Microbiota composition at six sites within the female reproductive tract

To explore the microbiota beyond the vagina, we collected samples from six locations (CL, lower third of vagina; CU, posterior fornix; CV, cervical mucus drawn from the cervical canal; ET, endometrium; FLL and FRL, left and right fallopian tubes; PF, peritoneal fluid from the pouch of Douglas) throughout the female reproductive tract from an initial study cohort of 95 Chinese women submitted to surgery for conditions not known to involve infections (Fig. 1). These conditions included hysteromyoma (also known as uterine fibroids), adenomyosis, endometriosis and salpingemphraxis, which are to our knowledge the best proxies for the upper reproductive tract in healthy young women (Supplementary Data 1). Samples from the vagina and the cervical mucus (CL, CU, CV) were taken upon visit to the clinic (without any prior disturbance). Samples from the peritoneal and uterine sites (PF, FL, ET) were taken during laparoscopy or laparotomy from minimally invasive surgery cuts to avoid possible contamination from the vaginal microbiota if the samples were to go through the cervical os (Fig. 1, Supplementary Fig. 1). The samples were subjected to 16S rRNA gene amplicon sequencing, quality-controlled and clustered into operational taxonomic units (OTUs) (Supplementary Data 2, 3).

Fig. 1 Composition of the vagino-uterine microbiota. a, b PCoA on the samples based on weighted (a) and unweighted (b) UniFrac distances. Samples were taken from CL, CU and CV before operation, and from ET, FLL, FRL and PF during operation. Each dot represents one sample. c Pie chart for the microbial genera at each body site according to the median relative abundance. Genera that took up <1% of the microbiota were labelled together as ‘others’. A pink rod represents about 102 copies/sample, according to the qPCR results in Supplementary Fig. 5e. Samples derive from the study cohort of 95 reproductive-age women (n = 94 CL, 95 CU, 95 CV, 80 ET, 93 PF, 9 FLL and 10 FRL, Supplementary Data 1) Full size image

The lower (CL, CU, CV) and upper (ET, FL, PF) reproductive tract samples separated when subjected to principal coordinate analyses (PCoA) based on weighted and unweighted UniFrac distances (Fig. 1a, b). The FLL and FRL showed essentially the same microbiota (Fig. 1a, b), and were therefore clustered into a common FL category for further analysis (except for calculation of intra-individual and inter-individual differences, referred to later).

Consistent with previous reports3,4,5, the CL as well as the CU were dominated by the Lactobacillus genus (Fig. 1c), and exhibited a low α-diversity (Supplementary Fig. 2a). At the species level, the CL and CU samples contained L. crispatus, L. iners and other Lactobacillus spp. (Supplementary Fig. 2b), similar to previous reports from the US3,4,5. Notably, CV samples generally contained a lower proportion of Lactobacillus than the vaginal samples (Fig. 1c), and varied among the sampled individuals (Supplementary Fig. 3a). In the ET samples, Lactobacillus no longer dominated, and bacteria such as Pseudomonas, Acinetobacter, Vagococcus and Sphingobium constituted a notable fraction of the microbiota (Fig. 1c, Supplementary Fig. 3b). The proportion of these bacteria further increased at the openings of the FL, leaving a median relative abundance of 1.69% for Lactobacillus (Fig. 1c, Supplementary Fig. 3d). The PF samples generally lacked the presence of Lactobacillus, but otherwise harboured a microbiota as diverse but not completely the same as the FL samples (Fig. 1, Supplementary Fig. 3c).

At the phylum level, the Firmicutes-dominated lower reproductive tract microbiota contrasted the large proportions of Proteobacteria, Actinobacteria and Bacteroidetes in the upper reproductive tract (Supplementary Fig. 3e).

