Association between co-infection with Chlamydia trachomatis or Mycoplasma genitalium and cervical lesions in HPV-positive population in Hunan, China: a cross-sectional study

Abstract

Objectives

The aim of this study was to determine the prevalence of Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) among HPV-positive women undergoing colposcopy at the Second Xiangya Hospital of Central South University, Hunan, China. Additionally, we aimed to assess the impact of C. trachomatis or M. genitalium co-infection with HPV on the severity of cervical lesions.

Methods

We collected HPV data, cervical cytology results, and demographic information from 439 women attending colposcopy. Cervical swabs were obtained for simultaneous amplification testing (SAT) of C. trachomatis and M. genitalium. Multivariate logistic regression analyses were performed to examine the association between sexually transmitted pathogens and cervical lesions.

Results

Among the participants, C. trachomatis was detected in 17 (3.87%) individuals, and M. genitalium in 16 (3.64%) individuals. There was no co-infection of C. trachomatis and M. genitalium. The highest prevalence of M. genitalium was observed in women aged 19–30 years (10.20%; 95% CI, 1.41-18.99%), with a subsequent decline in prevalence with increasing age (Ptrend = 0.014). The most common HPV subtype in our study was HPV52 (30.79%), followed by HPV16 (18.62%), HPV58 (16.95%), and HPV53 (10.02%). Infection with HPV16 (OR = 3.43, 95% CI, 2.13–5.53), HPV31 (OR = 3.70, 95% CI, 1.44–9.50), and HPV33 (OR = 3.71, 95% CI, 1.43–9.67) was associated with an increased severity of cervical lesions, while HPV53 infection was not likely to lead to advanced cervical lesions (OR = 0.45, 95% CI, 0.23–0.89). The leukocyte level in vaginal secretions (P = 0.042) and cervical cytology results (P < 0.001) showed associations with the degree of cervical lesions. However, there was no significant association between C. trachomatis or M. genitalium infection and the severity of cervical lesions, nor with their co-infection with HPV16.

Conclusions

There was no correlation between co-infection of Chlamydia trachomatis or Mycoplasma genitalium and the degree of cervical lesions in HPV-positive population in Hunan, China. Our findings emphasized the need to pay more attention to M. genitalium infection among young women. Increased levels of leukocytes in vaginal secretions may be linked to cervical lesions. HPV16, HPV31, and HPV33 in Hunan province, China, may exhibit higher cervical pathogenicity.

Introduction

In early 2021, the GLOBOCAN 2020 database, produced by the International Agency for Research on Cancer (IARC), revealed that cervical cancer (CC) ranks as the fourth most common cancer worldwide among women in terms of both incidence and mortality [1]. In China, it stands as the ninth most common cancer type [2]. Persistent infection with high-risk human papillomavirus (HPV) is a key factor contributing to the development and progression of cervical cancer [3]. While the majority of women contract HPV at some point in their lives, most cases resolve spontaneously, and only a small percentage of persistent infections lead to cervical lesions or cancer [4]. Therefore, it is important to focus on the risk factors that contribute to the progression of HPV infection to cervical cancer. The prevalence and pathogenicity of different HPV subtypes that lead to precancerous lesions and cervical cancer can vary among countries and populations [5]. Hence, it is crucial to determine the prevalence and impact of each HPV type in a specific region to effectively control and eliminate HPV infections, as well as prevent cervical lesions.

Sexually transmitted pathogens pose a significant public health concern worldwide, particularly for women’s health. Chlamydia trachomatis (CT) is the most common causative agent of bacterial sexually transmitted infections (STIs) and can result in various complications in women, including pelvic inflammatory disease (PID), hydrosalpinx, and infertility [6]. Some studies have suggested an association between C. trachomatis infection and HPV infection, indicating they are mutual risk factors [7, 8]. However, conflicting conclusions have also been reported. A cross-sectional study conducted in Beijing, China, found no significant difference in C. trachomatis infection rates between HPV-infected and non-HPV-infected groups or between groups with different cervical biopsy results [9]. Similarly, a retrospective analysis conducted in Sichuan, China, found no association between C. trachomatis infection and high-risk HPV infection [10]. Therefore, the relationship between C. trachomatis and HPV infection remains controversial and requires further investigation. Additionally, most existing studies focus solely on the relationship between C. trachomatis infection and HPV infection, disregarding the fact that HPV infection does not necessarily lead to cervical lesions [3]. Therefore, exploring the relationship between C. trachomatis infection and cervical lesions directly reflects the impact of C. trachomatis on female cervical health, yet few relevant studies have been conducted.

