Changes of the vaginal microbiota in HPV infection and cervical intraepithelial neoplasia: a cross-sectional analysis

This study aimed to explore the changes of the vaginal microbiota and enzymes in the women with high-risk human papillomavirus (HR-HPV) infection and cervical lesions. A total of 448 participants were carried out HPV genotyping, cytology tests, and microecology tests, and 28 participants were treated as sub-samples, in which vaginal samples were characterized by sequencing the bacterial 16S V4 ribosomal RNA (rRNA) gene region. The study found the prevalence of HR-HPV was higher in patients with BV (P = 0.036). The HR-HPV infection rate was 72.73% in G. vaginalis women, which was significantly higher than that of women with lactobacillus as the dominant microbiota (44.72%) (P = 0.04). The positive rate of sialidase (SNA) was higher in women with HR-HPV infection (P = 0.004) and women diagnosed with cervical intraepithelial neoplasia (CIN) (P = 0.041). In HPV (+) women, the α-diversity was significantly higher than that in HPV (−) women. The 16S rRNA gene-based amplicon sequencing results showed that Lactobacillus was the dominant bacteria in the normal vaginal microbiota. However, the proportion of Gardnerella and Prevotella were markedly increased in HPV (+) patients. Gardnerella and Prevotella are the most high-risk combination for the development of HPV (+) women. The SNA secreted by Gardnerella and Prevotella may play a significant role in HPV infection progress to cervical lesions.

Human papillomavirus (HPV) infections gave rise to over 600,000 cases of cancer in a year 1 . Most women will have been infected with HPV by intercourse during their lives, most HPV infections fade away by themselves in a few months, a few HPV infections persist and cause lesions 2 . Although most instantaneous HPV infections are cleared by the immune system, persistent infections can cause viral gene integration into the host genome and lead to HPV-related cancer 3 . Previous studies have found that local microbiota, epithelial surface integrity, immune regulation were synergistic factors in the progression of HPV to cancer. Nevertheless, little is known about the functional composition of the local microbiota and how it varies by cervicovaginal syndromes, infections, and diseases 4 . The vaginal microenvironment can be categorized into five kinds of community state types (CSTs), Lactobacillus spp. was the dominant microbiota in CST I, II, III, and V. And the CSTs have different Lactobacillus species types, such as Lactobacillus-crispatus, Lactobacillus-iners, Lactobacillus-jensenii, Lactobacillus-gasseri, and so on 5 .
Notably, The features of CST IV are higher vaginal pH (> 4.5), lack of Lactobacillus, and abundance of Gardnerella. These features are also features of bacterial vaginosis (BV) 6 . BV is a kind of mixed infection characterized by the reduction of Lactobacillus and the multiplication of pathogen, mainly Gardnerella vaginalis (G. vaginalis), accompanied by increased vaginal pH. It has been reported that BV infection in the Chinese population ranges from 10.5 to 51.6% 7 . Previous studies indicate that there is a close correlation between BV infection and HR-HPV persistence. A study by Gillet 8 showed that patients with BV are more prone to HR-HPV infections, while Guo 9 further found that a BV infection prolongs the duration and the regression time of HR-HPV infections.
As the main pathogen of BV, the detection rate of Gardnerella was significantly increased in HR-HPV-positive women 10 . In a 2-year longitudinal prospective study, the presence of specific anaerobic groups, including Gardnerella, was associated with the persistence and slow degradation of CIN2 11 . Meanwhile, different dominant bacterial communities produce different metabolomes. Gardnerella secretes SNA while elevated SNA concentration was associated with increased risk for cervical lesion 12 . The vaginal metabolome of HPV (−) women differed from HPV (+) women in terms of several metabolites, including biogenic amines, glutathione, and lipid-related metabolites 13 . L. crispatus producing hydrogen peroxide (H 2 O 2 ) show the strongest associations with vaginal health and are depleted in dysbiosis 14 . With the development of high-throughput sequencing, there is still a lack of systematic and comprehensive studies to investigate the types and enzymes of vaginal microbiota and their relationship with HPV infection and cervical lesions.
To solve the above problems, this study started from clinical data, with the help of high throughput sequencing, aimed to explore the changes of the vaginal microbiota and enzymes in the HR-HPV infection progress to cervical lesions and provide ideas for further exploration of the interaction mechanism between vaginal microbiota and HPV infection.

