Tebuconazole-induced ferroptosis impairs human trophoblast function via GPX4: A multi-omics study on unexplained recurrent pregnancy loss.

Abstract

Tebuconazole (TEB) is a widely used triazole fungicide with suspected endocrine-disrupting and reproductive toxicity. However, its effects on human placental trophoblasts and its association with unexplained recurrent pregnancy loss (RPL) remain poorly defined. Chorionic villi from 27 women with unexplained RPL and 25 women with normal pregnancies were analyzed by HPLC-MS/MS, and TEB effects were examined in BeWo and HTR-8/SVneo trophoblasts. Villus TEB residues were 1.82-fold higher in the RPL group (mean 0.0356 versus 0.0196 ng/g; P < 0.001), whereas TEB-OH did not differ significantly. In HTR-8/SVneo cells, 48-h TEB exposure reduced estradiol secretion at 20-80 µM and impaired migration at 40-80 µM without a significant loss of CCK-8 viability, and increased intracellular labile Fe^2 +. Transcriptome profiling at 80 µM identified 1680 differentially expressed genes (797 up-regulated, 883 down-regulated), and the downregulation of GPX4, TFRC, and SLC40A1 was confirmed by RT-qPCR and Western blotting. Network toxicology identified 72 core targets shared with RPL and fetal growth restriction, and docking predicted GPX4 as a candidate binding target of TEB (Ki= 44.74 µM). Consistent with a ferroptotic mechanism, ferrostatin-1 (2 µM) attenuated TEB-induced lipid peroxidation and Fe²⁺accumulation and partially restored GPX4, SLC40A1, and TFRC abundance, trophoblast migration, and estradiol secretion. These findings suggest that ferroptosis-related lipid peroxidation contributes to TEB-induced trophoblast dysfunction and links chorionic villus TEB residues to unexplained RPL. Because the effective in vitro concentrations exceed typical human exposure levels, the results provide mechanistic evidence of hazard and warrant further risk assessment.