The female genital tract is home to trillions of bacteria that usually exist in a mutualistic relationship with their host (Younge et al., 2017; Elovitz et al., 2019). Intriguingly, the characteristics of vaginal and cervical canal bacterial communities are even more conspicuous and different from gut bacteria, displaying a high relative abundance of Lactobacillus and low bacterial diversity (Kervinen et al., 2019; Villani et al., 2022). Individual differences in the bacterial landscape of the vaginal and cervical canals are dramatically influenced by host genetics and environment, also being impacted by lifestyle factors (Moosa et al., 2020). Recent evidence supports the notion that the bacterial communities of the vagina and cervix are strongly associated with health status, with changes associated with pH, urinary tract infections, sexually transmitted infections including bacterial and viral infections such as HIV (human immunodeficiency virus) along with pregnancy outcomes (Watts et al., 2005; Flaviani et al., 2021; Lykke et al., 2021). Moreover, certain bacterial profiles are associated with adverse obstetric outcomes such as preterm birth, potentially leading to neonatal morbidity and mortality (Elovitz et al., 2019; Torcia, 2019; Tsonis et al., 2020; Tu et al., 2020; Flaviani et al., 2021).

In the female reproductive system, the vagina, cervical canal, fallopian tubes and uterus form the reproductive tract (McDonald, 1980; Chen et al., 2017). In general, the fallopian tubes and uterus are believed to be sterile, which requires the cervix to be a perfect barrier (Chen et al., 2017). The cervix does indeed provide a physical barrier to pathogens, and for example, the fetus is protected from the vaginal pathogens during healthy pregnancies (Hein et al., 2002; Goldenberg, et al., 2008; Aagaard et al., 2014; Liu et al., 2015; Kindinger et al., 2016). However, in some cases bacteria and other pathogens can ascend along the mucosal surfaces of the vagina to the fallopian tubes or the uterus (Liu et al., 2015; Chen et al., 2017; Brown et al., 2019).

Cervical incompetence is defined as the inability of the uterine cervix to retain a pregnancy in the second trimester in the absence of uterine contractions (Boelig et al., 2019). About 1% of females experience painless cervical dilation causing preterm birth in the second trimester, making this one of the most common diseases in reproductive-aged women (Brown et al., 2019; Pilarski et al., 2021). Building evidence over recent years implicates bacteria as the major cause of cervical incompetence, especially the composition and structures of bacteria which are highly correlated with preterm birth (Ravel et al., 2011; Smith and Ravel, 2017; Brown et al., 2019). Nonetheless, the bacterial composition and structures of the vagina and cervical canal in pregnant women suffering from cervical incompetence are yet to be fully characterized (Koedooder et al., 2019; Hao et al., 2021). Focusing on pregnant women with cervical incompetence, this study aimed to expand the current knowledgebase regarding the bacterial profiles of the vagina and cervical canal compared to normal pregnant woman using a high throughput sequencing approach. The results uncover potential new insights into the pathogenesis of cervical incompetence with implications for the prevention and treatment of the associated complications.

The human body contains trillions of bacteria inhabiting body surfaces and cavities which can be exchanged with those in the external environment (Ravel et al., 2011). Recent studies have indicated that the bacteria from vaginal and cervical canal play important roles in maintaining a healthy female reproductive system, and moreover, may be mechanistically linked to the pathogenesis of preterm birth (Ansari et al., 2021; Villani et al., 2022). Different bacterial communities or abnormal levels of bacteria populating the vaginal tract and cervical canal during late pregnancy can produce infections or dysfunction, which can adversely affect pregnancy outcomes (Perino et al., 2011; Ravel et al., 2011; Villani et al., 2022). Here, we compared the vaginal and cervical canal bacterial communities between healthy pregnant women and those with cervical incompetence, a condition which may disrupt the distribution of bacteria in the female reproductive tract. Our analysis results for alpha and beta diversity were similar to that of previous studies (Chen et al., 2017).

The bacteria inhabiting the vagina are considered to originate from the upper genital tract (Khan et al., 2016; Tu et al., 2020). In normal pregnant women, the vaginal and cervical canal bacterial communities were different due to the obstruction of the cervix (Chen et al., 2017). In this study we found the bacterial communities of the cervical canal in pregnant women with cervical incompetence were significantly different from that in the normal pregnant women. However, we found no differences in bacterial composition between the vagina and cervical canal of the pregnant women with cervical incompetence, suggesting cervical incompetence promotes bacterial exchange between the vaginal and cervical canal communities. One of the most important risk factors for preterm birth is cervical incompetence in the second trimester of pregnancy. In support of a prior study (Kindinger et al., 2016), we found associations between vaginal and cervical bacteria diversity with preterm delivery. However, larger sample size is needed if we aim to understand the relationships between the vaginal and cervical bacteria diversity and preterm birth.

Lactobacillus are the unequivocal dominant bacteria in the vaginal and cervical canal of healthy women. Their production of lactic acid serves to maintain the acidic pH of the vagina and cervical canal, acting as a barrier against pathogens and protects them from genital infection (Petricevic et al., 2014; Chen et al., 2017; Pace et al., 2021). Deficiencies in Lactobacillus could disturb the vaginal bacterial balance, resulting in the syndrome of bacterial vaginosis (Petricevic et al., 2014; Lozano et al., 2021). Nonetheless, Lactobacillus was reported to remain as the dominant species in pregnant women with cervical incompetence, and no differences were found in the abundance of OTUs compared to healthy pregnant women (Lozano et al., 2021). The vaginal and cervical canal bacteria are dynamic communities owing to physiological, pathological, environmental, and nutritional factors (D’Alterio et al., 2021). Bacterial dysbiosis may contribute to an inflammatory response in the vagina and cervical canal, also predisposing pregnant woman to a higher risk of infection (D’Alterio et al., 2021). Bacterial imbalance of the vaginal and cervical canal is correlated with higher genital pro-inflammatory cytokine concentrations and increased APC (antigen-presenting cell) activation through LPS pathways. Notably, triggering local immunity to increase the concentration of inflammatory factors including TNF-α, NF-κB and COX-2 may affect pregnancy outcomes (Ravel et al., 2011; Smith and Ravel, 2017; D’Alterio et al., 2021).

