Susceptibility loci identified in Han Chinese influence genetic predisposition of PCOS in Indian women.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a multifactorial disorder characterized by a broad spectrum of reproductive and metabolic perturbations, necessitating early timely diagnosis and management. PCOS is a multigenic disorder and ample evidence from family based, candidate gene and genome-wide association studies (GWAS) has implicated genetic factors in development and progression of PCOS. The first GWASs in Han Chinese population revealed prominent gene loci to be strong contenders in the etiopathogenesis of PCOS. However, different ethnic and geographical settings impact the genetic association pattern of PCOS. METHODS AND RESULTS: In the current case-control replication study, we have genotyped previously identified polymorphisms viz. rs2479106 and rs10818854 of DENND1A and rs13405728 of LHCGR, rs4385527 and rs3802457 of c9orf3, rs705702 of RAB5B and rs1894116 of YAP1 in control (N = 247) and PCOS (N = 504) women by Sanger sequencing, and their association with PCOS susceptibility and its related traits was investigated. We found significant association of rs4385527 of c9orf3 and rs1894116 of YAP1 with decreased and increased PCOS susceptibility respectively in non-hyperandrogenic women. Trend towards association was also noted for rs2479106 of DENND1A and rs705702 of RAB5B. Additionally, polymorphisms also showed association with metabolic and androgen related traits in both controls and hyper- and non-hyperandrogenic women with PCOS. CONCLUSIONS: Thus, this study shows that some, but not all polymorphisms previously identified in Han Chinese women, could contribute to the genetic pathophysiology of PCOS in Indian women, accentuating essentiality of conducting replication studies to elucidate the genetic predisposition profile of PCOS.

Investigating the association of previously identified genome-wide significant loci (rs10739076 and rs1784692) with PCOS susceptibility and its related traits in Indian women.

OBJECTIVE(S): Polycystic ovary syndrome (PCOS) is a multifactorial endocrinopathy with an enigmatic etiology. Hallmark features include irregular menstrual cycles, insulin resistance and hyperandrogenemia and affected women are prone to development of adverse reproductive and cardiometabolic outcomes like anovulatory infertility, impaired glucose tolerance, type 2 diabetes, dyslipidemia, metabolic syndrome and cardiovascular disease. Genetic underpinnings of PCOS have been investigated extensively using genome-wide association studies, which have led to the identification of several novel susceptibility loci. However, as ethnic diversity contributes to phenotypic and genetic heterogeneity, we undertook the first genetic association study to determine the association of rs10739076 of PLGRKT and rs1784692 of ZBTB16 with PCOS susceptibility and its related traits in Indian women. STUDY DESIGN: The present case-control study comprised 497 women with PCOS diagnosed according to the Rotterdam criteria and 233 age matched healthy women as controls. All participants were characterized in terms of anthropometric, hormonal and metabolic parameters and the variants were investigated by direct sequencing. Genotypic and genotype-phenotype association of these variants with PCOS susceptibility and its related biochemical and hormonal traits was analyzed with appropriate statistical tests. RESULTS: The genotypic and allelic frequencies of rs1784692 of ZBTB16 were significantly decreased in lean women with PCOS only, and this variant was associated with lowered insulin levels, HOMA-IR, LH:FSH ratio along with increased ApoA1 levels and QUICKI in them. Although, the PLGRKT variant, rs10739076, showed similar frequency distribution in both lean and obese groups, it was found to be associated with reduced fasting glucose in all women with PCOS. CONCLUSION(S): To the best of our knowledge, this is the first study to demonstrate that ZBTB16 variant showed significant association with reduced PCOS susceptibility in lean rather than obese Indian women, highlighting the impact of obesity on determining genetic predisposition to PCOS in Indian women. In contrast, PLGRKT variant did not influence PCOS risk in lean or obese women. Importantly, both variants exerted a protective effect on glucose metabolism, insulin resistance, gonadotropin and lipid levels in women with PCOS. Determination of susceptibility variants for PCOS demand population specific replication studies to ascertain best candidate loci for PCOS.

Transcriptomic profile of GLCs of PCOS women highlights metabolic dysregulation as a plausible contributor to PCOS pathophysiology.

