Colonization of the cervicovaginal space with Gardnerella vaginalis leads to local inflammation and cervical remodeling in pregnant mice
Colonization of the cervicovaginal space with Gardnerella vaginalis leads to local inflammation and cervical remodeling in pregnant mice
Luz-Jeannette Sierra 0 1 2
Amy G. Brown 0 1 2
Guillermo O. BarilaÂ 0 1 2
Lauren Anton 0 1 2
Carrie E. Barnum 0 2
Snehal S. Shetye 0 2
Louis J. Soslowsky 0 2
Michal A. Elovitz 0 1 2
0 Funding: This paper was supported in part by the
1 Maternal Child Health Research Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 McKay Orthopedic Research Laboratory, University of Pennsylvania , Philadelphia, Pennsylvania , United States of America
2 Editor: David N Fredricks, Fred Hutchinson Cancer Research Center , UNITED STATES
The role of the cervicovaginal (CV) microbiome in regulating cervical function during pregnancy is poorly understood. Gardnerella vaginalis (G. vaginalis) is the most common bacteria associated with the diagnosis of bacterial vaginosis (BV). While BV has been associated with preterm birth (PTB), clinical trials targeting BV do not decrease PTB rates. It remains unknown if G. vaginalis is capable of triggering molecular, biomechanical and cellular events that could lead to PTB. The objective of this study was to determine if cervicovaginal colonization with G. vaginalis, in pregnant mice, induced cervical remodeling and modified cervical function. CD-1 timed-pregnant mice received a 5X108 CFU/mL intravaginal inoculation of G. vaginalis or control on embryonic day 12 (E12) and E13. On E15, the mice were sacrificed and cervicovaginal fluid (CVF), amniotic fluid (AF), cervix, uterus, placentas and fetal membranes (FM) were collected. Genomic DNA was isolated from the CVF, placenta, uterus and FM and QPCR was performed to confirm colonization. IL-6 was measured in the CVF and AF and soluble e-cadherin (seCAD) was assessed in the CVF by ELISA. RNA was extracted from the cervices to evaluate IL-10, IL-8, IL-1β, TNF-α, Tff-1, SPINK-5, HAS-1 and LOX expression via QPCR. Mucicarmine and trichrome staining was used to assess cervical mucin and collagen. Biomechanical properties of the cervix were studied using quasi-static tensile load-to-failure biomechanical tests. G. vaginalis successfully colonized the CV space. This colonization induced immune responses (increased IL-6 levels in CVF and AF, increased mRNA expression of cervical cytokines), altered the epithelial barrier (increased seCAD in the CVF), induced cervical remodeling (increased mucin production, altered collagen) and altered cervical biomechanical properties (a decrease in biomechanical modulus and an increase in maximum strain). The ability of G. vaginalis to induce these molecular, immune, cellular and biomechanical changes suggests that this bacterium may play a pathogenic role in premature cervical remodeling leading to PTB.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
role in study design, data collection and analysis,
decision to publish, or preparation of the
Preterm birth (PTB) is the leading cause of perinatal morbidity and mortality worldwide. In
the United States, one in nine babies is born prematurely and over 11 million PTB cases were
reported last year [
]. Premature babies have a higher incidence of developing medical
complications  resulting in a financial cost of over 26 billion dollars a year in the United States
]. Despite ongoing research, there are no effective strategies to predict or prevent the
majority of preterm births. Recently, studies focusing on the causes of PTB have shown that
women with bacterial vaginosis (BV) are at higher risk for spontaneous PTB (sPTB) [4±6]. BV
is the most common genital tract infection affecting women worldwide [
]. This disease is
characterized by a polymicrobial imbalance, or dysbiosis, of the natural microflora of the
cervicovaginal (CV) space. While studies have shown an association between BV and PTB, [
clinical trials targeting treatment of BV have failed to show differences in PTB rates [9±11]. The
human microbiome project has provided valuable information about the diverse bacteria
subspecies that make up a BV-like state [
]. CV microflora mainly composed of Lactobacilli
subspecies (spp.) is considered to be associated with a healthy CV space [8, 10, 14±24] while
the lack of these species is integral to the diagnosis of BV and is considered a marker of an
unhealthy CV space. Many BV cases are characterized by a decrease of Lactobacillus subspecies
(spp.) and an increase in biofilms that may include Mobilincus spp., Mycoplasma hominis,
Atopobium vaginae, Bacterioides spp. and Prevotellla spp. and Gardnerella vaginalis (G. vaginalis)
]. The conflicting data regarding BV and sPTB may be due, in part, to the role and/or
pathogenicity of the different organisms that may compose the clinical diagnosis of BV [
]. Since G. vaginalis is predominantly found in most cases of BV [29±31], the ability of this
bacterium to induce cellular and molecular changes in the CV space, during pregnancy, is of
scientific and clinical interest .
Recent studies have shown that disruption of the cervical epithelial barrier appears to be an
important primary step critical to the initiation of cervical remodeling [32±34]. Cervical
remodeling is a process that starts weeks, if not months, prior to parturition [
32, 33, 35
During this cervical remodeling process, cervical tissue undergoes robust changes at the molecular,
histological and biomechanical levels to allow for delivery of a fetus [
]. A clinical study
attempting to identify biomarkers for cervical epithelial remodeling found increased levels of
soluble epithelial-cadherin (seCAD) within the CV space . seCAD, a soluble byproduct of
proteolytic cleavage of e-cadherin (a member of the adherens junction complex), which may
act as a physiologically relevant biomarker capable of predicting sPTB.[
]. Additionally, there
is enhanced expression of several genes in the mouse cervix near delivery. These genes are
responsible for cervical distensibility (Hyaluronan, HA) [
], collagen disorganization (lysyl
oxidase or LOX) [
], and initiate changes to the cervical extracellular matrix (trefoil factor
I (Tff-1) and a Serine protein inhibitor Kasal 5 (SPINK-5)) [
35, 41, 42
]. Histology of the mouse
cervix has revealed evidence of collagen rearrangement which is consistent with previous
studies showing that the stiffness of the cervix decreases as parturition approaches .
Concordantly, cervical biomechanical studies in the mouse have provided valuable information to
understanding how important structural mechanical properties, such as cervical load and
stiffness, change at different embryonic stages [
]. Recently, Barnum et al evaluated the
intrinsic material properties of the mouse cervix during pregnancy . This study provides
evidence that alterations to the biomechanical properties of the cervix are likely part of the
process of cervical remodeling.
While some of the processes governing cervical remodeling at term have been revealed,
how specific bacteria, associated with BV, modify these processes have not been studied.
Therefore, the objective of this study was to determine if specific BV-associated bacteria can be
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pathogenic during pregnancy and induce premature cervical remodeling. We hypothesize that
G. vaginalis colonization of the CV space alters cervical function, contributes to dysfunction of
the cervical epithelial barrier and, consequently, initiates cervical remodeling. We created a
humanized pregnant mouse model of G. vaginalis colonization to determine if G. vaginalis
colonization altered the local immune response, induced cervical remodeling and/or altered
cervical biomechanics. Our results provide evidence that G. vaginalis colonization of the CV
space leads to inflammation, cervical remodeling and altered cervical biomechanics.
Materials and methods
CD-1 timed-pregnant mice were purchased from Charles River Laboratories (Wilmington,
MA). We considered E0 as mating day and E1 was determined based on presence of
copulatory plug. Animals were shipped on day 10 after mating, and housed individually in our
facilities. These animals were acclimated for 4 days before performing experiments. All the
experiments were performed in accordance with the National Institutes of Health
Guidelines on Laboratory Animals and with approval from the University of Pennsylvania's
Institutional Animal Care and Use Committee (IACUC #:805513).
