Hyperglycemia induced during oral glucose tolerance test (OGTT) triggers spontaneous neutrophil extracellular trap (NET) formation. (A) Spontaneous NET formation in 3-h culture of neutrophils isolated from controls, pregnancy, and gestational diabetes mellitus (GDM) at the 60 min time point of blood collection during OGTT, stained with SytoxGreen (left panel) and immunofluorescence for MPO (red), citH3 (green), and DAPI (blue). Scale bars: 100 µm. (B) Fluorimetric analysis with SytoxGreen of NET formation, (C) morphometric analysis pro-NETotic primed citH3+ and (D) NETotic MPO+/DAPI+ neutrophils isolated during OGTT (60 min). (E) mRNA expression of neutrophil elastase (NE) from neutrophils isolated during the OGTT (at 0, 60, and 120 min). 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 in triplicates with consistent results.