Results
Thirty-two women were enrolled in the study, nine of whom underwent salpingectomy due to hydrosalpinx (“cases”), and 23 due to other indications (“controls”). One patient in the “case” group was later excluded from the analysis due to low material yield from all samples taken, as described below, leaving 8 patients in the case group. Table 1 describes the baseline characteristics of the study participants. The mean age of patients was 41, with women in the case group being slightly younger than controls (40 vs. 41.5). Of cases, 88% were premenopausal, vs. 91% of controls. Two ethnic groups were represented in this study, Jewish and Arabs, with no differences in their distribution between groups (25% Arabs in the cases and 26% in controls). Indications for surgery in the case group were either infection (4/8) or infertility (4/8), and in the control group indications varied, the most common being sterilization (11/23) and leiomyoma (8/23).  All of the women in the control group, and 75% of women in the case group underwent bilateral salpingectomy, and samples were taken from both fallopian tubes.
Ninety-four samples from 32 participants were available for sequencing, producing a total of 2,310,035 raw reads (Supplementary Table 1). After QC and in silico decontamination  872,742 were available for analysis. After decontamination as described in the methods, 31/32 vaginal (V) samples (8/9 cases, and 23/23 controls) were available for further analysis, while only 10/62 fallopian tube (FT) samples belonging to 8/32 women (1/9 cases and 7/23 controls) produced sufficient genetic material for the final analysis (Supplementary Table 1).  The total read count of the samples included in the final analysis was 787,362, with an average read count of 22,847 for V samples and an average read count of 7909 for FT samples (p<0.001). The average read count for V samples of controls was 24,016, while the average read count for V sample of cases was 19,485 (p=0.064). Since read counts are a surrogate marker for bacterial load, we further analyzed the average read count of case-patients who received antibiotic treatment prior to sample collection and those who didn’t (24,005 vs. 14964, p=0.205).To account for the possible relation between vaginal read count and positive FT samples we compared average vaginal read counts of patients with positive and negative FT samples, and found no difference (25, 091 vs. 23,569 respectively p= 0.079).
For analysis of OTUs and diversity measures, rarefaction was performed to a depth of 4,000 sequences/sample as described in the methods, producing 145 total observed OTUs, ranging from 1 to 76 OTUs per sample, with an average of 8.8 OTUs for V samples and 28.2 OTUs for FT samples (p<0.001). Looking into the differences between vaginal samples of cases and controls, we examined the alpha diversity at the genus level, as measured using the Shannon index, and while the difference nearly reached statistical significance (p=0.0641, Figure 1a), the difference was further accentuated by comparing only vaginal case samples taken from patients who were operated due to infection, to control samples (none of the latter were operated due to infection) (p=0.0243). These results remaned statistically significant when analysed at the phylum level.  As most FT samples available after decontamination and rarefaction were from controls, we analyzed the differences in measures of diversity between matched vaginal samples of control patients to samples taken only from FT of the same control patients. Figure 1 illustrates the significant differences in both alpha (measured using the Shannon index, p=0.003, Figure 1b) and beta diversity (measured using Bray–Curtis dissimilarity index, p=0.004, Figure 1c) between both groups.
At the phylum level, both V and FT samples were composed mostly of Firmicutes, (83.1% in vaginal samples vs. 65.4% in FT samples p=0.073), though the relative abundances of Proteobacteria and (18.3% vs. 2.16% respectively p=0.011) and Cyanobacteria (0% in V, 2.73% in FT p=0.011) in FT samples were significantly higher, as shown in Figure 2.
Vaginal samples of both cases and controls contained mostly Firmicutes. While no significant difference was found in relative abundances of different genera, using linear discriminant analysis effect size (LEfSe) to account for differences in specific bacteria associated with vaginal dysbiosis, we found bacteria such as Prevotella ,Gardnerella, and Atopobium , to be more prevalent in case patients (p=0.015, p=0.013, p=0.024 respectivly), whereas Lactobacilli were more dominant in controls (p=0.005), as shown in Figure 3.