells of small bovine follicles in culture and could possibly represent a mechanistic origin of PCOS [65]. Ovarian inhibins are expressed in ovaries and act as modulators to suppress FSH levels. As a response to increased FSH levels inhibin B is increasingly expressed during early follicular phase in small developing follicles, while inhibin A is selectively produced in the dominant follicle. Inhibin B therefore correlates to total follicle number and may be a marker of follicle quality [66]. But inhibins also have a local effect to stimulate androgen production synthesis in theca cells for estradiol production [66][67][68]. Most PCOS studies find no differences in basal circulating inhibin B, but an abnormal and increased response to FSH. The normal basal levels may be explained by the diminished FSH secretion in PCOS or that the follicle quality is lower. The increased response to FSH could simply be due to their increased number of preantral and small antral follicles [67][68][69][70]. Moreover, follicular arrest in PCOS is associated with reduced levels of both inhibin A and inhibin B in follicular fluid, which both could explain the normal circulating levels although their increased pool of growing follicles, but also making these to possible actors in the impaired follicle development [71]. There is low evidence of any diagnostic value of circulating basal inhibin B measurements in PCOS [67,72].
Taken together, impaired folliculogenesis and steroidogenesis in PCOS seem to be multifactorial and are probably influenced by extra ovarian factors such as androgens, insulin, neuroendocrine alterations, and intraovarian local and intrinsic factors.

Increased Sympathetic
Activity. The autonomic nervous system consists of two divisions: the sympathetic and the parasympathetic nervous systems, and is controlled by the neurotransmitters noradrenaline and adrenaline and activation of adrenergic receptors. In a normal, healthy state, a fine balance between these two divisions, ensures homeostasis. Many of the classical components of PCOS such as polycystic ovaries, insulin resistance with related hyperinsulinemia, central obesity, and hypertension are associated with increased sympathetic activity [73][74][75][76]. It has therefore been suggested to account for at least a part of the syndrome etiology [74,75,77]. That increased sympathetic innervation of the ovaries might contribute to the impaired follicular development in PCOS that is supported by clinical evidence such as increased density catecholaminergic nerve fibers, increased NGF production, and altered catecholamine metabolism and/or uptake in PCOS ovaries [75,78,79]. Heart-rate recovery after a bout of exercise and heart-rate variability can be used as noninvasive markers of autonomic function. Measures in PCOS women indicate that they have decreased dynamic activity in their autonomic function, possibly by decreased activity in the parasympathetic component and increased in the sympathetic component [80][81][82][83]. These are though indirect measures and their accuracy may be questionable. We have though demonstrated by microneurography, which is a direct and reliable measure of muscle sympathetic nerve activity (MSNA), that women with PCOS have an increased sympathetic nerve activity that is correlated to high levels of testosterone [30].

PCOS: A Well-Orchestrated Pathology
Androgens play a central part in the pathology of PCOS. Androgens alone can affect many of the systems that are impaired in the syndrome and are sufficient to cause PCOSlike states in both animal models and female-to-male transsexuals [14,15,[17][18][19][84][85][86]. But these alterations Evidence-Based Complementary and Alternative Medicine 5 can themselves further increase hyperandrogenemia. Consequently, a vicious circle is created where the individual pieces may augment each other, although it is not clear where it started. Hyperandrogenemia in PCOS originates mainly from the ovaries and can have central effects by increasing gonadotropin secretion via affecting sex-steroid feed-back systems as well as enhancing the effect on ovarian gonadotropin stimulated sex steroid production [12,42,43]. Androgens also directly impair follicle development and maturation and thereby contribute both to the PCO morphology and to the ovarian pool of androgen producing cells [12]. Both of these will further drive ovarian androgen production and increase levels of free circulating androgens. Additionally, although the mechanism is not completely clear, increased adrenal androgen production contributes to the androgen excess in PCOS [87,88].
Androgens are also associated with an atherogenic blood lipid profile, enlarged adipocyte size, and peripheral insulin resistance although the androgen excess may not be the primary cause of their insulin resistance [89][90][91]. Moreover, together with obesity, this increases the risk of type 2 diabetes and cardiovascular disease (CVD) [27]. Similar to androgens, insulin resistance and hyperinsulinemia enhance ovarian gonadotropin stimulated sex steroid production [12,[44][45][46] and may contribute to the abnormal gonadotropin secretion although the mechanism is not clear [44][45][46]. Hyperinsulinemia also decreases liver production of sex hormone binding globuline (SHBG) which increases the amount of bioavailable free circulating sex steroids [13].
PCOS is related to increased MSNA and of special interest is that testosterone concentration was found to be a strong independent predictor [30]. Increased sympathetic nerve activity is related to insulin resistance with related hyperinsulinemia, central obesity, and hypertension [73][74][75][76] and might contribute to the increased cardiovascular risk [30]. There is also evidence that supports an increased sympathetic nerve activity to the ovaries [75,78,79,92] that may further drive androgen production and PCO morphology [93]. Apart from the previously described factors, there is a strong genetic component with familial aggregation of cases and symptoms [94,95] that probably is involved in the etiology of PCOS. Altogether, PCOS is a coordinated pathology containing factors that strongly influence each other in a viscous circle, making it difficult to separate the etiology.

