Introduction
Cervical cancer (CC) ranks as the fourth most frequently diagnosed cancer and the third leading cause of cancer death in the women globally. More than half a million CC cases are annually linked to (human papillomavirus) HPV infection, resulting in 250,000 deaths for per year [
1]. HPV infection is recognized as one of the major causes of CC, as well as other malignancies including anogenital tumors, anal, vulvar, penile, vaginal and oral cancers [
2,
3]. Recent studies suggest that 90% of oral squamous cell carcinomas are caused by HPV infections [
4].
The oral cavity is a natural open system composed of a complex microbiome consisting of more than 800 bacterial species [
5]. This oral microbiota has been implicated not only in periodontal disease but also in systemic conditions including haematological diseases, and lymphatic, lung, pancreatic and breast cancers [
6‐
10].
Changes in the composition of the oral microbiome are now recognized as potential biomarkers for cancers
, including colorectal cancer (CRC)
, marked by increased
Fusobacterium nucleatum abundance [
11,
12]. Similarly, shifts favouring oral pathogens such as the genera
Porphyromonas,
Fusobacterium and
Prevotella have been correlated with the incidence of CC, although the underlying mechanisms remain poorly understood [
13], these oral pathogens also cause the periodontal disease [
14]. Previous studies have shown a correlation between vaginal bacteria and gingival inflammation [
15], HPV not only invades the basal cells of the vaginal epithelium, but also infects the periodontal tissue and keeps the virus in a latent state [
5]. The oral and vaginal environments may provide similar colonization and growth conditions, resulting in a significantly increased risk of periodontal disease and cancer [
16]. However, relatively few studies exist on changes in the oral microbiome when the vaginal microbiota is transformed during the course of HPV infection to CC development, suggesting that more research is needed in this area.
Population-based CC screening is implemented as a public health priority in China [
17], and the best strategy is the use of a liquid-based cytology test (TCT) combined with HPV screening [
18,
19]. The combined screening technology, although advanced and effective, is costly and suitable for areas with adequate medical and health care. However, there are still many less economically developed areas (rural areas) in China, and there is an urgent need to find a high-quality and inexpensive method. Changes in oral microbial diversity that are detectable through low-cost profiling may offer additional value as biomarkers for early screening, diagnosis and monitoring of HPV infection and even CC. The aim of this study was to evaluate the differences and associations between vaginal and oral microorganisms in HPV-infected patients and CC patients. An increased understanding of microbial ecology may contribute to improving the accuracy of CC screening and providing life-saving interventions to vulnerable groups.
Discussions
In this study, the composition and changes in the vaginal and oral microbiotas of women who experienced abortion, HPV infection or cervical cancer were evaluated. In addition to the different vaginal microecological environments of the four groups, we determined that there were significant differences in the composition, abundance, diversity, marker genera, and functional pathways of oral microorganisms between CC patients and healthy controls, which further proves that vaginal and oral microbes are not independent entities and that there is flora transfer between different parts of the body, which may be associated with systemic metabolism [
22]. To our knowledge, this is the first study to explore the role of the oral microbiome in patients with CC, improving the accuracy of the CC screening process and broadening the scope of universal screening through insights gained from changes in the oral microbiome. Our team previously reported a meta-analysis and systematic review of CC specimens, which included cervical, vaginal, rectal, faecal, and urine samples without oral subgingival plaque (unpublished).
In exploring vaginal microecological shifts, there was no significant difference in the microbial composition between the AB group and the control group, which was dominated by
Lactobacillus [
23]. The effect of recurrent abortion on the vaginal flora is transient, regardless of cervical lesion type [
24]. As long as surgery does not cause substantial damage to vaginal tissues, the equilibrium between commensal bacteria and opportunistic pathogens remains the same. With the occurrence of HPV infection and CC, the species diversity increased, the proportion of Lactobacillus gradually decreased, and the internal structure of the microbiome became more complex, similar to previous findings [
25,
26]. The beta diversity of the Z group, AB group and HP group largely overlapped, which was significantly different from that of the CC group. It was further confirmed that the depletion of
Lactobacillus and the increase in specific anaerobes (such as
Megasphaera,
Prevotella and
Gardnerella) were related to cervical lesions [
24]. LEfSe analysis identified cancer-specific vaginal biomarkers, including
Mycoplasma,
Bacillus,
Bacteroides,
Dialister,
Peptoniphilus,
Porphyromonas,
Anaerococcus,
Prevotella, and
Sneathia, and the HPV + biomarker
Bifidobacterium distinguished them from the control; the reliability of the experimental findings has been confirmed by other studies [
13,
27‐
29].