Polycystic ovary syndrome (PCOS) is a complex heterogeneous disorder with reproductive and metabolic consequences whose aetiology is still elusive. To understand the cellular mechanisms that potentially govern follicular defect in women with PCOS, we performed transcriptomic profiles of granulosa-lutein cells (GLCs) by RNA-Seq analysis. We found differential expression of 876 genes in GLCs between PCOS and controls that belonged to various processes such as cell cycle, extracellular matrix organization, angiogenesis, oxidative stress, metabolism, etc. that support folliculogenesis, oocyte development, and maturation. The cross-talk between oocyte and GLCs is a fundamental cornerstone in determining oocyte quality and highly interlinked pathways of metabolism and redox homeostasis may influence this. We found several genes involved in the metabolism of carbohydrates, nucleotides, cholesterol, and lipids were dysregulated, which may impair the supply of metabolites to the growing oocyte, affecting oocyte development and competence. Additionally, high metabolic activity during folliculogenesis may augment oxidative damage to cells and macromolecules if not counter-balanced. We observed dysregulation of redox homeostasis and AGE-RAGE signalling in the follicular environment. Among the validated genes, prokineticin-1 and growth differentiation factor-15 were found to be negatively regulated, while, S100, calcium-binding protein A9 and angiomotin-like-2 were positively regulated in GLCs of women with PCOS. Comparing our data with previously published relevant transcriptomic studies showed metabolic, cytokine-cytokine receptor interaction, IL-17, and chemokine signalling pathways were most commonly affected in PCOS. Overall, this data can provide insights into mechanisms contributing to PCOS pathophysiology and can be explored as potential indicators for oocyte/embryo quality in IVF settings.

Molecular characterization of variants in mitochondrial DNA encoded genes using next generation sequencing analysis and mitochondrial dysfunction in women with PCOS.

Emerging studies indicates mitochondrial dysfunction and involvement of mitochondrial DNA (mtDNA) variants in the pathogenesis of polycystic ovary syndrome (PCOS). Cumulative effect of mtDNA rare variants are now gaining considerable interest apart from common variants in the pathogenesis of complex diseases. Rare variants may modify the effect of polymorphism or in combination with the common variants may affect the risk of disease. With the evolution of high throughput sequencing techniques, which can be utilized to identify common as well as rare variants along with heteroplasmy levels, comprehensive characterization of the mtDNA variants is possible. Till date, few studies reported common mtDNA variants using traditional sequencing techniques but rare variants in mtDNA encoding genes remain unexplored in women with PCOS. These mtDNA variants may be responsible for mitochondrial dysfunction and may contribute in PCOS pathogenesis. In this study we determined mtDNA copy number, a biomarker of mitochondrial dysfunction and first time analysed variants in mtDNA encoded genes in women with PCOS using mitochondrial Next Generation sequencing (NGS) approach and compared allele frequency from mitochondrial 1000 genome dataset. Variant annotation and prioritization was done using highly automated pipeline, MToolBox that excludes reads mapped from nuclear mitochondrial DNA sequences (NumtS) to identify unique mtDNA reads. The present study identified significant reduction in mtDNA copy number in women with PCOS compared to non-PCOS women. A total of unique 214 prioritized common to rare variants were identified in mtDNA encoded genes, 183 variants in OXPHOS complexes, 14 variants in MT-tRNA and 17 variants in MT-rRNA genes that may be involved in mitochondrial dysfunction in PCOS. Numerous variants were heteroplasmic, pathogenic in nature and occurred in evolutionary conserved region. Heteroplasmic variants were more frequently occurred in MT-CO3 gene. Non-synonymous variants were more than synonymous variants and mainly occurred in OXPHOS complex I and IV. Few variants were found to be associated with diseases in MITOMAP database. The study provides a better understanding towards pathogenesis of PCOS from novel aspects focusing on mitochondrial genetic defects as underlying cause for contributing mitochondrial dysfunction in women with PCOS.

Evidence for TET-mediated DNA demethylation as an epigenetic alteration in cumulus granulosa cells of women with polycystic ovary syndrome.

Peripheral and tissue-specific alterations in global DNA methylation (5-methylcytosine (5mC)) and DNA hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiles have been identified as both biomarkers for disease prediction and as hallmarks of dysregulated localized gene networks. Global and gene-specific epigenetic alterations in the 5mC profiles have shown widespread implications in the etiology of polycystic ovary syndrome (PCOS). However, there has been no study in PCOS that integrates the quantification of 5mC and 5hmC signatures alongside the expression levels of DNA methylating and demethylating enzymes as respective indicators of methylation and demethylation pathways. Having previously shown that the 5mC signatures are not substantially altered in PCOS, we assessed the global 5hmC levels in peripheral blood leukocytes and cumulus granulosa cells (CGCs) of 40 controls and 40 women with PCOS. This analysis revealed higher 5hmC levels in CGCs of PCOS women, indicating a more dominant demethylation pathway. Furthermore, we assessed the transcript and protein expression levels of DNA demethylating and methylating enzymes, i.e. ten-eleven translocation methylcytosine dioxygenases (TET1, TET2, TET3) and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), respectively, in CGCs. The relative transcript and protein expression levels of all three TETs were found to be higher in women with PCOS, and the TET mRNA expression profiles were positively correlated with 5hmC levels in CGCs. Also, all three DNMT genes showed altered transcript expression in PCOS, although only the downregulated DNMT3A transcript was correlated with decreasing 5mC levels. At the protein level, the expression of DNMT1 (maintenance methylation enzyme) was higher, while that of DNMT3A (de novo methylation enzyme) was found to be lower in PCOS compared to controls. Overall, these results indicate that DNA methylation changes in CGCs of PCOS women may arise partly due to intrinsic alterations in the transcriptional regulation of TETs and DNMT3A.

