Immune regulation of androgen signaling in PCOS

Polycystic ovary syndrome (PCOS) affects 10-15% of reproductive-age women and is the leading cause of anovulatory infertility. PCOS is characterized by hyperandrogenism and pathologically elevated luteinizing hormone (LH). All direct experimental evidence demonstrates that testosterone suppresses LH secretion, yet women with PCOS exhibit both elevated testosterone and increased LH. This paradox indicates fundamental disruption in androgen negative feedback mechanisms that remain unexplained. Without elucidating these mechanisms, curative therapies cannot be developed. PCOS pathophysiology has long been proposed to involve immune activation. Emerging evidence demonstrates that local tissue immune dysfunction drives disease pathogenesis. The cytokine tumor necrosis factor beta (TNFβ) is associated with PCOS symptom induction in mouse models and moderates the relationship between testosterone and phenotypic outcomes in women with PCOS. TNFβ induces androgen-independent androgen receptor (AR) translocation in gonadotropes, the source of LH, and alters expression of AR splice variants capable of androgen-independent action. The long-term goal of this proposal is to establish TNFβ-mediated immune-endocrine crosstalk as a therapeutic target for PCOS. This proposal tests the overarching hypothesis that immune dysregulation in PCOS generates local pituitary TNFβ signaling that disrupts normal testosterone-mediated negative feedback through AR splice variant expression. In Aim 1, the cell-specific mechanisms of TNFβ action on LH regulation will be determined using tissue flow cytometry, intracellular cytokine staining, and conditional knockout mice (T cell-specific TNFβ deletion, gonadotrope-specific TNFR1 deletion) in a letrozole-induced PCOS model. In Aim 2, the functional consequences of TNFβ-induced AR splice variants will be delineated using CRISPR knock-in stable cell lines, chromatin immunoprecipitation sequencing, RNA sequencing, and primary human pituitary cells to map variant-specific genomic binding and transcriptional programs driving LH hypersecretion. This innovative investigation combines conditional mouse genetics, single-cell approaches, and primary human tissue validation to reveal novel therapeutic targets including TNFβ neutralizing antibodies, TNFR1 antagonists, or AR splice variant-specific interventions that could restore normal reproductive function in PCOS.