Zearalenone impairs trophoblast function via ROS-mediated apoptosis and autophagy: A mechanistic insight into unexplained recurrent spontaneous abortion.

The impact of zearalenone (ZEN) on miscarriage, especially unexplained recurrent spontaneous abortion (URSA), remains unclear. We collected follicular fluid from URSA patients and found significantly elevated ZEN levels. This study further investigated ZEN's cytotoxic effects on human (HTR-8) and primary rat trophoblast cells. In HTR-8 cells, ZEN exposure (0.05-50 μM) induced time-dose-dependent suppression of cell viability, with concomitant reductions in human chorionic gonadotrophin (hCG) levels. ZEN (0.05-50 μM) downregulated the expression of antioxidants (SOD1, SOD2, CAT1 and GPX1) and anti-apoptotic gene (BCL2), and upregulated apoptotic genes (P53 and CASP9). ZEN affected the expression of nutrient transporters, including CD36 (at ≥ 50 μM), LDLR (at ≥50 μM), and SLC38A2 (at ≥ 5 μM). Flow cytometry analysis demonstrated that ZEN, at concentrations of ≥ 10 μM, induced reactive oxygen species (ROS) production, reduced mitochondrial membrane potential (MMP), and promoted apoptosis in HTR-8 cells. These effects were attenuated by acetylcysteine (NAC). In primary rat trophoblast cells, ZEN at ≥ 50 μM similarly triggered ROS generation, decreased MMP, and enhanced apoptosis, which were partially reversed by NAC. Western blot analysis further revealed that ZEN at concentrations as low as 5 μM induced autophagy in HTR-8 cells, as evidenced by increased levels of BECLIN1, LC3B, and p62. Notably, the ZEN-induced upregulation of LC3B protein was attenuated by NAC. ZEN remarkably reduced the phosphorylation of AKT1, AKT2, ERK1/2, and mTOR. These findings suggest that ZEN may harm pregnancy health through ROS-mediated apoptosis and autophagy in trophoblast cells.