Ovarian granulosa cells from women with PCOS express low levels of SARS-CoV-2 receptors and co-factors.

PURPOSE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is global pandemic with more than 5 million deaths so far. Female reproductive tract organs express coronavirus-associated receptors and factors (SCARFs), suggesting they may be susceptible to SARS-CoV-2 infection; however, the susceptibility of ovary/follicle/oocyte to the same is still elusive. Co-morbidities like obesity, type-2 diabetes mellitus, cardiovascular disease, etc. increase the risk of SARS-CoV-2 infection. These features are common in women with polycystic ovary syndrome (PCOS), warranting further scope to study SCARFs expression in ovary of these women. MATERIALS AND METHODS: SCARFs expression in ovary and ovarian tissues of women with PCOS and healthy women was explored by analyzing publically available microarray datasets. Transcript expressions of SCARFs were investigated in mural and cumulus granulosa cells (MGCs and CGCs) from control and PCOS women undergoing in vitro fertilization (IVF). RESULTS: Microarray data revealed that ovary expresses all genes necessary for SARS-CoV-2 infection. PCOS women mostly showed down-regulated/unchanged levels of SCARFs. MGCs and CGCs from PCOS women showed lower expression of receptors ACE2, BSG and DPP4 and protease CTSB than in controls. MGCs showed lower expression of protease CTSL in PCOS than in controls. Expression of TMPRSS2 was not detected in both cell types. CONCLUSION: Human ovarian follicle may be susceptible to SARS-CoV-2 infection. Lower expression of SCARFs in PCOS indicates that the risk of SARS-CoV-2 infection to the ovary may be lesser in these women than controls. This knowledge may help in safe practices at IVF settings in the current pandemic.

An integrated in silico analysis highlighted angiogenesis regulating miRNA-mRNA network in PCOS pathophysiology.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrinopathy and a leading cause of anovulatory infertility. Angiogenesis is vital for ovarian folliculogenesis. The expression of angiogenesis-associated genes/proteins is altered in the ovary of PCOS women. However, information on microRNAs (miRNAs) regulating their expression is limited. This study aims to identify dysregulated angiogenesis-related genes in the ovary of women with PCOS, to identify miRNAs regulating them, and to construct a miRNA-mRNA network associated with angiogenesis. METHODS: A comprehensive literature search and reanalysis of seven ovarian GEO microarray datasets were performed to identify differentially expressed angiogenesis-related genes in PCOS. These target genes were used to predict their regulating miRNAs by querying miRNA databases and their expression in the ovary was verified. Panther and STRING database were used for functional enrichment. Gene expression of shortlisted miRNAs was studied in granulosa cells using digital droplet PCR. RESULTS: The miRNAs expressed in the ovary and potentially targeting dysregulated angiogenesis-related genes in PCOS were identified and those enriched in angiogenesis-related pathways, like VEGF, FGF, PI3K/Akt, Notch signaling, and ECM interaction were shortlisted. Analysis showed PI3K/Akt signaling was the most enriched pathway. MiR-218-5p, miR-214-3p, miR-20a-5p, and miR-140-3p associated with the PI3K/Akt pathway were found to be up-regulated in granulosa cells of women with PCOS. CONCLUSIONS: By in silico analysis, we identified crucial dysregulated angiogenesis-related genes, the miRNA-mRNA interactions, and signaling pathways involved in impaired follicular angiogenesis in PCOS. This work provides a novel insight into the mechanism of aberrant ovarian angiogenesis contributing to PCOS pathophysiology.

An integrated multidisciplinary model of care for addressing comorbidities beyond reproductive health among women with polycystic ovary syndrome in India.

