Cervical cancer (CC) is one of the significant public health problems in the world. Globally, it represents the seventh most common cancer in humans and the fourth most common cancer in women, but it reaches second place in incidence and mortality in settings with a low human development index (Bray et al., 2018). In Mexico, this condition is aggravated because the diagnosis mainly occurs in locally advanced stages (IB-2 to IV-A), placing the pathology as the second leading cause of cancer death in women (Baezconde-Garbanati et al., 2019; Martínez-Rodríguez et al., 2021).

Recent studies have evaluated the relationship between the cervicovaginal microbiome and gynecological cancer to elucidate the involvement of bacterial communities in the establishment, progression, or cure of the disease. Dysbiosis due to the presence of bacterial vaginosis is the most studied entity, found to have a possible association with the induction of local inflammation and lactic acid depletion (Anahtar et al., 2015; Łaniewski et al., 2018), persistent high-risk Human Papillomavirus (HPV-Hr) infection (Jørgensen et al., 2009; Gao et al., 2013; Piyathilake et al., 2016; Ilhan et al., 2019; Norenhag et al., 2020; Wei et al., 2020), Cervical Intraepithelial Neoplasia (CIN) (Gillet et al., 2012; Huang et al., 2020) and the establishment of cervical cancer (Kwasniewski et al., 2018; Huang et al., 2020; Kang et al., 2021).

Among the main bacterial genera reported so far concerning CC are Gardnerella, Prevotella, Dialister, Slackia, Actinomyces, Porphyromonas, Peptoniphilus, Anaerococcus, Peptostreptococcus, Streptococcus, Ureaplasma, Megasphaera, Mycoplasma, Sneathia, Mobiluncus, Fusobacterium, and Atopobium (Anahtar et al., 2015; Di Paola et al., 2017; Wei et al., 2020), all of which are facultative anaerobic or strict anaerobic bacteria.

In 2002, Donders et al. (2002) reported the existence of a pathological entity called aerobic vaginitis, characterized by a high abundance and diversity of aerobic, enteric bacteria and inflammatory symptoms. In 2019, Wang et al. (2020) evaluated the bacterial profiles of aerobic vaginitis by both culture-dependent and molecular methods. They found no single profile to describe the vaginal bacteriome in patients with aerobic vaginitis. In 2021, Plisko et al. (2021) published a paper whose objective was to analyze the association between the vaginal microbiota of women with aerobic vaginitis and the presence of cervical intraepithelial neoplasia. In this study, aerobic microbiota was detected by wet-mount microscopy. The authors concluded that there might be a relationship between aerobic dysbiotic cervicovaginal microbiota and the development of cervical intraepithelial lesions.

Currently, to our knowledge, the aerobic microbiota present in women with established cervical cancer has not been evaluated. Therefore, this study aimed to identify the culturable aerobic bacterial microbiota in women with locally advanced cervical cancer as an approach before the metagenomic study of the cervicovaginal microbiome associated with cervical cancer in Mexican women.

Cervical cancer is one of the leading health problems in the world; there are multiple reports about the risk factors associated with HPV infection, its persistence, and cancer development. We have detected four risk factors in the Mexican population analyzed in this study. Education level, which can be related to socioeconomic status, was lower in the group of women with cervical cancer, coinciding with previous studies showing that detection of the disease is later as schooling level decreases (Chen et al., 2012; Thulaseedharan et al., 2012; Akinlotan et al., 2017; Nessa et al., 2020). The onset of active sexual life was at an earlier age in women with CC, and it has been reported that early age at first intercourse may predispose women to HPV infection (Creatsas and Deligeoroglou, 2012; Dahiya et al., 2017). Parity, which was higher in women with CC. This risk factor may be related to hormonal changes and epithelial modifications caused by repeated cervical trauma (Thulaseedharan et al., 2012; Nessa et al., 2020). And finally, the use of contraceptive methods, in which the higher use of hormonal methods by women with CC is observed.

