Characteristics of Vaginal Microbiome in Reproductive-Age Females with HPV Infection in Xinjiang, China

Objective We investigated the characteristics of vaginal microbiome in reproductive-age females with HPV infection in Xinjiang, China. Methods A total of 135 females of reproductive age were enrolled. There were 43 healthy HPV-negative females in control group (N group), 58 HPV-positive females in nonlesion group (P1 group), and 34 HPV-positive females in low-grade squamous intraepithelial lesion group (P2 group). DNA was extracted from the vaginal secretions, and V3–V4 regions of bacterial 16S rDNA were amplified and sequenced by NovaSeq. QIIME2 and R software were used to perform diversity analysis of bacteria. PICRUSt2 was used to predict the function of the vaginal microbiota. Results Lactobacillus was the main genus of vaginal microbiota in asymptomatic reproductive-age females with or without HPV in Xinjiang. The diversity of vaginal microbiota in the P1 group was significantly higher than that in the N group, and the proportion of Gardnerella increased significantly. The vaginal microbiota structure of the P2 group was different from the N group, characterized by the decrease of Lactobacillus crispatus and the increase of Shuttleworthia. The function of the inordinate microbiome may play a role in accelerating HPV replication and integration. Conclusion The structure of vaginal microbiota alters under persistent HPV infection in asymptomatic females of reproductive age in Xinjiang. The Gardnerella increase is associated with increased susceptibility to HPV infection, and Lactobacillus iners predominance and Shuttleworthia presence may be a signature of HPV infection with low-grade squamous intraepithelial lesion.


Introduction
Cervical cancer (CC) is a common malignant tumor in females, and its incidence is second only to breast cancer in genitourinary system. According to statistics, about 604,000 new cases and 342,000 deaths occurred worldwide in 2020 [1]. Females with sexual experience have an 80% chance of being infected with human papilloma virus (HPV) in their lifetime. Among the infected cases, 70%-80% of them would regress spontaneously in 12 months, and only 10% of them would develop into HPV persistent infection after 24 months, consequently resulting in CIN (cervical intraepithelial neoplasm) and even CC [2]. Te vaginal microbiome has become one of the important infuencing factors of HPV infection and development of cervical precancerous lesions and CC [3][4][5].
Clinical studies have observed that the diversity of the vaginal microbiome increases signifcantly with the severity of cervical precancerous lesions [6,7]. Mechanistically, it is confrmed [8] that HPV E7 protein reduces the secretion of vaginal mucosal defense peptides, which are the energy source of amino acid metabolism of Lactobacillus spp. Te decreasing Lactobacillus spp. may contribute to vaginal pH increase, which may favor the growth of vaginal pathogenic bacteria and result in structural changes in the vaginal microbiome ultimately. Tis suggests that HPV can regulate the reproduction of the vaginal microbiome and alter the vaginal environment by inhibiting Lactobacillus spp.
After DNA integration into the host genes, the HPV E6/E7 protein is highly expressed, which binds to tumor suppressor   gene p53 and Rb to inhibit apoptosis and fnally induces CIN and CC [9,10]. E6/E7 protein/RNA is rarely expressed in the low-grade squamous intraepithelial lesion (LSIL) [11] but is highly expressed in tissues of high-grade squamous intraepithelial lesions (HSIL) and CC [12,13]. Investigators have paid more attention to the association of vaginal microbiome and grades of squamous intraepithelial lesion and revealed many diferences in HSIL and CC. However, the characteristics of the vaginal microbiome in HPV-infected patients with or without LSIL are less known.
Herein, we investigated that if the characteristics of the vaginal microbiome in healthy reproductive-age females and reproductive-age females with HPV infection accompanied with LSIL or not, in Xinjiang, China. Our fndings may help the clinical diagnosis and treatment of HPV infection and cervical precancerous lesions. Patients had complications of severe heart, liver, and kidney insufciency. (8) Patients had mental disorders. (9) Patients sufered with malignant tumors. Age-matched healthy females who received physical examination during the same period were recruited as control. All participants received HPV testing and thin-layerliquid-based cytology testing (TCT). Participants who were HPV16/18-positive or were positive for the other 12 high-risk HPVs accompanied with the atypical squamous cell of undetermined signifcance (ASCUS) or above received colposcopy examination and biopsy when it was necessary. Tis study was approved by the Ethics Committee of Urumqi Maternal and Child Health Hospital (approval number: XJFYLL2021006). All participants signed the informed consent form. and 805R (5′-GACTACHVGGGTATCTAATCC-3′) were used as primers to amplify V3-V4 hypervariable regions of the bacterial 16S rDNA. Te PCR amplifcation system (25 μL) included 12.5 μL PusionHot Start Flex 2X Master Mix (M0536L, Yitao Biological Instrument Co., Shanghai, China), 2.5 μL upstream and 2.5 μL downstream primer, and 50 ng DNA template. Te PCR amplifcation procedure was as follows: predenaturation at 98°C for 30 s; 35 cycles of denaturation at 98°C for 10 s, annealing at 54°C for 30 s, and extension at 72°C for 45 s; and fnal extension at 72°C for last 10 min. Ten, PCR products were confrmed with 2% agarose gel electrophoresis and purifed by AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA). Te concentration was measured by Qubit (Invitrogen, USA). Te size and quantity of amplicon libraries were assessed on Agilent 2100 Bioanalyzer (Agilent, USA) and with the Library Quantifcation Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. Te DNA library with concentration above 2 nM was considered qualifed. Finally, 25 ng qualifed library was sequenced by LC-Bio Technology Co., Ltd.

