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\begin{document}

\title{Alterations in Vaginal Microbiota among Pregnant Women with COVID-19}



\author[1]{Ebru Celik}%
\author[2]{Gulin Ozcan}%
\author[2]{Cansel Vatansever}%
\author[3]{Erxiati  Paerhati }%
\author[4]{Mert Kuskucu}%
\author[2]{Ozlem Dogan}%
\author[2]{Sebile Guler Cekic}%
\author[2]{Onder Ergonul}%
\author[3]{Attila Gursoy}%
\author[3]{Ozlem Keskin}%
\author[2]{Fusun Can}%
\affil[1]{Koc Universitesi}%
\affil[2]{Koc University School of Medicine}%
\affil[3]{Koc University College of Engineering}%
\affil[4]{Istanbul University-Cerrahpasa Cerrahpasa Faculty of Medicine}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objective: To describe the alterations of the composition of vaginal
microbiota in pregnant women with COVID-19. Design: Prospective
observational single-centre study Setting: Tertiary referral hospital
Participants: Pregnant women with COVID-19 Methods: The vaginal swabs
were collected during the active phase of infection and consecutively,
within a month after recovering from infection. In three patients,
longitudinal samples before, in the course, and after infection were
also obtained. The microbiome alterations were examined by 16S rRNA gene
sequencing. Main outcome measures: Vaginal microbiota profiles in
pregnant women with COVID-19 Results: Nineteen pregnant women with
COVID-19 and 28 healthy controls who were matched according to the
maternal age and gestational week were recruited. Shannon index and
inverse Simpson index for cross-sectional cohort indicate that alpha
diversity is significantly higher in women with COVID-19 (P=0.007 and
P=0.006, respectively). There was a significantly decrease in Firmicutes
(P=0.007) and Lactobacillus (P=0.019) with an increase in Bacteroidetes
(P=0.024) in women with COVID-19 when compared to those of healthy
controls. The higher amounts of Ureaplasma were found in women with the
moderate/severe disease, compared to those of the asymptomatic/mild
disease (P=0.001). Lactobacillus gasseri disappeared in women with the
moderate/severe disease. Prevotella timonensis was identified only in
the COVID-19 group. In longitudinal analysis, Actinobacteria was
elevated, Firmicutes and Bacteroides depleted during the active phase.
Conclusion: The study revealed that vaginal dysbiosis with a low
abundance of Lactobacillus and an increase in Bacteroidetes is
associated with COVID-19.%
\end{abstract}%



\sloppy


\textbf{Alterations in Vaginal Microbiota among Pregnant Women with
COVID-19}

Ebru Celik \textsuperscript{1*},M.D., Gulin
Ozcan\textsuperscript{2,3*},Mrs, Cansel Vatansever
\textsuperscript{2,3},Mrs, Erxiati Paerhati \textsuperscript{4},Mr, Mert
Ahmet Ku\selectlanguage{polish}ş\selectlanguage{english}kucu\textsuperscript{3,5}, M.D., Ozlem Dogan
\textsuperscript{2,3},Prof, Sebile Guler Cekic \textsuperscript{1},
M.D.,Onder Ergonul\textsuperscript{3,6}, Prof, Attila G\selectlanguage{ngerman}ürsoy
\textsuperscript{4}, Prof, Özlem Keskin \textsuperscript{4}, M.D., Fusun
Can\textsuperscript{2,3**}, Prof.

\textsuperscript{1} Koc University, School of Medicine, Department of
Obstetrics and Gynecology, Istanbul, Turkey

\textsuperscript{2} Koc University, School of Medicine, Department of
Medical Microbiology, Istanbul, Turkey

\textsuperscript{3} Koç University IsBank Research Center for Infectious
Diseases (KUISCID), Istanbul, Turkey

\textsuperscript{4}Koç University, College of Engineering, Istanbul,
Turkey

\textsuperscript{5} Cerrahpa\selectlanguage{polish}ş\selectlanguage{english}a Medical School of Istanbul
University-Cerrahpa\selectlanguage{polish}ş\selectlanguage{english}a, Medical Microbiology, Istanbul, Turkey

\textsuperscript{6}Ko\selectlanguage{ngerman}ç University, School of Medicine, Department of
Infectious Diseases and Clinical Microbiology, Istanbul, Turkey

\textbf{Short Title:} Vaginal Microbiota in COVID-19

* These authors are equally contributing first authors.

