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\begin{document}

\title{Investigation of the Female Genital Tract Microbiome and its Association
with Hydrosalpinx in Women Undergoing Salpingectomy.}



\author[1]{Hadar Klapper-Goldstein}%
\author[2]{Yael Yagel}%
\author[2]{Yair Motro}%
\author[3]{Stefan Green}%
\author[1]{Ella Pardo}%
\author[2]{Jacob Moran-Gilad}%
\author[1]{Adi Weintraub}%
\affil[1]{Ben-Gurion University of the Negev Faculty of Health Sciences}%
\affil[2]{Ben-Gurion University of the Negev Department of Health Policy and Management}%
\affil[3]{Rush University Prevention Center}%


\vspace{-1em}



  
  \date{May 20, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objective: To describe the microbiome of the vagina and fallopian tubes,
and its relation with hydrosalpinx. Design: Prospective case-control
study. Population and Settings: Women who underwent salpingectomy for
hydrosalpinx or other indications at our medical center . Main Outcome
Measures: Samples obtained during surgery and were subjected to 16S rRNA
amplicon sequencing, and analyses of alpha diversity and beta diversity
meseaures were compared between sites and groups. Differential
abundances of bacteria associated with vaginal dysbiosis was compared
between cases and controls. Results: Nine women with hydrosalipnx, and
23 women without hydrosalpinx were included in the study. The mean age
of studied women was 41 (range: 29-54), and most (89\%) were
premenopausal. After in silico decontamination, only 30\% of control
fallopian tubes samples, and 10\% of case fallopian tubes samples, had
evidence of bacterial presence. The vaginal microbiota of control
patients showed greater abundance of lactobacilli whereas the vaginal
microbiome of case patients contained relatively more bacterial
vaginosis associated bacteria such as \emph{Prevotella},
\emph{Gardenrella} and \emph{Atopobium}. A significant difference was
found in alpha and beta diversity between the vaginal and FT microbiomes
in control patients, as fallopian tubes samples were more diverse. We
found that women with hydrosalpinx had a more ``dysbiotic'' vaginal
microbiome, and in women without hydrosalpinx, microbial composition
within the vagina and FT differed, possibly representing two distinct
ecological environments. Conclusion: women undergoing salpingectomy for
various reasons harbored bacteria within their FT, women with
hydrosalpinx generally did not. This suggests that even though infection
may be an underlying cause of hydrosalpinx, bacteria may not be present
by the time patients require surgery.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The microbiota of the female genital tract and its role in health and
disease has extensively been studied. The vaginal microbiome has been
well described both in healthy reproductive-age women (1), and
postmenopausal women (2). Various community state types (CST) exist
within the vaginal microbiome, and while most states are dominated by
lactobacilli, other diverse, non-lactobacilli-based microbiome profiles
have been described, sometimes associated with the presence of bacterial
vaginosis (BV) (1,3,4).

Together with the well-characterized lower genital tract microbiome, the
presence of bacteria in the upper reproductive tract including the
uterine cavity,~ the placenta, and fallopian tubes, has been more
frequently investigated in recent years~ (5). Studies examining the
presence of bacteria in the fallopian tubes found a diverse microbial
environment, albeit in very low quantities (6--8). A study examining the
ovarian microbiome found significant differences between cancer patients
and healthy controls (9). As these sites are accessible only via a
surgical procedure, there is an obvious obstacle in studying and
establishing a healthy microbial fingerprint, if one exists

Hydrosalpinx denotes a blocked edematous fallopian tube that is filled
with fluid. Tubal blockage and tubal infertility usually result from
previous pelvic infection such as pelvic inflammatory disease (PID), but
damage to the fallopian tube from previous surgery, endometriosis or
adhesions can also result in hydrosalpinx (10,11). The association of
hydrosalpinx with decreased pregnancy and implantation rates in \emph{in
vitro} fertilization (IVF) cycles has been widely reported, and value
for surgical treatments for tubal disease prior to IVF has been
previously established (12).

Microbiome studies examining infertility found different endometrial and
vaginal bacterial compositions associated with implantation and
pregnancy rates (13) as well as different lactobacilli-dominated vaginal
compositions, associated with pregnancy rates in women with unexplained
infertility (14). A recent meta-analysis also pointed to the association
between BV, a marker of vaginal dysbiosis, with tubal factor infertility
(15). BV has also been shown to be associated with increased risk for
sexually transmitted infections~ (16) pelvic inflammatory disease (17),
and preterm labor (18). However, the association between the vaginal and
fallopian tube microbiomes and their relation to hydrosalpinx has yet to
be elucidated.

In this study, we aim to describe the vaginal microbiome of women
undergoing salpingectomy for hydrosalpinx and compare it with those
undergoing salpingectomy for various other indications. Furthermore, we
analyze the microbiome of the resected fallopian tubes and its
association with hydrosalpinx.

