Vaginal microbiome and epithelial gene array in post-menopausal women with moderate to severe dryness

Abstract

After menopause, many women experience vaginal dryness and atrophy of tissue, often attributed to the loss of estrogen. An understudied aspect of vaginal health in women who experience dryness due to atrophy is the role of the resident microbes. It is known that the microbiota has an important role in healthy vaginal homeostasis, including maintaining the pH balance and excluding pathogens. The objectives of this study were twofold: first to identify the microbiome of post-menopausal women with and without vaginal dryness and symptoms of atrophy; and secondly to examine any differences in epithelial gene expression associated with atrophy. The vaginal microbiome of 32 post-menopausal women was profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Sixteen subjects were selected for follow-up sampling every two weeks for 10 weeks. In addition, 10 epithelial RNA samples (6 healthy and 4 experiencing vaginal dryness) were acquired for gene expression analysis by Affymetrix Human Gene array. The microbiota abundance profiles were relatively stable over 10 weeks compared to previously published data on premenopausal women. There was an inverse correlation between Lactobacillus ratio and dryness and an increased bacterial diversity in women experiencing moderate to severe vaginal dryness. In healthy participants, Lactobacillus iners and L. crispatus were generally the most abundant, countering the long-held view that lactobacilli are absent or depleted in menopause. Vaginal dryness and atrophy were associated with down-regulation of human genes involved in maintenance of epithelial structure and barrier function, while those associated with inflammation were up-regulated consistent with the adverse clinical presentation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist, with the exception of BV and DK. All authors have read the journal′s policy and have the following conflicts. BV and DK are scientists employed by Kimberly Clark, the company that provided a grant-in-aid for the study. They support the objective and complete presentation of all the results obtained in the study, plus the peer review, editorial decision making, and publication of the research. The stated competing interest did not alter the authors′ adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Microbiota profiles for 32 post-menopausal women clustered by biota similarity.

Each bar represents a single vaginal sample, and the colored segments represent the relative fraction of each bacterial taxon detected at 1% relative abundance or greater in any one sample. Sequences at less than 1% abundance have been included in the “remainder” fraction at the top of the bar (see color legend of bacterial taxa). The microbiota are clustered by similarity as represented in the dendogram above. The sample name (participant ID-time point) is labeled in the dendogram and corresponds to the bar below. The dryness score as observed by the examining nurse is represented below each microbiota bar.

Figure 2. Time-series microbiota profiles for 16 post-menopausal sampled every 2 weeks.

Each bar represents a single vaginal sample, and each cluster of bars is a single participant (starting at time 0 and sampled every 2 weeks for up to 10 weeks total). The colored segments represent the relative fraction of each bacterial taxon detected at 1% relative abundance or greater. Sequences at less than 1% abundance have been included in the “remainder” fraction at the top of the bar (see color legend of bacterial taxa). The dryness score as observed by the examining nurse is represented below each microbiota bar. Sample time points that were included in the microarray analysis are marked with an arrowhead: the first six green arrows are controls (no or mild dryness), and the last four red arrows are women experiencing moderate to severe dryness.

Figure 3. Heatmap of vaginal epithelial gene expression of 10 samples.

A clustered heatmap of differential gene expression (>2-fold change, p<0.05) between the control and dryness groups. Samples are labeled as participant number-time point (week). Samples assigned to the dryness or control groups based on physiological examination of the vagina are well separated by gene expression differences with exception of 4–8 which has an intermediate gene expression profile and clusters closer to the dryness group.