Prevalence of High-Risk Human Papillomavirus Infection, Associated Risk Factors, and Relationship With Cervical Precancerous Lesions in Perimenopausal and Older Women in an Area With High Cervical Cancer Incidence in China.

Cervical cancer is the fourth most common cancer diagnosed globally. In 2020, it was estimated that there were approximately 604,000 new cases, leading to 342,000 fatalities [ 1 ]. The incidence of cervical cancer varies by region and age group. In more affluent countries, incidence peaks at around 40 years of age. However, in less economically developed countries, the incidence continues to increase until the age of 55-69 years [ 2 ]. A strong association exists between cervical cancer and persistent infection with high-risk human papillomavirus (HR-HPV), with the risk of persistence increasing with age [ 3 ]. In China, individuals aged 60 years and over represent 23.5% of the total cervical cancer incidence rate [ 4 ], with the highest rate of squamous cell carcinoma seen in those aged 50 years and over [ 5 ]. With the global population aging rapidly, the number of older women is increasing every day [ 6 ]. Older women diagnosed with cervical cancer have a higher risk of mortality within three years compared to younger women [ 7 ]. They are also more likely to choose primary radiotherapy or to forgo treatment entirely [ 8 ]. Thus, older women represent a significant public health challenge in achieving the World Health Organization's global strategy for the elimination of cervical cancer [ 2 ]. Prior research has highlighted the dominance of certain HR-HPV genotypes, such as HPV-16 and HPV-18, in the etiology of cervical cancer and underscored the importance of early detection and treatment of high-grade precancerous lesions to reduce cancer incidence [ 9 ]. However, the epidemiology of HR-HPV infections and the link between specific genotypes and the development of precancerous lesions in older women have received less attention. Although the incidence of cervical cancer is declining in China [ 10 ], Lueyang County in Shaanxi Province continues to report one of the highest cervical cancer rates, while its average income remains one of the lowest in the country [ 11 ]. This underscores the need for focused research in areas like Lueyang County, where cervical cancer incidence is high and economic levels are low. This study aims to address these gaps by exploring the epidemiology and factors influencing HR-HPV infection and the relationship between HR-HPV genotypes and precancerous lesions among perimenopausal and older women in Lueyang County. By shedding light on these associations, the study seeks to contribute valuable insights towards the development and refinement of a standardized, holistic management model for HR-HPV infections and cervical-related diseases in the region, ultimately aiming to reduce morbidity and mortality associated with cervical cancer. This article was previously published on 20 September 2023 on the Research Square preprint server.

Age-specific prevalence of HPV infection Overall, 2,008 women aged 40-65 years were enrolled in this study, with an overall HPV prevalence of 21.71% (95% CI 19.93%-23.58%). Specifically, the prevalence rates for HR-HPV, Low-risk human papillomavirus (LR-HPV), and mixed-risk HPV infections were 18.43%, 6.32%, and 3.04%, respectively. The prevalence of HPV, HR-HPV, and LR-HPV infections increased with age, peaking at 60-65 years (HPV, 33.30%; HR-HPV, 28.37%; and LR-HPV, 10.74%). The prevalence of HPV, HR-HPV, LR-HPV, and mixed-risk HPV infections was significantly higher in the older group than in the perimenopausal group (P<0.001). The Cochran-Armitage trend test revealed a clear linear trend in the prevalence of HR-HPV infection with age (P<0.001) (Figure 1 , Table 1 ). A: Any, high-risk, low-risk, and mixed-risk HPV infections; B: Single, dual, and multiple HR-HPV infections; C: TBS diagnostic results. HPV: Human Papillomavirus; TBS: The Bethesda System; HR-HPV: High-risk Human Papillomavirus; ASC: Atypical Squamous Cells; LSIL: Low-grade Squamous Intraepithelial Lesion; HSIL: High-grade Squamous Intraepithelial Lesion. a Percentage of all women in study; b Percentage of women in each group; c The perimenopausal group compared with the elderly group; HPV: Human Papillomavirus; HR-HPV: High-risk Human Papillomavirus; LR-HPV: Low-risk Human Papillomavirus. The prevalence rates for single, dual, and multiple HR-HPV infections were 13.55% (95% CI 12.08%-15.12%), 3.93% (95% CI 3.13%-4.88%), and 0.95% (95% CI 0.57%-1.47%), respectively. The prevalence of single (P=0.045), double (P< 0.001), and multiple HR-HPV (P<0.001) infections was significantly higher in the older age group than in the perimenopausal group (Figure 1 , Table 1 ). Prevalence of HPV infection by genotype groups The top three HR-HPV genotypes with the highest infection rates in the overall, perimenopausal, and elderly groups were as follows: HPV-52 (3.78%), -53 (2.84%), and -58 (2.74%); HPV-52 (3.34%), -53 (2.49%), and -16 (2.37%); and HPV-58 (6.61%), -52 (5.79%), and -53 (4.41%), respectively (Figure 2 ). Moreover, the infection rates of HPV-31, -33, -39, -42, -51, -53, -58, -66, and -82 were notably higher in the elderly group than in the perimenopausal group (P<0.05). HPV: Human Papillomavirus; HR-HPV: High-risk human papillomavirus; LR-HPV: low-risk human papillomavirus. HR-HPV genotype distribution and number of HR-HPV infections across different TBS diagnostic outcomes and age groups Overall, 342 individuals underwent thin-layer cytological testing, and the prevalence of HR-HPV infection was 89.77% (n=307). Based on the The Bethesda System (TBS) diagnosis, participants were divided into four groups: normal (n=306), atypical squamous cells (ASC) (ASC-US (n=3); ASC-H (n=1)), low-grade squamous intraepithelial lesion (LSIL) (n=24), and high-grade squamous intraepithelial lesion (HSIL) groups (n=8). The age group with the highest incidence of abnormal TBS was 60-65 years (19.57%), while the age group with the lowest incidence was 40-44 years (4.55%). The incidence of ASC, LSIL, and HSIL peaked in the age group 60-65 years (ASC: 2.17%; LSIL: 11.96%; HSIL: 5.43%), while the lowest values were in the age group 40-49 years (0%), 40-44 years (2.27%), and 55-59 years (0%) (Figure 1 , Table 2 ). Significant differences were observed in the prevalence of abnormal (P<0.001), LSIL (P=0.030), and HSIL (P=0.035) between the perimenopausal and older age groups. The Cochran-Armitage trend test revealed a linear trend between abnormalities and age (P=0.005). a The perimenopausal group compared with the elderly group; b Cochran-Armitage trend test of TBS results with age. TBS: The Bethesda System; ASC: Atypical Squamous Cells; LSIL: Low-grade Squamous Intraepithelial Lesion; HSIL: High-grade Squamous Intraepithelial Lesion. The prevalence of HR-HPV infection in patients with abnormal TBS results (ASC, LSIL, and HSIL) was 100%. Additionally, the prevalence and number of infections for each HR-HPV genotype did not differ significantly between the older and perimenopausal groups. The three most common HR-HPV genotypes among patients with abnormal TBS outcomes in the overall, perimenopausal, and older groups were HPV-16 (33.33%), -52 (22.22%), -33 (19.44%), and -58 (19.44%); HPV-16 (38.89%), -52 (22.22%), and -58 (22.22%); and HPV-16 (27.78%), -33 (22.22%), and -52 (22.22%) (Table 3 ). a The perimenopausal group compared with the elderly group; b Comparison between different TBS results in the overall group; c Comparison between different TBS outcomes in the perimenopausal group; d Comparison between different TBS outcomes in the elderly group; HPV: Human Papillomavirus; HR-HPV: High-risk Human Papillomavirus; TBS: The Bethesda System; ASC: Atypical Squamous Cells; LSIL: Low-grade Squamous Intraepithelial Lesion; HSIL: High-grade Squamous Intraepithelial Lesion. Significant variations in infection rates for specific HR-HPV genotypes were observed between groups with different TBS outcomes (normal, ASC, LSIL, and HSIL) in the overall, perimenopausal, and elderly groups. The overall group, HPV-16 (P<0.001), -18 (P=0.004), -33 (P=0.019), and multiple HR-HPV (P=0.003) showed varying infection rates. In the perimenopausal group, HPV-16 (P<0.001) exhibited a significant difference, while in the elderly group, HPV-16 (P<0.001), -18 (P=0.024), and multiple HR-HPV infections (P=0.008) differ significantly (Table 3 ). Univariate logistic regression analysis of factors influencing HR-HPV infection This was conducted to identify the factors influencing HR-HPV infection in three different age groups. A total of 60 variables were included in the analysis, and we identified 26 (e.g. Age (years), Profession, and Educational level, etc.), 26 (e.g. Profession, Educational level, and Native place, etc.) and 8 (e.g. Educational level, Living environment and living conditions and Age of menarche, etc.) P<0.10 factors for the overall, perimenopausal, and older age groups, respectively with P<0.10 (Tables 5 - 10 in the Appendices). Proportion and type of missing data For all three groups, the missing data type was missing at random. Figure 3 illustrates the pattern of missing