Analysis of risk factors for persistent infection of asymptomatic women with high-risk human papilloma virus

ABSTRACT

Objective: This study aims to prevent persistentinfection, reduce the incidence of cervical cancer, and improve women's health by understanding the theoretical basis of the risk factors for continuous infection of asymptomatic women with high-risk human papilloma virus (HPV) strains via information collected, which includes the persistent infection rate and the most prevalent HPV strain types of high risk to asymptomatic women in the high-risk area of cervical cancer in Linfen, Shanxi Province.

Methods: Based on the method of cluster sampling, locations were chosen from the industrial county and agricultural county of Linfen, Shanxi Province, namely the Xiangfen and Quwo counties. Use of the convenience sampling (CS) method enables the identification of women who have sex but without symptoms of abnormal cervix for analyzing risk factors of HPV-DNA detection and performing a retrospective questionnaire survey in these 2 counties. Firstly, cervical exfoliated cell samples were collected for thin-layer liquid-based cytology test (TCT), and simultaneously testing high-risk type HPV DNA, then samples with positive testing results were retested to identify the infected HPV types. The 6-month period of testing was done to derive the 6-month persistent infection rate. The retrospective survey included concepts addressed in the questionnaire: basic situation of the research objects, menstrual history, marital status, pregnancy history, sexual habits and other aspects. The questionnaire was divided into a case group and a comparison group, which are based on the high-risk HPV-DNA testing result to ascertain whether or not there is persistent infection. Statistical analysis employed Epidate3.1 software for date entry, SPSS17.0 for date statistical analysis. Select statistic charts, Chi-Square Analysis, single-factor analysis and multivariate Logistic regression analysis to analyze the protective factors and risk factors of high-risk HPV infection. Risk factors are predicted by using the classification tree.

Results: 3000 women participated in the study. The high-risk type HPV infection rate was 8.7%, the persistent infection rate was 7.5%. The persistent infection rates for the 2 age groups (ages 18–26 and 27–30) were 6.9% and 8.7%. The persistent infection rates of Xiangfen county were 7.4% and 7.4% respectively, and those of Quwo county were 7.8% and 11.6% respectively; there was no significant difference between each pair of groups. Single risk-factor analysis showed that first-time sex at age under 20, high school/technical secondary school education or above, multiple sexual partners, having more than 2 sexual partners in the past 6 months, oral sex, and colitis are the risk factors of high-risk type HPV infection. Multivariate analysis showed that the number of sexual partners, smoking and oral sex had an effect on HPV infection. The risk of HPV infection from smoking was 5.0-fold higher, and the risk of HPV infection from oral sex was 6.1-fold higher. Having more than 2 sexual partners increase the risk of HPV infection. By the predicated model analysis, the probability of HPV conveyed by oral sex was 14.8%; if the sexual companion number was zero or more than 2 without oral sex, the probability of HPV infection was 12.1%; if there was one sexual partner who smokes without oral sex, the probability of infection was 18.6%; if there was one sexual partner who does not smoke and without oral sex, the probability of infection was 3.6%.

Conclusion: The persistent infection rate of asymptomatic women for high-risk type HPV is lower than those women in all ages. High-risk type HPV infection risk factors include the number of sexual partners, oral sex and smoking. Thus, young women may be able to reduce the risk of infection with high-risk type HPV by reducing the number of sexual partners, forming a correct sexual life habit, and avoiding smoking.

KEYWORDS:

• asymptomatic women
• human papilloma virus
• influence factors
• persistent infection

Introduction

HPV is a group of epithelial viruses with similar morphology and distribution, which has high specificity and no specific cross infection.¹ At present, WHO has defined more than 100 types of HPV, which are divided into skin type and genital tract epithelium type according to different infection sites.² High-risk types of HPV are related to the cervical cancer, precancerous lesions and other genital tract tumors; and the low-risk types of HPV often cause benign lesion such as genital warts.

