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Rosiglitazone and a β3-Adrenoceptor Agonist Are Both Required for Functional Browning of White Adipocytes in Culture

The recruitment of brite (or beige) adipocytes has been advocated as a means to combat obesity, due to their ability to phenotypically resemble brown adipocytes (BA). Lineage studies indicate that brite adipocytes are formed by differentiation of precursor cells or by direct conversion of existing white adipocytes, depending on the adipose depot examined. We have systematically compared the gene expression profile and a functional output (oxygen consumption) in mouse adipocytes cultured from two contrasting depots, namely interscapular brown adipose tissue, and inguinal white adipose tissue (iWAT), following treatment with a known browning agent, the peroxisome proliferator-activated receptor (PPARγ) activator rosiglitazone. Prototypical BA readily express uncoupling protein (UCP)1, and upstream regulators including the β3-adrenoceptor and transcription factors involved in energy homeostasis. Adipocytes from inguinal WAT display maximal UCP1 expression and mitochondrial uncoupling only when treated with a combination of the PPARγ activator rosiglitazone and a β3-adrenoceptor agonist. In conclusion, brite adipocytes are fully activated only when a browning agent (rosiglitazone) and a thermogenic agent (β3-adrenoceptor agonist) are added in combination. The presence of rosiglitazone throughout the 7-day culture period partially masks the effects of β3-adrenoceptor signaling in inguinal white adipocyte cultures, whereas including rosiglitazone only for the first 3 days promotes robust β3-adrenoceptor expression and provides an improved window for detection of β3-adrenoceptor responses.

The recruitment of brite (or beige) adipocytes has been advocated as a means to combat obesity, due to their ability to phenotypically resemble brown adipocytes (BA). Lineage studies indicate that brite adipocytes are formed by differentiation of precursor cells or by direct conversion of existing white adipocytes, depending on the adipose depot examined. We have systematically compared the gene expression profile and a functional output (oxygen consumption) in mouse adipocytes cultured from two contrasting depots, namely interscapular brown adipose tissue, and inguinal white adipose tissue (iWAT), following treatment with a known browning agent, the peroxisome proliferator-activated receptor (PPARγ) activator rosiglitazone. Prototypical BA readily express uncoupling protein (UCP)1, and upstream regulators including the β3-adrenoceptor and transcription factors involved in energy homeostasis. Adipocytes from inguinal WAT display maximal UCP1 expression and mitochondrial uncoupling only when treated with a combination of the PPARγ activator rosiglitazone and a β3-adrenoceptor agonist. In conclusion, brite adipocytes are fully activated only when a browning agent (rosiglitazone) and a thermogenic agent (β3-adrenoceptor agonist) are added in combination. The presence of rosiglitazone throughout the 7-day culture period partially masks the effects of β3-adrenoceptor signaling in inguinal white adipocyte cultures, whereas including rosiglitazone only for the first 3 days promotes robust β3-adrenoceptor expression and provides an improved window for detection of β3-adrenoceptor responses.
Keywords: adipocyte, beta adrenergic receptors, uncoupling protein 1, adrenoceptor, seahorse xf96 analysis, rosiglitazone, cl316243 inTrODUcTiOn The remarkable prevalence of obesity worldwide has sparked considerable interest in therapeutic strategies that are effective and safe in promoting weight loss. In conjunction with lifestyle modification, such strategies range from surgical intervention to reduce stomach capacity, through to pharmaceutical interventions aimed primarily at modulating neural pathways that affect food/ caloric intake. There are no pharmacological interventions that primarily act on adipose tissue, despite its obvious role in obesity. There are two types of adipose tissue with distinct functions: white adipose tissue (WAT) that stores chemical energy in the form of triacylglycerol, and brown adipose tissue (BAT) that releases chemical energy in the form of heat (thermogenesis).
Classical BAT depots have been studied extensively in adult rodents. They are highly innervated and are activated by centers in the brain responsive to cold exposure, leading to the release of norepinephrine (NE) from sympathetic nerves. Upon binding of NE to BAT β3-adrenoceptors (β3-ARs), increased levels of intracellular cyclic AMP (cAMP) promote lipolysis, and this breakdown of triglycerides leads to release of free fatty acids that upregulate and activate uncoupling protein 1 (UCP1). Activated UCP1 uncouples mitochondrial respiration leading to heat generation, thus β3-AR signaling increases respiration and nonshivering thermogenesis, with prototypical BAT adipocytes being remarkably rich in mitochondria (1).
β3-AR signaling in response to NE or synthetic receptor agonists occurs almost exclusively in peripheral tissues, including adipose tissue/adipocytes, bladder, and gastrointestinal tract. While there is detectable expression in the rodent brain (2), and central administration of β3-AR agonists directly into hypothalamic/third ventricle regions can reduce food intake and weight (3,4) and increase c-fos immunoreactivity (5), it is unlikely that peripheral administration of β3-AR agonists such as CL316243 and mirabegron would have direct central effects, since they do not readily cross the blood-brain barrier (6,7). Any effects that peripheral administration of β3-AR agonists has centrally are thought to occur indirectly through fatty acids liberated from lipolysis in peripheral adipose tissues.
In addition to prototypical white or brown adipocytes (BA), beige or brite adipocytes have been described (8,9). These cells reside in WAT depots but can be "browned" by various stimuli, most notably cold exposure or activation of β-AR signaling, and by the peroxisome proliferator-activated receptor (PPARγ) agonist rosiglitazone. Activation of brite/beige adipocytes leads to an increase in mitochondrial uncoupling similar to that occurring in BAT. Two studies indicate that brite or beige adipocytes contribute significantly to whole body energy expenditure: mouse models that have increased beige/brite adipocytes in WAT are protected from diet-induced obesity (10), and browning of WAT contributes to non-shivering adaptive thermogenesis in the absence of classical BA (11). Adult humans possess both brown and beige/brite adipose tissue. Humans were thought to lose BAT after infancy, but phenotypically beige/brite adipocytes have been isolated from human supraclavicular fat depots and neck biopsies (9,12,13).
There is considerable interest in identifying additional agents that promote browning of adipose tissue, as increased expenditure of energy as heat would be of therapeutic utility in obesity and type 2 diabetes. To date, agents and processes with browning potential fall into a number of classes, namely (i) cold exposure, activation of the sympathetic nervous system (SNS), and β3-AR agonists; (ii) G protein-coupled receptor (GPCR), ion channel, and signaling pathway modulators; (iii) exercise and associated factors; (iv) growth factors and cytokines; (v) nutritional and dietary factors; and (vi) PPAR agonists. The evidence for these as browning agents has been reviewed elsewhere (14).
Many studies of browning agents have employed a combination of cultured adipocytes and whole animal experiments. Clearly in vivo experiments have the advantage that test compounds are acting on cell populations as they exist in whole animals, thereby providing valid information on predicted clinical efficacy. It is important to understand the mechanism of action, however, particularly in relation to the precise cells targeted by browning agents. In the whole animal, such agents could be acting directly on adipocytes, but it is equally possible that they are targeting the central nervous system (15), or indirectly the sympathetic nervous system (16).
Cultured adipocytes thus offer a system for characterizing the direct effect of browning agents, and also have advantages in facilitating high-throughput screening of compounds. The ideal model system would be cultured human adipocytes with the potential to undergo browning, however, there have been difficulties in using human cultures or cell lines-(i) even in human subjects with highly inducible BAT (17), beige/brite adipocytes are localized to the neck and supraclavicular regions, and presumably arise from specialized cells within these adipose depots, and (ii) human primary cultures or immortalized lines such as SGBS cells require strongly adipogenic media in order to differentiate, including, for example, rosiglitazone, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), cortisol, transferrin, triiodothyronine, and insulin (9,18). In particular, the inclusion of rosiglitazone and IBMX (to increase cAMP), is highly likely to promote browning in conjunction with differentiation.
The primary cultured adipocytes most often utilized to study browning are differentiated from the stromal vascular fraction (SVF) of mouse inguinal WAT (iWAT) depots. Again these cultures generally include rosiglitazone at least for the first 2-4 days of culture (19)(20)(21)(22)(23), thus the mature adipocytes are likely to have undergone browning as well as differentiation. The aim of our study was to systematically clarify the effect of rosiglitazone on cultured adipocytes in the presence or absence of recognized browning agents targeting the β3-AR. We have examined adipocytes isolated from FVB/N mouse interscapular brown and inguinal white depots and cultured in a minimal medium consisting of DMEM supplemented with 10% newborn calf serum, 4.5 g/l glucose, and 2.4 nM insulin (8). We tested the effect of 1 µM rosiglitazone added to the culture medium for the entire 7 days or for the first 3 days only. The adipocyte cultures were treated for a further 24 h with CL316243 (in the absence of rosiglitazone), as a recognized browning agent. We find that BA cultures differentiate well even in the absence of rosiglitazone, whereas inguinal white adipocytes (iWA) require rosiglitazone for at least the first 3 days of culture. Substantial browning occurs only after 7-day rosiglitazone treatment in iWA, though induction of UCP1 and the thermogenic gene Cpt1b can be induced by CL316243 after 3 days of rosiglitazone. The highest levels of UCP1 mRNA occur following 7-day rosiglitazone combined with CL316243 treatment, and the vast majority of BA and iWA cells become positive for UCP1 immunostaining under these conditions. The top layer of the suspension was removed and the remaining cell suspension filtered through a 25-µm nylon mesh filter and centrifuged (700 × g, 10 min). The pellet containing preadipocytes was resuspended in DMEM and centrifuged (700 × g, 10 min). The pellet was suspended in culture medium (6 ml/animal for WAT, 4.8 ml/animal for BAT) and plated in either Seahorse cell culture plates (100 µl/well), 8-well chamber slides (200 µl/well), or 6-well plates (2 ml/well). The culture medium consisted of DMEM containing 4.5 g/l glucose, 10% (vol/vol) newborn calf serum, 2.4 nM insulin, 25 µg/ml sodium ascorbate, 10 mM HEPES, 4 mM l-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin, and supplemented where indicated with 1 µM rosiglitazone. Adipocytes were grown at 37°C in 8% CO2. Cells were washed in pre-warmed DMEM and medium renewed on day 1, then every second day. All experiments were conducted on day 7. For experiments treated for 7 days with 1 µM rosiglitazone (7-day Rosi), rosiglitazone was included in control media from day 1 to 7 (the day of experiment). In some experiments as indicated, 1 µM rosiglitazone was only included in the culture media from day 1 to 3, whereupon the cells were cultured in the absence of rosiglitazone until use on day 7.
reverse Transcription-qPcr Cells were serum starved on day 6 in DMEM/Nutrient Mix F-12 (1:1) with 4 mM l-glutamine, 0.5% BSA, 2.4 nM insulin, 10 mM Hepes, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 50 µg/ml sodium ascorbate, with rosiglitazone (1 µM) as indicated. Media was replaced with DMEM containing 4.5 g/l glucose, 0.5% BSA, 25 µg/ml sodium ascorbate, 10 mM HEPES, 4 mM l-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin, and supplemented with norepinephrine (1 µM), or CL316243 (1 µM) as indicated. Media was aspirated, the cells washed in warmed PBS, and plates rapidly frozen at −80°C until use. Total RNA was extracted using RNeasy Plus Mini Kits (QIAGEN), as per the manufacturer's instructions (samples in Figure 5 were extracted using TriReagent (Sigma-Aldrich), according to the manufacturer's instructions). RNA samples were DNAse treated using DNA-free DNA Removal Kit (Invitrogen), according to manufacturer's instructions. Where tissues were used, interscapular BAT or inguinal WAT were excised from similarly aged (3-to 4-week-old FVB/N mice of either sex) and housed mice (room temperature 22°C), and tissues rapidly frozen (−80°C). RNA from tissues was extracted using TriReagent according to manufacturer's instructions (Sigma-Aldrich). For preparation of cDNA, 0.5 µg of RNA was reversetranscribed using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad), in a total volume of 10 µl. For each independent sample, qPCR was performed in duplicate using TaqMan Gene Expression assays (Life Technologies) for Prdm16, Hoxc9, Ppara, Ppargc1a, Pparg, Ppargc1b, Fabp4, Adipoq, Slc27a1, Fasn, Pck1, Acaca, Fabp3, Acox1, Sirt3, Cpt1b, Cox4i1, Prdx3 Acadl, Vdac1, Pdk4, Slc2a1, Slc2a4, Hk2, Pfkm, Gapdh, Pgk1, Ucp1, Adrb3, and the reference gene Actb. The cDNA was diluted to the equivalent of 2.5 ng/µl of starting RNA and 4 µl added to 6 µl reaction mix comprising 1× TaqMan Gene Expression Assay and 1× TaqMan Fast Advanced Master Mix dispensed in 96-well plate, as per manufacturer's instructions. After initial denaturation at 95°C for 30 s, fluorescence was detected over 40 cycles (95°C for 5 s, 60°C for 30 s). Cq values were automatically calculated by the Realplex analysis module. qPCR presented in Figure 5 was performed on a CFX Connect™ real-time PCR detection system (BioRad) and samples were initially denatured at 50°C for 2 min, 95°C for 10 min, and fluorescence detected over 40 cycles (95°C for 15 s, 60°C for 1 min), and Cq values automatically calculated by the BioRad analysis module. All data are expressed as expression of the gene of interest relative to Actb, calculated as ( )* , 2 1000 −∆C q . Multiplication of all values by 1,000 does not change the relative expression levels and was done for two reasons; (i) it facilitates viewing of the data because even values for poorly expressed genes are greater than 1.0, and (ii) it positions expression values in the same range as RPKM or FPKM values obtained in RNA-Seq studies, as the average value for Actb in numerous cell and tissue types is 1,000. All statistics for gene expression were performed on ΔCq values, as these data are normally distributed. For the purpose of statistics, where genes were not detected within 40 cycles (and were, therefore, not detectably expressed), an over-assumption Cq value of 40 was used.