data for the overall group (see Figures 4 - 5 in the Appendices for the missing data pattern in the perimenopausal and elderly groups). The numbers below each pattern indicate the proportions of missing data for each condition. Specifically, the variables associated with sexual hygiene practices and contraceptive methods showed high rates of missing data. HPV: Human Papillomavirus Results of multivariable logistic regression analysis following multiple interpolation To address missing data, which were determined as missing at random, a multiple logistic regression analysis (Table 4 ) was conducted after multiple imputations (Table 11 in the Appendices) for each age group. The outcomes of the analysis without multiple imputations are provided in the Supplementary Material (Table 12 in the Appendices). HR-HPV: High-risk Human Papillomavirus. The factors influencing HR-HPV infection in the overall group (40-65 years) remained consistent with the results obtained without multiple imputations. These factors included a history of lower genital tract disease, multiple pregnancies, and contraception methods without tubal ligation. Additionally, we identified several new independent protective factors, including age 18 years, and the education level of spouse (high school or above). The factors influencing HR-HPV infection in the perimenopausal group remained consistent with multivariate logistic regression results without multiple imputations. These factors included multiple pregnancies and contraception without tubal ligation. Additionally, we observed a history of lower genital tract disease, nonmenopausal status, avoidance of other contraceptive methods, and never cleaning their vulva after sex were independent factors. Consistent with the multivariate logistic regression findings excluding multiple imputations, we observed that educational attainment beyond junior high school and practicing abstinence emerged as distinct protective factors against HR-HPV infection in the elderly group. Additionally, multiple imputation analyses revealed contraception without tubal ligation as an independent influencing factor.

In this study, the prevalence of HR-HPV infection among women aged 40-65 years in Lueyang County was 18.43%. The prevalence of any HPV infection, abnormal TBS results, LSIL and HSIL was significantly higher in older women compared to the perimenopausal group. The top three genotypes with the highest prevalence of infection and the most common genotypes in patients with abnormal TBS results differed in the overall, perimenopausal, and older age groups. The prevalence of HPV-16, -18, -33, and multiple HR-HPV infections increased with the severity of cervical squamous intraepithelial lesions. The independent influences on HR-HPV infections were mainly related to sexual behaviors and educational attainment. Our study revealed that the prevalence of HR-HPV infection (18.43%) was higher than that in Western countries such as Chile (9.1%) [ 16 ], but lower than the prevalence in African countries (32.3%) [ 17 ]. In addition, the prevalence of HR-HPV infection in this study was higher than that observed in the national screening population (17.7%) [ 18 ]. However, it was comparable to that in the national rural screening population (18.0%) [ 18 ]. Based on these findings, the disparity between HR HPV infection rates in Lueyang County and the western and national average rates highlights the potential impact of socioeconomics and health awareness on HPV infection rates. Lower economic status may increase the risk of HR HPV infection by limiting access to health education and screening services [ 19 ]. In addition, lack of health awareness may lead to behaviours that increase the risk of infection, such as infrequent use of protection during sexual activity [ 20 ]. Given these observations, addressing higher rates of HR HPV infection in areas such as Lueyang County requires targeted interventions to enhance services such as health education and HPV screening. In this study, the peaks of HPV infection and precancerous lesions were observed in older women, consistent with findings from those in less-developed countries [ 21 ]. While some studies have reported a decreased incidence of cervical intraepithelial neoplasia in older adults, they were primarily conducted in developed countries where older women are more likely to have undergone prior cervical cancer screening [ 2 ]. The elevated incidence of HPV infection and precancerous lesions in older women might be attributed to cohort effects. This cohort was born in the 1950s and 1960s, experiencing