Real-world river water organic pollutant mixture induces ovarian hyperandrogenism via aryl hydrocarbon receptor activation.

Humans are exposed to complex mixtures of endocrine-disrupting chemicals (EDCs) in the real world. Most prior studies, however, focus on individual EDCs. Here, we investigated the ovarian impact of organic pollutant mixtures extracted from the Raritan River, a heavily industrialized watershed in central New Jersey, using a three-dimensional (3D) in vitro ovarian follicle culture system. Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) identified organic pollutant mixtures dominated by alkylphenols, pharmaceuticals, per- and polyfluoroalkyl substances (PFAS), and phthalates, together with previous reports demonstrating that the Raritan River also contains hydrophobic persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and dioxins. In vitro exposure to the Raritan River-derived EDC mixture did not affect follicle growth and ovulation but dose-dependently increased preovulatory testosterone (T) secretion while suppressing postovulatory progesterone (P4) production. Transcriptomic analysis, Gene set enrichment analysis (GSEA), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and in situ RNA hybridization revealed widespread reprogramming of follicular somatic cell gene expression, including altered steroidogenic pathways and activation of aryl hydrocarbon receptor (AhR) signaling. An established AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), reproduced the phenotype of EDC mixture-induced hyperandrogenism, which was reversed by a selective AhR inhibitor. Together, this study demonstrates that real-world EDC mixtures, particularly AhR-activating chemicals, disrupt ovarian steroidogenesis and induce hyperandrogenism, highlighting EDC mixtures as an underrecognized mechanism potentially linking aquatic pollution to impaired women's reproductive health.