Candidate Gene For Polycystic Ovary Syndrome Research Articles

Loci on chromosome 12q13.2 encompassing ERBB3, PA2G4 and RAB5B are associated with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenemia of ovarian theca cell origin. We report significant association of androgen production with 15 single nucleotide variants (SNVs) identified by exome sequencing of theca cells from women with PCOS and normal ovulatory women. Ten SNVs are located within a 150 kbp region on 12q13.2 which encompasses loci identified in PCOS genome-wide association studies (GWAS) and contains PCOS candidate genes ERBB3 and RAB5B. The region also contains PA2G4 which encodes a transcriptional corepressor of androgen receptor and androgen receptor-regulated genes. PA2G4 has not previously been recognized as related to PCOS in published GWAS studies. Two of the SNVs are predicted to have functional consequences (ERBB3 missense SNV, PA2G4 promoter SNV). PA2G4 interacts with the ERBB3 cytoplasmic domain containing the missense variant, suggesting a potential signaling pathway disruption that could lead to the PCOS ovarian phenotype. Single cell RNA sequencing of theca cells showed significantly less expression of PA2G4 after forskolin treatment in PCOS cells compared to normal cells (padj = 3.82E-30) and in cells heterozygous for the PA2G4 promoter SNV compared to those without the SNV (padj = 2.16E-11). This is consistent with a functional effect of the PA2G4 promoter SNV. No individual SNV was significantly associated with PCOS in an independent family cohort, but a haplotype with minor alleles of three SNVs was found preferentially in women with PCOS. These findings suggest a functional role for 12q13.2 variants in PCOS and implicate variants in ERBB3 and PA2G4 in the pathophysiology of PCOS.

Expression of PCOS candidate genes in bovine fetal and adult ovarian somatic cells.

Polycystic ovary syndrome (PCOS) is an endocrine metabolic disorder that appears to have a genetic predisposition and a fetal origin. The fetal ovary has two major somatic cell types shown previously to be of different cellular origins, different morphologies and to differentially express 15 genes. We isolated the somatic gonadal ridge epithelial-like (GREL) cells (n = 7) and ovarian fetal fibroblasts (n = 6) by clonal expansion. Using qRT-PCR, we compared the gene expression levels of PCOS candidate genes with previous data on the expression levels in whole fetal ovaries across gestation. We also compared these levels with those in bovine adult ovarian cells including fibroblasts (n = 4), granulosa cells (n = 5) and surface epithelial cells (n = 5). Adult cell types exhibited clear differences in the expression of most genes. In fetal ovarian cells, DENND1A and ERBB3 had significantly higher expression in GREL cells. HMGA2 and TGFB1I1 tended to have higher expression in fetal fibroblasts than GREL cells. Another 19 genes did not exhibit differences between GREL cells and fetal fibroblasts and FBN3, FSHB, LHCGR, FSHR and ZBTB16 were very lowly expressed in GREL cells and fibroblasts. The culture of fetal fibroblasts in EGF-containing medium resulted in lower expression of NEIL2, but higher expression of MAPRE1 compared to culture in the absence of EGF. Thus, the two fetal ovarian somatic cell types mostly lacked differential expression of PCOS candidate genes.

Candidate genes for polycystic ovary syndrome are regulated by TGFβ in the bovine foetal ovary.