** Corresponding Author

Corresponding Author: Prof Dr Fusun Can

E-mail: fucan@ku.edu.tr

Koç University, School of Medicine, Department of Microbiology

34450 Sar\selectlanguage{polish}ı\selectlanguage{english}yer/\selectlanguage{polish}İ\selectlanguage{english}stanbul

\textbf{ABSTRACT}

\textbf{Objective:} To describe the alterations of the composition of
vaginal microbiota in pregnant women with COVID-19.

\textbf{Design:} Prospective observational single-centre study

\textbf{Setting:} Tertiary referral hospital

\textbf{Participants:} Pregnant women with COVID-19

\textbf{Methods:} The vaginal swabs were collected during the active
phase of infection and consecutively, within a month after recovering
from infection. In three patients, longitudinal samples before, in the
course, and after infection were also obtained. The microbiome
alterations were examined by 16S rRNA gene sequencing.

\textbf{Main outcome measures:} Vaginal microbiota profiles in pregnant
women with COVID-19

\textbf{Results:} Nineteen pregnant women with COVID-19 and 28 healthy
controls who were matched according to the maternal age and gestational
week were recruited. Shannon index and inverse Simpson index for
cross-sectional cohort indicate that alpha diversity is significantly
higher in women with COVID-19 (P=0.007 and P=0.006, respectively). There
was a significantly decrease in Firmicutes (P=0.007) and Lactobacillus
(P=0.019) with an increase in Bacteroidetes (P=0.024) in women with
COVID-19 when compared to those of healthy controls. The higher amounts
of Ureaplasma were found in women with the moderate/severe disease,
compared to those of the asymptomatic/mild disease (P=0.001).
Lactobacillus gasseri disappeared in women with the moderate/severe
disease. Prevotella timonensis was identified only in the COVID-19
group. In longitudinal analysis, Actinobacteria was elevated, Firmicutes
and Bacteroides depleted during the active phase.

\textbf{Conclusion:} The study revealed that vaginal dysbiosis with a
low abundance of Lactobacillus and an increase in Bacteroidetes is
associated with COVID-19.

\textbf{Funding source:} This study was funded by The Scientific and
Technological Research Council of Turkey (TUBITAK, Project no:119S463).

\textbf{Keywords:} Vaginal microbiome, vaginal microbiota, COVID-19,
pregnancy

\textbf{Introduction}

Coronavirus disease 2019 (COVID-19) has emerged worldwide while a subset
of people was infected severely, the others recovered from the disease
without symptoms. COVID-19 has been shown to have potential adverse
effects on pregnancy and neonatal outcomes. Pregnancy itself is a
risk~factor for the severity of COVID-19 disease, with an increased risk
of ICU admission, maternal morbidity, and
mortality\textsuperscript{1,2}. Furthermore, pregnancy complications
such as preeclampsia and preterm birth are more likely to occur in women
diagnosed with COVID-19 \textsuperscript{3,4}.

The predominance of Lactobacillus species (spp.) plays a key role in the
inhibition of binding non-domestic and potentially harmful
microorganisms to epithelial cells
\textsuperscript{5--7}.\emph{Lactobacillus} maintains the protective low
vaginal pH through secreting of lactic acid \textsuperscript{8}.
Pregnant women with decreased amounts of \emph{Lactobacillus crispatus,
Lactobacillus gasseri,} and \emph{Lactobacillus jensenii} in the vaginal
microbiota are more likely to deliver preterm
\textsuperscript{9}.~Likewise, the abundance of \emph{Gardnerella
vaginalis} increases the risk of preterm birth (PTB)
\textsuperscript{5,10}.

The mechanism of action of severe acute respiratory syndrome
coronavirus-2 (SARS-CoV-2) on pregnancy varies and remains unknown. The
SARS-CoV-2 genome has been identified in the vaginal mucosa of a
pregnant woman \textsuperscript{11}. Indeed, the role of SARS-CoV-2
infection in vaginal microbiome composition in pregnant women with
COVID-19 has not yet been investigated. Therefore, we anticipate that
COVID-19 may unfavorably affect the composition of the vaginal
microbiota, resulting in adverse pregnancy outcomes. We aimed to
describe the alterations of the composition of vaginal microbiota in
pregnant women with COVID-19.