After zur Hausen from German raised the hypothesis in 1970s that the onset of cervical cancer might be related to the infection of HPV,³ a great deal of researches have proved that there is an positive correlation between the content of DNA in HPV and the level of cervical lesions.^4-5 The circumcision and condoms are also related to the risk decline for male and his sexual partner. A report claims that smoking, using oral contraceptive, homosexual behavior, other sexual transmission factors, first sexual age and genetic predisposition may affect the risk of HPV infection,⁵ but there are still different epidemiological evidences currently.

At present, there are a lot of risk factors researches for cervical cancer and infection on different countries and different groups of people, but most of them focus on purely infection or other risk factors out of infection. Most researches aim at cervical cancer patients or female over 30 y old starting with the infection and continuous infection, the researches which discuss the affects to cervical cancer with the combined action of sexual activities, sexual health habits and diseases history (particularly research for female under 30 y old) are still less common. According to one report, Shanxi province is a high incidence area of cervical cancer, but there is still no studies on the risk factors of persistent infection for asymptomatic female under 30 y with high-risk type HPV in Linfen city. This study was based on the age group of 30 y old, to discuss the risk factors of persistent infection in asymptomatic women under 30 y of age and the risk factors for persistent infection in this area. Providing theoretical basis and baseline data for the prevention of cervical cancer in said area.

The current main types of HPV vaccines, also related to this study, are exclusively for HPV infection for asymptomatic female in Europe and America. HPV infection types for Chinese female are unknown, and similar research are rarely conducted in China, in where, HPV vaccines is still in a research and developing phase. Thus, as an innovation, this study aims to exploring HPV infection type in China, and provide theory basis for such HPV vaccine exclusively for China.

Results

Basic information

In this research, the age distribution of all females are between 18–30 y old, among them there are sub-totally 2000 females between 18–26 y old, 1320 in Xiangfen, 680 in Quwo. There are sub-totally 1000 females between 27–30 y old, 680 in Xiangfen and 320 in Quwo. 3000 females in total.

High-risk HPV persistent infection status

Age distribution of continuous infection

In this research, 54.8% of positive infectors were distributed between 23–26 y old, there are 88 female high-risk HPV persistent infectors between 18–26 y old which have a 4.4% persistent infection rate in said age group, 52 female high-risk HPV persistent infectors between 27–30 y old which have a 5.2% female positive rate. Through chi-square test, there is no significant difference on the high-risk HPV positive rate between the 2 age groups (chi-square = 2.764, P = 0.251). Among them, there are 57 persistent infectors between 18–26 y old with a persistent infection rate of 4.3%, and 32 female high-risk HPV persistent infectors between 27–30 y old with a persistent infection rate of 4.7%, there is no significant difference on the persistent infection rate between the 2 age groups (chi-square = 1.572, P = 0.456) in Xiangfen County; there are 31 continuous infector between 18–26 y old with a continuous infection rate of 4.6%, 20 female high-risk HPV continuous infector with a continuous infection rate of 6.3%. There is no significant difference on the continuous infection rate between the 2 age groups (chi-square = 1.634, P = 0.442) in Quwo County (Table 1).

Table 1. Difference analysis on the high-risk HPV continuous infection situation between different age groups in the two areas.

		Age Group
Area	High-risk HPV Continuous Infection Situation	18–26 y (%)	27–30 y (%)	Total (%)	χ^2	P
Xiangfen	Continuous Infection	57 (4.32)	32 (4.71)	89 (4.45)	1.572	0.456
	Temporary infection	53 (4.02)	35 (5.15)	88 (4.40)
	Negative	1210 (91.66)	613 (90.14)	1823 (91.15)
	Total	1320	680	2000
Quwo	Continuous Infection	31 (4.56)	20 (6.25)	51 (5.10)	1.634	0.442
	Temporary infection	23 (3.38)	13 (4.06)	36 (3.60)
	Negative	626 (92.06)	287 (89.69)	913 (9.13)
	Total	680	320	1000
Total	Continuous Infection	88 (4.40)	52(5.20)	140 (4.67)	2.764	0.251
	Temporary infection	76 (3.80)	48(4.80)	124 (4.13)
	Negative	1836 (91.80)	900 (90.00)	2736 (9.12)
	Total	2000	1000	3000