Results
Socio-demographic characteristics of participants. Characteristics of 448 participants were analyzed. Participants with normal cervical pathology or negative cytology and HPV (−) were defined as the normal group. Our study indicated that there were no significant differences in terms of age, nationality, marital status, and reproductive history among the groups (P > 0.05; Table 1).
The relationship between vaginal microenvironment and HPV infection, cervical lesions. In this study, the infection rate of BV was the highest, accounting for 41.96% (188/448), followed by vulvovaginal candidiasis (VVC), aerobic vaginitis (AV), cytolytic vaginosis (CV), and trichomonal vaginitis (TV), accounting for 12.05% (54/448), 5.13% (23/448) and 1.78% (8/448) and 0.89% (4/448). BV and HR-HPV infections were associated with each other. The difference was statistically significant (P = 0.036). The HR-HPV infection rate was 72.73% in G. vaginalis women, which was significantly higher than that of women with lactobacillus as the dominant microbiota (44.72%) (P = 0.04). However, no obvious correlation with HR-HPV infection was found between other dominant microbiota. We also found that the positive rate of SNA was higher in HR-HPV infection women (P = 0.004). The results are shown in Table 3. www.nature.com/scientificreports/ Among the 448 participants in this study, 18 were excluded because they did not undergo cytological or pathological examination. The remaining participants were in a ratio of approximately 1:2 according to HR-HPV infection or not. The pathological results of NILM and ≥ CIN I patients were matched, and they were divided into a case group (≥ CIN I) and a control group. Ultimately, a total of 177 patients were included in the second part of this study, including 51 cases and 126 controls. In women diagnosed with CIN, the positive rate of SNA increased (31.37% vs 17.46%, P = 0.041). However, catalase or leukocyte esterase (LE) were not significantly associated with cervical lesions (Table 4).   www.nature.com/scientificreports/

Changes of vaginal microbial diversity and microbiota in HPV infection women.
In our study, 28 samples were carried out high-throughput sequencing. Twenty-three HPV (+) and five HPV (−) samples were collected for the study and the control groups, respectively. There was no significant difference in age between HPV (+) and HPV (−) groups (P = 0.666). The curve plateaued (Fig. 1a,b) when the sample size was approximately 20, indicating that although 28 cases seem small, it was enough for data analysis. Therefore, the sample size was ample in this study. The evolutionary classification tree of the top 100 species with their abundance, and the corresponding phylum or genus of the top 20 species with their abundance (marked with asterisks) are marked in different colors ( Fig. 1c-f). The distribution of phylum, class, order and genus of vaginal microbiota in HPV infected women were shown in Fig. 2. Most of the vaginal microbial communities in the overall samples belong to the following phylum: Table 3. The changes of micro-environment factors between different HR-HPV infection women. Normal pH values between 3.8-4.5, and the rest are regarded as abnormal pH values. The normal flora diversity is the flora diversity ++ to +++, the normal flora density is the flora density ++ to +++, the rest is regarded as the flora diversity and the flora density abnormality. G+ Gram-positive bacteria, G− Gram-negative bacteria, GV Gardnerella vaginalis, AV aerobic vaginitis, BV bacterial vaginosis, CV cytolytic vaginosis, TV trichomonas vaginitis, VVC vulvovaginal candidiasis. § Is Fisher's exact test method. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Firmicutes, Actinobacteria, Bacteroidetes, Fusobacteria, Proteobacteria (Fig. 2a). Firmicutes accounted for 97.38% among total microbiota, which was the main vaginal microbiota in the HPV (−) women. However, the proportion of Firmicutes decreased (68.26%), and the proportions of Actinobacteria and Bacteroides increased (respectively 15.45%, 4.58%) in the HPV (+) women. The composition and differences of vaginal microbiota between the two groups were further analyzed at the genus level (Fig. 2d). In the HPV (−) women, Lactobacillus was the dominant bacteria (95.73%), with a small amount of Gardnerella (1.39%), Atopobium (0.03%) and other genera. In the HPV (+) women, the composition of the vaginal bacterial community structure had significant changes, mainly reflected in the decrease of Lactobacillus (62.91%), and the increase of Gardnerella (12.22%) and Prevotella (6.78%).