\textbf{Materials and Methods}

\textbf{Study population:}

A prospective study was conducted at Koc University Hospital between
August 2020 and August 2021. Pregnant women with active or recently
infected within one month with SARS-CoV-2 were included in the study.
SARS-CoV-2 infection was confirmed by a positive nasopharyngeal
polymerase chain reaction (PCR) test. The signs and symptoms of COVID-19
were evaluated in all women who had positive PCR tests for SARS-CoV-2
infection. COVID-19 was classified according to NIH COVID-19 clinical
guidelines \textsuperscript{12}. Patients with symptoms including fever,
myalgia, or gastrointestinal system symptoms were categorized as a mild
disease, those who required oxygen (O\textsubscript{2}) supplementation
as moderate or severe disease (MSD). Recovery was defined as clinical
improvement in combination with a negative nasopharyngeal PCR test.
Single vaginal swab was collected from each participant at the time of
COVID-19 and within one month after recovery from COVID-19. In three
patients, longitudinal microbiota analysis was performed with a
collection of vaginal swabs before, during active infection, and two
months after recovery. The overview of patients was presented in Figure
1.

The healthy controls (HC) were recruited from the prospective study
entitled ``Vaginal, Placental and Neonatal Buccal Mycobiota, and
Microbiome in Preterm Birth''. The study was initiated in April 2020
(ClinicalTrials.gov Identifier: NCT04165252). Maternal age and
gestational week-matched pregnant women were selected for the healthy
controls. Inclusion criteria for the healthy controls are as follows:
age older than 18 years with a singleton pregnancy. The exclusion
criteria were multiple pregnancies, major fetal structural defects
and/or chromosomal abnormalities, stillbirth, having used antibiotics
and/or antifungal medication within two weeks at the time of sample
collection, the presence of vaginal bleeding at the time of sample
collection, and sexual intercourse within 72 hours of sample collection.

Maternal characteristics, medical and obstetrical history were recorded
for all participants. Maternal height and weight were measured at the
same time with vaginal swabs collection. Gestational age was determined
from the last menstrual period and confirmed from the measurement of
fetal crown-rump length at the first-trimester scan. The written
informed consent was obtained from all participants. Ko\selectlanguage{ngerman}ç University
Research Ethics Board approved the study protocol. The study complies
with the decleration of Helsinki.

\textbf{Sample collection, processing, and sequencing}

Vaginal samples were collected with REMEL ESwabs. Vaginal swabs were
placed into the sterile tube then stored at \selectlanguage{english}-80degC until DNA extraction.
Frozen vaginal swabs were immersed in sterile PBS then DNA extraction
was performed using the Qiagen DNeasy PowerSoil Kit (Qiagen, Hilden,
Germany), as described by the manufacturer. DNA concentration was
quantified by Qubit (ThermoFisher Scientific, Waltham MA).

Library preparation was performed using QIAseq 16S/ITS Panel Kit
(Qiagen, Hilden, Germany) for sequencing the V1--V9 region of the 16S
rRNA genebacterial gene. Library quantification was done using QIAseq
Library Quant Assay (Qiagen, Hilden, Germany) kit following the
manufacturer's instructions with Applied Biosystems QuantStudio 7 Flex
Real-Time PCR (Applied Biosystems Inc., Foster City, California).
Sequencing was performed with the Illumina MiSeq platform using the
MiSeq v3 Reagent Kit (Illumina, San Diego, CA, United States).

\textbf{Bioinformatics}

FASTQ files were demultiplexed by different regions using the module in
the GeneGlobe Data Analysis Center.
(https://geneglobe.qiagen.com/tr/analyze). The resulting paired-end
FASTQ files were used to profile the microbiota of the samples with
DADA2 using default parameters. Sequencing of 16S rRNA gene(V1-V2)
regions was performed \textsuperscript{13}. Amplicon sequence variants
(ASV) were picked out and assigned with taxonomic annotations using the
naive Bayesian classifier method embedded in DADA2 \textsuperscript{14}.
The Silva (v138.1) was used as a reference database
(v138.1)\textsuperscript{15}.

\textbf{Statistical analysis}

Continuous variables were expressed as a median and interquartile range
(IQR), whereas categorical variables were expressed as percentages.
Mann-Whitney U and Fisher exact tests were applied for comparison of
maternal demographic and clinical characteristics between COVID-19
patients with healthy controls. Data were analyzed using IBM SPSS
Statistics for Windows, version 26.0 (Armonk, NY: IBM Corp.).