Background & objectives: Polycystic Ovary Syndrome (PCOS) is becoming an area of global and national health concern. It requires a life cycle approach from adolescence to menopause. To comprehensively address the wide spectrum of this disorder, a multidisciplinary model of care was established for women with PCOS in a government setting in India with an objective to screen and manage multifaceted manifestations of PCOS and to diagnose and treat associated comorbidities such as metabolic syndrome, dermatologic manifestations and psychological issues. Methods: A model of integrated multidisciplinary PCOS clinic was implemented for services and research at ICMR-National Institute for Research in Reproductive and Child Health (NIRRCH), Mumbai Maharashtra, India. This is a one-stop holistic centre for managing menstrual, cosmetic, infertility, obesity, metabolic and psychological concerns of women affected with PCOS. Two hundred and twenty six women diagnosed with PCOS using the Rotterdam criteria were screened for metabolic comorbidities with anthropometry, ultrasonography, hormonal and biochemical tests and for psychological problems. Analysis was performed using SPSS version 19.0. Results: Mean body mass index (BMI) was 26.1 kg/m2, higher for Asians. Hirsutism was observed in 53.6 per cent of women. Metabolic syndrome was seen among 35.3 per cent and non-alcoholic fatty liver in 18.3 per cent. Psychological issues such as anxiety and depression were identified in majority of the women 31.4 per cent of women could achieve pregnancy at the end of one year of multidisciplinary management. Interpretation & conclusions: The results of the present study suggest that an integrated multidisciplinary approach led to the early identification and treatment of comorbidities of PCOS, especially metabolic syndrome. There is hence an urgent need to implement multidisciplinary PCOS clinics in government health facilities.

Compromised Cumulus-Oocyte Complex Matrix Organization and Expansion in Women with PCOS.

The cumulus-oocyte complex (COC) matrix plays a critical role in the ovulation and fertilization process and a major predictor of oocyte quality. Proteomics studies of follicular fluid showed differential expression of COC matrix proteins in women with polycystic ovary syndrome (PCOS), indicating altered COC matrix in these women. In the present study, we aimed to understand COC matrix gene induction in humans and its probable dysfunction in women with PCOS. Animal studies have shown that amphiregulin (AREG) and growth differentiation factor-9 (GDF-9) are important in the induction of COC matrix genes which are involved in cumulus expansion. The effects of AREG and GDF-9 on expression of tumor necrosis factor alpha induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) on human cumulus granulosa cells (CGCs) and murine COC expansion were evaluated. Further time-dependent effects of growth factor supplementation on these gene expressions in CGCs from PCOS and control women were compared. Follicular fluid from PCOS showed reduced COC matrix expansion capacity, using murine COCs. Expression of COC matrix genes TNFAIP6 and HAS2 were significantly reduced in CGCs of PCOS. Treatment of CGCs with AREG and GDF-9 together induced expression of both these genes in controls and could only restore HAS2 but not TNFAIP6 expression in PCOS. Our results suggest that the reduced potential of follicular fluid to support COC expansion, altered expression of structural constituents, and intrinsic defects in granulosa cells of women with PCOS may contribute to the aberrant COC organization and expansion in PCOS, thus affecting fertilization.

Altered redox status may contribute to aberrant folliculogenesis and poor reproductive outcomes in women with polycystic ovary syndrome.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) are often infertile and opt for artificial reproductive techniques (ART) to conceive. Disrupted pro-/antioxidant balance in oocyte microenvironment may contribute towards sub-optimal oocyte/embryo quality and poor ART outcome in them. METHODS: Activities/levels of redox markers and their transcript expression were investigated in follicular fluid and granulosa cells respectively, in women with PCOS (n = 71) and controls (n = 50) undergoing in vitro fertilization (IVF). Correlation analysis of redox markers and IVF parameters was performed. RESULTS: Activities of superoxide dismutase, glutathione reductase, glutathione peroxidase, and paraoxonase1 were significantly lower in follicular fluid of PCOS women than in controls. Levels of lipid peroxidation, oxidative protein modification, and oxidized glutathione were higher, whereas those of total antioxidant capacity, total thiols, and reduced glutathione were lower in follicular fluid of PCOS women than in controls. Further, comparison of redox markers based on insulin resistance and BMI status of study participants showed similar trends, indicating that PCOS pathophysiology is a significant contributor to oxidative stress irrespective of insulin resistance and BMI. Transcript levels of antioxidant enzymes were lower in granulosa cells from PCOS women than in controls, and they accorded with their activities in follicular fluid. Moreover, few redox markers showed significant correlations with oocyte/embryo quality and pregnancy outcome. CONCLUSION: Our data indicates disrupted redox homeostasis in follicular environment in PCOS which may negatively influence oocyte/embryo quality. Further, granulosa cells may play crucial role in maintaining follicular redox homeostasis. Glutathione system and paraoxonase1 could be explored further as surrogates for IVF prognosis/outcome.

Replication study of THADA rs13429458 variant with PCOS susceptibility and its related traits in Indian women.