Similar to the results of previous studies we found differential genera in pregnant women with cervical incompetence including Staphylococcus, Actinomyces, Aerococcus, Clostridium_sensu_stricto, and Anoxybacillus (Flynn et al., 2013; Chen et al., 2017; Brown et al., 2019; Lozano et al., 2021). We identified Groups A and C -specific shifts in bacterial composition reflected by the enrichment of five species, including Staphylococcus petrasii, Bacteroides plebeius, Bifidobacterium pseudolongum, Anoxybacillus flavithermus, and Actinomyces neuii. Bacteroides plebeius has been found to be more significantly frequent in HPV-positive women, and Actinomyces neuii was reported in bacterial vaginosis (Chao et al., 2019; Castro et al., 2019) while the other species are unique to this study. Furthermore, we found the abundances of Sutterella stercoricanis, Methanobacterium congolense, Pseudomonas chlororaphis, Psychrobacter nivimaris, Nocardioides pacificus, Senegalimassilia anaerobia, Fusobacterium nucleatum, Clostridium fimetarium, Methylobacterium tardum were dramatically different between Groups B and D. Collectively, these results suggest that these species represent “biomarker” bacteria for differences between healthy pregnant women and those with cervical incompetence (Agarwal et al., 2020).

As an important potential anaerobic pathogen and Gram-negative bacterium, Fusobacterium nucleatum colonizes the female reproductive tract, digestive tract and oral cavity (Agarwal et al., 2020). This species has been associated with numerous human diseases, including oral infections, colorectal cancer, respiratory tract infections, and Alzheimer’s disease (Allen-Vercoe et al., 2011; Liu et al., 2014; Han, 2015; Agarwal et al., 2020). Fusobacterium nucleatum was also detected in a wide spectrum of fetal membranes, amniotic fluid, neonatal gastric aspirates, chorioamnionitis, and was frequently detected in the cord blood and amniotic fluid of premature infants, being associated with intrauterine infection and preterm birth (Han et al., 2010; Wang et al., 2013; Han, 2015). Additional research also indicated that the mother’s subgingival plaque in oral cavity may transfer Fusobacterium nucleatum to the placenta or fetus during pregnancy period, leading to acute inflammation, even leading to fetal stillbirth (Han, 2011; Wang et al., 2013). However, further validation is still needed to confirm the utility of the biomarker bacteria identified here and in other studies.

Preterm birth occurs in 5–20% of pregnant women and is defined as delivery before 37 weeks of gestation. Preterm birth is influenced by maternal, fetal and environmental factors, and is associated with a high risk of neonatal morbidity and mortality (Terzic et al., 2021). Some researches have indicated that cervical incompetence is related to preterm birth, although other potential risk factors can influence the incidence of preterm delivery, including diabetes, hypertension, thyroid disease, chronic renal disease and tobacco smoking (Guerra et al., 2006; Goldenberg et al., 2008; Vogel et al., 2018; Miklavcic et al., 2021). In addition, pre-pregnancy body mass index, advanced maternal age (more than 35 years old), endometriosis, polycystic ovary syndrome, and pre-pregnancy diabetes are also considered possible risk factors to preterm birth (Molin, 1993; Vyas et al., 2006; Ciancimino et al., 2014; Miklavcic et al., 2021; Pilarski et al., 2021). Other studies suggest that bacterial vaginal infection and maternal periodontal diseases increase the risk of preterm birth, with bacterial transfer from the vagina to the uterus through the cervix (D’Alterio et al., 2021; Terzic et al., 2021). Pregnant women with intrauterine infection could also spread bacteria to the amniotic cavity to cause preterm birth (Angioni et al., 2015). Some studies reported that periodontal diseases of pregnant women were associated with preterm birth, the proposed reason being gingival bacterium transfer to the uterine cavity and placenta via the bloodstream, resulting in an intra-amniotic infection (Offenbacher et al., 2006; Ren and Du, 2017).

In summary, we investigated the bacterial compositions and structures of the vagina and cervical canal from healthy pregnant women and pregnant women with cervical incompetence by high-throughput sequencing technology. The overall bacteria were not significantly different in Lactobacillus in the vaginal and cervical canal of healthy pregnant women and pregnant women with cervical incompetence. However, the alpha and beta diversities were significantly different between vaginal samples and cervical canal samples of normal pregnant women. We also found differences in the bacterial communities of cervical canal samples between normal pregnant women and those with cervical incompetence. Several specific bacteria were identified, suggesting that these bacteria may act as potential biomarkers. However, we must also acknowledge certain limitations regarding our study. First, the sample size is relatively small. Second, further investigations are required to elaborate the functional consequences of the “biomarker” bacteria. Third, more experimental technologies are necessary to reveal the correlation between bacteria and clinical features and elucidate the molecular mechanism.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The studies involving human participants were reviewed and approved by Anhui Medical University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

MS, GL and XL designed the experiments of this manuscript. MS, HG, JF, and GL performed the experiments. MS, NX, YL, and GL analyzed the data. MS, YL, and GL wrote the manuscript. All authors contributed to the article and approved the submitted version.

This research was supported by the National Natural Science Foundation of China (grant nos. 31702030 and 81772908) and the Natural Science Foundation for the Higher Education Institutions of Anhui Province of China (grant no. KJ2021A0246).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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