STUDY QUESTIONCould changes in transforming growth factor β (TGFβ) signalling during foetal ovary development alter the expression of polycystic ovary syndrome (PCOS) candidate genes leading to a predisposition to PCOS?SUMMARY ANSWERTGFβ signalling molecules are dynamically expressed during foetal ovary development and TGFβ1 inhibits expression of the androgen receptor (AR) and 7 (INSR, C8H9orf3, RAD50, ERBB3, NEIL2, IRF1 and ZBTB16) of the 25 PCOS candidate genes in foetal ovarian fibroblasts in vitro, whilst increasing expression of the AR cofactor TGFβ-induced transcript 1 (TGFB1I1 or Hic5).WHAT IS KNOWN ALREADYThe ovarian stroma arises from the mesonephros during foetal ovary development. Changes in the morphology of the ovarian stroma are cardinal features of PCOS. The ovary is more fibrous and has more tunica and cortical and subcortical stroma. It is not known why this is and when this arises. PCOS has a foetal origin and perhaps ovarian stroma development is altered during foetal life to determine the formation of a polycystic ovary later in life. PCOS also has a genetic origin with 19 loci containing 25 PCOS candidate genes. In many adult tissues, TGFβ is known to stimulate fibroblast replication and collagen deposition in stroma, though it has the opposite effect in the non-scaring foetal tissues. Our previous studies showed that TGFβ signalling molecules [TGFβs and their receptors, latent TGFβ binding proteins (LTBPs) and fibrillins, which are extracellular matrix proteins that bind LTBPs] are expressed in foetal ovaries. Also, we previously showed that TGFβ1 inhibited expression of AR and 3 PCOS candidate genes (INSR, C8H9orf3 and RAD50) and stimulated expression of TGFB1I1 in cultured foetal ovarian fibroblasts.STUDY DESIGN, SIZE, DURATIONWe used Bos taurus for this study as we can ethically collect foetal ovaries from across the full 9-month gestational period. Foetal ovaries (62–276 days, n = 19) from across gestation were collected from pregnant B. taurus cows for RNA-sequencing (RNA-seq) analyses. Foetal ovaries from B. taurus cows were collected (160–198 days, n = 6) for culture of ovarian fibroblasts.PARTICIPANTS/MATERIALS, SETTING, METHODSRNA-seq transcriptome profiling was performed on foetal ovaries and the data on genes involved in TGFβ signalling were extracted. Cells were dispersed from foetal ovaries and fibroblasts cultured and treated with TGFβ1. The effects of TGFβ regulation on the remaining eight PCOS candidate genes not previously studied (ERBB3, MAPRE1, FDFT1, NEIL2, ARL14EP, PLGRKT, IRF1 and ZBTB16) were examined.MAIN RESULTS AND THE ROLE OF CHANCEMany TGFβ signalling molecules are expressed in the foetal ovary, and for most, their expression levels increased accross gestation (LTBP1/2/3/4, FBN1, TGFB2/3, TGFBR2/3 and TGFB1I1), while a few decreased (FBN3, TGFBR3L, TGFBI and TGFB1) and others remained relatively constant (TGFBRAP1, TGFBR1 and FBN2). TGFβ1 significantly decreased expression of PCOS candidate genes ERBB3, NEIL2, IRF1 and ZBTB16 in cultured foetal ovarian fibroblasts.LARGE SCALE DATAThe FASTQ files, normalized data and experimental information have been deposited in the Gene Expression Omnibus (GEO) accessible by accession number GSE178450.LIMITATIONS, REASONS FOR CAUTIONRegulation of PCOS candidate genes by TGFβ was carried out in vitro and further studies in vivo are required. This study was carried out in bovine where foetal ovaries from across all of the 9-month gestational period were available, unlike in the human where it is not ethically possible to obtain ovaries from the second half of gestation.WIDER IMPLICATIONS OF THE FINDINGSFrom our current and previous results we speculate that inhibition of TGFβ signalling in the foetal ovary is likely to (i) increase androgen sensitivity by enhancing expression of AR, (ii) increase stromal activity by stimulating expression of COL1A1 and COL3A1 and (iii) increase the expression of 7 of the 25 PCOS candidate genes. Thus inhibition of TGFβ signalling could be part of the aetiology of PCOS or at least the aetiology of polycystic ovaries.STUDY FUNDING/COMPETING INTEREST(S)Funding was received from Adelaide University China Fee Scholarship (M.L.), Australian Research Training Program (R.A.) and the Faculty of Health and Medical Science Divisional Scholarship (R.A.), Adelaide Graduate Research Scholarships (R.A. and N.A.B.), Australia Awards Scholarship (M.D.H.), Robinson Research Institute Career Development Fellowship (K.H.) and Building On Ideas Grant (K.H.), National Health and Medical Research Council of Australia Centre for Research Excellence in the Evaluation, Management and Health Care Needs of Polycystic Ovary Syndrome (N.A.B., M.D.H. and R.J.R.; GTN1078444) and the Centre for Research Excellence on Women’s Health in Reproductive life (R.A., R.J.R. and K.H.; GTN1171592) and the UK Medical Research Council (R.A.A.; grant no. G1100357). The funders did not play any role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors of this manuscript have nothing to declare and no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Analysis of Upstream Regulators, Networks, and Pathways Associated With the Expression Patterns of Polycystic Ovary Syndrome Candidate Genes During Fetal Ovary Development.

