Dynamics of Extracellular Traps in Vaginal Dysbiosis Associated with Gardnerella vaginalis: Ex Vivo Evidence from Neutrophils and Monocytes

Vaginal dysbiosis, particularly bacterial vaginosis (BV), is associated with altered immune responses that may influence the formation of extracellular traps (ETs). This study aimed to characterize neutrophil and macrophage extracellular traps (NETs and METs) in vaginal discharge samples obtained from women with normal microbiota (NM) and BV, with particular emphasis on Gardnerella vaginalis (GV) detection. An ex vivo analysis was performed using vaginal smears from 14 patients previously classified according to the Nugent criteria. Immunofluorescence assays targeting neutrophil elastase (NE), citrullinated histone H3 (Citr-H3), CD15, and CD68 were conducted, and quantitative image analysis was performed using the TissueFAXS and StrataQuest platforms. NETs were classified into three morphotypes: spread (spr), diffuse (diff), and aggregated (agg). BV samples exhibited a substantially higher mean NET count than NM samples (842.43 vs. 91.86). The number of diffNETs was significantly higher in BV samples than in NM samples (p = 0.004), whereas GV-positive samples showed increased sprNET abundance compared with that in negative samples (248 vs. 8; p < 0.05). CD68+ cell counts were significantly higher in BV samples (p = 0.026), whereas no significant differences in NE or Citr-H3 fluorescence intensity were observed between groups. MET structures were also identified, suggesting macrophage involvement in the local immune response. Collectively, these findings indicate that vaginal dysbiosis and GV presence are associated with enhanced NET formation and distinct morphotype distributions, supporting a role for ETs in the immunopathology of BV.