Area distribution of continuous infection

Among the 2000 young women in Xiangfen County, there are 89 high-risk persistent infectors with an infection rate of 4.5%; among the 1000 young women in Quwo County, there are 51 high-risk HPV positive infectors with an infection rate of 5.1%. Through the chi-square test, there is no statistical difference on high-risk HPV infection rate between the 2 areas (P = 0.426) (Table 2).

Table 2. Difference analysis on high-risk HPV infection in the two area.

	Continuous Infection	Non-continuous /Negative	Total	Chi-square	P
Xiangfen	89	1911	2000	0.633	0.426
Quwo	51	949	1000
Total	140	2860	3000

Typing distribution of high-risk HPV

Baseline survey of typing distribution

This research made a typing detection of samples collected detection in 0 month, results shown that the infection rates from high to low of the HPV typing are, respectively, HPV58(35 cases, 1.8%), HPV16(30 cases, 1.5%), HPV66(24 cases, 1.2%), HPV52(20 cases, 1.0%), HPV39(16 cases, 0.8%), HPV51(15 cases, 0.8%), HPV18(13 cases, 0.7%), HPV59(13 cases, 0.7%), HPV68(12 cases, 0.6%), HPV56(10 cases, 0.5%), HPV31(9 cases, 0.5%), HPV33(5 cases, 0.3%), HPV35(5 cases, 0.3%) and HPV45(4 cases, 0.2%)(Table 3).

Table 3. Risk typing HPV infection status in quo.

High-risk HPV Typing	Infection Cases	Infection Rate (%)
HPV58	35	1.75
HPV16	30	1.50
HPV66	24	1.20
HPV52	20	1.00
HPV39	16	0.80
HPV51	15	0.75
HPV18	13	0.65
HPV59	13	0.65
HPV68	12	0.60
HPV56	10	0.50
HPV31	9	0.45
HPV33	5	0.25
HPV35	5	0.25
HPV45	4	0.20

Merged non-high-risk HPV infection rate and typing distribution

Among the 177 high-risk HPV infectors in Xiangfen County, 52 infectors are detected to have low-risk HPV infection at the same time, the infection rate is 2.6%. Among them there are 43 infectors with only one low-risk type of HPV, 9 infectors with 2 or more types which contributes 17.3%. The type of highest infection rate is HPV 61 with an infection rate of 0.5%, then HPV81, 54, 53 with the infection rate of 0.5%, 0.4%, 0.3% respectively (Table 4).

Table 4. Low-risk HPV infection status in quo.

Low-risk HPV Typing	Infection Cases	Infection Rate (%)
61	10	0.50
81	9	0.45
54	7	0.35
53	6	0.3
84	5	0.25
62	5	0.25
70	4	0.2
55	4	0.2
73	2	0.1
42	2	0.1
6	1	0.05
67	1	0.05
82	1	0.05
83	1	0.05
42	1	0.05
40	1	0.05

Analysis for influence factors of persistent infection

Sociology status

This research involves 1738 young females range with an average of 25.36±3.071; among all these respondents, 913 people have junior high school and lower education level taking a percentage of 52.5%; 801 people have senior high school, technical secondary school and higher education level taking a percentage of 46.1%; marriage status: 3 people are unmarried, 1687 (97.1%) people are married or living together, 11 people (0.6%) are divorced or living apart and 1 person is widowed; pregnancy times: 685 people for once or less (39.4%); 1022 people for twice to 4 times, (58.8%); vaginal delivery times: 1301 people for once or less (74.9%), 398 people for more than twice (22.9%); induced abortion times: 842 people for 0 time (48.4%), 627 people for once (36.1%); 222 people for twice and above (12.8%).