Bifidobacterium is a beneficial microorganism of the intestinal flora that has many functions, such as resisting harmful bacteria, exerting antitumour, increasing immunity and improving gastrointestinal function, and it also exists in the oral cavity and vagina [
30]. This research revealed that the use of
Bifidobacterium species to distinguish cervical lesions is highly important for diagnosing women’s health conditions. The abundance of
Bifidobacterium decreased, which is associated with high-grade squamous intraepithelial lesions (HSILs) [
31,
32]. Wang et al. reported that the increased abundance of
Bifidobacterium in the vaginal microbiome may be related to the clearance of HR-HPV infection, and focused ultrasound (FU) treatment may help to increase the abundance of
Bifidobacterium [
33], possibly because
Bifidobacterium can survive in acidic environments and produce lactic acid and hydrogen peroxide, which have a protective effect on the vaginal environment [
34].
Additionally, the shared periodontal pathogens
Porphyromonas and
Prevotella were identified as CC biomarkers, possibly because of the dynamic colonization of opportunistic bacterial pathogens on squamous epithelial cells in the oral or vaginal cavities and communication with the external environment. Oral pathogens can be transmitted from the gastrointestinal tract or through blood transmission to the vaginal cavity, and they can also be transmitted through person-to-person oral–genital contact [
35]. This suggests that the oral cavity and vagina share a common microbial community and that there is also a reciprocal exchange of related microbial communities. The salivary microbiota of participants with bacterial vaginosis (BV) was more diverse than that of BV-negative participants [
36], and
Prevotella intermedia and
Porphyromonas endodontalis were enriched in the subgingival gingival microbiota of BV-infected women compared to women without BV, which indicates the presence of a vagino–oral axis [
37]. Moreover,
Porphyromonas and
Prevotella have been proven to have carcinogenic potential via several different mechanisms. For example,
Porphyromonas can maintain chronic periodontal infection, leading to increased expression of proinflammatory molecules such as IL-6, IL-8, IL-1β, and TNF-α; activation of Toll-like receptors (TLRs) and antiapoptotic pathways (JAK/STAT and MAPK pathways); decreased expression of proapoptotic proteins; and increased cancer cell migration and invasion [
38]. The gingipain protease
Porphyromonas gingivalis activates NF-κB and MMP-9 in oral squamous cells, which are important for cancer cell invasion and metastasis [
39]. Similarly,
Prevotella produces virulence factors, fimbriae adhesins, lipopolysaccharides (LPSs), peptidoglycan and lipoteichoic acid, which induce the release of proinflammatory cytokines [
40].
Prevotella can also stimulate tyrosine kinase receptors, degrade immunoglobulin, exert toxic effects on fibroblasts, and coordinate with other pathogens to promote the migration and invasion of cancer cells [
41,
42]. Therefore, the pathogenic mechanisms of these periodontal pathogens are summarized as follows: they can stimulate chronic inflammation, inhibit cell apoptosis, activate cell proliferation and promote cell invasion, resulting in cancer [
43].
Vaginal microbiome transformations along the cervical carcinogenesis route have been characterized, but the associated impacts on the oral niche remain underexplored. This study revealed that the oral cavity contains a significantly greater number of bacterial species than does the vagina and revealed extensive bidirectional sharing of microbial communities between the vaginal and oral cavities through an emerging vagino–oral axis [
37]. This study provides insight into the microbial community shared between the oral and vaginal parts of the human body, as well as the exchange of related microbial communities. This study revealed that vaginal HPV infection and multiple abortions had no obvious impact on the oral flora of patients. However, the presence of CC can cause significant changes in the composition and abundance of oral microorganisms, leading to a lower diversity of the oral microbiome compared to that of the normal population, which is contrary to the changes in the vaginal microbiota. The prevalence of periodontal pathogens significantly increased in patients with CC. According to the results of both LEfSe and random forest analysis,
Fusobacterium,
Campylobacter,
Capnocytophaga,
Veillonella,
Streptococcus,
Lachnoanaerobaculum,
Propionibacterium,
Prevotella,
Lactobacillus and
Neisser were identified as oral bacterial markers for CC. Changes in the proportions of these bacteria can cause oral microflora dysbiosis and are also associated with all systemic diseases, including cancer [
5,
38,
40,
44‐
47]. Different bacteria, such as
Fusobacterium nucleatum,
Periodonticum,
Streptococcus salivarius,
Porphyromonas, and different
Lactobacillus subspecies, are associated with the diagnosis of this type of cancer. The periodontal pathogens
Fusobacterium nucleatum,
Campylobacter,
Pseudomonas aeruginosa and
Porphyromonas are considered “mobile microbiota” because they originate in the oral cavity but are also associated with extraoral infections and inflammation [
45]. There are various tumorigenesis mechanisms associated with the oral microbiome, mainly including increased cell factors and inflammatory factors, chronic inflammation, cell proliferation, metabolic pathway changes, pathogenic bacterial metabolites, suppression of the immune response, induction of tumour genetic damage, and alteration of epithelial barriers [
47‐
49]. Most of the current research focuses on how dysfunction of oral microorganisms affects major organs and systems of the whole body. However, there is a bidirectional relationship between oral and general health, and how systemic diseases adversely affect oral microorganisms needs further exploration.