Gardnerella vaginalis was purchased from the ATCC depository (ATCC# 14019) and grown
anaerobically at 37ÊC with 5% CO2 in Tryptic Soy Broth (TSB) (Becton, Dickinson and
Company, Sparks MD, USA) or Tryptic Soy Agar (TSA) (Becton, Dickinson and Company, Sparks
MD, USA) supplemented with 5% horse serum (Gibco, Thermo Fisher Scientific). Efficient
bacteria growth was measured and quantified by colony forming unit (CFU) assays. Bacteria
were centrifuged twice to remove the growth media and the final pellet was resuspended in
sterile filtered sugar water (10% fructose, 10% maltose, 10% glucose in sterile H2O
(SigmaAldrich, Saint Louis MO, USA)) for use in animal experiments. This sugar water was used as
the control in our animal trials.
Cervicovaginal colonization with G. vaginalis
We created a pregnant mouse model of G. vaginalis colonization as follows. CD-1 embryonic
day 12 (E12) timed-pregnant mice were anaesthetized with isoflurane and five cervicovaginal
lavages were performed with 100 μL of sterile PBS prior to control treatment or bacterial
inoculation. Bacterial doses were determined using published data in a non-pregnant mouse
], and then recapitulating similar G. vaginalis loads in pregnant mice. The animals
then received an intravaginal inoculation of G. vaginalis by inserting a sterile pipette tip and
injecting 50 μL of 5X108 CFU/ml or sugar water. The inoculations were performed on E12 and
repeated on E13 for both the G. vaginalis and the control group. This time point was chosen to
mimic a change in the cervicovaginal microflora early in pregnancy. Immediately
post-inoculation, each animal was positioned in dorsal decubitus under isoflurane anesthesia for 3
minutes and 100% pure petroleum jelly (Vaseline, Unilever USA) was added with a sterile swab to
ensure the inoculum would remain within the cervicovaginal space. Animals were observed
for 48 hours and specimens were collected on E15.
Using this protocol, we performed five separate trials. The first three trials were performed
to 1) determine the preterm birth rate and 2) to collect fluids/tissues for assessment of
inflammation, cervical remodeling and bacterial colonization. A fourth trial was performed to assess
the effects of higher CFU dose, where we increase the bacteria load to 5X1010 CFU/mL. A final
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trial was performed to collect whole cervices for biomechanical testing (N = 12 animals in each
experimental group) and cervical histology (N = 4 animals in each experimental group). In the
first trial, on E15, a subset of the G. vaginalis (N = 12) and control (N = 8) inoculated animals
were sacrificed to collect tissues for downstream assays (described below). The remaining
animals (G. vaginalis, N = 4 and control, N = 4) were monitored for preterm birth and allowed to
deliver to record pup weight and size. Preterm birth was defined as delivery prior to E18.
Around 24 hours post-delivery, we counted and weighed individual pups in each litter. For the
remaining trials, dams were sacrificed on E15 and the following specimens were collected
from both G. vaginalis (N = 10 per trial) and control (N = 8±12 per trial) groups: cervicovaginal
fluid (CVF), amniotic fluid (AF), cervix, lower uterus, placentas and fetal membranes. To
assess for active colonization of G. vaginalis, on E15, immediately following CVF collection
(N = 12), 50 μL of CVF was spread on Tryptic Soy Agar supplemented with 5% rabbit blood
and incubated for 48 hours under the conditions mentioned above.
Tissue and specimen collection
From the first three animal trials, on E15 CVF, AF, cervix, lower uterus, placenta and fetal
membranes were collected. CVF was collected by gently rinsing the cervicovaginal space
(pipetting in and out seven times) with 100 μL of sterile PBS. The washes were pooled together
into one sterile tube for each dam. AF was collected by aspirating the fluid out of the fetal sacs
with a 19 gauge needle. AF from all pups per dam were collected and pooled into one tube
and spun at 1,500 rpm for 5 minutes at 4ÊC. The AF supernatants were stored at -80ÊC until
needed. The cervix was dissected away from the vagina and the lower uterus and was collected
after removing the bladder, adipose tissue and rectum. A total of four placentas and their
respective fetal membranes were collected from the four fetuses closest to the cervix. The
cervices, lower uterus, placentas and fetal membranes were collected and flash frozen in liquid
nitrogen and stored at -80ÊC until needed for downstream assays.
From the fourth animal trial, cervices were collected and placed in 10 mL of 4% formalin,
stored for 48±72 hours and used for tissue histology and staining as described below. For
biomechanical testing, a second cohort of mice from the same animal trial were sacrificed and
stored at -20ÊC and cervical tissues were harvested at the time of testing as described below.
Genomic DNA isolation and QPCR
Genomic DNA (gDNA) was isolated and purified from the CVF with the ZR fecal MiniPrep
DNA extraction kit (Zymo Research, Irvine, CA, USA). To purify gDNA from placenta, uterus
and fetal membranes we used the DNeasy Blood and Tissue mini column DNA extraction kit
(Qiagen, Germantown, MD, USA) following the manufacture's protocol. To quantify the
amount of G. vaginalis gDNA, we used a 16S specific probe to this bacterium (Applied
Biosystems, Foster City, CA, USA). gDNA from the CVF, fetal membranes, uterus and placenta was
quantified by QPCR to determine tissue specific colonization. A standard curve was created
from serially diluted gDNA from G. vaginalis to quantify the amplification. This standard
curve was used for relative quantification of G. vaginalis abundance using the 7900HT
RealTime PCR System (Applied Biosystems). The results were analyzed using the RQ manager
software v2.4 (Applied Biosystems).
Amniotic fluid was used for measurement of IL-6. Cervicovaginal fluid was used for
measurement of IL-6 and soluble E-cadherin (seCAD) using commercially available ELISA assay kits
following the manufacturer's protocol (R&D Systems, Minneapolis, MN, USA).
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RNA isolation from cervix
To isolate RNA from the previously collected cervices, we placed the cervix in a round bottom
2.0 mL Eppendorf tube with TRIzol (Invitrogen, Thermo-Fisher Scientific). The cervices were
then mechanically homogenized with stainless steel beads (5mm, Qiagen) at a frequency of 30
Hz/sec for 10 minutes in a TissueLyser II (Qiagen) and underwent phenol-chloroform
extraction. RNA concentration was determined via a NanoDrop 2000 Spectrophotometer
(Nanodrop™ Rockland, DE) prior to the generation of cDNA.
cDNA generation and QPCR
cDNA was generated from 1 μg of isolated RNA from cervical tissue using the high capacity
cDNA reverse transcription kit (Applied Biosystems, Thermo-Fisher Scientific). QPCR was
performed on the 7900HT Real-Time PCR System (Applied Biosystems) using the TaqMan
Universal PCR Master Mix (Applied Biosystems) according to the manufacturers' protocols.
The standard curve method was used for relative expression quantification using the RQ
manager software v2.4 (Applied Biosystems). In TaqMan QPCR assays, the relative abundance of
the target of interest was divided by the relative abundance of 18S in each sample to generate a
standardized abundance for the target transcript of interest. All mRNA primers were
purchased from Applied Biosystems: IL-10, IL-8, IL-1β, TNF-α, Tff-1, SPINK-5, HAS-1, LOX and
18S (TaqMan gene expression assays, Applied Biosystems, Thermo-Fisher).
Trichrome and mucicarmine assay
At E15, post G. vaginalis inoculation, the cervices were harvested as noted above, and placed in
formalin for 48±72 hours and paraffin embedded. The cervices were sectioned (10 μm) and
mounted onto glass microscope slides. These sections were stained using hematoxylin and
eosin (H&E) (ScyTech, Logan Utah, USA), trichrome and mucicarmine staining kits (Abcam,
Cambridge, MA, USA), following the manufacturer's instructions and as previously reported
]. Pictures were taken with a Nikon Eclipse microscope (Nikon Instruments Inc., NY,
USA) with a 1394 color digital camera (Scion corp. Model 1310, NY, USA), and Image J
software (Version 1.34s Wayne Rasband, Java 1.5.0_19) was used to analyze the pictures.