HR-HPV
The diversity indicators of the two group samples were shown in Fig. 3a-d. In HPV (+) women, the Shannon index was higher than the HPV (−) women (F = 6.14, P = 0.023), indicating that the number of microbiota in HPV (+) women was more. In HPV (+) women, the Simpson index was lower (F = 9.494, P = 0.006), suggesting that the complexity of the vaginal microbiota was increased in patients with HPV infection and decreased in cases of vaginal health. However, the Chao index or ACE index were not related to HPV infection (P > 0.5).
To discover the changes of vaginal microbiota in HPV infection women, LEfSe analysis was performed on the two groups (Fig. 3e,f). The results showed that Lactobacillus was the main dominant bacteria in the HPV (−) www.nature.com/scientificreports/ women, and Prevotella was a distinct microbiota that plays a significant role in the HPV (+) women according to the Linear discriminant analysis (LDA) scores.

Discussion
The emerging studies suggest that vaginal microenvironment plays an essential role in women's health, specifically in sexually transmitted diseases. This is a cross-sectional study to explore the changes of the vaginal microbiota and enzymes in the HPV infection and cervical lesions. Overall, a significantly higher microbiota diversity was www.nature.com/scientificreports/ observed in HPV (+) women than that in HPV (−) women. The increase of Gardnerella and Prevotella and the decrease of Lactobacillus are closely associated with HPV infection. Previous studies indicated Lactobacillus is the dominant bacteria, which play important role in protecting the health of women's lower reproductive tract 15 . The vaginal microbiota is primarily dominated by one of the four most common Lactobacillus species: Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii 5 . Lee et al. 16 found the proportion of Lactobacillus was lower in HPV (+) patients. On the one hand, Lactobacillus maintains the weak acid environment of the vagina through its own lactic acid. On the other hand, a large quantity of Lactobacillus can reduce and inhibit the planting and growth of some opportunistic pathogenic bacteria to protect the lower reproductive tract from infection 17,18 . The presence of the signature "abnormal vaginal microbiota" in CIN was found by a laboratory culture in 1992 and confirmed in subsequent studies 19 . When the vagina's protective microbiota was destroyed, the defense against pathogen infection was weakened.
In this study, We have identified vaginal microenvironment disorder was bound up with HPV infection. Compared with BV (−) women, the HR-HPV infection rate in BV (+) patients increased. BV is associated with an increased risk of detection of HPV, and HPV infections are associated with an increased risk of BV 20 . Vaginal microenvironment disturbance was associated with increased inflammatory cytokines, mucosal injury and chronic inflammation. In order to investigate the mechanism of BV and HPV infection, Rodriguez-Cerdeira et al. suggested that G. vaginalis is one of the common microbiota in HPV (+) women 21 , and this has been reported in other researches that using next-generation sequencing 22,23 . Women with BV had higher levels of the cytokine interleukin (IL)-1β and lower levels of IL-17 24,25 .
As an important component of vaginal microenvironment, metabolomes play an important role in the pathogenicity of microbiota. The H 2 O 2 produced by Lactobacillus can catalyze peroxidase and further produce hypochlorite. It is a process that can prevent HPV from invading into cervical epithelial cells and prevent cervical lesions 26 . LE is an intracellular enzyme. When vaginal inflammation occurs, a large number of white blood cells can gather to engulf pathogens, resulting in the destruction of white cell membrane, and thus LE can be detected. However, no studies have linked LE to any type of vaginal inflammation. Similarly, this study also showed that there was no statistical significance between LE and HPV infection or cervical intraepithelial neoplasia. Gardnerella adheres tightly to the surface of vaginal epithelial cells, forms a dense biofilm, and can release vaginal cytolysin, which may inhibit the effect of vaginal mucosal barrier immunoglobulin A. Gardnerella can produce SNA, which can degrade mucosal protective factors (such as mucin) and causes vaginal epithelial cells to dissolve and expel 27 . SNA is an enzyme that cleaves terminal sialic acid residues and is associated with tissue destruction, immune response evasion, bacterial invasion, and access to bacterial-associated nutrients 28 . In addition to Gardnerella bacteria, such as Prevotella bacteria, Bacteroides bacteria and Mobiluncus bacteria also produce SNA 29 . SNA usually occupies terminal positions attached to mucosal defense factors, such as secretory IgA, secretory components, lactoferrin, and secretory leukocyte protease inhibitors 30 .
In order to investigate the mechanism of cervical lesions caused by vaginal microbiota and metabolomes, Zariffard et al. found that 31 the expression level of Toll-like receptor-(TLR) 4 mRNA in vaginal and cervical epithelial cells was significantly increased in patients with G. vaginalis-infected BV. Previous studies showed that TLR9 recognizes HPV infection and initiates immune response. Experiments at the transcriptional level confirmed that E6 and E7 oncoproteins directly downregulate TLR9 32 . However, whether Gardnerella activates the TLRs-related pathways through SNA to cause HPV infection is still unknown and needs to be assessed. All of these mucosal, bacterial, and immune activations associated with BV and G. vaginalis may lead to progression of HPV infection to cervical cancer.
This study also has some limitations. First, this is a cross-sectional study to explore the changes of the vaginal microbiota and enzymes in women with HPV infection and cervical lesions, further experiments are needed to confirm that SNA secreted by Gardnerella and Prevotella contributes to HPV causing cervical lesions. Secondly, our study only focused on the distribution of different microbiota, ignoring the identification of Lactobacillus species. Recent researches suggest that Lactobacillus iners is a transitional species that colonize after the vaginal environment is disturbed and leads to BV, sexually transmitted infections, and adverse pregnancy outcomes 33 . Further studies are necessary to identify the exact role of different Lactobacillus species in larger samples.
Our research demonstrates vaginal microenvironment, especially BV was closely related to HPV infection. So more attention should be paid to the prevention, discovery and proper management of BV in HPV infection women. Particularly, compared to HPV (−) women, Gardnerella and Prevodella are the most high-risk combination for the development of HPV (+) women. The SNA secreted by Gardnerella and Prevodella may play a significant role in HPV infection progress to cervical lesions. This finding provides ideas for further exploration of the interaction mechanism between vaginal microbiota and HPV infection.