poorer economic conditions in China during their upbringing. This often resulted in lower educational levels, limited knowledge about HPV and cervical cancer, and historically low participation in cervical cancer screening [ 22 ]. Additionally, the increased HPV infection rate in older women may be associated with a higher incidence of persistent infection and lower clearance rates [ 23 ]. A study found that the correlation between the prevalence of HR-HPV infection and cervical cancer incidence increased with age [ 24 ]. The elevated rate of persistent HR-HPV infection and diminished clearance in older women might be attributed to age-related changes in the cervicovaginal epithelium and increased reactivation of latent infections due to age-related immune decline [ 25 ]. The distribution of HR-HPV genotypes varies significantly across regions worldwide [ 21 ]. In China, HPV-52, -16, and -58 are the most prevalent genotypes in multiple regions [ 26 ]. The three HR-HPV genotypes (HPV-58, -52, and -53) that exhibit the highest prevalence of infection in the elderly group in our study may be associated with their persistence, especially HPV-58, which was prominent. A retrospective cohort study conducted in Heilongjiang Province found HPV-58 and -53 as the most persistent genotypes, followed by HPV-52 and HPV-16 [ 27 ]. A study in the United States reported high type-specific persistence rates (> 30%) for HPV-31, -16, -58, -52, and -53 [ 28 ]. Additionally, a Finnish study revealed that HPV types 35, 58, and 52 exhibited the longest persistence [ 29 ]. HPV-31 and -35 are more prevalent in Europe and the United States but less common in China [ 30 ]. The most common HR-HPV genotypes found in patients with abnormal TBS results in this study were highly consistent with the regional distribution observed in Beijing (HPV-16, -52, -58, and -33) [ 31 ] and rural areas of Shanxi Province (HPV-16, -52, -33, -31, and -58) [ 32 ]. The findings of Song et al. align with those of this study, revealing a significant increase in HPV-16, -18, and -33 infection rates with a higher degree of cytologic abnormalities [ 33 ]. Multiple HR-HPV infections have been associated with an elevated risk of cervical intraepithelial neoplasia and cervical cancer [ 34 ]. Several studies have highlighted the major link between HR-HPV infection and sexual behaviour [ 14 ]. In addition, these studies have highlighted the multifactorial nature of the risk factors contributing to infection rates [ 15 ]. Among these, smoking is considered an important causative factor, not only because of its carcinogenic properties, but also because it may impair the immune response to HPV [ 35 ]. Early marriage and multiple pregnancies are also considered key risk factors for HR-HPV infection. These conditions are often associated with earlier initiation of sexual activity and a larger number of lifelong sexual partners, increasing the likelihood of exposure and infection with HR-HPV [ 36 ]. In addition, a history of lower genital tract disease, including sexually transmitted infections (STIs) other than HPV, has been associated with an increased risk of HR-HPV infection. The presence of STIs may lead to mucosal damage, facilitating HPV entry and infection. Low literacy has also been identified as an important risk factor for HR-HPV infection [ 37 ]. Limited health literacy can affect an individual's ability to access, understand, and use information related to HPV prevention and treatment, including vaccination and screening programmes. Additionally, a spouse with an educational level below high school was identified as an independent risk factor for HR-HPV infection in women, highlighting the importance of health education on HPV and cervical cancer for both sexes. Abstinence serves as a protective factor against HR-HPV infection in older individuals since sexual contact is the primary mode of transmission [ 12 ]. In our study, we discovered that tubal ligation contraception and other contraceptive methods, such as menopause and in vitro ejaculation, were independent risk factors for HR-HPV infection. Women who have undergone tubal ligation may undergo cervical screening less frequently owing to the convenience of this method, which does not necessitate regular monitoring by healthcare providers [ 38 ]. Moreover, women who have opted for these long-acting contraceptive methods are less likely to use condoms during intercourse, leading to the potential transmission of HPV through sexual activity and an increased risk of cervical damage [ 39 ]. Several studies have indicated a decline in condom use