Multimodal single-cell protein and RNA profiling unveils dysregulated immature neutrophil dynamics in gestational diabetes mellitus

Neutrophils are the most abundant leukocytes in human peripheral blood, yet their heterogeneity in pregnancy, especially in gestational diabetes mellitus (GDM), remains incompletely understood. Here, we employed InfinityFlow-based surface marker profiling and single-cell RNA sequencing (scRNA-seq) to delineate neutrophil subsets in healthy and GDM pregnancies. We identified a low-density, immature subgroup (CD10⁻CD49d⁺Ig κ⁺) with distinctive morphology and transcriptomic profiles, contrasted against the CD10⁺ segmented mature neutrophils. In healthy pregnancy, these immature low-density neutrophils (LDNs) expanded by mid-gestation, elevating the immature-to-mature (I–M) neutrophil ratio and circulating myeloid progenitor levels throughout gestation. In GDM, this expansion was markedly blunted, with a consistently lower abundance of immature LDNs and progenitors. Flow cytometry and correlation analyses further linked the lower I–M ratio to impaired insulin sensitivity, underscoring a potential immune-metabolic axis in GDM. Collectively, our study provides a framework for neutrophil phenotyping in pregnancy and links disrupted neutrophil equilibrium and pregnancy complications. Integrated InfinityFlow and scRNA-seq profiling reveal a disrupted neutrophil landscape in GDM: blunted expansion of immature cells and progenitors and a lower I-M ratio linked to metabolic dysfunction, highlighting an immunometabolic axis.