custom Pcr array
We designed a custom mouse qPCR array in 384-well plates that comprised 2 sets of 192 genes, including Actb as a reference gene, a genomic DNA control (Lonza) and 190 genes representing a broad cross-section of targets downstream of 25 different transcription factors ( Table 1). Each array plate was used to analyze expression in a control and rosiglitazone-treated adipocyte culture from BA or iWA. cDNA was prepared as described above, then 4 µl equivalent to 10 ng of starting RNA was added to each well containing 1× SYBR Green PCR Master Mix (Applied Biosystems) and Lonza-dispensed primer sets in a total volume of 10 µl. qPCR reactions were carried out at the Australian Genome Research Facility (Parkville, VIC, USA) on a 7900HT Real-Time PCR System (Applied Biosystems). ingenuity Pathway analysis (iPa) Patterns of gene expression were examined using IPA software. For each of the adipocyte datasets, we uploaded fold-change between rosiglitazone treated and control cultures, along with the final expression value in rosiglitazone-treated cultures and P values for significant differential expression. The datasets were filtered for fold-changes of at least 2 in either direction, expression values normalized to Actb (as described above) of at least 1.0 in at least one culture condition, and P-values <0.05 (Student's t-test), leaving 171 genes. IPA core analysis was used to determine enrichment of molecular and cellular functions or upstream regulators predicted from observed patterns of up or down-regulation among the expressed genes. IPA uses Fisher's Exact Test to test the hypothesis that patterns of gene expression related to functions or to upstream regulators are not due to chance. This analysis provides P values that signify the degree of enrichment of differentially expressed genes, and activation z-scores that take into account the direction of change compared to effects predicted from the IPA knowledge base (25). A z-score of at least 2.0 is considered significant.

external Data
We reanalyzed publically available RNA sequencing data obtained from Hao et al. (26) (upstream analysis shown in Figure 6). In this published study 8-week-old male C57BL/6J mice were housed at thermoneutrality (28-30°C) on a 12:12 h light-dark cycle and fed standard chow diet ad libitum. After 8 days, mice were either retained at thermoneutrality or transferred to 4°C for 2 or 4 days. Mice were killed by cervical dislocation for isolation of total RNA from interscapular BAT and inguinal WAT. Data consisting of tag counts per million were generated by digital gene expression profiling, mapped to NCBI RefSeq mRNAs, and deposited in Gene Expression Omnibus (accession number GSE63031). We uploaded the 0-and 2-day cold exposure datasets to IPA to compare upstream regulators between BAT and iWAT, as described in the legend to immunocytochemistry for Detection of UcP-1 Cells were seeded in 8-well culture chamber slides (BD Biosciences, Franklin Lakes, BJ). On day 7, cells were fixed with 4% formaldehyde in PBS for 15 min, and quenched with 150 mM Tris pH 8.0 for 10 min. Cells were permeablized with 0.1% Triton X-100 for 10 min, blocked for 1 h at room temperature (5% BSA in PBS), and incubated with UCP1 (Abcam) primary antibody solution (diluted 1:1,000 in 1% BSA in PBS) overnight at 4°C. Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000 dilution in 1% BSA in PBS) was added and incubated at room temperature for 2 h. Images were observed on a Leica DMLB epifluorescence microscope. Images were acquired using a DC350F camera with IM500 software (Leica Microsystems AB; Kista, Sweden). Quantification was performed using Fiji (ImageJ version 1.50 g) (27), using a script developed by C.J. Nowell. Cells identified by DAPI staining in images were judged to be negative/positive for the protein of interest (UCP-1) in a blinded manner and counted using ImageJ software, performed by J. Merlin. No distinction was made in the relative intensities of staining within images.

Measurement of Oxygen consumption rates
Oxygen consumption rates (OCR) were measured using the Seahorse xF96 (Seahorse Bioscience). On day 7, adipocytes were washed twice in XF assay media (Seahorse Bioscience) supplemented with 25 mM glucose, 0.5 mM sodium pyruvate, 2 mM l-glutamine and 1% fatty free BSA, and 160 µl added/ well. OCR was measured as described in detail (28) with some modifications. 6 baseline rate measurements were made using a 2 min mix, 5 min measure cycle. After 6 basal measurements, oligomycin A (5 µM) was added for 6 measurements, followed by a combination of antimycin A (1 µM) and rotenone (0.1 µM) for 6 measurements. OCR rates immediately prior to oligomycin A injection at measurement number 6 were used as the basal rates and defined as 100%.

