The Risk Factors for Cervical Cytological Abnormalities Among Women Infected With Non-16/18 High-Risk Human Papillomavirus: Cross-sectional Study

Background High-risk human papillomavirus (hrHPV) infection is a necessary cause of almost all cervical cancers. Relative to hrHPV 16/18 infection, non-16/18 hrHPV infection is of less concern. However, the increasing prevalence of non-16/18 hrHPV infections has become an important public health issue. The early identification and treatment of cervical cytological abnormalities in women infected with non-16/18 hrHPV reduces the incidence of cervical cancer. To date, no study has examined the risk factors for cytological abnormalities in this high-risk population. Objective This population-based, cross-sectional study aimed to identify the risk factors for cervical cytological abnormalities in women infected with non-16/18 hrHPV. Methods A total of 314,587 women from the general population were recruited for cervical cancer screening at 136 primary care hospitals in Xiangyang, China. Of these, 311,604 women underwent HPV genotyping, and 17,523 non-16/18 hrHPV–positive women were referred for cytological screening according to the screening program. A logistic regression model was used to assess the risk factors for cytological abnormalities among these non-16/18 hrHPV–positive women. A separate analysis was performed to determine the factors influencing high-grade cytological abnormalities. Results The non-16/18 hrHPV infection rate was 5.88% (18,323/311,604), which was 3-fold higher than that of hrHPV 16/18 (6068/311,604, 1.95%). Among the non-16/18 hrHPV–positive women who underwent ThinPrep cytologic test, the overall prevalence rates of cervical cytological abnormalities and high-grade cytological abnormalities were 13.46% (2359/17,523) and 1.18% (206/17,523), respectively. Multivariate logistic regression analysis revealed that women with middle or high school educational attainment were at a higher risk of having cytological abnormalities than those who received primary education (odds ratio [OR] 1.31, 95% CI 1.17-1.45; P<.001, and OR 1.32, 95% CI 1.14-1.53; P<.001, respectively). Living in rural areas (OR 2.58, 95% CI 2.29-2.90; P<.001), gravidity ≥3 (OR 2.77, 95% CI 1.19-6.45; P=.02), cervix abnormalities detected in pelvic examination (OR 1.22, 95% CI 1.11-1.34; P<.001), and having a cervical cancer screening 3 years ago (OR 0.79, 95% CI 0.62-1.00; P=.048) were associated with cytological abnormalities. The risk factors for high-grade cytological abnormalities included middle school education (OR 1.45, 95% CI 1.07-1.98; P=.02), living in rural regions (OR 1.52, 95% CI 1.10-2.10; P=.01), and cervix abnormality (OR 1.72, 95% CI 1.30-2.26; P<.001). Conclusions The dominant epidemic of non-16/18 hrHPV infection is revealed in Chinese women. Multiple risk factors for cervical cytological abnormalities have been identified in women infected with non-16/18 hrHPV. These findings can provide important information for clinically actionable decisions for the screening, early diagnosis, intervention, and prevention of cervical cancer in non-16/18 hrHPV–positive women.