Hypothetical Mechanism of Acupuncture
Acupuncture is widely practiced and is now also accepted in the western world for the treatment or adjunct treatment for more and more conditions [96]. It is a relatively safe treatment with few side effects [97].
Acupuncture originates from traditional Chinese medicine (TCM) where fine needles are placed in the skin and underly muscle tissue at specific areas of the body, socalled acu-or acupuncture points. When placed, needles are stimulated manually by rotating or perturbing needles up and down, so-called manual acupuncture. Needles may also be stimulated electrically by applying an electrical field and passing an electrical current between two needles, socalled electroacupuncture (EA). Low-frequency (1-15 Hz) electroacupuncture with an intensity that evokes muscle contractions is believed to achieve biological processes that resemble the effects of exercise. Acupuncture from a western scientific perspective cannot confirm point specificity and instead explain the effect with the activation of afferent sensory nerve fibers at appropriate segmental level [98,99]. Acupuncture activates and modulates nervous pathways at peripheral (local), segmental (in the spinal cord), and supraspinal levels within central nervous system (CNS). Figure 3 illustrates the hypothetical mechanism explaining the effects of acupuncture in PCOS.
Starting at a peripheral level, both manual and electrical stimulations increase glucose uptake and microcirculation [100][101][102][103]. When needles are inserted and stimulated, peripheral nerve terminals release several neuropeptides, such as neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P, and calcitonin gene-related peptide (CGRP), and give an immediate local reaction, by which the two latter are probably involved, with an increase in microcirculation [100,104,105]. Moreover, data from administration of the opioid antagonist naloxone also suggest low-frequency EA stimulated peripheral opioid release [106].
Mechanoreceptors, responding to mechanic pressure or distortion, are activated by muscle contractions and are suggested to be involved in the somatic response of both manual and electrical stimulations [107,108]. Activation of mechanoreceptors by manual or electrical stimulation activates sensory nerve fibers, myelinated A , , , and unmyelinated C-fibers [109,110]. These signals are transmitted to the spinal cord (segmental level) where they through spinal reflexes may modulate the sympathetic output to the target organs in the same area of innervation as where needles are placed [111]. Importantly, these signals are also controlled via supraspinal pathways within the central nervous system (CNS) [112].
The efficacy of acupuncture for analgesia has been extensively studied and widely used. The involvement of endogenous opioids within the central nervous system has been suggested to mediate the effect of acupuncture-induced analgesia and lowering of blood pressure [113][114][115][116]. There are three opioid receptors , , and found in peripheral afferent nerve terminals and within the central nervous system in areas related to nociception and pain [117,118]. -endorphin, one of the cleavage products of proopiomelanocortin (POMC), binds to the -receptor with high affinity and has received special attention [117]. -endorphin produced in the arcuate nucleus of the medial basal hypothalamus is released within CNS, but it is also produced in the pituitary where it is released into the peripheral circulation [119][120][121]. The -endorphin is produced in hypothalamus project to the midbrain (especially the periaqueductal grey) and brainstem nuclei where it can influence pain sensitivity and autonomic function [120,121]. The effect on autonomic function includes effects on the vasomotor centre with regulation of blood pressure and muscle sympathetic nerve activity (markers of sympathetic tone) [122]. The other system is also under hypothalamic control, by corticotrophin releasing hormone (CRH), and involves the anterior pituitary lobe where POMC is cleaved into equal amounts of -endorphin, melanocyte stimulating hormone, and adrenocorticotrophic hormone (ACTH) that is released into circulation [123]. The two systems work independently, but both can be activated by afferent nerve activity such as manual and low-frequency electrical stimulation of acupuncture needles and exercise [120]. The -endorphinergic system is involved in many physiological effects, both centrally and peripherally, such as reproductive function, analgesia, stress response, and carbohydrate metabolism [124]. Both circulating and central levels of -endorphin have been shown to be modulated by acupuncture [125][126][127]. Further implications of acupuncture involving modulation of autonomic function are the reducing effect on MSNA and blood pressure [128,129]. The pain reliving effect of acupuncture was further blocked by low doses of naloxone, an opioid receptor antagonist, and the blood pressure suppression by high doses of naloxone [129,130].
The connection between central -endorphin and reproductive function involves both direct and indirect tonic inhibitory effects on GnRH and subsequent LH release and possibly also GnRH biosynthesis [121,131]. It is generally accepted that opioids mediate the inhibitory effect of estrogen [132]. Moreover, a reduction in opioid inhibitory tone amplifies and is essential, for generation of the LH surge preceding ovulation [121,133].
Circulating -endorphin is considered to be more related to stress stimuli than actually reflecting central opioid activity and should not be used as a marker of central activity [134]. Still, the changes in plasma -endorphin provide a link to the hypothalamus pituitary (HPA) axis, since -endorphin is coreleased with ACTH from the pituitary [123]. The controlling factor CRH is released by stress, but it is also known to decrease GnRH secretion, linking it back to the reproductive axis [135]. Acupuncture has been shown to decrease levels of CRH within hypothalamus and may therefore present effects on both HPO and HPA axes [136].

Control Situations of Acupuncture?
In general the effect of acupuncture may also be influenced by the patient's own expectations, since there is a strong psychological component Evidence-Based Complementary and Alternative Medicine 7 to it [137]. The use of placebo in acupuncture experiments is therefore always under debate, and efforts to develop suitable control situations have been made. However, the use of socalled placebo needles or minimal/sham acupuncture may not be a true control since clinical studies suggest that they are not inert treatments [138,139].

Implications of Acupuncture for Ovulation Induction in PCOS.
Since many of the features of PCOS are associated with disturbed opioid or sympathetic tone, including disturbed gonadotropin secretion, insulin resistance, and central obesity [74,124], opioid and/or sympathetic tone may be implicated in the pathogenesis of the condition. Women with PCOS have higher levels of circulating -endorphin which may contribute to the pathogenesis, possibly by insufficient inhibition of GnRH by -endorphins in the CNS [124,[140][141][142]. This is supported by studies in which the -receptor antagonist naltrexone improves cyclicity and SHBG, reduces androgen levels, LH/FSH, and LH response to GnRH [143][144][145].
The relation between acupuncture, -endorphin, and sympathetic activity seems to be sustained also in PCOS. Acupuncture treatment has been shown to reduce both high plasma -endorphin and sympathetic nerve activity and increase low hand-skin temperature [30,128,146]. The involvement of sympathetic nervous system and an alteration in neurogenic control of the ovary have been implicated in the etiology of PCOS [111,112,147,148]. One hypothetical mediator for that effect, combining an autonomic and ovarian effect, is nerve growth factor (NGF). Ovarian NGF production is increased in the follicular fluid in women with PCOS and in an estradiol-valerate-(EV-) induced rat PCOS model [92,149,150]. Over expressing NGF in the ovaries of a transgenic mice model results in ovarian hyperinnervation, arrested follicle growth, and increased ovarian steroidal responsiveness to gonadotropins [92]. Also ovarian adrenergic receptors have been shown to be increased in the EV-induced rat PCOS model further indicating involvement of sympathetic system in the regulation of ovarian function [151]. Electroacupuncture has been shown to decrease high ovarian NGF [149,150] and adrenergic receptors [152] in the EV-induced PCOS model and to decrease high mRNA expression of NGF and several markers of sympathetic activity in adipose in the DHT-induced PCOS model [153]. Together with microneurography measurements demonstrating a decrease of high MSNA in women with PCOS after 14 acupuncture treatments, this indicates an involvement of the sympathetic nervous system in both PCOS etiology and the mechanistic effect of acupuncture [30]. That naltrexone, a -receptor that antagonist induces ovulation and decreases LH concentration, indicates also a role of -endorphin in PCOS [143][144][145]. That similar effects are also mediated by electroacupuncture [146,154,155] concurrent with a lowering of -endorphin levels [146] implying the involvement of the opioid system in the underlying mechanism. This is also supported in our recent experimental study where electrical stimulation of the acupuncture needles affected the expression of opioid receptors and in rat hypothalamus, together with improved cyclicity and reduction of circulating testosterone levels [156].

