Cervical mucus in linked human Cervix and Vagina Chips modulates vaginal dysbiosis

Abstract

This study explores the protective role of cervicovaginal mucus in maintaining vaginal health, particularly in relation to bacterial vaginosis (BV), using organ chip technology. By integrating human Cervix and Vagina Chips, we demonstrated that cervical mucus significantly reduces inflammation and epithelial damage caused by a dysbiotic microbiome commonly associated with BV. Proteomic analysis of the Vagina Chip, following exposure to mucus from the Cervix Chip, revealed differentially abundant proteins, suggesting potential biomarkers and therapeutic targets for BV management. Our findings highlight the essential function of cervical mucus in preserving vaginal health and underscore the value of organ chip models for studying complex interactions within the female reproductive tract. This research provides new insights into the mechanisms underlying vaginal dysbiosis and opens avenues for developing targeted therapies and diagnostic tools to enhance women's reproductive health.

Keywords: Physiology; Reproductive biology.

Fig. 1. Effects of cervical mucus on cytokine production by the Vagina Chip.

A Schematic diagrams of Cervix and Vagina Chips and the transfer of cervical mucus between chips (Created with BioRender.com). B Cytokine protein levels for IL-1α, IL-1β, MIP-1β, and IL-10 measured in effluents of Vagina Chips cultured with (light gray bars) without (dark gray bars) cervical mucus for 1 day. Each data point indicates one chip; data shown are from 3 different experiments and are presented as mean ± sd; significance was calculated by unpaired t-test; ***P < 0.0001; **P < 0.001.

Fig. 2. Modulation of dysbiotic vaginal microbiome by cervical mucus introduced to the Vagina Chip.

Vaginal epithelium cultured on-chip for 72 h in the absence (Control: gray with stripes bars) or presence of BVC1 consortium (black bars) perfused either with media alone or with mucus-containing effluents from Cervix Chips that were added 1 day prior to the addition of bacteria (Pre; dark gray bar), 1 day after BVC 1 addition (Post; light gray bar), or continuously for the entire 3-day culture starting 1 day prior to addition of bacteria (Pre+Post; white bar). A Total non-adherent bacterial cell number (CFU) per chip determined by quantification of bacteria collected in effluents from the apical epithelial channel during 72 h of co-culture with BVC1 in Vagina Chips. B Total adherent CFU/chip determined by quantification of bacteria retained within epithelial tissue digests after 72 h of culture. C Bright-field microsopic image showing Clue-like cells and live epithelial cells using Trypan blue stain. D Ratio of Clue-like cells to live cells detected as described in (C). E Quantification of vaginal epithelial cell injury (percent cell viability) assessed by calculating the number of live cells relative to control using Trypan blue exclusion assay. In all graphs, results were obtained from at least 2 different experiments; each data point indicates one chip. Data are presented as mean ± sd; significance was calculated by unpaired t-test; ***P < 0.0001; **P < 0.001.

Fig. 3. Cytokine production by the Vagina Chip exposed to a dysbiotic consortium.

Heat map showing the innate immune response of vaginal epithelium cultured on-chip for 72 h in the absence (Control) or presence of BVC1 consortium perfused either without or with mucus-containing effluents from Cervix Chips that were added 1 day prior to the addition of bacteria (Pre), 1 day after BVC 1 addition (Post), or continuously for the entire 3-day culture starting 1 day prior to the addition of bacteria (Pre+Post). IL-10, RANTES(CCL5), TNF-α, MIP-1β, and IL-1α protein levels in the epithelial channel effluents were normalized for cell number. IFN-γ, IL-1β, IL-8, IL-6, MIP-1α, and IP-10 did not show a statistically significant difference between the groups. The grayscale represents the fold-change in cytokine levels relative to untreated control chips, and the statistical analysis was performed by comparing the BVC1 chips (n = 4–10 individual chips for each group from 4 independent experiments; significance was calculated by unpaired t-test ; *P < 0.05, ***P < 0.001, ****P < 0.0001 compared to BVC1).

Fig. 4. Suppression of G. vaginalis growth in Vagina Chip effluents in 2D culture.

A G. vaginalis growth in 2D culture wells by optical density (OD) measurement every 30 min for 24 h; (M) within mucus-containing Cervix Chip effluent; (V + M) similar effluent perfused through a Vagina Chip for 1 day; and (HBSS) control. B Logistic growth rate constant (k) for bacterial growth in (A). C Bacterial growth in permissive conditions in which the effluents shown in (A) were supplemented with 50% bacterial broth (PYT). While permissive conditions allowed for growth in the HBSS control group, G. vaginalis growth remained suppressed in the V + M group. D Logistic growth rate constant (k) for bacterial growth in (C). n = 5; *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 5. Cervical mucus alters the vaginal secretome.

Mass spectrometry analysis of Vagina Chip effluents pre-exposed to Cervix Chip mucus-containing effluent for 1 day (V + M) compared to Vagina Chip effluent alone (V) and Cervix Chip mucus-containing effluent (M). A Upregulated and B Downregulated proteins in (V + M) compared (V) and (M), determined by fold change ( | log2 fc | > = 1, p_adj ≤ 0.05). C Volcano plot showing differentially expressed proteins in (V + M) compared to (V) and (M). The plot was constructed using the normalized protein expression data with the negative logarithm of the adjusted p value represented on the y-axis and the log2 fold change represented on the x-axis. Each dot on the plot corresponds to a protein, with color coding used to indicate the statistical significance of differential expression. Benjamini-Hochberg (BH) method was used for multiple testing correction and plotted adjusted P values. Proteins with a p < 0.05 and fold change >2 are colored red, while proteins with a p < 0.05, and fold change < −2 are colored blue (Gray, proteins with p > 0.05). D PCA plot of the cervix mucus (M, red), vagina chip (V, green) and vagina chip with cervix mucus (V+M, blue) secretome.