Biomechanical testing was performed using methods previously described [
]. At the time of
testing, female reproductive tissues were carefully harvested, removing all musculature and
surrounding soft tissue, and hydrated in phosphate buffered saline (PBS) (N = 12 animals of
each experimental group). Orientation of the cervix was noted to ensure consistency
throughout the biomechanical experiments. The cervix was dissected free of any extra soft tissue and
the uterus and vagina were carefully removed. The cervix was laid flat to expose the lumen.
The ends were affixed between two pieces of sandpaper for gripping, such that a uniaxial
tensile load on the grips would simulate dilation of the cervical canal (loading occurred
perpendicular to the proximal-distal direction). The prepared sample was continually immersed in
PBS until the start of mechanical testing. A custom laser device was used to measure the cross
sectional area at a minimum of two locations, which took less than 60 seconds [
]. The cervix
was then placed in custom fixtures to grip it at both ends. The cervix was then tested under
uniaxial tension using an Instron 5848 testing system (Instron Corp., Norwood, MA). The
testing protocol consisted of a preload of 0.005N followed by a hold of 5 minutes and then a
ramp to failure at a rate of 1mm/minute. The entire test was performed in a saline bath at
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room temperature. The location of failure was recorded for each sample. Samples were
excluded from further analysis if failure did not occur within the mid-substance of the tissue.
Statistical analyses were performed for all experiments with the GraphPad Prism Software
(Version 4.0, La Jolla, CA, USA). For data that were normally distributed, an unpaired t-test
was used. If data were not normally distributed, then the unpaired t-test with Welch's
correction was used. For biomechanical testing, t-test analyses were used to compare between groups
for mechanical parameters. One-way ANOVA with Bonferroni-corrected post hoc tests were
used to evaluate differences in fiber re-alignment. P<0.05 was considered to be statistically
significant. P<0.1 was considered to be a trend.
G. vaginalis colonization of the CV space of timed-pregnant CD-1 mice
G. vaginalis successfully colonized the CV space (Fig 1, p<0.0001) and was not detected in the
uterus or placentas (S1 Methods; S1±S3 Figs). The most effective colonization was achieved
using a single dose of 5x108 CFU/mL of bacteria inoculated into the CV space for two
consecutive days. In this study, we confirmed that live G. vaginalis was present in the CV space 48
hours after first inoculation (S4 Fig). Importantly, G. vaginalis colonization did not affect the
litter size or the pup weight (S5 Fig). Animals treated with 5x108 CFU/mL G. vaginalis had a
PTB rate that ranged from 0 to 20 percent in three independent experiments (3 out of 12, 1 out
of 10 and 0 out of 12 animals) delivered before E18,which is our metric standard to define PTB
Fig 1. G. vaginalis colonization of the CV space of timed-pregnant CD-1 mice. Quantification of the 16S gene of G.
vaginalis in the CVF of animals inoculated with 5X108 CFU/mL, was performed via qPCR using a specific G. vaginalis
16S probe. Graphs shows the average quantity mean detected by qPCR of N = 8 Control and N = 12 G. vaginalis group.
T-test analyses with Welch's correction between these groups was performed ( p<0.0001). Values are mean ± SD.
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(data not shown). The CV space of the animals treated with 5x1010 CFU/mL G. vaginalis were
adequately colonized (S6 Fig) and had a PTB rate of 0 percent (0 out of 10) (data not shown).
Elevated inflammation in the cervix of G. vaginalis colonized animals
In order to determine if G. vaginalis colonization of the CV space results in a local
inflammatory response, we assessed IL-6 protein levels as a marker of generalized inflammation. IL-6
was significantly increased in the CVF of animals inoculated with G. vaginalis in comparison
to the samples in the control group (Fig 2A, p = 0.0007). We also observed a significant
increase in IL-6 in the amniotic fluid (Fig 2B, p = 0.0008) of animals colonized by G. vaginalis,
despite the absence of ascending bacteria into the fetal membranes, placenta or uterus.
Additionally, we quantified the gene expression of other known pro-inflammatory cytokines and
chemokines in the cervix including TNF-α, IL-10, IL1β and IL-8. Cervical gene expression of
IL-8 (p = 0.0055), IL-1β (p = 0.0120) and IL-10 (p = 0.0140) were significantly enhanced (Fig
3A, 3B and 3C respectively). TNF-α was not significantly altered (Fig 3D, p = 0.0842).
Colonization of the CV space with G. vaginalis induces cervical remodeling
As we have shown previously that increased seCAD is a molecular marker of cervical epithelial
barrier disruption, [
] we assessed if seCAD was altered in this model. Levels of seCAD were
significantly increased in dams colonized with G. vaginalis compared to controls (Fig 4,
p<0.0001). In addition to seCAD, we measured the gene expression of LOX, HAS-2, Tff-1 and
SPINK-5 which have all been previously reported to be involved in cervical remodeling [
We observed increased expression of Tff-1 (Table 1, p = 0.026), whereas the gene expression
levels of SPINK-5 (Table 1, p = 0.080), HAS-2 (Table 1, p = 0.076) and LOX (Table 1,
p = 0.3625) were not significantly different.
Prior work has demonstrated that histological changes within the cervix are consistent with
cervical remodeling [
]. Mucicarmine and trichrome stainings were performed to assess
for the presence of mucin and collagen in cervices of G. vaginalis colonized compared to
control animals (Fig 5). We observed increased expression of mucin in the cervices from dams
Fig 2. G. vaginalis increases levels of IL-6 in the cervicovaginal space and amniotic fluid. Levels of IL-6 in the CVF (A) and in the AF (B) were measured via ELISA.
T-test analysis with Welch's correction was performed to determine statistical significance between two groups ( p = 0.0007 in the CVF and p = 0.0008 in the AF
analysis) (N = 8 Control and N = 12 G. vaginalis group). Values are mean ± SD.
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Fig 3. Increased gene expression of IL-1β, IL-8 and IL-10 in the cervix of G. vaginalis colonized animals. Gene expression levels of IL-8 (A), IL-1-β (B), IL-10 (C),
and TNF-α (D) were measured by QPCR. T-test with Welch's correction was performed in each group (N = 8 Control and N = 11 G. vaginalis group) ( IL-8
(p = 0.0055), IL-1-β (p = 0.0120) and IL-10 (p = 0.0140)). Values are mean ± SD.
colonized with G. vaginalis compared to controls, consistent with cervical ripening (Fig 5A
and 5B). Additionally, we observed a dispersion of collagen fibers in animals colonized with
G. vaginalis in comparison to animals treated with sugar water (Fig 5C and 5D).
G. vaginalis colonization alters the cervical biomechanics
Using previously described [
] techniques and metrics, we found that colonization with G.
vaginalis demonstrated a decrease in modulus (Fig 6E, p< 0.05) as well as an increase in
maximum strain (Fig 6F, p< 0.05) but no change in tissue cross-sectional area (Fig 6A),
maximum load (Fig 6B), stiffness (Fig 6C, p<0.1) or maximum stress (Fig 6D). No difference in
collagen fiber re-alignment was observed during cervical mechanical testing between groups
(S1 Methods; S7 Fig).
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Fig 4. G. vaginalis increases soluble E-cadherin in the CV space. Levels of soluble E-cadherin were measured by
ELISA. T-test analysis with Welch's correction was calculated for significance of protein expression between the
groups ( p<0.0001). Values are mean ± SD.
As bacterial infection is thought to be one of the predominate factors associated with
spontaneous PTB [
] this study has provided novel insight into the role of cervicovaginal (CV)
bacteria in the CV space and, specifically, on cervical function. The results from this study suggest
that G. vaginalis, the most common bacteria associated with BV [
], has the ability to initiate
cervical remodeling through multiple biological, molecular and biomechanical mechanisms.
In this study, we successfully generated a novel pregnant mouse model with a CV space
colonized with G. vaginalis. This new model allowed us to study the effects of this bacterium within
the CV space and its association with premature cervical remodeling. This study provides
evidence that G. vaginalis colonization, localized to the CV space, was able to alter cervical
function through multiple biological mechanisms including activating a cervical immune response,
initiating cervical remodeling and modifying the material biomechanical properties of the
cervix. Therefore, these results suggest that the presence of G. vaginalis within the CV space
during pregnancy has the ability to directly alter many of the biological mechanisms regulating
cervical remodeling and, hence, could contribute to the pathogenesis of PTB.