As noted above, we took samples from the uterine and peritoneal sites without going through the cervical os to address a major concern in the field that transcervical samples might be contaminated by the vaginal microbiota. We nonetheless tested the specificity of sampling route by collection of ET samples via the uterus as well as through the cervical os, and of CV from the cervical os as well as from the uterine end. The distribution of bacteria in the samples taken through the cervical os showed high similarity to that in samples taken by opening the uterus during surgery (Supplementary Fig. 4a, b), suggesting that both the uterine and cervical microbiota could be readily accessed and analysed in women not undergoing surgery.

Estimation of bacterial biomass in the female reproductive tract

In order to provide some absolute measure of the microbiota beyond the vagina, we developed a species-specific real-time qPCR approach, focusing on the dominant vaginal bacteria Lactobacilli. The abundances of L. crispatus, L. iners, L. gasseri and L. jensenii monotonously decreased from CL, CU, CV, to ET and PF (Supplementary Fig. 5a–d), consistent with genus level data from 16S rRNA gene amplicon sequencing (Fig. 1). The concordance between qPCR and amplicon sequencing results were further confirmed by Spearman’s correlation, which showed a correlation coefficient of 0.72 for L. crispatus and 0.56 for L. iners, the two dominant Lactobacilli species (Supplementary Fig. 2b).

The total bacterial biomass within each site was then calculated based on the copy number from qPCR, divided by the corresponding relative abundance in the sample according to 16S rRNA gene sequencing. This calculation revealed that, while the vaginal sites contained about four orders of magnitude more bacteria (1010–1011), the PF samples contained similar bacterial biomass as the ET samples (Supplementary Fig. 5e), which are orders of magnitude above potential background noise9. The negative controls (sterile phosphate-buffered saline (PBS), sterile saline and ultrapure water) showed much higher cycle threshold (Ct values) than the low biomass ET and PF samples (Supplementary Table 1, Supplementary Fig. 1), signifying that much less bacterial DNA was detected in non-bio control samples. It is thus important to bear in mind that the higher bacterial diversity in the upper reproductive tract corresponds to a lower bacterial biomass than the much better established vaginal microbiota (Fig. 1c).

Cultivation of live bacteria from the upper reproductive tract

Another important question is whether live bacteria rather than debris makes up the bacterial DNA signal in the upper reproductive tract samples. Hence, in a validation study we performed 16S rRNA gene amplicon sequencing along with culturing of live bacteria from an additional cohort of 15 women.

Of the 21 diluent negative controls, 19 had lower DNA concentrations and library concentrations (close to zero) than their respective tissue samples (Supplementary Data 4), again confirming that there was little DNA in the non-bio control samples. In PCoA, microbial profiles differed between the low biomass upper reproductive tract samples and diluent controls (Supplementary Fig. 6, sterile saline for the peritoneal samples and PBS for the remaining samples).

The same samples extracted at different times, diluted or undiluted before amplification, or sequenced in different runs showed high reproducibility, indicating that random contamination had no major impact on bacterial community structure detected in the upper reproductive tract (Supplementary Table 2, Supplementary Data 5).

We further attempted to culture and isolate live bacteria from the freshly collected samples. PF samples were plated on peptone yeast extract-glucose (PYG) agar with 5% horse blood and incubated aerobically or anaerobically. Positive bacterial cultures were obtained from 5 out of 15 subjects, resulting in 8 different isolates belonging to 7 genera such as Lactobacillus, Staphylococcus and Actinomyces (Supplementary Table 3). These genera were also detected in our 16S rRNA gene amplicon sequencing data, and supported by previous cultivation of vaginal or amniotic samples10,11,12,13,14,15,16 (Supplementary Table 3). Reassuringly, no bacteria were isolated from the diluent-negative controls (including swabs from sterilised skin samples from the patients and the gloves used by doctors). Thus, live bacteria do exist in the upper reproductive tract, of which we succeeded in isolation of some using conventional culturing methods.