It has been commonly reported that the composition of “healthy” or “normal” cervicovaginal bacterial communities is based on the presence or absence of lactobacilli. Ravel et al. (2011) have classified it into multiple categories or “clusters” (I-V) based on the dominant Lactobacillus species and the ethnic group in which they are found. Bacterial communities I, II III and V, are dominated by Lactobacillus crispatus, L. gasseri, L. iners, and L. jensenii, respectively, and were found more frequently in Asian and white women. Bacterial community IV is not dominated by any lactobacillus species and has a high presence of anaerobic bacteria. This group was overrepresented in Hispanic and black women, concluding that vaginal bacterial communities not dominated by Lactobacillus species are common and appear normal in these ethnic groups (Ravel et al., 2011; Kroon et al., 2018). However, according to Martínez-Peña et al. (Martínez-Peña et al., 2013), the healthy cervicovaginal microbiota of Mexican women is mainly composed of L. acidophilus group lactobacilli, namely L. gasseri, L. fermentum, L. rhamnosus, L. jensenii, L. crispatus and L. brevis, which is in contrast to that described by Ravel et al. (Ravel et al., 2011). Likewise, our results also show the presence of critical aerobic bacteria in women without CC. We believe that further studies are needed to establish the normal parameters of this bacterial community and to determine with greater certainty the variation according to population, genotype, or the presence of disease.

Several studies have already exposed the link of cervicovaginal bacterial microbiota and CC during the natural history of the disease, where it is shown that from HPV infection and up to the presence of a cervical intraepithelial lesion, there is an association between cervical dysbiosis and disease (Mitra et al., 2015; Usyk et al., 2020; Onywera et al., 2021). The most studied entity is bacterial vaginosis, in which a higher diversity of strict anaerobic species and a considerable decrease in lactobacilli are observed (Sodhani et al., 2017; Brusselaers et al., 2019; Kovachev, 2020). However, current reports suggest that aerobic bacteria may also play an essential role in this pathology (Vieira-Baptista et al., 2016; Plisko et al., 2021).

This study aimed to detect aerobic bacteria related to the presence of CC. Our findings show that most isolates corresponded with facultative aerobic bacteria, which may demonstrate their high prevalence both in the bacterial communities present in women with CC and those without the pathology. According to Donders et al. (2017), one of the essential elements in aerobic vaginitis is colonization for Escherichia coli, Staphylococcus aureus, S. epidermidis, Streptococcus agalactiae, and Enterococcus faecalis. In our study, both groups presented bacteria typical of aerobic vaginitis. However, there were more strains isolated in women with cancer. This finding could contribute to the observation of Plisko et al. (Plisko et al., 2021), who concluded that changes in the microbiota associated with aerobic vaginitis are related to pre-invasive lesions. With our results, we can consider that these changes may also be related to cancer establishment.

Regarding the presence of this same group of bacteria in women without CC, although with lower strains isolated and fewer species, we can elucidate that this may represent infection or dysbiosis, but HPV infection is probably necessary for neoplastic changes to develop. According to Vieira-Baptista et al. (Vieira-Baptista et al., 2016), aerobic vaginitis is not associated with an increased risk of acquiring HPV, but it has been associated with the persistence of infection. It can therefore be considered a risk factor. The consequences of this and why it may or may not be related to the development of neoplasia should be studied under future designs and should also be studied considering the presence or not of HPV infection. These are two aspects that we did not consider in our study, representing points for improvement in subsequent projects.

Concerning the clinical stage of the disease, we found that more advanced stages presented a higher number of isolates and a more significant number of species coincident with aerobic vaginitis. On the contrary, less extended clinical stages showed a lower number of species and a more consistent bacterial community, with a better local ecology; this is consistent with previous studies (Audirac-Chalifour et al., 2016; García-García et al., 2017).

About the presence of these species and the severity of the disease, we know that aerobic vaginitis is an entity that by itself generates discomfort by producing a variable amount of inflammation and thinning of the vaginal epithelium (Oerlemans et al., 2020). We can elucidate that this dysbiotic microbiota could drive the pathology by promoting immune evasion, favoring tumor cell survival. It could also act by producing an inflammatory environment that in the long term promotes tissue damage resulting in an ecosystem more conducive to disease aggravation. These two aspects have been previously addressed to relate carcinogenesis to aerobic vaginosis, but not to explain the severity of the disease (Vieira-Baptista et al., 2016; Donders et al., 2017; Plisko et al., 2021). We suggest studies whose aim is directed to the analysis of these variables.