Polycystic Ovary Syndrome (PCOS) is a multifactorial syndrome with reproductive, endocrine, and metabolic symptoms, affecting about 10% women of reproductive age. Pathogenesis of the syndrome is poorly understood with genetic and fetal origins being the focus of the conundrum. Genetic predisposition of PCOS has been confirmed by candidate gene studies and Genome-Wide Association Studies (GWAS). Recently, the expression of PCOS candidate genes across gestation has been studied in human and bovine fetal ovaries. The current study sought to identify potential upstream regulators and mechanisms associated with PCOS candidate genes. Using RNA sequencing data of bovine fetal ovaries (62–276 days, n = 19), expression of PCOS candidate genes across gestation was analysed using Partek Flow. A supervised heatmap of the expression data of all 24,889 genes across gestation was generated. Most of the PCOS genes fell into one of four clusters according to their expression patterns. Some genes correlated negatively (early genes; C8H9orf3, TOX3, FBN3, GATA4, HMGA2, and DENND1A) and others positively (late genes; FDFT1, LHCGR, AMH, FSHR, ZBTB16, and PLGRKT) with gestational age. Pathways associated with PCOS candidate genes and genes co-expressed with them were determined using Ingenuity pathway analysis (IPA) software as well as DAVID Bioinformatics Resources for KEGG pathway analysis and Gene Ontology databases. Genes expressed in the early cluster were mainly involved in mitochondrial function and oxidative phosphorylation and their upstream regulators included PTEN, ESRRG/A and MYC. Genes in the late cluster were involved in stromal expansion, cholesterol biosynthesis and steroidogenesis and their upstream regulators included TGFB1/2/3, TNF, ERBB2/3, VEGF, INSIG1, POR, and IL25. These findings provide insight into ovarian development of relevance to the origins of PCOS, and suggest that multiple aetiological pathways might exist for the development of PCOS.

Molecular Features of Polycystic Ovary Syndrome Revealed by Transcriptome Analysis of Oocytes and Cumulus Cells.

Polycystic ovary syndrome (PCOS) is typically characterized by a polycystic ovarian morphology, hyperandrogenism, ovulatory dysfunction, and infertility. Furthermore, PCOS patients undergoing ovarian stimulation have more oocytes; however, the poor quality of oocytes leads to lower fertilization and implantation rates, decreased pregnancy rates, and increased miscarriage rates. The complex molecular mechanisms underlying PCOS and the poor quality of oocytes remain to be elucidated. We obtained matched oocytes and cumulus cells (CCs) from PCOS patients, compared them with age-matched controls, and performed RNA sequencing analysis to explore the transcriptional characteristics of their oocytes and CCs. Moreover, we validated our newly confirmed candidate genes for PCOS by immunofluorescence. Unsupervised clustering analysis showed that the overall global gene expression patterns and transposable element (TE) expression profiles of PCOS patients tightly clustered together, clearly distinct from those of controls. Abnormalities in functionally important pathways are found in PCOS oocytes. Notably, genes involved in microtubule processes, TUBB8 and TUBA1C, are overexpressed in PCOS oocytes. The metabolic and oxidative phosphorylation pathways are also dysregulated in both oocytes and CCs from PCOS patients. Moreover, in oocytes, differentially expressed TEs are not uniformly dispersed in human chromosomes. Endogenous retrovirus 1 (ERV1) elements located on chromosomes 2, 3, 4, and 5 are rather highly upregulated. Interestingly, these correlate with the most highly expressed protein-coding genes, including tubulin-associated genes TUBA1C, TUBB8P8, and TUBB8, linking the ERV1 elements to the occurrence of PCOS. Our comprehensive analysis of gene expression in oocytes and CCs, including TE expression, revealed the specific molecular features of PCOS. The aberrantly elevated expression of TUBB8 and TUBA1C and ERV1 provides additional markers for PCOS and may contribute to the compromised oocyte developmental competence in PCOS patients. Our findings may also have implications for treatment strategies to improve oocyte maturation and the pregnancy outcomes for women with PCOS.