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Fig 5. G. vaginalis colonization increased expression of mucin and decreased collagen dispersion within cervical tissues. Representative cervical sections from
Control (A, C) or G. vaginalis (B, D) treated animlas (N = 4 in each group). Cervices were stained with mucicarmine (A, B) for analysis of mucin production while
trichrome stain (C, D) shows collagen dispersion. Pictures were taken at a 10X magnification. Trichrome stains collagen blue, muscle fibers red and nuclei black-blue.
Mucicarmine stains mucin pink/red, the nuclei blue and any other tissue component yellow.
A previous study investigating the effects of G. vaginalis in the CV space of a non-pregnant
mouse model showed that G. vaginalis was able to replicate within the CV space and ascend
into the uterine horns [
]. Additionally, this same study showed that CV colonization of G.
vaginalis resulted in many of the hallmark symptoms typically associated with clinical BV such
as increased sialidase activity and vaginal epithelial exfoliation similar to phenotypes observed
in clue cells in BV cases [
]. Similarly, in our G. vaginalis colonized pregnant mouse model,
we observed high levels of G. vaginalis 16S in the CV space 48 hours post inoculation. It is
important to point out that G. vaginalis inoculated into the CV space remained alive 48 hours
post inoculation as evidenced by the growth of G. vaginalis colonies from CVF lavages (S6
Fig). G. vaginalis 16S was not detected within the uterus, placenta or fetal membranes
suggesting that, in our model, G. vaginalis primarily colonizes the CV space with no ascension into
the uterus. In contrast, it has been shown that G. vaginalis is capable of ascending into the
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Fig 6. Mechanical properties of G. vaginalis colonized cervices. Area (A), Max Load (B), Stiffness (C), Max Stress (D), Modulus (E),
and Max Strain (F) are shown for both control and G. vaginalis colonized cervices. All data is presented as means with standard
deviations and significance noted at p< 0.05, (n = 10±11 in each group).
uterus of a non-pregnant mouse model [
]. The ability of G. vaginalis to ascend into the
uterus in the non-pregnant model but not in our pregnant model is biologically important.
Due to the presence of increased cervicovaginal mucus known to be associated with
pregnancy, it is biologically plausible to suggest that the presence of increased cervical mucus (and
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its associated mucosal immunity) may play a significant role in preventing the ascension of
G. vaginalis into the uterus. Indeed, it has been observed that non pregnant mice have
ascending bacteria, but this does not occur in pregnant animals as we have demonstrated. The
inability of bacteria to ascend in the pregnant state may be due to expression changes of toll-like
receptors and anti-microbial peptides (eg mucin) on the cervix during pregnancy [
absence of detectable bacteria in the uterus correlates with the fact that G. vaginalis
colonization did not affect litter size or pup weight (S4 Fig). To our knowledge this study is the first to
show G. vaginalis colonization in pregnant mice.
The varied PTB rate animals treated with G. vaginalis may be attributed to the out-bred
genetic background of our mouse model which inherently adds to a variable outcome and
response to the colonization of G. vaginalis. Studies showing varying associations between BV
treatment and PTB rates [
] may suggest that, in human pregnancy, G. vaginalis alone may not
be sufficient to cause sPTB. Instead, G. vaginalis in combination with other BV-associated (or
other non-BV) bacteria may be needed in order to initiate a more consistent preterm birth
phenotype. Additionally, it is possible that the exposure time of G. vaginalis in the CV space
could have significant effects on the cervix. In our model the animals were only exposed to G.
vaginalis beginning on embryonic day 12, for a maximum time ranging from 48 hours to 5
days. Therefore, we cannot rule out the duration of exposure to G. vaginalis as being a
contributing factor to the pathogenesis of preterm birth. Finally, it is important to note that the mouse
epithelium is keratinized in contrast to the human, therefore G. vaginalis colonization and
adherence might be different in our mouse model as compared to the human population.
The increased expression of IL-6 in the CVF of animals colonized with G. vaginalis
indicated that the presence of this bacterium was able to initiate a localized immune response
within the CV space. Interestingly, even in the absence of ascending bacteria, elevated IL-6 in
the AF of animals inoculated with G. vaginalis suggests that increased cytokines localized to
the CV space might have the ability to further activate an inflammatory response within the
uterine cavity. Furthermore, when we analyzed the gene expression of these
cytokines/chemokines in the cervix of G. vaginalis colonized animals, we observed an increase in
PTB-associated cytokines [
] such as IL-8, IL-10 and IL-1β. In a reported non-pregnant model, G.
vaginalis colonization showed no histological inflammation , however cytokine and
chemokine expression levels were not assessed after CV infection. Therefore, our results provide
pertinent information about the inflammatory pathways induced by G. vaginalis colonization
of the CV space during pregnancy. Our study is the first to show that G. vaginalis has the ability
to induce an inflammatory response within the CV space of pregnant mice and it is
biologically plausible that this inflammation may be capable of altering cervical function and
Previous work from our laboratory has demonstrated that an inflammatory insult leads to
breakdown of the cervical epithelial barrier [
]. These observations are also present in other
mucosal epithelial tissues such as the gut [53±59]. In the gut, inflammation leads to the
activation of matrix metalloproteases (MMPs) that lead to disruption of the epithelial junctional
proteins including epithelial-cadherin (e-cadherin) [
]. Specifically, MMPs and other serine
proteases cleave the extracellular domain of e-cadherin resulting in the release of soluble
e-cadherin (seCAD) into the extracellular spaces. Thus, in the presence of G. vaginalis colonization,
increased seCAD in the CVF indicates a breakdown of the adherens junctions within the
cervical epithelial barrier, as we have demonstrated in in vitro studies with cervical epithelial cells
].Therefore, in our pregnant animal model, the fact that G. vaginalis colonization leads to
increased levels of seCAD suggests that inflammation within the CV space has the ability to
initiate the breakdown of the cervical epithelial barrier leading to cervical remodeling.
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Based on these results, we aimed to further demonstrate that colonization of the CV space
with G. vaginalis results in cervical remodeling. Previous work has demonstrated numerous
molecular markers associated with cervical ripening and remodeling in different embryonic
stages of mouse pregnancy [
]. To confirm cervical remodeling, we assessed LOX, Tff-1 and
SPINK-5 gene expression as they have been reported to be associated with cervical ripening
and dilation [
]. Interestingly, increases in Tff-1 have been previously associated with
increased internalization of e-cadherin, a process that occurs upon cleavage of the extracellular
domain of e-cadherin resulting in an elevation of seCAD, as was observed in this study .
Thus, increased Tff-1 gene expression levels in the cervix along with the significantly elevated
levels of seCAD in the CVF, provide evidence that G. vaginalis has the ability to alter the
process of cervical remodeling. There are characteristic histological changes of the mouse cervix
indicative of cervical remodeling; especially as parturition approaches there is evidence of
collagen rearrangement and a decrease in cervical stiffness [
]. In addition to the observed
changes in gene expression, mucicarmine and trichrome staining showed an increase in
mucin expression as well as a dispersion of collagen fibers in the cervix of animals colonized
with G. vaginalis providing additional evidence of cervical remodeling. While it is unknown if
the alterations in histological cervical remodeling or an activated immune response occurs
first, it is interesting to note, that increased mucin production has been linked to activation of
the innate immune system as a response to bacterial infection [
]. Therefore, it is plausible to
hypothesize that mucin is increased, in part, through the host's biological mechanisms to
protect the cervical epithelial cells from G. vaginalis infection. These results agree with previous
studies showing an activated inflammatory response causes an increase in histological mucin
expression and collagen dispersion [
45, 50, 62
]. While the exact pathological mechanisms
leading to histological cervical remodeling remain unclear, the results from this study provide
evidence that G. vaginalis has the ability to increase cervical remodeling.