AIM: The aetiopathogenesis of the multigenic multifactorial endocrinopathy, polycystic ovary syndrome (PCOS) has been explored using linkage, candidate gene and genome-wide association studies. Contradictory reports of replication studies attributed to phenotypic, ethnic and geographic variations are available. In this study, we investigated the association of Han Chinese GWAS polymorphism (rs13429458) in thyroid adenoma-associated gene (THADA) with PCOS susceptibility and its related traits in Indian women. METHODS: We genotyped rs13429458 of THADA by direct sequencing and investigated its association with PCOS and its related traits in controls (N = 150) and PCOS women (N = 348). All women were extensively phenotyped in terms of anthropometric, hormonal and metabolic parameters. Association of polymorphism with PCOS risk and its related traits was carried out by regression analysis. RESULTS: Genotypic and allele frequencies for rs13429458 were not different between controls and PCOS. Women with PCOS carrying variant allele showed significantly reduced fasting glucose levels, and decreased free and bioavailable testosterone and free androgen index. CONCLUSION: To our knowledge, this is the first study to demonstrate that although this polymorphism does not alter PCOS susceptibility, it favorably impacts glucose metabolism and hyperandrogenism in Indian women with PCOS only. This study highlights that genetic predisposition markers for PCOS may differ with ethnicity and phenotypic variations.

Elucidating the impact of obesity on hormonal and metabolic perturbations in polycystic ovary syndrome phenotypes in Indian women.

Polycystic ovary syndrome is a complex endocrinopathy with heterogeneous presentation and multifactorial etiology. We have undertaken this case-control study to compare metabolic and endocrine characteristics in different phenotypic subgroups of women with PCOS and the impact of obesity on them. Women with PCOS (n = 489) were classified into 4 phenotypes according to Rotterdam criteria. Comparisons of clinical, biochemical and hormonal parameters were performed across all phenotypic groups of PCOS and with controls (n = 270) by Welch's ANOVA with subsequent Games-Howell post-hoc test. We found maximum prevalence of normoandrogenic phenotype D, which is milder form of PCOS in terms of insulin resistance, gonadotropin levels and dyslipidemia, followed by phenotype A, in our total study population. After classification of the study group into lean and obese groups, only few insulin and lipid-related traits showed marked differences between phenotypes. Further, we noted that obese women showed adverse metabolic but not androgenic traits compared to lean counterparts in the same phenotype. Metabolic syndrome frequency is increased in hyperandrogenic phenotypes with HDL-C and waist circumference being most predominant contributing factors in total, lean and obese groups. We demonstrate that in our study population there is greater occurrence of phenotype D of PCOS. Our study highlights the importance of clinicians concurrently employing Rotterdam criteria along with obesity status for ascertaining accurate PCOS status and formulating suitable therapeutic intervention.

Alteration in angiogenic potential of granulosa-lutein cells and follicular fluid contributes to luteal defects in polycystic ovary syndrome.