Treatment of Reproductive Function in PCOS
Due to the uncertainty of the etiology of PCOS as well as the heterogeneity of the syndrome no cure can currently be offered. Women are instead treated in a symptom-oriented manner, often for long duration, with associated adverse effects. Lifestyle modifications, including diet and exercise, are frequently recommended as a first-line treatment in the large population of overweight and obese PCOS women. It improves several of the key features of PCOS such as body composition, hyperandrogenism, and cardiometabolic profile, including insulin sensitivity and blood lipids, but also autonomic function and inflammatory pattern [157][158][159][160][161][162][163][164][165][166]. The effect may also include improved ovulatory function and pregnancy [158,160,163,167,168] alone or together with clomiphene citrate (CC) [162], and even a modest (5%) weight reduction has been reported to improve metabolic and reproductive functions [169].
For women who do not wish to conceive, combined oral contraception is frequently used for regulation of menstrual bleeding patterns and reduction of androgen levels [25]. However, use of oral contraceptives may be contraindicatory in women with PCOS suffering from obesity and/or insulin resistance or risk of cardiovascular disease [25].
Nonrandomized studies on acupuncture for women with PCOS as well as with undefined anovulation show improvements of menstruation pattern, LH/FSH ratio, estrogen, and testosterone [146,154,155]. The effect on ovarian function was first demonstrated in an RCT in which acupuncture with combined manual and low-frequency (2 Hz) electrical stimulation of needles during 30 minutes per treatment was superior to 16 weeks of exercise according to the Randeva protocol [170] and no intervention in improving menstrual frequency and lowering circulating levels of testosterone in PCOS women [166]. Needles were placed in abdominal and leg muscles in the same somatic segment as the innervation of the ovaries. The treatment period was 16 weeks and women received in total 14 treatments: twice a week during 2 weeks, once a week during 8 weeks, and thereafter every other week. Concurrently, Pastore et al. compared the effectiveness of true acupuncture and sham acupuncture using the Park device [171]. They received 12 treatments over 14 weeks and the needle placement and stimulation in the true acupuncture group followed an old protocol [172]. Both groups improved their menstrual frequency, and no difference between the groups was observed on ovulation rate during or after treatment, thus indicating that the sham device is not completely inert. In a more recent RCT, we further determined ovulation frequency in PCOS women, during a 3-month long acupuncture or attention control treatment period, by weekly progesterone measurement as well as menstrual bleeding registrations. In this study, women received acupuncture twice a week during the study period with similar needles placement as in the previous study, thus increased intensity in terms of more frequent treatment. The group who were allocated to acupuncture treatment had higher ovulation frequency compared with the attention control group [173], an effect 8 Evidence-Based Complementary and Alternative Medicine that was augmented compared to the previous clinical studies on PCOS with fewer acupuncture treatments, indicating a dose-response effect [166,171,172]. In addition, the ovulation frequency in the attention control group [173] was comparable with ovulation frequency during CC stimulation [174], as well as the one in a previous acupuncture study on PCOS women [171]. Supporting animal data include improved ovarian morphology [153] and restored estrous cyclicity [47,156] with increased progesterone levels [156] after acupuncture in a DHT-induced rat PCOS model [22]. Moreover, there were no significant differences in estrous cyclicity improvement between the two stimulation modalities: electrical and manual needle stimulations [156]. The parallel effect regarding ovulation in the rat and human studies strengthens the result by the translational nature. Proceeding to ovarian morphology in the recent RCT, no group differences were observed when counting antral follicles ≤9 mm or ovarian volume [173]. However, a tendency to reduced number of antral follicle and ovarian volume was observed within the acupuncture group [173]. This is also in line with previous animal studies where electroacupuncture improved ovarian morphology in DHT-induced PCOS rats [153]. In addition, we found reduced circulating levels of inhibin B after acupuncture, without changes in AMH [173] which was in line with a previous study [60]. This may either represent a decreased size of the follicular pool or mimic an improved follicular development and reduced levels of ovarian sex steroids [66][67][68]71]. Moreover, delta changes of most circulating sex steroids (E 1 , E 1 -S, E 2 , T, free-T, DHT), sex steroid precursors (DHEA, DHEA-S) and glucuronidated androgen metabolites (ADT-G, AD3G, and AD17G), as determined by mass spectrometry differed between the intervention groups. Differences in E 1 -S, E 2 , DHEA, free-T, and ADT-G held for bonferroni correction. Acupuncture reduced levels of E 1 , E 1 -S, E 2 , DHEA, DHEA-S, 4-DIONE, T, and free T from baseline to end of treatment [173]. This is in line with, and extends, our previous RCT where several of these steroids were reduced [166]. Also here the clinical data is in line with the experimental data, where acupuncture with low-frequency electrical stimulation reduced levels of testosterone in DHT-induced PCOS rats [156]. The enhanced effect regarding both ovulation and sex steroids may be explained by increased number of treatments, more than 20 treatments, compared to the previous study comparing 14 treatments with acupuncture and exercise, as our animal studies infer dose-responsive effects [166,173,175,176].

Gap in Knowledge.
The present studies on menstrual and ovulation frequency have limited sample size and lack longitudinal followup for more than 16 weeks. It is also of importance to describe the most effective treatment regimen including maintenance treatment.
Treatment of first choice for ovulation induction in women with PCOS attempting to conceive is lifestyle interventions [177] and thereafter clomiphene citrate (CC) [178]. Ovarian hyperstimulation, multiple gestations, and spontaneous abortions (∼20% instead of the normal 12-15%) belong to the adverse effects of CC stimulation, although rare [178,179]. Approximate 20% of PCOS women are designated to be CC resistant [180,181] and if CC fails, gonadotropin (FSH) therapy or laparoscopic ovarian surgery is considered as further treatment. The efficacy of gonadotropins is a 70% ovulation rate and a 20% pregnancy rate [178], but the treatment is costly and requires intensive ovarian monitoring [182]. Laparoscopic ovarian surgery (LOS), such as ovarian drilling by electrocautery or laser, has a pregnancy rate between 25-51% and a live birth between 24-44% [183]. However, these more invasive treatments are associated with side-effects such as adherence. Other used treatments for ovulation induction are considered in more detail in other review articles such as the ESHRE/ARSM sponsored PCOS consensus report from 2007 [178]. The earlier described acupuncture studies show improvement of menstrual and ovulatory function and may therefore also improve pregnancy rate in women with PCOS, either alone or in combination with traditional treatment [166,171,173].

Gap in Knowledge.
Previous studies indicate that acupuncture induces ovulation and thus may lead to pregnancy in women with PCOS [166,173]. However, none of these trials were designed to investigate pregnancy or live birth. This gap in knowledge is addressed in an ongoing, multicenter, headto-head RCT (ClinicalTrials.gov: NCT01573858) in China. This trial tests the following hypotheses in anovulatory women with PCOS: (1) true acupuncture and CC are more likely to result in ovulation and live birth than control acupuncture and CC, (2) control acupuncture and CC are more likely to result in ovulation than true acupuncture and placebo CC, and (3) true acupuncture and placebo CC are more likely to result in live birth than control acupuncture and placebo CC.

