Objective:This study aims to observe the ultrastructure of ferroptosis in placental trophoblast cells of gestational diabetes mellitus (GDM), and investigate the role of ferroptosis in the occurrence and development of GDM.Methods:Pregnant women who delivered in Changzhou Maternal and Child Health Hospital from September 2020 to December 2022 were selected . Oral glucose tolerance tests were performed during 24-28 weeks of pregnancy.Pregnant women diagnosed with GDM were categorized based on interventions:GDM women controlled by diet therapy (G1 group), those controlled by oral metformin (G2 group), those requiring insulin for blood glucose control (G3 group), and those with unsatisfactory blood glucose control refusing medication (G4 group), each comprising 30 cases. Normal pregnant women (N group) served as the control. The ultrastructure of placental trophoblast cells was observed using transmission electron microscopy.The placental iron deposition was detected by Prussian blue staining.The localizations and expressions of Acyl-CoA synthetase long chain family member 4(ACSL4) and glutathione peroxidase 4(GPX4) in the syncytiotrophoblast layer were detected by immunohistochemistry.The protein expressions and the mRNA transcription levels of ACSL4 and GPX4 in placental tissues were detected by Western blotting and qRT-PCR,respectively.Results:Transmission electron microscopy revealed varying degrees of mitochondrial morphological changes in all four GDM placental groups.G1, G2, and G3 groups showed milder mitochondrial changes, while the G4 group exhibited typical ferroptosis with shrunken mitochondria.Prussian blue staining demonstrated iron particles in all five groups, with a significant increase in iron particles in the GDM placental groups compared to the N group. The iron particles in the G1,G2 and G3 groups were fewer than the G4 group.ACSL4 and GPX4 were predominantly expressed in the cytoplasm of syncytiotrophoblast cells.Compared to the N group, GDM placental tissues in all four groups showed increased ACSL4 protein expression and decreased GPX4 protein expression, with no significant differences in ACSL4 and GPX4 mRNA transcription levels.Conclusion:Ferroptosis exists in placental trophoblast cells of GDM patients, and it is correlated with the severity of the disease and medication.Placental ferroptosis in GDM patients is associated with abnormal expression of ACSL4 and GPX4.Metformin and insulin have a role in improving ferroptosis of placental trophoblast cells.