0.2 μm cellulose acetate filters

Six Lactobacillus isolates [2 L. jensenii (LJ2 and 5), 1 L. mucosae (LM2), 1 L. crispatus (LC2), 1 L. gasseri (LG1), 1 L. vaginalis, (LV6)] were cultured in MRS for 24 h under anaerobic conditions. Cultures were standardized to 4.18 × 10⁶ CFU/ml in MRS and incubated for an additional 20 h at 37 °C under anaerobic conditions, after which they were filtered using 0.2 μm cellulose acetate filters (Sigma-Aldrich, USA). Following 48 h incubation in brain heart infusion (BHI) broth at 37 °C under anaerobic conditions, G. vaginalis was standardized to 1 × 10⁸ CFU/ml and cultured with each lactobacilli cell-free supernatant anaerobically for 24 h at 37 °C. Absorbance at OD₆₀₀ was measured at baseline and 24 h after incubation. Following the 24 h incubation, G. vaginalis cultures were plated on BHI agar and incubated for 24 h at 37 °C under anaerobic conditions, after which CFUs were counted^{12 (link)}.

For cytokine measurement, MCs (Softcup®, Evofem Inc, USA) were inserted by the clinician and kept in place for an hour, after which they were transported to the lab at 4 °C and processed within 4 h of removal from the participants. MCs were centrifuged, and the cervicovaginal secretions were resuspended in phosphate-buffered saline (PBS) at a ratio of 1 ml of mucus: 4 ml of PBS and stored at − 80 °C until cytokine measurement. Prior to cytokine measurement, MC secretions were pre-filtered using 0.2-μm cellulose acetate filters (Sigma-Aldrich, MO, USA). The concentrations of 48 cytokines were measured in MC samples using Luminex (Bio-Rad Laboratories Inc®, CA, USA) [23 (link)]. K-means clustering was used to identify women with low, medium, and high pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-12p40, IL-12p70, tumor necrosis factor (TNF)-α, TNF-β, TNF-related apoptosis-inducing ligand (TRAIL), interferon (IFN)-γ] and chemokine profiles [cutaneous T cell-attracting chemokine (CTACK), eotaxin, growth regulated oncogene (GRO)-α, IL-8, IL-16, IFN-γ-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, MCP-3, monokine induced by IFN-γ (MIG), macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation, normal T cell expressed and secreted (RANTES)] (Additional file 1: Fig. S2).