Understanding Polycystic Ovary Syndrome: A Multifactorial Endocrine Disorder

Polycystic Ovary Syndrome (PCOS) is a common endocrine disorder affecting a significant population of women of reproductive age group worldwide. Due to the complex pathophysiology and overlapping symptoms, this disorder is often difficult to diagnose. Genome-wide association studies have identified several new risk loci and candidate genes for PCOS. However, currently, there are no standard genetic markers for PCOS susceptibility testing owing to the inconsistent findings. Despite the advent of the genomic era, the challenge to identify and pinpoint the heritable genetic basis of PCOS still exists. This mini-review explores the basic definition and phenotypes of PCOS, the different criteria for the diagnosis, the incidence, gestational complications associated with it, the basis of genetic heritability, and the influence of various gene polymorphisms. Also, this review briefly summarises the reports of genome-wide association studies conducted to identify candidate genetic markers to aid in understanding the complex pathophysiology of PCOS.

Could perturbed fetal development of the ovary contribute to the development of polycystic ovary syndrome in later life?

Polycystic ovary syndrome (PCOS) affects around 10% of young women, with adverse consequences on fertility and cardiometabolic outcomes. PCOS appears to result from a genetic predisposition interacting with developmental events during fetal or perinatal life. We hypothesised that PCOS candidate genes might be expressed in the fetal ovary when the stroma develops; mechanistically linking the genetics, fetal origins and adult ovarian phenotype of PCOS. In bovine fetal ovaries (n = 37) of 18 PCOS candidate genes only SUMO1P1 was not expressed. Three patterns of expression were observed: early gestation (FBN3, GATA4, HMGA2, TOX3, DENND1A, LHCGR and FSHB), late gestation (INSR, FSHR, and LHCGR) and throughout gestation (THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX and KRR1). A splice variant of FSHB exon 3 was also detected early in the bovine ovaries, but exon 2 was not detected. Three other genes, likely to be related to the PCOS aetiology (AMH, AR and TGFB1I1), were also expressed late in gestation. Significantly within each of the three gene groups, the mRNA levels of many genes were highly correlated with each other, despite, in some instances, being expressed in different cell types. TGFβ is a well-known stimulator of stromal cell replication and collagen synthesis and TGFβ treatment of cultured fetal ovarian stromal cells inhibited the expression of INSR, AR, C8H9orf3 and RAD50 and stimulated the expression of TGFB1I1. In human ovaries (n = 15, < 150 days gestation) many of the same genes as in bovine (FBN3, GATA4, HMGA2, FSHR, DENND1A and LHCGR but not TOX3 or FSHB) were expressed and correlated with each other. With so many relationships between PCOS candidate genes during development of the fetal ovary, including TGFβ and androgen signalling, we suggest that future studies should determine if perturbations of these genes in the fetal ovary can lead to PCOS in later life.

Naturally Occurring and Experimentally Induced Rhesus Macaque Models for Polycystic Ovary Syndrome: Translational Gateways to Clinical Application.

Indian rhesus macaque nonhuman primate models for polycystic ovary syndrome (PCOS) implicate both female hyperandrogenism and developmental molecular origins as core components of PCOS etiopathogenesis. Establishing and exploiting macaque models for translational impact into the clinic, however, has required multi-year, integrated basic-clinical science collaborations. Paradigm shifting insight has accrued from such concerted investment, leading to novel mechanistic understanding of PCOS, including hyperandrogenic fetal and peripubertal origins, epigenetic programming, altered neural function, defective oocytes and embryos, adipogenic constraint enhancing progression to insulin resistance, pancreatic decompensation and type 2 diabetes, together with placental compromise, all contributing to transgenerational transmission of traits likely to manifest in adult PCOS phenotypes. Our recent demonstration of PCOS-related traits in naturally hyperandrogenic (High T) female macaques additionally creates opportunities to employ whole genome sequencing to enable exploration of gene variants within human PCOS candidate genes contributing to PCOS-related traits in macaque models. This review will therefore consider Indian macaque model contributions to various aspects of PCOS-related pathophysiology, as well as the benefits of using macaque models with compellingly close homologies to the human genome, phenotype, development and aging.

Colocalization of Polycystic Ovary Syndrome Candidate Gene Products in Theca Cells Suggests Novel Signaling Pathways.