Introduction
Globally, cervical cancer is one of the most serious threats to the lives of women. Cervical cancer ranks fourth in terms of both incidence and mortality among women, with an estimated 604,000 new cases and 342,000 deaths globally in 2020 [1]. In China, cervical cancer is a major public health concern because of its high incidence and heavy economic burden [2]. In 2020, it was estimated that there were approximately 110,000 new cases and 59,000 deaths from cervical cancer in China. It is the sixth most frequently diagnosed cancer and the seventh leading cause of cancer-related deaths among Chinese women [3].
Cervical cancer is the most preventable and treatable form of cancer via human papillomavirus (HPV) vaccination, early diagnosis, and effective management. Persistent infection with high-risk HPV (hrHPV) is a necessary but not sufficient cause of almost all cervical cancers [4,5]. There are 14 hrHPV genotypes (16,18,31,33,35,39,45,51,52,56,58,59,66, and 68) that can be detected by real-time polymerase chain reaction assays [6], which are classified as hrHPV 16/18 and non-16/18 hrHPV by current diagnostic paradigms. The majority of cervical cancers are from infection with hrHPV 16, followed by hrHPV 18 [7]. Therefore, hrHPV 16/18 have been recognized as dominant risk factors for cervical cancer and are the focus of medical research, clinical diagnosis, and intervention. As a result, the prevalence of hrHPV 16/18 has significantly decreased over the years [8]. Researchers and the public are relatively less concerned about non-16/18 hrHPV because these infections are considered to be less prevalent and less risky than type 16/18 infections. However, recent studies have reported an increasing prevalence of non-16/18 hrHPV [9,10]. For example, a recent population-based study in China reported a prevalence of 2.2% and 15.3% for hrHPV 16/18 and non-16/18 hrHPV, respectively [11]. The prevalence of non-16/18 hrHPV infection is also a strong predictor of the persistence and progression of cervical diseases [12][13][14][15].
Women with cytological abnormalities in the cervix have a relatively high risk of cervical cancer [16]. Early identification and treatment of cervical abnormalities in the early stages or precursor phases of the neoplasm increases the likelihood of lesion regression and reduces the incidence of cervical cancer [17,18]. According to the guidelines of the American Society for Colposcopy and Cervical Pathology [19] and the Chinese Society for Colposcopy and Cervical Pathology [20], women infected with hrHPV 16/18 were directly subjected to colposcopy without cytological screening. Only the women with positive hrHPV genotypes were referred for ThinPrep cytologic test (TCT) followed by colposcopy among those with TCT-positive results. Therefore, following the detection of a non-16/18 hrHPV infection, cytological screening is a useful tool for the selection of women at risk of cervical cancer while reducing the colposcopy burden. A meta-analysis showed that cytological testing in women infected with non-16/18 hrHPV had an overall sensitivity of 69.6% and specificity of 90.2% for detecting cervical intraepithelial neoplasia or worse [21]. However, some women infected with non-16/18 hrHPV may not undergo cytological screening because of inadequate perception of the hazards associated with non-16/18 hrHPV infection or the lack of free screening programs, especially in resource-limited countries. Therefore, identifying the risk factors for cytological abnormalities among those with non-16/18 hrHPV infections will provide important information for impelling those at high risk to undergo screening and ultimately guide clinically actionable decisions for early diagnosis, monitoring, and intervention.
Nevertheless, no previous study has investigated the risk factors for abnormal cytological outcomes in individuals with non-16/18 hrHPV infections [8]. The majority of the previous studies were conducted on the whole population without considering HPV test results, and the factors under study and the conclusions were inconsistent. For example, an observational study in China showed that the risk of cytological abnormalities was associated with HPV genotype [22]. A population-based study in Nigeria showed that demographic characteristics, menopause, gravidity, parity, marital status, and education were associated with cytological abnormalities [23]. Moreover, some previous studies did not find an effect of age on cytological abnormalities in all women or those positive for HPV [24,25]. However, in some studies, the risk of cytological abnormalities significantly increased with age [22,26]. Two studies focused on individuals infected with HPV, among whom education level, years of sexually active life, and parity were risk factors for cytological abnormalities [27,28]. Besides these factors, recent studies have shown an association between cervical cancer and vaginal microbial infection [29,30]. Cervical cancer symptoms, such as bleeding after sex, abnormal vaginal discharge, and pelvic discomfort, may affect the timely diagnosis of cervical cancer [31]. The effects of these factors on cervical cytological abnormalities in individuals with non-16/18 hrHPV infections remain unknown. In particular, the potential impacts of some important factors, including vaginal microbial infection and pelvic examination (PE), on cervical cytological abnormalities have not been investigated previously.
This large population-based study of cervical cancer screening in Chinese women aimed to identify risk factors for cervical cytological abnormalities as well as high-grade cytological abnormalities among women with non-16/18 hrHPV infections, which would provide important information for the screening, early diagnosis, management, and prevention of cervical cancer in the target population (ie, non-16/18 hrHPV-positive women).