Mycoplasma genitalium (MG) is an emerging sexually transmitted pathogen that causes genitourinary tract diseases and has recently gained attention [11]. Research has indicated that M. genitalium can cause cervicitis [6] and is associated with various gynecological conditions, including PID [12] and infertility [13]. Furthermore, most M. genitalium infections are asymptomatic. The increasing prevalence and antimicrobial resistance of M. genitalium are concerning [6]. However, routine testing for M. genitalium is not commonly conducted in most hospitals in China, and epidemiological data on M. genitalium in Hunan Province is lacking. The relationship between M. genitalium, HPV progression, and cervical lesions remains unknown but is of significant importance.

Currently, there is inconsistency in the association between C. trachomatis infection and HPV infection. Moreover, epidemiological data on M. genitalium and its correlation with HPV infection are limited. Therefore, we conducted a study among HPV-positive individuals who underwent colposcopy in Hunan Province, China. This study aimed to investigate the distribution of HPV subtypes, as well as the prevalence and age distribution of C. trachomatis and M. genitalium. Additionally, it analyzed the risk factors for cervical lesions and examined the influence of co-infection with C. trachomatis or M. genitalium on cervical lesions in HPV-infected individuals. These findings have important implications for the prevention and control of infections caused by these pathogens, as well as the management of cervical lesions.

Materials and methods

Study design

A cross-sectional study was carried out at the Second Xiangya Hospital of Central South University from July to September 2022. A population of 500 women visiting the hospital’s gynecology department and undergoing colposcopy was invited to participate in the study. The inclusion criteria were as follows: (1) women with a history of sexual activity, (2) positive HPV test results, and (3) informed consent. The exclusion criteria included: (1) women infected with bacterial vaginosis (BV), fungi, or Trichomonas vaginalis (TV) at the time of examination, (2) women had sexual activity or gynecological examinations within the past 24 h, (3) women who are menstruating, (4) pregnant women, (5) women who had vaginal douching or used vaginal medications within the past 48 h, (6) women who had undergone hysterectomy, (7) women who had undergone cervical loop electrosurgical excision procedure (LEEP), laser treatment, or cold knife conization (CKC) within the past year, and (8) women with incomplete laboratory measurements. Following the application of these criteria, a total of 439 women (aged 19 to 74 years) were included in the final analysis. The study protocol was approved by the institutional review board of the Second Xiangya Hospital of Central South University (approval number: LYF2022113) and by the Chinese medical research registration information system (Ref number: MR-43-23-027426). Written informed consent was ensured from all study participants to take part in the study voluntarily after they get informed about the objective and purpose of the study.

Study procedure and sample collection

The study participants underwent colposcopy performed by a single trained gynecologist. After inserting a vaginal dilator to fully expose the cervix, a flocked swab (SanEn biotechnology Co., Ltd, Tianjin, China) was inserted into the cervix and rotated several times to collect cervical secretions for the detection of C. trachomatis and M. genitalium. The cervical swab was then placed into 2 ml of a transport and preservation medium (Rendu Biotechnology, Shanghai, China) and immediately frozen at -80℃. Acetic acid and Lugol iodine were applied to examine the cervix and vaginal walls. Any suspicious areas were biopsied for histopathological assessment. The histopathological grading included no squamous intraepithelial lesions (NO SIL), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cervical cancer. If multiple biopsy samples were taken simultaneously, the sample with the highest grade was considered for analysis. The researchers will track and record the biopsy results of each patient in the hospital’s medical record system using the patient’s unique card number.

Before undergoing colposcopy, the patient’s laboratory measurements, including HPV types, cervical cytology tests, and the level of vaginal leukocytes, as well as demographic information such as age, pregnancy history, reproductive history, and menopause, were recorded.

Laboratory measurements

Laboratory examination data were collected when the subjects underwent colposcopy.

HPV testing was performed using the Human Papillomavirus nucleic acid typing test kit (Toujing, Shanghai, China), which allowed for the detection of 21 HPV types. Among these, 15 HPV types were classified as high-risk HPV (HR-HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68), and 6 HPV types were classified as low-risk HPV (LR-HPV: 6, 11, 42, 43, 44, and 81).

