Polycystic ovarian syndrome (PCOS) is the most common gynecologic-endocrine disorder and leading cause of anovulatory infertility. Ovarian function is shaped by complex interaction of genetic, hormonal and environmental factors, which trigger epigenetic modifications. DNA methylation and miRNAs are critical epigenetic regulators of hormone synthesis and folliculogenesis. Beyond these, mRNA undergoes epitranscriptomic N6-methyladenosine (m6A) methylation, adding further complexity to the regulation of gene expression in PCOS. These epigenetic modifications have been independently associated with PCOS; however, their cross-layer interaction is sparsely investigated.
We investigated how DNA methylation influences miRNA regulation in dysregulated pathways associated with PCOS. Through in-silico reanalysis of seven GEO microarray datasets, we identified differentially expressed genes (DEGs) and curated differentially expressed miRNAs (DE-miRNAs) from studies focused on the follicular environment in PCOS. Functional enrichment analysis identified “Regulation of Actin Cytoskeleton” as the top disrupted pathway, and further network analysis revealed hub genes ARPC1B, ACTR2, RAC2, PIP5K1B, and IQGAP. In granulosa cells (GCs) of women with PCOS, we found significantly altered expression of hub genes and their regulating miRNAs: miR-196a-5p, miR-377-5p, and miR-424-5p. Further, using pyrosequencing, we found these miRNAs are regulated by differential DNA methylation. Disrupted cytoskeletal dynamics could impair steroidogenesis and cumulus expansion, contributing to aberrant folliculogenesis.
The interplay between m6A modification and miRNAs in PCOS is largely unexplored. We observed increased global m6A levels, with upregulation of “writers” like METTL3, METTL14, WTAP, VIRMA, downregulation of “erasers” such as FTO, ALKBH5 and dysregulation of readers (YTHDF1/2/3, YTHDC1) in GCs of PCOS. Notably, miR-20b and miR-607, which target METTL3 and FTO, showed differential expression, suggesting altered miRNA expressions regulate m6A modifications in PCOS. The strong correlations we observed between m6A levels, METTL3/FTO expression, and androgen indices indicate an androgen-m6A regulatory loop in PCOS.
Our study sheds light on the complex epi-regulatory network underlying ovarian dysfunction in PCOS.