Population
The cervical cancer screening program was conducted at 136 primary care hospitals in Xiangyang, China. Participants aged ≥30 years were recruited through media publicity and government notices between January 2017 and February 2018. Women who had received HPV vaccination, were pregnant, had no sexual history, had a hysterectomy, or had a history of pelvic radiotherapy were excluded. All participants were interviewed using questionnaires and underwent PE, vaginal microenvironment test, and HPV genotyping. Women infected with hrHPV 16/18 were directly subjected to colposcopy, whereas women positive for other hrHPV genotypes were referred for TCT, followed by colposcopy in women with TCT-positive results. Histopathological diagnosis was performed if the colposcopy was abnormal or if abnormalities were suspected. A technical manual was developed to regulate the screening process, and the medical staff were trained before the project began.

Questionnaires
The questionnaire, designed by gynecological oncologists, included age, educational level, residential type (rural or urban), whether the patient is in menopause, age at menopause, family history of cancer, gravidity, parity, contraceptive methods, personal history of other cancers, cervical cancer screening history, and presence of postcoital bleeding and abnormal leucorrhea (Multimedia Appendix 1). Professionally trained clinical staff distributed the questionnaires to the participants and collected data via face-to-face interviews. All data were inputted using the double-entry method.

PE and Vagina Microenvironment Test
All recruited women underwent routine PE and vaginal microenvironment test. The purpose of the PE was not only to assess pain, bleeding, and vaginal secretions but also to screen for cervical cancer and reproductive tract infections. The PE involved the visual inspection of the vulva, internal speculum examination of the vagina and cervix, and bimanual palpation of the adnexa and uterus. Vaginal secretions were collected with high-vaginal swabs and observed under a microscope to evaluate the vaginal microecosystem, including Trichomonas vaginalis, Candida, and Gardnerella [30].

HPV Genotyping
HPV genotyping was performed using the Cobas HPV test with the Cobas 4800 (Roche Molecular Systems) system, which is approved by the US Food and Drug Administration [6]. Specimens were collected using a cervical brush and sent to the laboratory for professional examination. The Cobas HPV test can provide individual results for hrHPV 16 and hrHPV 18 and simultaneously provide the pooled results for the other 12 non-16/18 hrHPV genotypes (31,33,35,39,45,51,52,56,58,59, 66, and 68).

Ethics Approval
Ethical approval was obtained from the Ethics Review Committee of Xiangyang Central Hospital, and all procedures followed the ethical standards specified by the institution (approval 2017-004). Written informed consent was obtained from all participants. All examinations complied with the routine medical requirements, and there were provisions for patient safety.

Statistical Analysis
The enrolled participants were divided into 2 groups based on the TCT results: NILM and ASC-US+. ASC-US+ was considered to be a cervical cytological abnormality. Participants' characteristics were summarized as counts and percentages, and the chi-square or Fisher exact tests were used to compare whether there were statistical differences in the characteristics between the 2 groups.
Based on the literature and clinical knowledge about the risk factors for cytological abnormalities or cervical cancer, we considered 16 factors that may be associated with cervical cancer. Univariate logistic regression was used to quantify the effect of each factor on the TCT results. Multivariate logistic regression was subsequently performed for all included variables. The generalized variance inflation factor (GVIF) for each variable was calculated to estimate the existence of multicollinearity, and the variable with the largest GVIF [1 / (2 ×df)] was removed at each step until the GVIF [1 / (2 ×df)] for all remaining variables was less than 2.24 (ie, 5 1/2 ) [33]. Odds ratios (ORs) and their 95% CIs were also calculated. Missing data were not inputted in this study because the rate was low, with 3.41% (597/17,523) of participants having missing values for at least one variable under study.
Since high-grade cytological abnormalities closely associated with cervical cancer require more attention, we specifically identified potential risk factors for high-grade cytological abnormalities (ASC-H, HSIL, AGC, and squamous cell carcinoma) [34] using univariate logistic regression. In the multivariate logistic regression analysis, only variables with P<.10 were considered independent variables due to the small sample size. All statistical analyses were performed using R statistical software (version 4.1.1; R Foundation for Statistical Computing). Two-sided statistical tests were used in all analyses, and P<.05 was considered statistically significant. Figure 1 shows the flow of the identification and selection of participants in the study. A total of 311,604 participants in the study underwent HPV genotyping, among which 6068 (1.95%) were infected with hrHPV 16/18, and 18,323 (5.88%) were infected with non-16/18 hrHPV. Of the 18,323 non-16/18 hrHPV-positive participants, 780 (4.26%) promised to receive TCT but did not come back until the end of the program; 20 (0.11%) did not comply with the screening process and underwent colposcopy directly rather than TCT first. As a result, 17,523 participants who were infected with non-16/18 hrHPV and underwent TCT were included in the final analysis of factors associated with cervical abnormalities. The TCT results illustrated that, among them, 15,164 participants (86.54%) had NILM and 2359 (13.46%) had cytologically abnormal findings (ASC-US+). Of the 2359 cytologically abnormal findings, ASC-US was the primary abnormality in TCT (n=1775, 75.25%), followed by LSIL (n=378, 16.02%), ASC-H (n=127, 5.38%), HSIL (n=65, 2.76%), and AGC (n=14, 0.59%).  Table 1 presents the demographic characteristics and diagnosis-related variables among participants with non-16/18 hrHPV infections. We stratified the participants into 4 groups based on age, and the ages of participants were mainly concentrated in the 40-60 years age range (12, Figure  2).     (18.5-25), and overweight (≥ 25). g PE: pelvic examination.