Materials and Methods
(Hangzhou, China) on the NovaSeq 6000 platform.

Sequencing Data Processing and Analysis.
We used QIIME2 [14,15] to extract high-quality sequences. Te raw data obtained by sequencing were subjected to overlap splice, quality control, and chimera fltering, and then highquality clean data were obtained. After dereplication by DADA2 (Divisive Amplicon Denoising Algorithm 2) [16], we obtained amplicon sequence variant (ASV) feature tables and feature sequences. Taxonomic identifcation of ASVs was performed using Silva (Release 138) database. Te α-diversity (Chao1 and Shannon index) and β-diversity were calculated by R software (V3.4.4), which was also used for graph plotting. Diferences in species abundance between groups were detected using Kruskal-Wallis and Wilcoxon rank sum tests. Te linear discriminant analysis (LDA) coupled with efect size measurements (LEfSe) online analysis tool (https://www.omstudio.cn/tool/) was used to analyze the bacterial genera diferences between groups.

Statistical
Analysis. SPSS 20.0 software was used. Te measurement data were tested for normality by the Kolmogorov-Smirnov test. Data with normal distribution were expressed as mean ± standard deviation and analyzed with one-way ANOVA. Non-normally distributed data were expressed as median and interquartile range and were compared using multisample nonparametric tests. Enumeration data were expressed as percentages or rates (%) and were analyzed using the chi-square test or Fisher's exact test. P < 0.05 was statistically signifcant.

Characteristics of Participants.
In total, 92 high-riskHPV-positive patients and 43 high-riskHPV-negative healthy females were enrolled in this study. Among the 92 patients, 58 cases were with normal TCT or normal colposcopy or with chronic cervicitis by histopathological diagnosis Evidence-Based Complementary and Alternative Medicine of biopsy, and they were classifed as the P1 group. Tirty-four patients with LSIL were classifed as the P2 group. Te 43 HPV-negative healthy females with normal TCT served as the control group (N group). Tere were no signifcant differences among groups in terms of age, ethnicity, body mass index, number of pregnancies, number of deliveries, phase of menstrual cycle, contraceptive method, age at frst sex, number of lifetime sexual partners, frequency of sex in the past year, and smoking status (P > 0.05, Table 1). However, signifcant diference was presented in the HPV infection status between P1 and P2 groups (P < 0.05). Te P1 group had more patients infected with HPV16/18 types, while the P2 group had more patients with HPV multi-infection.

Sequencing
Results. We obtained 10,791,360 assembled clean reads from 135 samples, with an average of 67,572.48 ± 7121.18 reads per sample. A total of 3195 ASVs were obtained, and 543 ASVs were shared among the three groups. Te P1 group had the richest ASVs while the N group had the least (Figure 1(a)). After annotation, a total of 30 phyla, 226 families, 485 genera, and 657 species were found. Te sequence dilution curves of the three groups' samples indicated that the sequencing depths were reasonable (Figure 1(b)).

Abundance and Diversity of Vaginal Microbiome.
Species abundance and diversity of the vaginal microbiome were assessed with Chao1 and Shannon indices, respectively. Chao1 index representing the average species abundance in the P1 group (105.01 ± 45.38) was signifcantly higher than that in the N group (80.18 ± 35.74, P < 0.01) and P2 group (90.33 ± 63.95, p < 0.05) (Figure 1(c)). Shannon index that comprehensively assessed the abundance and evenness of species in the P1 group (1.61 ± 1.07) was signifcantly higher than that in the N group (1.15 ± 0.74, P < 0.05), but there was  (Figure 1(d)).

