Association between vaginal microbiota and vaginal immune markers in postmenopausal people

Objectives: In premenopausal individuals, vaginal microbiota diversity is associated with greater mucosal inflammation, which is linked to higher risk of cervical dysplasia and infections. It is not known if this association is present after menopause, when the microbiota is likely to have high diversity.

Methods: This is a post-hoc analysis of a subset of postmenopausal individuals with moderate-severe vulvovaginal discomfort enrolled in a randomized trial of vaginal estradiol tablet + placebo gel, vaginal moisturizer + placebo tablet or dual placebo. Individuals with other explanations for symptoms were excluded (e.g. vaginal infections, lichen sclerosis). Vaginal fluid samples from 0, 4 and 12 weeks were characterized using 16S rRNA gene sequencing (microbiota) and MesoScale Discovery (vaginal fluid immune markers: IL1β, IL1α, IL2, IL6, IL18, IL10, IL9, IL13, IL8, IP10, MIP1α, MIP1β, MIP3α). Global associations between cytokines (individual values, or first Principal Component [PC1] of cytokines) and microbiota (assessed by relative abundance of individual taxa, Shannon index for alpha diversity, and beta diversity measured by Bray-Curtis distance) were explored, adjusting for treatment arm, using linear mixed models or GLMM MiRKAT.

Results: A total of 119 individuals with mean age 61 were included, 40 estradiol + placebo, 37 moisturizer + placebo and 39 dual placebo. Across all timepoints, beta diversity (p< 0.00001) and alpha diversity (Shannon index, p 0.003;Figure 1) were highly associated with PC1 of immune markers. Over 12 weeks, change in alpha diversity was associated with change in PC1 (Shannon, p=0.018). However, change in beta diversity characterized by Bray-Curtis distance was not associated with a change in PC1 (p=0.15). Of the top 30 taxa associated with PC1, 7 were Lactobacillus or Bifidobacterium species. Although L. crispatus and L. iners had similar prevalence (detected in 11% and 22% of samples, respectively, at an abundance of >1%), L. iners was not found to be associated with PC1. A diverse community at week 0 shifted to Lactobacillus/Bifidobacterium-dominant community at week 12 in 66 people, but this transition was not associated with a change in PC1. Individual markers that were significantly lower in people with Lactobacillus dominance vs. more diverse communities were similar to those observed in premenopausal people: IL1b, IL6, IL8, IL10 (p< 0.001, FDR< 0.01 for all; Figure 2), IL13 (p=0.002, FDR=0.02) and IL2 (p=0.01, FDR=0.09).

Conclusions: In this cohort of postmenopausal individuals, Lactobacillus dominance and lower alpha diversity are associated with lower concentrations of inflammatory immune markers, as has been reported in premenopausal people. This suggests that after menopause lactobacilli continue to have beneficial effects on mucosal immunity, despite lower prevalence.