Univariate analysis

The results of univariate analysis indicate that dangerous factors for high-risk HPV persistent infections include those the age for first sexual activity is below 20, education background is senior high school, technical secondary school or above, multiple sexual partners, more than 2 sexual partners in past 6 months, the sexual activity is oral sex and having colitis.(Table 5)

Table 5. Univariate analysis of high-risk HPV infection influence factors (statistically significant factors).

Variable	Case group	Control group	Chi-square	P value
First sexual activity age
Under 20 y old	18	196	4.727	0.030
20 and above	73	1431
Education background
Junior high school and lower	50	865	55.012 < 0.001
Senior high school/technical secondary school and above	148	762
If the sexual partners have other sexual partners
yes	8	74	4.594	0.032
no	63	1326
The quantity of sexual partners in past 6 months
1 or 0	87	1623	8.913	0.003
2 and above	4	12
Sexual activity is oral sex
yes	4	21	4.264	0.039
no	81	1582
Had colpitis or not
Yes	28	300	6.600	0.01
No	33	693

Multivariate analysis results

The variables with statistical significance in logistic regression analysis is presented in Table 6. Three factors risk ratio or value are shown in Table 7, Table 8 and Table 9. It is indicated that the quantity of sex partners, oral sex and smoking have influence on HPV infection. From the results, we can see that the infection risk ratio of smoking is 5.01 times than that of non-smoking; the infection risk ratio of having oral sex is 6.09 times than that of no oral sex. For the factor of sex partner quantity, 1 sex partner is the best and the infection risk ratio of HPV is increasing for 0 sex partner or more than 2 partners.

Table 6. Logistic multivariate analysis results.

Item	Estimated value	Standard tolerance	Chi-square	probability> chi-square
How many sex partners currently[2]	0.29641129	0.2857416	1.08	0.2996
How many sex partners currently[1]	−0.7908955	0.2196735	12.96	0.0003*
Do you smoke (yes)	0.80560336	0.4005007	4.05	0.0443*
Oral sex	0.9033678	0.2489635	13.17	0.0003*

Table 7. The danger ratio or value of different sex partner quantity resulting HPV infection.

Level 1	/level 2	Disadvantage ratio	probability >chi-square	Lower limit 95%	Upper limit 95%
2	0	0.82031	0.7448	0.2600188	2.8968452
1	0	0.2765456	0.0280	0.1096621	0.8547372

Table 8. The danger ratio or value of smoking resulting HPV infection.

level1	/level2	Disadvantage ratio	probability >chi-square	Lower limit 95%	Upper limit 95%
yes	no	5.0088518	0.0880	0.7438243	20.292796

Table 9. The danger ratio or value that oral sex resulting HPV infection.

Level 1	/level 2	Disadvantage ratio	probability >chi-square	Lower limit 95%	Upper limit 95%
Yes	No	6.090533	0.0017	2.1082159	15.318452

Creating prediction model by applying logistic regression and classification tree model

Through prediction model, the probability of HPV for oral sex people is 14.8%; the probability of HPV for people without oral sex but with 0 or more than 2 sex partners is 12.1%; the probability of HPV for smoking people with 1 sex partner is 18.6%; the probability of HPV for non-smoking people with 1 sex partner is 3.6% (Table 10).

Table 10. Classification tree prediction model results.