Since both molecular and histological differences were observed in the cervix due to
colonization of the CV space with G. vaginalis, cervical biomechanical parameters were also assessed.
There was a significant decrease in tissue modulus of cervices colonized with G. vaginalis,
when compared with the controls, indicating a change in its inherent material response.
Concomitantly, a trend towards decreased stiffness of the cervical tissue points towards a structural
(size) increase. This indicates a clear differential mechanical response of the murine cervix to
colonization with G. vaginalis. Further, we have previously observed similar material and
structural changes in the normal pregnant cervix immediately prior to parturition (E18.5).
Interestingly, although the modulus values fall to similar levels as those observed in the normal
E18.5 cervices, stiffness of the G. vaginalis samples remains appreciably higher. This finding
suggests that some of the mechanical mechanisms contributing to cervical remodeling are
different in animals colonized with G. vaginalis opposed to term parturition [
]. It is important
to note that some of the observed mechanical changes such as modulus and stiffness in the G.
vaginalis cervices could indicate more rapid cervical remodeling. In our model, colonization of
the CV space with G. vaginalis provides evidence that cervical softening is occurring
faster/earlier in comparison to our control group, as well as, in normal gestation [
One limitation of this animal model is that, unlike the human, mice are quadrupedal not
bipedal. The load of pregnancy would be divergently distributed in the mouse compared to the
human and could affect cervical biomechanics. Despite this limitation, in the future it is
imperative to continue to define how both structural and material mechanical properties of the
cervix work in tandem with other biological and mechanical factors to regulate both term and
We did not observe drastic differences in collagen fiber re-alignment between cervices
colonized with G. vaginalis versus control (S5 Fig.) indicating that the decrease in stiffness and
13 / 19
modulus of cervical tissue in the G. vaginalis samples was not due to any reorganization of the
load-bearing response of cervical collagen fibers. However, other factors such as collagen
cross-linking, density of collagen fibers, or change in collagen fiber diameter could also explain
the observed decrease in tissue mechanical properties [
]. Both the dispersed collagen
observed histologically combined with the increase of mucin expression could indicate other
mechanisms of cervical remodeling through activation of immunological pathways.
Collectively, the results of this study show that colonization of G. vaginalis in the
cervicovaginal space of pregnant mice has the ability to significantly alter cervical function. By
evaluating multiple biological mechanisms known to be associated with the cervical remodeling
process, we demonstrated that colonization of G. vaginalis in the cervicovaginal space can
induce local inflammation, damage the cervical epithelial barrier, initiate cervical remodeling
and alter the biomechanical characteristics of the cervix. The ability of G. vaginalis to induce
these molecular, immune and cellular changes suggests that this bacterium could play a
mechanistic role in sPTB in which cervical remodeling is the initiating event. Additionally, this
study demonstrates the feasibility of mimicking the human CV microbiota in a pregnant
mouse model. As shown in a recent study, G. vaginalis colonization has implications for other
lower genitourinary tract conditions, such as the case of recurrent E.coli infections in the
]. By elucidating the mechanisms by which G. vaginalis alters the epithelium,
underlying tissue and immune responses in the CV space, we might provide increased understanding
of conditions associated with G. vaginalis such as HIV [65±70], UTI [
], recurrent pregnancy
loss and preterm birth [
]. These findings have broader implications. An increased
understanding of the role of the cervicovaginal microbiome and how they might mitigate or modify
molecular, biomechanical and immune function in the CV space will be essential to
developing future therapeutic options for preventing sPTB.
S1 Fig. Presence of G. vaginalis in the CVF. gDNA from the CVF was used with a G. vaginalis
specific primer set to amplify G. vaginalis via PCR. PCR reactions were run on a 1% agarose
gel with ethidium bromide and exposed to UV light to capture DNA bands. G. vaginalis
positive bands were expected at an amplicon of 206 bp. As a positive control we included a PCR
sample of gDNA isolated directly from G. vaginalis cultures. To determine the PCR product
sizes we included wells with 1Kb and 100 bp ladders on each side of the gel.
S2 Fig. Presence of G. vaginalis in the uterus. gDNA from the uterus was used with a G.
vaginalis specific primer set to amplify G. vaginalis via PCR. PCR reactions were run on a 1%
agarose gel with ethidium bromide and exposed to UV light to capture DNA bands. G. vaginalis
positive bands were expected at an amplicon of 206 bp. As a positive control we included a
PCR sample of gDNA isolated directly from G. vaginalis cultures. To determine the PCR
product sizes we included wells with 1Kb and 100 bp ladders on each side of the gel.
S3 Fig. Presence of G. vaginalis in placenta. gDNA from the placenta was used with a G.
vaginalis specific primer set to amplify G. vaginalis via PCR. PCR reactions were run on a 1%
agarose gel with ethidium bromide and exposed to UV light to capture DNA bands. G. vaginalis
positive bands were expected at an amplicon of 206 bp. As a positive control we included a
PCR sample with gDNA isolated directly from G. vaginalis cultures. To determine the PCR
product sizes we included wells with 1Kb and 100 bp ladders on each side of the gel.
14 / 19
S4 Fig. G. vaginalis live bacteria in the CVF 48 hours post inoculation. Tryptic Soy Agar
plates supplemented with 5% defibrillated rabbit blood were inoculated with 50μL of CVF
collected from mice 48 hours post-inoculation and incubated for 72 hours in an anaerobic jar at
37ÊC and 5% CO2. After incubation, the numbers of colonies were counted on each plate.
S5 Fig. Treatment with G. vaginalis does not affect pup weight or litter size. Dams treated
with sugar water or 5X108 CFU/mL of G. vaginalis were allowed to deliver (N = 8 in each
group). The individual pup weights (A) and the number of pups per litter (B) were recorded.
T-test with Mann-Whitney nonparametric correction analysis was performed to determine
statistical significance between the two groups (pup weight: p = 0.8785 and Litter size:
p = 0.6454). Values are mean ± SD.
S6 Fig. G. vaginalis colonization of the CV space of timed-pregnant CD-1 mice using a
higher bacterial dose. Quantification of the 16S gene of G. vaginalis in the CVF of animals
inoculated with 5X1010 CFU/mL was performed via qPCR using a specific G. vaginalis 16S
probe. Graphs shows the average quantity mean detected by qPCR of N = 10 Control and
N = 10 G. vaginalis group. T-test analyses with Welch's correction between these groups was
performed (p = 0.0002). Values are mean ± SD.
S7 Fig. Collagen fiber re-alignment measured by polarized light analysis during cervical
mechanical testing. Representative plots of polarized light analysis at toe, end of toe, 45% of
maximum load, and 90% of maximum load. The control group is shown on the left (A) and G.
vaginalis colonized cervices are shown on the right (B). Lines represent significance of p< 0.05
(n = 10±11).
S1 Methods. G. vaginalis PCR and cervix collagen fiber alignment methodology.
We would like to thank the Children's Hospital of Philadelphia Pathology core, especially
Socrates Agrio and Elizabeth A. Tomeski. This paper was supported in part by the March of
Dimes Prematurity Research Center at the University of Pennsylvania (22-FY17-890), The
Maternity and Child Health Research Center at the University of Pennsylvania and the Penn
Center for Musculoskeletal Disorders (P30 AR069619).
Conceptualization: Louis J. Soslowsky, Michal A. Elovitz.
Formal analysis: Luz-Jeannette Sierra.
Funding acquisition: Michal A. Elovitz.
Investigation: Luz-Jeannette Sierra, Guillermo O. BarilaÂ, Carrie E. Barnum, Snehal S. Shetye.
Methodology: Luz-Jeannette Sierra, Guillermo O. BarilaÂ.
Project administration: Amy G. Brown.
Supervision: Amy G. Brown, Louis J. Soslowsky, Michal A. Elovitz.
15 / 19
Writing ± original draft: Luz-Jeannette Sierra.
Writing ± review & editing: Amy G. Brown, Lauren Anton, Michal A. Elovitz.