statistical analysis
All data are expressed as mean ± SEM of n. Statistical significance was determined by Student's t-test, multiple comparisons one-way ANOVA with Tukey's test, or multiple comparisons Kruskal-Wallis test (for non-parametric analyses), as indicated in Figure legends. P < 0.05 was considered significant. resUlTs rosiglitazone Treatment of cultures From inguinal WaT increases Differentiation and the expression of genes related to Thermogenesis 7-day rosiglitazone treatment (1 µM) promoted expression of genes associated with both maturation of white adipocytes, and thermogenic re-programming (Figure 1; Table 2). While BA cultures differentiate in the absence of rosiglitazone, displaying robust expression of the mature adipocyte marker Fabp4 (Figure 1B), iWA cultures express lower levels of Fabp4 (36% relative to BA), indicating that a much lower proportion of cells undergo differentiation under control conditions (in DMEM containing 4.5 g/l glucose, 10% (vol/vol) newborn calf serum, and 2.4 nM insulin). However, following 7d of rosiglitazone treatment, Fabp4 mRNA levels are similar to that in iWAT tissues, and also to BA tissue and cultures (Figure 1; Table 2). Likewise, the genes Acaca (acetyl CoA carboxylase), Fasn (fatty acid synthase), Slc27a1 (FATP1), Pck1 (phosphoenolpyruvate carboxykinase), and Adipoq (adiponectin), all characteristic of mature adipocytes, are expressed in control and treated BAs, whereas all of these genes are low in control iWA, and significantly increased by rosiglitazone (Figure 1B). Conversely, the white adipocyte marker Hoxc9 is expressed to some extent in both control and rosiglitazone-treated iWA cultures but is negligible in BA under any conditions (Figure 1).
The BA transcriptional regulator Prdm16 is expressed in both control and rosiglitazone-treated BA cultures, and is substantially increased in iWA cultures treated with rosiglitazone ( Figure 1A), suggesting browning of iWA under 7-day rosiglitazone treatment as we have shown previously (29). Pparg, encoding the rosiglitazone-targeted nuclear receptor PPARγ, a major regulator of adipocyte differentiation, is expressed in control white adipocyte cultures but also significantly increased by rosiglitazone in treated iWA. These findings indicate that control iWA cultures contain few fully differentiated adipocytes, but a significant population of Hoxc9-positive preadipocytes ( Figure 1A). Upon rosiglitazone treatment, levels of Fabp4, Adipoq, Fasn, Slc27a1, and Acaca expression in iWA equal or exceed those found in treated BA cultures, indicating a high degree of differentiation. Figure 1D shows genes involved in fatty acid metabolism and mitochondrial function. In particular, Cpt1b (carnitine palmitoyltransferase 1B) is the rate-limiting step for fatty acid oxidation and consequent stimulation of mitochondrial respiration, while Fabp3 is also required for efficient fatty acid oxidation (30). Cpt1b mRNA is 65 times higher in control BA than in iWA cultures, and in BA, undergoes 6.4-fold induction in the presence of rosiglitazone. iWA display a striking 455-fold induction, reaching levels similar to those in BA. Similarly, Fabp3 is induced by 630-fold in iWA cultures following rosiglitazone treatment. Additional genes related to thermogenesis (Acadl, Vdac1, Acox1, Sirt3, Pdk4, Prdx3, Cox4i1) display significantly higher expression in rosiglitazone-treated iWA than in control cultures. Ppara (PPARα), which promotes expression of genes essential for many aspects of fatty acid metabolism, and Ppargc1a (PGC-1α), a master regulator of adipocyte browning and mitochondrial biogenesis, are induced by 77-and 83-fold, respectively, in adipocytes from iWAT following rosiglitazone treatment ( Figure 1A).
Analysis of genes related to glucose handling ( Figure 1C) showed significant increases in the insulin responsive GLUT transporter Slc2a4 (GLUT4), and glucose metabolism enzymes Hk2, Pfkm, and Gapdh, in iWA cultures treated with rosiglitazone, further indicating the differentiation of iWA cultures toward an insulin-sensitive mature adipocyte population. Rosiglitazone treatment also increased the expression of Slc2a4, Gapdh, and Pgk1 in BAs, likewise suggesting changes in insulinmediated glucose handling, but also a significant increase in Slc2a1 (GLUT1), which has been shown to be the major GLUT involved in β3-AR-mediated glucose uptake in BA (31).
In parallel with individual qPCR assays, we obtained a comprehensive view of rosiglitazone-induced changes in cultured mouse adipocytes by analyzing mRNA levels of 160 expressed genes quantified using a custom qPCR array (categorized in Table 1). Products of these genes participate in a broad cross-section of molecular and cellular functions, and represent targets of diverse upstream transcriptional regulators. We assessed the expression of 4 genes from both the qPCR array and TaqMan assays (Cpt1b, Fabp3, Ppargc1a, and Sirt3). Because expression is normalized to the reference gene Actb, the two methods give closely corresponding values for a given sample, showing less inter-assay variation than the inter-sample differences seen using either method. Genes displaying greater than twofold change of expression in either direction (P < 0.05) were subject to enrichment analysis using IPA software (Qiagen). The significantly enriched molecular and cellular functions are similar between BA and iWA (Figure 2A). The top score is for molecular transport (which encompasses a wide array of cellular processes), but the remaining significant functions are related largely to energy homeostasis, fatty acid metabolism, and ATP generation. Figure 2B shows the upregulated genes expected to contribute to these functions in rosiglitazone-treated iWAs.
As shown in Figure 2A, there are four functions that show an apparent difference between BA and iWA. Increases in the "transport of lipid, " "metabolism of nucleic acid component, " and "transport of carbohydrate" functions are significant in only one of iWA or BA, but in each of these three cases the number of genes showing a direction of expression change consistent with increased function is small. For example, significantly increased transport of carbohydrate is predicted in BA, based on enhanced expression of 10 genes positively linked to this function (Hk2, Ppargc1a, Ppargc1b, Ptgs2, Slc2a1, Slc2a4, Sod1, Tnf, Trib3, and UCP1). In iWA, only seven of these genes display increased expression, so in these cells transport of carbohydrate is no longer predicted to be significantly affected. This finding is not borne out by the functional phenotype of iWA, as rosiglitazone treatment  (29). All data are expressed relative to Actb expression*1,000 ± SEM of three independent experiments (as per Figure 1).
significantly increases glucose uptake in response to norepinephrine and CL316243 (29). This reflects our observation that the limiting step in agonist-stimulated glucose uptake is the abundance of the β3-AR, which is markedly enhanced when iWAs are cultured in the presence of rosiglitazone [ Table 2; (29)]. Interestingly, the function "synthesis of reactive oxygen species" is predicted to be decreased in iWA (z-score of −2.2, based on 23 differentially expressed genes), while this function is not significantly affected by rosiglitazone treatment in BA (z-score of +0.27, 16 genes). Genes displaying changes in expression expected to enhance or inhibit synthesis of reactive oxygen species (ROS) are shown in Figure 2C (iWA) and Figure 2D (BA). The overlapping genes all display the same direction of change in expression in iWA and BA, but in iWA cultures there are 15 genes for which up-or down-regulation is expected to reduce ROS. The induction of genes encoding anti-oxidant enzymes, including superoxide dismutase SOD1 and SOD2 in iWAT also occurs in vivo following rosiglitazone treatment (32).

Brite adipocytes Display increased UcP1
Protein and Uncoupling capacity in response to 24 h Treatment With the β 3 -ar agonist cl316243 As chronic β-AR treatment of BA upregulates UCP-1 mRNA and protein levels (1), we treated BA and iWA cultures in the presence/ absence of 7-d rosiglitazone (1 µM) with norepinephrine (1 µM, 24 h) or CL316243 (1 µM, 24 h). Norepinephrine and CL316243 caused a small increase in UCP1 mRNA expression ( Figure 3B) and immunoreactivity (Figures 3A,C) in control BA, but this response was significantly amplified in rosiglitazone-treated BA cultures. The effect of combined rosiglitazone and β-AR agonist treatment was even more evident in iWA cultures. These data are consistent with the presence or absence of β3-ARs in these cultures (29). There were significant changes in the proportion of UCP1-positive cells between all control cultures and those treated with rosiglitazone alone (Figures 3A,C). Following an additional 24 h in the presence of CL316243, there were also significant differences in UCP1-positive cells between control and rosiglitazone-treated iWA cultures ( Figure 3C).
In order to investigate whether the increase in UCP1 mRNA/ protein levels following rosiglitazone, norepinephrine, or CL316243 treatment affected mitochondrial uncoupling in white adipocytes, we measured OCR in the presence of the ATP synthase inhibitor oligomycin (5 µM), and in the presence of rotenone (0.1 µM) and antimycin A (1 µM), that define non-mitochondrial sources of OCR. In control iWA cells, oligomycin inhibited OCR by ~50% (Figure 4). In iWA cells treated 7-day with rosiglitazone (1 µM), oligomycin inhibited OCR by only 34%. The insensitivity to oligomycin was enhanced when rosiglitazone treated cells were also treated with either NE (1 µM, 2 and 24 h) or CL316243 (1 µM, 2 and 24 h). This suggests an increase in uncoupled respiration. This was further assessed by treating the cells with a combination of rotenone (0.1 µM) and antimycin A (1 µM), that define non-mitochondrial sources of OCR (~36% of OCR was due to non-mitochondrial sources; Figure 4). After correction for non-mitochondrial OCR (indicated in dotted arrows in Figures 4C,D, and illustrated as % OCR due to proton leak in Figures 4E,F), we can see that mitochondrial uncoupling (proton leak) accounts for 14% of OCR in control iWA cells, 27% in rosiglitazone-treated cells, and this level rises to 40-50% in rosiglitazone-treated iWA cells further treated with NE or CL316243. These results suggest that rosiglitazone-induced brite adipocytes possess a greater mitochondrial uncoupling capacity, and that β3-AR treatment of brite adipocytes significantly increases this capacity.
The effects of rosiglitazone on adipocyte Differentiation, and the Thermogenic Potential of iWa, are separable Following

3-Day Treatment
Our studies indicate that 7-d rosiglitazone treatment promotes both differentiation and browning of iWA cultures. It is common practice to induce adipocyte differentiation using an adipogenic cocktail, generally containing IBMX, dexamethasone, insulin, high glucose, and often 1 µM rosiglitazone, particularly in studies involving human adipocytes. However, the presence of rosiglitazone, and perhaps IBMX that increases cellular cAMP and thus mimics the effect of β-AR agonists, are confounding factors in any attempt to attribute browning capacity to agents being tested using cultures derived from the stromal vascular fraction (14). Several published studies have included rosiglitazone in mouse adipocyte cultures only for the first 2-4 days before removing rosiglitazone  (25). Network of genes upregulated in iWAT that contribute to key non-redundant cellular functions (oxidation of fatty acid, concentration of ATP and energy homeostasis) (B). Differentially expressed genes identified by IPA that contribute to synthesis of reactive oxygen species (ROS) in iWA (c) and BA (D). In both panels, coloring of genes red or pink denotes upregulation and green denotes downregulation. In panel (c), blue lines signify genes showing a change in expression consistent with reduced ROS synthesis, while yellow lines signify genes that are not consistent with this function. The z-score for iWA is −2.2, below the significance threshold of |2|, whereas that for BA is +0.27 and not significant. iWA cultures treated with rosiglitazone display fourfold increases in expression of the key anti-oxidant genes Sod1 and Sod2 relative to control cultures. from the media for the remainder of the differentiation protocol, instead of including rosiglitazone for the entire differentiation period [e.g., Ref. (19,(21)(22)(23)33)]. We therefore examined changes in gene expression in both BA and iWA cultures treated with rosiglitazone for the entire 7 days compared to the first 3 days only (Figure 5). In iWA cultures, 3-d rosiglitazone promoted increases in Adrb3 mRNA that were 50% of those seen after the full 7-d treatment. 1-3 day rosiglitazone did not significantly alter expression of UCP1, Fabp3, Fabp4, or Cpt1b, in contrast to the substantial increases seen in 7d-treated cultures. We also tested the effect of CL316243 (1 µM, 24 h) on cultures differentiated with rosiglitazone for 3 or 7 days. CL316243 increased the expression of Ucp1 in 1-3 day cultures, whereas it had no effect on control cultures, indicating that 3 days does provide a suitable window to test for browning capacity. After 7 days in the presence of rosiglitazone, CL316243 treatment for 24 h did increase UCP1 mRNA further (although not significant in this cohort of adipocytes due to high variation observed), and also seen in Figure 3, however, there was no increase in Fabp3 or Cpt1b expression associated with 24 h CL316243. Thus longer exposure to rosiglitazone essentially masks some of the effects of CL316243.

