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Cervicovaginal (CV) microbiota is associated with vaginal health and disease in non-pregnant women. Recent studies in pregnant women suggest that specific CV microbes are associated with preterm birth (PTB). While the associations between CV microbiota and adverse outcomes have been demonstrated, the mechanisms regulating the associations remain unclear. As the CV space contains an epithelial barrier, we postulate that CV microbiota can alter the epithelial barrier function. We investigated the biological, molecular, and epigenetic effects of  Lactobacillus crispatus, Lactobacillus iners , and  Gardnerella vaginalis  on the cervical epithelial barrier function and determined whether  L. crispatus  mitigates the effects of lipopolysaccharide (LPS) and  G. vaginalis  on the cervical epithelial barrier as a possible mechanism by which CV microbiota mitigates disease risk. Ectocervical and endocervical cells treated with  L. crispatus, L. iners , and  G. vaginalis  bacteria-free supernatants alone or combined were used to measure cell permeability, adherens junction proteins, inflammatory mediators, and miRNAs. Ectocervical and endocervical permeability increased after  L. iners  and  G. vaginalis  exposure. Soluble epithelial cadherin increased after exposure to  L. iners  but not  G. vaginalis  or  L. crispatus . A Luminex cytokine/chemokine panel revealed increased proinflammatory mediators in all three bacteria-free supernatants with  L. iners  and  G. vaginalis  having more diverse inflammatory effects.  L. iners  and  G. vaginalis  altered the expression of cervical-, microbial-, and inflammatory-associated miRNAs.  L. crispatus  mitigated the LPS or  G. vaginalis -induced disruption of the cervical epithelial barrier and reversed the  G. vaginalis -mediated increase in miRNA expression.  G. vaginalis  colonization of the CV space of a pregnant C57/B6 mouse resulted in 100% PTB. These findings demonstrate that  L. iners  and  G. vaginalis  alter the cervical epithelial barrier by regulating adherens junction proteins, cervical immune responses, and miRNA expressions. These results provide evidence that  L. crispatus  confers protection to the cervical epithelial barrier by mitigating LPS- or  G. vaginalis -induced miRNAs associated with cervical remodeling, inflammation, and PTB. This study provides further evidence that the CV microbiota plays a role in cervical function by altering the cervical epithelial barrier and initiating PTB. Thus, targeting the CV microbiota and/or its effects on the cervical epithelium may be a potential therapeutic strategy to prevent PTB.

frontiers in microbiology

Common Cervicovaginal Microbial Supernatants Alter Cervical Epithelial Function: Mechanisms by Which Lactobacillus crispatus Contributes to Cervical Health