DEHP-induced male reproductive toxicity: Evidence from population studies, animal experiments, and multi-omics profiling.

OBJECTIVE: To evaluate the association between di(2-ethylhexyl) phthalate (DEHP) exposure and testosterone deficiency in adult men, and to explore molecular changes and candidate compounds related to DEHP-associated male reproductive toxicity. METHODS: We used a multi-level integrative strategy. Computational toxicology predicted DEHP endocrine-disrupting potential. In NHANES 2013-2016 adult males, survey-weighted logistic regression and restricted cubic splines assessed associations between urinary DEHP metabolites and testosterone deficiency. Male Sprague-Dawley rats were exposed to DEHP to evaluate sex hormones, testicular/epididymal histopathology, oxidative stress, inflammation, and CD68⁺ macrophage infiltration. Integrated infertility transcriptomes with ensemble machine learning identified hub genes, which were mapped in testicular single-cell RNA-seq with virtual knockout-based network inference. Connectivity Map screening plus molecular docking and molecular dynamics prioritized candidate compounds. RESULTS: In NHANES, oxidative DEHP metabolites (MEHHP, MEOHP, and MECPP) were associated with increased odds of testosterone deficiency and showed monotonic dose-response increases. Rat experiments showed that DEHP reduced testosterone accompanied by decreased FSH/LH levels, disrupted testicular structure and spermatogenesis, and increased oxidative stress, inflammatory markers, and macrophage infiltration. Hub genes (NNT, ACSS1, NANOG, S100A16, and CELF4) were mainly involved in mitochondrial energy metabolism and redox homeostasis. CMap screening and in silico analyses prioritized ursolic acid as a candidate compound, with the predicted ursolic acid-ACSS1 complex showing relative stability in molecular dynamics simulation. CONCLUSION: DEHP exposure was associated with testosterone deficiency in men and induced reproductive toxicity in rats. Integrated analyses highlighted candidate metabolic, redox, inflammatory, and Sertoli cell-related network alterations. Ursolic acid was computationally prioritized as a candidate compound, but experimental validation is required.