Acupuncture on Neuroendocrine Function in PCOS.
To elucidate possible explanations for the effect of acupuncture on ovulation in women with PCOS and DHT-induced PCOS rats studies measuring LH pulsatility and experimental studies exploring other neuroendocrine functions have been conducted with varying results [156,173,184].
In the rat, the highest density of GnRH neurons is located in the rostral medium septum (MS), diagonal band of Broca, and medial preoptic area (MPO) [185]. Most GnRH neurons send projections via mediobasal hypothalamus down to the median eminence where GnRH is released into pituitary portal blood [186,187]. In DHT-induced PCOS rats, there seem to be more GnRH-immunoreactive (GnRH-ir) cells in the MPO and horizontal limb of the diagonal band (HDB) than in control rats [184]. Additionally, compared to controls, the PCOS rats displayed increased levels of hypothalamic functionally active androgen receptor (AR) and AR-ir cells in the MPO as determined by western blot and immunohistochemistry (IHC). This implies that androgens may have a regulatory effect on the GnRH neurons. Moreover, the increased levels of GnRH-ir cells and AR in hypothalamus were reduced after electroacupuncture treatment [184]. Estrogen receptor has previously been shown to be colocalized with GnRH neurons, thereby indicating a direct action of estrogens on GnRH regulation [188]. It has also demonstrated a colocalization of AR and GnRH neurons that further strengthens the hypothesis of a direct androgenic regulatory control on GnRH neurons [184]. Hence, we have indications that both the GnRH abnormalities and the effect of electroacupuncture could be mediated via the AR in hypothalamus. Moreover, previous experiments have demonstrated that levels of circulating estradiol are not altered in the DHT-induced rat PCOS model, which further supports the conclusion that this is an androgenic effect [22].
Although the arcuate nucleus is considered to be the principal location of the GnRH pulse generator in the rat brain, the preoptic area regulates the GnRH secretion surge controlling the preovulatory LH surge [132,189] making the findings in MPO even more interesting. Administration of androgens prenatally has previously been shown to increase LH pulsatility, without affecting pituitary responsiveness to GnRH, and to abolish the estrogen-induced LH surge, which is indicative of acceleration in the GnRH pulse generator. A contemporary reduction of estradiol-induced progesterone receptor (PR) expression in the preoptic area was then considered as evidence of how androgens could mediate this effect [190]. 4-day administration of androgens to adult rats produced similar results on the LH surge and PR expression but with the contrast of an instead decreased LH and possibly GnRH secretion, similar to the male response [191,192] and in oppose to human PCOS. These results are indicative of a programming effect (defect) of androgens during prenatal development that is different from the direct effects of hyperandrogenemia in adult life. Since in the DHT-induced PCOS model, rats are continuous exposed to androgens from day 21 until adult life, it is possible that they also have reduced GnRH secretion, with abolished GnRH/LH surges [191,192]. One might therefore speculate that restoration of the GnRH and LH surge by effects on the androgen receptor in the MPO could be at least part of the mechanism behind the improved estrous cyclicity after electroacupuncture. A succeeding study could not confirm the affected hypothalamic GnRH and AR expression; however, these measurements were made at mRNA level [156]. This could mean that the effect is related to posttranslational events rather than gene expression changes. However, no measurements of LH pulsatility have been performed in the DHT-induced rat PCOS model, either basally or after electroacupuncture, to confirm a physiological effect.
The hypothesis that the higher ovulation frequency by acupuncture in women with PCOS is due to restoration of gonadotropin secretion was tested in the recent RCT [173]. However, none of the LH pulsatility measures were affected by acupuncture treatment twice a week during 3 months. The major effect accompanying the higher ovulation frequency and reduced circulating inhibin B levels in the acupuncture group was the general decrease in circulating sex steroids, androgen precursors, and glucuronidated androgen metabolites after treatment, pointing towards an effect at ovarian and adrenal levels. However, it does not exclude involvement of central control mechanisms [173].
In an earlier RCT comparing sham and true acupuncture in PCOS women the LH : FSH ratio was decreased in both groups together with rather high ovulation frequencies [171].
The lack of effect on LH pulsatility in the latest RCT [173] may be due to the sampling timing in the menstrual cycle, which could be a confounding factor [44,193,194]. The majority of endpoint overnight blood sampling was performed during cycle day 8-10 (mid to late follicular phase). It is possible that an effect might have been seen if blood samplings were instead performed in early follicular phase [173].

Discussion
The viscous circle of PCOS features aggravating each other may be driven by androgens, insulin, or other factors but must be broken to improve the health status of women with PCOS. Although pharmacological treatment may be effective, they are also associated with negative side-effects [97]. This review addresses acupuncture as a potential treatment option for reproductive and endocrine disturbances in women with PCOS. Several clinical and animal experimental studies indicate that acupuncture is beneficial for ovulatory dysfunction in PCOS. This is also related to decreased levels of sex steroids and possibly inhibin B. Although the clinical data does not support changes in LH pulsatility/secretion pattern as a possible mediator for this effect [82], there is strong evidence of central components that probably involve both opioid and sympathetic activities, an effect that may be mediated via the androgen receptor. However, the causality of changes in ovulation and sex steroids is difficult to determine. The reduction of androgen levels by normalization of intrinsic ovarian aberrations may calm the exaggerated follicular growth, restore the follicle maturation processes, and hence lead to ovulation [12]. It could also be the other way around that improved ovulation, possibly caused by external factors, restoring follicular growth and/or aberrations and thereby reducing androgen levels due to the lower pool and/or activity of androgen producing cells [32].
Improved menstrual and ovulatory patterns are indeed beneficial, both for women not trying and women who are attempting to conceive. Further studies are needed to investigate whether acupuncture improves pregnancy and live birth rate in women with PCOS.
For the purpose of implementing acupuncture in the conventional treatment strategy, though, it is also of importance to compare it with the first-line pharmaceutical options. This is important for the support and sanction of acupuncture as a treatment method. To further investigate the underlying mechanisms of the effect of acupuncture may also be supportive in the search for other possible alternative treatments, including pharmacological. [13] R. Pasquali and F. Casimirri, "The impact of obesity on hyperandrogenism and polycystic ovary syndrome in premenopausal women, " Clinical Endocrinology, vol. 39, no. 1, pp. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]1993.


extfieldsofstudy: Medicine Biology

text:
The Influence of Mucus Microstructure and Rheology in Helicobacter pylori Infection

The bacterium Helicobacter pylori (H. pylori), has evolved to survive in the highly acidic environment of the stomach and colonize on the epithelial surface of the gastric mucosa. Its pathogenic effects are well known to cause gastritis, peptic ulcers, and gastric cancer. In order to infect the stomach and establish colonies on the mucus epithelial surface, the bacterium has to move across the gel-like gastric mucus lining of the stomach under acidic conditions. In this review we address the question of how the bacterium gets past the protective mucus barrier from a biophysical perspective. We begin by reviewing the molecular structure of gastric mucin and discuss the current state of understanding concerning mucin polymerization and low pH induced gelation. We then focus on the viscoelasticity of mucin in view of its relevance to the transport of particles and bacteria across mucus, the key first step in H. pylori infection. The second part of the review focuses on the motility of H. pylori in mucin solutions and gels, and how infection with H. pylori in turn impacts the viscoelastic properties of mucin. We present recent microscopic results tracking the motion of H. pylori in mucin solutions and gels. We then discuss how the biochemical strategy of urea hydrolysis required for survival in the acid is also relevant to the mechanism that enables flagella-driven swimming across the mucus gel layer. Other aspects of the influence of H. pylori infection such as, altering gastric mucin expression, its rate of production and its composition, and the influence of mucin on factors controlling H. pylori virulence and proliferation are briefly discussed with references to relevant literature.

