Objective Characterise the vaginal metabolome of cervical HPVinfected and uninfected women.Design Cross-sectional.Sample Thirty-nine participants, 13 categorised as HPV-negative and 26 as HPV-positive (any genotype; HPV + ), 14 of whom were positive with at least one high-risk HPV strain (hrHPV).Method Self-collected mid-vaginal swabs were profiled for bacterial composition by 16S rRNA gene amplicon sequencing, metabolites by both gas and liquid chromatography mass spectrometry, and 37 types of HPV DNA. Main outcome measures Metabolite abundances.Results Vaginal microbiota clustered into Community State Type (CST) I (Lactobacillus crispatus-dominated), CST III (Lactobacillus iners-dominated), and CST IV (low-Lactobacillus, 'molecular-BV'). HPV + women had higher biogenic amine and phospholipid concentrations compared with HPVwomen after adjustment for CST and cigarette smoking. Metabolomic profiles of HPV + and HPV À women differed in strata of CST. In CST III, there were higher concentrations of biogenic amines and glycogen-related metabolites in HPV + women than in HPVwomen. In CST IV, there were lower concentrations of glutathione, glycogen, and phospholipid-related metabolites in HPV + participants than in HPVparticipants. Across all CSTs, women with hrHPV strains had lower concentrations of amino acids, lipids, and peptides compared with women who had only low-risk HPV (lrHPV). ConclusionsThe vaginal metabolome of HPV + women differed from HPV À women in terms of several metabolites, including biogenic amines, glutathione, and lipid-related metabolites. If the temporal relation between increased levels of reduced glutathione and oxidised glutathione and HPV incidence/persistence is confirmed in future studies, anti-oxidant therapies may be considered as a non-surgical HPV control intervention. Keywords 16S rRNA gene amplicon sequencing, human papillomavirus, vaginal metabolome, vaginal microbiota. Tweetable abstract Metabolomics study: Vaginal microenvironment of HPV + women may be informative for nonsurgical interventions. Please cite this paper as: Borgogna JC, Shardell MD, Santori EK, Nelson TM, Rath JM, Glover ED, Ravel J, Gravitt PE, Yeoman CJ, Brotman RM. The vaginal metabolome and microbiota of cervical HPV-positive and HPV-negative women: a cross-sectional analysis.

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Introduction Cigarette smoking is strongly associated with bacterial vaginosis (BV) and a low-Lactobacillus vaginal community state type (CST). Metabolite profiles have previously been shown to delineate BV and non-BV women and appear to be influenced by microbial composition. Therefore, we sought to determine if vaginal metabolites varied between smokers and non-smokers within each stratum of CST. Methods Forty reproductive-aged women (20 smokers/20 non-smokers) were recruited. Vaginal bacterial composition was char-acterised by 16S rRNA gene analysis. Metabolic profiles were determined by GC/MS and LC/MS and compared to libraries of known metabolites. Data were analysed with Random Forests, a method that uses decision trees to rank metabolite importance. Results We identified 619 metabolites from mid-vaginal swab eluates-threefold more than previously described in the vagina. Women were categorised into CST-I (L. crispatus-dominated), CST-III (L. iners-dominated) and CST-IV (low-Lactobacillus/high anaerobes). Metabolites were strongly separated by CST (F = 3.0855, P PERM = 0.0001). Within each CST, significant differences in metabolic profiles of smokers and non-smokers were evident. Nicotine and the breakdown metabolite cotinine was higher in smokers versus non-smokers from all CSTs. Smokers in CST-I had higher concentrations of xanthosine and paraxanthine. Smokers in CST-III were reduced in their relative concentrations of N-stearoyltaurine and 4-methylcatechol sulfate. Biogenic amines agmatine, cadaverine and spermidine were increased in smokers from CST-III and IV. Smokers from CST-IV had higher concentrations of kynurenate, 3-methyl-2-oxovalerate and palmitoyl ethanolamide. Conclusion The metabolite profile of the vaginal tract is strongly influenced by the vaginal microbiota. Detection of nicotine and breakdown products in the vagina may serve as molecular bio-markers of smoking. Kynurenate and biogenic amines have known roles in lymphocyte proliferation and inflammation and may indicate bacterial immune-and stress-resistance. Our results suggest that smoking affects several important metabolites present in the vagina that may have implications for women's health. Disclosure of interest statement We declare no conflict of interest.