STUDY QUESTION: Is angiogenic potential of follicular fluid (FF) and granulosa-lutein cells (GLCs) altered in polycystic ovary syndrome (PCOS) and does it play a role in corpus luteum (CL) defect observed in them? SUMMARY ANSWER: FF and GLCs of women with PCOS show reduced expression of pro-angiogenic factors compared to controls and exhibit a diminished capacity to induce angiogenesis. WHAT IS KNOWN ALREADY: In women with PCOS, CL insufficiency and frequent miscarriage are reported, which may be due to defect in CL. The development of new blood vessels is essential to promote ovarian folliculogenesis and functional CL formation. The vasculature formation in CL which is important for its function is still unexplored in these women. STUDY DESIGN, SIZE, DURATION: This case-control study was conducted in 30 healthy control women and 30 women with PCOS undergoing controlled ovarian hyperstimulation for IVF. The FF, GLCs and serum were collected from all participants during ovum pick up. PARTICIPANTS/MATERIALS, SETTING, METHODS: The capacity of FF to induce angiogenesis was assessed by measuring levels of pro-angiogenic factors vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) and its tube formation and wound healing potential using human umbilical vein endothelial cells (HUVECs). We investigated the angiogenic potential and endothelial cell-like nature of GLCs using several approaches such as the expression of angiogenic genes by quantitative PCR, DiI-conjugated acetylated low-density lipoproteins (Dil-Ac-LDL) internalization assay, tube formation assay, expression of endothelial cell markers by immunofluorescence analysis. In addition, correlation of transcript levels of angiogenic genes with oocyte parameters was studied. MAIN RESULTS AND THE ROLE OF CHANCE: FF and serum levels of VEGF and FGF2 were significantly higher and lower, respectively, in PCOS compared to controls. The tube formation and wound healing capacity of HUVECs was found to be reduced when measured after supplementation with FF of women with PCOS compared to controls. This suggests a decreased angiogenic capacity of FF in women with PCOS. Tube formation (P = 0.003) and Dil-Ac-LDL internalization (P = 0.03) ability of GLCs were significantly reduced in women with PCOS compared to controls. Protein expression levels of endothelial markers, vascular endothelial growth factor A (VEGFA) (P = 0.004), vascular endothelial growth factor receptor 2 (VEGFR2) (P = 0.011), TEK Receptor Tyrosine Kinase (Tie-2) (P = 0.026), fibroblast growth factor receptor 1 (FGFR1) (P = 0.026) and CD31 (P = 0.035) and transcript levels of angiogenic genes VEGFA (P = 0.042), hypoxia inducing factor 1A (HIF1A) (P = 0.025), FGF2 (P = 0.038), angiopoietin 1 (ANGPT1) (P = 0.028), heparin sulfate proteoglycan 2 (HSPG2) (P = 0.016), ADAM metallopeptidase with thrombospondin type1 motif, 1 (ADAMTS1) (P = 0.027) and fibronectin 1 (FN1) (P = 0.016) were found to be low in GLCs of PCOS compared to controls. Thus, the findings of this study indicate that endothelial cell-like characteristics of GLCs were significantly decreased in PCOS. Furthermore, transcript levels of VEGFA (r = 0.46, P = 0.009), ADAMTS1 (r = 0.55, P = 0.001), FGF2 (r = 0.42, P = 0.022) and ANGPT2 (r = 0.47, P = 0.008) showed a positive correlation with oocyte fertilization rate. LIMITATIONS, REASONS FOR CAUTION: The vasculature formation in CL is not possible to study in women, but we explored the angiogenic characteristics of FF and GLC obtained from women with PCOS to speculate any vascularization defect of CL in these women. The FF and GLCs were obtained from the stimulated cycle during oocyte retrieval, which may not exactly mimic the in-vivo condition. The small sample size is another limitation of this study. Larger sample size and support by color Doppler studies on CL blood flow would help to strengthen our findings. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that the altered angiogenic potential of FF and GLCs may affect vasculature development required for CL formation and function in PCOS. These findings pave the way to devise therapeutic strategies to support angiogenesis process in follicle of women with PCOS, which may improve CL insufficiency, progesterone levels and prevent frequent miscarriages in these women. Furthermore, our study also hypothesizes that the vascularization around the ovarian follicles is also compromised which may lead to the growth arrest of the follicles in PCOS, however, this needs thorough investigations. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grant BT/PR16524/MED/97/346/2016 from the Department of Biotechnology, Government of India. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Identification of Variants in Mitochondrial D-Loop and OriL Region and Analysis of Mitochondrial DNA Copy Number in Women with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a multifactorial disorder characterized by irregular menstrual problems, hyperandrogenism, and presence of polycystic ovaries. Till date, molecular mechanism underlying PCOS remains elusive. Recently mitochondrial displacement loop (D-loop) variants have been identified to be novel players in the pathogenesis of PCOS. At present, rare variants, besides common variants, are also the focus of research as it is believed to make essential contribution to the risk of complex diseases. However, rare and low hetroplasmic variants in mitochondrial D-loop are still not investigated in PCOS women. Furthermore, variants in light-strand origin of DNA replication (OriL) of mitochondrial DNA (mtDNA) have not been explored in PCOS. Hence, in this study, we investigated rare to common mitochondrial D-loop and OriL region variants obtained using mtDNA next-generation sequencing in women with PCOS. Furthermore, we also assessed mtDNA copy number, a biomarker of mitochondrial dysfunction (MD) in women with PCOS, as the variants in mtDNA are known to be associated with low mtDNA copy number in PCOS women. A total of 67 D-loop variants including 6 novel variants were identified in 30 PCOS women. Among 67 variants, 29 variants were reported in PCOS women. A single variant, 5746A was found in OriL region in two PCOS women. Both transition and transversion variants were found but transition variants occur at very high frequency compared with transversions (82.35% vs. 17.64%, respectively). As transition variants in mtDNA are known to arise because of polymerase γ errors, occurrence of high transition rates indicates that most mutation arises because of defect in replication errors that causes mtDNA damage leading to MD. Furthermore, mtDNA copy number was found to be low in women with PCOS compared with healthy control women suggesting that MD may be the contributing factor in the pathogenesis of PCOS.

