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. Electronic supplementary material The online version of this article (10.1186/s13148-019-0657-6) contains supplementary material, which is available to authorized users.

D-Chiro-Inositol Treatment Affects Oocyte and Embryo Quality and Improves Glucose Intolerance in Both Aged Mice and Mouse Models of Polycystic Ovarian Syndrome

“…We were unable to obtain sufficient numbers of blastocysts for mRNA expression analysis, but COCs treated with DCI in this TP model showed higher expression of Gdf9 and Tnf, and lower expression of Akr1c3 and Hapln1. In agreement with our results, transcripts of AKR1C3 and HAPLN1 have been found upregulated, while Tnf was downregulated in the granulosa cells of women with PCOS [ 49 ]. The pro-inflammatory cytokine Tnf suppresses FSH-induced Lhcgr promoter activation and could be a factor contributing to hyperandrogenemia.…”

“…DCI has been shown to have insulin-like bioactivity and to reduce meal-induced hyperglycemia [ 48 ]. Moreover, DCI has been described to decrease the expression of Akr1c3 , a gene found upregulated in the granulosa cells of women with PCOS [ 49 ]. ADR1C3 is a steroidogenic enzyme that converts androstenedione (A4) into biologically active testosterone in non-testicular tissues [ 50 ].…”

“…ADR1C3 is a steroidogenic enzyme that converts androstenedione (A4) into biologically active testosterone in non-testicular tissues [ 50 ]. Moreover, AKR1C3 also acts as a prostaglandin F synthase to facilitate luteolysis [ 50 ], and luteal insufficiency and premature luteolysis are frequent in PCOS [ 49 ]. Interestingly, a relationship has been detected between Sirt3 and Sod2 expression and AKT phosphorylation, protecting oocytes against oxidative stress in diabetic mice [ 35 ].…”

“…The pro-inflammatory cytokine Tnf suppresses FSH-induced Lhcgr promoter activation and could be a factor contributing to hyperandrogenemia. Moreover, optimal TNF levels have been reported to confer a protective function in the maintenance of bovine granulosa cells and oocytes and to facilitate ovulation [ 49 , 59 ], while lowered TNF in the granulosa cells of women with PCOS may hamper COC expansion, compromising ovulation [ 49 ]. AKR1C3 is a steroidogenic enzyme that converts androstenedione into testosterone in non-testicular tissues, and is expressed by the granulosa cells of periovulatory follicles [ 60 ].…”

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

“…In considering developmental origins for PCOS, maternal-fetal environmental modification of the fetal female epigenome contributes to its transgenerational transmission [52][53][54] and can provide an additional mechanism beyond inheritance of gene variants to PCOS-like trait heritability. Amniotic fluid from daughters of women with PCOS exhibit male-similar T levels during mid-gestation, exceeding levels in mid-gestation daughters of women without PCOS [55].…”