Over the last decades or so, female reproductive health has been at the forefront of research as strong evidence supports bidirectional relationships between the cervicovaginal microenvironment and cervical cancer, viral acquisition and persistence, gynecologic and obstetric diseases and other benign conditions like cervical ectopy (Laniewski et al., 2020). During adolescence, pregnancy, and in women who take hormonal contraceptives, eversion of the endocervix exposes the columnar epithelium to the vaginal environment, forming a cervical ectopy (Jacobson et al., 2000; Reich and Fritsch, 2014). Cervical ectopy tissue is eventually replaced by newly-formed squamous epithelium through an irreversible physiological process of squamous metaplasia (Jacobson et al., 2000). Cervical ectopy is generally considered a benign condition, however, there is evidence linking cervical ectopy to an increased risk of acquisition of sexually transmitted diseases (STDs), like Chlamydia trachomatis (Lee et al., 2006), Human Immunodeficiency Virus (HIV) (Myer et al., 2006), and Human Papillomavirus (HPV) (Monroy et al., 2010). In a previous work, we reported that a high proportion of women with cervical ectopy, who went on to receive treatment, were positive for the presence of high-risk HPV types (HR-HPV) (Monroy et al., 2010). Persistent infection with HR-HPV has been associated with cervical cancer (Walboomers et al., 1999). The clinical relevance of HPV infection in women with cervical ectopy remains unclear as cervical ectopy is not considered a malignant condition. Also, a number of women with cervical ectopy suffer from bothersome symptoms like abundant leucorrhoea, postcoital and intermenstrual bleeding, pelvic pain and recurrent cervicitis, that can impact their quality of life (Harry et al., 2007; Hua et al., 2012; Cekmez et al., 2016). The presence of chronic symptoms, along with the risk of further infections, warrants treatment of cervical ectopy, mainly cauterization of the area (Machado et al., 2008).

The natural history of cervical ectopy is influenced by the vaginal microenvironment and modified by the presence of reproductive hormones (estrogen), trauma (sexual activity), and the pH of the cervicovaginal mucus. Indeed, squamous metaplastic activity is stimulated by the acidic pH of the vagina (Jacobson et al., 2000), which is in turn regulated by the vaginal microbiota. Low vaginal pH (< 4.5) is maintained by the activity of most species of Lactobacillus (L. crispatus, L. iners, L. jensenii, and L. gasseri), which colonize the cervicovaginal mucus (Lamont et al., 2011) and constitute what is considered an optimal vaginal microbiota. The interaction between epithelial cells, cervicovaginal mucus and vaginal microbiota, is important to preserve female reproductive health, by providing a biological barrier that prevents acquisition of STDs (Amabebe and Anumba, 2018), among other functions. Accordingly, recent studies have demonstrated that infection of the squamous epithelium with HR-HPV was associated with a vaginal microbiota dominated by bacterial vaginosis-associated bacteria (Wei et al., 2021) and depleted in Lactobacillus spp. (Andrade Pessoa Morales et al., 2021). Moreover, HPV-associated precancerous cervical lesions are also linked with changes in the vaginal microbial composition (Nieves-Ramírez et al., 2021). Very little is known about the vaginal microbial ecology in women with symptomatic cervical ectopy, as a result, the potential interactions between the vaginal microbiota and HPV infection are also currently unknown. This is possibly related to the fact that cervical ectopy is considered a common condition that is often overlooked and understudied.

A general feature of women with symptomatic cervical ectopy is the abnormal discharge of cervicovaginal mucus, which is produced by the everted glandular cells. Cervicovaginal mucus contributes to mucosal immune responses, because it harbors both cytokines and antibodies which are mediators of innate and adaptive immunity. Genital inflammation, often determined by levels of pro-inflammatory cytokines in cervicovaginal mucus, has been extensively studied in women with HPV-associated squamous lesions and tumors (Iwata et al., 2015; Daniilidis et al., 2016; Otani et al., 2019), but lesser so in women with cervical ectopy (Hwang et al., 2011; Kyongo et al., 2012). Our group has shown the presence of mucosal IgA anti-HPV antibodies in a proportion of HPV-infected women with cervical ectopy (Monroy et al., 2010). Although cervical ectopy has been associated with increased levels of pro-inflammatory cytokines, the potential impact of HPV infection in the development of mucosal innate and inflammatory responses in women with symptomatic cervical ectopy has not been fully explored. Moreover, local immune responses are thought to be regulated by the vaginal microbiota (Anahtar et al., 2018). For instance, the presence of Gram-negative bacteria in a Lactobacilli-enriched genital tract induces a Th17 response and the production of IL-23 (Witkin et al., 2011). To our knowledge, the impact of the vaginal microbiota in regulating inflammatory and cellular immune responses to HPV in women with cervical ectopy is unknown.