Modulation of gene expression in Ba and iWa cultured From the sVF compared to In Vivo adipose Depots
Our gene expression data and previous functional studies (29) indicate that although BA and iWA cultures are derived from adipocyte precursors in the SVF, the mature adipocytes retain distinct properties according to their site of origin. It has been shown recently that in vivo treatment of mice with rosiglitazone induces a population of mature UCP1-positive inguinal adipocytes that have a distinct profile compared to populations induced by in vivo treatment with CL316243 (34). This differs from our findings based on adipocytes derived from the SVF, as in our cultures prior differentiation with rosiglitazone was required in order for cells to induce expression of the β3-AR and thereby become responsive to CL316243. This suggests that the mature adipocytes residing in iWAT depots differ from those derived from differentiation of the SVF. We compared the regulation of gene expression by rosiglitazone in our cultures with that demonstrated by transcriptome sequencing of RNA from BAT and iWAT subjected to sympathetic stimulation due to cold exposure Arrows indicate the addition of oligomycin (5 µM) or the combination of rotenone (0.1 µM) and antimycin A (1 µM), with basal OCR set to 100% before the addition of oligomycin at rate 6 to account for variations in the raw data between adipocyte cultures made on different days. (e,F) The relative changes in OCR between oligomycin (defining OCR due to ATP synthase) and rotenone/antimycin A (defining OCR due to non-mitochondrial sources) are expressed as % OCR due to proton leak [calculated from the results presented in (c,D) indicated with the dotted arrows]. Data are non-parametrically distributed and, therefore, statistically analyzed by non-parametrically analysis. *P < 0.05, **P < 0.01, ***P < 0.001 indicate statistical significance from control cells, # P < 0.05 indicates statistical significance from rosiglitazone-treated cells, α P < 0.05 indicates statistical significance from adrenergic treatment alone (multiple comparisons Kruskal-Wallis test, one-way ANOVA). of mice for 2 days (26). We also compared the expression of key adipocyte and thermogenic genes in our cultures with that in freshly isolated BAT and iWAT ( Table 2). These tissues were isolated from mice subject to mild thermal stress by housing at 22°C, rather than at thermoneutrality (35,36).
We used IPA to compare upstream regulators associated with all BA and iWA genes differentially expressed in the presence of rosiglitazone (twofold change in either direction, P < 0.05). As would be expected for cells treated with rosiglitazone, high-scoring upstream regulators across all cultures were closely associated with the PPARγ/PGC-1α network previously implicated in activating thermogenesis (Figure 6A). We also performed IPA upstream regulator analysis on the data from RNA sequencing of adipose depots from cold-exposed mice  Figure 6B]. In BAT and iWAT, seven of the highest scoring upstream regulators from the in vivo study corresponded to those seen in our rosiglitazone-treated cultures. This highlights the previously characterized thermogenic pathway involving activation of PPARγ by endogenous fatty acids released due to sympathetic stimulation of lipolysis (37). When we compared expression of key adipocyte and thermogenic genes in control and rosiglitazone-treated cultures with that in native AT depots, many showed similar levels in rosiglitazone-treated cultures and in BAT or iWAT. There are, however, striking differences in levels of UCP1, Acot11, and Pck1 mRNA, which each display over 10-fold higher expression in native BAT and iWAT than in the corresponding rosiglitazone-treated cultures (Table 2; Figure 6).

DiscUssiOn
The presence of active BAT in humans (38)(39)(40)(41) has reignited research into ways to promote increased thermogenesis in humans as a strategy to combat obesity and its related disorders such as type 2 diabetes. This is in part due to the overwhelming  Fabp3 (g,h), or Cpt1b (i,J). Data represent mean ± SEM of 6 (BA) or 7-8 (iWA) independent experiments, performed in duplicate, relative to β-actin (Actb) expression. *P < 0.05, **P < 0.01, ***P < 0.001 indicate statistical significance between all treatments and the control cultures. # P < 0.05, ## P < 0.01, ### P < 0.001 indicate statistical significance between all treatments and the Rosi (3d) treated cultures. ∞ P < 0.05, ∞∞ P < 0.01, ∞∞∞ P < 0.001 between all treatments and the Rosi (7 days) treated cultures. Data analyzed by Tukey's multiple comparisons of one-way ANOVA performed on the ΔCt values, which are normally distributed. One sample (iWA treated with rosiglitazone for 3 days) was excluded from all analysis due to poor integrity of its RNA. A single measurement of Adrb3 for iWA treated with CL316243, and of Fabp4 for BA treated with Rosi 7 days, was excluded from the analysis due to failure of the qPCR reaction.
evidence that activation of β3-ARs leading to increased UCP1 expression and function in BAT can reverse obesity, and prevent the development of insulin resistance and diabetes in rodents (42)(43)(44)(45). However, translation of these responses observed in rodents has not led to effective treatments in humans (46,47). This may in part be due to human tissue comprising adipocytes that are more akin to mouse beige or brite adipocytes rather than classical BAs, based on gene expression profiling (9,48). Thus research into beige/brite adipocytes, and how they can be induced by different stimuli [reviewed in Ref. (14)] may offer greater therapeutic potential. It is critical to this research that model systems comprising cultured adipocytes respond in the same way to browning agents as endogenous cells in vivo.
We have characterized FVB/N mouse adipocytes induced by rosiglitazone in culture, utilizing a method for in vitro differentiation (8) that in control cultures lacks potential brite-inducing agents commonly used in adipocyte differentiation cocktails (such as triiodothyronine, IBMX, and rosiglitazone itself). We show that BA undergo differentiation in control cultures, but still display increased expression of UCP1 mRNA and protein in the presence of rosiglitazone. iWA cultures undergo both differentiation and browning due to inclusion of rosiglitazone. In BA cultures treated with rosiglitazone, 80% of cells become UCP1-positive, while this figure is 45% in iWA cultures ( Figure 3B). These findings align with our observation that the key transcriptional regulators Prdm16 and PPARα are expressed at somewhat lower levels in iWA. Importantly, PPARγ and PGC-1α show comparable robust expression in rosiglitazone-treated BA and iWA (Figure 1). cultured adipocytes From iWaT Depots require Priming by rosiglitazone for induction of UcP1 and Thermogenic genes by the β 3

-ar agonist cl316243
We showed previously that the majority of BA cells express β3-AR protein in control and rosiglitazone-treated cultures, whereas in iWA cultures the proportion of β3-AR-positive cells increase markedly in the presence of rosiglitazone (29), concomitant with cold-exposed mice. IPA software was used to determine upstream regulators consistent with observed patterns of target gene up-or down-regulation in adipocyte cultures treated with rosiglitazone relative to vehicle-treated control cultures (a). Transcriptional regulators are ranked according to the sum of activation z-scores across brown adipocyte and inguinal white adipocytes (iWA) cultures, irrespective of positive or negative direction. Z-scores ≥|2| describe statistically significant matches between known regulators and observed patterns of up-and down-regulated genes, and also predict the activation state of each putative regulator (activated shown in orange, inhibited shown in blue) (25). As an example, the top transcriptional regulators in rosiglitazone-treated iWAs are PGC-1α (z = + 4.88, P value = 1.83E-46), PPARγ (z = + 4.35, P value = 5.89E-44), and PPARα (z = + 3.38, P value = 1.19E-32). These are predicted to be activated based on z-score, and are themselves upregulated in the treated cultures (83-, 3-, and 77-fold, respectively). P values (Fisher's Exact Test) describe the significance of overlap between the observed differentially expressed genes and all genes associated with a particular upstream regulator in the Ingenuity database, curated from published literature. As an additional comparison, we re-analyzed literature-derived RNA sequencing data for adipose depots from cold-exposed mice (26) (B). 8-week-old mice were housed at 28-30°C for 8 days, then one group maintained at thermoneutrality and a second group housed at 4°C for 2 days. Fold-change values were calculated for all genes expressed in iWAT and brown adipose tissue (BAT) of mice at 4°C compared to control mice at 28-30°C. These fold changes were analyzed using IPA and upstream regulators compared by z-score between BAT and iWAT. (c) Shows the predicted upstream regulators across the entire set of 171 genes found to be expressed in adipocyte cultures, ranked by log(P-value of overlap), demonstrating that the dataset is not biased toward the regulators predicted to govern differential expression in adipocyte cultures.
increased levels of β3-AR mRNA (Figure 5). This in turn facilitates the response to β3-AR agonists (29). This may represent a special case, however, it is equally likely that receptors targeted by other agents are similarly induced during adipocyte differentiation and/ or browning. In mice, the β3-AR is expressed in native BAT and iWAT at levels similar to those in rosiglitazone-treated cultures ( Table 2), consistent with widespread findings that in vivo treatment with CL316243 can induce browning in iWAT depots (9,35,49). The utility of cultured adipocytes as a screening platform is clearly dependent on the presence of the target receptors.
Wu et al. (9) suggested that the inherent capacity for brite adipogenesis in vivo is independent of external factors, such as innervation, blood flow, oxygen supply, and nutrients. While differentiation of cultured adipocytes from the SVF does occur in a cell-autonomous manner, properties of the precursor cells appear to be specified by the environment from which they were derived, with a significant contribution from SNS innervation. In vivo, BAs possess a well-established ability to adapt to chronic β-AR activation; prolonged exposure to cold increases UCP1 expression in BAT (50,51), which is inhibited by sympathetic denervation of BAT (37,52). This upregulation in UCP1 occurs via β3-ARs and can be inhibited by β3-AR selective antagonists (53,54). The requirement for intact sympathetic innervation has also been demonstrated in iWAT. For example, 3-week-old mice have high levels of UCP1 expression in iWAT, but this is markedly reduced by 8 weeks of age (35). At 8 weeks, mature adipocytes within iWAT re-express UCP1 in response to CL316243 stimulation, but this effect is abolished in mice undergoing prior surgical denervation of iWAT depots at 3 weeks. Thus sympathetic tone due to the mild cold-stress associated with housing mice at room temperature (55) maintains browning capacity in iWAT. We observed high UCP1 expression in native iWAT from animals housed at 22°C, reflecting this thermal stress ( Table 2). Conversely, UCP1 mRNA is undetectable in the iWAT of mice housed at thermoneutrality [28-30°C (56)].
Importantly, reports of CL316243-induced browning have used mice housed at room temperature rather than at thermoneutrality (9,35,49). Previous studies have demonstrated an in vitro effect of CL316243 on UCP1 expression and function only in white adipocytes cultured in the presence of rosiglitazone or adipogenic cocktails (9,49). Likewise, we found that while 24 h β3-AR stimulation (NE or CL316243) increased the proportion of UCP1-expressing cells in both control and treated BA cultures, neither agent alone induced significant browning of iWA cultures (Figure 3). These findings may recapitulate the requirement for intact SNS innervation of WAT seen in vivo, as sympathetic tone and consequent β-AR activation would be expected to drive lipolysis and subsequent activation of PPARγ by endogenous fatty acids. From our overall profiling of gene expression using the custom qPCR array, we examined predicted upstream regulators (IPA) based on genes differentially expressed following 7d rosiglitazone in cultured adipocytes, and these regulators were compared to those predicted in BAT and iWAT of mice subject to cold exposure for 2 days (26). In both cases, the top upstream regulators included PGC-1α, PPARγ, PPARα, PPARδ, CEBPα, ERRα (estrogen-related receptor α), and KLF15 (Figure 6), consistent with a common mechanism of induction that is dependent on PPARγ.