The alpha diversity, beta diversity, and vaginal microbiota composition
of the COVID-19 group, and healthy control were compared.
Alpha-diversity metrics were calculated as the number of phyla, genus,
and species. The vaginal microbiota of asymptomatic or mild, moderate,
or severe cases and healthy controls were compared. Beta-diversity was
defined by using Bray-Curtis distance. The differences in alpha
diversity metrics were performed by Wilcoxon signed-rank test using
Python 3.7 (32-bit) and statistical significance was set as P value
\textless{}.05. Statistical data were visualized with GraphPad Prism
8.0.2.

\textbf{Data availability:} All data generated or analyzed during this
study are included in this published article.

\textbf{Results}

\textbf{Study cohort demographics}

There were no differences in maternal age, BMI, gestational age at
delivery, and birth weight between women with the COVID-19 group with
healthy controls. (Table 1). Of 19 women with COVID-19, 13 women had the
asymptomatic or mild disease (AMD) (68.4\%), and 6 of those had moderate
or severe disease (31.6\%). The rate of preterm birth (PTB) was 15.3\%
(n=3) in the COVID-19 group; 2 of 6 women (33.3\%) with moderate or
severe disease and 1 of 13 women (7.7\%) with asymptomatic or mild
disease (Table 2).

In asymptomatic or mild disease, 61.5\% of those had infection in the
second trimester and 38.5\% in the third trimester (Table 2). Two women
with moderate or severe disease received both antibiotic and antiviral
medications. Seven patients received low molecular weight heparin during
the active period of COVID-19 disease (Table 2).

\textbf{The composition of vaginal microbiota composition in the healthy
controls and women with COVID-19}

Alpha diversity was evaluated by using Shannon and Simpson Index. In the
COVID-19 group, the Shannon index was significantly elevated, compared
to those of the healthy controls (1.09 vs. 0.75; P=0.007), and the
Simpson index was significantly lower in the COVID-19 group than those
of healthy controls (0.4 vs. 0.61; P=0.006) (Figure 2A).~In pregnant
women with asymptomatic/mild and moderate/severe disease, the Shannon
index was found to be significantly higher than those of the healthy
controls (1.06 and 1.16 vs. 0.75; P\textless{}0.05), whereas the Simpson
index was significantly decreased, compared to the healthy controls
(0.42 and 0.49 respectively, vs. 0.61; P\textless{}0.05) (Figure 2A).
Beta diversity (Bray-Curtis) results indicated that there were no
compositional differences between COVID-19 status and healthy controls
(P=0.49) (Figure 2B).

There were no statistically significant differences in the alpha
diversity of vaginal microbiota between the second and third trimesters
of healthy controls according to Shannon index and Simpson index (0.41
vs. 0.5; P= 0.22 and, 0.8 vs. 0.75; P= 0.08, respectively).

The vaginal microbiota composition and relative abundances of the
bacterial phylum, genera and species for two groups were summarized in
Figure 3. The three phyla; Firmicutes (86.02\%), Actinobacteria
(12.12\%) and Bacteriodata (Bacteroidetes) (0.54\%) accounted for 99.8\%
of the bacterial species in COVID-19 group. In a comparison of COVID-19
group with healthy controls, Firmicutes significantly decreased (85.9\%
vs. 96.11, P=0.049) while the amounts of Bacteroidetes significantly
increased (0.53\% vs. 0.44\%; P=0.024) (Figure 3A).

At the genus level, \emph{Lactobacillus sp.} significantly decreased in
the COVID-19 group compared to the healthy controls (80.6\% vs. 93.98\%;
P=0.019).~ Particularly, \emph{Lactobacillus delbrueckii (L.
delbrueckii) was not detected} in the COVID-19 group, although its
amount was 1.5\% in the healthy controls (P=0.046) (Figure 3B).

\emph{Lactobacillus iners (L.iners)} , \emph{Lactobacillus crispatus (L.
crispatus)} , and \emph{Lactobacillus jensenii (L. jensenii)} showed
trends towards a decline in COVID-19 group (26.5\%, 3.2\%, and 7.1\%,
respectively) when compared to the healthy controls (32.8\%, 3.9\%, and
7.5\%, respectively) but the differences were not statistically
significant (P\textgreater{}0.05) (Figure 3C).