Mitochondrial dysfunction: An emerging link in the pathophysiology of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by irregular menstrual cycles, hyperandrogenism and subfertility. Due to its complex manifestation, the pathogenic mechanism of PCOS is not well defined. Cumulative effect of altered genetic and epigenetic factors along with environmental factors may play a role in the manifestation of PCOS leading to systemic malfunction. With failure of genome-wide association study (GWAS) and other studies performed on nuclear genome to provide any clue for precise mechanism of PCOS pathogenesis, attention has been diverted to mitochondria. Mitochondrion plays an important role in cellular metabolic functions and is linked to Insulin Resistance (IR). Recently, increasing reports suggest that mitochondrial dysfunction may be a contributing factor in the pathogenesis of PCOS. Hence, in this review, we have discussed mitochondrial biology in brief and emphasizes on genetic and epigenetic aspects of mitochondrial dysfunction studied in PCOS women and PCOS-like animal models. We also highlight underlying mechanism behind mitochondrial dysfunction contributing to PCOS and its related complications such as obesity, diabetes, cardiovascular diseases, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD) and cancer. Furthermore, contrasting remarks against involvement of mitochondrial dysfunction in PCOS pathophysiology have also been presented. This review enhances our understanding in relation to mitochondrial dysfunction in the etiology of PCOS and stimulates further research to explore a clear link between mitochondrial dysfunction and PCOS pathogenesis and progression. Understanding pathogenic mechanisms underlying PCOS will open new windows to develop promising therapeutic strategies against PCOS.

DNA methylome profiling of granulosa cells reveals altered methylation in genes regulating vital ovarian functions in polycystic ovary syndrome.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) manifest a host of ovarian defects like impaired folliculogenesis, anovulation, and poor oocyte quality, which grossly affect their reproductive health. Addressing the putative epigenetic anomalies that tightly regulate these events is of foremost importance in this disorder. We therefore aimed to carry out DNA methylome profiling of cumulus granulosa cells and assess the methylation and transcript expression profiles of a few differentially methylated genes contributing to ovarian defects in PCOS. A total of 20 controls and 20 women with PCOS were selected from a larger cohort of women undergoing IVF, after carefully screening their sera and follicular fluids for hormonal and biochemical parameters. DNA extracted from cumulus granulosa cells of three women each, from control and PCOS groups was subjected to high-throughput, next generation bisulfite sequencing, using the Illumina HiSeq 2500® platform. Remaining samples were used for the validation of methylation status of some identified genes by pyrosequencing, and the transcript expression profiles of these genes were assessed by quantitative real-time PCR. RESULTS: In all, 6486 CpG sites representing 3840 genes associated with Wnt signaling, G protein receptor, endothelin/integrin signaling, angiogenesis, chemokine/cytokine-mediated inflammation, etc., showed differential methylation in PCOS. Hypomethylation was noted in 2977 CpGs representing 2063 genes while 2509 CpGs within 1777 genes showed hypermethylation. Methylation differences were also noted in noncoding RNAs regulating several ovarian functions that are dysregulated in PCOS. Few differentially methylated genes such as aldo-keto reductase family 1 member C3, calcium-sensing receptor, resistin, mastermind-like domain 1, growth hormone-releasing hormone receptor and tumor necrosis factor, which predominantly contribute to hyperandrogenism, premature luteolysis, and oocyte development defects, were explored as novel epigenetic candidates in mediating ovarian dysfunction. Methylation profiles of these genes matched with our NGS findings, and their transcript expression patterns correlated with the gene hypo- or hypermethylation status. CONCLUSION: Our findings suggest that the epigenetic dysregulation of genes involved in important processes associated with follicular development may contribute to ovarian defects observed in women with PCOS.

Quantitative mass spectrometric analysis to unravel glycoproteomic signature of follicular fluid in women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex endocrinopathy affecting women of reproductive age, and whose etiology is not well understood yet. In these women, the follicular growth is arrested at preantral stage leading to cyst formation, consequently resulting in anovulatory infertility in these women. As the follicular fluid provides the conducive microenvironment for the growth of oocytes, molecular profiling of the fluid may provide unique information about pathophysiology associated with follicular development in PCOS. Post-translational addition of oligosaccharide residues is one of the many modifications of secreted proteins influencing their functions. These glycoproteins play a significant role in disease pathology. Despite glycoproteins having such essential functions, very limited information is available on their profiling in human reproductive system, and glycoproteomic profile of follicular fluid of women with PCOS is yet unexplored. In the present study, we performed a comparative glycoproteomic analysis of follicular fluid between women with PCOS and controls undergoing in vitro fertilization, by enrichment of glycoproteins using three different lectins viz. concanavalin A, wheat germ agglutinin and Jacalin. Peptides generated by trypsin digestion were labeled with isobaric tags for relative and absolute quantification reagents and analyzed by liquid chromatography tandem mass spectrometry. We identified 10 differentially expressed glycoproteins, in the follicular fluid of women with PCOS compared to controls. Two important differentially expressed proteins- SERPINA1 and ITIH4, were consistently upregulated and downregulated respectively, upon validation by immunoblotting in follicular fluid and real-time polymerase chain reaction in granulosa cells. These proteins play a role in angiogenesis and extracellular matrix stabilization, vital for follicle maturation. In conclusion, a comparative glycoproteomic profiling of follicular fluid from women with PCOS and controls revealed an altered expression of proteins which may contribute to the defects in follicle development in PCOS pathophysiology.

