Characteristics of high-risk human papillomavirus infection in women with abnormal cervical cytology: a population-based study in Shanxi Province, China


 Background: High-risk human papillomavirus (HR-HPV) infection is widely known as the major cause of cervical intraepithelial neoplasia (CIN) and cervical cancer and it’s characteristics vary greatly in different population. Women with abnormal cervical cytology could increase the risk of cervical cancer, however, HR-HPV infection characteristics in women with abnormal cervical cytology remains unclear. Methods: This study was based on baseline survey of the CIN Cohort established in Shanxi Province, China. A total number of 2300 women with cervical abnormalities were enrolled in this study. All participants gave informed consent and agreed to HPV and thinprepcytologic test (TCT). Each individual completed a questionnaire about characteristics related to HPV infection. Results: The overall prevalence of HR-HPV in 2300 women was 32.0%, and the proportion of single and multiple HR-HPV infections were 70.2% and 29.8% in HR-HPV infection women, respectively. The top five HR-HPV genotypes were ranked as HPV16, HPV58, HPV52, HPV53 and HPV51. The prevalence of HR-HPV in atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion and above(HSIL+) were 30.8%, 36.5% and 54.9%, respectively, showing an increasing trend with the severity of cervical cytology ( χ 2 trend =13.952; p <0.001). The women aged 35~45 years, with lower education level, less frequency of bathing, multiple gravidity, multiple parity, history of gynecological diseases and premenopausal women were prone to HR-HPV infection. Conclusions: We defined the characteristics related to HR-HPV infection in abnormal cervical cytology women, and provided an insight for the development and deeply research of HPV vaccine.


Background
Human papillomavirus (HPV) infection has been identi ed as de nite human carcinogen [1], and account for more than 50% of infection-linked cancers in females [2]. Cervical cancer ranks as the fourth most common cancer amongst women worldwide, with an estimated 570,000 new cases and 311,000 deaths in 2018 worldwide [3]. High risk human papillomavirus (HR-HPV) has been identi ed as an etiological factor for cervical cancer and precancerous lesions [4].
To date, more than 100 HPV genotypes have been identi ed [5], of which approximately 40 HPV types are associated with the genital tract infection [6]. Among the numerous members of the HPV family, 15 HR-HPV genotypes (types 16,18,31,33,35,39,45,51,52,53,56,58,59,66 and 68) are etiologically associated with more than 98% of cervical cancers. Effective prophylactic vaccines against the most important carcinogenic HPV types are available, but they do not cover main types of HPV in different countries and regions [7]. Moreover, the prevalence and genotype distribution of HPV substantially vary with ethnicities, demographic and behavioral characteristics, as well as health status [8][9][10]. However, few studies have been reported in women with abnormal cervical cytology.
Cervical cytology test had been used as a diagnostic aid for earlier detection of cervical cancer [11]. Some studies found that the incidence of cervical intraepithelial neoplasia (CIN) 2~3 and cervical cancer in women with smears revealing atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) was 56.5% and 9.3%, respectively [12], the incidence of CIN 2~3 and cervical cancer in women with high-grade squamous intraepithelial lesion (HSIL) smears was 62.9% and 25.8% [13]. HPV-positive women with atypical squamous cells of undetermined signi cance (ASC-US)/ lowgrade squamous intraepithelial lesion (LSIL) cytology had a 5~year CIN3+ risk of 22.2%, for those with normal cytology this risk was 7.9% [14].These studies clearly demonstrate that women with abnormal cervical cytology had high rate of signi cant cervical lesions particularly invasive cervical disease.
However, women with what characteristics would be more susceptible to HR-HPV infection remains unclear.
It was reported that the prevalence of HPV infection in women with abnormal cervical cytology was 47.0% to 72.8% [15,16]. HPV prevalence increased with increasing severity of cervical lesions from 12% in normal cytology to 89% in invasive cervical cancer [17]. Compared to the researches of general population, women with abnormal cervical cytology might be more susceptible to HR-HPV. However, the distribution of HR-HPV infection varied in different countries and regions, and weather demographicbehavioral characteristics related to HR-HPV infection in women with cervical abnormalities are poorly understood. The primary aims of this study were to describe the characteristics of HR-HPV infection in women with abnormal cervical cytology, so as to provide foundation for preventing and controlling cervical lesions.

Study Population
This study was based on data obtained from the baseline survey of the CIN Cohort established during June 2014 to December 2014 in Shanxi Province, China. Brie y, the present survey was conducted in two counties of Shanxi province. A woman was considered eligible to enter the study if she a) had resided in Shanxi province for at least 1 year; b) had current or past sexual activity; c) not pregnant; d) no prior history of cervical cancer or precancerous lesions; e) no prior history of treatments for cervix such as Loop Electrosurgical Excision Procedure (LEEP), conization, and adnexectomy; f) agreed to participate in the present study. A total of 39,988 women aged 19~65 years were included. All participants completed a demographic and behavioral characteristic related questionnaire, medical examination and Thinprepcytological test (TCT). Of them, 37,219 women were negative for intraepithelial lesion or malignancy (NILM), 2,769 women showed abnormal cervical cytology, including 2,305 with ASC-US, 82 with ASC-H, 316 with LSIL, 54 with HSIL, 10 with squamous cervical carcinoma (SCC), and 2 with atypical glandular cells (AGC). Then, 2769 participants with abnormal cervical cytology were referred to HPV testing. Of them, 68 refused, 387 had inadequate investigation, 8 had not fully completed medical examination, 6 specimens were not suitable for detection, At last, 2,300 women were included in the present study (Fig.1). Informed consents have been signed by all the participants and this study has been approved by the Institutional Review Board of Shanxi Medical University (No: 2013-003).

