Hyperandrogenism in polycystic ovary syndrome augments Estrogen synthesis through AR-FOXL2-mediated activation of the aromatase gene in granulosa cells.

backgroundPolycystic ovary syndrome (PCOS) is a complex disorder characterized by various reproductive, metabolic, and endocrine abnormalities. Hyperandrogenism is a key feature of PCOS that significantly impacts ovarian function. However, its effects on granulosa cells (GCs) function for estrogen production in PCOS remains limited.

methodsMouse PCOS-like models were used to investigate the effects of androgen treatments on androgen receptor (AR) downstream gene expression in preantral follicles and primary GCs by qPCR, immunohistochemistry. The expression pattern of AR and FOXL2 was analyzed by single-cell RNA-sequencing analysis, immunohistochemical, and immunofluorescence staining. The AR-FOXL2 interaction was characterized using molecular docking and immunoprecipitation. The functional regulation of the CYP19A1 promoter by AR-FOXL2 was further analyzed using chromatin immunoprecipitation and dual-luciferase reporter assay. Effects of AR knockdown and inhibitors on estrogen secretion were evaluated in cultured follicles and GCs. Clinical specimens from PCOS patients with hyperandrogenism were analyzed for estrone and estradiol levels in follicular fluid and gene expression in GCs.

resultsSingle-cell RNA sequencing revealed similar expression patterns of AR and FOXL2 across ovarian cell populations. Dihydrotestosterone increased AR protein expression, particularly in preantral follicles, and upregulated both AR and FOXL2 target genes. CYP19A1 gene that encodes aromatase was significantly elevated in androgens-treated ovaries, follicles, and GCs. Immunofluorescence and co-immunoprecipitation demonstrated that androgen treatment promotes AR-FOXL2 complex formation, enhancing their binding to the CYP19A1 promoter to directly regulate aromatase transcription in GCs. ChIP-PCR confirmed increased AR binding to three AREs in the promoter II region of CYP19A1, with FOXL2 overexpression further enhancing this binding. Elevated estrogen secretion was observed in GCs and cultured follicles, which was attenuated by AR knockdown or inhibition. Clinical samples showed increased estrone, estradiol, and AR, CYP19A1, and FST mRNA expression, with CYP19A1 positively correlating with AR, FOXL2, and estrogen levels.

conclusionsHyperandrogenism in PCOS augments estrogen synthesis in GCs by enhancing AR-FOXL2 interactions, which activate CYP19A1 gene transcription and thereby increase aromatase expression. This androgen-driven mechanism leads to elevated estrogen levels, offering new insights into the complex pathophysiology of PCOS and strengthening the rationale for targeting the AR-FOXL2-aromatase axis as a therapeutic strategy for ovulation induction in PCOS patients. CLINICAL TRIAL NUMBER: Not applicable.