Structure and β-diversity of the Vaginal Microbiome.
PCoA (principal co-ordinates analysis) was performed using a weighted UniFrac matrix (Figure 2(a)), and the two principal components explained 71.07% and 13.53%, respectively. Te N group overlapped with the P2 group, and both N group and P2 group overlapped with the P1 group. At the phylum level, the top fve phyla in the three groups were Firmicutes, Actinobacteriota, Fusobacteriota, Bacteroidota, and Proteobacteria (Figure 2(b)). However, the relative abundance of each phylum varied among groups. Te  Evidence-Based Complementary and Alternative Medicine relative abundances of Firmicutes in N, P1, and P2 groups were 92.09%, 76.84%, and 88.52%, respectively (P � 0.003). Te relative abundances of Actinobacteriota in the three groups were 7.19%, 19.4%, and 8.52% (P � 0.009). Te relative abundance of Fusobacteriota in the P1 group (2.34%) was higher than that in the N group (0.20%) and in the P2 group (1.17%), but there was no statistical signifcance (p � 0.365). Te relative abundance of Bacteroidota in the P2 group (1.03%) was higher than that in the N group (0.11%) and in the P1 group (0.52%), but there was no statistical signifcance (P � 0.087). Tere was no signifcant diference in the relative abundance of Proteobacteria among three groups. At the genus level, the top fve genera in the three groups are shown in Figure 2(c). Te genera with relative abundance ≥1% in the three groups are shown in Table 2. Lactobacillus was the dominant genus in all three groups, with signifcant diferences among groups. It is worth noting that Shuttleworthia was only present in the P1 (0.01%) and P2 groups (1.01%) (P1 group vs. N group, P � 0.014; P2 group vs. N group, P � 0.004; P1 group vs. P2 group, P � 0.383).
At the species level of Lactobacillus ( Figure 3 and Table 2), Lactobacillus iners were the most abundant bacteria among the three groups and was also the dominant bacteria in the P2 group (P2 vs. P1, P � 0.024). Te relative abundance of Lactobacillus crispatus in the three groups showed a decreasing trend (P2 group vs. N group, P � 0.020). Te relative abundance of Lactobacillus gasseri and Lactobacillus jensenii was signifcantly lower than Lactobacillus iners and Lactobacillus crispatus. However, there was no signifcant diference in Lactobacillus gasseri and Lactobacillus jensenii among groups.

Identifcation of Signature Vaginal Microbes.
Due to the limitations of 16S rDNA amplicon sequencing technology, not all bacteria can be typed to the species level. Tus, we performed the analysis from the phylum to the genus level. Phylogenetic tree of species (Figure 4(a)) showed the different microbes among the three groups from phylum to genus level. Analysis by LEfSe showed that there were signature microbes in each group (P < 0.05, LDA score ≥ 3.0) (Figure 4(b)). Te key genus with the most signifcant difference in the N group was Lactobacillus, while in the P1 group was Gardnerella, and in the P2 group was Shuttleworthia.  Reconstruction of Unobserved States) based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database was used to predict the function of microbes. When comparing between the groups, we found out the metabolic and functional diferences caused by vaginal microbes. At the KEGG pathway level 2, there were 39 functional gene pathways with diferent microbial gene abundances in the three groups, and 14 pathways were signifcantly diferent among groups ( Figure 5), mainly on metabolism and gene information processing. Compared with the P1 group, the proportion of functional gene pathways of enzyme family, infectious diseases, replication, and repair in the N group was signifcantly increased. Te amino acid metabolism, energy metabolism, metabolism of cofactor, and vitamin and cell motility functional gene pathways in the P1 group were signifcantly increased compared with the N group. By contrast, the genes of immune system diseases and metabolic pathways in the P2 group were also signifcantly richer than those in the P1 group.

