Cell-resolved genetic risk reveals hyperandrogenism-associated immune and metabolic rewiring in the PMOS follicular microenvironment.

BACKGROUND: Polyendocrine metabolic ovarian syndrome (PMOS) is a leading cause of anovulatory infertility. Although follicular developmental arrest is a defining feature of PMOS, the cell-type-specific mechanisms linking genetic susceptibility, endocrine imbalance and follicular microenvironmental dysfunction remain incompletely understood, particularly across clinical subtypes.

METHODS: We performed single-cell RNA sequencing of preovulatory follicular-fluid cells from healthy controls and women with uncomplicated PMOS, hyperandrogenic PMOS and metformin-treated insulin-resistant PMOS. We integrated these data with GWAS-derived disease relevance scores and ovarian chromatin-accessibility annotations to map genetic risk to follicular cell populations and regulatory programmes. Ligand-receptor analyses were used to infer intercellular communication networks. Transcriptomic metabolic findings were further assessed by follicular-fluid metabolic and steroid-hormone measurements, DHT-treated granulosa-cell functional assays, and reanalysis of a DHEA-induced mouse PMOS-like ovarian dataset.

RESULTS: We generated a single-cell atlas of the PMOS follicular microenvironment comprising 220,983 high-quality cells. Granulosa cells showed the strongest enrichment of PMOS-related genetic risk, with regulatory-genetic support from ovarian snATAC-seq integration. Among granulosa cell states, a GC2_CYP19A1⁺ population was preferentially associated with hyperandrogenic PMOS and displayed a transcriptional programme characterized by steroidogenic remodelling coupled to reduced energy-metabolic activity. Biochemical profiling supported this endocrine-metabolic imbalance, showing increased follicular-fluid androstenedione (ASD) and estrone, reduced estradiol and E2/ASD ratio, decreased ATP, citrate and fumarate hydratase activity, and increased lactate and β-hydroxybutyrate. Prolonged DHT exposure impaired granulosa-cell mitochondrial respiration, ATP production and mitochondrial membrane potential. In parallel, PMOS subtypes showed immune remodelling and altered ligand-receptor signalling, with hyperandrogenic PMOS associated with increased inflammatory signalling and reduced homeostatic or tissue-repair pathways. Reanalysis of a DHEA-induced mouse ovarian dataset provided supportive evidence for androgen-associated steroidogenic pathway perturbation.

CONCLUSIONS: This study provides a cell-resolved framework linking PMOS genetic susceptibility to endocrine-metabolic and immune remodelling in the preovulatory follicular microenvironment. The identification of a hyperandrogen-associated GC2_CYP19A1⁺ state highlights a candidate cellular programme in which steroidogenic imbalance is coupled to impaired granulosa-cell bioenergetics. These findings provide a resource for future validation and may inform microenvironment-guided stratification of PMOS and the development of strategies aimed at restoring follicular function.