16 / 19
17 / 19
18 / 19
1. In: Behrman RE , Butler AS , editors. Preterm Birth: Causes , Consequences, and Prevention. The National Academies Collection: Reports funded by National Institutes of Health . Washington (DC) 2007 .
2. 2016 Premature Birth Report Card . http://www.marchofdimes.org/materials/premature-birth -reportcard-united-states . pdf2016 .
3. Romero R , Dey SK , Fisher SJ . Preterm labor: one syndrome, many causes . Science . 2014 ; 345 ( 6198 ): 760 ±5. https://doi.org/10.1126/science.1251816 PMID: 25124429 .
4. Brabant G. [ Bacterial vaginosis and spontaneous preterm birth] . Journal de gynecologie, obstetrique et biologie de la reproduction. 2016 ; 45 ( 10 ): 1247 ± 60 . https://doi.org/10.1016/j.jgyn. 2016 . 09 .014 PMID: 27793493 .
5. Luong ML , Libman M , Dahhou M , Chen MF , Kahn SR , Goulet L , et al. Vaginal douching, bacterial vaginosis, and spontaneous preterm birth . Journal of obstetrics and gynaecology Canada: JOGC = Journal d'obstetrique et gynecologie du Canada: JOGC . 2010 ; 32 ( 4 ): 313 ± 20 . https://doi.org/10.1016/S1701- 2163 ( 16 ) 34474 - 7 PMID: 20500937 .
6. Thorsen P , Vogel I , Olsen J , Jeune B , Westergaard JG , Jacobsson B , et al. Bacterial vaginosis in early pregnancy is associated with low birth weight and small for gestational age, but not with spontaneous preterm birth: a population-based study on Danish women. The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies , the International Society of Perinatal Obstet . 2006 ; 19 ( 1):1±7 . https:// doi.org/10.1080/14767050500361604 PMID: 16492583 .
7. Gilbert NM , Lewis WG , Lewis AL . Clinical Features of Bacterial Vaginosis in a Murine Model of Vaginal Infection with Gardnerella vaginalis . PloS one . 2013 ; 8 ( 3 ). https://doi.org/10.1371/journal.pone. 0059539 PMID: 23527214
8. Nelson DB , Hanlon A , Hassan S , Britto J , Geifman-Holtzman O , Haggerty C , et al. Preterm labor and bacterial vaginosis-associated bacteria among urban women . Journal of perinatal medicine . 2009 ; 37 ( 2 ): 130 ±4. https://doi.org/10.1515/JPM. 2009 .026 PMID: 18999913 .
9. Abramovici A , Lobashevsky E , Cliver SP , Edwards RK , Hauth JC , Biggio JR . Quantitative Polymerase Chain Reaction to Assess Response to Treatment of Bacterial Vaginosis and Risk of Preterm Birth . American journal of perinatology . 2015 ; 32 ( 12 ): 1119 ± 25 . https://doi.org/10.1055/s-0035-1549294 PMID: 26023904 .
10. Reid G , Bocking A . The potential for probiotics to prevent bacterial vaginosis and preterm labor . American journal of obstetrics and gynecology . 2003 ; 189 ( 4 ): 1202 ± 8 . PMID: 14586379 .
11. Brocklehurst P , Gordon A , Heatley E , Milan SJ . Antibiotics for treating bacterial vaginosis in pregnancy. The Cochrane database of systematic reviews . 2013 ; (1):CD000262 . https://doi.org/10.1002/14651858. CD000262.pub4 PMID: 23440777 .
12. Srinivasan S , Hoffman NG , Morgan MT , Matsen FA , Fiedler TL , Hall RW , et al. Bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria . PloS one . 2012 ; 7 ( 6 ):e37818. https://doi.org/10.1371/journal.pone.0037818 PMID: 22719852 .
13. Nelson DB , Rockwell LC , Prioleau MD , Goetzl L . The role of the bacterial microbiota on reproductive and pregnancy health . Anaerobe . 2016 ; 42 : 67 ± 73 . https://doi.org/10.1016/j.anaerobe. 2016 . 09 .001 PMID: 27612939 .
14. Breshears LM , Edwards VL , Ravel J , Peterson ML . Lactobacillus crispatus inhibits growth of Gardnerella vaginalis and Neisseria gonorrhoeae on a porcine vaginal mucosa model . BMC microbiology . 2015 ; 15 : 276 . https://doi.org/10.1186/s12866-015-0608-0 PMID: 26652855 .
15. Romero R , Hassan SS , Gajer P , Tarca AL , Fadrosh DW , Bieda J , et al. The vaginal microbiota of pregnant women who subsequently have spontaneous preterm labor and delivery and those with a normal delivery at term . Microbiome . 2014 ; 2 : 18 . https://doi.org/10.1186/2049-2618-2-18 PMID: 24987521 .
16. Ma B , Forney LJ , Ravel J . Vaginal microbiome: rethinking health and disease . Annual review of microbiology . 2012 ; 66 : 371 ± 89 . https://doi.org/10.1146/annurev-micro- 092611 -150157 PMID: 22746335 .
17. Bai G , Gajer P , Nandy M , Ma B , Yang H , Sakamoto J , et al. Comparison of storage conditions for human vaginal microbiome studies . PloS one . 2012 ; 7 ( 5 ):e36934. https://doi.org/10.1371/journal.pone. 0036934 PMID: 22655031 .
18. Ravel J , Gajer P , Abdo Z , Schneider GM , Koenig SS , McCulle SL , et al. Vaginal microbiome of reproductive-age women . Proceedings of the National Academy of Sciences of the United States of America . 2011 ; 108 Suppl 1 : 4680 ±7. https://doi.org/10.1073/pnas.1002611107 PMID: 20534435 .
19. Mielczarek E , Blaszkowska J . Trichomonas vaginalis: pathogenicity and potential role in human reproductive failure . Infection . 2016 ; 44 ( 4 ): 447 ± 58 . https://doi.org/10.1007/s15010-015-0860-0 PMID: 26546373 .
20. Burgmeier C , Dreyhaupt J , Schier F . Gender-related differences of inguinal hernia and asymptomatic patent processus vaginalis in term and preterm infants . Journal of pediatric surgery . 2015 ; 50 ( 3 ): 478 ± 80 . https://doi.org/10.1016/j.jpedsurg. 2014 . 08 .015 PMID: 25746711 .
21. Holst E , Goffeng AR , Andersch B . Bacterial vaginosis and vaginal microorganisms in idiopathic premature labor and association with pregnancy outcome . Journal of clinical microbiology . 1994 ; 32 ( 1 ): 176 ± 86 . PMID: 8126176 .
22. Kimberlin DF , Andrews WW . Bacterial vaginosis: association with adverse pregnancy outcome . Seminars in perinatology. 1998 ; 22 ( 4 ): 242 ± 50 . PMID: 9738988 .
23. Martinez de Tejada B , Coll O , de Flores M , Hillier SL , Landers DV . [Prevalence of bacterial vaginosis in an obstetric population of Barcelona] . Medicina clinica . 1998 ; 110 ( 6 ): 201 ± 4 . PMID: 9547730 .
24. McDonald HM , O'Loughlin JA , Vigneswaran R , Jolley PT , Harvey JA , Bof A , et al. Impact of metronidazole therapy on preterm birth in women with bacterial vaginosis flora (Gardnerella vaginalis): a randomised, placebo controlled trial . British journal of obstetrics and gynaecology . 1997 ; 104 ( 12 ): 1391 ± 7 . PMID: 9422018 .
25. Robinson LS , Perry J , Lek S , Wollam A , Sodergren E , Weinstock G , et al. Genome Sequences of 15 Gardnerella vaginalis Strains Isolated from the Vaginas of Women with and without Bacterial Vaginosis . Genome announcements. 2016 ; 4 ( 5 ). https://doi.org/10.1128/genomeA. 00879 -16 PMID: 27688326 .