Page label	Yes	Yes	No	No
Oral sex (no) and how many sex partners currently(., 1)andsmoking or not(.no)	0.0360		0.9640
Oral sex (no) and how many sex partners currently(., 1)andsmoking or not(.yes)	0.1861		0.8139
Oral sex (no) and how many sex partners currently (0. ,2)	0.1211		0.8789
Oral sex (yes)	0.1483		0.8517

Discussion

The research results indicate that the persistent infection rate of high-risk HPV among asymptomatic female in Linfen City Shanxi Province is lower than that of other researches⁶; the infection rate is also lower compared to the overall high-risk HPV infection rate (around 10.0%) in the world,⁷ the possible reason for this may be that the objects of this research are asymptomatic people while most researches are focusing on cervical cancer patients or elder age people, so the results are different. It is reported by reference documents that the fluctuation range of HPV infection rate among people with normal cytology in different countries is 1.4–25.6%,⁸ the fluctuation range in Asian area is 1.6%−14.2%, the possible reason is that the research objects of this research are young female in sexual active phase and don't have cervix abnormal symptoms or other related medical history. The high-risk HPV types with the highest infection rate in the world are HPV16 and HPV18, compared to them, the HPV58 and HPV16 types have higher infection rates in this research; the infection rate of HPV16 is ranking in the second place as 1.5%, the infection rate of HPV58 is in the first place which is 2.9%, indicating the importance of HPV58 infection among the asymptomatic female in this area. The research of Wang Xin in Shandong can prove that the detection rate of HPV58 in uterus exfoliative cells of cervical cancer females is the highest⁹ and much Chinese research indicates that the HPV58 is an important type for young female infection. Japanese scholars found out that the positive rate of HPV58 type in tumor-like lesion of the cervix uteri epithelium and infiltrating carcinoma tissues is 32.0% while there is no large-scale research in other Southeast Asia countries; since the latitude of Japan is close to the latitude of this research, it is suggested to pay close attention to the function of HPV58 in North China cervical cancer. According to the statistics, the cervix cancer prevalence rate is 13.3/100000 in China ranking in the second place of gynecological oncology while HPV persistent infection is the necessary pathogen of the generation and development of cervix cancer. Chinese female's infection types are mainly HPV16, 58, 52, 18. Currently among all the females in China with different cervix uterus statuses as ICC, HSIL and LSIL, the infection rate of HPV16/18 is 69.7%, 44.6% and 28.2% successively; in the ICC people group samples, the 5 top infection rate types are HPVl6, 18, 58, 52 and 33, for HSIL they are HPVl6, 58, 52, 18 and 33; for LSIL, they are HPVl6, 58, 52, 18 and 33; for normal cervical tissues, they are HPVl6, 58, 52, 18 and 39. The world's highest high-risk type HPV were HPV16, HPV18, HPV31, HPV58, and HPV52, compared to the study result HPV58, HPV16, HPV66, HPV52. The infection rate of HPV58 was the highest, infection rate of HPV16 came in second, the fourth with the HPV52 infection rate. Some domestic research results is relatively consistent,¹⁰ so it can be seen that HPV16 infection rates were high in the whole world, but HPV58 and HPV52 infection is higher in the region's women, prompt the area should pay attention to HPV58 and HPV52 screening and prevention. The world of HPV vaccine including 16 and 18, including high-risk type such as HPV16, HPV18, HPV52, HPV58 and so on. For infection type distribution, Chinese researchers mainly focus on females with higher age or cervix cancer patients, currently; several researches have indicated that the cervix cancer and infection of HPV became younger in average age while there is barely any research of infection type distribution and HPV risk factors research for young females from 18 to 30. So under the situation of severe cervix cancer morbidity which is becoming younger in average age, it is necessary to strengthen the prevention and control of HPV infection among young females. This study provides the basic data for the HPV infection type distribution in China.

