In an exploratory examination of the NETotic activity of freshly isolated neutrophils in vitro, we observed that NET formation was significantly elevated in a case with GDM when compared to a matching second trimester sample (Figure 1A). This feature was confirmed in a larger series of GDM cases (n = 9) and matching healthy control pregnancies (n = 10) and non-pregnant donors (n = 10) (Table S1 in Supplementary Material; Figure 1B). In these analyzes, it was noted that GDM samples contained more neutrophils activated to undergo NETosis as apparent from the presence of citrullinated histone H3 (citH3 +) moieties (Figure 1B), a key feature in the NETotic signaling cascade. This was mirrored in a greater tendency to undergo NETosis in vitro, as indicated by the quantity of extracellular NE/DNA (total area) (Figure 1B, lower panel). Furthermore, this NETosis increased from the time of diagnosis (24–28 weeks of gestation) throughout the second and third trimesters of gestation as measured with cell-free nucleosomes in the supernatant of freshly isolated neutrophils (Figure S1 in Supplementary Material).