The pathologists categorized the results of the cervical cytology as follows: Negative for intraepithelial lesion or malignancy (NILM), Atypical squamous cells of undetermined significance (ASC-US), Atypical squamous cells: cannot exclude high-grade squamous intraepithelial lesion (ASC-H), Low-grade squamous intraepithelial lesion (LSIL), High-grade squamous intraepithelial lesion (HSIL), Atypical glandular cells (AGC) and Squamous cell carcinoma (SCC). None of the patients participating in this study had been diagnosed with AGC. Therefore, the AGC cytological grade is not listed in Table 1.

The levels of leukocytes in the vaginal secretions of the participants were examined by gynecologists. Based on the number of leukocytes observed in each high-power field, the examination results were classified into three categories: 0–15/HP, 16–30/HP, and > 30/HP.

All laboratory measurements were obtained from the Second Xiangya Hospital, Central South University.

Simultaneous Amplification and Testing (SAT) assay for C. trachomatis and M. genitalium detection

In a biosafety cabinet, the swab soaked in the preservation solution was fully ground and then discarded. The same volume of normal saline was added and thoroughly mixed. The target RNA was extracted using magnetic beads by adding nucleic acid extraction solution to the appropriate samples, following the instructions provided by the C. trachomatis and M. genitalium commercial test kits (Rendu Biotechnology, Shanghai, China). The magnetic beads containing the target nucleic acid were mixed with the prepared amplification detection liquid. A 30µL magnetic bead suspension was placed into each microreaction tube, and 10µL of preheated enzyme solution was added to start the reaction. The reaction process was set at 42℃ for 1 min per cycle, with a total of 40 cycles. The fluorescence signals in the fluorescein FAM channel were recorded every minute. A Ct value of ≤ 35 indicated a positive result for C. trachomatis RNA or M. genitalium RNA. For samples with Ct values between 35 and 40, retesting was required. A Ct value of < 40 in the retesting was considered positive.

Statistical analyses

All statistical analyses were conducted using SPSS version 25.0 (Chicago, IL, United States). The Shapiro-Wilk test was employed to assess the normal distribution of variables. Continuous variables that were not normally distributed are presented as the Median (interquartile range, IQR: Q1-Q3), while categorical variables are presented as percentages. The Chi-square test or nonparametric Kruskal-Wallis test was used to compare the three groups statistically. Chi-square trend tests were used to examine changes in the prevalence of C. trachomatis and M. genitalium by age. A multivariate logistic regression model was employed to evaluate factors associated with cervical lesions using variables with a P-value of less than 0.1 in univariate analyses. Throughout the study, results were considered statistically significant when P ≤ 0.05.

Fig. 1

Flow chart of the study procedure

Results

Socio-demographic characteristics among participants

According to the inclusion and exclusion criteria, a total of 439 out of the 500 individuals who underwent colposcopy were included in our study (Fig. 1). The median age of the participants was 45 years (IQR 36–53). Based on cervical biopsy results, the 439 HPV-positive patients were categorized into three groups: no squamous intraepithelial lesions group (NO SIL, n = 204), low-grade squamous intraepithelial lesion (LSIL) group (n = 127), and high-grade squamous intraepithelial lesion (≥ HSIL) group (n = 108). Table 1 presents the baseline characteristics of the participants. Among the participants, C. trachomatis was detected in 17 individuals (3.87%), M. genitalium in 16 individuals (3.64%), and there was no co-infection of C. trachomatis and M. genitalium. Significant differences were observed in the leukocyte level of vaginal secretions among the groups (P = 0.042), with the highest leukocyte level (> 30/HP) observed in the HSIL group (12.38%). Cervical cytology results also showed significant differences among the groups (P < 0.001), with the NILM grade being more common in the group without complicated lesions (81.86%), while the ASC-H (10.19%) and HSIL (15.74%) grades were more common in the HSIL group. No significant differences were found in age, pregnancy history, reproductive history, menopause, HPV single or mixed infection, or the positive rate of C. trachomatis or M. genitalium among the groups.