\textbf{Methods}

\emph{Study design and population}

The study was a prospective cohort study, performed at the Soroka
University Medical Center, an 1100-bed tertiary hospital in southern
Israel, and was authorized by the institutional research ethics
committee (0228-17-SOR). All women who planned to undergo elective
salpingectomy at our center were offered the opportunity to participate
in this study. Women receiving immunosuppressive treatments were
excluded from the study, and women with overt, active infections were
excluded from the control group. After obtaining informed consent from
willing participants, a vaginal swab was taken before local cleansing in
the operating room following anesthesia. All women were treated with
prophylactic antibiotics 30-60 minutes before the surgical incision.
Samples were obtained by introducing nylon flocked swab (ESwab, Copan,
Italy) to the vagina up to 5 cm from the introits via a speculum and
rotational swabbing of the vaginal walls for 5-10 seconds. After
performing the salpingectomy procedure, the fallopian tube was
sagittally incised using a strict aseptic technique and a swab was taken
from each of the tube's lumen at the ampullary region. Samples were
immediately refrigerated and then frozen at -80\selectlanguage{ngerman}°C within 24 hours of
sampling until further analysis. Metadata including: age, ethnicity,
menopausal status and prior antibiotic treatments were extracted form
patients' medical records.

\emph{DNA extraction, PCR amplification and sequencing}

All samples were subjected to genomic DNA extraction, followed by 16S
ribosomal RNA based PCR amplification to allow for bacterial DNA
sequencing and analysis. Genomic DNA was extracted from swabs using a
LEV blood kit (Promega, Madison, WI) implemented on a Maxwell 16
instrument, following manufacturer's protocol with several
modifications. Modifications included a lysozyme incubation (10 ng/\selectlanguage{greek}µ\selectlanguage{english}L
lysozyme; Thermo Fisher Scientific) for 30 min at 37\selectlanguage{ngerman}°C, followed by
bead-beating (40 s at 6 m/s) using a FastPrep-24 System (MP
Biomedicals). Homogenized samples were transferred to the Maxwell
cartridges for final purification of DNA.

Genomic DNA was prepared for sequencing using a two-stage amplicon
sequencing workflow, as described previously (19). Initially, genomic
DNA was PCR amplified using primers targeting the V4 region of microbial
16S ribosomal RNA (rRNA) genes. The primers, 515F modified and 806R
modified (20), contained 5' linker sequences compatible with Access
Array primers for Illumina sequencers (Fluidigm, South San Francisco,
CA). PCRs were performed in a total volume of 10 microliters using
MyTaq\selectlanguage{english} HS 2X Mix (Bioline), primers at 500 nM concentration and
approximately 1,000 copies per reaction of a synthetic double-stranded
DNA template (described below). Thermocycling conditions were as
follows: 95degC for 5' (initial denaturation), followed by 28 cycles of
95degC for 30 sec, 55deg for 45 sec, and 72degC for 30 sec. One microliter of
PCR product from each reaction was transferred to the second-stage PCR
reaction. Each reaction was conducted in a final volume of 10
microliters using MyTaq HS 2X mix, and each well contained a unique
primer pair of Access Array primers containing Illumina sequencing
adapters, single index sample-specific barcode, and linker sequences.
Thermocycling conditions were as follows: 95degC for 5' (initial
denaturation), followed by 8 cycles of 95degC for 30 sec, 60deg for 30 sec,
and 72degC for 30 sec. Libraries were pooled and purified using 0.6X
concentration of AMPure XP beads to remove short fragments below 300 bp.

\emph{Bioinformatics analysis}

Raw sequence data underwent QC, trimming, and removal of the synthetic
DNA template using FastQC (21), Trimmomatic (22) and bbmap (BBTools
v37.28; (23)), respectively. The sequence data that passed QC was
imported into the QIIME2 package (v2019.10) (24)) for analysis using the
deblur pipeline (25) with the SILVA DB (v128 at 97\%, (26)). Following
filtering of chimeric sequences and taxonomic assignments, OTUs that
were not assigned as bacteria were removed. Due to the nature of the
samples (i.e. low biomass), additional filtering was conducted using
decontam (27). Finally, the samples were rarefied at a sampling depth of
4,000 using the diversity plugin (diversity core-metrics-phylogenetic).
Differences in the relative abundance of microbial taxa between vaginal
and fallopian tube samples were assessed using the~ R package microeco
(28), with statistical significance measured with the Kruskal-Wallis
statistical test (with Benjamini-Hochberg adjusted p-value \textless{}
0.05 considered significant). Alpha diversity (Shannon index) and beta
diversity (Bray-Curtis dissimilarity index) metrices were measured using
the QIIME2 diversity plugin (diversity alpha and diversity beta
respectively). Comparisons of alpha diversity (Shannon index) between
groups of interest (i.e. case vs. control, or vaginal vs. fallopian tube
were conducted using the QIIME2 diversity plugin (diversity
alpha-group-significance), with the Kruskal-Wallis statistical test
(with Benjamini-Hochberg adjusted p-values considered significant at
\textless{}0.05). Comparisons of beta diversity (Bray-Curtis
dissimilarity) between groups of interest (as above) were conducted
using the QIIME2 diversity plugin (diversity beta-group-significance),
with the PERMANOVA statistical test (using 999 permutations, with
Benjamini-Hochberg adjusted p-values considered significant at
\textless{}0.05). The linear discriminant analysis effect size (LEfSE)
biomarker discovery tool (29) {[}used as part of the R package
microeco{]} was used to identify differentially abundant OTUs, where
variation in abundance of taxa (with relative abundance greater than
0.01\%) was considered significant where the LDA score was greater than
3 and/or the p-value from the LefSe test was \textless{} 0.05.