Thus, in the present work we determined the prevalence of HPV genotypes in glandular tissue of women with symptomatic cervical ectopy. Mucosal immune responses were evaluated in cervicovaginal mucus of a selected group of HPV-positive and HPV-negative women, in order to determine their genital inflammation profile (cytokines and chemokines). Next, we also characterized the vaginal microbiota by 16S rRNA sequencing. Finally, associations between HPV infection, vaginal microbial communities and genital inflammation were explored. Our study aims at improving our understanding of the complex interactions of distinct elements within the cervicovaginal environment of women suffering from a common, but scarcely explored condition of the female genital tract.

Here, we present data on the prevalence of HPV infection and distribution of HR-HPV types, the presence and magnitude of genital inflammation in cervicovaginal mucus, the diversity and taxonomic composition of the vaginal microbiota, and associations in women with symptomatic cervical ectopy, a common gynaecological condition, associated with hormonal changes, that either resolves by itself or requires treatment when symptoms (bleeding and recurrent vaginal infections) persist.

The presence of cervical ectopy has been associated with an increased risk of sexually transmitted infections (Lee et al., 2006; Monroy et al., 2010). Here, we detected the presence of HPV in more than half of women with symptomatic cervical ectopy, in particular in younger women (20-30 years of age). Our results are comparable to other studies (Nielsen et al., 2008; Bruni et al., 2010). We found high prevalence of single and multiple HR-HPV and pHR-HPV types, including HPV16, HPV31 and HPV18. We and others have previously reported the presence of oncogenic HPV types in women with cervical ectopy (Monroy et al., 2010; Hwang et al., 2011), however, since endocervical cells are unable to sustain productive HPV life cycle, the clinical relevance and implications of HR-HPV infection in everted endocervical epithelium remain controversial. Interestingly, a refined model of cervical carcinogenesis, that considers the participation of endocervical cells as potential precursors of cervical cancer has been proposed (Doorbar and Griffin, 2019). In line with this thinking, cervical ectopy represents a challenge, because in this condition endocervical cells are exposed to the complex vaginal microenvironment containing chemical, immune and biological elements that may alter the natural evolution of columnar epithelium to metaplastic squamous tissue.

In women with cervical ectopy, endocervical cells protruding onto the ectocervix create an additional mucosal barrier with a natural vulnerability to physical trauma that may lead to increased genital inflammation in sexually active women (Kyongo et al., 2012). Accordingly, we found the co-expression of five important mediators of inflammation, IL-1β, IL-22, IL-8, CCL17, and CCL3 in the vast majority of women included in this study. Our results are consistent with previous reports showing increased levels of cervicovaginal pro-inflammatory cytokines in healthy women with cervical ectopy (Hwang et al., 2011; Kyongo et al., 2012). However, the impact of HPV infection on the inflammatory responses in women with symptomatic cervical ectopy has not been previously evaluated. We could not detect differences in pro-inflammatory cytokines between HPV-pos and HPV-neg women, suggesting that HPV infection does not increase inflammation further. Our results agree with Kriek and colleagues (Kriek et al., 2016), who did not find an association between pro-inflammatory cytokines and HPV in endocervical samples. In contrast, a pro-inflammatory microenvironment has been identified in squamous epithelium persistently infected by HPV, where E5, E6 and E7 oncoproteins were found to be the main activator of inflammation-associated pathways (Hemmat and Bannazadeh, 2019). Glandular cells are unable to sustain HPV replication cycle, and expression of oncoproteins. This might explain why the presence of HPV is not associated with increased genital inflammation in our cohort.