In the COVID-19 group, among anaerobe taxa, particularly\emph{Prevotella
timonensis} (0.04\%) and \emph{Dialister propionicifaciens (0.02\%)}
were identified but none of these were detected in the healthy controls.
In addition to these, the COVID-19-infected group showed a higher
abundance of Gardnerella\emph{vaginalis} (6.4\% vs. 1.98\%; P=0.213).
Also, \emph{Ureaplasma}(0.69\% vs. 0.04\%; P=0.054) was found to be
higher in the COVID-19 group compared to the healthy controls.

\textbf{The variations of vaginal microbiota composition in relation to
the severity of COVID-19}

\emph{Mycoplasma hominis} , \emph{Fusobacterium nucleatum}
and\emph{Anaerococcus tetradius} were only identified in patients with
moderate or severe disease (8\%, 0.03\%, and 0.003\%, respectively)
(Figure 4). In a comparison of the patients with asymptomatic or mild
and moderate or severe disease, there was no significant difference in
abundance of \emph{Prevotella timonensis} (0.05\% and 0.02\%, p=0.379);
however, \emph{Ureaplasma spp.} was significantly higher in the moderate
or severe group (2.09\%) than those of the asymptomatic or mild (
0.04\%, p=0.005).~In addition, \emph{Gardnerella vaginalis} showed a
gradual increase in both two groups (1.99\% and 5.11\%) compared to the
healthy controls (9.33\%) (Figure 4).

In the longitudinal vaginal microbiota analysis of the three pregnant
women, the relative abundance of Actinobacteria increased from 10.8\% to
59.2\% during active infection and returned to 10.8\% in the
post-COVID-19 phase. There is a declining trend in the abundance of
Bacteroidata (Bacteroidetes) (from 65.4\% to 17.9\%) and Firmicutes
(from 59.2\% to 28.4\%) during the phase of active COVID-19 with no
regrowth in the post-COVID-19 phase (17.9\% and 10.8\%, respectively)
(Figure 5).

\textbf{Discussion}

\textbf{Main Findings}

The findings of our study indicated that the composition of vaginal
microbiota was unfavorably affected by COVID-19 disease and there was a
prominent dysbiosis during active COVID-19 infection. Intrauterine
infection is a well-established reason for preterm birth. The most
common and genuine pathway is that microorganisms can access the
amniotic cavity by ascending from the vagina and cervix, resulting in
the development of intrauterine infection and subsequent inflammatory
response in fetoplacental tissues that eventuates preterm
birth\textsuperscript{16,17}.~Since evidence on the relation of
dysbiosis in vaginal microbiota and preterm birth is accumulating, we
can speculate that one of the mechanisms for the explanation of
increased rate of COVID-19-associated PTB may be vaginal
dysbiosis\textsuperscript{5,18,19}.

Vaginal dysbiosis is defined as an increase of alpha diversity in
vaginal microbiome communities \textsuperscript{20}. The study found
that the Shannon index was remarkably high (1.16) in the COVID-19 group
compared to the healthy controls (0.75). Recent studies have revealed
that vaginal dysbiosis has a negative impact on vaginal protective
mechanisms via increasing local pro-inflammatory
effectors\textsuperscript{21,22}.

We identified diminished \emph{Lactobacillus} communities in women with
COVID-19 disease, particularly more profound in those with moderate or
severe disease (77\%) when compared to the healthy controls (93\%;
P=0.04). \emph{Lactobacillus delbrueckii} (P=0.046) significantly
decreased among women with COVID-19. Within-subgroup
analysis,\emph{Lactobacillus gasseri (L. gasseri)} disappeared in
patients with moderate or severe disease. It is well-established that
pregnant women with low amounts of \emph{L. crispatus, L. gasseri, or L.
jensenii} in their vaginal microbiota are more likely to deliver before
term\textsuperscript{9,23}. In a case-control study, the abundance
of\emph{L. gasseri} was found to be associated with decreased risk of
early spontaneous preterm birth \textsuperscript{24}.

