Unraveling the Toxicity and Mechanisms of PFAS in Female Diminished Ovarian Reserve: Novel Insights from Integrated Network Toxicology, Machine Learning, Molecul ar Dynamics Simulations, and In Vitro Experimental Validation

Per- and polyfluoroalkyl substances (PFAS) are a class of anthropogenic organic compounds ubiquitous in the environment. Growing evidence has underscored these associated health risks. Diminished ovarian reserve (DOR), which is characterized by a decline in oocyte quantity and quality, is a significant cause of female infertility. However, the role of PFAS in the pathogenesis of DOR remains unclear. This study integrated network toxicology, machine learning, molecular docking, and molecular dynamics (MD) simulations to investigate the mechanisms of PFAS on DOR, with subsequent validation using KGN cells. Multi-database screening identified 35 candidate targets. Gene Ontology and KEGG enrichment analyses revealed that PFAS perturb pathways governing cell proliferation and differentiation, immune-inflammatory responses, and hormone signaling. Furthermore, we identified three core targets by integrating the GEO database analysis, and machine learning. Single-gene GSEA elucidated multi-pathway regulatory mechanisms. Molecular docking demonstrated strong binding affinities between PFAS and these targets, while MD simulations confirmed the structural stability of the resulting complexes. PFAS exposure induces dose- and time-dependent cytotoxicity and oxidative stress in KGN cells in vitro. It activates apoptotic signaling via BAX upregulation and Bcl-2 downregulation and accelerates oxidative stress by suppressing glutathione S-transferase P1 (GSTP1) and peroxiredoxin 5 (PRDX5) expression. Additionally, PFAS impair ovarian reserve function by inhibiting the synthesis and secretion of anti-Müllerian hormone (AMH) and inhibin B (INHB). Notably, TGFBR1 upregulation promotes ovarian fibrosis, further compromising ovarian function. Our findings establish a comprehensive mechanistic framework wherein PFAS accelerate DOR progression by promoting apoptosis, inducing oxidative stress, suppressing reserve markers, and activating fibrotic pathways. This study provides novel insights into the pathogenesis of PFAS-associated DOR.