HPV is one of the most common pathogens affecting the cervical canal. Cellular immune responses are decisive for the resolution of HPV infections (Stanley, 2006). Here, we detected increased levels of IL-21 and CXCL9 in HPV-pos women. IL-21 is a crucial factor for the immunological control of viral infections. IL-21 is produced by CD4+ T lymphocytes and natural killer T (NKT) cells, and it modulates the anti-viral activities of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (Spolski and Leonard, 2008). IL-21 may contribute to both initial immune responses and control of HPV-associated genital warts (Abu El-Hamd et al., 2019). Thus, it is reasonable to assume that elevated concentrations of IL-21 detected in our cohort, might reflect a cellular immune response against HPV. As already stated, IL-21 is produced by NKT cells. Migration of NKT cells to sites of infection and inflammation is mediated by chemokines such as CXCL9 (Ding et al., 2016). We detected increased levels of CXCL9 in HPV-pos women, which may be stimulating the recruitment of IL-21-producing NKT cells. An association of HPV infection with elevated levels of cervicovaginal CXCL9 was also reported by Shannon and colleagues (Shannon et al., 2017), however, gynecological conditions of women included in that study were not reported. Interestingly, the expression of CXCL9 can also be induced by bacterial lipopolysaccharide (LPS) (Bandow et al., 2012), suggesting that some bacteria within the vaginal microbiota may also influence the expression of chemokines and as a consequence the recruitment of cells to this site.

As shown by others, bacterial communities in the vagina are able to influence host mucosal immune responses (Agostinis et al., 2019). A vaginal microbiota is considered optimal if dominated by Lactobacillus species and a low microbial diversity. We found that the vaginal microbiota of HPV-pos women was highly diverse and differed in community composition (beta diversity) with a decrease in Lactobacillus dominance compared to HPV-neg women. We also found a strong correlation between alpha diversity metrics and IL-1β and IL-22, both of which are produced at sites of inflammation. These results are in line with a previous study showing correlations between highly diverse bacterial communities and genital pro-inflammatory cytokines (Anahtar et al., 2015). Also, changes in vaginal microbial composition have been shown to associate with female genital tract conditions including pelvic inflammatory disease (Ness et al., 2005). To our knowledge, this is the first report showing associations between specific pro-inflammatory cytokines, and increased bacterial diversity in women with symptomatic cervical ectopy in the context of HPV infection. Our results show that high bacterial diversity correlates with both IL-1β and IL-22. Also, NKT-recruiting factor CXCL9 was increased in the cervical mucus of HPV-pos women. Both results are of great interest to us. IL-1β induces the production of IL-22 by NKT cells as a response to bacterial invasion (Doisne et al., 2011). Likewise, levels of IL-22 are increased in women with HPV-associated genital warts (Radwa et al., 2021), thus it is likely that both, the altered bacterial communities and the presence of HPV may contribute to the elevated levels of IL-22 detected in our cohort.

The link between vaginal dysbiosis and HPV infection, persistence, premalignant lesions (Laniewski et al., 2018; Usyk et al., 2020), and ultimately cervical cancer has been clearly established (Brusselaers et al., 2019; Xie et al., 2020). Given that cervical ectopy is not considered a malignant condition, the potential association between HPV and vaginal dysbiosis in women with this condition remains unclear. As stated previously, a Lactobacillus-dominant is considered an optimal vaginal microbiota, whereas the presence of strict anaerobes is considered a state of dysbiosis (Pramanick et al., 2022). In our cohort, HPV-neg women were mostly Lactobacillus dominant, whereas HPV-pos women showed a significant decreased in Lactobacilli and increase in anaerobes such as Sneathia, Pseudomonas, Megasphaera, Atopobium, Shuttleworthia, Prevotella, and Clostridium. The presence of anaerobes is associated with high rates of bacterial vaginosis recurrence (Onderdonk et al., 2016) and cervicovaginal inflammation (Mitchell and Marrazzo, 2014). Furthermore, byproducts of dysbiotic bacteria metabolism may cause degradation of the mucosal surfaces of the glandular epithelium (Africa et al., 2014), and increase the vaginal pH in a number of women (Donders et al., 2016). In contrast, most Lactobacillus spp. are known to produce lactic acid and favor a low pH in the vagina. Importantly, low pH (pH 4.5) is a critical factor for the natural replacement of ectopic tissue with metaplastic squamous epithelium. Thus, the loss or decrease in the relative abundance of Lactobacilli may delay the recovery of the external stratified epithelium in women with cervical ectopy, possibly increasing their risk of acquiring STDs, including HPV and HIV.