Functional Demonstration of iWa Browning
The therapeutic potential of adipocyte browning is contingent upon the dissipation of energy through mitochondrial uncoupling becoming dominant over energy storage as triglycerides. We previously have shown that increased UCP1 function in brite adipocytes (defined as the percentage of OCR due to proton leak) is increased in brite adipocytes as compared to white adipocytes (29), which correlates with an increase in the expression of several genes involved in energy production (Figure 1). To assess whether combined treatment of iWA cells with rosiglitazone and a β-AR agonist (2 or 24 h treatment with either NE or CL316243) could further increase UCP1 function, we analyzed our OCR data, as described by Collins and colleagues (57), by defining the mitochondrial capacity that is not coupled to ATP synthesis. Rosiglitazone treatment of iWA induces a significant increase in this non-coupled spare capacity at the level of mitochondria, which is further increased in iWA also treated with either NE or CL316243 (Figure 4). These results indicate that the combination of both a browning agent (rosiglitazone) and a β3-AR agonist are required for maximal effects on mitochondrial function in brite adipocytes. This demonstrates that inguinal-derived brite adipocytes are geared toward mitochondrial uncoupling (and energy expenditure), consistent with a previous report that brite adipocytes are thermogenically competent, in that their mitochondria are functional and able to uncouple via UCP1 (58).
UCP1 is activated upon release of fatty acids generated following adrenergic stimulation of lipolysis, however, a recent study has also demonstrated that UCP1 activity is sensitized in the presence of ROS due to sulfenylation at Cys253 (59). In vivo, increased ROS due to deletion of Sod2 (encoding Mn-superoxide dismutase) leads to enhanced expression of key fatty acid oxidation genes in iWAT, and elevated mitochondrial oxygen consumption (60). Furthermore, UCP1 plays a central role in protecting adipocytes from mitochondrial dysfunction in the presence of high ROS and calcium overload (61). Thus, UCP1 is both activated by and protects against the effects of increased mitochondrial ROS. Our gene expression profiling and IPA indicates that rosiglitazone treatment may have dual protective actions in iWA, by increasing expression of UCP1 and at the same time inducing Sod1, Sod2, and other genes implicated in reduced ROS synthesis ( Figure 2C).

culture conditions That Promote adipogenesis but not Browning
Another issue raised by our study is whether it is possible to separate the effects of rosiglitazone on adipocyte differentiation from those on thermogenic programming in iWA. Certainly 7d rosiglitazone treatment promotes induction of UCP1, with further substantial increases following an additional 24 h in the presence of CL316243 (Figures 3 and 5). In contrast, Fabp3 and Cpt1b are increased by 7d rosiglitazone but there is no potentiation of expression by CL316243, indicating that responses to the β3-AR agonist are in part masked by prolonged rosiglitazone treatment. The magnitude of responses was smaller when rosiglitazone was included in the cultures only for the first 3 days of the 7-day culture period, however, this protocol did provide an improved window for observing the browning effect of 24 h CL316243 over and above rosiglitazone alone, with significant increases in expression of UCP1 (96-fold). The capacity to respond to CL316243 in these 3d cultures may be dictated solely by rosiglitazone-induced expression of the β3-AR, or it may reflect other aspects of the differentiation process. We cannot say at this time whether the 3d iWA most closely resemble the brite adipocytes seen in vivo (34), though profiling both 3d and 7d cultures would be worthwhile as it would facilitate a better understanding of the relationship between these adipocytes and those induced by PPARγ activation in mice.
cOnclUsiOn While there is a wealth of data on the gene expression of brite adipocytes (8,9,56,(62)(63)(64)(65)(66), these studies have generally aimed to identify differential genetic markers between brown, white, and brite adipocytes. We have instead sought to determine the similarities of brite adipocytes to conventional BAs in metabolic responses and profiles of thermogenic gene expression. We have demonstrated that the induction of brite adipocytes involves the upregulation of metabolic genes associated with thermogenesis, along with expression of β3-ARs, and this allows for adrenergically mediated activation of UCP1. Furthermore, brite adipocytes display the adaptive capacity observed in BAs, capable of responding to sustained adrenergic stimuli. Our findings indicate that cultured white adipocytes derived from regions such as iWAT with adequate sympathetic innervation respond efficiently to agonists, but only in combination with an additional priming stimulus such as rosiglitazone. This contrasts with in vivo iWAT depots in which chronic sympathetic tone is sufficient to maintain the capacity for brite activation. Our results emphasize the importance of activating both browning and thermogenic programs in cultured white adipocytes in order to reach maximum browning capacity. This understanding will translate into better model systems for the screening of beige or brite capacity to combat obesity and type 2 diabetes.


Comparative phase I randomized open-label pilot clinical trial of Gynophilus® (Lcr regenerans®) immediate release capsules versus slow release muco-adhesive tablets

Gynophilus® (Lcr regenerans®) is a live biotherapeutic product (LBP) that contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35®, which is indicated to restore vaginal health. The aim of the study was to compare the safety, ease of use, and compliance of two formulations (immediate release: IR capsule and slow release: SR muco-adhesive tablets) as well as the colonization of Lcr35® in healthy women. This phase I study (Comprigel) is a parallel, randomized, 4-arm, and open-label clinical trial evaluating an IR daily capsule formulation vs. a SR tablet administered every 3, 4, or 5 days for 21 days. Self-collected vaginal swabs were used to quantify Lcr35® and characterize the composition and structure of the vaginal microbiota. Both LBPs were well-tolerated, and no severe adverse effects were reported. All groups had Lcr35® vaginal concentrations over 107 colony forming unit per milliliter of vaginal secretion on each day in the study. The new Gynophilus® slow release tablets administered either every 3, 4, or 5 days provided vaginal concentrations that were not significantly different from those of classic Gynophilus® (capsule) once-a-day regimen. The LBPs and the different regimens did not adversely influence the abundance of native Lactobacillus spp. and indeed tended to favor their growth and reduce colonization by non-Lactobacillus spp. This study illustrates that the SR muco-adhesive LBP tablet (Gynophilus® SR) administered every 3 or 4 days as a safe, well-tolerated, and efficacious alternative to a more demanding IR daily capsule and could protect women’s healthy vaginal microbiome by promoting endogenous Lactobacillus spp.

Introduction
The healthy vaginal microbiota is dominated by Lactobacillus spp. which play important roles in protecting women, particularly against vaginal infections [1,2]. Bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) are the most prevalent vaginal infections in women of reproductive age [3,4]. These infections have a negative impact on a woman's quality of life and represent a significant cost to the healthcare system, especially due to medical consultations and antimicrobial treatments. In addition, recurrence is common and can lead to multiple relapses over time [5,6]. The use of probiotic products or live biotherapeutic products (LBPs) represent an alternative or complement to traditional treatment regimens to help control and/or re-establish a beneficial vaginal microbiota [7,8]. Indeed, LBPs are thought to stimulate the Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10096-018-3321-8) contains supplementary material, which is available to authorized users. recolonization of endogenous Lactobacillus spp., which can inhibit the growth of pathogens such as Gardnerella vaginalis and Candida albicans [7,8]. LBPs have been used successfully as an adjunct to traditional antibiotics for BV and as an antifungal for VVC to improve the cure rates and prevent recurrence by driving the restoration of beneficial commensal microbiota [3,9,10].
LBPs were first delivered in the form of vaginal dosage capsules because vaginal administration affects vaginal health more quickly than does oral LBP and enhances the viability of the live biotherapeutic microorganism (LBM) [14]. To be efficacious, a sufficient amount of the probiotic bacteria must be delivered locally. Previous studies have shown that in healthy women, the concentration of Lactobacillus spp. is greater than or equal to 10 7 CFU/ml of vaginal secretion [15,16]. Capsule administration results in an immediate release of all the LBMs that are linked to the natural vaginal secretions induced by the daily elimination of approximately 10 7 to 10 8 CFU per day and therefore requires daily administrations [17]. To limit this rapid loss, Gynophilus ® SR tablet was developed to ensure the continuous release and presence of the LBM in sufficient quantities for many days while conserving its in vitro intrinsic characteristics (viability, stability, growth, and pathogen inhibition) [18,19]. For the first time, a LBM was formulated as slow release tablet for local vaginal administration to enhance women's quality of life by spacing LBP intake [14,20].
The objective of this pilot phase I clinical trial was to determine the optimal posology for the new tablet form (SR) compared to the daily capsule (IR). The following criteria were analyzed (i) safety, (ii) ease of use of two galenic forms, (iii) vaginal concentration of the LBM L. rhamnosus Lcr35 ® in healthy women, and (iv) impact of the products on the vaginal microbiota.