Genome-wide association studies identified loci associated with polycystic ovary syndrome (PCOS), including those near the LH receptor gene (LHCGR), a clathrin-binding protein (DENND1A) that functions as a guanine nucleotide exchange factor, and the gene encoding RAB5B, a GTPase involved in vesicular trafficking. We proposed that these three PCOS loci could be assembled into a functional network that contributes to altered gene expression in theca cells, resulting in increased androgen synthesis. The functional significance of this network was supported by our discovery that a truncated protein splice variant of the DENND1A gene, termed DENND1A.V2, is elevated in PCOS theca cells, and that forced expression of DENND1A.V2 in normal theca cells increased CYP11A1 and CYP17A1 expression and androgen synthesis, a hallmark of PCOS. In this study, we demonstrate the colocalization of LHCGR, DENND1AV.2, and RAB5B proteins in various cellular compartments in normal and PCOS theca cells by immunofluorescence. Human chorionic gonadotropin and forskolin stimulation was shown to affect the cytoplasmic distribution of LHCGR, DENND1A.V2, and RAB5B. DENND1A.V2 accumulated in the nuclei of the theca cells. Moreover, PCOS theca cells, following forskolin treatment, had a significantly greater relative abundance of nuclear DENND1A.V2. RAB5B also accumulated in the nuclei of PCOS theca cells treated with forskolin. In contrast, LHCGR did not enter the nucleus. This cytological evidence, and the previously reported increase in androgen biosynthesis with forced expression of DENND1A.V2 in normal theca cells, raises the possibility that DENND1A.V2 and RAB5B participate in increasing transcription of genes involved in androgen synthesis.

Hypothalamic DNA methylation in rats with dihydrotestosterone-induced polycystic ovary syndrome: effects of low-frequency electro-acupuncture.

What is the central question of this study? What is the role of hypothalamic DNA methylation in the development of polycystic ovary syndrome (PCOS) and the response to electro-acupuncture treatment. What is the main finding and its importance? Global DNA methylation and expression of DNA methyltransferases (DNMTs) were increased in PCOS-like rats, and electro-acupuncture (EA) decreased global DNA methylation and DNMT3b expression. Pyrosequencing showed that the DNA methylation of some PCOS candidate genes was changed in the PCOS and PCOS+EA groups, suggesting that hypothalamic DNA methylation plays an important role in the development of PCOS and in mediating the effects of electro-acupuncture treatment. Polycystic ovary syndrome (PCOS) is a common reproductive and endocrine disease of unknown aetiology. Recently, epigenetic studies focusing on DNA methylation in PCOS have received much attention, but the mechanisms are still unclear. In the present study, we used the 5α-dihydrotestosterone-induced PCOS-like rat model and treated the rats with electro-acupuncture (EA). Rats were randomly divided into four groups- controls, diet-induced obesity, PCOS and PCOS+EA. We examined the reproductive, metabolic and behavioural phenotypes, validated the effect of EA, and explored the role of hypothalamic DNA methylation by analysing the methylation of global DNA and selected candidate genes. The PCOS rats presented with reproductive dysfunctions such as lack of regular oestrous cyclicity, metabolic disorders such as increased body weight and insulin resistance, and depression and anxiety-like behaviours. EA improved the reproductive functions, decreased body weight and improved experimental depressive behaviour. Furthermore, global DNA methylation and the expression of DNA methyltransferases (DNMTs) were increased in PCOS rats compared to the control group, and EA decreased the global DNA methylation and the expression of DNMT3b. In addition, pyrosequencing showed that the DNA methylation of certain CpG sites in targeted genes (Plcg1, Camk2b, Esr2 and Pgr) was increased in the PCOS group, but the DNA methylation of Camk2b and Ar was decreased after EA treatment. These results indicate that hypothalamic DNA methylation might be correlated with the development of PCOS and that EA has an effect on hypothalamic DNA methylation in PCOS rats.

An update of genetic basis of PCOS pathogenesis.

Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder that affects 5-20% of reproductive age women. PCOS clinical symptoms include hirsutism, menstrual dysfunction, infertility, obesity and metabolic syndrome. There is a wide heterogeneity in clinical manifestations and metabolic complications. The pathogenesis of PCOS is not fully elucidated, but four aspects seem to contribute to the syndrome to different degrees: increased ovarian and/or adrenal androgen secretion, partial folliculogenesis arrest, insulin resistance and neuroendocrine axis dysfunction. A definitive etiology remains to be elucidated, but PCOS has a strong heritable component indicated by familial clustering and twin studies. Genome Wide Association Studies (GWAS) have identified several new risk loci and candidate genes for PCOS. Despite these findings, the association studies have explained less than 10% of heritability. Therefore, we could speculate that different phenotypes and subphenotypes are caused by rare private genetic variants. Modern genetic studies, such as whole exome and genome sequencing, will help to clarify the contribution of these rare genetic variants on different PCOS phenotypes. Arch Endocrinol Metab. 2018;62(3):352-61.