Our results should be interpreted with caution due to the limitations in statistical power inherent to small cohort sizes like ours. We also acknowledge that we did not adjust for confounding variables; when comparing clinical and demographic variables between these two groups, we found that the number of sexual partners was significantly higher in HPV-positive women as previously reported. All other variables were not significant, with the exception of age. There was a tendency for HPV-negative women to be older than HPV-positive women.

We are aware that our study has important limitations; first, the inclusion of a control group of women without cervical ectopy was not possible, so we cannot compare our results with those of age-matched women without cervical ectopy. However, previous reports show that cytokines, chemokines and vaginal microbial community profiles are different in women with cervical ectopy compared with women with a normal ectocervix (Hwang et al., 2011; Kyongo et al., 2012). Taking this latter in consideration, we hypothesize that our results might have been in line with those previously published. Another limitation is the conceivable presence of HPV-infected squamous cells, in the biopsy used for HPV DNA determination. Although we performed an inspection of the cervix after acetic acid application, and we separated women showing an acetowhite lesion, we cannot exclude the possibility of an ongoing exocervical HPV infection with no clinical manifestations in some women. We are aware that recovering pure glandular tissue from biopsies using microdissection would be the ideal methodology. The cross-sectional nature of our study prevents us from proposing any associated mechanisms or causative relationships. To this end, longitudinal studies are in process. Finally, we are aware of the difficulty of classifying to species level with this approach (16S), so we only reported taxonomic profiles at the genus level.

In conclusion, we showed the presence of oncogenic HPV types in a high proportion of women with symptomatic cervical ectopy. Notably, HPV was associated with increased levels of IL-21 and CXCL9 suggesting an active cellular immune response. We also found the co-expression of inflammation markers in these women irrespective of HPV status. We identified that changes in vaginal microbiota composition were associated with the presence of HPV. Lactobacillus dominance decreased in HPV-pos women, and bacterial vaginosis-associated anaerobes were increased. Correlations between inflammation-associated cytokines IL-1β and IL-22 and the vaginal microbiota were exclusively found in HPV-pos women. Taken together, our observations suggest that HR-HPV infection and vaginal dysbiosis coexist in women with symptomatic cervical ectopy. These women might be more susceptible to chronic inflammation and epithelial damage, and could benefit from a closer follow-up to improve their sexual and reproductive health. Our study provides a deeper understanding of the cervicovaginal microenvironment of women with symptomatic cervical ectopy, a benign gynecological condition.

The raw 16S sequences have been deposited at the National Center for Biotechnology Information (NCBI)-Sequence Read Archive (SRA) under project PRJNA763215.

The studies involving human participants were reviewed and approved by Local Ethics Committee of the Colposcopy Clinic of the “Cruz-Talonia” Foundation (Reference Number: 17/02/2017/LRZ/CE05). All women gave written informed consent to participate in this study.

LR-Z conceived the project, designed experiments, coordinated study operation, supervised laboratory staff, analyzed and discussed data, and wrote the manuscript. SP-C conducted 16S rRNA gene sequencing analysis, designed figures, analyzed and discussed data, and wrote the manuscript. ML-F performed flow cytometry analysis, sequencing reactions, analyzed data, designed figures and wrote the manuscript. FC generated clinical data base, processed tissue samples, extracted and analyzed DNA, and designed tables. TG conducted the E7-MPG assay to determine HPV genotypes. OC and FC-T coordinated study operation ant the Colposcopy Clinic of the “Cruz-Talonia” Foundation, and obtained tissue samples. MC-T extracted DNA from cervicovaginal mucus, constructed bacterial libraries, and performed 16S rRNA sequencing. CA-G analyzed cases and contributed to diagnosis confirmation. IM-H performed flow cytometry for chemokine determination. JM and VC-M performed flow cytometry analysis of cytokines. CA participated in the selection of patients, collection of patient data and processing of patient samples. MT coordinated study operation at the International Agency for Research in Cancer, analyzed and discussed data. All authors contributed to the article and approved the submitted version.

This work was supported by a grant from PAPIIT, UNAM (grant number IN204419). LR-Z received a fellowship from Programa de Apoyos para la Superación del Personal Académico de la UNAM (PASPA. Number 523.01/2299DFA/2017). Grants from CONACyT (grant numbers 620702, 464745) were awarded to FC. SP-C was supported by funds from the Mexican Government (Programa Presupuestal P016, Anexo 13 del Decreto del Presupuesto de Egresos de la Federación).

Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization.

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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