INTRODUCTION
As is well known, Helicobacter pylori (H. pylori), the most abundant pathogen in the stomach causes gastritis, peptic ulcers, and gastric cancer by establishing colonies on the epithelial surface of the stomach that generate a host-immune response. It has three major pathogenic effects on its host: gastric inflammation, disruption of the gastric mucosal barrier, and alteration of gastric physiology (1)(2)(3)(4). The virulence factors that are responsible for the pathogenic effects also enable the bacteria to manipulate the host-immune response and support its long-term survival in the stomach (5)(6)(7)(8)(9). The question of how the bacterium initially breaches the protective mucus barrier to reach the epithelial cell surface and colonize in the extreme acidic environment of the stomach (10) is particularly intriguing from a physical perspective. Bacterial motility in aqueous solutions is well understood (11, 12), and motility through viscous polymeric solutions has been investigated for many years (13-18). However, much less is understood about how a bacterium moves through a gel, and how the motion depends on, and in turn, influences the structure and dynamic properties of the mucus gel. Recently, some theoretical advances have been made on the related problem of motility of sperm [for a recent review see Ref. (19)], and on the swimming of helical objects in a viscoelastic medium (20). The presence of an elastic network with fluid filled pores in a gel raises questions such as: (i) "Can a bacterium move through a gel, and if so, how does it move?" (ii) "Is it possible for the bacterium flagella motors to exert sufficient force and torque to deform a gel and enable it to move?" (iii) How do the speed and torque depend on the rheological parameters of the gel? Conversely, does the bacterium alter the physical and chemical properties of the gel? In this review we describe the physical characteristics of the gastric mucus lining, and discuss the structure and gelation of gastric mucin, which is the gel forming component of mucus. We then address the question of the motility of H. pylori in the acidic environment of the stomach, and its influence on the structure and mechanical properties of the mucus gel.
The gastrointestinal (GI) tract, like other physiological systems with a cavity open to the environment, is lined with a protective mucus layer [for reviews see Ref. (21,22)]. The high molecular weight glycoprotein, mucin, secreted by cells in the lining of these organs is responsible for giving mucus the physical characteristics of a viscoelastic fluid and a hydrogel (21). This physical state presents a unique environment to the more than a trillion bacteria that live or move in the GI tract. In the stomach, like the intestine, bacteria are found in the viscous fluid-like outer mucus layer but not in the dense mucus gel layer that adheres www.frontiersin.org to the cell surface (23, 24). Compared to the intestine, relatively few bacteria inhabit the mucus layer of the stomach, as most are unable to survive in the acidic environment of the stomach (25). H. pylori, the most abundant, and the most long-term inhabitant of gastric mucosa, copes with the acidic environment by secreting urease to hydrolyze urea and produce ammonia to neutralize the acid (26, 27). How it gets across the mucosal barrier in the stomach to colonize on the gastric epithelium (10, 28, 29) is a largely unsolved problem. The commonly held view that it bores its way through the mucus gel-like a corkscrew (10, 30) is probably not valid, in light of observations showing that H. pylori are immobile in porcine gastric mucin (PGM) gels under acidic conditions, although their flagella rotate and they wiggle in-place. Instead, we show that motility across the gel is achieved due to the same biochemical mechanism that H. pylori uses for surviving in the acid, namely urea hydrolysis to elevate the pH of its environment. The elevation of pH to neutral transforms the viscoelastic mucin gel to a viscous liquid, enabling the bacterium to swim in the viscous solution (31). Additionally, the helical cell-shape may enable it to swim faster in the viscous solution, as implied by the theoretical prediction of (20). The helical cell-shape has also been shown to be of importance in colonization, as rod-and C-shaped mutants of H. pylori, although motile in soft agar, are not as effective in establishing colonies (32, 33).
Helicobacter pylori infection is also known to impair mucin production, and alter the composition of mucins in the gastric mucus (34 , observed a reduction in the thickness of the adherent mucus layer in mice. Mucins also bind to H. pylori (24, 40), the binding is pH dependent (41, 42) and the bacterium is chemotactic toward mucin. Conversely, mucins influence the proliferation, gene expression, and virulence of H. pylori, implying a dynamic interplay between the bacterium and its host (43). These aspects will not be further addressed in this review; the reader is referred McGuckin et al. (24) for the complex interplay between mucins and bacterial pathogens. In the remainder of this review we focus on the physical properties of mucus relevant to the transport of H. pylori across the mucus barrier, and provide some insight into the mechanism of H. pylori motility. We also address the impact of H. pylori infection on mucus structure and rheology.

STRUCTURE OF MUCUS
Of all the organs, it is in the stomach that mucus faces its severest challenges from secreted HCl, digestive enzymes, alcohol, drugs, and bacteria such as H. pylori (10, 44). Gastric mucus is a highly hydrated (swollen to ∼95% water), viscoelastic substance containing 3% of mucin glycoprotein mixed with about 2% low molecular weight lipids, electrolytes, other small molecules, and other proteins such as trefoil factors (21, 45). The mucin glycoprotein is responsible for the remarkable hydration, viscoelastic, and mucoadhesive properties of the protective mucus layer (21, 46). These properties are primarily related to the ability of mucin to polymerize to high molecular weight [for reviews of biophysical properties of mucin see Ref. (47,48)]. At the typical concentrations found in mucus secretions in mammalian stomachs, mucin further aggregates, and gels under acidic pH.
The gastric mucosal surface is coated with mucus, about 200-400 µm in thickness, consisting of an adherent layer of mucus on the epithelial surface covered with loosely attached, mobile mucus on the luminal side (49). Atuma et al. (50) reported in vivo measurements of the thickness of the mucus lining from stomach to colon in anesthetized rats. They observed that the mucus lining is continuous and consists of two layers, a loosely adherent outer layer which can be easily removed by suction and a firmly adherent layer attached to the epithelial surface. In the rat stomach the loose layer varies from 100 to 120 µm and the firmly adherent layer ranges from 80 to about 150 µm.
Several investigators have used scanning electron microscopy (SEM) to visualize the stomach mucosal surface [see for e.g., Ref. (49, 51-53)], and numerous images and beautiful illustrations of the microstructure of the inner surface of the stomach, with and without bacteria, are readily available see for e.g., http://katierosejohnston.blogspot.com/2011/09/ research-images.html. These pictures reveal a highly convoluted, self-similar, or fractal surface with numerous folds of stomach epithelia forming gastric glands (also called gastric pits) which open on the luminal surface of the stomach. Mucus secretions can be seen as wispy, fibrous material on the surface on many of these images. Forte (52) reported low-resolution images of bullfrog mucosa where the residual mucus secretion left on the SEM specimen was visible as bright white, coagulated strands. In this review, we reproduce an image from Nunn et al. (51) showing surface mucous cells, covered with sheets of mucus (Figure 1).
The shiny, translucent film of mucus, visible to the naked eye, can be removed by gentle scraping and further processed to prepare purified mucin (54). To avoid the perturbative effects of SEM preparation we examined the structure of hydrated mucin and mucus on the sub-micron length scale in vitro by atomic force microscopy (AFM) in a liquid cell under appropriate buffers. A tapping mode AFM measurement of a wet sample of human mucus taken from the discarded material obtained in the lavage following a gastric biopsy reveals a swollen network (Figure 2) formed by the glycoprotein mucin (55). The mucin appears to form aggregates that are connected as in a "pearl-necklace" and enclose aqueous pores of about 200-300 nm in diameter.