Alternative splicing of DENND1A, a PCOS candidate gene, generates variant 2

Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20.

Common Variants in the Sex Hormone-Binding Globulin (SHBG) Gene Influence SHBG Levels in Women with Polycystic Ovary Syndrome

Background: Decreased sex hormone-binding globulin (SHBG) levels were associated with polycystic ovary syndrome (PCOS). SHBG polymorphisms associated with reduced SHBG production were tested for their association with PCOS, but with inconclusive results. We tested whether altered SHBG levels and SHBG variants were associated with PCOS. Methods: The study subjects included 242 women with PCOS and 238 control women. SHBG genotyping was done by real-time PCR. Results: Higher minor allele frequency of rs13894, rs858521 and rs727428 was seen in PCOS cases, and significant differences in rs858521 and rs727428 genotypes distribution were seen between PCOS cases and controls. Multivariate regression analysis confirmed the association of only rs727428 with PCOS. Though it was not statistically significant, serum SHBG levels were reduced according to rs727428 genotypes in PCOS cases than in controls. Carriage of rs727428 minor allele was associated with significant increases in free/bioactive testosterone in PCOS cases. Seven-locus (rs9898876-rs13894-rs858521-rs1799941-rs6257-rs6259-rs727428) haploview analysis showed increased frequency of GCCGTGA, GTCGTGA and GTCATGG, and reduced frequency of GTCGTGG haplotypes in PCOS cases than in controls, thus conferring disease susceptibility and protective nature to these haplotypes, respectively. Conclusion: Specific SHBG variants affecting serum SHBG levels and SHBG haplotypes are associated with PCOS, suggesting the role for SHBG as PCOS candidate gene.

Regulation of human (adrenal) androgen biosynthesis—New insights from novel throughput technology studies

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38α (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.

Common Variant rs9939609 in Gene FTO Confers Risk to Polycystic Ovary Syndrome

BackgroundFat mass and obesity-associated gene (FTO) has been associated with obesity, especially the common variant rs9939609. Polycystic ovary syndrome (PCOS) is a complex endocrine-metabolic disorder and over 50% of patients are overweight/obese. Thus FTO is a potential candidate gene for PCOS but their relationship is confusing and remains to be clarified in different population with a large sample size. MethodThis study was performed adopting a two-stage design by genotyping SNP rs9939609. The first set comprise of 741 PCOS and 704 control subjects, with data from our previous GWAS. The second phase of replication study was performed among another independent group of 2858 PCOS and 2358 control subjects using TaqMan-MGB probe assay. All subjects are from Han Chinese. ResultsThe less meaningful association of FTO rs9939609 and PCOS discovered in GWAS (P = 2.47E-03), was further confirmed in the replication study (P = 1.86E-09). Using meta-analysis, the P-meta value has reached 6.89E-12, over-exceeding the genome-wide association level of 5.00E-8. By combination, the P value was 1.26E-11 and after BMI adjustment it remained significant(P = 1.82E-06). To further elucidate whether this association is resulted from obesity or PCOS per se, the samples were divided into two groups–obese and non-obese PCOS, and the results were still positive in obese group (P obese = 5.81E-05, OR = 1.55), as well as in non-obese PCOS group (P non-obese = 7.06E-04, OR = 1.28). ConclusionVariant rs9939609 in FTO is associated with PCOS in Chinese women, not only in obese PCOS subjects, but also in non-obese cases.

