During pregnancy, the vaginal microbiome plays an important role in both maternal and neonatal health outcomes. Throughout pregnancy, the vaginal microbial composition undergoes significant changes, including a decrease in overall diversity and enrichment with Lactobacillus spp. In turn, the modifications in the microbial profiles are associated with shifts in the composition of vaginal metabolites. In this study, we characterized the vaginal metabolic profiles throughout pregnancy at two different gestational ages, correlating them with a microscopic evaluation of the vaginal bacterial composition. A total of 67 Caucasian pregnant women presenting to the Family Advisory Health Centres of Ravenna (Italy) were enrolled and a vaginal swab was collected at gestational ages 9-13 weeks (first trimester) and 20-24 weeks (second trimester). The composition of the vaginal microbiome was assessed by Nugent score and women were divided in 'H' (normal lactobacilli-dominated microbiota), 'I' (intermediate microbiota), and 'BV' (bacterial vaginosis) groups. Starting from the cell-free supernatants of the vaginal swabs, a metabolomic analysis was performed by means of a 1H-NMR spectroscopy. From the first to the second trimester, a greater number of women showed a normal lactobacilli-dominated microbiota, with a reduction of cases of dysbiosis. These microbial shifts were associated with profound changes in the vaginal metabolic profiles. Over the weeks, a significant reduction in the levels of BV-associated metabolites (e.g. acetate, propionate, tyramine, methylamine, putrescine) was observed. At the same time, the vaginal metabolome was characterized by higher concentrations of lactate and of several amino acids (e.g. tryptophan, threonine, isoleucine, leucine), typically found in healthy vaginal conditions. Over time, the vaginal metabolome became less diverse and more homogeneous: in the second trimester, women with BV showed metabolic profiles more similar to the healthy/intermediate groups, compared to the first trimester. Our data could help unravel the role of vaginal metabolites in the pathophysiology of pregnancy., Competing Interests: The authors have declared that no competing interests exist.