Discussion
Tis is the frst study to use 16S rDNA amplicon sequencing technology based on the NovaSeq 6000 platform to explore the characteristics of vaginal microbes in healthy and HPVinfected females of reproductive-age in Xinjiang, China. Te sequencing results were processed by DADA2. Te advanced sequencing platforms and processing methods make the obtained data more accurate [14]. Among asymptomatic females of reproductive-age, vaginal microbes are dominated by Lactobacillus spp. Ravel et al. said that Lactobacillus iners were more common in Asian females [16]. Consistently, this study found that Lactobacillus iners had the highest proportion in vaginal microbes of healthy females in  Figure 4: Identifcation of signature vaginal microbes. (a) Phylogenetic tree of species. Diferent circle layers radiate from the inside to the outside to represent the seven taxonomic levels of kingdom, phylum, class, order, family, genus, and species. Each node represents a species classifcation at this taxonomic level. Te higher the abundance of the species, the larger the node. Te yellow node means that the species has no signifcant diference among groups, and other color node means that the species has a signifcant diference among groups. (b) Histogram shows the LEfSe (LDA efect size) analysis of signifcantly diferential bacteria from phylum to species level among groups (LDA score ≥ 3.0). Xinjiang, China. However, the proportion was no more than 50%.
We found that compared with healthy females, the species abundance and diversity of vaginal microbes in HPV-infected patients without LSIL were signifcantly increased, which suggests that there are alterations in vaginal microbes in the early stage of HPV infection. Tis is consistent with the fndings of majority studies [17][18][19] but not consistent with that by Wu et al. [20]. Tis may be caused by the regional diferences of the participants. Moreover, we found that in the early stage of HPV infection, vaginal microbes were mainly characterized by a decreasing proportion of Lactobacillus crispatus and an increasing proportion of anaerobic bacteria, especially the Gardnerella, which is similar to the results of Yang et al. [21]. Te structure of vaginal microbiome in HPV-positive females was similar to bacterial vaginosis. Te latter has been reported to be associated with sexually transmitted diseases (such as HIV), preterm labor, pelvic infammatory disease, etc. [22]. Such vaginal microbiome structure may enhance the susceptibility of cervical epithelium to HPV.
Our study showed that the proportion of HPV multiple infections in the P2 group was signifcantly increased. Tis indicates that multiple infections may promote lesion development, as we all know. However, the vaginal microbial abundance and diversity of the P2 group were slightly higher than those in the N group, without signifcant diference. Tis is similar to the results of a previous study [23]. Nevertheless, Chen et al. showed that the diference between LSIL group and healthy group was obvious [6]. Tey included postmenopausal females in their study. Tus, the diference may be related to the diferent subjects.
Additionally, there were signifcant diferences in microbial composition in the P2 group compared to that in the N group, with the highest abundance of Lactobacillus iners and the lowest abundance of Lactobacillus crispatus in the three groups, which is similar to the fndings of Chao et al. in persistently infected HPV patients [24]. Tis is also consistent to the meta-analysis by Norenhag et al. [5], which concluded that compared with vaginal microbiota dominated by Lactobacillus crispatus, precancerous lesions are more likely to occur in patients with Lactobacillus inersdominated vaginal microbiota.
Tis study also found that the unique microbe of the vaginal microbiota in the P2 group was the genus Shuttleworthia. Shuttleworthia is a Gram-positive anaerobic bacilli. Downes frst reported the isolation of Shuttleworthia and Shuttleworthia satelles from human oral secretions in 2002 [25]. Ten, investigators have noticed that they were enriched in the oral microbes of periodontitis [26] and childhood caries [27]. Recently, Eun et al. showed that in patients with oral squamous cell carcinoma, the level of Shuttleworthia in the oral microbiota of patients with lymph node metastasis was signifcantly enriched [28], indicating that it may be involved in the malignant transfer. Recently, this genus has also been found in the microbes of the female reproductive tract. In 2019, Onywera et al. reported that Shuttleworthia was common in bacterial vaginosis [29]. In 2021, Yuan and colleagues also showed that eight bacterial genera, including Shuttleworthia, were strongly associated with bacterial vaginosis [30]. Te association of Shuttleworthia with HPV infection and cervical lesions is also being studied. Chorna [32]. Te discrepancy may be related to the diferent inclusion criteria of participants, regions, and sample sizes. Te participants of this study were females of reproductive age who lived in Xinjiang, China. It showed that the genus Shuttleworthia was signifcantly enriched in females who were infected with HPV and were with LSIL. Te increase of Shuttleworthia may accelerate cervical epithelial lesions, but the specifc pathogenic mechanism remains to be elucidated. In this study, the PICRUSt functional prediction showed that the abundance of pathways of energy metabolism, metabolism of amino acids, biosynthesis of secondary metabolites, and metabolism of cofactors and vitamin of vaginal microbiome in the P1 group was signifcantly higher than those in the N group. However, the abundance of pathway of replication and repair in P1 and P2 groups was signifcantly lower than that in the N group. Tese results are consistent with those by Yang et al. [21], indicating that inordinate vaginal microbiome may facilitate HPV replication and transmission and contribute to HPV integration into host genes. Interestingly, there was a signifcant increase in cell motility function gene pathways in P1 and P2 groups, which may be associated with good outcomes after HPV infection according to Usyk et al.'s study [19].
Tere are some limitations in this study. First, this is a cross-sectional study, which cannot clarify the causal relationship between diferent bacterial genera and clinical diagnosis. Second, clinical indicators that might be related to HPV infection, such as infammation index, vaginal PH, and serum estrogen level, were not included for further analysis. Tird, the sample size was small. Further studies are warranted.

Conclusions
In conclusion, this study revealed that the vaginal microbiota of asymptomatic females of reproductive-age in Xinjiang was dominated by Lactobacillus, among which the relative abundance of Lactobacillus crispatus was higher in healthy females. Te signifcant increase in Gardnerella may enhance the susceptibility of cervical epithelium to HPV infection. Te predominance of Lactobacillus iners and the presence of Shuttleworthia may be a signature for HPV-infected females with LSIL.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding authors upon reasonable request.