Association between per- and polyfluoroalkyl substances exposure and recurrent pregnancy loss: The potential role of oxidative stress and the HIF1α‑GLUT1/VEGFA axis.

Recurrent pregnancy loss (RPL) is a prevalent reproductive disorder affecting women of childbearing age globally, and a substantial proportion of cases remain idiopathic with unclear etiology. Per- and polyfluoroalkyl substances (PFAS) are ubiquitous persistent environmental pollutants with potential reproductive toxicity; however, the association between PFAS exposure and RPL as well as the underlying mechanisms remain poorly characterized. This study aimed to investigate the independent association between PFAS exposure and RPL risk, and to explore the potential mediating role of oxidative stress and the HIF1α‑GLUT1/VEGFA signaling axis in PFAS‑related reproductive impairment. A multi‑level research design was adopted, including a case‑control study of 50 RPL patients and 100 healthy controls, in vivo mouse exposure experiments, and in vitro cellular functional assays. Serum PFAS levels were measured by LC‑MS/MS, and multi‑omics profiling was performed to screen candidate pathological pathways. Female mice were exposed to PFOA or PFPeA via drinking water, and reproductive outcomes, tissue histology, and pathway expression were evaluated. Human reproductive cell lines were treated with PFAS, and N‑acetylcysteine (NAC) rescue experiments were conducted to verify the upstream role of oxidative stress. Clinical results showed that serum concentrations of multiple long‑chain PFAS were significantly higher in RPL patients, and PFAS exposure was independently associated with increased RPL risk after adjustment for confounding factors. Untargeted serum metabolomics and exploratory endometrial proteomics identified RPL-associated disturbances in glycolysis and HIF-1α/VEGF signaling as candidate pathological pathways; these RPL-associated signatures were taken forward as mechanistic hypotheses for testing the causal role of PFAS in subsequent animal and cell models. Animal experiments showed that exposure to long-chain PFOA disrupted estrous cycles, reduced pregnancy rates, and increased embryo resorption, accompanied by dysregulated expression of HIF1α, GLUT1 and VEGFA in reproductive tissues. In vitro cellular assays consistently demonstrated that PFOA treatment suppressed cell viability, promoted intracellular ROS overproduction, impaired trophoblast migration and endothelial angiogenesis, and perturbed the HIF1α‑GLUT1/VEGFA axis. Parallel experiments with short-chain PFPeA provided exploratory comparative toxicity data across both in vivo and in vitro models. NAC pretreatment partially attenuated PFAS-induced intracellular ROS accumulation and loss of cell viability in HTR-8/SVneo cells, supporting oxidative stress as a potential upstream mediator of PFAS-associated cytotoxicity. Reversal of HIF1α-axis dysregulation and functional impairment (migration/angiogenesis) was not assessed in NAC rescue experiments. In conclusion, serum PFAS exposure is independently and robustly associated with elevated RPL risk. Oxidative stress‑mediated dysregulation of the HIF1α‑GLUT1/VEGFA axis may underlie PFAS‑related reproductive dysfunction and adverse pregnancy outcomes. These findings provide supporting evidence for the reproductive health risks of ubiquitous PFAS exposure and offer preliminary mechanistic clues for etiological research on idiopathic RPL.