In the longitudinal study with three patients, we didn't perform
analysis at the species level because of the small sample size. There
was approximately a 40\% decline in the relative abundance of Firmicutes
and Bacteroides during the active COVID-19 period, which was sustained
after recovery. In addition, the abundance of Actinobacteria was the
highest in the active disease stage compared to the pre and post
COVID-19 periods. Ceccarani et al. revealed that the vaginal flora of
healthy women was constituted of mainly Firmicutes and Bacteroidetes,
albeit with a low abundance of Actinobacteria \textsuperscript{25}. In
view of our findings, it would be considered that SARS-CoV-2 infection
negatively affects the vaginal compositions of pregnant women.

\textbf{Strengths and Limitations}

The most important strength of the study is the first trial, which
elucidated the relation of SARS-CoV-2 infection with the composition of
vaginal flora. The vaginal samples were comprehensively collected from
the healthy controls and women with COVID-19 disease. The samples from
vaginal flora were analyzed exclusively by the three doctors in the
group. Furthermore, we had longitudinal samples from three pregnant
women, which provided an opportunity for comparison of vaginal
composition of pregnant women before and after the disease. Further,
detailed clinical data were collected from each participant and reported
to consider for an evaluation of each subject.

There are several limitations. Our sample size was small to detect the
statistical significance between women with severe COVID-19 and women
with asymptomatic or mild disease, albeit with some significant
differences. Secondly, a potential confounding factor that may differ
among the groups was the use of antibiotics in the severe cases during
the active stage at the time of sample collection.

\textbf{Interpretations}

Disruption of balance of vaginal microbiota causes invades of several
facultative or strict anaerobes, including \emph{Gardneralla vaginalis,
Mycoplasma hominis, Prevotella spp., Fusobacterium spp} ., Ureoplasma
spp., and \emph{Porphyromonas spp.} and replacement
of\emph{Lactobacilli} \textsuperscript{26--28}. Aligning with the
aforementioned results, we identified a significantly higher abundance
of Bacteroidetes in COVID-19 group. In particular, \emph{Prevotella
timonensis was only identified} in women with COVID-19 (0.04\%).
Likewise, \emph{Dialister propionicifaciens} appeared in the COVID-19
group (0.02\%). The SARS-CoV-2 infection triggers the production of
prostaglandins and pro-inflammatory mediators to confer ischemia
resulting in widespread tissues \textsuperscript{29,30}. Notably, we
determined an increase of anaerobic species such as \emph{Gardnerella
vaginalis} , \emph{Anaerococcus tetradius} , \emph{Fusobacterium
nucleatum} , \emph{Prevotella timonensis} abundance in women with severe
disease. Based on these results, we postulated that ischemia in
genito-urinary compartments could be a predisposing factor for
overgrowth of anaerobes in vaginal microbiota.

Pregnant women are more susceptible to SARS-CoV-2 infection due to
physiological, mechanical, and immunological changes during
pregnancy\textsuperscript{2,31}. Data supported that pregnancy is a risk
factor for severe disease related to COVID-19 \textsuperscript{4,32}.
Recently, in a large population-based cohort study, fetal death and
preterm birth occurred more frequently in women with SARS-CoV-2
infection than non-infected women (adjusted odds ratio (aOR), 2.21; 95\%
confidence interval (CI), 1.58 -- 3.11; P\textless{} 0.001 and~OR, 2.17;
95\% CI, 1.96 -- 2.4; P\textless{}0.001, respectively)
\textsuperscript{1}. In the study, the prevalence of preterm birth
(15.3\%) was high, especially in the severe COVID-19 (2 out of 3 preterm
birth), as compared to the prevalence reported before COVID-19 (9.6\%)
\textsuperscript{33}. Even with the small sample size of our study, the
rate of preterm birth increased in women with severe COVID-19 disease
than those with asymptomatic or mild disease, aligning with a recent
meta-analysis\textsuperscript{34}. The abundance of \emph{Ureaplasma}
and\emph{Mycoplasma} species increased the risk of preterm delivery
through chorioamnionitis, salpingitis, bacterial vaginosis, and
postpartum endometritis \textsuperscript{20,35}. We found that
\emph{Mycoplasma hominis} (8\% vs. 0\%; P=0.01) and \emph{Ureaplasma}
spp. (2.09\% vs. 0.04\%, p=0.001) showed significantly higher abundance
in moderate or severe cases compared to those of the healthy controls.
Of note, the abundance of \emph{Ureaplasma spp.} was significantly
higher in women with moderate or severe disease than those of
asymptomatic or mild disease (p=0.005). We claim in light of these
findings and previous evidence from microbiota studies that the
subsequent preterm birth in women with severe disease could be a
consequence of impaired vaginal composition.