Table 1 Baseline characteristics of 439 women undergoing colposcopy in the Second Xiangya Hospital of Central South University according to the degree of cervical lesions

The prevalence of M. genitalium decreased with increasing age, but the prevalence of C. trachomatis did not

Subjects in our study were stratified by age (ranging from 19 to 74 years), and we examined the changes in the prevalence of C. trachomatis and M. genitalium with respect to age. The overall prevalence of M. genitalium was 3.64% (95% CI, 1.88-5.40%), with statistically significant differences observed among different age groups. The highest prevalence was found in women aged 19–30 years (10.20%; 95% CI, 1.41-18.99%), and it steadily decreased as women aged, reaching 1.94% (95% CI, 0.26-4.13%) in women over 50 years old (Ptrend = 0.014; Table 2). Surprisingly, the prevalence of C. trachomatis was higher in women aged 41–50 years (4.20%; 95% CI, 0.54-7.86%) and in women over 50 years old (5.16%; 95% CI, 1.64-8.68%), and it did not show a trend with increasing age (Ptrend = 0.301).

Table 2 The prevalence of Mycoplasma genitalium and Chlamydia trachomatis changes with age

The prevalence of C. trachomatis or M. genitalium was not associated with the degree of cervical lesions

Multivariate logistic regression analyses were conducted to investigate the association between C. trachomatis or M. genitalium and the severity of cervical lesions (Table 3). The results showed that neither LSIL nor HSIL phenotypes were associated with either C. trachomatis or M. genitalium in any of the models, using the NO SIL phenotype as the reference. After adjusting for all confounding factors, the prevalence of C. trachomatis or M. genitalium was not found to be associated with the severity of cervical lesions (all P > 0.05).

Table 3 Multivariable logistic regression analysis of the association between C. trachomatis or M. genitalium and the degree of cervical lesions

Association of human papillomavirus with the degree of cervical lesions

Out of the 439 women included in our study, 20 had untyped HPV test results, so we analyzed accurate HPV typing results for 419 women. The most prevalent HPV genotype was HPV52 in 129 subjects (30.79%), followed by HPV16 in 78 subjects (18.62%), HPV58 in 71 subjects (16.95%), and HPV53 in 42 subjects (10.02%). In the univariate analysis, HPV genotypes associated with cervical lesions were HPV16 (OR 3.35, 95% CI 2.10–5.33; P = 0.000), HPV31 (OR 2.67, 95% CI 1.05–6.76; P = 0.039), and HPV33 (OR 3.17, 95% CI 1.24–8.13; P = 0.016), indicating that they increased the severity of cervical lesions. Surprisingly, patients infected with HPV53 were less likely to develop advanced cervical lesions (OR 0.39, 95% CI 0.20–0.76; P = 0.006; Table 4). Due to the low number of detected cases of HPV45 and HPV11, which only appeared in the NO SIL group but not in the LSIL and HSIL groups, statistical analysis could not be conducted. In the multivariate analysis including HPV genotypes with a P-value < 0.10, the severity of cervical lesions was associated with HPV16, HPV31, HPV33, and HPV53.

Table 4 Relationship of human papillomavirus and the degree of cervical lesions by univariate and multivariate logistic regression analysis

Association of sexually transmitted infections and human papillomavirus infection with the degree of cervical lesions

Having identified the correlation between HPV16, HPV31, HPV33, and HPV53 with the extent of cervical lesions (Table 4), we further explored the combined effects of sexually transmitted infections (STIs) and HPV genotypes on the severity of cervical lesions. Due to the small number of positive cases of C. trachomatis and M. genitalium, and the fact that none of the patients infected with HPV31 and HPV33 were infected with C. trachomatis or M. genitalium, we only investigated the relationship between HPV16 or HPV53 and C. trachomatis or M. genitalium (Table 5). In the univariate logistic regression analysis, we did not observe any significant interactions between C. trachomatis or M. genitalium and HPV16 (all P > 0.05). Since patients infected with HPV53 rarely had C. trachomatis infection, and co-infection was only present in the NO SIL group but not in the LSIL and HSIL groups, statistical analysis could not be performed. Furthermore, no significant interaction was found between HPV53 and M. genitalium (P > 0.05).

