Pronounced neutrophil extracellular trap (NET) formation in pregnancies is affected by gestational diabetes mellitus (GDM). (A) In vitro spontaneous NET formation assay performed with neutrophils isolated from non-pregnant (controls), healthy pregnant (pregnancy), and GDM patients during a 3-h time course. NETs are detected by immunofluorescence staining for neutrophil elastase (NE) (green) and DNA-DAPI (blue). Scale bars: 50 µm. (B) Morphometric analysis of extracellular NETs with citH3 staining (% of positive cells) and NE+/DAPI+ staining (% positive area) from controls, pregnancy, and GDM neutrophils. (C–E) Measurement of cell-free nucleosomes of control, IInd, and IIId trimester pooled pregnancy samples, IInd and IIId trimester pooled GDM samples, type 1 diabetes mellitus (T1DM), and type 2 diabetes mellitus (T2DM) serum samples (C), myeloperoxidase (D) and NE (E). (F) Quantitation of serum A1AT levels in controls, pregnancy, GDM, T1DM, or T2DM. (G) Serum NE enzymatic activity in controls, pregnancy, and GDM. (B–G) 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 six times with consistent results.