Live biotherapeutic products
Two industrial LBPs, provided by BIOSE (Aurillac, France), were used for this study: Gynophilus ® (immediate release capsule), and Gynophilus ® SR (slow release tablet), both of which were administered vaginally. Gynophilus ® capsule contains 350 mg administered daily and the new mucoadhesive Gynophilus ® SR vaginal tablet contains 1000 mg. LBPs batch release correspond to L. rhamnosus Lcr35 ® concentration greater than 10 9 CFU per dosage forms (capsule and tablet). The LBPs and their corresponding characteristics are shown in Table 1. In this article, the LPBs are called by their respective commercial names: Gynophilus ® and Gynophilus ® SR.

Study design
The study was designed to include at least 32 female volunteers. Following the management of the recruitment centers, the cohort comprised of 35 healthy reproductive age women (> 18 years old). All women were not pregnant (negative urine pregnancy test) and used adapted contraception throughout the trial. Exclusion criteria included, in addition to the classical criteria, gynecological bacterial, fungal, or viral infection in the month prior to or at time of enrollment; use of vaginal or oral probiotics in the last 30 days; or allergy to one of the active ingredients or one of the excipients in the products. Further, additional specific exclusion criteria included an inability to comply with the constraints of the protocol, currently breastfeeding, being post-menopausal (no menstruation in the last 6 months), menstrual bleeding lasting more than 8 days, participated in a clinical study in the last 3 months, severe acute or chronic conditions deemed incompatible with participation in the trial by the clinician, and immunosuppression.
At enrollment, 1 day after the last day of menstruation, each participant completed a medical history questionnaire and was examined by a clinician who collected cervico-vaginal samples to test for Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Candida spp. A clinical evaluation was performed at Alfred Fournier Institute according to the Amsel criteria [21]. A second vaginal swab was also collected using the Copan ESwab system (Cat# 480C) by the clinician and stored at BIOSE at − 80°C in 1 ml of Amies transport medium for L. rhamnosus Lcr35 ® quantification and vaginal microbiota analysis by 16S rRNA gene amplicon sequencing.
Among the 35 volunteer women, one of them had taken a prohibited treatment before the inclusion, and one other woman did not take the test drug after inclusion because of menstruation. Therefore, after obtaining written consent and enrollment, 33 women were randomized to one of four arms: Gynophilus ® capsules administered daily which served as the reference arm (REF, n = 9) or Gynophilus ® SR tablets administered either every 3 (TRT1, n = 8), 4 (TRT2, n = 9), or 5 (TRT3, n = 7) days. Randomization was stratified within enrollment centers (Centre Hospitalier of Aurillac or Vichy, France). The randomization list was prepared using the SAS ® software version 9.3. Women self-administered the first LBPs according the product package insert on the day of enrollment and continued for 21 days according to their assigned regimen. Each day, women self-collected a vaginal swab (Copan ESwab system) and stored the sample at 4°C in their home fridge. Volunteers delivered all self-collected vaginal swabs to the clinic at the second visit. This second clinical visit occurred within 4 days of the last administration of the assigned LBP. A clinical evaluation similar to that performed at enrollment was performed and a vaginal swab was collected by the clinician using a Copan ESwab. All sample collected were stored at − 80°C at BIOSE until additional analysis.

DNA extraction from vaginal samples
An optimized and standardized DNA extraction protocol dedicated to bacterial DNA extraction from swab samples has been used (Genoscreen, Lille, France). ESwabs were thawed on ice and a total of 500 μl of cell suspension in Amies transport medium was processed using the Nucleospin 96 tissue kit (Macherey Nagel, Germany) including a bead-beating step, as recommended by the manufacturer. DNA quantification was performed using SYBR green (Life Technologie, Paisley, UK). A 16S rRNA gene PCR amplification was performed using 2 μl of extracted DNA to confirm the presence of bacterial DNA using standard 16S rRNA gene PCR. DNA extraction and quantification steps were performed by GenoScreen Lille, France.

Quantification of L. rhamnosus Lcr35 ® by qPCR
Quantification of L. rhamnosus Lcr35 ® was estimated by a specific qPCR run in triplicate as previously described (Darbaky et al. 2016). L. rhamnosus Lcr35 ® quantities were expressed in CFU/ml using a standard curve correlating Ct values to colony forming unit (CFU) from in vitro culture in De Man, Rogosa, and Sharpe (MRS) medium.

Quantification of total 16S rRNA copies
The number of 16S rRNA gene copies was measured using the BactQuant qPCR assay as previously reported [22]. Estimates of taxa absolute abundance were obtained by multiplying the relative abundance of a specific taxa with the 16S rRNA gene copy number in a sample (Online Resources 1 and 2).

Vaginal microbiota characterization
The V3-V4 regions of the 16S rRNA gene were amplified from 50 ng of DNA from each sample using the methods of Fadrosh et al. [23] and sequenced on an Illumina MiSeq (Illumina, San Diego, CA, USA) using the 300-bp pairedend protocol. Raw sequence reads were processed using in house scripts and QIIME version 1.6.0 [24] to remove the amplification primer sequences and quality screen was performed according the following criteria: sequence reads with average quality score of less than 20 over a 30-bp sliding window were truncated at the first base pair of the window and evaluated for length. If the trimmed read was < 75% of its original length, it was discarded. Paired sequences were assembled according to Fadrosh et al. [23] and each sequence was assigned to a bacterial taxonomy using the PECAN software [25]. Community state types (CST) according to the previously reported method [26], taxa relative abundance and CST assignments are shown in Online Resources 1 and 2.

Statistical analysis
Data are presented as the mean and standard deviation for continuous variables and as numbers or proportions (%) for categorical variables. Comparisons between groups were performed by using the Kruskal-Wallis test or Fisher exact test for categorical variables. To compare the mean concentration of Lcr35 ® over time between treatment and reference arms, the Wilcoxon test was used. All clinical statistical analyses were performed using the SAS software (SAS ® institute Cary, NC, USA, www.sas.com); p values < 0.05 were considered statistically significant.
Inter-group differences in numbers of 16S rRNA copies and microbiota were assessed using a Kruskal-Wallis test. Theses statistical analyses were carried out using the GraphPad Prism 5 software (www.graphpad.com/prism); p values < 0.05 were considered statistically significant.
As a classic pilot study, small arm population induces a low power calculation so the statistical analysis was focused on midpoint and variability.

Volunteer population
A total of 35 volunteer women were enrolled, and 34 were randomized into 4 treatment arms; 33 women completed the study (Fig. 1). Two women were excluded because of use of treatment before product initiation or menstruation during the treatment.
The population characteristics and statistic results are shown in Table 2. The median participant age was 32 years (32.3 ± 7.7 years). A variability was observed in the median participant's age among the 4 treatment arms (TRT1, TRT2, TRT3, and REF) (p = 0.02). However, all women were between 21 and 52 years of age. Body mass index (BMI) was not significantly different (p = 0.60) between each arm, with a mean of 23 ± 5 (kg/m 2 ). Overall, 54.5% of the participants had at least one previous live birth. The mean number of children was 2 for the entire cohort, varying from 1 in TRT2 to 3 in TRT1 ( Table 2).

Safety of Lcr regenerans ® products
No severe adverse events were observed (Table 3). Ten adverse events (AE) were reported, but no differences (p = 0.40) in the frequency of AEs were detected among the 4 arms ( Table 3). All AEs were considered not serious. One of the AEs in TRT2 was deemed to be of severe intensity with vulvar itching. Other reported AEs included three that were linked to product use, and the women reported vulvar itching, brown discharges, and vulvovaginal discomfort after administration of the first tablet. However, none of the AEs led to permanent or temporary withdrawal from the study (Table 3).

Tolerance/acceptance
Clinical tolerance was evaluated at the last visit by the study clinician through questioning and examination of the participants. Factors evaluated were onset of clinical signs and/or urogenital symptoms ( Table 4). The products were welltolerated (score: excellent) by half the subjects and were tolerated in the other half (score: good). Specific criteria evaluation (tingling, dryness, burning, itching, and pelvic pain) was rarely reported, and no difference in these reported factors between arms was observed ( Table 5). Administration and dosage regimen demonstrated strong satisfaction ( Table 6). The tablet regimen showed significantly higher acceptance after the 3-week administration, especially in the less restrictive arms, in which the tablets were taken every 5 days compared to the daily capsule (Table 6).

Lcr35 ® colonization
Women self-collected a daily vaginal swab that was used to measure the concentration of Lactobacillus rhamnosus Lcr35 ® in each arm (Fig. 2). The average vaginal Lcr35 ® concentration showed no major difference among the four arms (p = 0.77). A decrease in the daily Lcr35 ® concentration was observed post-tablet administration as the frequency of administration increased from every 3 to every 5 days (Fig. 2). Although the daily Lcr35 ® concentration decreased post-administration, it remained greater than 10 7 CFU/ml in TRT1 and TRT2, with the exception of 1 day in TRT2. In TRT3, 5 days post-administration, the Lcr35 ® concentration was approximately 10 7 CFU/ml.