Data and Sample Collection
The information of demographic characteristics, history of gynecological diseases (including vaginitis, pelvic in ammation disease, uterine broids, hyperplasia endometria, and ovarian tumors), lifestyle habits, sexual and reproductive information was collected by trained interviewers using structured questionnaire. Two samples of exfoliated cervical cells were collected after visualization of the cervix. One sample was for routine TCT, another sample was for HPV testing.

TCT test
Exfoliated cervical cells were obtained from each participant using a cytobrush. All liquid-based cytology specimens were independently diagnosed by two experienced cytopathologists. If the diagnosis differed between the 2 cytopathologists, the sample was reviewed by a third cytopathologist, and a consensus diagnosis was obtained by the three cytopathologist together. The cytology results were diagnosed according to the 2001 Bethesda System and classi ed as NILM, ASC-US, ASC-H, LSIL, HSIL, SCC and AGC. A diagnosis of ASC-US or higher was considered cytological abnormality.

HR-HPV Detection
HPV detection and genotyping was tested using HybriMax HPV Geno-Array kit (Hybribio Biotechnology Limited Corp, Chaozhou, China) according to the manufacturer's instruction. 21 types of HPV, including 15 HR-HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68) and 6 low-risk human papillomavirus (LR-HPV) (6, 11, 42, 43, 44 and CP8304) were identi ed by the ow-through hybridization technique using a TC-96/G/H6 HPV DNA Ampli cation Analyzer and an HMM-2 fast nucleic acid molecule hybridization instrument (HybriBio Ltd). Brie y, PCR was performed in a 25μL reaction mixture containing 5μL extracted DNA,0.75μL DNA Taq polymerase and 19.25μL PCR-mix solution containing MY09/11 primer system. The PCR protocol was: denaturing at 95°C for 9 min, followed by 40 cycles of 20s at 95°C, 30s at 55°C, and 30s at 72°C, at last a nally extension at 72°C for 5 min. A positive control and a negative control were run in each PCR process. The HPV-genotype result was determined by the position of the HPV-genotype probes on the microarray chip. Adding NBT/BCIP solution to display the results, a positive result was indicated by a clearly visible indigo dot. Multiple dots indicated multiple infections. The HR-HPV single infection or multiple infections was de ned as HR-HPV infection in the study.

Statistical Analyses
Statistical analyses were performed by using Statistical Package for the Social Sciences (SPSS) software version 22.0 (IBM Corporation, Armonk, New York, USA) for Windows. Count data were examined by Chisquare tests, odds ratios (ORs) and 95% con dence interval (CI) were calculated using unconditional logistic regression. Graphics were used GraphPad Prism 7 (GraphPad Software Inc, CA, USA). All reported p values were two-sided, and statistical signi cance was de ned as p<0.05.

Results
Demographic characteristics of participants A total of 2300 women were included in the study. Demographic characteristics were summarized in Table 1. The mean age was 50±13 (range 19~65) years, 1,462 (63.6%) women received education in junior high school and below, and only 36.4% with senior high school and above. 66.3% were housewives or farmers and 96 (4.2%) women with divorced, widowed, or separated. The average yearly income was under 10000 Chinese Yuan Renminbi (RMB).
Demographic-behavioral characteristics related to HR-HPV infection HR-HPV infection peak age was aged 35 to 45 years, while 60 years old and over with the lowest HR-HPV infection rate (22.7%). The women with 35~45 years old, lower education level, less frequency of bathing, multiple gravidity (number of pregnancies), multiple parity (number of deliveries), history of gynecological diseases and premenopausal women have high risk to HR-HPV infection( Table 3, (Table 4).