Principal Findings
Middle or high school education, living in rural areas, gravidity ≥3, and cervix abnormalities detected in PE were the risk factors for ASC-US+ in this study. In addition, receiving cervical screening >3 years ago was negatively associated with the prevalence of ASC-US+ among women with non-16/18 hrHPV infections. Our findings may have important implications for the prevention and control of cervical cancer in non-16/18 hrHPV-positive individuals. High-risk groups identified by their risk factors should be carefully diagnosed and treated according to medical advice to prevent adverse outcomes.
We observed that age had no effect in this study. Considering the large sample size of this study (n=17,523) and broad age range (from 30 to 78 years old), we believe that the result of null effect of age on cytological abnormalities in women infected with non-16/18 hrHPV is reliable. Some previous studies also did not find an effect of age on cytological abnormalities in all women or those infected with HPV [24,25,28]. However, in some studies, the risk of cytological abnormalities increased significantly with age [22,26]. This inconsistency may be due to differences in race, social environment, behavior, and habits in different areas.
Education was an important risk factor for cytological abnormalities. Women with middle and high school education were more likely to have cytological abnormalities than those with primary school education. Previous studies have also shown that women with middle and high school education are at a higher risk for cervical cancer [4,27,35]. The reason may be that women with primary school education tend to marry earlier and have more stable sexual partners. Previous studies have reported that both women and their husbands' lifetime number of sexual partners were significantly positively correlated with cervical cancer risk [36]. Women in rural areas had a higher probability of cytological abnormalities. Poor sanitation, insufficient knowledge about cervical cancer, and poor awareness of prevention in rural areas [37] could increase vulnerability to cervical cancer. In addition, women in rural areas have a lower frequency of gynecologic examination and cervical cancer screening than those in urban areas [38], resulting in an inability to detect abnormalities and receive timely treatment. Therefore, efforts should be intensified in rural areas to popularize cervical cancer prevention knowledge and reduce the incidence of cervical cancer. Furthermore, risk factors for cytological abnormalities differ in rural and urban areas. Among rural women, middle or high school education and gravidity ≥3 were associated with an increased risk of cytological abnormalities, whereas such results were not observed in urban women. This finding means that narrowing and eventually addressing the socioeconomic gap is imperative for cervical cancer prevention.
The prevalence of cytological abnormalities significantly increased when gravidity was ≥3, which may be related to hormonal changes during pregnancy [39]. Female sex hormones (estrogen and progesterone) may affect immune function [40].
Unstable sex hormone levels reduce immunity in women, thus lowering the resistance to hrHPV, weakening the ability to clear hrHPV, and resulting in an increased probability of cytological abnormalities. Women with high gravidity who are infected with hrHPV are recommended to consult their physician for further diagnosis in a timely manner. In addition to complying with the cervical cancer screening guidelines [41], it is recommended that women who are infected with non-16/18 hrHPV undergo HPV examination and cytology test again 1 year later, even if their TCT results were NILM.
Women with cervix abnormalities in PE are more likely to have cytological abnormalities. Previous studies have shown that the appearance of the cervix is correlated with the incidence of cervical cancer [42]. In the United Kingdom, both clinical practice guidelines on the diagnosis of cancer [43] and the National Institute for Health and Care Excellence guidelines [44] recommend visualizing the cervix to facilitate timely diagnosis of women with cervical cancer. Although no such guidelines exist in the United States, the American College of Obstetricians and Gynecologists Committee on Gynecologic Practice suggests a similar approach [45]. Therefore, PE is recommended to be added to the physical examination in women to detect the abnormal appearance of the cervix and facilitate early treatment, thereby lowering the incidence of cervical cancer.
Some cohort studies have shown that cervical cytology screening can reduce the incidence of cervical cancer by detecting precancerous lesions and early-stage cancer [18,46]. We found that cervical screening performed >3 years ago was a protective factor against cytological abnormalities. However, such protective effects were not observed when screening was performed within 3 years. Women with cytological abnormalities are particularly recommended to undergo regular follow-up cytological screening to monitor the progression or regression of cervical abnormalities. Women who screened for cervical cancer within 3 years were more likely to have previous cervical abnormalities than those screened >3 years ago. Further, women who were screened for cervical cancer >3 years ago were likely to have normal results on their last cervical cancer examination, indicating a low risk of current cytological abnormalities. Undoubtedly, well-organized screening programs have been documented to reduce the incidence and mortality of cervical cancer [17,47,48]. Women are advised to adhere to the Cervical Cancer Screening Program, which is expected to expand worldwide. It is recommended that women with non-16/18 hrHPV-positive status undergo regular cervical cancer screenings regardless of disease status and follow up with doctors if abnormalities are detected upon screening.