The persistent infection of HPV is a necessary factor to cause cervix cancer and the generation of precancerous lesions, but it is not the sufficient condition of cervix cancer; infection of HPV doesn't mean there must be the generation of cervix cancer; 70% HPV infected person will eliminate it by themselves, 90% infected person will eliminate it within 2 y and only minority high-risk HPV infected people will develop into persistent infection and finally lead to cervix cancer.¹¹

Concerning the relevant factors analysis of high-risk HPV persistent infection of females without cervix abnormal symptoms, current researches have indicated that the high-risk sexual life manners (including young age for first sexual activity, multiple sex partners and having partners with HPV) are the main dangerous factors for female high-risk HPV infection.¹² But domestic researches on HPV persistent infection dangerous factors mainly focus on cervix cancer patients, there are less researches focusing on asymptomatic females, while this study firstly carries out the research on the asymptomatic females in Linfen city Shanxi province and the research results showed that first time sex partners quantity, smoking and taking oral sex as the sexual activity are the risk factors for the high-risk HPV infection. From the results, we can see that the infection danger ratio of smoking people is 5.01 times than that of non-smoking people; the infection danger ratio of having oral sex is 6.09 times than that of no oral sex. For the factor of sex partner quantity, 1 sex partner is the best and the infection risk ratio of HPV is increasing for 0 sex partner or more than 2 partners. It is indicated that unhealthy sex life habit and smoking play an important role in the process of high-risk HPV persistent infection for young females which keeps in accordance with research results of many domestic and foreign researchers.^13-16 Ever less discussed in the literature of oral sex as a risk factor, but now more and more studies focus on the relationship between HPV and oropharyngeal cancer in recent year.^17-19 As one route of HPV infection, oral sex have being a new research point and risk factors. Since the local females usually apply tubal ligation for contraception, not so many oral contraceptive practices, so this result is different from that the oral contraceptive is a risk factor for high-risk HPV infection in domestic research.^14,20 It is indicated by the prediction tree model that the HPV development of oral sex people is 14.8%; it is suggested that local females should reduce oral sex in order to prevent HPV infection as much as possible. Based on the HPV rate of people with 0 or more than 2 sex partners but without oral sex is 12.1%, it is suggested to keep marriage sex activity and avoid extramarital sex. For smokers, the HPV rate of people with 1 sex partner and without oral sex is 18.6%, so it is reminded that smoking will increase the rate of HPV infection, and it is necessary to reduce smoking or avoid smoking as possible. For females who don't smoke, if they have 1 sex partner and don't have oral sex, HPV rate is 3.6% much lower than all the other above-mentioned situations, so it is necessary to keep a good sexual life habit.

This paper applies CS sampling method to carry out the research about privacy questions; CS is also called accidental or convenience sampling, meaning choosing people met by chance or choosing people close or people who would like to cooperate, people who are easiest to find as the research objects. Compared with random sampling, the representativeness is relatively low but considering the large amount of samples in this research and privacy questions, if acquiring research objects by random sampling method, some people may not want to answer the privacy questions or give the true answers. So the convenience sampling research results reflect the true situations of the researched people and the results analysis is possessed with more facticity avoiding information bias. Furthermore, the case group and the control group come from the same group of people avoiding selection bias; the classification of case group and control group is made after the questionnaire survey, namely the investigators and respondents don't know which group they belong to, so the survey is carried out under double-blind conditions, there is no exposure suspicion bias and recall bias of the research objects. In this research, the inspection of high-risk HPV is carried out by automatic sample preparation and real-time polymerase chain reaction technology which can avoid the influence on inspection results from the experiment technology. So the result of this research is dependable.

In summary, the persistent infection rate of asymptomatic female high-risk HPV in Linfen City Shanxi Province is lower than that of other age groups; the prevalence rate of cervical cancer and the rate of precancerous lesions are lower than that of other age groups. There are 4 common infection types of high-risk HPV in this area: HPV58, 16, 66, 52; the infection is mainly unitary type while the infection rates of dual type and triple type are relatively low. The univariate analysis indicates that: the age for first sexual activity is below 20, education background is senior high school, technical secondary school or above, multiple sexual partners, more than 2 sexual partners in past 6 months, the sexual activity is oral sex and having colitis are risk factors for high-risk HPV persistent infections. The multivariate analysis indicates that quantity of sex partners, oral sex and smoking have influence on HPV infection. From the results, we can see that the infection risk ratio of smoking people is 5.01 times that of non-smoking people; the infection risk ratio of having oral sex is 6.09 times that of no oral sex. For the factor of sex partner quantity, 1 sex partner is the best and the infection danger ratio of HPV is increasing for o sex partner or more than 2 partners.