Signature species for each site of the reproductive tract

To get more insight into the ecology of the vagino-uterine communities, we defined signature OTUs for each body site considering both the occurrence and abundance of the given OTUs (P < 0.05 in multi-level pattern analysis, and indicator value index (IndVal) >0.5, Fig. 2, Supplementary Data 6). L. iners and L. crispatus were identified as signature species of the vaginal sites (CL and CU). No signature OTUs with IndVal >0.5 was identified for CV or ET, suggesting that they are bacterial transition zones between the vagina and the upper anatomical sites. The FL had a number of signature OTUs including Pseudomonas, Erysipelothrix and Facklamia (Fig. 2, Supplementary Data 6). The PF featured many signature OTUs, including Pseudomonas, Morganella, Sphingobium and Vagococcus, all of which increased monotonously from the CU and the CV to the ET and PF (Supplementary Data 6). In support of the existence of these taxa in the upper reproductive tract, they have been reported in previous studies of the vaginal microbiota in humans or animals17,18,19,20,21. Overall, we observed the female reproductive tract as a habitat for facultative anaerobes or aerobes; the upper sites contained a variety of bacteria that grow in mildly alkaline conditions, contrasting the Lactobacillus-dominated low pH environment of the vagina.

Fig. 2 Signature species for each body site. Heatmap for the relative abundances of signature OTUs from each body site (P < 0.05, multi-level pattern analysis, and IndVal >0.5). Each bar represents one sample (n = 94 CL, 95 CU, 95 CV, 80 ET, 93 PF, 9 FLL and 10 FRL, Supplementary Data 1) Full size image

Intra-individual and inter-individual similarity in the vagino-uterine microbiota

To examine the microbiota relationship at the six sites of the reproductive tract, we computed the intra-individual and inter-individual distances between samples from each individual. Weighted intra-individual UniFrac distance relative to CL samples gradually increased from CU and CV to ET and PF (Fig. 3a), consistent with the anatomical contingency of these sites (Fig. 1). The same was also true in reverse order, i.e. weighted intra-individual UniFrac distance relative to PF gradually increased from ET, CV, to CU and CL (Fig. 3b). Samples from the same individual were highly correlated, and the Sorenson indices between body sites were consistent with their anatomical contingency (Fig. 3c). The intra-individual correlation was notable even between the less invasive sampling site of CV and the less reachable PF (median of Sorenson index = 0.255, Fig. 3c), hinting at the possibility of minimally invasive surveys of the uterine and peritoneal microbiota by cervical mucus sampling in the general population.

Fig. 3 Similarity of the vagino-uterine microbiota within and between individuals. a, b Weighted UniFrac distance of the microbiota at each body site relative to the CL (a) and PF (b) in the same individual. Boxes denote the interquartile range (IQR) between the first and third quartiles (25th and 75th percentiles, respectively), and the line inside the boxes denote the median. The whiskers denote the lowest and highest values within 1.5 times the IQR from the first and third quartiles, respectively. Circles represent data points beyond the whiskers. c Intra-individual Sorenson indices between different body sites. d Inter-individual Sorenson indices between different body sites. Samples derive from the study cohort of 95 reproductive-age women (n = 94 CL, 95 CU, 95 CV, 80 ET, 93 PF, 9 FLL and 10 FRL, Supplementary Data 1) Full size image

Of note, the clear distinction between the upper and lower reproductive tract, intersecting at CV and ET, remained intact even when performing an inter-individual comparison (Fig. 3d) (although the level of the inter-individual indices diminished from that of the intra-individual (Fig. 3c)), hence underscoring both anatomical as well as individual-specific features of the vagino-uterine microbiota.

Site-specific community types in the vagino-uterine microbiota

To further understand the relationship between communities at each site, we examined community types in all samples using the Dirichlet multinomial mixture (DMM) model5. Of the five community types detected by DMM (Supplementary Fig. 7), Type 1 and 5 were dominated by the Lactobacillus genus, which was present in notable amounts also in Type 2, 3 and 4 (Fig. 4a, b, Supplementary Data 7). Type 4, which contained a higher proportion of Prevotella and might correspond to the more diverse vaginal microbiota type reported previously3,4,5, was detected in some of the CV and ET samples but was not found in the FL and PF samples of this cohort (Fig. 4, Supplementary Fig. 7). The major type in PF, Type 2 was also present in CV, while Type 3 was present at all sites, yet dominating in the FL samples (Fig. 4, Supplementary Fig. 7).