Materials and methods
Study population. The study participants were selected from the Fujian Cervical Lesions Screening Cohorts (FCLSCs), China. A total of 448 participants have carried out microecology tests, HPV genotyping and cytology tests, and 28 participants were treated as sub-samples, in which vaginal microecological samples were characterized by sequencing the region of bacterial 16S V4 ribosomal RNA (rRNA) gene (Fig. 4) Vaginal microbiological metabolites detection. The Vaginal secretions were obtained on 1/3 of the vaginal sidewall. Check whether there is trichomonad, mycelium, clue cells under the microscope after daubing on clean slide. H 2 O 2 , LE and SNA in secretions were detected by bPR-2014A vaginitis automatic detector and supporting detection kit (Master Biotechnology Co., Ltd, Jiangsu, China). Vaginal PH value was determined by color strips. If the PH value was no more than 4.5 (pH ≤ 4.5), the result was defined as normal. On the contrary, pH > 4.5 was defined as abnormal. Vagina cleanness was diagnosed in accordance with the standard of National Clinical Laboratory Practice Guideline 18: I-II were defined as normal vagina cleanness, and vagina cleanness III-IV defined as abnormal. AV, BV, CV, TV and VVC were all negative or positive. The Nugent scoring method was used to diagnose BV. The Nugent score was calculated by assessing the numbers of Lactobacillus morphotypes (scored as 0-4), G. vaginalis morphotypes (scored as 0-4), and Mobiluncus morphotypes (scored as 0-2). A Nugent score of 7-10 was interpreted as consistent with BV and a score of 4-6 as intermediate, while a score of 0-3 was interpreted as negative for BV. SNA colorless is normal (−), red or purple is positive (+). LE colorless is normal (−), and green or blue is positive (+). H 2 O 2 > 2 mmol/L is red or purple, negative (−), H 2 O 2 < 2 mmol/L is positive (+), blue. All laboratory procedures were conducted according to the manufacturer's instructions.
HPV genotyping. The HPV genotypes (16, 18, 31, 33,  Liquid-based cytology. The cytological samples were blinded and independently evaluated by two experienced cytopathologists and re-evaluated until reach a consensus when the diagnoses were different. Samples www.nature.com/scientificreports/ were classified as NILM, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), atypical squamous cells, and it was not possible to exclude high-grade squamous intraepithelial lesions (ASC-H), squamous cervical cancer (SCC) and atypical glandular cells (AGC).
Histology. According to the cervical cancer screening procedure, women with HR-HPV infection or abnormal cytology results may be referred for colposcopy or biopsy. When biopsy diagnosis results in ≥ HSIL, patients underwent a loop electrosurgical excision procedure cone or conization by cold knife to biopsy. Formalin (10%) was used to fix specimens, which were routinely processed for paraffin embedding. Then, 4 µm thick histological sections were cut and stained with hematoxylin and eosin using standard methods. Cervical biopsy specimens were examined and diagnosed according to the CIN system. If the review reading is inconsistent, conduct a second histological review. If two-thirds of the diagnoses are the same, the result is considered the final result. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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