Abstract
BACKGROUND: Gestational diabetes mellitus (GDM) affects up to 14% of pregnancies globally, with insulin resistance (IR) playing a critical but often underappreciated role in its pathogenesis. Yet the specific impact of insulin at IR levels on mitochondrial function and pyroptosis in first-trimester trophoblasts remains unclear. Metformin use in GDM pregnancies is rising, but its impact on placental mitochondrial function is uncertain. This study aimed to investigate the impact of IR, a key feature of GDM, on mitochondrial dysfunction and pyroptosis in trophoblasts and to evaluate the protective effects of metformin. METHODS: Dual staining assays using TUNEL and caspase-1, and enzyme-linked immunosorbent assay were conducted to assess pyroptosis and pyroptosis-related inflammatory markers in placentas from 42 GDM patients and 39 controls. In vitro, HTR-8/SVneo trophoblast cells were treated with IR-level insulin concentrations, and a concentration gradient of metformin to evaluate the mitochondrial damage, pyroptosis, and cell viability. RESULTS: There was a significant increase in pyroptosis in GDM placenta, as well as pyroptosis-related inflammatory markers, IL-1β and IL-18. Placental IL-1β and IL-18 levels were strongly correlated with IR indices, especially in GDM cases. Moreover, IR-level insulin concentrations induced mitochondrial dysfunction and activated the NLRP3 inflammasome, triggering pyroptosis in HTR-8/SVneo trophoblasts. Metformin, particularly at therapeutic doses (10-100 µM), mitigated IR-induced mitochondrial damage by promoting mitochondrial biogenesis and reducing pyroptosis via suppressing the ROS/TXNIP/NLRP3 pathway. Metformin-treated cells exhibited enhanced mitochondrial respiration, restored membrane potential homeostasis, and reduced oxidative stress. CONCLUSION: IR, independent of hyperglycemia, drives placental inflammation and trophoblastic injury via pyroptosis. Targeting the ROS/TXNIP/NLRP3 pathway with metformin or other therapeutic agents offers potential therapeutic value in managing IR-related complications in GDM.