Interpretation
Several studies have shown the presence of bacterial DNA within the upper female reproductive tract (6–9), however a debate still exists regarding the presence of bacteria in sites considered normally sterile such as the human placenta (30,31). A previous study comparing vaginal samples to FT samples found that the vagina contained four orders of magnitude more bacteria than the FT (7). Another study found 20% of FT samples harbored no bacterial signal after PCR amplification, and other samples contained varying and sometimes very low amounts of bacterial reads (8).
In our study, we found lower rates of positive FT samples in patients with hydrosalpinx. As this condition is sometimes associated with an infectious etiology, administration of extended antibiotic regimens instead of surgical resection has been attempted. Pathogenic bacterial presence has been hypothesized to be the cause of the deleterious effect of the stagnant fluid on fertility (33). Our study results do not support this hypothesis, since FT fluid of case-patients didn’t contain bacteria, as opposed to controls, even in case-patients who underwent surgery due to prior infection. One explanation for this phenomenon may be the fact that case-patients were more exposed to antibiotic treatments than controls, which altered the amount and composition of bacterial communities, especially that of the vaginal ”reservoir”. When we compared vaginal samples of patients with and without prior antibiotic treatment, we did find a non-significant difference in read counts. Nevertheless, when we compared vaginal samples of patients with positive and negative FT samples, we did not find any differences in average read counts, undermining the “quantitative” argument for this difference in bacterial presence. We believe that the vagina is the most important, if not the sole source of bacterial colonization of the FT. Hence, we hypothesize that tubal occlusion resulting the hydrosalpinx, is the cause of the paucity of bacterial presence within the fallopian tubes, by blocking the anatomical pathway of bacterial migration. As this study included only nine patients with hydrosalpinx, it was underpowered to compare the results of women with or without prior antibiotic exposure. Such a comparison would have provided important data regarding the contribution of antibiotic treatment to FT bacterial colonization in the setting of tubal occlusion and should be attempted in future studies.
It is well accepted that different microbial profiles of the vagina are associated with disease states (4). Looking into the differential abundance of the vaginal microbiota, we found marked differences in the bacterial composition of cases and controls. Figure 3 depicts the differences in the presence of certain bacteria associated with a dysbiotic vaginal environment between the two groups. Of these,Gardnerella, Atopobium, Prevotella, and  Ureaplasma , bacteria known to be dominant in bacterial vaginosis (34), and therefore infertility, and adverse pregnancy outcomes, were more prevalent in samples from case-patients. Moreover, samples from case-patients lacked the presence of lactobacilli, the most important genus associated with the normal vaginal microbiota, compared with controls. As stated above, case-patients had higher rates of antibiotic exposure, which cannot be excluded as a contributing factor to the results. These findings, while not providing an answer to whether they are the cause or effect of the tubal occlusion, attest to the fact that women with hydrosalpinx are more likely to have a dysbiotic vaginal environment.
As samples that were taken from occluded FT in case patients did not contain enough bacterial DNA after the decontamination process, we compared the microbiome of the vagina and fallopian tubes only of matched control patients. As described in previously cited studies, these two environments were different in both alpha and beta diversity. The FT microbiome was found to be more diverse, and while Firmicutes was the predominant phylum in both groups, FT samples were more abundant in Proteobacteria, similar to findings in previous studies. This highlights the fact that although bacteria in the FT may largely originate from the vagina, both sites may still represent different microbial niches.
In our study, we used stringent criteria to filter out possible contamination which led to the exclusion of 70% of FT samples in the control and 90% of FT samples in the case groups. These figures are in line with rates of culture-positive samples in other studies (7). Exploring low-biomass samples is always subject to various contamination mechanisms (32), and the lower the amount of bacterial DNA present, the higher the relative weight any contamination bears on sample analysis. This was previously described by Miles et al. (6) who compared vaginal to FT samples, before and after exclusion of samples with containing low numbers of sequence reads. The exclusion of these samples enabled clustering of upper and lower genital tract samples more closely together. This may imply that some of the less abundant samples did not necessarily belong to actual anatomical microbiota. Furthermore, the genera described as more abundant in FT than in vaginal samples in other studies showed a clear trend towards environmental bacteria such asAcinetobacter, Pseudomonas, and Methylobacterium . That is in contrast to culture-positive samples, which mostly contained bacteria which are typical residents of the vaginal microbiota. We believe that in the normal female reproductive tract, the major biological pathway of bacterial presence in the FT is the translocation of the vaginal microbiota through the upper genital pathway. This may account for the very low amounts of bacteria that were described. Thus, decontamination should be strongly considered when non-vaginal bacteria are found in FT samples.