In this study, a positive correlation was observed between vaginal and oral microorganisms, where certain bacteria exhibited connections with many others. Many studies have shown the synergistic effect of pathogenic microorganisms. Lo et al. reported that
Prevotella intermedia is enriched in patients with CRC and enhances the migration and invasion of cancer cells; moreover,
Prevotella intermedia and
Fusobacterium nucleatum collectively contribute to the malignant transformation of colorectal adenoma into carcinoma [
41].
Streptococcus gordonii,
Fusobacterium nucleatum and
Porphyromonas gingivalis synergistically promote the formation and proliferation of plaque biofilms, inhibit the growth of dendritic cells [
50], and cause peripheral blood infection via bacterial tyrosine (BY) kinase (Ptk1), which is an important part of the signalling pathway that controls the synergistic interaction between
Porphyromonas gingivalis and
Streptococcus gordonii [
51]. The colonization of vaginal epithelial cells by
Atopobium enhances the virulence of
Gardnerella, and biofilms formed by
Gardnerella also dominate other bacteria for colonization [
52]. The interactions of a variety of microbial synergy mechanisms are very complex. These mechanisms include not only metabolite cross-feeding but also a large number of microbiota quorum-sensing signals that result in enhanced resistance against the immune system, antibiotics, or direct contact between microorganisms to promote synergy [
53]. Thus, the comprehensive impact of two or more microorganisms on disease is more severe than that of a single microorganism, and the complex interaction network may enhance the pathogenicity of multiple microbial infections, ultimately affecting disease initiation and progression. Notably, these studies are based on different bacteria at the same site, while the composition of the vaginal microbiome is different from that of the oral microbiome, and more studies are needed to clarify the mechanisms of action of the microbiome at different sites.
Systemic inflammation (such as cervical lesions and HPV infection) caused significant variation in the CRP concentration across all the groups. An increase in the number of pregnancies, childbirths and abortions can easily disturb the vaginal microenvironment, decrease the abundance of
Lactobacillus, and increase the proportions of the anaerobic bacteria
Prevotella and
Gardnerella. Severe systemic and local inflammation are closely related to imbalances in the vaginal microbiome [
54]. Similarly, when oral hygiene was good and brushing frequency was high, the abundance of pathogenic microorganisms associated with periodontal disease, such as
Selenomonas,
Prevotella,
Treponema, and
Aggregatibacter, decreased; this is due to oral microorganisms or their metabolites directly migrating through the blood or indirectly affecting the inflammatory mediators produced in the oral cavity [
55].
Changes in microbial diversity in CC patients are consistent with changes in amino acid metabolism. Cancer cells grow rapidly, metabolites are overexpressed, and glycogen is consumed to produce large amounts of energy and intermediates [
56]. It has been verified by metabolomics and transcriptomics that the occurrence of CC is significantly related to metabolism, proteolysis or proteoglycans [
57]. With increasing cervical lesion severity, the consumption of lactic acid also gradually peaks, metabolic characteristics increase the consumption of glutamine after the development of CC, and these metabolite changes are negatively correlated with the abundance of
Lactobacillus [
58]. Studies in humans have shown that CC has distinct metabolic fingerprints in blood, tumour tissue, faeces, and urine [
59]. However, metabolic maps of the oral cavity are lacking, and the PICRUST predictive analysis of this study provides additional data. Cervical lesions also affect oral microbial metabolites, and the amino acid metabolism of oral bacteria in patients with CC is greater than that in controls because of the presence of the corresponding pathogenic microorganisms.
Capnocytophaga grows in places with high glycogen consumption [
60], and
Porphyromonas,
Prevotella and
Fusobacterium are closely related to differences in metabolites. Pyrimidine metabolism is significantly increased in patients with CC and is positively correlated with periodontal disease [
61]. Glutamate, histidine, and tyrosine metabolism are also involved in the development of periodontitis. Cervical lesions lead to changes in the oral microenvironment, which may increase the pathogenic effect of periodontal bacteria, induce the selective growth and reproduction of oral pathogens, produce a more pathogenic microbiome, and even produce the “Warburg effect”.