\textbf{Conclusions}

COVID-19 disease in pregnant women causes dysbiosis in vaginal
microbiota with a significant reduction in the abundance of
Lactobacillus combined with an increase of Bacteroidetes, especially
in\emph{Prevotella timonensis} . We detected that the severity of the
disease was associated with increased \emph{Ureaplasma spp.} Based on
these findings, we suggest that COVID-19 promotes an unfavorable vaginal
microenvironment, which may provide insight into the risk of adverse
pregnancy outcomes such as preterm birth. These results raise clinically
relevant questions regarding the use of microbiome associated biomarkers
as a risk assessment tool for preterm birth in pregnant women during
COVID-19. The implication of findings would postulate possible targeted
therapy, comprising modification of the vaginal microbiota composition
in pregnant women with COVID-19.

\textbf{Disclosure of interests:} The authors declare that they have no
competing interests.

\textbf{Contribution to authorship:} F.C., E.C., G.O. and C.V. designed
and conducted the research. F.C., M. K., O.D., E.C., O.E., G.O., C.V.,
A.G. and O.K. interpreted the results and wrote the manuscript. S.G.C
and E.C. collected the samples and clinical outcomes. G.O. and C.V.
processed the samples, prepared the DNA for sequencing, and conducted
the experiments. E.P., G.O., C.V., E.C. analyzed the data and prepared
the figures and tables. E.P. processed the bioinformatic analysis. A.G.
and O.K. supervised the bioinformatics analysis.

\textbf{Details of ethics approval:} The ethical approval was obtained
for the healthy controls and pregnant women with COVID-19 from Koc
University Research Ethics Board (No:2019.093IRB2.030 and
No:2020.138.IRB1.028). The written consent forms were obtained from all
participants.

\textbf{Funding:} This study was funded by The Scientific and
Technological Research Council of Turkey (TUBITAK, Project no:119S463).



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\textbf{Figure legends}

\textbf{Figure 1.} An overview of study design

The cohort includes pregnant women with COVID-19 (n=19) and the healthy
controls (n=28), recruited from Clinical Trials. NCT04165252 study.
Longitudinal study cases were shown in purple. The three preterm birth
cases were shown in black circles.

\textbf{Figure 2.} The comparison of alpha diversity of the vaginal
microbiota between healthy controls and pregnant women with COVID-19

TC and HC refer to total COVID-19 patients (n=19) and healthy controls
(n=28), respectively. Pregnant women with COVID-19 were divided into two
sub-groups according to severity of disease as Asymptomatic/Mild (AMC)
(n=13) and Moderate/Severe (MSC) (n=6). Mann Whitney U rank test is
performed for comparison of alpha diversity metrics. Data are presented
as mean The P value less than 0.05 is accepted as significant (*
P\textless{}0.01, ** P\textless{}0.05). \textbf{B.} Beta diversity was
analyzed with Bray-Curtis distances (P=0.49). Blue color refers to the
healthy controls and pregnant women with COVID-19 were presented in red
colour.

\textbf{Figure 3.} The vaginal microbiome composition of healthy
controls and women with COVID-19

Healthy control (n=28) and women with COVID-19 (n=19); A. The most
abundant bacteria at the phylum level are represented for two groups.
B.The most abundant bacteria at the genus level are represented for two
groups. C. The relative abundance of different bacteria species in two
groups. Only phyla and genera present at relative abundances
\textgreater{}0.01\% are reported. Remaining taxa are grouped as
``Others''.

\textbf{Figure 4.} The comparison of abundance of vaginal microbiota
species among Healthy Controls, Asymptomatic/Mild and Moderate/Severe
cases

* refers to statistical significance P\textless{}0.05; ** represents
statistical significance P\textless{}0.01; P1 refers the comparison of
bacteria species between the healthy controls with women with
asymptomatic/mild disease; P2 refers the comparison of bacteria species
between the healthy controls with women with moderate/severe disease. P3
refers to the comparison of bacteria species between women with
asymptomatic/mild disease with those of moderate/severe disease.

\textbf{Figure 5.} The longitudinal analysis of vaginal microbiota

The abundance rate of Phyla in vaginal microbiota before COVID-19,
during acute phase and after recovery in three patients
(asymptomatic/mild {[}n=2{]} and moderate/severe {[}n=1{]})



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