PON1 promoter polymorphisms contribute to PCOS susceptibility and phenotypic outcomes in Indian women.

Polycystic ovary syndrome is a common endocrinopathy characterized by anovulatory infertility, hyperandrogenism, insulin resistance and oxidative stress, which predisposes affected women to reproductive and cardiometabolic complications in later life. We have investigated the association of PON1 promoter polymorphisms with PCOS susceptibility, PON1 activity and its related traits in Indian women. The genotypic and allelic frequency distribution of only -907G/C polymorphism in PON1 promoter showed significant difference between non-hyperandrogenic control and PCOS women, and was significantly associated with reduced susceptibility to PCOS, considering the recessive model. PON1 lactonase and arylesterase activities were also significantly decreased in women with PCOS compared to controls. Further, PON1 promoter polymorphisms were linked to altered insulin and testosterone levels in hyperandrogenic and non-hyperandrogenic women with PCOS. This study highlights PON1 as an important candidate gene influencing genetic pathophysiology of PCOS.

Pathomechanisms of polycystic ovary syndrome: Multidimensional approaches.

Polycystic ovary syndrome is a complex endocrine disorder affecting numerous women of reproductive age across the globe. Characterized mainly by irregular menses, hirsutism, skewed LH: FSH ratios and bulky polycystic ovaries, this multifactorial endocrinopathy results in unfavorable reproductive and metabolic sequelae, including anovulatory infertility, type 2 diabetes, metabolic syndrome and cardiovascular disease in later years. Increasing evidence has shown that the manifestation of polycystic ovary syndrome (PCOS) is attributable to a cumulative impact of altered genetic, epigenetic and protein profiles which bring about a systemic dysfunction. While genetic approaches help ascertain role of causal variants in its etiology, tissue-specific epigenetic patterns help in deciphering the auxiliary role of environmental, nutritional and behavioral factors. Proteomics is advantageous, linking both genotype and phenotype and contributing to biomarker discovery. Investigating molecular mechanism underlying PCOS is imperative in order to gain insight into the pathophysiology of PCOS and formulate novel diagnostic and treatment strategies. In this review we have summarized these three aspects, which have been successfully utilized to delineate the pathomechanisms of PCOS.

LINE1 CpG-DNA Hypomethylation in Granulosa Cells and Blood Leukocytes Is Associated With PCOS and Related Traits.

Context: Altered global DNA methylation is indicative of epigenomic instability concerning chronic diseases. Investigating its incidence and association with polycystic ovary syndrome (PCOS) is essential to understand the etiopathogenesis of this disorder. Objectives: We assessed global DNA methylation differences in peripheral blood leukocytes (PBLs) and cumulus granulosa cells (CGCs) of controls and women with PCOS; and their association with PCOS and its traits. Design, Setting, Participants, Main Outcome Measure: This study included a total of 102 controls and women with PCOS. Forty-one women undergoing controlled ovarian hyperstimulation (COH) and 61 women not undergoing COH were recruited from in vitro fertilization (IVF) and infertility clinics. DNA methylation was measured by ELISA for 5'-methyl-cytosine content and bisulfite sequencing of 5'-untranslated region (5'-UTR) of long interspersed nucleotide element-1 (LINE1/L1). Results: Total 5'-methyl-cytosine and L1 methylation levels in PBLs and CGCs were similar between controls and women with PCOS. Methylation assessed at CpG sites of L1 5'-UTR revealed a single CpG-site (CpG-4) to be consistently hypomethylated in PBLs of both PCOS groups and CGCs of stimulated PCOS group. In unstimulated women, hypomethylation at CpG-4 was strongly associated with PCOS susceptibility, whereas in stimulated group it showed strong associations with PCOS and its hormonal traits. Furthermore, CGCs demonstrated consistent global and CpG-DNA hypomethylation relative to PBLs, irrespective of normal or disease states. Conclusion: Our study revealed strong association of single hypomethylated CpG-site with PCOS. Identification and characterization of more such methyl-CpG signatures in repetitive elements in larger study populations would provide valuable epigenetic insights into PCOS.