Quantitative analysis of follistatin (FST) promoter methylation in peripheral blood of patients with polycystic ovary syndrome

Epigenetic mechanisms may contribute to polycystic ovary syndrome (PCOS). To date, however, no studies have associated CpG methylation levels of any candidate gene with PCOS susceptibility. Follistatin (FST), an activin-binding protein, is expressed in numerous tissues and is shown to have linkage with PCOS. However, results from case–control association analyses between this gene and PCOS are inconsistent. Thus, this study investigated possible association of methylation levels in the promoter and 5′-untranscribed region (UTR) of the FST gene with PCOS incidence in peripheral blood leukocytes and endometrial tissue. Using mass array quantitative methylation analysis, first the 5′-UTR methylation in FST was analysed in 130 PCOS patients and 120 controls. The methylation level of the FST gene was further studied in endometrium from 24 controls and 24 PCOS patients. This study demonstrates that methylation levels of CpG sites in the FST promoter and 5′-UTR are not associated with PCOS. Nonetheless, this was the first study to quantitatively evaluate the methylation levels of a candidate gene in association with PCOS. Further studies should be performed to examine methylation in other candidate genes. Understanding the epigenetic mechanisms involved in PCOS may yield new insights into the pathophysiology of the disorder.Animal models demonstrate that epigenetic reprogramming may contribute to polycystic ovary syndrome (PCOS). To date, however, no studies have associated CpG methylation levels of any candidate gene with PCOS susceptibility. Follistatin (FST), an activin-binding protein, is expressed in numerous tissues and is a PCOS candidate gene. However, results from association analyses between this gene and PCOS are inconsistent. Thus, we investigated possible association of methylation levels in the promoter and 5′-UTR of the FST gene with PCOS incidence in peripheral blood leukocytes and endometrial tissue. Using mass array quantitative methylation analysis, we firstly analysed 5′-UTR methylation in 40 PCOS patients and 40 controls. We then validated results in a second sample consisting of 90 PCOS patients and 80 controls. The methylation level of the FST gene was further studied in endometrium from 24 controls and 24 PCOS patients. Finally, we quantitatively analysed FST expression in the endometrium using real-time PCR. Our study demonstrated that methylation levels of CpG sites in the FST promoter and 5′-UTR are not associated with PCOS. Nonetheless, as far as is known, this is the first study to quantitatively evaluate the methylation levels of a candidate gene in association with PCOS. Further studies should be performed to examine methylation in other candidate genes. Understanding the epigenetic mechanisms involved in PCOS may yield new insights into the pathophysiology of the disorder.

Common variants in the sex hormone-binding globulin gene (SHBG) and polycystic ovary syndrome (PCOS) in Mediterranean women

Is there an association between polycystic ovary syndrome (PCOS) and the sex hormone-binding globulin (SHBG) rs1799941, rs6257, rs6259 and rs727428 variants in a large series of Mediterranean women? The rs727428 and rs6259 variants are associated with PCOS in Mediterranean women. The level of SHBG, the primary plasma transport protein for sex steroids, which regulates the bioavailability of these hormones to target tissues, is reduced in patients with PCOS. Single-nucleotide polymorphisms in the SHBG gene influence circulating SHBG levels in American patients with PCOS and may predict the development of type 2 diabetes. This was a genetic case-control association study including 1004 premenopausal Mediterranean women. In an Academic setting, we genotyped a clinical cohort consisting of 281 patients with PCOS and 142 women without any evidence of androgen excess, and a population-based cohort comprised of 581 unselected female blood donors from Spain and Italy. The latter included 31 patients with PCOS and 550 controls, of whom 298 had no evidence of any androgen excess disorder and were considered hyper-normal controls. Mutant alleles of the rs727428 variant were more frequent in patients with PCOS compared with controls and with hyper-normal controls. This association was independent of obesity. Carrying mutant alleles of rs727428 was found to be associated with a 1.29 odds ratio (OR) for PCOS, whereas carrying mutant alleles of rs6259 associated with a 0.68 OR for PCOS. The rs1799941 and rs6257 variants were not associated with PCOS. None of the SHBG variants influenced serum SHBG concentrations. The associations found here were relatively weak and, arising from a case-control study, do not necessarily indicate a causative role of the SHBG variants in the development of PCOS. Also, we studied different patients and controls from different sources, making some of the interpretations difficult. Finally, the rs1799941 variant was not in Hardy-Weinberg equilibrium in the small group of patients with PCOS recruited from the general population, yet this variant was not associated with PCOS. SHBG variants that influenced circulating SHBG levels in American patients with PCOS are also associated with this syndrome in Mediterranean women, pointing to SHBG as a candidate gene for PCOS. This study was supported by grants PI080944 and PI110357 from Instituto de Investigación Carlos III, Spanish Ministry of Economy and Competitiveness. CIBERDEM is also an initiative of Instituto de Investigación Carlos III. The Authors have no competing interests to declare.