No marked differences were observed in histological type for the microbiota associated with either of the two carcinomas found in this study group. In general, squamous cell carcinoma was related to a more significant bacterial presence, both in quantity and species number; this could be influenced by the anatomical characteristics of the tumor, which could provide a more favorable habitat for the installation of these species. A larger sample size regarding adenocarcinoma is likely to be necessary to obtain conclusive results.

Our study does not provide fundamentals to determine a causal association between the bacteria characteristic of aerobic vaginitis and cervicovaginal neoplasia; we do not have elements to clarify if aerobic vaginitis promotes carcinogenesis or if it is established as a consequence of cancer development due to the deprivation of the immune system when trying to counteract the neoplasia. But considering previous studies that have demonstrated a link between an altered vaginal microbiome and pre-invasive cervical disease, we propose that aerobic vaginitis and the inflammation with which it is accompanied are probably crucial for the progression of preneoplastic lesions to cancer (Vieira-Baptista et al., 2016; Plisko et al., 2021).

This cross-sectional study, developed as a preliminary experiment for further research, made it possible to explore the essential characteristics of the cervicovaginal microbiota associated with cervical cancer, the differences between women with and without the pathology, and the need for more in-depth studies. It also allowed us to establish variables for future analyses, funding needs, and sample collection and treatment methods.

Although the “omics” era has accelerated all aspects of biological research, it is advisable to integrate traditional microbiology methods to formulate optimal study designs. Conducting a research project depends primarily on the scientific objective and the availability of samples, the quality of the samples, and the cost of the experiment. Massive sequencing of 16S rRNA gene amplicons and metagenomic sequencing is used to obtain a broader view of bacterial communities and determine potential functions and genome recovery. However, it generally involves the development of large-scale research projects with sufficient funding. In developing countries, where financial resources in science and technology are scarce, it is essential to perform detailed designs that properly manage samples, variables, and financial resources, minimizing losses. Our preliminary identification of the microbiota as a preliminary approach to metagenomic studies can contribute to establishing this type of design.

We can conclude that we were able to establish the presence of bacteria typical of the entity known as aerobic vaginitis in women with CC and that it is crucial to carry out more in-depth studies. There is little information about this dysbiosis and its association with cervical cancer, so the reported findings are helpful to corroborate that there are bacterial communities that, although they may be abundant, so far have been little studied and whose influence on the establishment, development, or cure of CC are unknown. Our report shows that cross-sectional study designs and traditional microbiological methodology are helpful to determine whether there is a need for more complex studies and to know whether it is necessary to optimize the management of variables, samples, and financial resources.

Among the limitations of this study, we consider it necessary to report that we did not manage to extend the participants’ last use of antimicrobials and antifungals, so we limited ourselves to 30 days as exclusion criteria. We know that this period is short for the microbiota to recover satisfactorily and return to “normal”. However, we tried to lessen its impact by choosing a large sample size that balanced some of the participants’ confounding results. Subsequent studies should address this issue. Likewise, we could not diagnose aerobic vaginitis or identify HPV; however, this is a precedent for future work to determine the association between aerobic vaginitis and HPV infection or persistence. Finally, we consider it a limitation to have performed a cross-sectional study. However, this is an antecedent for future studies to address longitudinal designs in which the relationship between aerobic vaginitis and cervical cancer can be evaluated more effectively.

The obtained sequences are publicly available in the GenBank of the National Center for Biotechnology Information with access numbers MH108987-MH109026 (Group 1) and MH158254-MH158283 (Group 2). All other data in the study are available on request from the corresponding author.

The studies involving human participants were reviewed and approved by Ethics and Research Committee of the National Cancer Institute of Mexico (INCan, 016/11/ICI-CEI/1016). The patients/participants provided their written informed consent to participate in this study.

GM-L, JC-M, MR-M, LB-M, HS-T, and NR-D participated in the study design. JC-M, MR-M, and IR-C collected epidemiological data. GM-L, MR-M, and IR-C performed sample collection. GM-L, HS-T, and NR-D executed the experiment. GM-L and LB-M performed the analysis of results. All authors contributed to the article and approved the version presented.

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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