Materials and methods

Survey objects and HPV sample sources

Adopt the method of cluster sampling to extract one industrial county and one agricultural county in Linfen City of Shanxi Province, Xiangfen county and Quwo county; adopt the method of CS to extract female these having sexual life but without abnormal cervical symptoms for HPV DNA detection analysis; retrospective risk factors questionnaire survey. Obtain the final data of this research through laboratory detection results and questionnaire analysis results. We totally collect 3000 volunteers.

Risk factors questionnaire design

This questionnaire is summarized and made according to the obtained HPV infection risk factors by consulting domestic and overseas literatures, and then released after the expert review and approval. The questionnaire content includes that the basic information of the research object, history of menses, marriage, pregnancy and birth, sexual habits and health habits etc.; the investor makes survey to the research objects after training. There is a pre-survey before formal survey, the problems found in pre-survey are improved before executing the formal survey.

Statistical analysis method

Retrospective survey: reorganize and analyze the questionnaires which were divided into disease group and control group according to whether the infection of high-risk type HPV, use Epidata3.0 to input data, and then import into SPSS17.0 for statistical analysis, among which single factor analysis uses χ2 analysis, multiple-factor analysis uses Logistic regression analysis and the prediction of risk factors uses Logistic analysis and classification trees.

The variable in this questionnaire includes classified variable and continuous variable, the assignment situation of classified variable is shown in the following table; continuous variable includes: age, menarche age, first sex age, pregnant times, induced abortion times, vaginal delivery times, number of sexual partners in the past 6 months, number of sexual partners in the past 5 years, total number of sexual partners, frequency of cleaning vagina, length of sexual life, frequency of sexual life, age of sexual partner (Table S1).

Do initial analysis on the risk factors through single factor analysis, but since such single factor analysis itself hasn't considered the effects of interaction factors between each factor (namely one independent variable may affect the level of another independent variable), a non-conditional Logistic multiple-factor analysis has been conducted after the accomplishment of the single factor analysis.

Through the Logistic regression prediction model and classification tree prediction model, we can predict the possibility that the risk factors of this research cause the high-risk HPV continuous infection.

Logistic regression prediction model is as follows:$p=\frac{e^{-0.74+0.81*\text{smoking}+0.90*\text{oral}\text{sex}+0.30*\text{sex}\text{partners}\text{quantity}2-0.79*\text{sex}\text{partners}\text{quantity}1}}{1+e^{-0.74+0.81*\text{smoking}+0.90*oral\text{sex}+0.30*\text{sex}\text{partners}\text{quantity}2-0.79*\text{sex}\text{partners}\text{quantity}1}}$

As the questionnaire survey contents of this research are relatively intimate contents such as sexual life etc., before sampling, considering that the sampled research objects may be less willing to fill out the questionnaires or fill out untrue questionnaires, which may affect the authenticity of the questionnaires and obtain poor authenticity of risk factor. Therefore, after consulting statistics experts, we adopt the method of convenience sampling in this survey, in order to obtain more realistic results.