Effect of Gynophilus ® formulations on the vaginal microbiota
The daily composition and absolute abundance of the vaginal microbiota were evaluated using 16S rRNA gene amplicon sequencing and quantitative PCR. No significant difference (p = 0.12) in the concentration of non-Lcr35 ® Lactobacillus spp. was observed before treatment (D0), during treatment and at the end of treatment (D21) (Fig. 3a). The overall composition in Lactobacillus spp. remained consistent during and after treatment.
The vaginal microbiota was classified into six distinct community state types (CSTs) based on the microbiota bacterial composition and abundance. The before and after treatment CST frequencies are shown in Table 7, where CST-I is dominated by L. crispatus, CST-II is dominated by L. gasseri, CST-III is dominated by L. iners, CST-V is dominated by L. jensenii, CST-IV is characterized by a paucity of Lactobacillus spp. and a diverse set of strict and facultative anaerobes, and CST-VI is dominated by the LBM Lcr35 ® . The frequency of CSTs was not different between visit 1 and visit 2, indicating that Gynophilus ® treatments did not disturb the indigenous vaginal microbiota. Only one on 35 women is classified as CST-VI (dominance Lcr35 ® ) because the last tablet was administrated just before the V2. So, Lcr35 ® did not colonize and replace the endogenous microbiota. Moreover, the CST proportions were not drastically modified which suggest that Lcr35 ® did not promote only one CST type.   Fig. 4 and highlight the absence of a rapid change in the composition and structure of the vaginal microbiota during treatment. Of note, it was observed that the Escherichia coli present at visit 1 was rapidly eliminated after the initiation of treatment and that the indigenous Lactobacillus spp. re-colonized the vagina (Fig. 4a). Moreover, E. coli species was detected at least once during the study in vaginal samples of 28 women. For 21 of them, E. coli is eliminated quickly after treatment (Online Resource 3). Overall, after the initiation of Gynophilus ® treatments, Lactobacillus spp. abundance remained stable or promoted (Fig. 4).

Discussion
LBPs aim to re-establish a Lactobacillus-dominated vaginal microbiota by promoting the recolonization of the indigenous Lactobacillus spp., thereby restoring the protection against infections [7,8,27,28]. The LBP Gynophilus ® is vaginally delivered and is indicated to prevent recurrent VVC and BV by restoring vaginal health [11][12][13]. This LBP contains L. rhamnosus Lcr35 ® , which was previously reported to have potent antimicrobial properties [18,19,[29][30][31][32]. Gynophilus ® (Lcr regenerans ® ) LBP formulated in vaginal capsules was shown to be well-tolerated and significantly enhanced the quality of life of women after treatment [13]. A mucoadhesive slow release tablet formulation (SR) was developed to limit the rapid loss of the LBP associated with natural vaginal secretions and ultimately enhance the ease of use by minimizing the frequency of administration [14,[18][19][20] resulting in an increased compliance. CompriGel is a pilot phase I clinical trial, in which 35 volunteers were randomized to four arms to compare the LBP vaginal concentrations between daily IR capsule administration and SR tablets taken every 3, 4, or 5 days for 21 days. As most of the pilot clinical studies, the power calculation is limited due to the small size population but minimize the risks in healthy subjects while allowing selection of an interesting posology. So, this pilot study was designed to determine the more adapted posology of the new SR tablet.
In this study, we have shown no difference in Lcr35 ® colonization between SR tablets administered every 3 or 4 days and daily capsules. Importantly, the mean concentration of L. rhamnosus Lcr35 ® in all treatment arms remained greater than 10 7 CFU/ml, which is the necessary minimal concentration for activity and, ultimately, to prevent vaginal infections [17]. The Lcr35 ® colonization was quantified by molecular biology. This method was not used to differentiate viable and dead microorganisms and we know that the delivered LBM concentration is at least 1.10 9 CFU/posology and we observe a quickly remove of the LBM probably induced by the vaginal secretions [17]. So, administration of tablets every 3 or 4 days maintained a mean Lcr35 ® concentration greater than 10 9 CFU/ml, similar to that obtained with the daily capsules. This finding supports the possibility of a clinical regimen in which Gynophilus ® SR The formulation of the muco-adhesive Gynophilus ® SR tablet was developed to provide a three-fold increase in the quantity of LBM brought in one dosage form to reduce the rapid loss of LBM Lcr35 ® delivered by a capsule. Moreover, this new dosage form ultimately improved the comfort and ease of use, as the controlled muco-adhesive vaginal formulation affords a prolonged residence time of the LBM at efficacious concentrations with less frequent administration. Slow release delivery strategies have been successfully developed for antifungal and antibacterial drugs and for other vaginal LBPs using different muco-adhesive tablets or vaginal rings [33][34][35][36][37].
The main goal of slow release strategies is to promote compliance when the recommended treatment duration is longer than 2-3 days and, ultimately, to improve the comfort and ease of use by reducing the burden on women through spacing administrations. The results presented have confirmed that over a 3-week treatment period, Gynophilus ® SR tablets administered every 3 or 4 days were reported to be convenient and less burdensome than were daily Gynophilus ® capsules (Table 6). Daily treatment schedules generally induce poor compliance over long administration periods. Gynophilus ® SR promoted compliance and improved treatment adherence. This finding supports a benefit of the LBP for treating recurrent vulvovaginal infections, which have been shown to require a 3-week treatment [13]. While compliance and ease of use are important factors, the safety of the product is of the utmost importance. In this phase I clinical trial, no severe adverse events (AE) and only a few AEs were reported; these AEs were reported equally in all arms of t h e s t u d y. N o n e o f t h e A E s l e d t o t e m p o r a r y discontinuation or withdrawal from the study. Overall, the tolerance of the LBPs was reported to be excellent in half of the participants and good in the other half and was associated with an overall strong satisfaction with the use of the products. These results are in line with previous clinical trials for the prevention of recurrent VVC and BV with various Gynophilus ® formulations [11,13].
Gynophilus ® SR appears to be a well-accepted, safe, and easy-to-use LBP with high compliance that delivers an adequate concentration of LBM Lcr35 ® for up to 5 days post-administration. The efficacy of Gynophilus ® SR must be confirmed in a large-scale study with the final selected dose. The impact of Gynophilus ® LBP formulation and treatment frequency on the composition and structure of the vaginal microbiota was evaluated. The results showed that at study entry, 54.6% of the volunteer's vaginal microbiota harbored microorganisms that were phylogenetically related to Lactobacillus spp. as the numerically dominant members ( Table 7). The frequency of vaginal CSTs in these otherwise healthy French women is similar to that observed by Ravel et al. in North American women [26,38]. In this phase I clinical study, Gynophilus ® capsules and SR tablet administration did not disturb the endogenous Lactobacillus spp. and trends towards an increase in the abundance of indigenous Lactobacillus spp. without Lcr35 ® colonization ( Fig. 3 and Fig. 4). This finding supports a beneficial effect of treatment by restoring a healthy Lactobacillus-dominated vaginal microbiota. However, because all participants are healthy and disease free, a clinical trial designed to evaluate treatment efficacy in women with VVC or BV is warranted to confirm that Gynophilus ® SR administered every 3 or 4 days will also promote the restoration of a resilient Lactobacillus spp.dominated microbiota while efficiently treating the vaginal infections.
Even if statistical results showed no differences between both galenic forms, it has to be remind that low number of subject does not allow any extrapolation to the global population. This pilot clinical trial will afford In conclusion, this study showed that the SR formulation of Gynophilus ® administered every 3, 4, or 5 days is well-tolerated, easy-to-use, and less burdensome than the capsule form. More importantly, the findings support the conclusion that the muco-adhesive Gynophilus ® SR administered every 3, 4, or 5 days is a safe, well-tolerated, well-accepted, and efficient alternative to provide a daily  Informed consent In accordance with the law of 6 January 1978 modified, the woman volunteer was informed in the information leaflet and gave her written consent for the processing of her personal data implemented as part of this study and to enable analysis of the research results. Before inclusion in the study, the investigator provided the woman volunteer with all the information about the study. When the woman volunteer gives her consent, she and the investigator signed and dated the consent form.
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s2fieldsofstudy: Biology Medicine

text:
Genetic variations in the SULF1 gene alter the risk of cervical cancer and precancerous lesions

Human papillomavirus (HPV) infection alone is not sufficient to explain the development of cervical cancer. Genetic variants have been linked to the development of precancerous lesions and cervical cancer. In this study, we aimed to evaluate the association of 10 single nucleotide polymorphisms (SNPs) of the Fas cell surface death receptor (FAS), trinucleotide repeat containing 6C (TNRC6C), transmembrane channel like 8 (TMC8), DNA meiotic recombinase 1 (DMC1), deoxyuridine triphosphatase (DUT), sulfatase 1 (SULF1), 2′-5-oligoadenylate synthetase 3 (OAS3), general transcription factor IIH subunit 4 (GTF2H4) and interferon gamma (IFNG) genes with susceptibility to precancerous lesions and cervical cancer. In total, 608 female participants, consisting of 199 patients with persistent low-grade precancerous lesions (CIN1), 100 with high-grade precancerous lesions (CIN2/3), 17 patients with cervical cancer and 292 healthy controls, were enrolled in this study. SNPs were tested for associations with each of the above-mentioned cervical group lesions or when considering an overall patient group. A significant difference for rs4737999 was observed between the controls and the overall patient group considering the recessive mode of inheritance [odds ratio (OR), 0.48; 95% confidence interval (CI), 0.24–0.96; P=0.033]. This effect was even stronger on the risk of CIN1 lesions. Carriers of the rs4737999 AA genotype were almost 3-fold less likely of having low grade lesions compared to the other genotypes. On the whole, this study provides evidence of an influence of the SULF1 gene rs4737999 SNP in the development of precancerous lesions/cervical cancer.