as individuals age. Although condoms are effective in preventing the transmission of sexually transmitted infections, older adults may be unaware of the associated risks and may not choose to use them [ 40 ]. In our study, the univariate logistic regression analysis revealed that condom use was associated with a reduced risk of HR-HPV infection in the overall study group, consistent with the findings of Niu et al. [ 15 ] . However, the multivariate logistic regression analysis showed that the protective effect of condoms was insignificant. Therefore, factors like tubal ligation and other long-acting contraception (such as during menopause) may serve as instrumental variables in decision-making regarding condom use. Thus, efforts to prevent cervical cancer among perimenopausal and older women should prioritize increasing health awareness, improving cervical cancer screening rates, and encouraging condom use [ 22 ]. However, there were some limitations to this study that were worth mentioning. Firstly, the sample size and scope might not have been fully representative of the wider population, as the data came from only one hospital in Lueyang County. This suggested that future studies would need to incorporate a more diverse and broader sample pool from multiple locations to improve representativeness. Secondly, the focus of the study was on an older age group, limited to individuals aged 60-65 years, which might have affected the generalizability of the findings to a wider group of older women. Future studies could have addressed this limitation by expanding the age range of participants to include more older women, thus providing a more comprehensive understanding of the prevalence of HR-HPV in this population and its impact.

The higher rate of abnormal TBS results in older women, compared to perimenopausal women, may be attributed to factors such as a higher prevalence of HR-HPV genotypes prone to persistent infection, an increased likelihood of multiple infections, a heightened prevalence of highly oncogenic HR-HPV genotypes, lower educational attainment, lower participation in screening programs, and lower condom usage. These findings had important clinical and public health prevention implications. Specifically, the study highlighted the need for increased education about HR-HPV risk and the benefits of regular screening, as well as the need to increase access to these screenings for older women. In addition, the study highlighted the importance of promoting safe sex practices to reduce the risk of HR-HPV infection in this population. The results of the study advocated for targeted interventions that addressed the unique challenges and needs of perimenopausal and older women in order to reduce the incidence of cervical cancer in this population.

Study participants This study investigated all women who received HPV screening between September 2021 and January 2022 at the Maternal and Child Health and Family Planning Service Center in Lueyang County, Shaanxi Province. Inclusion criteria included individuals: (1) aged between 40 and 65 years, (2) residents of Lue Yang County for at least six months, (3) having engaged in sexual intercourse, (4) abstaining from sexual intercourse, vaginal douching, drug applications, and any form of transvaginal manipulation for three days prior to the examination, and (5) capable of fully understanding the study, signing the informed consent form, and voluntarily participating. Exclusion criteria included individuals: (1) who were pregnant or menstruating,(2) with a history of hysterectomy or cervical resection,(3) who had cognitive disorders preventing them from understanding or completing the study, or who refused to cooperate with the investigation. Specimen collection Before the examination, study participants were instructed to abstain from sexual intercourse, vaginal medications, and douching and should not have had menstruated in the last three days before the examination. Experienced gynecologists collected cervical and vaginal cell samples from the cervical canal using cell brushes. The collected samples were submerged in a fixative solution or coated on fixed pieces. Subsequently, they were stored in a specimen transport medium. To maintain temperature stability between 2-8℃, the specimens were stored in an incubator with ice. Subsequently, all specimens were transported within 24 h to a medical laboratory in Xi'an, Shaanxi Province, for HPV DNA genotyping. DNA extraction and HPV genotyping DNA was extracted using Yaneng Biotechnology's HPV Nucleic Acid Extraction Reagent (Yaneng