Cervicovaginal Microbiome Composition Is Associated with Metabolic Profiles in Healthy Pregnancy

Abstract

Microbes and their metabolic products influence early-life immune and microbiome development, yet remain understudied during pregnancy. Vaginal microbial communities are typically dominated by one or a few well-adapted microbes which are able to survive in a narrow pH range and are adapted to live on host-derived carbon sources, likely sourced from glycogen and mucin present in the vaginal environment. We characterized the cervicovaginal microbiomes of 16 healthy women throughout the three trimesters of pregnancy. Additionally, we analyzed saliva and urine metabolomes using gas chromatography-time of flight mass spectrometry (GC-TOF MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) lipidomics approaches for samples from mothers and their infants through the first year of life. Amplicon sequencing revealed most women had either a simple community with one highly abundant species of Lactobacillus or a more diverse community characterized by a high abundance of Gardnerella, as has also been previously described in several independent cohorts. Integrating GC-TOF MS and lipidomics data with amplicon sequencing, we found metabolites that distinctly associate with particular communities. For example, cervicovaginal microbial communities dominated by Lactobacillus crispatus have high mannitol levels, which is unexpected given the characterization of L. crispatus as a homofermentative Lactobacillus species. It may be that fluctuations in which Lactobacillus dominate a particular vaginal microbiome are dictated by the availability of host sugars, such as fructose, which is the most likely substrate being converted to mannitol. Overall, using a multi-"omic" approach, we begin to address the genetic and molecular means by which a particular vaginal microbiome becomes vulnerable to large changes in composition.IMPORTANCE Humans have a unique vaginal microbiome compared to other mammals, characterized by low diversity and often dominated by Lactobacillus spp. Dramatic shifts in vaginal microbial communities sometimes contribute to the presence of a polymicrobial overgrowth condition called bacterial vaginosis (BV). However, many healthy women lacking BV symptoms have vaginal microbiomes dominated by microbes associated with BV, resulting in debate about the definition of a healthy vaginal microbiome. Despite substantial evidence that the reproductive health of a woman depends on the vaginal microbiota, future therapies that may improve reproductive health outcomes are stalled due to limited understanding surrounding the ecology of the vaginal microbiome. Here, we use sequencing and metabolomic techniques to show novel associations between vaginal microbes and metabolites during healthy pregnancy. We speculate these associations underlie microbiome dynamics and may contribute to a better understanding of transitions between alternative vaginal microbiome compositions.

Keywords: Lactobacillus; longitudinal; metabolome; microbiome; pregnancy; vagina.

Copyright © 2020 Oliver et al.

Figures

FIG 1

Study outline. Eighteen women were sampled throughout pregnancy and their offspring were sampled at birth and 6 and 12 months of age. Samples collected were urine, saliva, and cervical vaginal fluid (CVF) from the mothers and urine and saliva from the children. CVF was sequenced using shotgun metagenomics and amplicon sequencing. All samples were analyzed using GC-TOF MS and lipidomics.

FIG 2

Taxonomy and alpha diversity of vaginal microbiomes during pregnancy. (A) Relative abundance plot of operational taxonomic units, from 16S amplicon data, grouped together by individual. Each individual is clustered into a larger category defined by the dominating microbe. (B) Presence or absence of fungi, at the genus level, per sample. Linear mixed-effects models (LME) were done on the alpha diversity metrics to account for repeated measures in the data. (C) Evenness between samples dominated by Lactobacillus (n = 34 samples) is significantly lower than samples dominated by Bifidobacteriaceae (n = 7 samples). (D) No significant change in the observed OTUs between the trimesters (n = 15, 13, and 14 samples, respectively) of pregnancy and likewise. (E and F) There was no change in evenness (E) or phylogenetic diversity (F) throughout pregnancy.

FIG 3

Ordination of vaginal microbiomes during pregnancy. (A) Nonmetric multidimensional scaling (nMDS) of Bray-Curtis dissimilarity between vaginal microbiomes (n = 42 samples) of mothers. Color indicates the most abundant microbe within the microbial community. The most abundant microbe in the community plays a statistically significant role in the composition of the community (LME; R2 = 69%; P < 0.0001). (B) Some participants (6/16 individuals) experienced large, significant shifts (LME; P = 0.0219) in their microbiomes throughout the trimesters of pregnancy.

FIG 4

Relationship between vaginal microbes and metabolites. (A) Distance-based linear model recapitulates the relationship between the vaginal microbiomes of these subjects (n = 42 samples). Superimposed are vectors showing which annotated GC-TOF MS molecules are best correlated with these microbial communities. Length and direction of vectors correspond to the strength of the association between the metabolite and the microbial communities. Box plots show the raw abundance (n = 45 samples) of indole-3-lactate (B) and mannitol (C).

FIG 5

Ordination of functional pathways within the vaginal microbiome. An nMDS of HUMAnN2 analysis, examining the abundance of pathways in each microbiome (n = 35 samples). Vaginal microbiomes have functions that are indicative of the most abundant microbe present in the samples.

FIG 6

Proposed vaginal microbial community model. Current hypothesized model of vaginal microbial community physiology, with gaps in understanding (denoted by question marks) where future work is needed. Our study indicates that mannitol production is associated with a high relative abundance of L. crispatus.