THBS1 Induces Dysfunction of Ovarian Granulosa Cells in Patients with Polycystic Ovary Syndrome by Activating the TGF-β/Smad Pathway

Objective: This study aims to investigate the role of thrombospondin-1 (THBS1) in polycystic ovary syndrome (PCOS) pathogenesis and its mechanism in regulating granulosa cell (GC) function. Methods: Follicular fluid and granulosa cells from 21 PCOS patients and 21 age-matched non-PCOS controls were analysed for THBS1 expression and clinical correlations. A dehydroepiandrosterone (DHEA)-induced PCOS rat model with adeno-associated virus serotype 9 (AAV9)-mediated THBS1 knockdown was used to assess phenotypic changes. The KGN human granulosa-like cell line was employed to evaluate THBS1 overexpression effects on proliferation, apoptosis, and steroidogenesis. Mechanistic studies included RNA sequencing with Gene Set Enrichment Analysis (GSEA), co-immunoprecipitation, molecular docking against the latent TGF-β1 crystal structure (PDB 9VJJ), molecular dynamics simulation, an active/total TGF-β1 ELISA, and pharmacological TGF-β receptor inhibition. Results: THBS1 was elevated in PCOS follicular fluid and granulosa cells and correlated positively with serum AMH and LH after Benjamini–Hochberg FDR correction. AAV9-mediated ovarian THBS1 knockdown (37.4% protein reduction, p = 0.006) ameliorated cystic morphology, restored estrous cyclicity, and normalised serum AMH/LH/T. In KGN cells, THBS1 overexpression suppressed proliferation, induced apoptosis and inflammatory cytokines, and dysregulated steroidogenic enzymes. Transcriptome analysis revealed upregulation of canonical TGF-β/Smad pathway components (SERPINE1, SMAD7, TGFB2, INHBA, CCN2, COL1A1/2). Molecular docking and 100-ns dynamics simulation supported a stable interaction between THBS1 and latent TGF-β1 (ΔG_TOTAL ≈ −120 kcal·mol⁻¹). Co-immunoprecipitation confirmed physical association in cells, and ELISA showed elevated TGF-β1 in PCOS follicular fluid and rat serum, both attenuated by THBS1 knockdown. Pharmacological TGF-β receptor inhibition with SB-431542 rescued THBS1-induced cellular dysfunction. Conclusions: THBS1 is associated with PCOS-related granulosa cell dysfunction through the TGF-β/Smad pathway and represents a candidate biomarker and exploratory therapeutic target that warrants validation in independent multicentre cohorts.