Laboratory detection

HPV infection status in quo adopts the method of laboratory detection, collecting the cervical exfoliated cells to make TCT, and making the high-risk HPV-DNA detection. The subjects with high-risk HPV-DNA positive or the cytology result greater than ACS-US will be recalled for a colposcopy fixed-point or randomly 4-quadrant living tissue sampling, and make the Endocervical Canal Curettage(ECC) for observing the lesion situation, if the cells are detected as ASC-H or HSIL, then recall the subjects for colposcopy examination and cervical 4-quadrant tissue sampling, and make the ECC; the extracted tissue samples will be proceeded the pathological diagnosis after the treatment of paraffin-embedded sections and HE staining. Cervical exfoliated cell sampling: all the subjects receive gynecologic examination and cervical exfoliated cell sampling by qualified gynecologists, the sample will be kept in cell preservation liquid. The sampling process is the same as the regular method of liquid -based cells collection. The cell preservation liquid is preserved and transported under indoor temperature (samples are not allowed to frozen in the preservation and transshipment period). Thinprep Cytology Test: the samples of cervical exfoliated cell are concentrated transshipped to selected central lab, and made cervical exfoliated cytology pathological examination by the Thinprep Cytologic System (USA Hologic Company). The Samples for cytology examination are not allowed to frozen during the preservation. HPV DNA and mRNA detection: The cervical exfoliated cell samples are examined the DNA of 14 types of high-risk HPV by adopting the Cobas 4800 HPV detection system (Swiss Roche Company), and typing HPV to 16 type and 18 subtype at the same time. During the detection process, it adopts fully automatic sample preparation and the technology of real-time polymerase chain reaction (PCR). All positive sample of high-risk HPV-DNA are typed by Linear array HPV genotyping test kit (Swiss Roche Company). The kit detects the samples by adopting the technology of PCR and Linear Blotting Hybridization, which can be divided into 37 low-risk type and high-risk type of HPV virus. This method has higher sensitivity than the traditional ELISA method and hybrid capture method, it can detect trace amounts of HPV DNA. Making 14 types of high-risk HPV E6/E7 mRNA detection on the back-up samples of the subjects with high-risk HPV-DNA positive or the cytology result is greater than ACS-US, this detection does not type on the HPV of samples. Tissue pathology sampling and detection: the subjects satisfying the biopsy tissue sampling standard are sampled living tissue from the cervical suspicious lesion part or specified part by qualified colposcopy physicians under the direction of colposcopy. Put the tissue into individual screw-top sealed bottle with 10% neutral formalin stationary liquid, and preserve it under indoor temperature. The biopsy tissue samples are transshipped to the research central lab together, the method of paraffin-embedded sections is adapted for treatment, and make regular HE (hematoxylin - eosin) staining after preparation, then make pathological diagnosis under microscope. Some of paraffin-embedded tissue sections will be preserved for HPV typing detection, to avoid cross contamination between samples as far as possible. The pathology samples determined as CIN2+ by central lab pathology experts need to be re-examined for confirmation by outside gynecological pathology experts. The paraffin-embedded tissue samples with microscopic diagnosis of CIN1 lesion and above conduct HPV typing detection by adopting the InnoLipa HPV genotyping extra kit (Belgian Fujirebio Europe Company). This kit makes detection by technology of PCR and Linear Blotting Hybridization, which can be divided into 28 low-risk type and high-risk type of HPV virus.

Adopt SPSS17.0 to do this research and statistical analysis, mainly use rate and constituent ratio to describe the infection status in quo and type distribution for on-site high-risk type of HPV. The differences detection of rate and constituent ratio adopt chi-square testing or Fisher's Exact Test for analysis.

Quality control

Field survey: Before formal survey, do training for investors, and do pre-survey according the local actual situation to get familiar with the survey items as soon as possible, make corrections on the impertinent places in questionnaire according to the results of pre-survey.

Conducted a preliminary audit for the survey form finished every day to mainly check whether the basic information is completed, supplement the unnecessary empty items and missing items, preliminarily review whether there is any obvious fill-in error or logical error. Exclude the unsatisfactory survey forms.

Laboratory test: all the laboratory test results and relevant detections are carried out under the condition of blind method, so as to ensure the truthfulness and objectivity of obtained data.

Abbreviations

CS	=	Convenience sampling
ECC	=	Endocervical Canal Curettage
HPV	=	human papilloma virus
PCR	=	polymerase chain reaction
TCT	=	cytology test

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.