Glucose and TNF-α induce neutrophil activation and neutrophil extracellular traps (NETs) formation in gestational diabetes mellitus (GDM). (A) TNF-α concentration in the supernatants from BeWo cells treated with/without 25 mM of d-glucose. (B) NET morphometry over a 3-h time course with SytoxGreen DNA-binding dye (left panel) and MPO+/DAPI+ (right panel) after in vitro incubation of control neutrophils with control, pregnancy, and GDM plasma with and without pretreatment with 5 µg/ml infliximab. (C) TNF-α concentration of controls, pregnancy, and GDM plasma. (D) Morphometric quantitation of NETs performed with MPO+/DAPI+ and citH3 of neutrophils from controls after exposure to 25 nM glucose, 25 nM Mannitol, 50 ng/ml TNF-α, and PMA 50 ng for 3 h. (A–D) Data are presented as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) (one-way ANOVA followed by Tukey’s multiple comparison post-test). All experiments were performed at least four times with consistent results.