Comparison With Prior Work
To the best of our knowledge, this is the first study investigating cytological abnormalities in women infected with non-16/18 hrHPV. A few previous studies have explored the influencing factors of cytological abnormalities in all women; however, they did not focus on this overlooked subpopulation of those infected with non-16/18 hrHPV. Compared with previous studies, one of the strengths of this study is the large sample size of 17,523 individuals collected from multiple centers, which guarantees high statistical power and good precision of the estimates. In addition, we considered other potential influencing factors, including demographic characteristics, menstruation and fertility, PE results, and vaginal microenvironment infection.

Limitations
Our study has some limitations. First, this was a cross-sectional study without detailed information from previous screening results, and all subjects were infected with non-16/18 hrHPV detected by the current screening. The HPV genotype was not considered in this study because the selected Cobas HPV test could not detect specific types of non-16/18 hrHPV. This information on specific HPV genotypes and the persistence of infection may have an impact on abnormalities according to previous research [27,49]. Second, this study included only Chinese women; the risk factors for cytological abnormalities may differ according to ethnicity, social environment, and behavioral habits. Therefore, caution should be exercised when extrapolating the conclusions to other populations. Third, personal behaviors, such as cigarette smoking and long-term oral contraceptive use, which have been proven to be cofactors in cervical cancer [50], were not controlled in our study. As a result, the relationship between these factors and cytological abnormalities could not be investigated. Finally, reporting and recall biases may exist because of the use of a self-reported questionnaire.

Conclusion
This large-scale, cross-sectional study assessed the prevalence and risk factors of cytological abnormalities in 17,523 Chinese women infected with non-16/18 hrHPV. Middle or high school education, living in rural areas, gravidity ≥3, and cervix abnormalities detected in PE were found to be risk factors for cytological abnormalities, whereas receiving cervical screening >3 years ago was associated with a reduced prevalence of cytological abnormalities. In addition, middle school education, living in rural regions, and cervix abnormality were risk factors for high-grade cytological abnormalities. More attention should be paid to improving diagnostic, management, and vaccination strategies among individuals with non-16/18 hrHPV infections.