EHDPP Exposure Drives Ovarian Reserve Decline via Metabolic Perturbations: Evidence from Epidemiological and In Vivo Studies

Abstract Exposure to organophosphate esters (OPEs) has been implicated in reproductive toxicity; however, evidence linking OPE exposure to diminished ovarian reserve (DOR) and the underlying mechanism remains limited. A nested case–control study (70 DOR cases and 70 controls) was conducted within a reproductive health cohort to quantify serum OPEs using ultrahigh-performance liquid chromatography–tandem mass spectrometry. Associations of individual OPEs and their mixtures with DOR were evaluated using generalized linear models and advanced Bayesian kernel machine regression. Untargeted metabolomics based on ultrahigh-performance liquid chromatography–high-resolution mass spectrometry, combined with metabolome-wide association analysis, a meet-in-the-middle framework, and mediation analysis, was used to identify pathways, which were then validated in a mouse model. Of the 12 OPEs, 2-ethylhexyl diphenyl phosphate (EHDPP) was higher in cases (1.22 vs 1.06 μg/g lipid; P = 0.025) and was associated with increased DOR risk (odds ratio = 3.59, 95% confidence interval [CI]: 1.44–9.86) and a lower antral follicle count (regression coefficient = −0.17, 95% CI: −0.30, −0.04). Mixture analysis identified EHDPP as the dominant contributor (posterior inclusion probability = 0.871). A total of 26 metabolites were jointly associated with EHDPP exposure and DOR, and 11 mediated 13.8%–26.5% of the association, primarily via alanine, aspartate, and glutamate metabolism. Although the overall OPEs mixture did not demonstrate a statistically significant association with DOR risk, mixture analysis identified EHDPP as the dominant contributor. EHDPP exposure induced ovarian dysfunction and consistent metabolic alterations in mice. These findings suggest that amino acid metabolism explains the association between EHDPP exposure and DOR.