Polymorphonuclear neutrophils lead to excessive neutrophil elastase (NE) release and IRS1 degradation in placenta during gestational diabetes mellitus (GDM). (A) H&E staining of placental tissue. (*): syncytial knotting. Scale bars: 50 µm. (B) Immunofluorescence staining for MPO (red), citH3 (green), and DNA (blue) of placental tissue collected at the term of pregnancy from controls and GDM women. Scale bars: 50 µm. (C,D) Morphometric analysis of extracellular citH3+ and MPO+ in neutrophils (suggestive of activated and NETing neutrophils) in tissue sections from term placentae of pregnancy and GDM. (E) Western blot and densitometric analysis of IRS1 and β-actin protein expression levels in placental tissue as in (A). (F) Immunofluorescence staining for NE (red), IRS1 (green), and DNA (blue) of placental tissue as in (A). Scale bars: 100 µm. (G) NE protein expression determined by morphometric analysis in placental tissue lysates from pregnancy and GDM. (H) mRNA expression level of NE determined by qPCR in placental tissue as in (G). AU, arbitrary unit. 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 three times with consistent results.