RNA m⁶A modification and its regulatory factors are altered in granulosa cells from women with PMOS.

PURPOSE: Polyendocrine metabolic ovarian syndrome (PMOS), an endocrine disorder with unknown aetiology is the leading cause of anovulatory infertility. N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, regulates multiple aspects of RNA metabolism and reproductive function. However, the contributions of m6A modification and its regulatory factors including microRNAs to the pathophysiology of PMOS remain poorly understood. We therefore investigated global m6A abundance, expression of key m6A regulatory genes, and selected experimentally validated m6A-associated microRNAs in granulosa cells (GCs) from women with PMOS. METHODS: GCs were collected from 23 women with PMOS and 23 age- and BMI- matched controls undergoing in vitro fertilization. Global m6A modification was quantified by ELISA, whereas the m6A writer, reader, eraser (WRE) protein-coding genes and selected microRNAs were assessed by RT-qPCR. RESULTS: The total m6A levels were significantly increased in GCs from women with PMOS. Transcript encoding the core writer complex (METTL3, METTL14, WTAP, RBM15, VIRMA) and reader proteins (YTHDF1, YTHDF3, YTHDC1) were upregulated, whereas transcripts encoding the eraser proteins (FTO, ALKBH5) were downregulated. Elevated m6A, METTL3, METTL14 and YTHDF1 levels positively correlated with androgen excess, anti-Müllerian hormone and reduced oocyte fertilization rates. Among the experimentally validated m6A-associated microRNAs, miR-20b, whose maturation is regulated by METTL3, was downregulated, whereas miR-607, a validated regulator of FTO, was upregulated in PMOS. CONCLUSION: Coordinated alterations in global m6A abundance, m6A regulatory genes and the selected microRNAs indicate widespread epitranscriptomic dysregulation in GCs from women with PMOS, providing a foundation for future mechanistic and therapeutic studies.