Fig. 4 Community types of the vagino-uterine microbiota. a Distribution of samples among the five community types at each body site (n = 94 CL, 95 CU, 95 CV, 80 ET, 93 PF, 9 FLL and 10 FRL, Supplementary Data 1). b Relative abundances of the top 8 most abundant genera in the 5 community types. The boxes denote the interquartile range (IQR) between the first and third quartiles (25th and 75th percentiles, respectively), and the line inside the boxes denote the median. The whiskers denote the lowest and highest values within 1.5 times the IQR from the first and third quartiles, respectively. Circles represent data points beyond the whiskers. c Community types connected between neighbouring sites according to individual subjects. Due to the small sample size, results for the fallopian tubes were plotted in a separate graph (Supplementary Fig. 7c). The width of each line indicates the number of subjects (n = 1–95) Full size image

When we examined transitions between community types at neighbouring sites, most subjects with Type 1 in the vagina (CL) remained Type 1 at the CU, and changed to Type 5 either at CV or at ET, and then turned to Type 2 in PF (Fig. 4c). By contrast, subjects with Type 3 or 4 at the vaginal sites rarely became Type 5 at the uterine sites, while some Type 4 subjects were found to switch to Type 3 in the upper part (Fig. 4b, Supplementary Fig. 7c). These findings corroborated our aforementioned results that there is an intra-individual continuum of the microbiota from the vagina to the peritoneal fluid that gradually changes according to the habitat.

Lifestyle and clinical factors associated with microbiota changes

The vaginal microbiota has previously been reported to vary between ethnic groups (in the US) and the menstrual cycle3, 4. With our comprehensive collection of metadata (Supplementary Data 1), we examined whether various clinical and lifestyle factors were associated with changes in the vagino-uterine microbiota in women of reproductive age. Factors such as age of initial sexual intercourse, duration of menstrual period, and gravida and para appeared associated with microbiota composition at one or more sites, but failed to reach statistical significance after controlling for multiple testing (permutational multivariate analysis of variance (PERMANOVA), P < 0.05 but q > 0.05, Supplementary Data 8 and 9). Having given birth (multipara versus nullipara) was associated with microbiota composition in CV and PF (q < 0.05, Supplementary Data 9). Herbal medication was associated with microbiota composition in the CV only (q < 0.05, Supplementary Data 9). Age, hysteromyoma, adenomyosis and endometrosis were also associated with microbiota composition in the PF (q < 0.05, Supplementary Data 8, 9). Therefore, variations in the composition of the vagino-uterine microbiota are associated with a number of natural and diseased physiological conditions important for women’s health.

Identification of diseases based on the vagino-uterine microbiota

We further examined whether variations in the vagino-uterine microbiota composition were associated with different physiological conditions and diseases of the reproductive tract.

In relation to hysteromyoma (benign tumours in the uterus), Lactobacillus sp. were found to be more abundant in the vaginal and cervical samples of individuals with no hysteromyoma, while L. iners was more abundant in the CV of individuals with hysteromyoma (Supplementary Fig. 8). Among the hysteromyoma-enriched OTUs, one OTU in the CU and 11 OTUs in the PF were also identified to be influenced by the stage in the menstrual cycle (Spearman’s correlation coefficient >0.3 or <−0.3, q < 0.05, Supplementary Fig. 8); however, none of these OTUs showed a correlation with age. Overall, the results were consistent with hysteromyoma being associated with a minimally altered vagino-uterine microbiota.