Table 5 Analysis of the joint effect of sexually transmitted infections and human papillomavirus genotypes on the degree of cervical lesions

Discussion

A cross-sectional study was conducted among 439 HPV-positive women who underwent colposcopy in Hunan province, China, to investigate the prevalence and age distribution of C. trachomatis and M. genitalium infections, as well as the impact of co-infection with HPV on cervical lesions. The aim of the study was to contribute to the existing research in this field. C. trachomatis was detected in 17 participants (3.87%), while M. genitalium was detected in 16 participants (3.64%). Co-infection of C. trachomatis and M. genitalium was not observed. The prevalence of M. genitalium was highest in women aged 19–30 years (10.20%; 95% CI, 1.41-18.99%) and decreased steadily with age (Ptrend = 0.014). It was observed that HPV16, HPV31, and HPV33 may exhibit higher cervical pathogenicity in Hunan province, China. Additionally, a surprising finding was the association between elevated levels of leukocytes in vaginal secretions and cervical lesions. However, neither C. trachomatis nor M. genitalium infection, alone or in co-infection with HPV16, were found to be associated with the severity of cervical lesions. This study provided insights into the pathogenicity of different HPV subtypes on cervical lesions and explored the age distribution of M. genitalium and its relationship with cervical lesions in the HPV-positive population in Hunan province, China. These findings are expected to contribute to the understanding of the epidemic pathology of sexually transmitted pathogens and aid in the prevention, screening, and control of such pathogens.

The population included in our study had certain unique characteristics. Samples were collected from the colposcopy laboratory, and patients who underwent colposcopy were excluded if they had bacterial vaginitis, fungal vaginitis, trichomonas vaginitis, HIV, or syphilis. This exclusion was done to prevent worsening of the patients’ condition and to avoid the risk of infection for healthcare workers. Therefore, bacterial, fungal, trichomonas, HIV, and syphilis infections were not considered in our study population to eliminate any potential synergistic pathogenic effects they might have with C. trachomatis or M. genitalium.

The prevalence of both C. trachomatis and M. genitalium was relatively low, with C. trachomatis often having a higher prevalence compared to M. genitalium. For instance, among the general female population visiting gynecology departments in Sichuan province, China, the infection rates of C. trachomatis and M. genitalium were reported as 6.5% and 2.6%, respectively [10]. In a study on women undergoing cervical cancer screening in Beijing, China, the infection rates of C. trachomatis and M. genitalium were 11.3% and 1.0%, respectively [9]. In our study, the infection rate of C. trachomatis (3.87%) was slightly lower than that reported in other studies, while the infection rate of M. genitalium (3.64%) was slightly higher. Co-infection of M. genitalium with other sexually transmitted pathogens has been reported. In young high-risk women with asymptomatic bacterial vaginosis in the United States, the prevalence of M. genitalium co-infection with C. trachomatis was 29.9%, and co-infection with Neisseria gonorrhoeae was 23.6% [14]. However, in a study conducted in Belgium, Germany, Spain, and the United Kingdom, the co-infection rate of M. genitalium with C. trachomatis was only 0.6%, and with N. gonorrhoeae was 0.1% [15]. In our study, co-infection of C. trachomatis and M. genitalium was not observed, likely due to differences in the study population and the low positive rates of C. trachomatis and M. genitalium.

Studies have indicated that sexually transmitted pathogen infections are more prevalent among young women [6]. Our study revealed that the highest prevalence of M. genitalium was observed in women aged 19 to 30 years (10.20%; 95% CI, 1.41–18.99%), and this prevalence declined steadily with age, consistent with previous research [16, 17]. However, the prevalence of C. trachomatis was higher among women over 50 years old (5.16%; 95% CI, 1.64–8.68%), but not in young women under 30 years of age. This finding contradicts Chen’s research, as Chen et al. discovered that the prevalence of C. trachomatis infection was highest among the group aged ≤ 25 years and gradually decreased with age [8]. However, a study conducted in Shenzhen, China, found that the prevalence of C. trachomatis infection was higher in the group aged > 35 years compared to the group aged ≤ 35 years [18]. This finding aligns with our study and suggests that C. trachomatis is not necessarily exclusive to young women. The inconsistent results may be attributed to differences in study areas and populations. In conclusion, our findings suggest that the growing openness towards sexual concepts and the younger age at first sexual intercourse among Chinese women may increase the likelihood of sexually transmitted pathogen infections in young women, but the health of elderly women should not be disregarded.