Introduction
Cervical cancer is estimated to be the fourth most frequent type of cancer among women worldwide (1) and the fourth leading cause of cancer-related mortality (1)(2)(3). Environmental factors, living habits and human papillomavirus (HPV) infection have been linked to the development of cervical cancer (4-6). Specifically, HPV infection is considered to be one of the most important causal factors related to cervical cancer (7). However, HPV alone, appears to not be sufficient for the development of cervical cancer (8), as only a small amount of HPV-infected women finally develop cervical cancer (3,9).
A number studies have provided evidence of familial clustering of cervical cancer, supporting the existence of genetic effects (10)(11)(12). Moreover, several association studies have demonstrated a number of genetic variants that possibly confer susceptibility to cervical cancer by affecting immune responses, DNA repair or viral cell entry and infection (3,13,14). However, uncertainty for the effect size of genetic variants, particularly in different ethnic backgrounds still exists, as the results of different genetic studies have been conflicting (13).
Recently, Wang et al, genotyped 7,140 SNPs across 305 genes that were involved in HPV infection, cell entry and DNA repair, and reported that genes, among which general transcription factor IIH subunit 4 (GTF2H4), deoxyuridine triphosphatase (DUT), DNA meiotic recombinase 1 (DMC1), 2'-5-oligoadenylate synthetase 3 (OAS3), sulfatase 1 (SULF1), interferon gamma (IFNG), transmembrane channel like 6 (TMC6) and transmembrane channel like 8 (TMC8) were associated with the risk of HPV persistence and cervical pre-cancer/cancer (14). The loss of the expression of DMC1 plays an important role in the development of cancers in human tissues, including cervical cancer lines (15). The DUT enzyme influences nucleotide metabolism by producing the immediate precursor of thymidine nucleotides, dUMP, and consequently decreasing the intracellular concentration of dUTP (16). As a result, uracil cannot be incorporated into DNA (16). SULF1 is a heparin-degrading endosulfatase, which desulfates heparan sulfate proteoglycans (HSPGs) and blocks the binding of growth factors and their receptors, inhibiting as a result, the activation of growth factors and signaling pathways (17,18). OAS3 is induced during viral infection and plays an important role on the antiviral intracellular innate immune response (19). GTF2H4 is a general transcription factor that interacts with factors important in carcinogenesis and is involved in processes of DNA repair and transcriptional control (20). IFNG is regulatory cytokine, released by lymphocytes, that enhances cellular immune responses via increased T-cell cytotoxicity and natural killer (NK)-cell activity (21). The TMC6 and TMC8 genes (also referred to as EVER1 and EVER2 genes), are known for the development of Epidermodysplasia verruciformis, which is associated with a high sensitivity to HPV infections (22). The TMC6 and TMC8 proteins appear to regulate cellular zinc homeostasis in keratinocytes and lymphocyte (23).
In a previous analysis in a population from Northern Greece (3), we failed to detect a significant effect of two SNPs of the EVER1/2 gene region (rs2290907 and rs16970849) and the FAS-670 polymorphism (rs1800682) on precancerous lesions and cervical cancer. This was in contrast to a previous positive study by Castro et al (24). FAS belongs to the family of tumor necrosis factor (TNF) receptors (25,26). The downregulation of FAS leads to resistance to death signals, a phenomenon that has been observed in cervical cancer (27)(28)(29)(30).

Materials and methods
Study population. A total of 608 women that had attended the Obstetrics and Gynaecology Clinic of the University Hospital of Larissa, Larissa, Greece participated in this study. The patient group consisted of 316 women with a histopathologically confirmed diagnosis of cervical cancer (n=17) or precancerous lesions, either high grade (CIN2/3, n=100) or persistent low grade (CIN1, n=199). The control group consisted of 292 age-matched women with normal annual cervical cytology screening.
The local Ethics Review Board of the University Hospital of Larissa approved the study protocol. Informed consent was obtained from all individual participants included in the study.
Isolation of DNA and genotyping. Genomic DNA was extracted from 200 µl of EDTA-anti-coagulated whole blood, using a QIAamp ® DNA Blood Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. SNPs were genotyped with TaqMan allele-specific PCR amplification technology on an ABI PRISM 7900 Sequence Detection System and analyzed with the Sequence Detection Software (SDS 2.1) (both from Applied Biosystems, Foster City, CA, USA) by laboratory personnel blinded to clinical status. In order to assess genotyping reproducibility, initially observed SNP allelic discrimination curves of all genotypes were confirmed by direct DNA sequencing on an ABI PRISM 3100 genetic analyzer (Applied Biosystems).
Statistical analysis. Hardy-Weinberg equilibrium was examined with the exact test Power calculation analysis performed using the CaTS Power Calculator (31). Genotype-disease association analysis was performed with binary logistic regression using the SNPStats platform (http://bioinfo.iconcologia.net/SNPstats/) (32). Odds ratios (ORs), 95% confidence intervals (CIs) and P-values were calculated assuming the co-dominant (genotypic) model (AA vs. Ab vs. bb) and the recessive (AA + Ab vs. bb) modes of inheritance. Four phenotypic groups were searched for the association with the analyzed SNPs compared to the healthy controls: i) The cervical cancer group; ii) the group of patients with high-grade precancerous lesions (CIN2/3); iii) the group of patients with low-grade precancerous lesions (CIN1); and iv) an overall patient group with abnormal cervical changes (either cervical cancer or any type of precancerous lesions).

Results
The characteristics of the 10 studied SNPs (gene, chromosome, chromosomal position, minor allele and minor allele frequencies) are presented in Table I. The genotype call rate was ≥98.85%. All studied SNPs were found to follow the Hardy-Weinberg equilibrium either in the cases or the controls (exact test, P>0.01) (33). Genotype call rate and P-value (exact test) for HWE, for each SNP, are presented and Table II.
The allelic and genotypic frequencies of the studied SNPs in the control and the overall patient group, as well as in the cervical cancer, high-grade precancerous lesion and low-grade precancerous lesion groups are presented in Table III. Of note, as regards rs9893818, all successfully genotyped participants (100%) carried the CC genotype, whereas as regards rs12302655, >99.0% of the participants carried the wild-type genotype.
Power analysis revealed that our study had a statistical power of >80.0% to detect an genetic association with an OR of 1.78, under the assumption of the multiplicative model, a minor allele frequency of 5% (the lowest in cases for the rs16970849), a type I error level of 0.05, in a sample size consisting of 292 controls and 316 cases (data not shown).
Binary logistic regression analysis demonstrated a significant effect of SULF1 rs4737999 on the risk of the abnormal cervical changes. In particular, a significant difference was observed between the controls and the overall patient group (low-grade, high-grade and cervical cancer) considering the recessive mode of inheritance (OR, 0.48; 95% CI, 0.24-0.96; P= 0.033). Individuals carrying the AA genotype had almost half a risk of having cervical cancer, and low-or high-grade lesions compared to those carrying either the GG or the GA genotypes. Moreover, this effect was even more potent on the risk of low-grade precancerous lesions (OR, 0.36; 95% CI, 0.14-0.92; P=0.042) and (OR, 0.35; 95% CI, 0.14-0.87; P=0.014) in the co-dominant and recessive models, respectively. Carriers of the rs4737999 AA genotype were almost 3-fold less likely of having low-grade lesions compared to carriers of the other genotypes. No other SNP was found to alter the risk of any examined phenotype (Table IV).
The main mechanism of SULF1 gene is presented in Fig. 1. SULF1 gene encodes the SULF1 protein. SULF1 is a heparin-degrading endosulfatase, which desulfates HSPGs and blocks the binding of growth factors and their receptors. Consequently, it inhibits the activation of growth factor and the signaling pathways.

Discussion
In the present study, we tried to replicate the findings of previous studies regarding the role of SNPs in DNA repair, viral infection and cell entry, and their effects on the risk of cervical cancer and precancerous lesions (14,24). In addition, we re-examined an independent Greek cohort in order to determine the influence of the rs1800682 (FAS), rs2290907 (TMC6) and rs16970849 (TMC8) gene variants (3). In the present study, we found that a specific variant of the SULF1 gene, rs4737999, was associated with a significantly decreased risk of developing precancerous lesions and cervical cancer. The SULF1 gene is located at the 8q13.3 region. It encodes the homonymous protein, a heparin-degrading endosulfatase, which desulfates HSPGs and blocks the binding of growth factors and their receptors and as a result it inhibits the activation of growth factor and the signaling pathways ( Fig. 1) (17,18). The expression of SULF1 appears to be stable in normal tissues, whereas it is downregulated in several tumor cells (34). Moreover, the proliferation and migration of tumor cells can be inhibited by the re-expression of the SULF1 gene (35).
In the study by Wang et al (14), the SULF1 gene reached a statistically significant threshold for the CIN3/Cancer group compared to the controls (P= 0.0030). Moreover, the SULF1 gene was also associated with HPV persistence (P= 0.005). Additionally, according to SNP-Based association analysis, when the CIN3/Cancer group was compared to the control group, 3 out of the 77 examined SULF1 SNPs (rs4737999, rs4284050 and rs10108002) achieved statistical significance (P-value trend <0.05). In this analysis of Wang et al, the strongest association was reported for the rs4737999; this polymorphism also was associated with precancerous lesions and cervical cancer in the present study.
A number of SNPs of the SULF1 gene have been found to influence the risk of cancer. The AA genotype of r3802278, a SNP located in the 3'-untranslated region (3'-UTR) of the SULF1 gene, was found to play a protective role against breast cancer (36). Moreover, rs2623047, a 5'-upstream gene variant in SULF1, has been associated with an increased risk of breast cancer, as well as with an earlier age of onset and the   (8) SNPs, single nucleotide polymorphisms. The rows indicating the 'missed' numbers indicate the number of failed samples (DNA from some participants failed to be genotyped and consequently there were a few missed genotypes). Percentages (%) have been calculated according to the total number of patients in each group.  All cases (n=316) vs.
Cancer (n=17)  survival of ovarian cancer (37). Finally, rs6990375, a 3' prime UTR variant, has been associated with earlier age of ovarian cancer (38). The SNP rs4737999, that reached a statistically significant threshold in the present study, is an intronic noncoding variant located between exons 13 and 14. Therefore, the SULF1 gene may represent an important locus linked to tumorigenesis, as SNPs located in the 5'-upstream region, in the 3'-UTR region or even in the middle of the gene have been found to alter the risk of cancer.
A number of studies have reported that the FAS-670 gene promoter polymorphism is associated with cervical carcinogenesis (39)(40)(41)(42)(43). Moreover, the expression of the FAS/FASL genes and the CD95-CD95 ligand (FAS/FASL) interaction seem to confer susceptibility to the development of cervical cancer (3). However, the present study failed to detect any significant effect of the FAS gene SNPs on the risk of cervical cancer or any precancerous lesion. This is in accordance with the results of our previous study in another Greek cohort (3). It is possible that ethnic differences in FAS gene variability may account for the different results among populations (44).
In conclusion, the present study confirms the finidngs of previous reports regarding the role of SULF1 in the risk of precancerous lesions and cervical cancer. This association may have prognostic and pharmacogenetic implications to precancerous lesions or cervical cancer, as SULF1 may be considered as a therapeutic target or biomarker (45,46). Our findings need to be replicated in other populations of other ethnic backgrouns and in experimental models, in order to elucidate the possible role of polymorphic variants of the SULF1 gene in the pathophysiology of mechanism of tumorigenesis.