MOLECULAR COMPOSITION OF GASTRIC MUCIN
For completeness we include a brief description of mucin composition, although this topic has been reviewed extensively. Gastric mucin, like other mucins is a very high molecular weight glycoprotein (2-20 million g/mol) with about 70-80% polysaccharides. The protein, which is quite unlike normal globular proteins, forms the linear core of the molecule on which the polysaccharide chains are radially arrayed similar to the bristles of a bottle-brush, as shown in Figure 3 [adapted from (48)].  Of the ∼20 mucin genes that have been identified with a similar sequence structure (56,57), gastric mucus contains only two secretory mucins MC5AC and MUC6, in addition to the ubiquitous cell surface mucin MUC1 (58). Dekker et al. (59) suggest that the mucin family be separated into two groups on the basis of sequence homology, one group representing those similar to the cell surface mucin MUC 1, and the other group comprising the secretory mucins similar to MUC2. Both MUC1 and MUC2 mucins contain large domains comprised of tandem repeating sequences of serine (S), threonine (T), and proline (P), located in the heavily O-glycosylated portion of the molecule. The S and T amino acids provide the O-glycosylation sites for the covalent attachment of the polysaccharide. The length and number of the STP repeat varies for the different mucins coded by different genes, and also varies between species. For example, MUC5AC contains 66-124 repeats of 8 amino acids with the consensus sequence TTSTTSAP (60) and MUC6 contains 15-30 repeats of 169 amino acids (61). In all MUC2 type secretory mucins, this glycosylated (g) domain occupies the central region of the apoprotein. It is flanked by cysteine rich domains and a Cystine Knot domain, with non-repeating sequences at the C-terminal (see Figure 3), and by domains similar to the von Willebrand Factor (vWF) C, D domains involved in blood clotting pathways, at the N-terminal. These weakly or non-glycosylated (ng) regions resemble typical secreted globular proteins in their amino acid composition and contain small amount of isolated N -glycosylated oligosaccharides, but do not form a bottle-brush.

POLYMERIZATION, AGGREGATION, AND GELATION OF MUCIN
Early studies of mucin were interpreted in terms of tetrameric wind-mill like structure (45). However, transmission electron microscopy (TEM) studies clearly established that gastric, cervical, and respiratory mucins are all linear polymers (62,63). AFM and dynamic light scattering (DLS) studies further show that even in solution, mucins are highly elongated, rod-like, or worm-like polymers [for detailed review and references see Ref. (47,48)]. AFM imaging of individual molecules of PGM in aqueous solution reveals long, curvilinear filaments ranging from 500 nm to 4 µm in length (55) and ∼1 nm in height, reflecting the height of the hydrated brush probably flattened down due to interactions with the AFM tip. The height and diameter reported by Hong et al. (55) were made in an aqueous environment, and thus provide a better estimate of the dimensions of the hydrated, native PGM molecule than that obtained by TEM measurements (63) or earlier AFM measurements (64) which were done on dried films. Hong et al. (55) also examined the pH dependence of PGM, observing in situ aggregation at pH < 4 with the formation of large, spherical, or oblong aggregates and a 5-to 10-fold increase in height.
The formation of large glycoproteins with molecular weight ranging from 2 to 20 million g/mol is generally believed to involve C-terminal dimer formation of the apoprotein via disulfide (S-S) linkages of the Cysteine knot domains (57,65). These dimers then further polymerize to form large multimers, as illustrated in Figure 3. There is also the possibility of the C-linked dimers to form trimers via N-terminal S-S linkages involving the vWF D domains. This has been observed in MUC2 (66,67) and in porcine submaxillary mucin (57) predominantly composed of MUC5B. This would imply the occurrence of tri-functional branches in mucin. It is not clear whether vWF D linked trimers are formed in MUC5AC, although other cleavage sites at the C-terminal have been noted (68,69). Since TEM and AFM images show www.frontiersin.org predominantly linear polymers, it is possible that trimers, if there, are present in small quantities, and the weak branching at the molecular level is not resolvable by TEM and AFM techniques.
The molecular mechanism of crosslinking in PGM gels is not fully understood. Using fluorescent dye binding (70) suggested that aggregation/gelation at low pH involves a complex interplay between electrostatic and hydrophobic interactions (48) with the formation of non-covalent crosslinks via the hydrophobic association of specific regions of ng domains, modulated by pH dependent changes in the electrostatic interactions of charged amino acids in the N-and C-terminal regions of the apoprotein (48). This network is schematically illustrated in Figure 3, which shows the mucin polymer as having long glycosylated (g), hydrophilic domains, alternating with short, and somewhat hydrophobic ng domains. The differential affinity for water of the alternating domains will stabilize non-covalent crosslinks formed by association of hydrophobic amino acids exposed at low pH in the ng domains in the C-and N-terminal regions, as illustrated in Figure 3. Exactly which domains are involved in these hydrophobic interactions, and whether it involves single domains from dimers or whether it involves the N-terminal tri-functional vWF units has not been explored. Discrete molecular dynamics simulations show pH induced changes in the folding of the PGM 2X domains that are not seen in vWF C domains (71). Further work, both theoretical and experimental, concerning the folding and association of the ng domains, and the swelling of the gel due to the electrostatic interactions of the negatively charged polysaccharide brush of the glycosylated domains, would be valuable in developing a detailed molecular model of pH induced gelation of mucin.