Subjects with adenomyosis (abnormal presence of endometrial tissue within the myometrium) showed depletion or enrichment of many bacteria throughout the reproductive tract, some of which overlapped with bacteria associated with anaemia (Supplementary Figs. 9 and 10), which was consistent with clinical links between the two conditions (P = 0.001062 between the two conditions in our cohort, Fisher’s exact test). Furthermore, cross-validated random forest models distinguished subjects with adenomyosis from those without according to OTUs from any of the sites (Supplementary Fig. 11, Supplementary Data 10).

The microbiota-based models also allowed us to distinguish subjects without and with infertility attributed to endometriosis, a condition where endometrial cells present abnormal growth outside the uterus, and a major cause of infertility (Fig. 5, Supplementary Data 11). Altogether, these results suggest that the composition of the vagino-uterine microbiota potentially could be used to detect a number of ailments common to reproductive age women.

Fig. 5 Microbiota-based classification of infertility due to endometriosis. a, b, c, d, e Distribution of 5 trials of 10-fold cross-validation error in random forest classification of fertile versus infertile samples as the number of OTUs increases (a, CL; b, CU; c, CV; d, ET; e, PF). The model was trained using relative abundance of the OTUs (present in at least 10% of the samples) in the samples (n = 16 without endometriosis (fertile), 32 with endometriosis (infertile)). Subjects with endometriosis who had given birth, and infertile subjects due to reasons other than endometriosis, e.g. salpingemphraxis, were not analysed (Supplementary Data 1). The red curve indicates an average of the five trials (pink lines). The grey line marks the number of OTUs in the optimal set. (f, g, h, i, j) Receiver operating curve (ROC) for the cross-validated sample set (f, CL; g, CU; h, CV; i, ET; j, PF). The area under receiver operating curve (AUC) is 0.8272, 0.5919, 0.8493, 0.8304 and 0.8613, respectively. The 95% confidence intervals (CI) are shown as shaded areas. The diagonal lines mark an AUC of 0.5 (i.e. random classification). Full size image

Functional inferences of the reproductive tract microbiota

To estimate the functional capacities of the vagino-uterine microbiota, we predicted KOs (Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology groups) from the 16S rRNA gene data.

In the case of adenomyosis, the analyses revealed that adenomyosis affected functions throughout the vagino-uterine microbiota (Supplementary Fig. 12). The microbiota in subjects with adenomyosis were especially enriched in pathways involved in flagella assembly and biosynthesis of aromatic amino acids, while the microbiota in subjects without adenomyosis were enriched in phosphotransferase system and fatty acid biosynthesis. These results suggest that functionally, the microbiota is also a continuum in the reproductive tract, and could be perturbed at multiple sites in a disease.

Menstrual cycle relates to the vagino-uterine microbiota and its function

Differences in the vagino-uterine microbiota were also identified between phases of the menstrual cycle, as was apparent from cross-validated random-forest models that classified samples into different phases of the cycle (Supplementary Fig. 13). OTUs that led to optimal classification included L. iners, and Lactobacillus sp. from vaginal sites, Sphingobium sp., Propionibacterium acnes and Pseudomonas sp. from the ET or PF, which were differentially enriched during the proliferative and secretory phases (Supplementary Fig. 13c). Notably, P. acnes was more abundant in the secretory phase in the ET (Supplementary Fig. 13l), and has previously been identified in the placenta and cultured from follicular fluid8, 22,23,24. Functionally, the proliferative phase appeared associated with increased bacterial proliferation in the vagina and ET compared to the secretory phase, seen as higher abundance of pathways for pyrimidine and purine metabolism, aminoacyl-tRNA biosynthesis, and peptidoglycan biosynthesis (Fig. 6). The secretory phase, on the other hand, showed higher abundance of pathways for porphyrin metabolism, arginine and proline metabolism, degradation of benzoate, nitrotoluene and biosynthesis of siderophore (Fig. 6). These results indicate that the vagino-uterine microbiota probably varies according to the menstrual cycle.