PROJECT SUMMARY Specific microbial and immune features of vaginal ecosystems, including a Lactobacillus-deplete microbiota, have been linked to spontaneous preterm birth (PTB). Mechanisms driving associations remain incompletely understood. Premature cervical remodeling, involving cervicovaginal (CV) epithelial barrier dysfunction, is a key biologic process on the pathway to PTB. In other systems, metabolomics has emerged as a lens through which phenotypes of microbial ecosystems can be more clearly elucidated. Microbial and host metabolite- modification of immune responses and epithelial barrier integrity has given rise to novel targeted postbiotic therapeutics. The bioactive potential of the vaginal metabolome remains under investigated. Our group recently identified a role for vaginal polyamines in discerning individuals at greatest risk of PTB in those with similar microbiota-related risk — among individuals with a Lactobacillus-deplete microbiota, those who delivered preterm had a 25-fold reduction in spermine. Polyamines spermidine and spermine play key roles in immunity and oxidative balance in other systems. The overall goal of this proposal is to elucidate their effects on barrier function in the CV space. Our central hypothesis is that vaginal spermidine and spermine maintain immune homeostasis and mitigate oxidative stress to enhance barrier function in the setting of a Lactobacillus-deplete microbiota. We further posit that inadequate host production of spermidine and spermine in response to microbial challenge results in CV barrier dysfunction, ascribing a mechanistic link between vaginal metabolites and cervical remodeling. We propose the following aims: 1) determine how polyamines modify immune responses in the CV space; 2) determine how polyamines regulate oxidative balance in the CV space; and 3) quantify associations of vaginal polyamines with premature cervical remodeling. In the first aim, we will use in vitro methodology to interrogate the role of polyamines in inflammasome activation, NFB-induction of immune mediators, macrophage differentiation, and epithelial-immune cell communication. For the second aim, we will use in vitro methodology to investigate polyamine effects on oxidative stress measured by lipid peroxidation, antioxidant enzyme expression, DNA and protein damage, as well as the ability of polyamine-mediated oxidative balance to modify host immune responses. In the third aim, we will leverage an ongoing pregnancy cohort to quantify associations between polyamines, lipid peroxidation markers, and short cervix to assess the potential impact of polyamines on tissue remodeling and PTB. We propose an innovative approach to define mechanisms by which the vaginal metabolome may modify cervical remodeling. Implications of this work extend beyond pregnancy, as immune perturbations and oxidative stress contribute to myriad adverse reproductive outcomes. Findings may be harnessed to modify aspects of the vaginal ecosystem...