Demographic and clinical characteristics of the study population
We enrolled 22 women with POF and 29 healthy controls for analysis. The clinical characteristics of the two groups are shown in Table 1. Among patients with POF, the mean age was 30.50 ± 3.17 years, body mass index (BMI) was 22.34 ± 3.32, waist-to-hip ratio was 0.83 ± 0.04. Among the healthy control group, the mean age was 29.79 ± 3.99 years, BMI was 23.47 ± 3.51, and waist-to-hip ratio was 0.84 ± 0.06. The age, BMI, and waist-to-hip ratio were not significantly different between the two groups (P > 0.05), while AMH and E2 were significantly lower in the POF group (P < 0.001). Levels of FSH and LH were higher among patients with POF compared with control (P < 0.001 and P < 0.01, respectively). Among menopausal women, the mean age was 57.96 ± 6.57 years, BMI was 23.57 ± 3.21, and menopause had been experienced for at least 1 year.
Microbial profiling
The mean community diversity indexes (alpha diversity, including Chao1, observed species, Shannon, and Simpson indices) were significantly higher in the POF group compared with the control group (Figure 1A, Supplementary Figure 2, P < 0.01). Beta diversity was also significantly different between groups according to the weighted UniFrac phylogenetic distance matrices (analysis of similarities, R = 0.175, P = 0.002), and showed in PCoA plots (Figure 1B and C). Thus, the vaginal microbiota of the POF group was significantly different to that of the control group . The detailed 16S rRNA raw sequence data were available in the NCBI Sequence Read Archive (SRA) under accession number SRP594533
Abundance of taxa in the two groups
By LEfSe analysis, we identified 51 genera-discriminative features (Figure 2A, LDA > 2, P < 0.05). Comparison of vaginal microbiota by the Mann-Whitney U test revealed 51 taxa that were differentially abundant between the groups (P < 0.05); the species of the top 20 are shown in Figure 2B. The agreement of results of the two analytical methods indicates the stability of the vaginal microbiological data.
The relative abundance of bacterial taxonomic groups at the genus level showed that ten genera including Gardnerella, Prevotella, Bacteroides, Sneathia, Dialister, and Anaerococcus were abundant in the POF group. Only Lactobacillus was found to be abundant in the control group (Supplementary Table 1). The Lactobacillus members are mainly grouped into two species: L. gallinarum was the most abundant, followed by L. iners and L.jensenii (Supplementary Table 2) in the control group. However, as our observation in women with POF, the relative abundance of L. iners increased as the colonization of L. gallinarum and L.jensenii dropped (Figure 2C).
A Spearman heatmap was constructed to identify correlations among the above-mentioned genera, which revealed Lactobacillus to be negatively correlated with other genera (Figure 2D). Lactobacillus is a probiotic which plays an important role in human health[31, 32]. Thus, studying the interactions of this genus with other genera could provide insight into the functions of these species in the development of POF.
Analyses of correlations between reproductive-related clinical indicators and vaginal flora
Redundancy analysis was used to produce a two-dimensional sorting map relating vaginal flora to reproductive-related clinical indicators. Serum FSH and LH levels showed the greatest association with female vaginal flora, and E2 had a significant effect. Gardnerella and Prevotella were positively correlated with serum FSH and LH, and negatively correlated with E2. Lactobacillus in the vagina were positively correlated with E2 and negatively correlated with FSH and LH. Similar to E2, AMH was positively correlated with Lactobacillus and negatively correlated with Gardnerella and Prevotella (Figure 3A). Subsequently, we analyzed the correlation between the Lactobacillus members and clinical indicators. The relative abundance of L. gallinarum was negatively corrected with FSH、LH and P level. Meanwhile, the abundance of L. gallinarum was positively related to E2 level and there was a significant difference(P<0.05). In addition, the relative abundance of L. iners was negatively related to E2 level. That means L. gallinarum and L. iners play a weighted part in female reproductive health (Figure 3B).
Next, the predictive model from the random forests system was based on the vaginal flora profile including the taxon, taxon that exhibited significantly different abundances at the genus level from Wilcoxon rank-sum test. We identified 34 genera that could be used to predict occurrence of POF with the random forests model (Figure 4A). A mean classification error of 0.382 was achieved, and the AUC was 0.841 (95% confidence interval [CI]: 0.618–1, sensitivity: 71.4%, specificity: 100%, cut-off rate: 43.2%; Figure 4B).
Functional alterations in the vaginal flora
Then, we analyse the metabolic pathways of the two groups of subjects. The predicted genome database has been greatly expanded such that in addition to metabolic pathways, related enzymes, genes and other information can be obtained. LEfSe analysis identified seventeen KO identifiers enriched in women with POF patients including K02014(iron complex outermembrane recepter protein),K07497(putative transposase),K00123(formate dehydrogenase major subunit),K00799(glutathione S-transferase),K01223(6-phospho-beta-glucosidase),etc.(Figure 5A).
Figure 5B shows the results of Metacyc, the predictive functions performed using PICRUSt2.0. The pathways that were significantly enriched in POF were gondoate biosynthesis, fatty acid elongation – saturated, palmitoleate biosynthesis I (from (5Z)-dodec-5-enoate), superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass, superpathway of tetrahydrofolate biosynthesis and salvage, pyridoxal 5'-phosphate biosynthesis I, superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis, D-galactarate degradation I and so on. Whereas the microbial functions related to aerobic respiration I (cytochrome c), myo-, chiro- and scillo-inositol degradation, adenosine nucleotides degradation II, superpathway of L-serine and glycine biosynthesis I were higher in the vaginal microbiota of the POF group(Supplemental Table 3).
Comparison of vaginal flora in the case of premature ovarian failure or menopause
Finally, we compared the vaginal microbial composition of patients with POF and menopausal women. The high abundance of Lactobacillus, Gardnerella, and Prevotella was confirmed from comparison of vaginal micribiota of women with POF and menopausal individuals. However, the flora of menopausal women exhibited increased diversity (Figure 6A and B). Differential species analysis showed Lactobacillus to be less abundant among menopausal women than those with POF (Figure 6C).