From association to mechanism: Prenatal PFAS Co-exposures induces fetal neural tube defects via autophagy-mediated ferroptosis.

Neural tube defects (NTDs) represent severe congenital malformations whose environmental determinants remain incompletely understood. Per- and polyfluoroalkyl substances (PFAS), persistent environmental contaminants capable of crossing the placental barrier during critical developmental windows, have been linked to various adverse birth outcomes, yet their association with NTDs remains unclear. We combined population-based, animal, and cellular approaches to establish associations and explore relevant mechanisms. In a case-control study of 271 NTD and 391 controls, placental PFAS concentrations were significantly elevated (41.29 vs. 22.36 ng/g, P < 0.001). High exposure was associated with markedly increased NTD risk (OR=14.13, 95 % CI: 4.39-45.49). Three mixture modeling approaches (BKMR, WQS, Qgcomp) consistently identified PFOS, PFDA, and PFHxS as dominant contributors, with weights of 35 %, 14 %, and 12 %, respectively. To validate causality, we exposed pregnant mice to this human-relevant PFAS mixture, which induced dose-dependent NTD phenotypes in the high-dose group (16.18 % vs. 2.67 %) accompanied by embryonic iron accumulation, oxidative stress, and dysregulation of ferroptosis and autophagy pathways, suggesting these pathways mediate PFAS developmental toxicity. Mechanistic validation in hPSCs confirmed that PFAS exposure reduced viability and suppressed GPX4 while increasing lipid peroxidation. Critically, pharmacological interventions revealed distinct rescue mechanisms. Ferrostatin-1 restored GPX4/SLC7A11 expression and reduced lipid peroxidation, while chloroquine blocked LC3B activation, prevented NCOA4-mediated ferritin degradation, and attenuated iron release, demonstrating that PFAS-induced ferroptosis operates through an autophagy-mediated mechanism wherein excessive autophagy promotes ferritinophagy and subsequent iron-driven cell death. This study identifies PFOS, PFDA, and PFHxS as potential developmental toxicants and highlight ferroptosis and autophagy as potential therapeutic targets, suggesting the need for further evaluation of PFAS regulation to protect prenatal health.