In this instance, clear differences in the NETotic capacity of isolated neutrophils were discernible between samples obtained from healthy non-pregnant women, healthy pregnant women, or cases with GDM (Figure 3A), which was most pronounced 60 min after the HG challenge (Figures S2B,C in Supplementary Material). This increase in NETosis was apparent using either Sytox Green to detect exogenous DNA extruded by isolated neutrophils in vitro (Figure 3A, left panel, Figure 3B; Figure S2B in Supplementary Material), fluorescent immunohistochemistry for MPO (Figure 3A, right panel, Figure 3D; Figure S2C in Supplementary Material), as well as by examining for NET products in matching serum samples (Figure S2E in Supplementary Material). As previously observed, this increase in spontaneous NETosis was paralleled by a similar increase in primed pro-NETotic neutrophils, identified by posttranslational histone modifications facilitating chromatin unraveling (citH3+) (Figure 3C; Figure S2D in Supplementary Material).