Discussion
In the present study, we found that women with abnormal cervical cytology had a relatively higher prevalence of HR-HPV with 32.0%, which was higher than population-based prevalence observed in Shanxi Province (15.2%) [18], Jiangxi Province (12.3%) [19], Guangdong Province (15.7%) [20] and Yunnan Province (18.1%) [21]. X. Castellsagué et al [22] reported that HPV16, 18, 31, 58, 52 were the top ve HR-HPV types in both women with normal and abnormal cervical cytology according to estimates from 193 countries. Hernan et al [23] found HPV16, 53, 52, 58 and 59 were the top ve genotypes in women with abnormal cervical cytology from Bogota, Columbia. Our nding indicated that the top ve HR-HPV genotypes were HPV16, 58, 52, 53 and 51 in women with abnormal cervical cytology in Shanxi Province, China, while HPV18 was in the 12th position. The distribution of HR-HPV genotypes vary greatly worldwide, and these differences might be related to the complex geographical and biological interplay between different HPV genotypes and host immunogenetic factors.
Our results showed that the proportion of single and multiple HR-HPV infections were 70.2% and 29.8%, respectively. HPV16, 58 and 39 were signi cantly more prevalent in women with single HR-HPV infection than multiple HR-HPV infections, suggested that HPV16, 58 and 39 might have stronger pathogenicity than other HR-HPV genotypes. A study conducted in Cyprus also showed that single HR-HPV infection accounted for 77.2% and multiple HR-HPV infections reached 22.8% in women HR-HPV infection combining with abnormal cervical cytology [16]. To date, there are three prophylactic vaccines targeting various HPV types, including the bivalent vaccine (HPV16/18), the quadrivalent vaccine (HPV16/18/6/11) and the nonavalent vaccine (HPV16/18/6/11/31/33/45/52/58) [24], but they do not cover the most frequent HR-HPV genotypes such as HR-HPV53, 51 in Shanxi province, China from our present study. Therefore, further work will be needed to develop the next generation vaccine which can target at more HR-HPV types.
Jing Li et al [25] reported the HR-HPV prevalence presented two age peaks in general population of China, one at age 15~24 years and the other in women aged 35~49 years, respectively. HR-HPV infection is a sexually transmitted disease, and the higher rate of infection in these women might have more frequent sexual intercourse, the cervical cells might be more vulnerable to damage and HR-HPV infection. In our study, the prevalence of HR-HPV was much higher in aged 35~45 years of women with abnormal cervical cytology. The results were inconsistent with those in the general women, suggested that women with aged 35~45 years should be regarded as high-risk groups and the focus of prevention among women with abnormal cervical cytology. Additionally, we observed that HPV16, 58 and 52 presented the highest prevalence in women aged 35~45 years, suggested that HPV16, 58 and 52 infection in this region should be given attention. Moreover, the nonavalent vaccine appears to be more valuable than quadrivalent vaccine in abnormal cervical cytology women.
Remarkably, it should be noted that HR-HPV infection rate decreased with increasing education level, the same result was demonstrated from study conducted in Yunnan Province, China [26]. The women with junior high school and below had limited knowledge about cervical cancer and its prevention. It will be necessary to improve education level of women to prevent HR-HPV infection and cervical lesions. Our ndings provided evidence that women bathing less frequent were more vulnerable to HR-HPV infection.
A possible reason was that in the surveyed area where lack of water resources. Bathing infrequently results in increasing viral or bacterial reproduction, which can be curbed by frequently cleaning the lower genital tract [27].
We found that the prevalence of HR-HPV signi cantly increased with increasing gravidity and parity, which might because of the hormonal changes of pregnancy. The female sex hormones, estrogen and progesterone, might in uence susceptibility for HR-HPV infection. The changes in susceptibility to HR-HPV might also be the result of hormone-induced changes in the host adaptive immune response [28]. Our previous studies have shown that menopause was a protective factor of HR-HPV infection, which could be ascribed to the decreased estrogen level of postmenopausal women [29][30][31]. These results indicated that HPV DNA testing and effective HPV vaccines should be implemented in premenopausal women.
Several studies reported that women with gynecological diseases were prone to HR-HPV infection due to unstable sexual hormone levels, poor function of immune system, lower resistance to HR-HPV and weak ability of viral clearance[32, 33].
Our nding indicated that HPV16 was the most frequently identi ed type in the women with ASC-US, LSIL and HSIL+, con rming that HPV16 was the main HR-HPV type associated with cervical lesions. Furthermore, we found that the infection rate of HPV16 and 33 increased signi cantly with the degree of cytological abnormality. Our analyses suggested HPV16 and HPV33 infection may be biomarker to predict cervical lesions progression in women with abnormal cervical cytology.

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In conclusion, our nding indicated that the top ve HR-HPV genotypes were HPV16, 58, 52, 53 and 51 in women with abnormal cervical cytology in Shanxi Province, China. High-risk individuals were aged 35~45 years, lower education level, less frequency of bathing, multiple gravidity, multiple parity, history of gynecological diseases and premenopausal women. Our results provided insight into HR-HPV infection related demographic-behavioral characteristics in women with abnormal cervical cytology. Based on these important leads, the prospective cohort studies are needed to provide powerful evidence.

Declarations
All data generated or analyzed during this study were included in this published article.

Ethical approval and consent to participate
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study has been approved by the Institutional Review Board of Shanxi Medical University (No: 2013-003). Informed consents have been signed by all the participants.

Consent for publication
All participants provided written and explicit consent for their anonymized data to be used in publications.
30.Wang J T GES, Ding L, Cheng Y Y, Yan J W. Association between endogenous hormones, hormone receptors and cervical cancer.      Association between demographic-behavioral characteristics and HR-HPV infection in women with abnormal cervical cytology. Women aged 35~45 years old, with lower education level, less frequency of bathing, multiple gravidity, multiple parity, history of gynecological diseases and premenopausal women have relatively higher risk for HR-HPV infection.