Biotechnology, Shenzhen, China). The method followed the manufacturer's protocol for cervical and vaginal cell samples. Polymerase chain reaction (PCR)-reverse dot blot hybridization was utilized to detect 23 HPV subtypes. The panel included 17 HR-HPV types: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, -59, -66, -68, -73, and -82. Additionally, six low-risk human papillomavirus (LR-HPV) genotypes were identified: HPV-6, -11, -42, -43, -81, and -83 [ 12 ]. ThinPrep cytologic test In this study, samples were obtained from the squamo-columnar junction, also known as the transformation zone of the cervix, using a specialized cervical cytology brush for meticulous examination of the cervical canal. Utmost precautions were taken to prevent any cervical injury, which could lead to bleeding and potentially compromise the results. For the liquid-based cytology method, the ThinPrep cytologic test (Shaanxi Fu'an Biotechnology Co. Ltd., Baoji City, China) was used. The collected cells were either immediately washed or the brush head was directly placed into a vial containing preservation solution, preparing the samples for subsequent analysis. HPV genotype categories We aimed to analyze HPV infection rates in different categories as follows comprehensively: (1) overall and genotype-specific infection rates; (2) HR-HPV, LR-HPV, and mixed-risk HPV categories (mixed high-risk and low-risk HPV infections), based on human carcinogenic factor classification; (3) single, dual, and multiple HR-HPV infection groups (infected with three or more genotypes); (4) age-related prevalence, with participants grouped into five categories: 40-44, 45-49, 50-54, 55-59, and 60-65 years; and (5) the perimenopausal (40-59 years) and older groups (60-65 years). Questionnaire design The questionnaire design was influenced by a comprehensive review of domestic and international literature [ 13 - 15 ]. We incorporated professional expertise and sought consultation from gynecologists, ensuring the questionnaire aligned with the specific characteristics of Lueyang County. The questionnaire included basic information, menstrual history, marital history, reproductive history, personal medical history, family oncological history, sexual behavior, hygienic behavior, cognitive situation, basic information about spouse or sexual partner, marital history, and circumcision history. The investigators were uniformly trained, standardized terminology was unified, and face-to-face conversations were used to conduct the survey, and the study participants and managing doctors were required to sign the Informed Consent Form. Ethics approval Ethical approval for this study was granted by the Maternity Service Center of Lueyang Maternal and Child Health Hospital on December 4, 2021 (Approval No. 2021-001). Statistical analysis Analyses were performed using specific software per test type: Chi-square tests and trend analyses with IBM SPSS (version 23.0, IBM Corp., Armonk, USA), logistic regression models in SAS (version 9.4), and the handling of missing data through multiple imputation techniques in R (version 4.2.1, R Foundation for Statistical Computing, Vienna, Austria). Count data rates (%) were analyzed using the Chi-square (χ2) test, which demonstrated the prevalence of specific types of HPV and cervical lesions. The Cochran-Armitage trend test was used to assess whether there was a linear relationship between the HR-HPV infection rate, histological findings, and age. Univariate and multivariate logistic regression analyses were performed to investigate the correlation between HR-HPV positivity and risk factors obtained from the questionnaire. An inverse stepwise likelihood ratio test was used for the multivariate logistic regression analysis to explore the factors influencing HR-HPV. Additionally, the Hosmer-Lemeshow test was employed to evaluate model fit. Risk factors with a p<0.10 in the univariate logistic regression analysis were included in the risk regression analysis. The statistical significance level was set at P<0.05. To address the missing data, the mice package, and Visualization and Imputation of Missing Values (VIM)   package of the R software was utilized to explore the proportion and type of missing data. The pattern of missing data was determined through a correlation matrix known as the shadow matrix. Multiple imputation techniques were employed to handle the missing data and subsequently evaluated the reliability of the interpolated results. Interpolated results with the highest Cronbach's alpha coefficients were used for the multifactor analysis following multiple imputations.