pH DEPENDENT VISCOELASTIC BEHAVIOR OF MUCIN
The formation of a network in gastric mucin at low pH has a profound influence on its rheological properties, i.e., its fluidlike flow behavior and response to mechanical deformation and shear forces. In view of the key role that these properties play in the transport of particles and bacteria across mucus, we discuss the underlying concepts before discussing the results. Like many other soft biological materials, the mucus layer exhibits a viscoelastic response to deformation that represents the combined effect of both its liquid-like and its solid-like features, arising from the presence of a polymer network filled with liquid. As illustrated in Figure 4A, a normal viscous solution, like water or glycerin, flows when subjected to a tangential shear force with a viscosity η, reflecting the resistance to flow, i.e., the dissipation or loss of energy. In contrast, an elastic solid stretches (or compresses) like a spring when subjected to tensile forces, and its shape deforms, like the spine of a hardcover book, when subjected to shear forces, but it does not flow in either case. A viscoelastic material exhibits both liquid-like flow and solid-like elasticity, as summarized in Figure 4B (72,73). One of the hallmarks of a viscoelastic material is that its response to deformation depends on the time for which the material is deformed, as illustrated by the familiar Silly Putty which bounces like a ball when dropped quickly (reflecting the solid-like response on short time scales), but flows like a liquid does not fall at all in mucin at pH 2, indicting the formation of a gel network at pH 2 (54).
Further insight into the rheological properties of a gel can be obtained from techniques such as oscillatory shear rheology which provides a direct measurement of the frequency dependent bulk viscoelastic moduli of a material. Oscillatory shear measurements clearly show that viscoelastic properties of gastric mucin are highly pH dependent, as shown in Figure 5 (74). At elevated pH (>4) gastric mucin flows like a viscous polymer solution, in which mucin glycoprotein macromolecules are in solution phase, with only transient intermolecular entanglement. In these conditions response to deformation is dominated by viscous flow (and viscous modulus G (ω) > elastic modulus G (ω), over a wide range of values of ω) with a small (but measurable) elasticity. Conversely, at pH < 4, associations between mucin domains give rise to a connected intermolecular network with significant elasticity and minimal flow [elastic modulus G (ω) > viscous modulus G (ω)] (see text box for definitions). Further insight is gained by examining the scaling relationship in plots of G (ω) and G (ω) versus ω and identifying the frequency at which the moduli cross over as described previously (74,75). This behavior is paralleled in a wide range of biopolymer systems driven by polymer concentration, solvent interactions, pH, salt concentration. Similar results on the pH dependence of PGM have been reported by Maleki et al. (76) using rheo-SALS, a rheometric technique coupled to small angle light scattering. Bulk rheology, as described above, provides the average viscoelastic response of a material. However, in a swollen gel such as mucin, the elastic, and viscous response is likely to be length scale dependent due to the inherently heterogeneous structure of a gel consisting of fluid filled pores and network strands. The www.frontiersin.org local, microrheological properties can be probed by tracking the hindered, Brownian motion of micron sized polystyrene latex particles in the medium of interest and calculating the complex elastic modulus G*(ω) from the mean square displacement ∆r 2 of the particle (77)(78)(79). Using this method, Celli (80) found that both G (ω) and G (ω), the elastic and viscous moduli, at pH 6 have similar magnitudes to those obtained in bulk rheology, as expected for particles moving in a viscous solution (80). In contrast, at pH 2 the moduli obtained by microrheology were significantly lower than those obtained by bulk rheology (Figure 5), indicating that, on small length scales, the particles sample a less viscous environment than the bulk gel. Similar results were obtained in a microscopic DLS study (81) in which the much smaller, 100 nm latex particles, appeared to probe two microenvironments; with some beads moving freely in large pores in the pH 2 gel, and others displaying the same slow relaxation as the signal from the dynamics of the gel itself. Lieleg et al. (82) also observed heterogeneities in their investigation of particle translocation through mucin hydrogels. Time-resolved microscopic particle tracking measurements (80) show that at pH 2 the probe particles move in an inhomogeneous micro-environment consisting of water filled pores in a gel and thus encounter reduced viscous damping. This is illustrated in Figure 6 showing the trajectory of the center of mass (c.m) of a 1 µm latex bead trapped in a pore in the pH 2 mucin network, and only moving at most a total distance of ∼0.1 µm. Rheological studies under non-linear deformations reveal an apparent yield stress, the stress at which the elasticity breaks down, that is also highly influenced by pH. The gel begins to flow just above 10 Pa (74). PGM exhibits a highly non-Newtonian shear thinning behavior, viscosity decreasing with increasing stress in steady shear flow tests, as was previously observed in commercially made PGM (83). The lower pH samples are dramatically shear thinning, decreasing in viscosity by about three orders of magnitude over four decades increase in shear rate and approaching a constant yield stress at low shear rates. These findings on purified mucin are consistent with bulk and microrheological measurements of mucus rheology (84), and provide a molecular basis for understanding the rheological properties of mucus. The shear thinning and yield stress may be relevant to mucus shedding during peristalsis, and could provide a physical mechanism for washing away bacteria that are not adhered to the epithelial surface. It would be interesting to investigate the rheological properties of the mucin from the two distinct mucus layers discussed earlier in this review (50).
We end this section by a cautionary remark concerning the use of commercial mucin preparations such as those obtained by Sigma Aldrich. These do not form a pH dependent gel, because the mucin has been proteolytically digested during purification (85). Such a reduced mucin does not gel upon lowering pH (54). The rheological behavior also depends on which mucin is the predominant component of the preparation. For example, the Orthana MUC6 mucin investigated by Yakubov et al. (86) and Di Cola et al. (87) has a linear dumbbell structure with a central glycosylated portion flanked by hydrophobic, ng regions, suggesting that it consists of monomeric apoprotein, and not the polymerized, gel forming mucin (c.f. Figure 3).

FIGURE 6 | (Top) the motion of the center of mass (c.m) of a 1 µm latex bead trapped in a pore in the mucin gel at pH 2.
The maximal excursion of the particle's c.m <0.1 µm, indicating that it is essentially immobile in the gel network. (Bottom) occasionally, a particle finds a channel in the gel network indicating that the network structure is heterogeneous. The length unit in both images is micrometer. Reproduced from Celli (80) with author's permission.

MOTILITY OF H. PYLORI
The work described above on particle diffusion in gels shows that the microstructure of mucin and mucus gels restricts the diffusional motion of micron sized particles in such gels. It is not a priori clear how the flagella-driven motion of H. pylori would be impacted by the size restriction due to the movement in a confined geometry. In the next part of this review we address how the helical shape of H. pylori and it's unique biochemical adaptation Frontiers in Immunology | Mucosal Immunity to survive in an acidic environment also enables it to go across the mucus gel.
Like most other bacteria that colonize the GI mucosa, H. pylori, too have evolved to be adapted to their unique niche of the stomach mucosa. These Gram-negative, helical-shaped bacteria, 2.5-5.0 µm in length and 0.5-1.0 µm wide (88) have four to six unipolar-sheathed flagella, which are essential for bacterial motility. Each flagellum, ∼3 µm long and 2.5 nm thick, has a characteristic terminal bulb, which is an extension of the flagellar sheath (88). The motility and the helical shape have both been shown to be of importance to the survival of these organisms. Eaton et al. (89) showed that flagellar mutants of H. pylori were unable to colonize the gastric mucosa of gnotobiotic piglets. More recent work by Ottemann and Lowenthal (90) establishes that mutants with non-motile but otherwise intact flagella also do not colonize.
Helicobacter pylori has also evolved to survive in the acidic environment of the stomach (91). It is well established that the ability of H. pylori to hydrolyze urea and elevate the pH of its surroundings is important in enabling it to escape the acidity of the gastric lumen (26,27,92,93), penetrate the thick mucosal gel and reach the surface epithelium (91). H. pylori, exhibits chemotaxis toward urea present in the epithelial cell surface and a pH tactic response toward elevated pH, both of which may also be crucial for survival in the stomach (94). H. pylori survival in acidic conditions is also stated conversely, namely that acidic conditions are required for H. pylori survival in the presence of urea because the subsequent rise in pH to highly alkaline levels is also toxic to the bacterium. To help avoid overproduction of ammonia, the urea channel is regulated by protons to open at low pH and close at high pH. H. pylori's TlpB receptors enable pH taxis (95,96). It uses the mucosal pH gradient which varies from low on the luminal surface to neutral on the cell surface (28, 97) to move away from the lumen toward the mucosal surface (98) where it attaches itself with adhesins (10, 99). Mutants lacking either the TlpA or TlpB receptors also show altered extent of inflammation (100). The growth of H. pylori in culture is also pH dependent (101).
The motility and chemotaxis of H. pylori and the related Campylobacter has been investigated by using microscopic tracking [for a recent review see Ref. (102)]. By detailed comparison of the motions of straight-rod E. coli and helical bacteria H. pylori and Campylobacter, in liquid cultures Karim et al. (103) showed that the helical bacteria swim faster than E. coli, presumably due to their helical body shape. Their finding is consistent with the idea of Berg and Turner (16) that a helical shape would result in additional screw-like propulsion for bacteria moving in viscous environments such as those faced by H. pylori in its native environment. However, differences between species cannot be ruled out in this comparison. Yoshiyama et al. (30,104) and Worku et al. (105) investigated the motility and chemotactic response of H. pylori in viscous synthetic polymer solutions, and found that swimming speeds decrease with increasing viscosity of the polymer solution, and the bacteria became immobile at very high viscosities.