Few studies have explored the relationship between leukocyte levels in vaginal secretions and the severity of cervical lesions. A study conducted in southwest China found that patients infected with C. trachomatis or M. genitalium were more likely to have elevated leukocyte levels in vaginal secretions [10]. In our study, we observed that increased leukocyte levels in vaginal secretions may be associated with cervical lesions. Leukocyte levels are often indicative of inflammation, but our study excluded patients with bacterial vaginitis, fungal vaginitis, and trichomonal vaginitis. The elevated leukocyte levels in the vaginal secretions of the subjects may have resulted from an imbalance in the vaginal microecology. In recent years, numerous studies have focused on the relationship between vaginal microbiota and female reproductive tract diseases [19, 20]. Some studies have revealed that an imbalance in vaginal microecology may be associated with persistent HPV infection and cervical lesions [21, 22]. In the future, metagenomics technology can be employed to further investigate the vaginal microbiota and its relationship with cervical lesions.

Persistent infection with high-risk HPV can result in cervical lesions and potentially cervical cancer. The prevalence and distribution of HPV subtypes vary across countries, races, and populations. In our study, the most prevalent HPV genotype was HPV52 (30.79%), followed by HPV16 (18.62%), HPV58 (16.95%), and HPV53 (10.02%). These results align with data from other Chinese populations. A study conducted in southern China identified HPV52, HPV16, and HPV58 as the three most common HPV subtypes [8]. In the Inner Mongolia region of China, HPV16 was the most prevalent genotype, followed by HPV58 and HPV52 [23]. This indicates that HPV16, HPV52, and HPV58 are predominant among the general Chinese population. However, a high prevalence of HPV subtypes does not necessarily imply their strong ability to cause cervical lesions. In our study, HPV16 (OR = 3.43, 95% CI, 2.13–5.53), HPV31 (OR = 3.70, 95% CI, 1.44–9.50), and HPV33 (OR = 3.71, 95% CI, 1.43–9.67) infections were associated with an increased severity of cervical lesions, whereas HPV53 infection was not likely to progress to advanced cervical lesions (OR = 0.45, 95% CI, 0.23–0.89). Therefore, it is recommended to pay more attention to patients infected with more pathogenic HPV subtypes based on local HPV epidemiological data when managing HPV-positive patients.

Many previous studies have primarily focused on the relationship between cervical cytology results and sexually transmitted pathogens [23, 24]. However, cervical cytology results do not fully represent the cervical status. Cervical biopsy serves as the gold standard for assessing cervical status, highlighting the importance of exploring the relationship between sexually transmitted pathogens, HPV infection, and cervical biopsy. In our study, we found no association between C. trachomatis and M. genitalium infection, either alone or in co-infection with HPV16, and the severity of cervical lesions. Regarding C. trachomatis infection, its association with HPV infection and cervical lesions is currently controversial. Several studies have indicated an association between C. trachomatis infection and HPV infection as well as cervical lesions [8, 23]. However, other studies have suggested that C. trachomatis infection does not increase the risk of HPV infection and cervical lesions [9, 10, 25], which aligns with our research findings. The conflicting results may stem from differences in cohorts or limitations in sample size. As for M. genitalium infection, there is limited research on its association with HPV infection and cervical lesions. A study by A et al. found no association between M. genitalium infection and HPV infection or cervical lesions [9], which is consistent with our findings. Currently, most studies are cross-sectional or retrospective. Large-scale, multicenter prospective cohort studies are warranted to explore the relationship between co-infection of sexually transmitted pathogens with HPV and cervical lesions.

This study had several limitations. Firstly, it was a cross-sectional study, which cannot accurately capture the dynamic changes in HPV infection and cervical lesions among the subjects. Secondly, the sample size was small, and due to the low infection rates of C. trachomatis and M. genitalium, the number of positive cases was insufficient to conduct comprehensive stratified analysis. Additionally, the samples collected were primarily from Changsha city, Hunan Province, which may affect the generalizability of the findings regarding the prevalence of sexually transmitted pathogens. Future research should be conducted using large longitudinal cohorts, including women from different locations, to identify and validate risk factors for HPV infection and the progression of cervical lesions, ultimately aiming to prevent cervical cancer.

In conclusion, our study suggests that greater attention should be given to M. genitalium infection among young women, while C. trachomatis infection in older women should not be overlooked. Increased levels of leukocytes in vaginal secretions may be associated with cervical lesions. In Hunan province of China, HPV16, HPV31, and HPV33 appear to have higher pathogenicity in relation to cervical lesions. The findings of this study highlight the ongoing controversy regarding whether C. trachomatis and M. genitalium infection, either alone or in co-infection with HPV, contribute to an increased severity of cervical lesions. Further clarification is needed through large longitudinal cohort studies in the future.