26. Fredricks DN , Fiedler TL , Thomas KK , Oakley BB , Marrazzo JM . Targeted PCR for detection of vaginal bacteria associated with bacterial vaginosis . Journal of clinical microbiology . 2007 ; 45 ( 10 ): 3270 ±6. https://doi.org/10.1128/JCM.01272-07 PMID: 17687006 .
27. DiGiulio DB , Callahan BJ , McMurdie PJ , Costello EK , Lyell DJ , Robaczewska A , et al. Temporal and spatial variation of the human microbiota during pregnancy . Proceedings of the National Academy of Sciences of the United States of America . 2015 ; 112 ( 35 ): 11060 ±5. https://doi.org/10.1073/pnas. 1502875112 PMID: 26283357 .
28. Callahan BJ , DiGiulio DB , Goltsman DSA , Sun CL , Costello EK , Jeganathan P , et al. Replication and refinement of a vaginal microbial signature of preterm birth in two racially distinct cohorts of US women . Proceedings of the National Academy of Sciences of the United States of America . 2017 ; 114 ( 37 ): 9966 ± 71 . https://doi.org/10.1073/pnas.1705899114 PMID: 28847941 .
29. Hilbert DW , Schuyler JA , Adelson ME , Mordechai E , Sobel JD , Gygax SE . Gardnerella vaginalis population dynamics in bacterial vaginosis . European journal of clinical microbiology & infectious diseases: official publication of the European Society of Clinical Microbiology . 2017 . https://doi.org/10.1007/ s10096-017-2933-8 PMID: 28197729 .
30. Vodstrcil LA , Twin J , Garland SM , Fairley CK , Hocking JS , Law MG , et al. The influence of sexual activity on the vaginal microbiota and Gardnerella vaginalis clade diversity in young women . PloS one . 2017 ; 12 ( 2 ):e0171856. https://doi.org/10.1371/journal.pone.0171856 PMID: 28234976 .
31. Hardy L , Jespers V , Van den Bulck M , Buyze J , Mwambarangwe L , Musengamana V , et al. The presence of the putative Gardnerella vaginalis sialidase A gene in vaginal specimens is associated with bacterial vaginosis biofilm . PloS one . 2017 ; 12 ( 2 ):e0172522. https://doi.org/10.1371/journal.pone.0172522 PMID: 28241058 .
32. Nallasamy S , Mahendroo M. Distinct Roles of Cervical Epithelia and Stroma in Pregnancy and Parturition . Seminars in reproductive medicine. 2017 ; 35 ( 2 ): 190 ± 200 . https://doi.org/10.1055/s-0037-1599091 PMID: 28278536 .
33. Nallasamy S , Yoshida K , Akins M , Myers K , Iozzo R , Mahendroo M. Steroid Hormones Are Key Modulators of Tissue Mechanical Function via Regulation of Collagen and Elastic Fibers . Endocrinology. 2017 ; 158 ( 4 ): 950 ± 62 . https://doi.org/10.1210/en.2016-1930 PMID: 28204185 .
34. Anton L , DeVine A , Sierra LJ , Brown AG , Elovitz MA . miR -143 and miR-145 disrupt the cervical epithelial barrier through dysregulation of cell adhesion, apoptosis and proliferation . Scientific reports . 2017 ; 7 ( 1 ): 3020 . https://doi.org/10.1038/s41598-017-03217-7 PMID: 28596604 .
35. Read CP , Word RA , Ruscheinsky MA , Timmons BC , Mahendroo MS . Cervical remodeling during pregnancy and parturition: molecular characterization of the softening phase in mice . Reproduction . 2007 ; 134 ( 2 ): 327 ± 40 . https://doi.org/10.1530/REP-07-0032 PMID: 17660242 .
36. Yoshida K , Mahendroo M , Vink J , Wapner R , Myers K. Material properties of mouse cervical tissue in normal gestation . Acta biomaterialia . 2016 ; 36 : 195 ± 209 . https://doi.org/10.1016/j.actbio. 2016 . 03 .005 PMID: 26961804 .
37. Bastek JA , Hirshberg A , Chandrasekaran S , Owen CM , Heiser LM , Araujo BA , et al. Biomarkers and cervical length to predict spontaneous preterm birth in asymptomatic high-risk women . Obstetrics and gynecology . 2013 ; 122 ( 2 Pt 1 ): 283 ±9. https://doi.org/10.1097/AOG.0b013e31829ab714 PMID: 23969796 .
38. Straach KJ , Shelton JM , Richardson JA , Hascall VC , Mahendroo MS . Regulation of hyaluronan expression during cervical ripening . Glycobiology . 2005 ; 15 ( 1 ): 55 ± 65 . https://doi.org/10.1093/glycob/cwh137 PMID: 15317739 .
39. Akins ML , Luby-Phelps K , Bank RA , Mahendroo M. Cervical softening during pregnancy: regulated changes in collagen cross-linking and composition of matricellular proteins in the mouse . Biology of reproduction . 2011 ; 84 ( 5 ): 1053 ± 62 . https://doi.org/10.1095/biolreprod.110.089599 PMID: 21248285 .
40. Yoshida K , Jiang H , Kim M , Vink J , Cremers S , Paik D , et al. Quantitative evaluation of collagen crosslinks and corresponding tensile mechanical properties in mouse cervical tissue during normal pregnancy . PloS one . 2014 ; 9 ( 11 ):e112391. https://doi.org/10.1371/journal.pone.0112391 PMID: 25397407 .
41. Timmons BC , Mahendroo M. Processes regulating cervical ripening differ from cervical dilation and postpartum repair: insights from gene expression studies . Reproductive sciences . 2007 ; 14 ( 8 Suppl) : 53 ± 62 . https://doi.org/10.1177/1933719107309587 PMID: 18089611 .
42. Timmons BC , Mitchell SM , Gilpin C , Mahendroo MS. Dynamic changes in the cervical epithelial tight junction complex and differentiation occur during cervical ripening and parturition . Endocrinology . 2007 ; 148 ( 3 ): 1278 ± 87 . https://doi.org/10.1210/en.2006-0851 PMID: 17138657 .
43. Barnum CE , Fey JL , Weiss SN , Barila G , Brown AG , Connizzo BK , et al. Tensile Mechanical Properties and Dynamic Collagen Fiber Re-Alignment of the Murine Cervix are Dramatically Altered Throughout Pregnancy . Journal of biomechanical engineering . 2017 ; 139 ( 6 ). https://doi.org/10.1115/1.4036473 PMID: 28418563 .
44. Gilbert NM , Lewis WG , Lewis AL . Clinical features of bacterial vaginosis in a murine model of vaginal infection with Gardnerella vaginalis . PloS one . 2013 ; 8 ( 3 ):e59539. https://doi.org/10.1371/journal.pone. 0059539 PMID: 23527214 .
45. Yellon SM , Ebner CA , Elovitz MA . Medroxyprogesterone acetate modulates remodeling, immune cell census, and nerve fibers in the cervix of a mouse model for inflammation-induced preterm birth . Reproductive sciences . 2009 ; 16 ( 3 ): 257 ± 64 . https://doi.org/10.1177/1933719108325757 PMID: 19087974 .
46. Favata M , Beredjiklian PK , Zgonis MH , Beason DP , Crombleholme TM , Jawad AF , et al. Regenerative properties of fetal sheep tendon are not adversely affected by transplantation into an adult environment . Journal of orthopaedic research: official publication of the Orthopaedic Research Society . 2006 ; 24 ( 11 ): 2124 ± 32 . https://doi.org/10.1002/jor.20271 PMID: 16944473 .
47. Nold C , Anton L , Brown A , Elovitz M. Inflammation promotes a cytokine response and disrupts the cervical epithelial barrier: a possible mechanism of premature cervical remodeling and preterm birth . American journal of obstetrics and gynecology . 2012 ; 206 ( 3 ): 208 e1± 7 . https://doi.org/10.1016/j.ajog. 2011 . 12 .036 PMID: 22285171 .