MOTILITY OF H. PYLORI IN MUCIN SOLUTIONS AND GELS
We have examined the motility of H. pylori in purified gastric mucin at different pH's mucin (31) using phase contrast, digital video microscopy to image live bacteria. Some typical results from analysis of the movies published by Celli et al. (31) are shown and discussed here. We observed that in PGM at neutral pH of 6 or 7 the bacteria swam considerable distances, along almost linear or curved tracks as shown in Figure 7. They exhibit a large spread in their speeds, reflecting both the variation in the size/shape and number of flagella of individual bacteria, as well as the variation in their speed as flagella motors fire asynchronously, and flagella bundle and unbundle. The mean speed averaged over all bacteria is about 16 µm/s which is qualitatively comparable to the speeds of about 20-30 µm/s reported by Worku et al. (105) in methylcellulose.
In contrast to the swimming behavior observed in PGM solution at pH 6-7, we noted that when bacteria were added to PGM gels buffered at low pH of 4 or 2 and deprived of urea, they were immobilized and did not move over any measurable distance. Rotation of the flagella and wiggling of the bacteria in-place could be observed at 60-100× magnification, but this did not displace the c.m of the bacterium. Using theoretical models based on resistive force theory of Magariyama et al. (106)(107)(108) we obtained the motor torque as 3.6 × 10 −18 Nm (31), which is about three times the torque of E. coli swimming in an aqueous environment. The higher torque maybe needed to rotate the flagella in a medium.
We also noticed that, although low pH impairs the flagella motors (109,110), the bacteria were alive at low pH over the duration of the experiment, as confirmed by increasing the pH back to neutral (see below) and observing the renewed motion www.frontiersin.org of bacteria that were stuck in the low pH environment. In our experiments with motility in PGM gels, we observed a dramatic difference depending on the presence of urea. In the absence of urea the bacteria remained immobile in the low pH gel. However, if urea was present and the sample was not buffered, then bacteria that were immobile in a low pH PGM gel, became mobile within a few minutes. Using two-photon fluorescence microscopy to image the bacterial in an initially low pH mucin gel containing urea we showed that the onset of bacterial movement is directly correlated with a rise in pH to near neutral values, as indicated by the pH sensitive fluorescent dye, BCECF (31). These observations in purified PGM gels are consistent with observations in anesthesized gerbils, which showed that H. pylori became immotile in <1 min at lumen pH values of 2 and 3, and in 2 min at pH 4, but remained motile for more than 15 min at pH 6 (111).

INFECTION WITH H. PYLORI AFFECTS THE RHEOLOGICAL PROPERTIES OF PURIFIED PGM
The results above show that the motility of H. pylori depends on the physical state of the mucin gel. To address the converse question, whether the infection by H. pylori impacts the physical properties of mucin or mucus, we performed measurements of the frequency dependent bulk viscoelasticity of purified PGM infected with the bacteria using oscillatory shear methods (31). In earlier studies with purified PGM (shown in Figure 5) we had shown that at pH 4 and 2 purified PGM is a gel with an elastic modulus that dominates over the viscous response, while at pH 6 it is a solution (74). A similar experiment on a 15 mg/ml PGM sample initially at pH 4 incubated with H. pylori for 24 h (Figure 8) clearly shows that the rheological parameters of the infected sample were close to those of pure PGM at pH 6-7 and it remained a solution even at the lowest stress values with a greatly reduced viscosity (31). The concurrent gel to solution transition with the onset of mobility in PGM containing urea suggests that H. pylori gets across the mucus gel using urease secretion to neutralize the acid, raising the pH, and triggering a gel-sol transition. This is illustrated in Figure 9.
Some cautionary remarks about motility and rheology measurements are in order here. Motility measurements in a population of bacteria are inherently polydisperse. Thus, characterizing the behavior by a single average velocity provides limited information and could lead to erroneous predictions; either several different statistical measures such as median, mean, maximal, and minimal speeds should be reported or better yet, the entire distribution. It is also important to analyze hundreds or thousands of tracks, as bacteria move out of the focal plane, and sometimes get stuck in air bubbles and similar defects. By the same token, measurements made very close to the surface of the slide are likely to be influenced by substrate interactions. We also note that bulk rheology provides the average dynamical response, which may not be the same as observed in microrheology, particularly in microstructured environments. The bacteria can also find channels and swim through other heterogeneities in the medium. Bulk rheology experiments require a large sample volume (1-5 ml), while microrheology done on a microscope slide requires only a few microliters. This is a great advantage when analyzing purified samples, mutants, or expressed proteins that are available only in small quantities.
The results discussed above suggest that other factors that can de-gel the mucin would also enable H. pylori to get across the mucus barrier. Worku et al. (105), had observed that H. pylori which were immobile in biopsy samples of mucus, became motile when saline was added to the mucus gel. On the basis of our experiments showing that mucin does not gel at low pH in high salt concentrations (54,70,74) we suggest that saline restores motility because it too triggers a gel to solution transition. Whether these observations have implications for identifying factors such as high salt diet, that may promote H. pylori infection in certain populations, remains to be seen.

SUMMARY AND FUTURE OUTLOOK
The review presented here focuses narrowly on the interplay between mucus structure and rheology and the motility of H. pylori. Among the many topics that we have not discussed are motility in a chemotactic environment, possible chemical interactions of the bacterium with mucin or other factors present in mucus, the binding of mucin to H. pylori, the role of H. pylori in altering mucin production or proteolytic digestion of mucin, the effect of the bacterium on mucous producing cells, the mucosal factors that are involved in the adhesion of the bacterium to the epithelial surface, and cell signaling in the mucus environment to form a colony. Some references on these topics have been provided.
The work discussed here however, does show the importance of physical limitations of the mucus gel microstructure and its pH dependence on the motility of this important pathogen. It also shows the usefulness of microscopic bacterial and particle tracking tools for examining the initial stage of bacterial penetration. We hope that this review encourages further theoretical investigations of the fundamental problem of addressing how a helical-shaped bacterium with helical flagella might propel itself in a viscoelastic medium. Further experimental investigations using microfluidics for investigating stomach mucus barrier (112) and single molecule imaging methods, combined with advances in Frontiers in Immunology | Mucosal Immunity FIGURE 9 | This cartoon illustrates a possible mechanism by which H. pylori gets across the mucus gel. According to Celli et al. (31), gastric mucin forms a gel at low pH < 4. The bacterium cannot move in mucin gels. It secretes urease that metabolizes urea, producing ammonia and elevating the pH. This de-gels the mucin, and enables the bacterium to swim in the resulting polymer solution. The picture, not copyrighted, is reproduced from an NSF press release 09-149. http://www.nsf.gov/news/news_summ.jsp? cntn_id=115409&org=NSF&from=news molecular biology, genetics, immunology, and the availability of mutants with specific molecular and functional alterations, will enable deeper understanding of this fascinating problem.