Alterations in vaginal microbiota among pregnant women with COVID‐19

Abstract The maintenance of vaginal microbiota is an important factor to achieve optimum pregnancy outcomes. The study aims to describe the alterations in the composition of vaginal microbiota in pregnant women with coronavirus disease 2019 (COVID‐19). This was a prospective case‐control study. Vaginal swabs were collected from uninfected pregnant women (n = 28) and pregnant women with COVID‐19 (n = 19) during the active phase of infection and within a month after recovering from infection. The vaginal microbiota on the swabs was examined by 16S rRNA gene sequencing. Shannon index indicates that alpha diversity is significantly higher in women with COVID‐19 (p = 0.012). There was a significant decrease in Firmicutes (p = 0.014) with an increase in Bacteroidota (p = 0.018) phyla and a decrease in Lactobacillus (p = 0.007) genus in women with COVID‐19 than those of uninfected pregnant women. The relative abundance of L. crispatus, L. iners, L. gasseri, and L. jensenii were lower in the COVID‐19 group than in uninfected pregnant women. In subgroup analysis, the amount of Ureaplasma spp. was higher in women with moderate/severe than those of asymptomatic/mild disease (p = 0.036). The study revealed that vaginal dysbiosis with low abundance of Lactobacillus species occurred in pregnant women infected with severe acute respiratory syndrome coronavirus‐2. These findings may lead to new studies to elucidate the risk of pregnancy adverse outcomes related to COVID‐19.

The predominance of Lactobacillus species (spp.) play a key role in inhibiting nondomestic and potentially harmful microorganisms to epithelial cells. [5][6][7] Lactobacilli maintain the protective low vaginal pH through secretion of lactic acid. 8 Pregnant women with decreased amounts of Lactobacillus crispatus, L. gasseri, and L. jensenii in the vaginal microbiota are more likely to deliver preterm. 9 Likewise, the abundance of Gardnerella vaginalis increases the risk of (PTB). 5,10 The mechanism of action of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in pregnancy varies and remains unknown. The SARS-CoV-2 genome has been identified in the vaginal mucosa of a pregnant woman. 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 in the composition of vaginal microbiota in pregnant women with COVID-19.

| Study population
A prospective study was conducted at Koc University Hospital between August 2020 and August 2021. Pregnant women (n = 19) with active or recently infected within 1 month with SARS-CoV-2 were included in the study. The 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 with positive PCR tests for SARS-CoV-2 infection. COVID-19 was classified according to NIH COVID-19 clinical guidelines. 12 Patients with symptoms including fever, myalgia, or gastrointestinal system symptoms were categorized as a mild disease and those who required oxygen (O 2 ) supplementation as moderate/severe disease (MSD).
Recovery was defined as clinical improvement in combination with a negative nasopharyngeal PCR test. The single vaginal swab was collected from each participant at the time of COVID-19 and within 1 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 2 months after recovery.
The overview of patients is presented in Figure 1.   13 High quality sequences were aligned with SILVA bacterial reference database (v.138.1). 14 Chimeric sequences were removed using the VSEARCH program embedded in the Mothur. Then, the sequences were assigned with taxonomic annotation using the Wang approach 15 implemented in the Mothur.

| Bioinformatics
Silva (v.138.1) was used as the reference database for the assignment. Finally, sequences with no more than 3% dissimilarity were clustered into one Operational Taxonomic Unit for the analysis of diversity and composition.

| Study population
There were no differences in maternal age, body mass index, gestational age at delivery, and birthweight between women in the COVID-19 group with uninfected pregnant women (Table 1). Of 19 women with COVID-19, 13 had asymptomatic/mild disease (68.4%), and 6 had MSD (31.6%). The rate of PTB was 15.3% (n = 3) in the COVID-19 group; 2 of 6 women (33.3%) with MSD and 1 of 13 women (7.7%) with asymptomatic/mild disease ( Table 2). Two patients had COVID-19 in the third trimester and 1 in the second trimester. No other risk factors for PTB were detected.
In asymptomatic/mild disease, 61.5% of those had infection in the second trimester and 38.5% in the third trimester (Table 2). Two women with MSD received both antibiotic and antiviral medications.
Seven patients received low molecular weight heparin during the active period of COVID-19 disease (Table 2).

| The composition of vaginal microbiota in the healthy controls and women with COVID-19
Alpha diversity was evaluated by using the Shannon index. In the COVID-19 group, the Shannon index was significantly elevated,   15.8%, and 7.6%, respectively) when compared to the uninfected pregnant women (32.6%, 36.9%, 16.9%, and 8%, respectively) but the differences were not statistically significant (p > 0.05) ( Figure 3C).
In pregnant women with COVID-19 disease, L. iners decreased the most among Lactabacillus species while the relative abundances of G. vaginalis, Mycoplasma hominis, and Ureaplasma spp. increased in this group of women (Figure 4).

| The variations of vaginal microbiota composition in relation to the severity of COVID-19
The amount of L. iners was higher in the moderate/severe group Several studies have shown that pregnant women with low amounts of L. crispatus, L. gasseri, or L. jensenii in their vaginal microbiota are more likely to deliver before term. 9,33 In a case-control study, the increased abundance of L. gasseri was found to be associated with decreased risk of early spontaneous PTB. 34 Disruption of the balance of vaginal microbiota leads to invasion of several facultative or strict anaerobes, including G. vaginalis, M.  In our longitudinal study of three patients, we were unable to perform analysis at the species level because of the small sample size.
We observed that, during the active phase of the disease, there was an increasing trend in the proportion of Actinobacteria and Bacteriodata that decreased to predisease levels. Ceccarani et al. 35 revealed that the vaginal flora of healthy women consisted of mainly Firmicutes and Bacteroidota, albeit with a low abundance of Actinobacteria.
There are several limitations to our study. First, our cohort sample size was too small to detect a statistically significant difference between women with severe COVID-19 and women with asymptomatic/mild disease, although we were able to show some significant differences between the groups of pregnant women.
Second, a potential confounding factor that may differ among the

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data supporting this study's findings are available from the corresponding author upon reasonable request. The data are not publicly available due to privacy or ethical restrictions.