48. Akgul Y , Word RA , Ensign LM , Yamaguchi Y , Lydon J , Hanes J , et al. Hyaluronan in cervical epithelia protects against infection-mediated preterm birth . The Journal of clinical investigation . 2014 ; 124 ( 12 ): 5481 ±9. https://doi.org/10.1172/JCI78765 PMID: 25384213 .
49. Gonzalez JM , Xu H , Chai J , Ofori E , Elovitz MA . Preterm and term cervical ripening in CD1 Mice (Mus musculus): similar or divergent molecular mechanisms? Biology of reproduction . 2009 ; 81 ( 6 ): 1226 ± 32 . https://doi.org/10.1095/biolreprod.108.075309 PMID: 19684330 .
50. Elovitz MA , Gonzalez J . Medroxyprogesterone acetate modulates the immune response in the uterus, cervix and placenta in a mouse model of preterm birth. The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies , the International Society of Perinatal Obstet . 2008 ; 21 ( 4 ): 223 ± 30 . https:// doi.org/10.1080/14767050801923680 PMID: 18330817 .
51. Racicot K , Cardenas I , Wunsche V , Aldo P , Guller S , Means RE , et al. Viral infection of the pregnant cervix predisposes to ascending bacterial infection . Journal of immunology . 2013 ; 191 ( 2 ): 934 ± 41 . https://doi.org/10.4049/jimmunol.1300661 PMID: 23752614 .
52. Romero R , Espinoza J , Goncalves LF , Kusanovic JP , Friel L , Hassan S. The role of inflammation and infection in preterm birth . Seminars in reproductive medicine . 2007 ; 25 ( 1 ): 21 ± 39 . https://doi.org/10. 1055/s-2006-956773 PMID: 17205421 .
53. Lechuga S , Ivanov AI . Disruption of the epithelial barrier during intestinal inflammation: Quest for new molecules and mechanisms . Biochimica et biophysica acta . 2017 ; 1864 (7): 1183 ± 94 . https://doi.org/10. 1016/j.bbamcr. 2017 . 03 .007 PMID: 28322932 .
54. McGuckin MA , Eri R , Simms LA , Florin TH , Radford-Smith G . Intestinal barrier dysfunction in inflammatory bowel diseases . Inflammatory bowel diseases . 2009 ; 15 ( 1 ): 100 ± 13 . https://doi.org/10.1002/ibd. 20539 PMID: 18623167 .
55. Barmeyer C , Erko I , Awad K , Fromm A , Bojarski C , Meissner S , et al. Epithelial barrier dysfunction in lymphocytic colitis through cytokine-dependent internalization of claudin-5 and -8 . Journal of gastroenterology. 2017 . https://doi.org/10.1007/s00535-017-1309-2 PMID: 28138755 .
56. Barmeyer C , Schulzke JD , Fromm M . Claudin-related intestinal diseases . Seminars in cell & developmental biology . 2015 ; 42 : 30 ±8. https://doi.org/10.1016/j.semcdb. 2015 . 05 .006 PMID: 25999319 .
57. Luettig J , Rosenthal R , Barmeyer C , Schulzke JD . Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation . Tissue barriers . 2015 ; 3 ( 1 ±2):e977176. https://doi.org/10.4161/21688370. 2014 .977176 PMID: 25838982 .
58. Pastorelli L , De Salvo C , Mercado JR , Vecchi M , Pizarro TT . Central role of the gut epithelial barrier in the pathogenesis of chronic intestinal inflammation: lessons learned from animal models and human genetics . Frontiers in immunology. 2013 ; 4 : 280 . https://doi.org/10.3389/fimmu. 2013 .00280 PMID: 24062746 .
59. Shen L , Weber CR , Raleigh DR , Yu D , Turner JR . Tight junction pore and leak pathways: a dynamic duo . Annual review of physiology . 2011 ; 73 : 283 ± 309 . https://doi.org/10.1146/annurev-physiol- 012110 - 142150 PMID: 20936941 .
60. Durer U , Hartig R , Bang S , Thim L , Hoffmann W. TFF3 and EGF induce different migration patterns of intestinal epithelial cells in vitro and trigger increased internalization of E-cadherin . Cellular physiology and biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology . 2007 ; 20 ( 5 ): 329 ± 46 . https://doi.org/10.1159/000107519 PMID: 17762162 .
61. Cornick S , Tawiah A , Chadee K. Roles and regulation of the mucus barrier in the gut . Tissue barriers . 2015 ; 3 ( 1 ±2):e982426. https://doi.org/10.4161/21688370. 2014 .982426 PMID: 25838985 .
62. Elovitz M , Wang Z . Medroxyprogesterone acetate, but not progesterone, protects against inflammationinduced parturition and intrauterine fetal demise . American journal of obstetrics and gynecology . 2004 ; 190 ( 3 ): 693 ± 701 . https://doi.org/10.1016/j.ajog. 2003 . 10 .693 PMID: 15042001 .
63. Timmons BC , Reese J , Socrate S , Ehinger N , Paria BC , Milne GL , et al. Prostaglandins are essential for cervical ripening in LPS-mediated preterm birth but not term or antiprogestin-driven preterm ripening . Endocrinology . 2014 ; 155 ( 1 ): 287 ± 98 . https://doi.org/10.1210/en.2013-1304 PMID: 24189143 .
64. Gilbert NM , O'Brien VP , Lewis AL . Transient microbiota exposures activate dormant Escherichia coli infection in the bladder and drive severe outcomes of recurrent disease . PLoS pathogens . 2017 ; 13 ( 3 ): e1006238. https://doi.org/10.1371/journal.ppat.1006238 PMID: 28358889 .
65. Sha BE , Chen HY , Wang QJ , Zariffard MR , Cohen MH , Spear GT . Utility of Amsel criteria, Nugent score, and quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp. for diagnosis of bacterial vaginosis in human immunodeficiency virus-infected women . Journal of clinical microbiology . 2005 ; 43 ( 9 ): 4607 ± 12 . https://doi.org/10.1128/JCM.43.9. 4607 - 4612 . 2005 PMID: 16145114 .
66. Sha BE , Zariffard MR , Wang QJ , Chen HY , Bremer J , Cohen MH , et al. Female genital-tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Mycoplasma hominis . The Journal of infectious diseases . 2005 ; 191 ( 1 ): 25 ± 32 . https://doi.org/10.1086/426394 PMID: 15592999 .
67. Cohn JA , Hashemi FB , Camarca M , Kong F , Xu J , Beckner SK , et al. HIV-inducing factor in cervicovaginal secretions is associated with bacterial vaginosis in HIV-1-infected women . Journal of acquired immune deficiency syndromes . 2005 ; 39 ( 3 ): 340 ± 6 . PMID: 15980696 .
68. Al-Harthi L , Roebuck KA , Olinger GG , Landay A , Sha BE , Hashemi FB , et al. Bacterial vaginosis-associated microflora isolated from the female genital tract activates HIV-1 expression . Journal of acquired immune deficiency syndromes . 1999 ; 21 ( 3 ): 194 ± 202 . PMID: 10421242 .
69. Al-Harthi L , Spear GT , Hashemi FB , Landay A , Sha BE , Roebuck KA . A human immunodeficiency virus (HIV)-inducing factor from the female genital tract activates HIV-1 gene expression through the kappaB enhancer . The Journal of infectious diseases . 1998 ; 178 ( 5 ): 1343 ± 51 . PMID: 9780254 .
70. St John EP , Zariffard MR , Martinson JA , Simoes JA , Landay AL , Spear GT . Effect of mucosal fluid from women with bacterial vaginosis on HIV trans-infection mediated by dendritic cells . Virology . 2009 ; 385 ( 1 ): 22 ±7. https://doi.org/10.1016/j.virol. 2008 . 08 .031 PMID: 19117586 .
71. Isik G , Demirezen S , Donmez HG , Beksac MS . Bacterial vaginosis in association with spontaneous abortion and recurrent pregnancy losses . Journal of cytology . 2016 ; 33 ( 3 ): 135 ± 40 . https://doi.org/10. 4103/ 0970 - 9371 .188050 PMID: 27756985 .