Targeted genome-wide DNA methylation profiling of ovarian granulosa cells from women with PCOS

Polycystic ovary syndrome (PCOS) is a complex endocrinopathy of obscure pathophysiologic origins, globally affecting 6-15% women of childbearing age. Emerging evidence on repercussions of environmental insults and changing lifestyles on fecundity and reproductive health have necessitated the study of tissue-specific epigenetic alterations in PCOS development. In semblance to follicular and oocyte defects observed in PCOS ovaries, targeted bisulfite sequencing was performed to generate the methylome signatures of ovarian granulosa cells (GCs) obtained from age-BMI matched women with PCOS (n=3) and healthy, regularly menstruating controls (n=3) using next generation sequencing approach. Paired end sequencing of samples was carried out on Illumina HiSeq 2500 ® platform and data were analyzed using the Bismark tool. Methylation levels of a few selected genes relevant to ovarian function were further validated in GCs obtained from 10 controls and 10 women with PCOS by pyrosequencing.  Relative transcript levels of these genes were assessed by q-RT PCR using Taqman assays. In the methylome analysis, a total of 6486 CpG sites representing 3840 genes associated with pathways such as Wnt signaling, G-protein receptor signaling, angiogenesis, chemokine and cytokine mediated inflammation and integrin signaling showed differential methylation in PCOS. Of these, a total of 2977 CpG sites representing 2063 genes were identified as hypomethylated while 3509 CpG sites in 1777 genes were found to be hypermethylated. Additionally, differential methylation was also noted in several non-coding RNAs regulating vital ovarian functions and which are reported to be dysregulated in PCOS. This data provides compelling evidence in support of epigenetic alterations as etiopathogenic factors associated with ovarian dysfunction in PCOS

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

Abstract 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