417 The diffusion of vaginal bacterial extracellular vesicles through cervicovaginal mucus facilitates inflammation in female reproductive tract cells

Objectives/Goals: To probe microbe and bacterial extracellular vesicle (bEV) mobility through biological barriers, we use novel multiple-particle tracking technology. The goal is to evaluate changes caused by extracellular vesicles relevant to placental function and neonatal development. Methods/Study Population: We conducted multiple particle tracking to assess whole bacterial and bEV mobility in cervicovaginal mucus. To accomplish this, cervicovaginal mucus was self-collected from 10 women. Mucus samples were characterized via wet mount, Nugent score, and pH measurements. In parallel, we cultured commercially available vaginal bacteria strains in anaerobic conditions. We isolated bEVs via ultracentrifugation, and subsequently characterized them via nanoparticle tracking analysis to measure size, ζ-potential, and concentration. We investigated reproductive tract tissues response to bEVs. We dosed vaginal, endometrial, myometrial, and placental cells lines with bEVs over a 24 h period and determined uptake, viability, and cytokine production. One-way analysis of variance was used for statistical analysis. Results/Anticipated Results: Based on our previous work, size and ζ-potential greatly affect particle mobility in mucus. G. vaginalis and M. mulieris were smaller than L. crispatus and L. iners . G. vaginalis had a more net-neutral ζ-potential compared to other bEVs. During multiple-particle tracking analysis, whole bacteria were unable to diffuse through vaginal mucus, while bEVs showed increased mobility. Through fluorescence levels, we determined M. mulieris bEVs reach >90% uptake at 24 h. Uptake was verified via microscopy. Across all strains, bEVs were not detrimental to placental viability. When investigating cytokine production in placental cells, an increase in IL-6 was seen after treatment with L. iners bEVs, while TNFα was increased after treatment with G. vaginalis bEVs. Discussion/Significance of Impact: Vaginal microbiome dysbiosis increases adverse obstetric indications. We demonstrate that bacteria are unable to ascend to reproductive tissues. We propose that bEVs travel through vaginal mucus, facilitating microbe–host communication. This impacts obstetric disease pathology and is relevant for diagnostic criteria during pregnancy.