\textbf{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\textless{}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\textless{}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 \emph{Prevotella}
,\emph{Gardnerella,} and \emph{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.



\textbf{Discussion}

\emph{Main findings}

In our study, salpingectomy for various reasons was relate to bacteria
within the FT, while hydrosalpinx generally did not. In addition, women
with hydrosalpinx show more ``dysbiotic'' vaginal microbiome, while in
women without hydrosalpinx, microbial composition within the vagina and
fallopian tubes differed both in composition and diversity of bacterial
communities.

\emph{~Strengths and Limitations}

Our study has several limitations. Firstly, the small sample size,
limited us from performing subgroup analysis to various conditions that
are known to affect microbial composition such as menopausal state, age,
and former use of antibiotics. The latter is also a limitation in terms
of the comparison of microbiota between cases and controls. While such
studies are difficult to perform on a prospective cohort, larger studies
are definitely needed.~ Another limitation may be our decontamination
protocols, which could be filtering out outputs that might have been of
importance, and the choice to set a high rarefaction cutoff in the
setting of a low biomass microbial environment causing many samples to
fail the positivity criterion. While our preference was to optimize our
analyzed data, it came with the cost of losing a substantial number of
eligible samples. The study was performed at a single center, by the
same surgical team, which is an advantage when considering the
consistency (internal validity) of the sampling technique in all
patients but its external validity and generalizability may be limited.

\emph{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,\emph{Gardnerella, Atopobium, Prevotella,} and~ \emph{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
as\emph{Acinetobacter, Pseudomonas,} and \emph{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.

\textbf{Conclusion}

We found that while some women undergoing salpingectomy for various
reasons harbored bacteria within their FT, women with hydrosalpinx
generally did not. This implies that even though infection may be the
cause of hydrosalpinx, bacteria are not present by the time patients
undergo surgery. We also found that women with hydrosalpinx had a more
``dysbiotic'' vaginal microbiome, and in women without hydrosalpinx,
microbial composition within the vagina and fallopian tubes differed
both in composition and diversity of bacterial communities, possibly
representing two ecological environments. The characteristics of the FT
microbiota and its role in the pathogenesis of female reproductive
pathologies should be further investigated.

\textbf{Disclosure Statement:} All authors have nothing to
disclose.\textbf{Funding Statement:} All authors state that they have no
financial disclosures that might lead to a conflict of
interests.\textbf{Attestation Statement:} The paper is our original
work, unpublished or under consideration for publication elsewhere, and
no other manuscript has been published by the authors overlapping with
this manuscript. The manuscript has been read and approved by all
authors.\textbf{Author's contribution:}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Author initials & Major contribution\tabularnewline
YY & Methodology, Writing - Original Draft, Writing - Review \&
Editing,\tabularnewline
YM & Methodology, Software, Formal analysis , Resources\tabularnewline
SG & Validation, Investigation, Resources\tabularnewline
EP & Investigation, Data Curation\tabularnewline
HKG & Investigation, Data Curation\tabularnewline
JMG & Conceptualization, Methodology, Writing - Review \& Editing,
Supervision\tabularnewline
AYW & Conceptualization, Methodology, Writing - Review \& Editing,
Supervision\tabularnewline
\bottomrule
\end{longtable}



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\textbf{Table 1.} \emph{Clinical and demographic characteristics of
study participants}

\textbf{Figure 1.} Diversity measures compared between sites and
groups.\textbf{a} . Alpha diversity plot for vaginal samples of cases
vs. controls at the genus level (using Shannon index). \textbf{b} .
Alpha diversity plot comparing vaginal and FT samples of controls at the
genus level (using Shannon index). \textbf{c} . Beta diversity plot
comparing vaginal and FT samples of controls at the genus level, (using
Bray--Curtis dissimilarity index).

\textbf{Figure 2} . Relative abundances of different phyla in vaginal
vs. FT samples

\textbf{Figure 3} . Differential abundances of bacterial genera in
vaginal samples of cases and controls (using lefse)

\textbf{Table S1.} \emph{Sample sequencing data}

\textbf{Hosted file}

\verb`Figures bjog.docx`  available at \url{https://authorea.com/users/754761/articles/724592-investigation-of-the-female-genital-tract-microbiome-and-its-association-with-hydrosalpinx-in-women-undergoing-salpingectomy}

\textbf{Hosted file}

\verb`Tables bjog.docx`  available at \url{https://authorea.com/users/754761/articles/724592-investigation-of-the-female-genital-tract-microbiome-and-its-association-with-hydrosalpinx-in-women-undergoing-salpingectomy}

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