Role of ferroptosis-related GPX4 signaling in the fusion of human trophoblast cells

The placenta is mainly composed of trophoblast cells which fuse to create multinucleated syncytiotrophoblasts in the process of syncytialization. Syncytiotrophoblasts secrete human chorionic gonadotropin (hCG) for maintaining pregnancy. Impaired syncytialization contributes to pregnancy complications such as preterm labor and fetal growth restriction. Ferroptosis, a form of regulated non-apoptotic cell death, is characterized by the iron-dependent accumulation of lipid peroxides. This process is triggered by inactivation of glutathione peroxidase 4 (GPX4)-dependent antioxidant system. During pregnancy, iron demand increases, accompanied by elevated transferrin expression in syncytiotrophoblasts. However, the physiological relevance of ferroptosis signaling in trophoblast fusion remains largely explored. To address the impact of ferroptosis signaling on syncytialization, forskolin-stimulated trophoblast BeWo cells were treated with GPX4 inhibitors RSL3, ML-210, or erastin. Both RSL3 and ML-210 increased hCGβ expression and increased the number of fusogenic cells, alongside elevated intracellular Fe²⁺ levels and lipid peroxidation. These effects were suppressed by deferoxamine, an iron chelator, and ferrostatin-1, a lipid peroxidation inhibitor, both of which significantly reduced hCGβ expression. Enrichment analyses with 1,306 transcripts upregulated by ML-210 indicated the involvement of oxidative stress and endoplasmic reticulum (ER) stress pathways. ML-210 further upregulated NRF2, HO-1 and KEAP1, along with ER stress markers. Inhibition of the ER stress sensors IRE1α and ATF6 attenuated hCGβ expression, implicating these pathways in the regulation of syncytialization. Collectively, these findings suggest that ferroptosis-related lipid peroxidation and iron signaling contribute to the regulation of trophoblast fusion, potentially physiological role in placental development.