Understanding polycystic ovarian syndrome pathogenesis: an updated of its genetic aspects.

Polycystic ovary syndrome (PCOS) is the most frequent cause of female infertility. It is also characterized by metabolic defects that raise the risk for cardiovascular disease. Despite the progress in the definition of the clinical aspects of the syndrome, only very few definite data are available about the ethiopathogenetic mechanisms that subtend PCOS. It is likely that the PCOS phenotype derives from the interaction between environmental and genetic factors. While environmental factors have easily been investigated, the individuation of the genetic factors seem to be more complex. Indeed, PCOS appears to be inherited as a complex, polygenic trait. Several family studies have been conducted with the aim to clarify the genetic aspects of PCOS, but their findings are often conflicting and not conclusive.Moreover, it is difficult to establish with certainty which genes are involved and their effective role in the development of the syndrome because in PCOS, genetic analysis is hampered by low fecundity, lack of a male phenotype, absence of an animal model, and dissimilarity of the diagnostic criteria used to select the patients. Since multiple biochemical pathways are implicated in PCOS pathogenesis, genes of steroid hormone metabolism, gonadotropin release and action, insulin secretion and action, adipose tissue metabolism and others have been investigated. Nevertheless, none of them seems to play a key role in the ethiopathogenesis of PCOS. This article reviews the large body of literature generated to support the presence of genetic abnormalities in PCOS women by taking in consideration the most important studies regarding PCOS candidate genes.

Family-Based Analysis of Candidate Genes for Polycystic Ovary Syndrome

Studies on familial aggregation of hyperandrogenemia in polycystic ovary syndrome (PCOS) have provided evidence consistent with a genetic contribution of hyperandrogenemia to risk of the disease. Numerous association studies have investigated possible PCOS candidate genes but there is no consensus on any proposed candidate gene. Few of the positive reported PCOS genetic associations have been confirmed or followed up in replication studies. Previous studies have provided strong evidence that D19S884, a polymorphic marker in fibrillin 3, or a nearby determinant, are associated with risk of PCOS. This strong association has been replicated in 3 independent association studies. The aim of this study was to replicate previous findings showing a strong association between single nucleotide polymorphisms (SNPs) in the region of D19S884 and PCOS and to confirm and extend published data on other putative candidate genes. The transmission disequilibrium test (TDT) was used to study the association and linkage of putative candidate genes with PCOS. Overexpression of a gene marker in TDT testing indicated positive association with PCOS. SNP markers were genotyped in 453 families having a proband with PCOS; the total number was 502, including 49 affected sisters. The primary study outcome was transmission frequency of SNP alleles. A 6-SNP haplotype allele (designated allele-C) identified within a 6.7 kb region including D19S884 was significantly associated with PCOS. Allele-C was overtransmitted to offspring with PCOS: allele-C TDTχ2 = 10.0 (P = 0.0016). Combination of allele-C with D19S884-allele 8 was also overtransmitted: allele-C/A8 TDTχ2 = 7.6 (P = 0.006). Of the other 26 putative candidate genes tested using the TDT, 4 SNPs had a nominally significant association (ACVR2A, POMC, FEM1B, and SGTA). After correction for multiple testing, only one SNP in POMC (rs12473543) remained significant (χ2 = 9.1; Pcorrected = 0.036). These findings confirm other studies showing that the D19S884 region is associated with risk of PCOS and suggest that the putative candidate gene POMC should be investigated.

A variant in the fibrillin-3 gene is associated with TGF-β and inhibin B levels in women with polycystic ovary syndrome

In an attempt to evaluate the association between allele 8 (A8) of D19S884 in the fibrillin-3 gene and circulating transforming growth factor (TGF) β and inhibin levels in women with polycystic ovary syndrome (PCOS), we studied 120 similarly aged women from families with PCOS and compared 40 women with PCOS who did not have A8 (A8- PCOS) with 40 women with PCOS who had A8 (A8+ PCOS) and 40 normally menstruating women who did not have either PCOS or A8 (A8- Non-PCOS). A8- PCOS is associated with higher levels of TGF-β1 compared with A8+ PCOS or A8- Non-PCOS, similar levels of TGF-β2 compared with A8+ PCOS but lower levels of TGF-β2 compared with A8- Non-PCOS, and lower levels of inhibin B and aldosterone compared with A8+ PCOS.